14 Chapter-IV NICARDIPINE INJECTION METHOD ...shodhganga.inflibnet.ac.in/bitstream/10603/19305/14/14...METHOD VALIDATION This chapter express about Identification followed by characterization

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CHAPTER IV

NICARDIPINE INJECTION METHOD DEVELOPMENT AND

METHOD VALIDATION

This chapter express about Identification followed by characterization of a novel

degradant. It also describes method validation specific RP-LC test procedure Nicardipine

impurities in injectable finished product in pharmaceutical formulations. This chapter also

describes about materials used and scope of the present study. It describes the reagents,

solvents, glass-ware, standards and test samples (dosage forms) used in this present study,

Brief description of equipment and methods employed in the present study.

4.1 REAGENTS AND CHEMICALS

Ammonium dihydrogen phosphate (NH4)2HPO4:

Merck, Darmstadt, Germany supplied ammonium dihydrogen phosphate was used for

this study.

Sodium hydroxide (NaOH):

Merck supplied AR grade Sodium hydroxide utilized for this study.

Hydrogen Peroxide (H2O2):

Merck supplied Analytical grade (AR) Hydrogen peroxide (30%) was used for this

study.

Ortho-phosphoric acid (H3PO4):

J.T.Baker, Mumbai supplied analytical grade (AR) of ortho-phosphoric acid was used

for the analysis.

Hydrochloric acid (HCl):

Rankem supplied hydrochloric acid was used for the analysis.

Tri-ethyl amine:

J. T. Baker, Mumbai supplied analytical grade (AR) of Tri-ethyl amine was used for

the analysis.

4.2 SOLVENTS

Acetonitrile (CH3CN):

HPLC grade solvent was used for the analysis, manufactured by Rankem and Merck

India Ltd.

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Methanol (CH3OH):

HPLC grade solvent was used for the analysis, manufactured by Merck India Ltd and

Rankem Ltd.

4.3 GLASSWARE:

Brand new of Borosil and Glaxo of class-A, grade glassware like pipette, volumetric

flask, conical flask, glass bottle, test tubes, measuring cylinder and glass beaker were used for

analysis.

4.4 STANDARDS:

Working standards: Nicardipine hydrochloride working standard and its impurity.

4.5 TEST SAMPLE (DOSAGE FORMS)

Finished products: Nicardipine hydrochloride Injection

4.6 EQUIPMENTS AND INSTRUMENTS

HPLC:

Waters HPLC, equipped with 2996 PDA detector which is used in forced degradation

studies and specificity and OR 2489 UV/Visible detector are used in the present study. The

Empower2 software was used to monitor and process the output signal.

Liquid Chromatography-Mass Spectroscopy (LC-MS):

To establish mass by charge ratio of potential degradant in specificity (forced

degradation) experiment the LC-MS/MS system was utilized. Agilent make LC system which

is connected to Biosystem 4000 Q trap used for study.

Nuclear magnetic resonance (NMR) and HRMS:

The proton and carbon Nuclear magnetic resonance (NMR) experiment were

performed for potential degradant in drug product. Deuterated methanol used as solvent.

Frequency for 1H and 13C was 500 megahertz and 125 megahertz correspondingly applied.

Bruker make INOVA was used for the spectrum.Tetramethylsilane is used as internal

standard which chemical shift considered as Zero ppm.

High resolution mass spectroscopy (HRMS) experiments were performed on Waters

LCT premier XE (TOF).

Analytical balance:

Mettler Toledo make AB265-5/fact model is used for this research work.

Centrifuge:

Remi limited make centrifuge instrument is used for samples preparation.

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pH Meter:

Mettler-Toledo, Switzerland make pH meter seven easy model is used to measure pH of the

solution for this work.

Sonicator:

Partech scientific suppliers manufactured 25 Liter sonicator and Life care

manufactured 10Litre was used for analysis.

