INTERNATIONAL JOURNAL OF PHARMACEUTICAL RESEARCH … 1424.pdf · The sample of Miconazole was observed for its color and texture. 2) Melting point The sample of Miconazole was taken

Research Article CODEN: IJPRNK Impact Factor: 5.567 ISSN: 2277-8713 Aseem Verma, IJPRBS, 2018; Volume 7(1): 56-86 IJPRBS

Available Online at www.ijprbs.com 56

METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION

OF MICONAZOLE AND ORNIDAZOLE IN PHARMACEUTICAL DOSAGE FORM BY

RP-HPLC

ASEEM VERMA 1, PROF. SUSHIL AGARWAL2 1. Student, M.Pharma (Quality Assurance) 4th Semester, Student of ,Mahatma Gandhi College of Pharmaceutical Sciences, Jaipur (Rajasthan)

2. M Pharma. Mahatma Gandhi College of Pharmaceutical Sciences, Jaipur (Rajasthan).

Accepted Date: 10/01/2018; Published Date: 27/02/2018

Abstract: The liposomes are concentric bilayered vesicle in which an aqueous volume is entirely enclosed by the membranous lipid bilayer mainly composed of the natural or synthetic phospholipids. The liposomes are one of unique drug delivery system which can be use in controlling & targeting drug delivery system. The liposomes are generally classified based upon the structure, method of preparation, composition and application, conventional liposome, and specialty liposome. The liposomes can be filled with drugs & used to deliver the drugs for cancer & other diseases. This review is focused on the various aspects of liposomes like classification, methods of preparation, characterization and applications of liposomes.

Keywords: Liposomes, Phospholipids, Targeting drug delivery system, Bilayered

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Aseem Verma, IJPRBS, 2018; Volume 7(1): 56-86



INTRODUCTION

Indian sub-continent, being Tropical region has high temperature and high humidity conditions,

which are favourable for the growth of fungal and other infections. Presently, the treatment is

the Azole group/antibiotics. With continuous efforts, the combination of Azole have become a

preferred choice for the better response and therapeutics efficacy. To develop separate

methods of quantification for two drugs requires more efforts for the chemist and less output

in terms of number of samples analysed. In lieu of above, efforts are now being put in

developing a common analytical method for multicomponent formulation analysis.

Thus, despite of various analytical methods developed, still no work has been done in

developing Simultaneous method of Quantification of these drugs. As there is no Stability

indicating HPLC method reported for these drugs in combined dosage forms.

Therefore it was wisely thought worthwhile to develop Stability indicating RP-HPLC method for

simultaneous estimation of Micronazole and Ornidazole in the combined Pharmaceutical

dosage form. Moreover, there are two methods develop for the simultaneous estimation of

Ornidazole and Miconazole by HPLC, one is for cream formulation and the other has been

developed for Tablet formulation. Moreover the run time for Chromatogram is higher, thus the

method is superior, accurate and more reliable than the developed one.

Looking from the QA point of view, the method can be successfully adopted for routine quality

analysis of Ornidazole and Miconazole in combined tablet dosage from without any

interferences from common excipients and impurities. Also method can be transferred to utilize

for routine Laboratory analysis and Assay of Ornidazole and Miconazole in their tablet dosage

form.

DRUG PROFILE9-10

1.4.1. MICONAZOLE9

INTRODUCTION

Name Miconazole

Official in IP-2010, BP-2009, USP30-NF25

Description Miconazole is an imidazole antifungal agent that is used topically and by intravenous infusion.



Structure

Chemical Formula

C18H14Cl4N2O

Mol. Weight 416.13

IUPAC Name 1-[2-(2,4-dichlorophenyl)-2-[(2,4-dichlorophenyl)methoxy]ethyl]-1-Himidazole

Categories Anti infective

Solubility Very slightly soluble in Water, freely soluble in Methanol, soluble in Ethanol (96 per cent)

PHARMACOLOGY

Classes Benzoids

Mechanism of action Miconazole interacts with 14αdemethylase, a cytochrome P450enzyme necessary to convert lanosterol to ergosterol. As ergosterol is an essential component membrane, inhibition of its synthesis results in increased cellular permeability causing leakage of cellular contents.

