STANDARDIZATION 5.1 Introduction - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/23763/8/07_chapter_5... · to chemical based medicine and pharmaceutical necessities by giving

Chapter 5 Standardization

Md Sarfaraj Hussain Ph D Thesis (2013) Integral University 134

STANDARDIZATION

51 Introduction

Plant materials and herbal remedies derived from them represent substantial portion of

global market and in this respect internationally recognized guidelines for their quality

control are necessary WHO has recognized the need to ensure quality control of

medicinal plant products by using modern techniques and by applying suitable standards

Several Pharmacopoeia including Indian Pharmacopoeia British Pharmacopoeia

Pharmacopoeia of Republic of China Japanese Pharmacopoeia United State

Pharmacopoeia do cover monograph and quality control test for few of medicinal plants

used in the respective countries but basically these pharmacopoeia are designed to cater

to chemical based medicine and pharmaceutical necessities by giving their standards test

methods For pharmaceutical purpose the quality of medicinal plant materials must be as

high as that of other medicinal preparations However it is impossible to assay for

specific chemical entity when the bioactive ingredient is not known Further problem

posed by those preparations which contain heterogeneous mixtures Directive on the

analytical control of vegetable drug must take account of the fact that material to be

examined has complex and inconsistent composition Therefore the analytical limits

cannot be as precise as for the pure chemical compound Vegetable drugs are inevitably

inconsistent because of their composition and hence the purity and authenticity of plant

material may be influenced by several factors such as age and origins harvesting period

method of drying and so on To eliminate some of the causes of inconsistency one

should use cultivated rather than wild plant which are often heterogeneous in respect of

the factors and consequently in their content of active principles Some problems facing

purity and authenticity of plant material of crude drugs include the confusion existing

over the identity of source material the impossibility to assay for a specific chemical

entity when the bioactive ingredient is not known and the problem posed by those

preparations which contain heterogeneous mixtures The purpose of purity and

authenticity of medicinal plant products is obviously to ensure therapeutic efficacy and to

check any adulteration or non-deliberate mixing in commercial batches [1] Following

protocol for purity and authenticity of raw material has been prepared

1 Authentication

2 Preliminary Phytochemical Screening

3 fluorescence analysis

4 Foreign matter

5 Ash value

Total ash

Acid insoluble ash

Water soluble ash

6 Extractive value

Alcohal soluble extractive value

Water soluble extractive

7 Loss on drying

8 Chromatographic profiles

9 Qunatitative estimation of phytoconstituents

Total phenol content

Total flavonoids content

Total proanthocyanidin content

Total saponin content

Total alkaloidal content

52 EXPERIMENTAL

MATERIALS amp METHODS

Collection and authentication of plant material

The fresh plants of Abutilon indicum Hygrophila auriculata and Trichosanthes dioica

were collected from the field area of Baryahi Saharsa District Bihar India in January

and May 2009 The plant specimens were authenticated by Prof (Dr) Anjani Kumar

Sinha Principal M L T Saharsa College Saharsa Bihar A voucher specimen no SHC

55012009 560509 and 570509 has been deposited at the herbarium Department of

Botany M L T Saharsa College Saharsa- 852201

Preparation of extract

The plants A indicum H auriculata and T dioica were air-dried and powdered 500 g of

the powdered material were packed in muslin cloth and subjected to soxhlet extractor for

continuous hot extraction with methanol for 72 hrs separately Thereafter methanolic

extracts of each plants were filtered through Whatman paper no 42 and the resultant

filtrates were concentrated under reduced pressure and finally vacuum dried The yields

of the methanolic extract were 132 173 and 143 ww respectively

Preliminary phytochemical screening

The preliminary phytochemical screening was carried out using different plant extracts

for their content of different classes of compounds The extract obtained then subjected to

qualitative chemical tests for identification of various plant constituents present in the

crude drug The extract should be subjected to preliminary phytochemical investigation

for detection of following [2-5]

Alkaloids

Carbohydrates

Glycosides

Phenolic compounds

Flavonoids

Protein and amino acids

Saponins

Sterols

Acidic compounds

Mucilage

Resins

LipidsFats

Tests for alkaloids

Each of the extract were taken separately in 5 ml of 15 hydrochloric acid and Filtered

The filtrate was then tested with following reagents

a) Dragendorffrsquos reagent

Few drop of dragendroff‟s reagent (potassium iodide+ bismuth nitrate) were

added in each of the extract and observed for formation of orange yellow

precipitate

b) Hagerrsquos reagent

Few drops of Hager‟s reagent (a saturated solution of picric acid in cold water)

were added in extract and observed for the formation of yellow precipitate

c) Wagnerrsquos reagent

Few drops of Wagner‟s reagent (iodine solution) were added in extract and

observed for formation of precipitate

d) Mayerrsquos reagent

Few drops of Mayer‟s reagent (potassium mercuric iodide solution) were added in

extract and observed for formation of white or cream colored precipitate

Test for Carbohydrates

a) Molisch test

Small quantities of alcoholic and aqueous extracts were dissolved separately in 5

ml of distilled water and filtered To this solution 2-3 drops of α-naphthol

solutions were added followed by about 1ml of concentrated sulphuric acid along

the sides of inclined test tube so as to form two layers and observed for formation

of violet colored ring at the interface

b) Fehling reagent (Detection of reducing sugar)

Few drops of Fehling solution (alkaline solution of cupric ion complexed with

tartrate ion) were added in dilute extracts and heated for 30 minutes and observed

for formation of brink red colored precipitate

Test for Glycosides

About 2 ml of methanolic extract were taken amp subjected to following tests

a) Killer-Killani test

One ml of glacial acetic acid containing traces of ferric chloride and one ml of

concentrated sulphuric acid were added to the extract and observed for formation

of reddish brown color at the junction of two layers and the upper layer turned

bluish green in presence of glycoside

b) Borntragerrsquos test

One ml of benzene and 05 ml of dilute ammonia solution were added to the

methanolic extract and observed for formation of reddish pink color

c) Legal test

Concentrated methanolic extract was made alkaline with few drops of 10

sodium hydroxide solution and then freshly prepared sodium nitroprusside

solution were added to the solution and observed for the formation of blue color

d) Baljet test

To the concentrated methanolic extract sodium picrate reagent were added and

observed for the formation of orange and yellow color

Test for phenolic compounds

a) Ferric chloride solution

The extracts were taken in water and warmed to this 2ml of ferric chloride

solution was added and observed for the formation of green and blue color

b) Lead acetate solution

To the extract (2 ml) lead acetate solution was added and observed for the

formation of precipitate

c) Gelatin solution

A few ml of Gelatin Solution was added to the aqueous extract and observed for

the formation of precipitate or turbidity

Test for flavonoids

a) Ammonia test

Filter paper strips were dipped in alcoholic solution of extract ammoniated and

observed for color change from white to yellow

b) Shinoda test

A small quantity of extract was dissolved in 5 ml of ethanol and treated with few

drops of concentrated hydrochloric acid 05 gm of magnesium turning and

observed for the formation of pink color

Test for protein and amino acids

a) MillIonrsquos test

To a few ml of methanolic extract 5 ml of distilled water was added and filtered

To two ml of this filtrate 5-6 drops of Million‟s reagent (Solution of mercury

nitrate and nitrous acid) were added and observed for the formation of red

precipitate

b) Xanthoprotein test

To 2 ml of extract few drops of nitric acid were added by the sides of test tube and

observed for formation of yellow color

c) Biuret test

To the ammoniated alkaline filtrate of the extract 2-4 drops of 002 copper

sulphate solution were added and observed for the formation of red and violet

colour

d) Ninhydrine test

To the extract lead acetate solution was added to precipitate tannins The

precipitate was spotted on a paper chromatogram sprayed with ninhydrin reagent

and heated at 110 degC for 5 minutes and observed for the formation of red and

violet colour

Test for Saponins

a) Foam test

Few mg of residue was taken in test tube with small amount of water and shaken

vigorously for one minute and observed for formation of rich lather which was

stable for more than ten minutes To the alcoholic extract few drops of sodium

bicarbonate were added shaken well and observed for formation of Honeycomb

like frothing

Test for Sterols

The methanolic extract was evaporated to dryness and the residue was extracted with

petroleum ether and acetone The insoluble residue left after extraction with

petroleum ether amp acetone was tested for sterols as

a) Salkowski reaction

To the extract two ml of concentrated sulphuric acid were added amp observed for

formation of yellow ring at the junction which turns red after one minute

b) Herchersquos reaction

To the residue 2-3 ml of trichloroacetic acid were added heated and observed for

the formation of red and violet color

c) Libermann - Burchards reaction

To a few mg of the residue in a test ndash tube few ml of acetic anhydride was added

and gently heated The contents of the test ndash tube were cooled Few drops of

concentrated sulphuric acid were added from the side of the test ndash tube A blue

colour gave the evidence of presence of sterols

Test for terpenoids

Nollers test

A few mg of extract is taken in a dry test tube and is treated with a bit of tin foil and 05

ml of thionyl chloride It is heated gently if necessary The formation of pink colour

shows the presence of terpenoids

Test for acidic compounds

a) To the methanolic extract sodium bicarbonate solution was added and observed

for the production of effervescence

b) A small amount of alcoholic extract was taken in warm water and filtered The

filtrate was then tested with litmus paper and methyl orange and observed for

appearance of blue color

Test for Mucilage

The extract was heated with ruthenium red solution in lead acetate and observed

for the formation of pink color

Test for Resins

a) Distilled water (50 ml) was added to the extract and observed for turbidity

b) A mixture of extract in acetone (3 ml) and HCl (3 ml) was heated on a water

bath for 30 minutes and observed for pink color

Fluorescence analysis

Many herbs fluoresce when cut surface or powder is exposed to UV light and this can

help in their identification The powder drug was treated with different reagents and was

examined under UV light (254 and 366 nm) [6 7]

Ash value

Ash value is an important parameter for the purpose of evaluation of crude drugs The ash

of any organic material is composed of their non-volatile inorganic components The ash

remaining following ignition of medicinal plant materials is determined by three different

methods which measure total ash acid-insoluble ash and water-soluble ash [8 9]

Total ash

1 The total ash is designed to measure the total amount of material remaining

after ignition

2 Indian pharmacopoeia 1996 and WHO prescribes suitable methods for

determination of ash values

3 About 2-3 gm of air dried crude drug was placed in the tarred platinum or

silica dish and was incinerated at a temperature not exceeding 450 degC until

free from carbon cooled and weighed to get the total ash content

Acid insoluble ash

1 Acid insoluble ash is the residue obtained after boiling the total ash with dilute

hydrochloric acid and igniting the remaining insoluble matter This measures

the amount of silica present especially as sand and siliceous earth

2 Ash was boiled with 25 ml of hydrochloric acid for 5 minutes The insoluble

matter was collected on ash less filter paper washed with hot water and

ignited at a temperature not exceeding 450 oC to a constant weight

Water soluble ash

Water soluble ash is the difference in weight between the total ash and the

residue after treatment of total ash with water It is a good indicator of either

previous extraction of water-soluble salts in the drug or in corrected

preparation Ash was dissolved in distilled water and the insoluble part

collected on an ash less filter paper and was ignited at 450 0C to a constant

weight By subtracting the weight of insoluble part from that of ash the

weight of the soluble part of ash was obtained

Extractive value

This method determines the amount of active constituents extracted with solvents

from a given amount of medicinal plant material According to Indian Pharmacopoeia

1996 British Pharmacopoeia 1980 and WHO guideline the determination of water

soluble and alcohol soluble extractives is used as a means of evaluating crude drugs

which are not readily estimated by other means The extraction of any crude drug

with particular solvent yields a solution containing different phyto-constituents that‟s

why such extractive value provide an indication of the extent of polar medium polar

and non polar components present in the plant material [8 9]

Loss on drying

This parameter determines the amount of moisture as well as volatile components

present in a particular sample The powdered drug sample (10 gm) was placed on

a tarred evaporating dish and dried at 105 degC for 6 hours and weighed The drying

was continued until two successive reading matches each other or the difference

between two successive weighing was not more than 025 of constant weight

Foreign matter

It is the matter present in the drug Its presence may be due to faulty collection of

crude drug or due to deliberate mixing It is separated from the drug so that results

obtained from analysis of the drug gives accuracy Its percentage in the crude

drug is calculated [11]

Determination of total phenolic content

Reagents -10 FC reagent in distilled water Na2CO3 (1 M) in distilled water

and Standard (Gallic acid) 1mgml solution in methanol was prepared Different

dilutions of standard gallic acid (25 μg ml to 300 μg ml) was made in methanol

Samples preparation ndash Prepared 10 mgml solution of drug in methanol

followed by addition of 5 ml FC reagent and 4 ml Na2CO3 solution Absorbance

was taken at 765 nm after 15 minutes

Preparation of standard- 05 ml of each std dilutions was taken and added 5 ml

FC reagent and 4 ml Na2CO3 solution Absorbance was taken at 765 nm after 15

minutes

Blank solutionndash Take 05 ml methanol and 5 ml FC reagent to this add 4 ml

Na2CO3 solution

After taking the absorbance of standard dilutions as mentioned above calibration

curve was plotted (Fig 51) Phenolic contents in drug were calculated by using

standard calibration [12]