Degasser:

Borosil manufactured glass degasser apparatus is used and Millipore 0.45micron filter

paper is used for degassing.

Milli-Q water purification system:

Milli pore manufactured water purification system which is having 0.22µm membrane

filter is used for analysis.

Water bath:

Water bath, make Cintex which is manufacture at India was utilized for hydrolysis

study.

Photo stability chamber:

Photo-stability study experiment was performed in photo-stability chamber which is

from Sanyo, Leicestershire, UK.

4.7 SCOPE OF THE PRESENT STUDY:

Keeping in view of the importance of active ingredients and related substances

(process and degradant related impurities) present in drug product to be monitored the scope

of the present study is designed. The current research study focuses on the identification of a

degradation product. Subsequent test procedure validation followed by development of

quantification of active ingredients and its impurities present in drug products by RP-LC

which are sophisticated analytical techniques. NIC is prescribing in the curing of high blood

pressure and angina. Nicardipine hydrochloride Injection for IV administration contains 0.1

milligram per milliliter NIC in either dextrose or NaCl.

The author selected the following active pharmaceutical drug substances and its

impurity for the present study. The Literature no proposed validated methods for separation

and Quantification of the below stated active ingredients & its impurities present in

pharmaceutical dosage forms by the proposed techniques.

Table- 4.7.a: Active Pharmaceutical Ingredients & Its Impurity Structures

4.8 INTRODUCTION:

Nicardipine hydrochloride, (±)

(Fig.4.8.a). Nicardipine is a Ca ion antagonist. Nicardipine hydrochloride intravenous (I.V.)

injection has 0.1 milligram per milliliter NIC

chloride. Its MW is C26H29N3O

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4.7.a: Active Pharmaceutical Ingredients & Its Impurity Structures

hydrochloride, (±)-2-(benzyl-methyl amino) ethyl mono hydrochloride


milligram per milliliter NIC hydrochloride either in dextrose or sodium

O6•HCl and MW is 515.99[1].

4.7.a: Active Pharmaceutical Ingredients & Its Impurity Structures

methyl amino) ethyl mono hydrochloride


hydrochloride either in dextrose or sodium

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Fig-

4.8.a:It shows the Structure and Chemical name of Nicardipine

In the literature survey there were few references available and discussed about

nicardipine degradation product identification and control of impurities. Structural

elucidation of NIC photo degradant by LC-MS and evaluation of photo stability of

nicardipine solution were reported [2, 3]. A hydrolytic degradation product of nicardipine and

the elucidation of nicardipine degradation product were reported in some of the literature [4, 5].

Method for estimation of nicardipine assay and impurity profile by HPLC in pharmaceutical

preparation were also discussed [6-8]. The Chromatographic method was also available in

literature for determination of assay of nicardipine in combination with other compound [9,

10].Determination of nicardipine in plasma sample using liquid chromatography has been

performed [11-14]. Besides the reported literature for nicardipine degradation product and

method as per our present knowledge no literature specifies about a new potential degradant

of nicardipine injection name as named as Methyl 3-methyl-3’-nitro-5-oxo-1,2,5,6-

tetrahydro-[1,1’-biphenyl]-2-carboxylate (Imp-A). This degradation product was formed in

formal stability study and forced degradation study and its structure was elucidated by

advanced spectroscopic techniques like NMR, LC/MS, and TOF-MS. The stability indicating

chromatographic method is not reported for this impurity. The current research study explain

about isolation and characterization of imp-A. It is also describes developament and

valiedation of selective RP-LC test procedure for the deteremination of degradants of

nicardipine and other relaited substanaces (Impu.-A, Impu.-B and Impu.-C and Impu.-D).

The optimized liquid chromatography procedure method validation was performed for

specificity, linearity, precision, recovery, LOD, LOQ and robustness. Force degradation study

in different stress conditions was performed on placebo and finished dosage forms to ensure

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the selectivity of the developed test procedure. These parameters were made in accordance

with ICH guidelines [16-18].