PROPERTIES

State Solid.

CAS NO. 22916-47-8

Melting point 159-163°C

Experimental properties

Property Value

Log P 5.86

PKa 6.77 (Basic)

http://www.sigmaaldrich.com/catalog/search?term=107724-20-9&interface=CAS%20No.&N=0&mode=partialmax&lang=en&region=IN&focus=product



1.4.2. ORNIDAZOLE10

INTRODUCTION

Name Ornidazole

Official in IP 2010

Description Ornidazole is a drug that cures some protozoan infections.It has been investigated for use in Crohn's disease after bowel resection

Structure

Chemical Formula

C7H10ClN3O3

Mol.Wt 219.625 g/mol

IUPAC 1-chloro-3-(2-methyl-5-nitro-1H-imidazol-1-yl)propan-2-ol

Categories Anti amoebiasis

Solubility Soluble in chloroform and in methanol.=, Practically insoluble in water

PHARMACOLOGY

Classes Tissue amoebecides

Mechanism of action

It causes the inhibition of DNA synthesis resulting in to loss of helical structure.

PROPERTIES

State Solid.

CAS NO. 16773-42-5

Melting point 77-78°C

Experimental Properties

Property Value

Log P 1.28

pKa 2.4



AIM: Literature review reveals that numbers of individual analytical methods available for

estimation of Miconazole and Ornidazole in their individual dosage forms. But no method has

been reported for simultaneous estimation of Miconazole and Ornidazole in combined

pharmaceutical dosage form by RP-HPLC. So it is develop reverse phase high performance liquid

chromatographic method for simultaneous estimation of Miconazole and Ornidazole in

Combined Dosage Form. So Aim of present work is to develop simple, accurate, precise, rapid,

specific, sensitive and selective Reverse Phase HPLC method for simultaneous estimation of

Miconazole and Ornidazole.

3.2 OBJECTIVE

1) To develop HPLC method for simultaneous estimation of, Miconazole and Ornidazole in

pharmaceutical dosage form.

2) Applying the newly developed, validated analytical method for the estimation of Miconazole

and Ornidazole in formulations.

The Premilinary Analysis:

MICONAZOLE:

1) Description

The sample of Miconazole was observed for its color and texture.

2) Melting point

The sample of Miconazole was taken in capillary and place into the melting point apparatus.

Observed the melting point and compared with the reference.

3) Identification Test

Potassium Bromide IR disc was prepared using 1mg of Miconazole on Hydraulic Pellet Press.

This disc was scanned in the region of 4000–400cm-1 in FTIR and obtained IR spectrum was

compared with the reference spectrum of Miconazole.

4) Solubility

The sample of Miconazole was taken in test tubes and observed for solubility in various

solvents like water, methanol, 0.1 N Hcl and 0.1 N NaOH.

ORNIDAZOLE:



1) Description

The sample of Ornidazole was observed for its and texture.

2) Melting point

The sample of Ornidazole was taken in capillary and place into the melting point apparatus.

Observed the melting point and compared with the reference.

3) Identification Test

Potassium Bromide IR disc was prepared using 1mg of Ornidazole on Hydraulic Pellet Press. This

disc was scanned in the region 0f 4000–400cm-1 in FTIR and obtained IR spectrum was

compared with the reference spectrum of Ornidazole.

4) Solubility

The sample of Ornidazole was taken in test tubes and observed for solubility in various solvents

like water, methanol, 0.1 N Hcl and 0.1 N NaOH.

>Development and Validation for Simultaneous Estimation of Miconazole and Ornidazole By

RP-HPLC.

Apparatus and Instruments:

Model: Shimadzu HPLC System

Pump:-LC-20 AT

Column: C18 (250 mm × 4.6 mm i.d.,5µm)

Injector: 20μL fixed loop.

Detector: UV Detector

Software: Spinchrom

Analytical balance: Electronic Balance (Shimadzu AUX220)

REAGENTS AND MATERIALS:

Miconazole was procured from Yash Pharma

Ornidazole was procured from Yash Pharma

Water



Methanol

Acetonitrile

Hydrogen phosphate

Preparation of standard solutions:

(A) Ornidazole standard stock solution: (500 μg/mL)

A 50 mg of Ornidazole was weighed and transferred to a 100 mL volumetric flask. Volume was

made up to the mark with methanol.