Determination of total flavonoid content

Reagents- AlCl3 (01 gmml) and CH3COONa (1M) were prepared Prepared

dilutions for quercetin (standard) from 10 μg ml to 100 μgml

Samples preparationndash 10 mgml solution of drug was prepared in methanol05

ml of solution was taken and added 15 ml methanol added To this 01 ml of

AlCl3 01 ml of CH3COONa reagents and 28 ml distilled water were added and

kept for 30 minutes After that absorbance was taken at 415 nm

Preparation of Standardndash 05 ml of standard dilution of quercetin were taken

and 15 ml methanol added To this added 01 ml of AlCl3 and 01 ml of

CH3COONa reagents were mixed followed by 28 ml distilled water and kept for

30 minutes after that absorbance was taken at 415 nm

Blank solutions- To 2 ml of methanol 01 ml of AlCl3 + 01 ml of CH3COONa

reagents and 28 ml distilled water were added

curve was plotted (Fig 52) Flavonoidal contents in drug were calculated by

using standard calibration curve [13]

Determination of total proanthocyanidins contents

Contents of proanthocyanidins were determined by the procedure of Sun et al

1998 Five hundred microliters of extract and different fractions solution were

mixed with 3 mL of 4 vanillinndashmethanol solution and 15 mL hydrochloric acid

The mixture was allowed to stand for 15 min The absorbance was measured at

500 nm while the final result was expressed as mg catechin equivalent (CE)g dry

basis [14]

Determination of total saponins content

The method used was that of Obadoni and Ochuko (2001) The samples were

ground and 20 g of each were put into a conical flask and 100 ml of 20 aqueous

ethanol were added The samples were heated over a hot water bath for 4 h with

continuous stirring at about 55 degC The mixture was filtered and the residue re-

extracted with another 200 ml 20 ethanol The combined extracts were reduced

to 40 ml over water bath at about 90degC The concentrate was transferred into a

250 ml separatory funnel and 20 ml of diethyl ether was added and shaken

vigorously The aqueous layer was recovered while the ether layer was discarded

The purification process was repeated 60 ml of n-butanol were added The

combined n-butanol extracts were washed twice with 10 ml of 5 aqueous

sodium chloride The remaining solution was heated in a water bath After

evaporation the samples were dried in the oven to a constant weight the saponin

content was calculated as percentage [15]

Determination of Total alkaloidal content

5 g of the sample were weighed into a 250 ml beaker and 200 ml of 10 acetic

acid in ethanol were added and covered and allowed to stand for 4 h This was

filtered and the extract was concentrated on a water bath to one-quarter of the

original volume Concentrated ammonium hydroxide was added dropwise to the

extract until the precipitation was complete The whole solution was allowed to

settle and the precipitated was collected and washed with dilute ammonium

hydroxide and then filtered The residue is the alkaloid which was dried and

weighed [16]

Thin Layer Chromatography (TLC profile)

The plant drug extracts were subjected to thin layer chromatography to find out the

number of compounds present in them The detail of the procedure followed is given

Preparation of the plates

The adsorbent used for thin layer chromatography was silica gel G 25 g of silica gel G

were taken in a glass mortar and about 35 ml of distilled water was added to it The

mixture was stirred with glass rod until it became homogeneous This mixture was then

allowed to swell for about 15 minutes Then an additional 15 ml of distilled water was

added to it with stirring The suspension was then transferred to a 150 ml flask fitting

with a plastic stopper and was shaken vigorously for about 2 minutes This suspension

was then spread immediately on thin layer chromatographic plates with the help of a thin

layer chromatography (TLC) applicator (SUPERFIT) of continental instruments

Bombay

Drying and storage of the plates

The freshly coated plates were then air dried until the transparence of the layer had

disappeared The plates were then stacked in a drying rack and were heated in an oven for

30 min at 110 0C The activated plates were kept in a desiccator till required for further

Application of the sample

For applying test samples on TLC plate glass capillaries were used The spots were

applied with the help of a transparent template keeping a minimum distance of 1 cm

between the two adjacent spots The spots of the samples were marked on the top of the

plate to know their identity

Chromatographic chamber conditions of saturation and the development of TLC

plates

Chromatographic rectangular glass chamber (165cm x 295cm) was used in the

experiments To avoid insufficient chamber saturation and the undesirable edge effect a

smooth sheet of filter paper approximately of 15 x 40 cm size was placed in the

chromatographic chamber in a bdquoU‟ shape and was allowed to be soaked in the developing

solvent After being thus moistened the paper was then pressed against the walls of the

chamber so that it adhered to the walls The experiments were carried out at room

temperature in diffused daylight

Spraying equipments

Compressed air sprayer with a fine nozzle was used to detect the different constituents

present on TLC plates Air compressor was attached to a glass sprayer The sprayer was

filled with about 50 ml of the detecting reagent and then used After each spray the

sprayer was washed separately with water chromic acid and distilled water and then

with acetone [17]

HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY

High performance thin layer chromatography (HPTLC) is a modern powerful analytical

technique with separation power performance and reproducibility superior to class TLC

HPTLC is still a better means to separate the various components of a mixture HPTLC is

rapidly gaining importance in biochemistry of natural products and in analysis of bio-

fluid in the field of pharmacokinetics The analytical profiles for carotenoids tropane

alkaloids flavonoids steroidal compounds anthracene aglycones and lipids have been

developed by using this technique TLCHPTLC technique is applied for the compilation

of profiles pertaining to varied range of bioconstituents such as berberine quinine and

related alkaloids opium alkaloids and eugenols [18]

For analysis of herbal drugs HPTLC offers a number of advantages the technique is

especially suitable for comparison of sample based on finger prints Fingerprint analysis

by HPTLC is one of the most powerful tools to link the botanical identity to the chemical

constituent‟s profile of the plants In combination with the microscopic investigations the

fingerprint provides the means for a convenient identity check It can be used to detect

adulteration in raw materials From the constituents profile a number of marker

compounds can be chosen which might be used to further describe the quality of the herb

or herbal preparation High performance thin layer chromatography can also be employed

for quantitative determination of such marker compounds

The production of most herbal preparation includes some extraction process It is

essential for quality assurance that this extraction is standardized The quantity of marker

compounds of their relative abundance assayed by HPTLC or HPLC is the principal

method of monitoring the production process When choosing marker compounds for a

particular herb or herbal preparation it is of critical importance that chemically well-

characterized standards are available for their quantification It is often impossible to

separate all components of a plant extract completely Therefore it must be proven with

an independent method that a given marker compound in the extract is not condasheluting

with any other substance Thus HPTLC is now a day applied to obtain fingerprint pattern

of herbal formulations quantification of active ingredients and also detection of

adulteration

Principle

High performance thin layer chromatography enables the most complicated separations

The HPTLC plates are prepared from optimized (eg particle size and particle size

distribution) absorbent layers and extremely even surfaces These plates offer greater

separation efficacy through smaller plate heights than the conventional TLC plates

Shorter analysis time detection limits in nanogram with UV absorption detection and in

pictogram range with fluorimetric detection are some additional advantages with these

plates The greater efficacy of these plates is demonstrated by fact that they can provide

typically about 4000 theoretical plates over a distance of 3 cm in ten minutes compared

with 2000 theoretical plates over 12 cm in 25 minutes for conventional TLC plates [19-

21] There are three main steps of TLC procedure (1) the samples to be analyzed are

applied to the chromatogram layer Volume precision and exact positioning are ensured

by the use of a suitable instrument (2) the solvent (mobile phase) migrates the planned

distance in the layer (stationary phase) by capillary action In the process the sample is

separated into its components After evaporation of the mobile phase the fraction remains

stored on the layer and (3) the separating track are scanned in the densitometer either in

the light beam in the visible or UV range of the spectrum Absorbance or florescence is

measured by the diffused reflectance

Steps involved in HPTLC

o Selection of chromatographic layer

o Sample and standard preparation

o Layer pre-washing

o Layer pre-conditioning

o Application of the sample and standard

o Chromatographic development

o Detection of the spots

o Scanning and documentation of chromatographic plates

53 QUANTITATIVE ESTIMATION OF BIOACTIVE

PHYTOPHARMACEUTICALS BY HPTLC METHOD

HPTLC is one of the most widely used modern sophisticated and automated form of

separation technique to establish reference fingerprints of herbs against which raw

materials can be evaluated and finished products can be assayed HPTLC provides the

means for not only for flexible screening procedures and qualitative analysis but also for

demanding quantitative determinations

531 Development and validation of HPTLC method for simultaneous estimation of

bioactive ldquoLupeol and Stigmasterolrdquo and ldquoQuercetin and Gallic acidrdquo in

methanolic extracts of A indicum H auriculata and T dioica separately

EXPERIMENTAL WORK

Materials and Equipments

Materials

Lupeol Natural Remedies Bangalore

Stigmasterol Natural Remedies Bangalore

Quercetin Natural Remedies Bangalore

Gallic acid Natural Remedies Bangalore

Ammonia Merck Ltd Mumbai

Acetone Merck Ltd Mumbai

Ethyl acetate Merck Ltd Mumbai

Methanol Merck Ltd Mumbai

Ethanol Merck Ltd Mumbai

Formic acid SD fine Chem Ltd Mumbai

Toluene SD fine Chem Ltd Mumbai

Diethyl ether SD fine Chem Ltd Mumbai

Chloroform

Acetonitrile

Merck Ltd Mumbai

Sisco Research Lab Ltd Mumbai

Dicholoromethane SD fine chem Ltd Mumbai

Acetic acid Merck India Ltd Mumbai

n-Hexane Merck India Ltd Mumbai

Equipments

Double beam UV spectrophotometer 1601 Shimadzu Japan

Melting point apparatus Remi equipments India

pH meter ndash HI84240 Microconm Italy

Stability oven Nirmal International India

Electronic balance Sartorious India

Micropippetes Tarsons India

Incubator Scientific laboratory India

Magnetic stirrer Metrex Delhi

Centrifuge Remi equipments Delhi

Deep freezer Westfrost India

Vortex mixer Nirmal international India

Viscometer Brookfield DV III ultra V60 RV

cone and plate rheometer

Applicator CAMAG Linomat V Switzerland

Scanner CAMAG TLC Scanner III

Switzerland

Pre-coated HPTLC plates E Merck India ltd Germany

Description of plate used

Sorbent Silica Gel 60

Support Aluminium sheets

Layer thickness 200 m

Pore size (nm) 6

Pore vol (mlg) 08

Particle size distribution (m) 4-8

Plate size 20 x 20 cm

F254 Fluroscent indicator

Preparation of standard and quality control (QC) samples

Stock solutions of standards Lupeol (LP) Stigmasterol (ST) Quercetin (QE) and Gallic

acid (GA) (10 mg mLminus1

) were separately prepared in methanol and dilutions were made

in the concentration range of 01 to 10 mg mLminus1

For calibration LP standard solution

(1-10 μL) was applied to a HPTLC plate to furnish amounts in the range 100ndash1000 ng

band-1

ST standard solution (05-5 μL) was applied to furnish amounts in the range 50ndash

500 ng band-1

Gallic acid (GA) standard solution (1-10 μL) was applied to a HPTLC

plate to furnish amounts in the range 100ndash1000 ng band-1

however Quercetin (QE)

standard solution (05-5 μL) was applied to furnish amounts in the range 150ndash900 ng

band-1

Peak area and amounts applied were treated by linear least-squares regression

Each amount was applied six times QC samples as low medium and high at

concentration level of 200 400 and 800 ng band-1

were taken for LP and 100 200 and

400 for ST and 150 300 and 600 ng band-1

were taken for Gallic acid (GA) and 200 400

and 800 were considered for Quercetin (QE) to carry out validation of the method

Chromatography

Chromatography was performed as described previously [22-26] on 20 cm times 10 cm

aluminum Lichrosphere HPTLC plates precoated with 200-μm layers of silica gel 60F254

(E Merck Darmstadt Germany) Samples were applied as bands 6 mm wide and 10 mm

apart by means of Camag (Muttenz Switzerland) Linomat V sample applicator equipped

with a 100-μL syringe The constant application rate was 160 nL sminus1

Linear ascending

development with toluenendashmethanolndashformic acid (702703 vvv) for LP and ST and

toulene ethyl acetate formic acid (541 vvv) for QE and GA as mobile phase was

performed in a 20 cm times 10 cm twin-trough glass chamber (Camag) previously saturated

with mobile phase for 15 min at room temperature (25plusmn2degC) and relative humidity

60plusmn5 The development distance was 8 cm (development time 10 min) and 20 mL

mobile phase was used The plates were dried at room temperature in air and derivatized

with anisaldehyde-sulphuric acid reagent for LP and ST and warmed (at 75degC for 5 min)

to identify compact bands Densitometric analysis for LP and ST was performed at 530

nm and 270 nm for QE and GA in reflectance mode with a Camag TLC scanner III

operated by WinCATS software (Version 120) The slit dimensions were 5 mm times 045

mm and the scanning speed of 20 mm sminus1

HPTLC-UV530nm fingerprinting and image analysis

The protocol for preparing sample solutions was optimized for high quality fingerprinting

and also to extract the marker compounds efficiently Since the marker compounds were

soluble in methanol therefore methanol was used for extraction The fingerprinting of

methanolic extracts of A indicum H auriculata and T dioica were executed by spotting

10 microL of suitably diluted sample solution of the methanolic extract of each plant on a

HPTLC plate Each amount was applied six times Peak area and amounts applied were

treated by linear least-squares regression The plates were developed and scanned as

same discussed above The peak areas were recorded and the amount of ST and LP and

QE and GA were calculated using the calibration curve respectively

Method validation

Validation of the developed method has been carried out as per ICH guidelines for

linearity range precision accuracy limits of detection (LOD) and quantification (LOQ)

and recovery

a) Precision and accuracy

Precision (inter and intraday) and accuracy of the assay were evaluated by performing

replicate analyses (n=6) of QC samples at low medium and high QC levels of 200 400

and 800 ng band-1

for LP 100 200 and 400 ng band-1

for ST and 150 300 and 600 ng

band-1

for GA and 200 400 and 800 ng band-1

for QE respectively Inter-day precision

and accuracy were determined by repeating the intra-day assay on three different days