4.9 OPTIMIZED TEST PROCEDURE

Chromatographic Conditions:

Column: ACE C8 column 25 X 0.46centimeter internal diameter by 5

microne particle size

Mobile phase :

Solvent-A : pH 6.0±0.05 of phosphate buffer adjusted with NaOH

(75%) and Acetonitrile (25%)

Solvent-B : water: Acetonitrile (25:75v/v)

Gradient program : 0-12min, sol-B: 25-35; 12-50min, sol-B: 35-45; 50-

60min, sol- B: 45-80; 60-67min, sol-B: 80-80; 67-69min,

sol-B: 80-25 and 69-75min, sol-B: 25-25.

Flow rate : 2.0 mL minute-1

UV detector Wavelength: 237nm

Injection volume : 50 microL

Column Temperature : 30°C

Diluent:

Solvent B used as diluent.

System suitability Solution:

Transferred an accurately weighed amount of 100 milligram Nicardipine

hydrochloride WS to 100 milliliter flaske and 70 milliliter of dilunt was addad. Further it is

sonicate t dissolved the material completely and added 5.0 mL of standard solution into 10

milliliter flask, then added 1.0 milliliter of impurity stock solution (0.05 milligram per

milliliter solution of impurity B, C and D) and make the volume of flask with diluent, mixed.

Standard Solution:

Standard (1000 μg/mL) was made in diluent. Weigh and transferred about 100mg of

Nicardipine hydrochloride working standard in 100 milliliter volumetric flask. Standard of

nicardipine hydrochloride was dissolved completely with in-between sonication by adding

diluent. After completion of sonication sample was diluted with diluent.This solution is 20

times diluted with diluent. Also, further it is 20 times diluted with diluent to obtained 2.5 μg

per mL concentration.

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Test Solution Preparation:

Dilute 5 mL of the sample (eq. to 12.5 milligram of nicardipine HCl ) in to 25ml flask

and added 15mL of diluent sonicate for ten minu then dilute to volum with dilunt.

Procedure:

Separately injected equal 50μL volume of blank, system suitability solution, standard

preparation, test solution in to the LC instrument and obtained the chromatograms and

assessed the areas of peaks in the chromatograms and calculated the results using below

formula.

System Suitability:

The tailing factor for Nicardipine peak was NMT 2.0 in standard solution, the

Resolution between Impurity B peak and Nicardipine peak from system suitability solution

was NLT 2.0 and the ratio of nicardipine peak response from two standard injections was

from 0.9 to 1.1.

Calculation:

Percentage (%) of Known of Impurity =

At * WS * 5 * 5 * 25 * P * 1 * 100 As * 100 * 100 * 100 * 5 * 2.5 * 100 * RRF

Where in “As” is Nicardipine peak response in STD preparation. “At” is the known

impurity peak response in sample preparation. “WS” stands for the Nicardipine hydrochloride

weight of standard which is for preparation standard solution in milligram. “P” is the potency

of Nicardipine as Nicardipine hydrochloride and ''RRF'' = relative response factor of each

known impurity.

Percentage (%) of Unknown of Impurity =

At * WS * 5 * 5 * 25 * P * 1 * 100 As * 100 * 100 * 100 * 5 * 2.5 * 100

Where in “As” is the Nicardipine peak response in STD preparation. “At” is the

unknown impurity peak response in test preparation. “WS” is the weight of Nicardipine

hydrochloride standard which is for STD in milligram and “P” represents the Nicardipine

potency as Nicardipine hydrochloride.

4.10 METHOD DEVELOPMENT:

Selection of Wavelength:

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Nicardipine hydrochloride and its impurities were scanned across the UV range from

200-400nm using 2996 PDA Detector. All Nicardipine hydrochloride and its impurities have

UV maximum absorbance. Spectrum represents the absorption spectra which are publicized

in Fig 4.10.a.