(B) Miconazole standard stock solution: (100 μg/mL)

A 10 mg of Miconazole was weighed and transferred to a 100 mL volumetric flask. Volume was

made up to the mark with methanol.

(C) Preparation of standard solution of binary mixtures of Ornidazole (50 μg/mL) and

Miconazole (10 μg/mL)

Take 1 mL from the Ornidazole stock solution and 1mL from Miconazole stock solution and

transferred to 10 mL volumetric flask and volume made up to the mark by mobile phase which

was used in particular trials.

Selection of wavelength:

Standard solution of Ornidazole (5 μg/mL) and Standard solution of Miconazole (10 μg/mL)

were scanned between 200-400 nm using UV-visible spectrophotometer.

Both solutions were scanned between 200 - 400 nm.

Wavelength was selected from the overlay spectra of above solutions.

Chromatographic separation:

Standard solutions of 25-75 μg/ml of Ornidazole and 5-15 μg/ml of Miconazole were injected in

column with 20 μl micro-syringes. The chromatogram was run for appropriate minutes with

mobile phase Buffer(pH 5): Methanol (50:50)

The detection was carried out at wavelength 240 nm. The chromatogram was stopped after

separation achieved completely. Data related to peak like area, height, retention time,

resolution etc were recorded using software.



System suitability test:

It is an integral part of chromatographic method. These tests are used to verify that the

resolution and reproducibility of the system are adequate for the analysis to be

performed. System suitability tests are based on the concept that the equipment,

electronics, analytical operations and samples constitute an integral system that can be

evaluated as a whole. System suitability testing provides assurance that the method will

provide accurate and precise data for its intended use.

Chromatographic conditions:

Column: C18 (250 mm × 4.6 mm i.d.,5µm)

Mobile Phase: Buffer (pH 5):Methanol(50:50)

Flow Rate : 1.0 ml/min

Detection Wavelength: 240 nm

Runtime: 10 min

Injection volume : 20.0 μl

Validation of Developed RP-HPLC method

Linearity

The linearity for Miconazole and Ornidazole were assessed by analysis of combined

standard solution in range of 5-15 μg/ml and 25-75 μg/ml respectively,

5,7.5,10,12.5,15 ml solutions were pipette out from the Stock solution of Ornidazole(500

μg/ml) and Miconazole(100 μg/ml) and transfer to 100 ml volumetric flask and make up with

mobile phase to obtain 5,7.5,10,12.5,15 μg/ml and 25,37.5,50,62.5 and 75 μg/ml for

Miconazole and Ornidazole respectively.In term of slope, intercept and correlation co-efficient

value. The graph of peak area obtained verses respective concentration was plotted.

Precision: Results should be expressed as Relative standard deviation (RSD) or coefficient

of variance.

A. Repeatability

Standard solution containing Ornidazole (50µg/ml) and Miconazole (10 µg/ml) was injected six

times and areas of peaks were measured and % R.S.D. was calculated.



B. Intra-day precision

Standard solution containing (25, 50, 75 µg/ml) of Ornidazole and (5, 10, 15 µg/ml) of

Miconazole were analyzed three times on the same day and % R.S.D was calculated.

C. Inter-day precision

Standard solution containing (25, 50, 75 µg/ml) of Ornidazole and (5, 10, 15 µg/ml) of

Miconazole were analyzed three times on the different day and % R.S.D was calculated.

Accuracy

For Miconazole

5 µg/ml drug solution was taken in three different flask label A, B and C. Spiked 80% , 100%,

120% of standard solution in it and diluted up to 10ml. The area of each solution peak was

measured at 240 nm. The amount of Miconazole was calculated at each level and % recoveries

were computed.

For Ornidazole

25 µg/ml drug solution was taken in three different flask label A, B and C. Spiked 80% , 100%,

120% of standard solution in it and diluted up to 10ml. The area of each solution peak was

measured at 240 nm. The amount of Ornidazole was calculated at each level and % recoveries

were computed.

LOD and LOQ

The LOD was estimated from the set of 3 calibration curves used to determination

method linearity. The LOD may be calculated as,

LOD = 3.3 × (SD/Slope)

Where,

SD= Standard deviation of Y-intercepts of 3 calibration curves.