Precision was expressed as the coefficient of variation (CV ) of measured

concentrations for each calibration level whereas accuracy was expressed as percentage

recovery [(Drug founddrug applied) times 100]

b) Robustness

Robustness was studied in triplicate at 400 ng bandminus1

by making small changes to mobile

phase composition mobile phase volume and duration of mobile phase saturation and

activation of TLC plates the effect on the results were examined by calculation of RSD

() and SE of peak areas Mobile phases prepared from toluenendashmethanolndashformic acid in

different proportions (65 32 03 vvv 68 29 03 vvv 72 25 03 vvv and 70

27 03 vvv) keeping the volume formic acid constant were used for chromatography

for LP and ST and for QE and GA toluene ethyl acetate formic acid (541 vvv) in

different proportions (55351 vvv 54505 vvv 55405 vvv and 631 vvv)

were used separately for chromatography Mobile phase volume and duration of

saturation investigated were 20 plusmn 2 mL (18 20 and 22 mL) and 20 plusmn 10 min (10 20 and

30 min) respectively The plates were activated at 60 plusmn 5degC for 2 5 and 7 min before

chromatography

c) Sensitivity

To estimate the limits of detection (LOD) and quantification (LOQ) blank methanol was

applied six times and the standard deviation (σ) of the analytical response was

determined The LOD was expressed as 3σslope of the calibration plot and LOQ was

expressed as 10σslope of the calibration plot

d) Recovery studies

Recovery was studied by applying the method to drug samples to which known amounts

of marker corresponding to 50 100 and 150 of the LP or ST and QE and GA had been

added Each level was analyzed in triplicates This was to check the recovery of LP or ST

and QE and GA at different levels in the extracts Recovery of the markers at different

levels in the samples was determined

54 RESULT AND DISCUSSION

Result The results of preliminary phytochemical investigation are summarized in Table

52 for A indicum H auriculata and T diocia

Table 51 Phytochemical Screening of A indicum Hauriculata and T dioica

SNo Constituents A indicum H auriculata T dioica

1 Alkaloids + + +

2 Carbohydrates + + -

3 Glycosides + + +

4 Phenolic compounds and tannins + + +

5 Flavonoids + + +

6 Terpenoids + + +

7 Saponins + + +

8 Sterols + + +

9 Proteins _ - +

10 Resins + - -

11 Lipids Fats - - -

12 Mucilage - - -

+ = Present - = Absent

Result The result of Fluorescence analysis for A indicum H auriculata and T diocia

are tabulated in Tables 52 53 and 54 respectively

Table 52 Flourescence analysis of A indicum

Solvent used Day light U V light

254 nm 366 nm

Powder as such Light green Green colour Green colour

1N HCl Dark green Green colour Cream colour

50 HCl Light green Green colour Dark green

50HNO3 Dark green Light green Buff colour

50H2SO4 Blackish brown Slight green Light green

1N NaOH Light yellow Greenish brown Moderate green

Alcoholic NaOH Light green Green colour Dark green

Methanol Light green Dark green Green colour

Benzene Yellow Slight buff Green colour

FeCl3 Brownish yellow Light green Green colour

1 KOH Light greenish Green Dark green

Lead acetate White White Florescent white

Distilled water Clear Green Green colour

Table 53 Flourescence analysis of H auriculata

254 nm 366 nm

Powder as such Light brown Light green Green

1N HCl Light green Green colour Dark green

50HNO3 Light brown Dark green Blackish green

50H2SO4 Slightly red Slight green Light green

1N NaOH Light yellow Dark green Moderate green

Methanol Light green Dark green Slightly pink

Benzene Yellow Slight buff Green

Ethyl acetate Light brown Dark green Light yellow

FeCl3 Brownish yellow White Green

1 KOH Brown Green Dark green

Lead acetate White Florescent white White

Distilled water Clear Green Green

Table 54 Flourescence analysis of T dioica

254 nm 366 nm

Powder as such Dark green Slight green Blackish green

1N HCl Greenish brown Brownish green Dark green

50HNO3 Green Brownish green Blackish green

50H2SO4 Slightly green Brownish green Light green

1N NaOH Light yellow Green colour Moderate green

Methanol Light green Dark green Dark green

Benzene Slightly yellow Slight buff Green

1 KOH Brownish black Light buff Dark green

Ash Value

Result The results of ash value of A indicum H auriculata and T diocia are

mentioned in Tables 55 56 and 57 respectively

Table 55 Ash value of A indicum

S No Total ash () Acid insoluble ash () Water soluble ash ()

1 95 28 865

2 95 27 865

3 93 27 860

Mean 943 273 863

Total ash acid insoluble and water soluble ash values of A indicum were found to be

943 273 and 863 ww respectively with respect to air-dried crude drug

Table 56 Ash value of H auriculata

1 99 147 85

2 98 146 80

3 99 149 81

Mean 986 147 82

Total ash acid insoluble and water soluble ash values of H auriculata were found to be

Table 57 Ash value of T dioica

1 655 171 798

2 665 185 795

3 665 175 795

Mean 661 177 796

Total ash acid insoluble and water soluble ash values of T dioica were found to be 661

177 and 796 ww respectively with respect to air-dried crude drug

Extractive value

Result Both alcohol soluble and water soluble extractive values of A indicum H

auriculata and T diocia have been determined and results are given in Tables 58 59

and 510 respectively

Table 58 Extractive values of A indicum

S No Alcohal soluble extractive () Water soluble extractive ()

1 352 114

2 352 112

3 360 112

Mean 352 112

Alcohal soluble and water soluble extractive values of A indicum were found to be 352

and 112 ww respectively

Table 59 Extractive values of H auriculata

1 780 1680

2 796 1600

3 796 1620

Mean 790 1600

Alcohal soluble and water soluble extractive values of H auriculata were found to be

790 and160 ww respectively

Table 510 Extractive values of T dioica

1 2232 2704

2 2192 2672

3 2224 2695

Mean 2216 2690

Alcohal soluble and water soluble extractive values of T dioica were found to be 2216

Foreign matter

Result The results of foreign matter are given in Tables 511 512 and 513

respectively A indicum H auriculata and T diocia

Table 511 Foreign matter of A indicum

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8390 0161 116

Mean 8390 0161 116

A indicum containing the foreign matter 116 ww

Table 512 Foreign matter of H auriculata

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 9890 0110 110

10000 9900 0100 100

10000 9850 0150 150

Mean 9880 0120 120

H auriculata containing the foreign matter 120 ww

Table 513 Foreign matter of T diocia

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8520 0148 148

Mean 8520 0148 148

T diocia containing the foreign matter 148 ww

Loss on drying

Result The Loss on drying of A indicum H auriculata and T diocia have been

determined and results were given in Table 514 515 and 516 respectively

Table 514 Loss on drying in A indicum powder (10 gm)

Wt of drug + Petri

dish (Before drying)

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 59740 0157 785

Moisture content of A indicum powder was found to be 785 ww

Table 515 Loss on drying in H auriculata powder (10 gm)

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

4174 +10000

Mean 51773 0629 630

Moisture content of H auriculata powder was found to be 630 ww

Table 516 Loss on drying in T dioica powder (10gm)

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 69524 0216 785

Moisture content of T dioica powder was found to be 785 ww

Result The of Total phenolic content in the methanolic extract of A indicum H

auriculata and T diocia have been estimated and results were given in Tables 517 518

and 519

Figure 51 Calibration curve of gallic acid for total phenolic content

Table 517 Content of total phenolic in methanolic extract of A indicum

S No Absorbance Intercept Slope Conc (microgmL) Mean

1 0785 0004 0008 9810

9859 plusmn0255

2 0792 0004 0008 9871

3 0796 0004 0008 9896

Total phenolic content in the methanolic extract of Aindicum was found to be 9859

plusmn0255 microgmL

Table 518 Content of total phenolic in methanolic extract of H auriculata

1 08032 0004 0008 10170

10111 plusmn72 2 07921 0004 0008 9967

3 08069 0004 0008 10197

Total phenolic content in the methanolic extract of H auriculata was found to be

10111plusmn072 microgmL

Table 519 Content of total phenolic in methanolic extract of T dioica

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

Total phenolic content in the methanolic extract of T dioica was found to be 10328

plusmn122microgmL

Total flavonoid content

Result The of Total flavonoidal content in the methanolic extract of A indicum H

Figure 52 Calibration curve of quercetin for total flavonoid content

Table 520 Content of total flavonoids in methanolic extract of A indicum

S No Absorbance Intercept Slope Conc (microgml) Mean

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

Total flavonoids content in the methanolic extract of A indicum was found to be

Table 521 Content of total flavonoids in methanolic extract of H auriculata

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

Total flavonoids content in the methanolic extract of H auriculata was found to be 9719

plusmn 239 microgmL

Table 522 Content of total flavonoids in methanolic extract of T dioica

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

Total flavonoids content in the methanolic extract of T dioica was found to be

10890plusmn377microgmL

Result The of Total proanthocyanidin content in the methanolic extract of A indicum

H auriculata and T diocia have been estimated and results were given in Tables 523

524 and 525 respectively

Figure 53 Calibration curve of catechin for total proanthocyanidin content

Table 523 Content of total proanthocyanidin in methanolic extract of A indicum

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

Total proanthocyanidin content in the methanolic extract of A indicum was found to be

1902 plusmn 0012 microgmL

Table 524 Content of total proanthocyanidin in methanolic extract of Hauriculata

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

Total proanthocyanidin content in the methanolic extract of H auriculata was found to

be 1677 plusmn 070 microgmL

Table 525 Content of total proanthocyanidin in methanolic extract of T dioica

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

Total proanthocyanidin content in the methanolic extract of T dioica was found to be

Total saponins content

Result The Total saponin of A indicum H auriculata and T diocia have been

estimated and results were given in Tables 526 527 and 528 respectively

Table 526 Total saponins content of A indicum

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

Total saponin content in A indicum was found to be 189plusmn0034 ww

Table 527 Total saponins content of H auriculata

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

Total saponin content in H auriculata was found to be 079plusmn0057 ww

Table 528 Total saponins content of Tdioica

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

Total saponin content in T dioica was found to be 088plusmn0003 ww

Result The Total alkaloidal content of A indicum H auriculata and T diocia have

been estimated and results were given in Tables 529 530 and 531 respectively

Table 529 Total alkaloidal content of A indicum

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

Total alkaloidal content in Aindicum was found to be 253plusmn0037 ww

Table 530 Total alkaloidal content of H auriculata

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

Total alkaloidal content in H auriculata was found to be 278plusmn0046 ww

Table 531 Total alkaloidal content of T dioica

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

Total alkaloidal content in T dioica was found to be 226plusmn0011 ww

TLC profile

Table 532 TLC profile of the methanolic extract of A indicum

Extract Solvent system No of spots Rf values Visualizing

Methanolic Dichloromethane

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Table 533 TLC profile of the methanolic extract of H auriculata

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Table 534 TLC profile of the methanolic extract of T dioica

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Development amp validation of HPTLC method for simultaneous estimation of

bioactive ldquoLupeol and Stigmasterolrdquo and ldquoQuercetin and Gallic acidrdquo in methanolic

extracts of H auriculata

Chromatography

Chromatogram were developed under chamber saturation conditions using toluenendash

methanolndashformic acid (702703 vvv) for LP and ST and toluene ethyl acetate

formic acid (541 vvv) for QE and GA as mobile phase or solvent system separately

(Fig 54 55 and 514 515) The both the mobile phases have been also employed for

the separation of methanolic extracts of A indicum H auriculata and T dioica

separately (Fig 58 510 512 and 514 515 516) The optimized saturation time was

found to be 10 min UV Densitometric analysis was performed at 530 nm for LP and ST

and 270 nm for QE and GA in the reflectance mode as HPTLC-UV Compact bands as

sharp symmetrical and with high resolution were obtained at RF 052plusmn002 and 028plusmn005

for LP and ST and RF 031plusmn002 and 050plusmn004 for QE and GA respectively (Fig 5) The

plates were visualized at different wavelengths 254 366 and 530 nm for LP and ST and

254 and 270 nm for QE and GA as the compounds were found to absorb at variable

spectrum range In addition this helped in the generating a better fingerprint data

whereby species could be well differentiated on enhanced visual identification of

individual compounds The methods developed here were found to be quite selective with

good baseline resolution of each compound (Fig 56 and 519) The identity of the bands

of compounds in the sample extracts was confirmed by overlaying their UV absorption

spectra with those of the standards at 530 nm and 270 nm separately (Tables 535 and

Calibration

Linearity of compounds LP and ST and QE and GA was validated by the linear

regression equation and correlation coefficient The six-point calibration curves for LP

and ST were found to be linear in the range of 100-1000 ng bandminus1

and 50-500 ng bandminus1

for QE and GA were found to be linear in the range of 100ndash1000 ng bandminus1

and 150ndash900

ng bandminus1

separately Regression equation and correlation coefficient for the reference

compound were Y = 00059x (r2=09994) for LP and Y = 0013x -0037 for ST

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

for QE which revealed a good linearity response for developed method and are presented

in Tables 535 and 541 The mean values (plusmn sd) of the slope were 00059 plusmn 00008 and

0013plusmn0006 and intercept was zero and 0037 plusmn 0004 for LP and ST and 00048plusmn00003

and 0033plusmn0008 and intercept were 0012plusmn0007 and 0017plusmn0002 for GA and QE

respectively No significant difference was observed in the slopes of standard plots

(ANOVA P gt 005)

Method validation

Intra-day and inter-day precision (as coefficient of variation CV) and accuracy of the

assay for LP and ST at three QC levels (200 400 and 800 ng band-1

) and (150 300 and

600 ng band-1

for GA and QE Intra-day precisions (n = 6) for LP and ST were le180

and le184 however the inter-day precisions were le218 and le191 and for GA and

QE intra-day precisions were le170 and le189 and the inter-day precisions were

le198 and le197 respectively which demonstrated the good precision of proposed

method Intra-day accuracy for LP and ST were 991ndash1002 and 975ndash1005 and

inter-day accuracy were 981ndash997 and 957ndash992 and intra-day accuracy for GA and

QE were 988ndash1000 and 987ndash1001 and inter-day accuracy were 994ndash997 and

988ndash998 of GA and QE respectively These values are within the acceptable range so

the method was accurate reliable and reproducible

b) Robustness

The SD and RSD was calculated for LP and ST and GA and QE separately The low

values of SD and RSD (˂2) were obtained after introducing small deliberate changes

in the method indicated that the method was robust (Table 538 and 544)

c) Sensitivity

LOD and LOQ values were 45 and 18 ng band-1

and 135 and 54 ng band-1

for LP and ST

for GA and QE respectively (Tables

536 and 542) indicating adequate assay sensitivity The LOD and LOQ were

determined from the slope of the lowest part of the calibration plot This indicated that

the proposed method exhibited a good sensitivity for the quantification of above

compounds

d) Recovery studies

Good recoveries were obtained by the fortification of the sample at three QC levels for

LP and ST The percent recoveries for both markers after sample processing and applying

were in the range of 982ndash997 (LP) and 972ndash996 (ST) and 994ndash999 (GA) and

987- 994 (QE) for as shown in Tables 539 and 545

HPTLC-UV530nm analysis of bioactive LP ST and GA QE in A indicum H

auriculata and T dioica extracts

The content of LP and ST and GA and QE was estimated in the A indicum H auriculata

and T dioica methanolic extracts by the proposed method and the results obtained are

summarized in tables 540 and 546 The percentage of LP and ST and GA and QE

obtained in the methanolic extract of each plant were given in tables 540 and 546

respectively with RSD It is for the first time a simple accurate and rapid HPTLC

method has been developed for the simultaneous quantification of bioactive compounds

in A indicum H auriculata and T dioica

Figure 54 Chromatogram of standard lupeol at RF 052

Figure 55 Chromatogram of standard stigmasterol at RF 028

Figure 56 Chromatogram of LP and ST simultaneously determined in methanolic

extract of each plant by using toluenendashmethanolndashformic acid (70 27 03 vvv) as

solvent system and scanned at 530 nm [LP (052) and ST (028)]

Figure 57 TLC image of methanolic extract of A indicum (Lanes 1ndash3) showing

superimposed tracks at of A indicum extracts at various concentrations

Figure 58 HPTLC chromatogram of methanolic extract of A indicum scanned at 530

nm [peak 1-11 LP (052) and ST (028)]

Figure 59 TLC image of methanolic extract of H auriculata (Lanes 1ndash3) showing

superimposed tracks at of HA extracts at various concentrations

Figure 510 HPTLC chromatogram of methanolic extract of H auriculata scanned at

530 nm [peak 1-9 LP (052) and ST (028)]

Figure 511 TLC image of methanolic extract of T dioica (Lanes 1ndash3) showing

superimposed tracks at of T dioica extracts at various concentrations

Figure 512 HPTLC chromatogram of methanolic extract of T dioica scanned at 530

nm [peak 1-10 LP (052) and ST (028)]

Table 535 TLC fingerprints (Rf value) of methanolic extracts of Aindicum H

auriculata and T dioica (

A Indicum H auriculata T dioica

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

Solvent system toluenendashmethanolndashformic acid (702703 vvv)

Table 536 Rf linear regression data for the calibration curve and sensitivity parameter

for LP and ST

Parameter LP ST

RF 052 028

Linearity range (ng band-1

) 100ndash1000 50ndash500

Regression equation Y=00059X+0 Y=0013-0037

Correlation coefficient (r2) 09994 09941

Slopeplusmnsd 00059plusmn00008 0013plusmn0006

Interceptplusmnsd Nil 0037plusmn0004

Standard error of slope 00011 0003

Standard error of intercept - 0014

LOD 45 18

LOQ 135 54

Table 537 Precision and accuracy of the method

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

aConcentration in ng band

Mean from six determinations (n=6) cPrecision as

coefficient of variation (CV ) = [(standard deviation)(concentration found)] times 100

dAccuracy () = [concentration found)(nominal concentration)] times 100

Table 538 Robustness of the method

Optimisation condition LP ST

SD RSD SD RSD

Mobile phase (toluenendashmethanolndashformic acid (65 32

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Mobile-phase volume (18 20 and 22 mL) 138 127 112 098

Duration of saturation (10 20 and 30 min) 192 183 107 091

Activation of TLC plates (2 5 and 7 min) 119 108 143 122

Table 539 Recovery studies of LP and ST

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Table 540 LP and ST contents estimated in methanolic extract of A indicum H

auriculata and Tdioica by developed method

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

) RSD Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Volume applied in each replicate was ten microlitres

Figure 513 Display of all tracks of methanolic extract of A indicum H auriculata and

Tdioica at 270 nm

Figure 514 HPTLC chromatogram of standard gallic acid at RF 031

Figure 515 HPTLC chromatogram of standard Quercetin at RF 050

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

Figure 518 HPTLC chromatogram of methanolic extract of T diocia scanned at 270

nm [peak 1-14 GA (031) and QA (050)]

Figure 519 Chromatogram of GA and QA simultaneously determined in by using

toluenendashethyl acetatendashformic acid (541 vvv) as solvent system scanned at 270 nm

[GA (031) and QA (050)]

Table 541 TLC fingerprints (RF value) of A Indicum H auriculata and T dioica

extracts at 270 nm for GA and QE

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Solvent system toluene ethyl acetate formic acid (541 vvv)

for gallic acid and quercetin

Parameters Gallic acid Quercetin

Rf 031 050

) 100ndash1000 150ndash900

Interceptplusmnsd 0012plusmn0007 0017plusmn0002

Standard error of intercept 00040 00011

LOD 23 41

LOQ 69 123

Gallic acid Quercetin

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

bMean from six determinations (n=6)

cPrecision as

Optimisation condition Gallic acid Quercetin

SD RSD SD RSD

Mobile phase

(Toulene ethyl acetateformic acid 55351

vvv 54505 vvv 55405 vvv and

631 vvv)

179 182 191 165

Table 545 Recovery studies of gallic acid and quercetin

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Gallic acid and quercetin contents estimated in A indicum H auriculata and T dioica

extracts by developed method

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

[1] Raskin I Ribnicky D M Komarnytsky S Ilic N Poulev A Borisjuk N

Brinker A Moreno D A Ripoll C Yakoby N O‟Neal J M Cornwell T

Pastor I and Fridlender B (2002) Plants and Human health in the twenty

first century Trends in Biotechnology 20(12) 522-531

[2] Ali M (1997) Textbook of Pharmacognosy CBS Publishers and Distributors

New Delhi

[3] Malome M H (1983) The pharmacological evaluation of natural products -

general and specific approaches to screening ethnopharmaceuticals Journal

of Ethnopharmacology 8 127

[4] Jeong Sei-Joon Miyamoto T Inagaki M Kim Youn-Chul and Higuchi

R(2000) Rotundines A-C three novel sesquiterpene alkaloids from Cyperus

rotundus Journal of Natural Product 65 616-618

[5] Trease G E and Evans M C (1983) Text book of Pharmacognosy 12th

Balliere Tindall London 343-383

[6] Chase J A and Pratt R J (1949) Fluorescence of powdered vegetable drugs

with particular reference to development of a system of identification Journal

of American Pharmaceutical Association 38 324-331

[7] Kokoshi C J Kokoski R J and Sharma P J (1958) Fluorescence of

powdered vegetable drugs under UV radiation Journal of American

Pharmaceutical Association 47 561-570

[8] Choudhry R D (1996) A Practical apporoach to industrial Pharmacognosy

Herbal drug Industries first edition 353-356

[9] The Ayurvedic Pharmacopoeia of India (1999) part ndashI Vol II 1st edition New

Delhi Government of India ministry of Health and Familr Welfare Department

of Indian system of Medicine amp Homeopathy 189-196

[10] Gupta A K (2003) Quality standards of Indian medicinal plants vol 1

published by ICMR 57-81

[11] Anonymous (1998) Quality control methods for medicinal plants WHO

Geneva

[12] Singleton V L Orthofer R and Lamuela-Raventos R M (1999) Analysis of

total phenols and other oxidation substrates and antioxidants by means of

FolinndashCiocalteau reagent Methods of Enzymology 299 152ndash178

[13] Chang C Yang M Wen H Chern J (2002) Estimation of total flavonoid

content in propolis by two complementary colorimetric methods Journal of

Food Drug Analysis 10 178-182

[14] Obdoni B O and Ochuko P O (2001) Phytochemical studies and

comparative efficacy of the crude extracts of some Homostatic plants in Edo

and Delta States of Nigeria Global Journal of Pure Appllied Sciences 8b 203-

[15] Harborne J B (1973) Phytochemical methods London Chapman and Hall

Ltd 49-188

[16] Sun B S Ricardo-Da-Silva J M and Spranger M I (1998) Critical factors of

vanillin assay for catechins and proanthocyanidins Journal of Agriculture and

Food Chemistry 46 4267ndash4274

[17] Stahl E (2005) Thin layer Chromatography-A Laboratory handbook

Springer India private limited 2nd

ed 53-83

[18] Mukherjee P K (2002) Quality Control of Herbal Drugs (Business Horizon‟s

Pharmaceutical Publishers New Delhi) 138ndash141

[19] Ali M (1998) High performance thin layer chromatography for herbal and

pharmaceutical product analysis Indian Journal of Pharmaceutical Education

32(1) 15-19

[20] Mukherjee P K (2002) Quality control of herbal drugs an application to

evaluation of botanical HPTLC Printed at syndicate binders New Delhi 494-

[21] Sethi P D (2006) HPTLC Quantitative analysis of pharmaceutical

formulations CBS publishers and distributers New Delhi 126-127 140-141

[22] Kaul N Agrawal H Patil B Kakad A and Dhaneshwar S R (2005)

Application of stability-indicating HPTLC method for quantitative

determination of metadoxine in pharmaceutical dosage form IL Farmaco 60

351ndash360

[23] Faiyazuddin Md Baboota S Ali J Ahuja A Ahmad S and Akhtar J (2009)

Validated HPTLC method for simultaneous quantitation of bioactive citral

isomers in lemongrass oil encapsulated solid lipid nanoparticle formulation

International Journal of Essential Oil Therapy 3 142ndash146

[24] Faiyazuddin M d Ahmad N Baboota S Ali J Ahmad S and Akhtar J

(2010) Chromatographic Analysis of trans and cis-Citral in Lemongrass Oil

and in a Topical Phytonanocosmeceutical Formulation and Validation of the

Method Journal of Planar Chromatography 23 233ndash236

[25] Faiyazuddin M d Ahmad S Iqbal Z Talegaonkar S Ahmad F J

Bhatnagar A Khar R K (2010) Stability indicating HPTLC method for

determination of terbutaline Sulfate in bulk and from Submicronized dry

Powder inhalers Analytical Science 26 1ndash5

[26] Faiyazuddin M d Rauf A Ahmad N Ahmad S Iqbal Z Talegaonkar S

Bhatnagar A Khar R K andAhmad F J (2011) A validated HPTLC

method for determination of terbutaline sulfate in biological samples

Application to pharmacokinetic study Saudi Pharmaceutical Journal 19 185ndash

bbib55

1 Authentication

2 Preliminary Phytochemical Screening

3 fluorescence analysis

4 Foreign matter

5 Ash value

Total ash

Acid insoluble ash

Water soluble ash

6 Extractive value

Alcohal soluble extractive value

Water soluble extractive

7 Loss on drying

8 Chromatographic profiles

9 Qunatitative estimation of phytoconstituents

Total flavonoids content

Total saponin content

52 EXPERIMENTAL

Alkaloids

Carbohydrates

Glycosides

Phenolic compounds

Flavonoids

Saponins

Sterols

Acidic compounds

Mucilage

Resins

LipidsFats

Tests for alkaloids

precipitate

a) Molisch test

Test for Glycosides

c) Legal test

d) Baljet test

c) Gelatin solution

Test for flavonoids

a) Ammonia test

b) Shinoda test

precipitate

c) Biuret test

colour

d) Ninhydrine test

violet colour

Test for Saponins

a) Foam test

like frothing

Test for Sterols

Test for terpenoids

Nollers test

Test for Mucilage

Test for Resins

Ash value

Total ash

after ignition

Acid insoluble ash

Water soluble ash

Extractive value

Loss on drying

Foreign matter

minutes

Na2CO3 solution

basis [14]

weighed [16]