Slope = Mean slope of the 3 calibration curves.

The LOQ was estimated from the set of 3 calibration curves used to determine method

linearity. The LOQ may be calculated as,

LOQ = 10 × (SD/Slope)



Where,

SD = Standard deviation of Y-intercepts of 3 calibration curves.

Slope = Mean slope of the 3 calibration curves.

Robustness:

Following parameters were changed one by one and their effect was observed on system

suitability for standard preparation.

1. Flow rate of mobile phase was changed (± 0.2 ml/min) 0.8 ml/min and 1.2 ml/min.

2. pH of Mobile phase was changed ( ± 0.2 ) 5.2 and 4.8.

3. Ratio of Mobile phase was changed (±2) Buffer: Methanol (48:52) and Buffer: Methanol

(52:48)

Analysis of formulation

Take Tablet Powder equivalent to 50 mg of Ornidazole and 10 mg of Miconazole was

transferred to a 100 ml volumetric flask, add 60 ml of Mobile phase and shake well till 15

minutes and made up volume up to the mark with mobile phase. The solution was filtered

through Whatman filter paper no. 42 and first few drops of filtrate were discarded. 1 ml of this

solution was diluted to 10 ml with mobile phase. The solution was injected 20 µl. The areas of

resulting peak were measured at 240 nm.

Identification of Drugs

Determination of Solubility

Drug Solubility

Miconazole Slightly Soluble in Water & Freely Soluble in Methanol

Ornidazole Insoluble in water in Soluble Methanol

Determination of Melting Point:

Drug Melting Point (Observed) Melting Point (Observed)

Miconazole 158-162°C 158-163°C

Ornidazole 76-78°C 76-78°C



Identification by IR

Fig: IR Spectra of Sample Ornidazole

Functional group Frequency

NO2 (Aromatic) 1500

C-N 1200

C=C 1250

C-Cl 750

Fig. : IR Spectra of sample Miconazole



Functional group Frequency

C-O 1050

C-N 1300

C=C 1690

C-Cl 730

Development of RP-HPLC Method

1 Selection of wavelength

The sensitivity of HPLC method that uses UV detection depends upon proper selection of

detection wavelength. An ideal wavelength is the one that gives good response for the drugs

that are to be detected. In the present study drug solutions of Miconazole (10 ppm) and

Ornidazole (50 ppm) were prepared in Methanol. These drug solutions were than scanned in

UV region of 200-400 nm and overlay spectrums were recorded.

Figure: UV Spectra of Miconazole (10 ppm) and Ornidazole (50 ppm) in Methanol

(Wavelength Taken 240 nm)

2 Selection of Mobile Phase

Trail contains various mobile phase which are considered of Methanol, Water and Acetonitrile

in different proportions and different volumes at different flow rate were tried. On the basis of



various trails the mixture of Buffer (pH 5.0): Methanol (40:60), at 1.0 mL/min flow rate, proved

to be better than the other mixture in terms of peak shape, theoretical plate and asymmetry.

Trials are summarizes in following table.

TABLE: List of Mobile Phase trials

Sr. Mobile Phase Remark

1 Water : Methanol (20:80) Two Peaks Observed

2 Water : Methanol (20:80) First peak was of Miconazole, Confirmed by injecting Miconazole (10ppm) individual

3 Water : Methanol (20:80) Second peak was of Ornidazole, Confirmed by injecting Ornidazole (50ppm) individual

4 Water : Methanol (30:70) Both Peak are merged and Peak shape of peak of Ornidazole is Irregular

5 Phosphate Buffer (pH 6.0): Methanol (50:50)

Peak Shape Became sharp but peak of Miconazole observed at Solvent peak time


Retention time of Second peak increased but peak of Miconazole still observed at Solvent peak time


Again Retention time of Peak of Ornidazole Increased by increasing the proportion of Buffer but Peak of Miconazole is as it is.