Bombay

plates

Spraying equipments

with acetone [17]

adulteration

Principle

o Layer pre-washing

EXPERIMENTAL WORK

Materials

Chloroform

Acetonitrile

Merck Ltd Mumbai

Equipments

Switzerland

Pore size (nm) 6

Pore vol (mlg) 08

band-1

500 ng band-1

band-1

Chromatography

Linear ascending

Method validation

and recovery

and 800 ng band-1

band-1

b) Robustness

chromatography

c) Sensitivity

d) Recovery studies

1 Alkaloids + + +

3 Glycosides + + +

5 Flavonoids + + +

6 Terpenoids + + +

7 Saponins + + +

8 Sterols + + +

9 Proteins _ - +

10 Resins + - -

12 Mucilage - - -

254 nm 366 nm

Ash Value

1 95 28 865

2 95 27 865

3 93 27 860

Mean 943 273 863

1 99 147 85

2 98 146 80

3 99 149 81

Mean 986 147 82

1 655 171 798

2 665 185 795

3 665 175 795

Mean 661 177 796

Extractive value

1 352 114

2 352 112

3 360 112

Mean 352 112

1 780 1680

2 796 1600

3 796 1620

Mean 790 1600

1 2232 2704

2 2192 2672

3 2224 2695

Mean 2216 2690

Foreign matter

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8390 0161 116

Mean 8390 0161 116

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 9890 0110 110

10000 9900 0100 100

10000 9850 0150 150

Mean 9880 0120 120

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8520 0148 148

Mean 8520 0148 148

Loss on drying

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 59740 0157 785

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

4174 +10000

Mean 51773 0629 630

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 69524 0216 785

and 519

1 0785 0004 0008 9810

9859 plusmn0255

2 0792 0004 0008 9871

3 0796 0004 0008 9896

plusmn0255 microgmL

1 08032 0004 0008 10170

10111 plusmn72 2 07921 0004 0008 9967

3 08069 0004 0008 10197

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

plusmn122microgmL

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

52 EXPERIMENTAL

Alkaloids

Carbohydrates

Glycosides

Phenolic compounds

Flavonoids

Saponins

Sterols

Acidic compounds

Mucilage

Resins

LipidsFats

Tests for alkaloids

precipitate

a) Molisch test

Test for Glycosides

c) Legal test

d) Baljet test

c) Gelatin solution

Test for flavonoids

a) Ammonia test

b) Shinoda test

precipitate

c) Biuret test

colour

d) Ninhydrine test

violet colour

Test for Saponins

a) Foam test

like frothing

Test for Sterols

Test for terpenoids

Nollers test

Test for Mucilage

Test for Resins

Ash value

Total ash

after ignition

Acid insoluble ash

Water soluble ash

Extractive value

Loss on drying

Foreign matter

minutes

Na2CO3 solution

basis [14]

weighed [16]

Bombay

plates

Spraying equipments

with acetone [17]

adulteration

Principle

o Layer pre-washing

EXPERIMENTAL WORK

Materials

Chloroform

Acetonitrile

Merck Ltd Mumbai

Equipments

Switzerland

Pore size (nm) 6

Pore vol (mlg) 08

band-1

500 ng band-1

band-1

Chromatography

Linear ascending

Method validation

and recovery

and 800 ng band-1

band-1

b) Robustness

chromatography

c) Sensitivity

d) Recovery studies

1 Alkaloids + + +

3 Glycosides + + +

5 Flavonoids + + +

6 Terpenoids + + +

7 Saponins + + +

8 Sterols + + +

9 Proteins _ - +

10 Resins + - -

12 Mucilage - - -

254 nm 366 nm

Ash Value

1 95 28 865

2 95 27 865

3 93 27 860

Mean 943 273 863

1 99 147 85

2 98 146 80

3 99 149 81

Mean 986 147 82

1 655 171 798

2 665 185 795

3 665 175 795

Mean 661 177 796

Extractive value

1 352 114

2 352 112

3 360 112

Mean 352 112

1 780 1680

2 796 1600

3 796 1620

Mean 790 1600

1 2232 2704

2 2192 2672

3 2224 2695

Mean 2216 2690

Foreign matter

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8390 0161 116

Mean 8390 0161 116

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 9890 0110 110

10000 9900 0100 100

10000 9850 0150 150

Mean 9880 0120 120

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8520 0148 148

Mean 8520 0148 148

Loss on drying

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 59740 0157 785

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

4174 +10000

Mean 51773 0629 630

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 69524 0216 785

and 519

1 0785 0004 0008 9810

9859 plusmn0255

2 0792 0004 0008 9871

3 0796 0004 0008 9896

plusmn0255 microgmL

1 08032 0004 0008 10170

10111 plusmn72 2 07921 0004 0008 9967

3 08069 0004 0008 10197

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

plusmn122microgmL

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

Saponins

Sterols

Acidic compounds

Mucilage

Resins

LipidsFats

Tests for alkaloids

precipitate

a) Molisch test

Test for Glycosides

c) Legal test

d) Baljet test

c) Gelatin solution

Test for flavonoids

a) Ammonia test

b) Shinoda test

precipitate

c) Biuret test

colour

d) Ninhydrine test

violet colour

Test for Saponins

a) Foam test

like frothing

Test for Sterols

Test for terpenoids

Nollers test

Test for Mucilage

Test for Resins

Ash value

Total ash

after ignition

Acid insoluble ash

Water soluble ash

Extractive value

Loss on drying

Foreign matter

minutes

Na2CO3 solution

basis [14]

weighed [16]

Bombay

plates

Spraying equipments

with acetone [17]

adulteration

Principle

o Layer pre-washing

EXPERIMENTAL WORK

Materials

Chloroform

Acetonitrile

Merck Ltd Mumbai

Equipments

Switzerland

Pore size (nm) 6

Pore vol (mlg) 08

band-1

500 ng band-1

band-1

Chromatography

Linear ascending

Method validation

and recovery

and 800 ng band-1

band-1

b) Robustness

chromatography

c) Sensitivity

d) Recovery studies

1 Alkaloids + + +

3 Glycosides + + +

5 Flavonoids + + +

6 Terpenoids + + +

7 Saponins + + +

8 Sterols + + +

9 Proteins _ - +

10 Resins + - -

12 Mucilage - - -

254 nm 366 nm

Ash Value

1 95 28 865

2 95 27 865

3 93 27 860

Mean 943 273 863

1 99 147 85

2 98 146 80

3 99 149 81

Mean 986 147 82

1 655 171 798

2 665 185 795

3 665 175 795

Mean 661 177 796

Extractive value

1 352 114

2 352 112

3 360 112

Mean 352 112

1 780 1680

2 796 1600

3 796 1620

Mean 790 1600

1 2232 2704

2 2192 2672

3 2224 2695

Mean 2216 2690

Foreign matter

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8390 0161 116

Mean 8390 0161 116

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 9890 0110 110

10000 9900 0100 100

10000 9850 0150 150

Mean 9880 0120 120

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8520 0148 148

Mean 8520 0148 148

Loss on drying

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 59740 0157 785

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

4174 +10000

Mean 51773 0629 630

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 69524 0216 785

and 519

1 0785 0004 0008 9810

9859 plusmn0255

2 0792 0004 0008 9871

3 0796 0004 0008 9896

plusmn0255 microgmL

1 08032 0004 0008 10170

10111 plusmn72 2 07921 0004 0008 9967

3 08069 0004 0008 10197

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

plusmn122microgmL

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

Test for Glycosides

c) Legal test

d) Baljet test

c) Gelatin solution

Test for flavonoids

a) Ammonia test

b) Shinoda test

precipitate

c) Biuret test

colour

d) Ninhydrine test

violet colour

Test for Saponins

a) Foam test

like frothing

Test for Sterols

Test for terpenoids

Nollers test

Test for Mucilage

Test for Resins

Ash value

Total ash

after ignition

Acid insoluble ash

Water soluble ash

Extractive value

Loss on drying

Foreign matter

minutes

Na2CO3 solution

basis [14]

weighed [16]

Bombay

plates

Spraying equipments

with acetone [17]

adulteration

Principle

o Layer pre-washing

EXPERIMENTAL WORK

Materials

Chloroform

Acetonitrile

Merck Ltd Mumbai

Equipments

Switzerland

Pore size (nm) 6

Pore vol (mlg) 08

band-1

500 ng band-1

band-1

Chromatography

Linear ascending

Method validation

and recovery

and 800 ng band-1

band-1

b) Robustness

chromatography

c) Sensitivity

d) Recovery studies

1 Alkaloids + + +

3 Glycosides + + +

5 Flavonoids + + +

6 Terpenoids + + +

7 Saponins + + +

8 Sterols + + +

9 Proteins _ - +

10 Resins + - -

12 Mucilage - - -

254 nm 366 nm

Ash Value

1 95 28 865

2 95 27 865

3 93 27 860

Mean 943 273 863

1 99 147 85

2 98 146 80

3 99 149 81

Mean 986 147 82

1 655 171 798

2 665 185 795

3 665 175 795

Mean 661 177 796

Extractive value

1 352 114

2 352 112

3 360 112

Mean 352 112

1 780 1680

2 796 1600

3 796 1620

Mean 790 1600

1 2232 2704

2 2192 2672

3 2224 2695

Mean 2216 2690

Foreign matter

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8390 0161 116

Mean 8390 0161 116

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 9890 0110 110

10000 9900 0100 100

10000 9850 0150 150

Mean 9880 0120 120

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8520 0148 148

Mean 8520 0148 148

Loss on drying

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 59740 0157 785

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

4174 +10000

Mean 51773 0629 630

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 69524 0216 785

and 519

1 0785 0004 0008 9810

9859 plusmn0255

2 0792 0004 0008 9871

3 0796 0004 0008 9896

plusmn0255 microgmL

1 08032 0004 0008 10170

10111 plusmn72 2 07921 0004 0008 9967

3 08069 0004 0008 10197

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

plusmn122microgmL

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

c) Gelatin solution

Test for flavonoids

a) Ammonia test

b) Shinoda test

precipitate

c) Biuret test

colour

d) Ninhydrine test

violet colour

Test for Saponins

a) Foam test

like frothing

Test for Sterols

Test for terpenoids

Nollers test

Test for Mucilage

Test for Resins

Ash value

Total ash

after ignition

Acid insoluble ash

Water soluble ash

Extractive value

Loss on drying

Foreign matter

minutes

Na2CO3 solution

basis [14]

weighed [16]

Bombay

plates

Spraying equipments

with acetone [17]

adulteration

Principle

o Layer pre-washing

EXPERIMENTAL WORK

Materials

Chloroform

Acetonitrile

Merck Ltd Mumbai

Equipments

Switzerland

Pore size (nm) 6

Pore vol (mlg) 08

band-1

500 ng band-1

band-1

Chromatography

Linear ascending

Method validation

and recovery

and 800 ng band-1

band-1

b) Robustness

chromatography

c) Sensitivity

d) Recovery studies

1 Alkaloids + + +

3 Glycosides + + +

5 Flavonoids + + +

6 Terpenoids + + +

7 Saponins + + +

8 Sterols + + +

9 Proteins _ - +

10 Resins + - -

12 Mucilage - - -

254 nm 366 nm

Ash Value

1 95 28 865

2 95 27 865

3 93 27 860

Mean 943 273 863

1 99 147 85

2 98 146 80

3 99 149 81

Mean 986 147 82

1 655 171 798

2 665 185 795

3 665 175 795

Mean 661 177 796

Extractive value

1 352 114

2 352 112

3 360 112

Mean 352 112

1 780 1680

2 796 1600

3 796 1620

Mean 790 1600

1 2232 2704

2 2192 2672

3 2224 2695

Mean 2216 2690

Foreign matter

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8390 0161 116

Mean 8390 0161 116

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 9890 0110 110

10000 9900 0100 100

10000 9850 0150 150

Mean 9880 0120 120

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8520 0148 148

Mean 8520 0148 148

Loss on drying

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 59740 0157 785

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

4174 +10000

Mean 51773 0629 630

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 69524 0216 785

and 519

1 0785 0004 0008 9810

9859 plusmn0255

2 0792 0004 0008 9871

3 0796 0004 0008 9896

plusmn0255 microgmL

1 08032 0004 0008 10170

10111 plusmn72 2 07921 0004 0008 9967

3 08069 0004 0008 10197

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

plusmn122microgmL

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

d) Ninhydrine test

violet colour

Test for Saponins

a) Foam test

like frothing

Test for Sterols

Test for terpenoids

Nollers test

Test for Mucilage

Test for Resins

Ash value

Total ash

after ignition

Acid insoluble ash

Water soluble ash

Extractive value

Loss on drying

Foreign matter

minutes

Na2CO3 solution

basis [14]

weighed [16]

Bombay

plates

Spraying equipments

with acetone [17]

adulteration

Principle

o Layer pre-washing

EXPERIMENTAL WORK

Materials

Chloroform

Acetonitrile

Merck Ltd Mumbai

Equipments

Switzerland

Pore size (nm) 6

Pore vol (mlg) 08

band-1

500 ng band-1

band-1

Chromatography

Linear ascending

Method validation

and recovery

and 800 ng band-1

band-1

b) Robustness

chromatography

c) Sensitivity

d) Recovery studies

1 Alkaloids + + +

3 Glycosides + + +

5 Flavonoids + + +

6 Terpenoids + + +

7 Saponins + + +

8 Sterols + + +

9 Proteins _ - +

10 Resins + - -

12 Mucilage - - -

254 nm 366 nm

Ash Value

1 95 28 865

2 95 27 865

3 93 27 860

Mean 943 273 863

1 99 147 85

2 98 146 80

3 99 149 81

Mean 986 147 82

1 655 171 798

2 665 185 795

3 665 175 795

Mean 661 177 796

Extractive value

1 352 114

2 352 112

3 360 112

Mean 352 112

1 780 1680

2 796 1600

3 796 1620

Mean 790 1600

1 2232 2704

2 2192 2672

3 2224 2695

Mean 2216 2690

Foreign matter

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8390 0161 116

Mean 8390 0161 116

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 9890 0110 110

10000 9900 0100 100

10000 9850 0150 150

Mean 9880 0120 120

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8520 0148 148

Mean 8520 0148 148

Loss on drying

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 59740 0157 785

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

4174 +10000

Mean 51773 0629 630

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 69524 0216 785

and 519

1 0785 0004 0008 9810

9859 plusmn0255

2 0792 0004 0008 9871

3 0796 0004 0008 9896

plusmn0255 microgmL

1 08032 0004 0008 10170

10111 plusmn72 2 07921 0004 0008 9967

3 08069 0004 0008 10197

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

plusmn122microgmL

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

Test for terpenoids

Nollers test

Test for Mucilage

Test for Resins

Ash value

Total ash

after ignition

Acid insoluble ash

Water soluble ash

Extractive value

Loss on drying

Foreign matter

minutes

Na2CO3 solution

basis [14]

weighed [16]