Now retention time of Peak of Miconazole increased,


Run time decreased

10 Phosphate Buffer (pH 5.0): Methanol

(50:50)

Again run time decreased

11 Phosphate Buffer (pH 5.0): Methanol

(40:60)

First peak observed at solvent peak time, so

above mobile phase is final



Figure: HPLC Chromatogram of Miconazole (10ppm) and Ornidazole (50ppm) in Water:

Methanol (20:80)

Figure 6.5: HPLC Chromatogram of Miconazole (10ppm) in Water : Methanol (50:50)

Figure : HPLC Chromatogram of Ornidazole (50ppm) in Water : Methanol (50:50)



Figure: HPLC Chromatogram of Miconazole (10ppm) and Ornidazole (50ppm) in Water :

Methanol (30:70)

Figure: HPLC Chromatogram of Miconazole (10ppm) and Ornidazole (50ppm) in Buffer (pH

6.0): Methanol (50:50)



Figure : HPLC Chromatogram of Miconazole (10ppm) and Ornidazole (50ppm) in Buffer (pH

6.0): Acetonitrile (60:40)


6.0): Methanol (70:30)




6.0):Methanol (70:30)


5.0):Methanol (60:40)




5.0):Methanol (50:50) (Final)


5.0):Methanol (40:60)

Mobile Phase was selected based on the review of literature. Various mobile phases were tried.

Trial contains various mobile phases which consisted of Methanol, Water, Triethylamine in

different proportions with various pH and different volumes at flow rate 1 ml/min were tried.

On the basis of various trials the mixture of Buffer (pH 5.0):Methanol (50:50)

Parameters Miconazole Ornidazole

Retention Time 3.320 4.707

Theoretical Plates 7539 7262



Asymmetry 1.350 1.400

Resolution 7.418

Optimization of flow rate

1ml/min flow rate, proved to be better than the other in terms of resolution, peak shape and

shorter retention time.

Table: RP-HPLC optimized chromatographic conditions

Parameters Chromatographic Condition

Mode of elution Isocratic

Mobile Phase Buffer (pH 5.0):Methanol (50:50)

Column C18 (25cm x 0.46 cm) Hypersil BDS

Flow rate 1ml/min

Runtime 6 min

Injection volume 20 µL

Detection wavelength 240 nm

Validation of RP-HPLC method:

Specificity:

Fig. :- Chromatogram of Miconazole and Ornidazole std



Fig. :- Chromatogram of Miconazole and Ornidazole sample

Fig. :- Chromatogram of Miconazole and Ornidazole Blank

The Chromatograms of Miconazole and Ornidazole standards and Miconazole and Ornidazole

sample show no interference with the Chromatogram of Miconazole and Ornidazole Blank, so

the Developed method is Specific.

Linearity:

The regression line equation for Ornidazole and Miconazole are as following:

For Ornidazoley = 98.439x - 0.8074 and For Miconazole: y = 84.221x + 0.6068

Table: Linearity data for Ornidazole

Sr.No Concentration(µg/ml) Area

1 25 2517.111

2 37.5 3636.106

3 50 4923.711

4 62.5 6082.409

5 75 7446.404



Sr.No Concentration(µg/ml) Area

1 5 430.105

2 7.5 621.961

3 10 847.982

4 12.5 1040.391

5 15 1273.655

Table: Linearity data for Miconazole.

Fig.: Overlay chromatogram of different concentrations of mixtures of Ornidazole and

Miconazole

Fig. : Calibration Curve of Miconazole (5-15μg/ml).



Fig. : Calibration Curve of Ornidazole (25-75 μg/ml)

Precision I. Repeatability

Ornidazole

Sr No. Conc (μg/ml) Area Mean ± S.D (n=6) % R.S.D

1.

50

4956.633 4962.454±26.202

0.528

4951.608

4986.296

4961.373

4996.102

4922.712

Table: Repeatability data for Ornidazole.

Miconazole

Sr No. Conc (μg/ml) Area Mean ± S.D (n=6) % R.S.D

1.

10

847.956 849.973 ±3.179

0.374

847.155

853.119

848.838

854.802

847.970

Table : repeatability data for Miconazole.