Bombay

plates

Spraying equipments

with acetone [17]

adulteration

Principle

o Layer pre-washing

EXPERIMENTAL WORK

Materials

Chloroform

Acetonitrile

Merck Ltd Mumbai

Equipments

Switzerland

Pore size (nm) 6

Pore vol (mlg) 08

band-1

500 ng band-1

band-1

Chromatography

Linear ascending

Method validation

and recovery

and 800 ng band-1

band-1

b) Robustness

chromatography

c) Sensitivity

d) Recovery studies

1 Alkaloids + + +

3 Glycosides + + +

5 Flavonoids + + +

6 Terpenoids + + +

7 Saponins + + +

8 Sterols + + +

9 Proteins _ - +

10 Resins + - -

12 Mucilage - - -

254 nm 366 nm

Ash Value

1 95 28 865

2 95 27 865

3 93 27 860

Mean 943 273 863

1 99 147 85

2 98 146 80

3 99 149 81

Mean 986 147 82

1 655 171 798

2 665 185 795

3 665 175 795

Mean 661 177 796

Extractive value

1 352 114

2 352 112

3 360 112

Mean 352 112

1 780 1680

2 796 1600

3 796 1620

Mean 790 1600

1 2232 2704

2 2192 2672

3 2224 2695

Mean 2216 2690

Foreign matter

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8390 0161 116

Mean 8390 0161 116

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 9890 0110 110

10000 9900 0100 100

10000 9850 0150 150

Mean 9880 0120 120

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8520 0148 148

Mean 8520 0148 148

Loss on drying

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 59740 0157 785

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

4174 +10000

Mean 51773 0629 630

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 69524 0216 785

and 519

1 0785 0004 0008 9810

9859 plusmn0255

2 0792 0004 0008 9871

3 0796 0004 0008 9896

plusmn0255 microgmL

1 08032 0004 0008 10170

10111 plusmn72 2 07921 0004 0008 9967

3 08069 0004 0008 10197

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

plusmn122microgmL

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

Total ash

after ignition

Acid insoluble ash

Water soluble ash

Extractive value

Loss on drying

Foreign matter

minutes

Na2CO3 solution

basis [14]

weighed [16]

Bombay

plates

Spraying equipments

with acetone [17]

adulteration

Principle

o Layer pre-washing

EXPERIMENTAL WORK

Materials

Chloroform

Acetonitrile

Merck Ltd Mumbai

Equipments

Switzerland

Pore size (nm) 6

Pore vol (mlg) 08

band-1

500 ng band-1

band-1

Chromatography

Linear ascending

Method validation

and recovery

and 800 ng band-1

band-1

b) Robustness

chromatography

c) Sensitivity

d) Recovery studies

1 Alkaloids + + +

3 Glycosides + + +

5 Flavonoids + + +

6 Terpenoids + + +

7 Saponins + + +

8 Sterols + + +

9 Proteins _ - +

10 Resins + - -

12 Mucilage - - -

254 nm 366 nm

Ash Value

1 95 28 865

2 95 27 865

3 93 27 860

Mean 943 273 863

1 99 147 85

2 98 146 80

3 99 149 81

Mean 986 147 82

1 655 171 798

2 665 185 795

3 665 175 795

Mean 661 177 796

Extractive value

1 352 114

2 352 112

3 360 112

Mean 352 112

1 780 1680

2 796 1600

3 796 1620

Mean 790 1600

1 2232 2704

2 2192 2672

3 2224 2695

Mean 2216 2690

Foreign matter

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8390 0161 116

Mean 8390 0161 116

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 9890 0110 110

10000 9900 0100 100

10000 9850 0150 150

Mean 9880 0120 120

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8520 0148 148

Mean 8520 0148 148

Loss on drying

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 59740 0157 785

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

4174 +10000

Mean 51773 0629 630

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 69524 0216 785

and 519

1 0785 0004 0008 9810

9859 plusmn0255

2 0792 0004 0008 9871

3 0796 0004 0008 9896

plusmn0255 microgmL

1 08032 0004 0008 10170

10111 plusmn72 2 07921 0004 0008 9967

3 08069 0004 0008 10197

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

plusmn122microgmL

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

Loss on drying

Foreign matter

minutes

Na2CO3 solution

basis [14]

weighed [16]

Bombay

plates

Spraying equipments

with acetone [17]

adulteration

Principle

o Layer pre-washing

EXPERIMENTAL WORK

Materials

Chloroform

Acetonitrile

Merck Ltd Mumbai

Equipments

Switzerland

Pore size (nm) 6

Pore vol (mlg) 08

band-1

500 ng band-1

band-1

Chromatography

Linear ascending

Method validation

and recovery

and 800 ng band-1

band-1

b) Robustness

chromatography

c) Sensitivity

d) Recovery studies

1 Alkaloids + + +

3 Glycosides + + +

5 Flavonoids + + +

6 Terpenoids + + +

7 Saponins + + +

8 Sterols + + +

9 Proteins _ - +

10 Resins + - -

12 Mucilage - - -

254 nm 366 nm

Ash Value

1 95 28 865

2 95 27 865

3 93 27 860

Mean 943 273 863

1 99 147 85

2 98 146 80

3 99 149 81

Mean 986 147 82

1 655 171 798

2 665 185 795

3 665 175 795

Mean 661 177 796

Extractive value

1 352 114

2 352 112

3 360 112

Mean 352 112

1 780 1680

2 796 1600

3 796 1620

Mean 790 1600

1 2232 2704

2 2192 2672

3 2224 2695

Mean 2216 2690

Foreign matter

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8390 0161 116

Mean 8390 0161 116

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 9890 0110 110

10000 9900 0100 100

10000 9850 0150 150

Mean 9880 0120 120

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8520 0148 148

Mean 8520 0148 148

Loss on drying

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 59740 0157 785

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

4174 +10000

Mean 51773 0629 630

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 69524 0216 785

and 519

1 0785 0004 0008 9810

9859 plusmn0255

2 0792 0004 0008 9871

3 0796 0004 0008 9896

plusmn0255 microgmL

1 08032 0004 0008 10170

10111 plusmn72 2 07921 0004 0008 9967

3 08069 0004 0008 10197

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

plusmn122microgmL

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

basis [14]

weighed [16]

Bombay

plates

Spraying equipments

with acetone [17]

adulteration

Principle

o Layer pre-washing

EXPERIMENTAL WORK

Materials

Chloroform

Acetonitrile

Merck Ltd Mumbai

Equipments

Switzerland

Pore size (nm) 6

Pore vol (mlg) 08

band-1

500 ng band-1

band-1

Chromatography

Linear ascending

Method validation

and recovery

and 800 ng band-1

band-1

b) Robustness

chromatography

c) Sensitivity

d) Recovery studies

1 Alkaloids + + +

3 Glycosides + + +

5 Flavonoids + + +

6 Terpenoids + + +

7 Saponins + + +

8 Sterols + + +

9 Proteins _ - +

10 Resins + - -

12 Mucilage - - -

254 nm 366 nm

Ash Value

1 95 28 865

2 95 27 865

3 93 27 860

Mean 943 273 863

1 99 147 85

2 98 146 80

3 99 149 81

Mean 986 147 82

1 655 171 798

2 665 185 795

3 665 175 795

Mean 661 177 796

Extractive value

1 352 114

2 352 112

3 360 112

Mean 352 112

1 780 1680

2 796 1600

3 796 1620

Mean 790 1600

1 2232 2704

2 2192 2672

3 2224 2695

Mean 2216 2690

Foreign matter

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8390 0161 116

Mean 8390 0161 116

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 9890 0110 110

10000 9900 0100 100

10000 9850 0150 150

Mean 9880 0120 120

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8520 0148 148

Mean 8520 0148 148

Loss on drying

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 59740 0157 785

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

4174 +10000

Mean 51773 0629 630

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 69524 0216 785

and 519

1 0785 0004 0008 9810

9859 plusmn0255

2 0792 0004 0008 9871

3 0796 0004 0008 9896

plusmn0255 microgmL

1 08032 0004 0008 10170

10111 plusmn72 2 07921 0004 0008 9967

3 08069 0004 0008 10197

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

plusmn122microgmL

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

weighed [16]

Bombay

plates

Spraying equipments

with acetone [17]

adulteration

Principle

o Layer pre-washing

EXPERIMENTAL WORK

Materials

Chloroform

Acetonitrile

Merck Ltd Mumbai

Equipments

Switzerland

Pore size (nm) 6

Pore vol (mlg) 08

band-1

500 ng band-1

band-1

Chromatography

Linear ascending

Method validation

and recovery

and 800 ng band-1

band-1

b) Robustness

chromatography

c) Sensitivity

d) Recovery studies

1 Alkaloids + + +

3 Glycosides + + +

5 Flavonoids + + +

6 Terpenoids + + +

7 Saponins + + +

8 Sterols + + +

9 Proteins _ - +

10 Resins + - -

12 Mucilage - - -

254 nm 366 nm

Ash Value

1 95 28 865

2 95 27 865

3 93 27 860

Mean 943 273 863

1 99 147 85

2 98 146 80

3 99 149 81

Mean 986 147 82

1 655 171 798

2 665 185 795

3 665 175 795

Mean 661 177 796

Extractive value

1 352 114

2 352 112

3 360 112

Mean 352 112

1 780 1680

2 796 1600

3 796 1620

Mean 790 1600

1 2232 2704

2 2192 2672

3 2224 2695

Mean 2216 2690

Foreign matter

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8390 0161 116

Mean 8390 0161 116

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 9890 0110 110

10000 9900 0100 100

10000 9850 0150 150

Mean 9880 0120 120

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8520 0148 148

Mean 8520 0148 148

Loss on drying

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 59740 0157 785

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

4174 +10000

Mean 51773 0629 630

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 69524 0216 785

and 519

1 0785 0004 0008 9810

9859 plusmn0255

2 0792 0004 0008 9871

3 0796 0004 0008 9896

plusmn0255 microgmL

1 08032 0004 0008 10170

10111 plusmn72 2 07921 0004 0008 9967

3 08069 0004 0008 10197

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

plusmn122microgmL

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

Bombay

plates

Spraying equipments

with acetone [17]

adulteration

Principle

o Layer pre-washing

EXPERIMENTAL WORK

Materials

Chloroform

Acetonitrile

Merck Ltd Mumbai

Equipments

Switzerland

Pore size (nm) 6

Pore vol (mlg) 08

band-1

500 ng band-1

band-1

Chromatography

Linear ascending

Method validation

and recovery

and 800 ng band-1

band-1

b) Robustness

chromatography

c) Sensitivity

d) Recovery studies

1 Alkaloids + + +

3 Glycosides + + +

5 Flavonoids + + +

6 Terpenoids + + +

7 Saponins + + +

8 Sterols + + +

9 Proteins _ - +

10 Resins + - -

12 Mucilage - - -

254 nm 366 nm

Ash Value

1 95 28 865

2 95 27 865

3 93 27 860

Mean 943 273 863

1 99 147 85

2 98 146 80

3 99 149 81

Mean 986 147 82

1 655 171 798

2 665 185 795

3 665 175 795

Mean 661 177 796

Extractive value

1 352 114

2 352 112

3 360 112

Mean 352 112

1 780 1680

2 796 1600

3 796 1620

Mean 790 1600

1 2232 2704

2 2192 2672

3 2224 2695

Mean 2216 2690

Foreign matter

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8390 0161 116

Mean 8390 0161 116

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 9890 0110 110

10000 9900 0100 100

10000 9850 0150 150

Mean 9880 0120 120

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8520 0148 148

Mean 8520 0148 148

Loss on drying

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 59740 0157 785

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

4174 +10000

Mean 51773 0629 630

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 69524 0216 785

and 519

1 0785 0004 0008 9810

9859 plusmn0255

2 0792 0004 0008 9871

3 0796 0004 0008 9896

plusmn0255 microgmL

1 08032 0004 0008 10170

10111 plusmn72 2 07921 0004 0008 9967

3 08069 0004 0008 10197

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

plusmn122microgmL

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

Spraying equipments

with acetone [17]

adulteration

Principle

o Layer pre-washing

EXPERIMENTAL WORK

Materials

Chloroform

Acetonitrile

Merck Ltd Mumbai

Equipments

Switzerland

Pore size (nm) 6

Pore vol (mlg) 08

band-1

500 ng band-1

band-1

Chromatography

Linear ascending

Method validation

and recovery

and 800 ng band-1

band-1

b) Robustness

chromatography

c) Sensitivity

d) Recovery studies

1 Alkaloids + + +

3 Glycosides + + +

5 Flavonoids + + +

6 Terpenoids + + +

7 Saponins + + +

8 Sterols + + +

9 Proteins _ - +

10 Resins + - -

12 Mucilage - - -

254 nm 366 nm

Ash Value

1 95 28 865

2 95 27 865

3 93 27 860

Mean 943 273 863

1 99 147 85

2 98 146 80

3 99 149 81

Mean 986 147 82

1 655 171 798

2 665 185 795

3 665 175 795

Mean 661 177 796

Extractive value

1 352 114

2 352 112

3 360 112

Mean 352 112

1 780 1680

2 796 1600

3 796 1620

Mean 790 1600

1 2232 2704

2 2192 2672

3 2224 2695

Mean 2216 2690

Foreign matter

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8390 0161 116

Mean 8390 0161 116

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 9890 0110 110

10000 9900 0100 100

10000 9850 0150 150

Mean 9880 0120 120

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8520 0148 148

Mean 8520 0148 148

Loss on drying

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 59740 0157 785

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

4174 +10000

Mean 51773 0629 630

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 69524 0216 785

and 519

1 0785 0004 0008 9810

9859 plusmn0255

2 0792 0004 0008 9871

3 0796 0004 0008 9896

plusmn0255 microgmL

1 08032 0004 0008 10170

10111 plusmn72 2 07921 0004 0008 9967

3 08069 0004 0008 10197

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

plusmn122microgmL

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

adulteration

Principle

o Layer pre-washing

EXPERIMENTAL WORK

Materials

Chloroform

Acetonitrile

Merck Ltd Mumbai

Equipments

Switzerland

Pore size (nm) 6

Pore vol (mlg) 08

band-1

500 ng band-1

band-1

Chromatography

Linear ascending

Method validation

and recovery

and 800 ng band-1

band-1

b) Robustness

chromatography

c) Sensitivity

d) Recovery studies

1 Alkaloids + + +

3 Glycosides + + +

5 Flavonoids + + +

6 Terpenoids + + +

7 Saponins + + +

8 Sterols + + +

9 Proteins _ - +

10 Resins + - -

12 Mucilage - - -

254 nm 366 nm

Ash Value

1 95 28 865

2 95 27 865

3 93 27 860

Mean 943 273 863

1 99 147 85

2 98 146 80

3 99 149 81

Mean 986 147 82

1 655 171 798

2 665 185 795

3 665 175 795

Mean 661 177 796

Extractive value

1 352 114

2 352 112

3 360 112

Mean 352 112

1 780 1680

2 796 1600

3 796 1620

Mean 790 1600

1 2232 2704

2 2192 2672

3 2224 2695

Mean 2216 2690

Foreign matter

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8390 0161 116

Mean 8390 0161 116

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 9890 0110 110

10000 9900 0100 100

10000 9850 0150 150

Mean 9880 0120 120

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8520 0148 148

Mean 8520 0148 148

Loss on drying

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 59740 0157 785

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

4174 +10000

Mean 51773 0629 630

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 69524 0216 785

and 519

1 0785 0004 0008 9810

9859 plusmn0255

2 0792 0004 0008 9871

3 0796 0004 0008 9896

plusmn0255 microgmL

1 08032 0004 0008 10170

10111 plusmn72 2 07921 0004 0008 9967

3 08069 0004 0008 10197

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

plusmn122microgmL

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

EXPERIMENTAL WORK

Materials

Chloroform

Acetonitrile

Merck Ltd Mumbai

Equipments

Switzerland

Pore size (nm) 6

Pore vol (mlg) 08

band-1

500 ng band-1

band-1

Chromatography

Linear ascending

Method validation

and recovery

and 800 ng band-1

band-1

b) Robustness

chromatography

c) Sensitivity

d) Recovery studies

1 Alkaloids + + +

3 Glycosides + + +

5 Flavonoids + + +

6 Terpenoids + + +

7 Saponins + + +

8 Sterols + + +

9 Proteins _ - +

10 Resins + - -

12 Mucilage - - -

254 nm 366 nm

Ash Value

1 95 28 865

2 95 27 865

3 93 27 860

Mean 943 273 863

1 99 147 85

2 98 146 80

3 99 149 81

Mean 986 147 82

1 655 171 798

2 665 185 795

3 665 175 795

Mean 661 177 796

Extractive value

1 352 114

2 352 112

3 360 112

Mean 352 112

1 780 1680

2 796 1600

3 796 1620

Mean 790 1600

1 2232 2704

2 2192 2672

3 2224 2695

Mean 2216 2690

Foreign matter

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8390 0161 116

Mean 8390 0161 116

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 9890 0110 110

10000 9900 0100 100

10000 9850 0150 150

Mean 9880 0120 120

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8520 0148 148

Mean 8520 0148 148

Loss on drying

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 59740 0157 785

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

4174 +10000

Mean 51773 0629 630

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 69524 0216 785

and 519

1 0785 0004 0008 9810

9859 plusmn0255

2 0792 0004 0008 9871

3 0796 0004 0008 9896

plusmn0255 microgmL

1 08032 0004 0008 10170

10111 plusmn72 2 07921 0004 0008 9967

3 08069 0004 0008 10197

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

plusmn122microgmL

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

Chloroform

Acetonitrile

Merck Ltd Mumbai

Equipments

Switzerland

Pore size (nm) 6

Pore vol (mlg) 08

band-1

500 ng band-1

band-1

Chromatography

Linear ascending

Method validation

and recovery

and 800 ng band-1

band-1

b) Robustness

chromatography

c) Sensitivity

d) Recovery studies

1 Alkaloids + + +

3 Glycosides + + +

5 Flavonoids + + +

6 Terpenoids + + +

7 Saponins + + +

8 Sterols + + +

9 Proteins _ - +

10 Resins + - -

12 Mucilage - - -

254 nm 366 nm

Ash Value

1 95 28 865

2 95 27 865

3 93 27 860

Mean 943 273 863

1 99 147 85

2 98 146 80

3 99 149 81

Mean 986 147 82

1 655 171 798

2 665 185 795

3 665 175 795

Mean 661 177 796

Extractive value

1 352 114

2 352 112

3 360 112

Mean 352 112

1 780 1680

2 796 1600

3 796 1620

Mean 790 1600

1 2232 2704

2 2192 2672

3 2224 2695

Mean 2216 2690

Foreign matter

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8390 0161 116

Mean 8390 0161 116

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 9890 0110 110

10000 9900 0100 100

10000 9850 0150 150

Mean 9880 0120 120

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8520 0148 148

Mean 8520 0148 148

Loss on drying

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 59740 0157 785

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

4174 +10000

Mean 51773 0629 630

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 69524 0216 785

and 519

1 0785 0004 0008 9810

9859 plusmn0255

2 0792 0004 0008 9871

3 0796 0004 0008 9896

plusmn0255 microgmL

1 08032 0004 0008 10170

10111 plusmn72 2 07921 0004 0008 9967

3 08069 0004 0008 10197

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

plusmn122microgmL

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

Pore size (nm) 6

Pore vol (mlg) 08

band-1

500 ng band-1

band-1

Chromatography

Linear ascending

Method validation

and recovery

and 800 ng band-1

band-1

b) Robustness

chromatography

c) Sensitivity

d) Recovery studies

1 Alkaloids + + +

3 Glycosides + + +

5 Flavonoids + + +

6 Terpenoids + + +

7 Saponins + + +

8 Sterols + + +

9 Proteins _ - +

10 Resins + - -

12 Mucilage - - -

254 nm 366 nm

Ash Value

1 95 28 865

2 95 27 865

3 93 27 860

Mean 943 273 863

1 99 147 85

2 98 146 80

3 99 149 81

Mean 986 147 82

1 655 171 798

2 665 185 795

3 665 175 795

Mean 661 177 796

Extractive value

1 352 114

2 352 112

3 360 112

Mean 352 112

1 780 1680

2 796 1600

3 796 1620

Mean 790 1600

1 2232 2704

2 2192 2672

3 2224 2695

Mean 2216 2690

Foreign matter

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8390 0161 116

Mean 8390 0161 116

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 9890 0110 110

10000 9900 0100 100

10000 9850 0150 150

Mean 9880 0120 120

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8520 0148 148

Mean 8520 0148 148

Loss on drying

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 59740 0157 785

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

4174 +10000

Mean 51773 0629 630

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 69524 0216 785

and 519

1 0785 0004 0008 9810

9859 plusmn0255

2 0792 0004 0008 9871

3 0796 0004 0008 9896

plusmn0255 microgmL

1 08032 0004 0008 10170

10111 plusmn72 2 07921 0004 0008 9967

3 08069 0004 0008 10197

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

plusmn122microgmL

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

Method validation

and recovery

and 800 ng band-1

band-1

b) Robustness

chromatography

c) Sensitivity

d) Recovery studies

1 Alkaloids + + +

3 Glycosides + + +

5 Flavonoids + + +

6 Terpenoids + + +

7 Saponins + + +

8 Sterols + + +

9 Proteins _ - +

10 Resins + - -

12 Mucilage - - -

254 nm 366 nm

Ash Value

1 95 28 865

2 95 27 865

3 93 27 860

Mean 943 273 863

1 99 147 85

2 98 146 80

3 99 149 81

Mean 986 147 82

1 655 171 798

2 665 185 795

3 665 175 795

Mean 661 177 796

Extractive value

1 352 114

2 352 112

3 360 112

Mean 352 112

1 780 1680

2 796 1600

3 796 1620

Mean 790 1600

1 2232 2704

2 2192 2672

3 2224 2695

Mean 2216 2690

Foreign matter

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8390 0161 116

Mean 8390 0161 116

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 9890 0110 110

10000 9900 0100 100

10000 9850 0150 150

Mean 9880 0120 120

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8520 0148 148

Mean 8520 0148 148

Loss on drying

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 59740 0157 785

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

4174 +10000

Mean 51773 0629 630

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 69524 0216 785

and 519

1 0785 0004 0008 9810

9859 plusmn0255

2 0792 0004 0008 9871

3 0796 0004 0008 9896

plusmn0255 microgmL

1 08032 0004 0008 10170

10111 plusmn72 2 07921 0004 0008 9967

3 08069 0004 0008 10197

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

plusmn122microgmL

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

b) Robustness

chromatography

c) Sensitivity

d) Recovery studies

1 Alkaloids + + +

3 Glycosides + + +

5 Flavonoids + + +

6 Terpenoids + + +

7 Saponins + + +

8 Sterols + + +

9 Proteins _ - +

10 Resins + - -

12 Mucilage - - -

254 nm 366 nm

Ash Value

1 95 28 865

2 95 27 865

3 93 27 860

Mean 943 273 863

1 99 147 85

2 98 146 80

3 99 149 81

Mean 986 147 82

1 655 171 798

2 665 185 795

3 665 175 795

Mean 661 177 796

Extractive value

1 352 114

2 352 112

3 360 112

Mean 352 112

1 780 1680

2 796 1600

3 796 1620

Mean 790 1600

1 2232 2704

2 2192 2672

3 2224 2695

Mean 2216 2690

Foreign matter

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8390 0161 116

Mean 8390 0161 116

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 9890 0110 110

10000 9900 0100 100

10000 9850 0150 150

Mean 9880 0120 120

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8520 0148 148

Mean 8520 0148 148

Loss on drying

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 59740 0157 785

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

4174 +10000

Mean 51773 0629 630

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 69524 0216 785

and 519

1 0785 0004 0008 9810

9859 plusmn0255

2 0792 0004 0008 9871

3 0796 0004 0008 9896

plusmn0255 microgmL

1 08032 0004 0008 10170

10111 plusmn72 2 07921 0004 0008 9967

3 08069 0004 0008 10197

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

plusmn122microgmL

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

1 Alkaloids + + +

3 Glycosides + + +

5 Flavonoids + + +

6 Terpenoids + + +

7 Saponins + + +

8 Sterols + + +

9 Proteins _ - +

10 Resins + - -

12 Mucilage - - -

254 nm 366 nm

Ash Value

1 95 28 865

2 95 27 865

3 93 27 860

Mean 943 273 863

1 99 147 85

2 98 146 80

3 99 149 81

Mean 986 147 82

1 655 171 798

2 665 185 795

3 665 175 795

Mean 661 177 796

Extractive value

1 352 114

2 352 112

3 360 112

Mean 352 112

1 780 1680

2 796 1600

3 796 1620

Mean 790 1600

1 2232 2704

2 2192 2672

3 2224 2695

Mean 2216 2690

Foreign matter

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8390 0161 116

Mean 8390 0161 116

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 9890 0110 110

10000 9900 0100 100

10000 9850 0150 150

Mean 9880 0120 120

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8520 0148 148

Mean 8520 0148 148

Loss on drying

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 59740 0157 785

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

4174 +10000

Mean 51773 0629 630

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 69524 0216 785

and 519

1 0785 0004 0008 9810

9859 plusmn0255

2 0792 0004 0008 9871

3 0796 0004 0008 9896

plusmn0255 microgmL

1 08032 0004 0008 10170

10111 plusmn72 2 07921 0004 0008 9967

3 08069 0004 0008 10197

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

plusmn122microgmL

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

254 nm 366 nm

Ash Value

1 95 28 865

2 95 27 865

3 93 27 860

Mean 943 273 863

1 99 147 85

2 98 146 80

3 99 149 81

Mean 986 147 82

1 655 171 798

2 665 185 795

3 665 175 795

Mean 661 177 796

Extractive value

1 352 114

2 352 112

3 360 112

Mean 352 112

1 780 1680

2 796 1600

3 796 1620

Mean 790 1600

1 2232 2704

2 2192 2672

3 2224 2695

Mean 2216 2690

Foreign matter

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8390 0161 116

Mean 8390 0161 116

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 9890 0110 110

10000 9900 0100 100

10000 9850 0150 150

Mean 9880 0120 120

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8520 0148 148

Mean 8520 0148 148

Loss on drying

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 59740 0157 785