II. Intraday precision

Ornidazole

SR. NO. Conc. (µg/ml)

Area Mean ± S.D. (n=3)

% R.S.D

1 25 2482.292 ± 3.734 0.150

2 50 5000.660 ± 28.846 0.577

3 75 7438.857± 30.997 0.417

Table: Intraday precision data for estimation of Ornidazole

Miconazole



% R.S.D

1 5 424.703 ± 1.082 0.255

2 10 856.778 ± 3.470 0.405

3 15 1272.391 ± 5.272 0.414

Table: Intraday precision data for estimation of Miconazole

III. Interday precision

Ornidazole



% R.S.D

1 25 2460.137 ± 31.438 1.277

2 50 4955.970± 61.494 1.241

3 75 7470.511± 37.323 0.500

Table: Interday precision data for estimation of Ornidazole.

Miconazole



% R.S.D

1 5 421.478 ± 3.597 1.049

2 10 849.944± 7.245 0.852

3 15 1278.500± 5.597 0.438

Table: Interday precision data for estimation of Miconazole.



Accuracy:

SR. NO.

Conc. Level (%)

Sample amount (μg/ml)

Amount Added (μg/ml)

Amount recovered (μg/ml)

% Recovery

% Mean Recovery ± S.D

1 80 % 25 20 20.136 100.681 99.679 ± 1.726

2 25 20 19.537 97.686

3 25 20 20.134 100.670

4 100 % 25 25 24.970 99.880 100.539 ± 0.977

` 25 25 25.019 100.074

6 25 25 25.415 101.662

7 120 % 25 30 29.891 99.638 99.589 ± 0.799

8 25 30 29.630 98.766

9 25 30 30.109 100.362

Table: Recovery data for Ornidazole

SR. NO.

Conc. Level (%)

Sample Amount

Amount Added

Amount recovered (μg/ml)

% Recovery

% Mean Recovery ± S.D

1 80 % 5 4 4.038 100.945 100.572 ± 0.648

2 5 4 3.993 99.824

3 5 4 4.038 100.947

4 100 % 5 5 5.006 100.112 100.326 ± 0.218

5 5 5 5.016 100.318

6 5 5 5.027 100.547

7 120 % 5 6 5.991 99.848 99.986 ± 0.545 8 5 6 5.971 99.524

9 5 6 6.035 100.587

Table: Recovery data for Miconazole



LOD and LOQ:

Calibration curve was repeated for five times and the standard deviation (SD) of the intercepts

was calculated. Then LOD and LOQ were calculated as follows:

LOD = 3.3 * SD/slope of calibration curve

LOQ = 10 * SD/slope of calibration curve

Where, SD = Standard deviation of intercepts

Limit of Detection:

Ornidazole Miconazole

LOD = 3.3 x (SD / Slope) = 3.3 x (71.061/98.43) = 2.382µg/ml

LOD = 3.3 x (SD / Slope) = 3.3 x (12.469/84.22) = 0.489 µg/ml

Table: Limit of Detection data for Miconazole and Ornidazole.

Limit of Quantitation:

Ornidazole Miconazole

LOQ = 10 x (SD / Slope) = 10 x (71.061/98.43) = 7.219 µg/ml

LOQ = 10 x ( SD / Slope ) = 10 x (12.469/84.22) = 1.481 µg/ml

Table: Limit of Quantitation data for Miconazole and Ornidazole.

Robustness:

Table: Robustness data for Ornidazole.

SR NO. Area at Flow rate (- 0.2 ml/min)

Area at Flow rate (+ 0.2 ml/min)

Area at pH (-0.2)

Area at pH (+0.2)

Area at Mobile phase(-2)

Area at Mobile phase(+2)

1 5136.090 3333.630 5076.642 5001.410 5031.404 4912.148

2 5115.490 3282.563 5042.385 5001.071 5021.296 4909.360

3 5115.459 3374.725 5081.618 4991.104 5021.230 4966.243

% R.S.D 0.232 1.386 0.422 0.117 0.117 0.651



SR NO. Area at Flow rate (- 0.2 ml/min)

Area at Flow rate (+ 0.2 ml/min)

Area at pH (- 0.2)

Area at pH (+ 0.2)

Area at Mobile phase(-2)

Area at Mobile phase(+2)

1 878.606 2282.685 868.529 855.633 860.790 840.382

2 875.087 2354.415 868.557 859.950 848.874 843.762

3 872.713 2368.847 869.428 853.941 859.081 849.686

% R.S.D 0.339 1.976 0.059 0.362 0.753 0.558

Table: Robustness data for Miconazole.