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

4174 +10000

Mean 51773 0629 630

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 69524 0216 785

and 519

1 0785 0004 0008 9810

9859 plusmn0255

2 0792 0004 0008 9871

3 0796 0004 0008 9896

plusmn0255 microgmL

1 08032 0004 0008 10170

10111 plusmn72 2 07921 0004 0008 9967

3 08069 0004 0008 10197

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

plusmn122microgmL

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

254 nm 366 nm

Ash Value

1 95 28 865

2 95 27 865

3 93 27 860

Mean 943 273 863

1 99 147 85

2 98 146 80

3 99 149 81

Mean 986 147 82

1 655 171 798

2 665 185 795

3 665 175 795

Mean 661 177 796

Extractive value

1 352 114

2 352 112

3 360 112

Mean 352 112

1 780 1680

2 796 1600

3 796 1620

Mean 790 1600

1 2232 2704

2 2192 2672

3 2224 2695

Mean 2216 2690

Foreign matter

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8390 0161 116

Mean 8390 0161 116

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 9890 0110 110

10000 9900 0100 100

10000 9850 0150 150

Mean 9880 0120 120

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8520 0148 148

Mean 8520 0148 148

Loss on drying

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 59740 0157 785

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

4174 +10000

Mean 51773 0629 630

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 69524 0216 785

and 519

1 0785 0004 0008 9810

9859 plusmn0255

2 0792 0004 0008 9871

3 0796 0004 0008 9896

plusmn0255 microgmL

1 08032 0004 0008 10170

10111 plusmn72 2 07921 0004 0008 9967

3 08069 0004 0008 10197

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

plusmn122microgmL

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

254 nm 366 nm

Ash Value

1 95 28 865

2 95 27 865

3 93 27 860

Mean 943 273 863

1 99 147 85

2 98 146 80

3 99 149 81

Mean 986 147 82

1 655 171 798

2 665 185 795

3 665 175 795

Mean 661 177 796

Extractive value

1 352 114

2 352 112

3 360 112

Mean 352 112

1 780 1680

2 796 1600

3 796 1620

Mean 790 1600

1 2232 2704

2 2192 2672

3 2224 2695

Mean 2216 2690

Foreign matter

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8390 0161 116

Mean 8390 0161 116

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 9890 0110 110

10000 9900 0100 100

10000 9850 0150 150

Mean 9880 0120 120

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8520 0148 148

Mean 8520 0148 148

Loss on drying

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 59740 0157 785

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

4174 +10000

Mean 51773 0629 630

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 69524 0216 785

and 519

1 0785 0004 0008 9810

9859 plusmn0255

2 0792 0004 0008 9871

3 0796 0004 0008 9896

plusmn0255 microgmL

1 08032 0004 0008 10170

10111 plusmn72 2 07921 0004 0008 9967

3 08069 0004 0008 10197

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

plusmn122microgmL

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

Ash Value

1 95 28 865

2 95 27 865

3 93 27 860

Mean 943 273 863

1 99 147 85

2 98 146 80

3 99 149 81

Mean 986 147 82

1 655 171 798

2 665 185 795

3 665 175 795

Mean 661 177 796

Extractive value

1 352 114

2 352 112

3 360 112

Mean 352 112

1 780 1680

2 796 1600

3 796 1620

Mean 790 1600

1 2232 2704

2 2192 2672

3 2224 2695

Mean 2216 2690

Foreign matter

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8390 0161 116

Mean 8390 0161 116

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 9890 0110 110

10000 9900 0100 100

10000 9850 0150 150

Mean 9880 0120 120

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8520 0148 148

Mean 8520 0148 148

Loss on drying

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 59740 0157 785

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

4174 +10000

Mean 51773 0629 630

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 69524 0216 785

and 519

1 0785 0004 0008 9810

9859 plusmn0255

2 0792 0004 0008 9871

3 0796 0004 0008 9896

plusmn0255 microgmL

1 08032 0004 0008 10170

10111 plusmn72 2 07921 0004 0008 9967

3 08069 0004 0008 10197

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

plusmn122microgmL

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

1 655 171 798

2 665 185 795

3 665 175 795

Mean 661 177 796

Extractive value

1 352 114

2 352 112

3 360 112

Mean 352 112

1 780 1680

2 796 1600

3 796 1620

Mean 790 1600

1 2232 2704

2 2192 2672

3 2224 2695

Mean 2216 2690

Foreign matter

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8390 0161 116

Mean 8390 0161 116

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 9890 0110 110

10000 9900 0100 100

10000 9850 0150 150

Mean 9880 0120 120

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8520 0148 148

Mean 8520 0148 148

Loss on drying

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 59740 0157 785

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

4174 +10000

Mean 51773 0629 630

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 69524 0216 785

and 519

1 0785 0004 0008 9810

9859 plusmn0255

2 0792 0004 0008 9871

3 0796 0004 0008 9896

plusmn0255 microgmL

1 08032 0004 0008 10170

10111 plusmn72 2 07921 0004 0008 9967

3 08069 0004 0008 10197

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

plusmn122microgmL

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

1 780 1680

2 796 1600

3 796 1620

Mean 790 1600

1 2232 2704

2 2192 2672

3 2224 2695

Mean 2216 2690

Foreign matter

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8390 0161 116

Mean 8390 0161 116

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 9890 0110 110

10000 9900 0100 100

10000 9850 0150 150

Mean 9880 0120 120

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8520 0148 148

Mean 8520 0148 148

Loss on drying

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 59740 0157 785

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

4174 +10000

Mean 51773 0629 630

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 69524 0216 785

and 519

1 0785 0004 0008 9810

9859 plusmn0255

2 0792 0004 0008 9871

3 0796 0004 0008 9896

plusmn0255 microgmL

1 08032 0004 0008 10170

10111 plusmn72 2 07921 0004 0008 9967

3 08069 0004 0008 10197

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

plusmn122microgmL

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8390 0161 116

Mean 8390 0161 116

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 9890 0110 110

10000 9900 0100 100

10000 9850 0150 150

Mean 9880 0120 120

Wt of Crude

Drug (g)

Wt of drug after

removal of foreign

matter (g)

Wt of foreign

matter (g)

Foreign matter

10000 8520 0148 148

Mean 8520 0148 148

Loss on drying

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 59740 0157 785

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

4174 +10000

Mean 51773 0629 630

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 69524 0216 785

and 519

1 0785 0004 0008 9810

9859 plusmn0255

2 0792 0004 0008 9871

3 0796 0004 0008 9896

plusmn0255 microgmL

1 08032 0004 0008 10170

10111 plusmn72 2 07921 0004 0008 9967

3 08069 0004 0008 10197

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

plusmn122microgmL

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

Loss on drying

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 59740 0157 785

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

4174 +10000

Mean 51773 0629 630

Wt of drug + Petri

Wt of drug+Petri

dish (After drying)

Loss on Drying ()

5974 +10000

Mean 69524 0216 785

and 519

1 0785 0004 0008 9810

9859 plusmn0255

2 0792 0004 0008 9871

3 0796 0004 0008 9896

plusmn0255 microgmL

1 08032 0004 0008 10170

10111 plusmn72 2 07921 0004 0008 9967

3 08069 0004 0008 10197

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

plusmn122microgmL

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

and 519

1 0785 0004 0008 9810

9859 plusmn0255

2 0792 0004 0008 9871

3 0796 0004 0008 9896

plusmn0255 microgmL

1 08032 0004 0008 10170

10111 plusmn72 2 07921 0004 0008 9967

3 08069 0004 0008 10197

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

plusmn122microgmL

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

1 08321 0004 0008 10531

10328 plusmn122 2 08034 0004 0008 10108

3 08219 0004 0008 10347

plusmn122microgmL

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

1 0541 0079 0006 10333

10238plusmn1007 2 0534 0079 0006 10037

3 0543 0079 0006 10345

1 05126 0079 0006 925

9719 plusmn 239 2 05341 0079 0006 10037

3 05213 0079 0006 9871

plusmn 239 microgmL

1 05636 0079 0006 10710

10890plusmn377 2 06219 0079 0006 11615

3 0543 0079 0006 10345

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

1 00629 0013 0004 1858

1902 plusmn 0012 2 00693 0013 0004 2032

3 00611 0013 0004 1817

1 00521 0013 0004 1629

1677 plusmn 070 2 00517 0013 0004 1587

3 00610 0013 0004 1817

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

1 004293 0013 0004 1353

1267 plusmn 0430 2 003997 0013 0004 1232

3 003945 0013 0004 1217

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0367 183

2 20 0391 195

3 20 0378 189

Mean 189plusmn0034

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0158 079

2 20 0157 078

3 20 0161 080

Mean 079plusmn0057

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Saponins content

1 20 0176 088

2 20 0177 088

3 20 0178 089

Mean 088plusmn0003

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0123 246

2 5 0129 258

3 5 0128 256

Mean 253plusmn0037

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0135 270

2 5 0139 278

3 5 0143 286

Mean 278plusmn0046

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

SNo Amount of drug

taken (g)

Amount of product

obtained (g)

Alkaloidal content

1 5 0113 226

2 5 0112 224

3 5 0114 228

Mean 226plusmn0011

TLC profile

Methanol (9505)

08 024 026 029

034 041 052

062 070

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 027 033 039

051 062 076

1 Sulpuhric

acid in

methanol

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

Methanol (9505)

07 025 030 041

053 056 065

1 Sulpuhric

acid in

methanol

Chromatography

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

Calibration

and 150ndash900

ng bandminus1

(r2=09941) and Y=00048X+0012 (r

2=09991) for GA and Y=0033-0017 (r

2=09941)

(ANOVA P gt 005)

Method validation

) and (150 300 and

600 ng band-1

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

b) Robustness

c) Sensitivity

for LP and ST

compounds

d) Recovery studies

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

nm [peak 1-11 LP (052) and ST (028)]

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

530 nm [peak 1-9 LP (052) and ST (028)]

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

nm [peak 1-10 LP (052) and ST (028)]

014 013 011

024 022 019

028 (ST) 028 (ST) 028 (ST)

036 039 039

039 052 (LP) 045

045 059 052 (LP)

052 (LP) 064 065

066 069 069

085 078 078

091 - 087

094 - -

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

for LP and ST

Parameter LP ST

RF 052 028

LOD 45 18

LOQ 135 54

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

Lupeol Stigmasterol

Nominal

Obtained

Precision

Recoveryd

Nominal

Obtained

Precisionc

Recovery

Intraday batch

200 1983 180 991 100 975 173 975

400 3968 175 992 200 1986 184 993

800 8014 153 1001 400 4022 137 1005

Interday batch

200 1962 218 981 100 957 191 957

400 3928 186 982 200 1969 178 984

800 7983 170 997 400 3967 155 992

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

SD RSD SD RSD

03 vvv 68 29 03 vvv 72 25 03 vvv and

70 27 03 vvv)

163 152 159 135

Conc added

to analyte ()

Theoretical

Added (ng) Detected

Recovery () RSD ()

Lupeol

200 5893 982 192

100 400 7936 992 151

150 600 9968 997 149

Stigmasterol

100 2915 972 114

100 200 3952 988 189

150 300 4978 996 117

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

Plant Drug

Lupeol Stigmasterol

Content (ng spot-1

A indicum 590 143 830 129

H auriculata 197 171 470 103

T dioica roxb 670 122 346 115

Tdioica at 270 nm

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

nm [peak 1-15 GA (031) and QA (050)]

270 nm [peak 1-15 GA (031) and QA (050)]

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

nm [peak 1-14 GA (031) and QA (050)]

[GA (031) and QA (050)]

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

001 003 003

006 006 015

010 010 023

015 014 028

019 020 031(GA)

023 023 037

031 (GA) 031 (GA) 046

037 043 050 (QE)

043 046 056

046 050 (QE) 065

050 (QE) 056 068

056 065 079

065 068 083

073 079 089

079 082 -

082 - -

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

Rf 031 050

) 100ndash1000 150ndash900

LOD 23 41

LOQ 69 123

Nominal

Obtained

Precision

Accuracy

Nominal

Obtained

Precisio

Accuracy

Intraday batch

150 1483 170 988 200 1975 173 987

300 2991 165 997 400 3986 189 996

600 6004 155 1000 800 8013 137 1001

Interday batch

150 1492 198 994 200 1977 194 988

300 2998 176 999 400 3969 197 992

600 5983 170 997 800 7988 195 998

cPrecision as

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

SD RSD SD RSD

Mobile phase

631 vvv)

179 182 191 165

Concentration added

to analyte ()

Theoretical

Detected

Recovery

Gallic acid

200 4973 994 192

100 400 6986 998 151

150 600 8991 999 149

Quercetin

100 1975 987 174

100 200 2982 994 192

150 300 3974 993 127

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

Table 546

Plant drug

Content (ng spot-1

A indicum 693 143 579 138

H auriculata 280 111 410 106

T dioica 317 112 396 115

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

55 References

New Delhi

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

Geneva

Ltd 49-188

ed 53-83

32(1) 15-19

351ndash360

bbib55

351ndash360

bbib55

Documents

STANDARDIZATION 5.1 Introduction - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/23763/8/07_chapter_5... · to chemical based medicine and pharmaceutical necessities by giving