Analysis of marketed formulation by developed method

Sample Stock Solution (Miconazole 100 μg/mL, and Ornidazole 500 μg/mL):

Take Tablet Powder equivalent to 10 mg of Miconazole, and 50 mg of Ornidazole was

transferred to a 100 ml volumetric flask, Add 60 ml Mobile phase and Shake for 15 min and

make up volume with Mobile phase. The solution was filtered through Whatman filter paper

no. 42.

Working Sample Preparation (Miconazole 10 μg/mL, and Ornidazole 50 μg/mL):

Take 1 mL from standard stock solution and transferred to 10 ml volumetric flask and made up

volume up to the mark with the mobile phase

Inject above Solution 20 μl for Assay Analysis.

Tablet Candifem

Label claim Miconazole (100mg) Ornidazole (500mg)

Assay (% of label claim*) Mean ± S. D.

97.402±0.885 97.214±1.180

Table: Analysis on marketed formulation

The assay results were comparable to labelled value of each drug in Combined dosage form.

These results indicate that the developed method is accurate, precise, simple and rapid. It can

be used in the routine quality control of dosage form in industries

SUMMARY AND CONCLUSION

RP-HPLC method was developed for simultaneous estimation Miconazole and Ornidazole. In

RP-HPLC method, good resolution and separation of two drugs was achieved. Buffer (pH

5.0):Methanol (50:50) was used as mobile phase. Retention time of Miconazole and

Ornidazole were found to be 3.320 min and 4.707 min respectively with a flow rate of 1



ml/min. The proposed method was accurate and precise. Therefore proposed method can

be used for routine analysis of Miconazole and Ornidazole in Combined Dosage form.

Validation parameters like Linearity, Accuracy, Precision, Robustness, System suitability,

Specificity were tested.

Observation of all these parameters leads to the point that developed RP-HPLC

method is linear, accurate, precise, specific and robust.

It can be successfully adopted for routine quality control analysis of Miconazole And

Ornidazole in combined Tablet dosage form without any interference from common

excipients and impurity.

This method can now transfer to utilize for routine laboratory analysis and assay of

Miconazole And Ornidazole In Their Tablet Dosage Form.

CONCLUSION:

RP-HPLC method was developed for simultaneous estimation Miconazole and Ornidazole. In

RP-HPLC method, good resolution and separation of two drugs was achieved. Buffer (pH

5.0):Methanol (50:50) was used as mobile phase. Retention time of Miconazole and

Ornidazole were found to be 3.320 min and 4.707 min respectively with a flow rate of 1

ml/min. The proposed method was accurate and precise. Therefore proposed method can

be used for routine analysis of Miconazole and Ornidazole in Combined Dosage form.

Validation parameters like Linearity, Accuracy, Precision, Robustness, System suitability,

Specificity were tested.

Observation of all these parameters leads to the point that developed RP-HPLC

method is linear, accurate, precise, specific and robust.

It can be successfully adopted for routine quality control analysis of Miconazole And

Ornidazole in combined Tablet dosage form without any interference from common

excipients and impurity.

This method can now transfer to utilize for routine laboratory analysis and assay of

Miconazole And Ornidazole In Their Tablet Dosage Form



COMPARISON WITH THE EXISTING METHOD:

Developed Method Existing Method

Mobile Phase: Phosphate Buffer (pH 5.0): Methanol 50:50

Mobile Phase: Acetonitrile: Methanol 80:20

Column: C18 (250 mm × 4.6 mm i.d.,5µm) Column:C18 (250 mm × 4.6 mm i.d.,5µm)

Detection Wavelength: 240nm Detection Wavelength: 219nm

Flow rate: 1ml/min Flow rate: 1ml/min

Injection Volume: 20 μl Injection Volume: 20 μl

Run time: 6min Run time: 10min

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3. FDA, "Guidance for Industry; Analytical Procedures and Methods Validation (Draft guidance),

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INTERNATIONAL JOURNAL OF PHARMACEUTICAL RESEARCH … 1424.pdf · The sample of Miconazole was observed for its color and texture. 2) Melting point The sample of Miconazole was taken