Fig 4.10.a: UV Spectrum of Nicardipine and its Impurities

The above UV spectra indicate that, nicardipine and its impurities have maximum

Absorbance at 237 nm. The results were found satisfactory and reproducible at this

wavelength.

Selection of HPLC Column & Mobile Phase:

Initially isocratic method was employed for separation of impurities, using 0.05M

potassium dihydrogen orthophosphate (62%) and 38% solvent mixture (90% ACN and 10%

Impurity –A UV spectra

201.9 218.3 240.7

351.2

AU

0.000

0.005

Impurity –B UV spectra

203.1 236.0

351.2

AU

0.02

0.04

Nicardipine UV spectra

191.4

237.2

352.4

AU

0.00

1.00

2.00

Impurity –C UV spectra

262.0372.5

AU

0.00

0.02

0.04

Impurity –D UV spectra

237.2352.4

AU

0.000

0.010

0.020

nm200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00

Page 9 of 36

CH3OH), pH adjusted to 5.2 using OPA, Waters Symmetry shield C18 5.0 micron column

(250 * 4.6 millimeter) FR kept as 1.20 mL per minute on HPLC-PDA detector. Peak shape of

Imp-D was not found symmetrical and baseline disturbance was observed. To get symmetric

peak of imp-D and baseline stabilization an attempt was made with different column and

different buffer by employing gradient program method, with modified solvent A (0.1M

Ammonium acetate contain 1mL tri ethylamine, adjusted the pH to 6.20 with AA, acetonitrile

mixed in proportion of 75:25) and solvent B (combination of 25% H2O and 75% ACN) and

Zorbax Stable Bond C8 (250 x 4.6 millimeter) 5 micron column. All known impurities peak

shape found to be symmetric but unknown impurity was merging with imp-C. Hence, further

pH of buffer, its concentration and column was optimized. On the optimization, nicardipine

and all four impurity peaks were separated.

Selection of Diluent:

Selection of diluent for Nicardipine and its impurity was a challenge in the method

development. Nicardipine hydrochloride is frely solubl in CH3OH and AA. In watar its

sligtly soluble. It is insoluble in hexane. All of molecules are sligtly solubl in H2O and solubl

in ACN and CH3OH. Hence, watar and acetonitrile mixture was used for dilution because of

peak shape of impurity.

Selection of Run Time and Flow Rate:

With this developed method the retention times for nicardipine and its impurities were

found within 75 minutes where flow of mobile phase was 2.0 mL per minute. Above 2.0 mL

per minute flow rates leads to early retention, merging of impurities and column high

backpressure problem, it causes quantification problems for nicardipine impurities.

Selection of Standard and Sample Solution Concentration:

The standard concentration was based on 0.5% concentration of test concentration.

The sample concentration selected as 0.5 mg/mL to get LOD and LOQ level of impurity

below reporting threshold as per ICH guideline

Finally Optimized Chromatographic Parameters:

ACE C8 (250 * 4.6 millimeter) column with 5 micron was applied. The MP-A 75%

of 10 MM ammonium dihydrogen phosphate buffer, pH 6.0 set with sodium hydroxide and

25% of acetonitrile mixture. MP-B ACN in the proportion of 25:75 v/v, optimized as 0/25,

12/35, 50/45, 60/80, 67/80, 69/25 and 75/25. The Mobile phase FR was 2.0 milliliter per

minut. The colum oven temp was set at 30° . The output signal was recorded at of 237

nanometer wavelength. The injection volume was 50 micro liter.

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4.11 IDENTIFICATION AND CHARACTERIZATION OF NEW IMPURITY:

Liqud Chromatogrphy-Mass Spectromatry (LC-MS) Conditions:

To determin the mass by charge ratio of potential degradant in specificity (forced

degradation) experiment the LC-MS/MS system was utilized. Agilent make LC system which

is connected to Biosystem 4000 Q trap used for study. The test procedure was optimized on

ACE C8, 250 X 4.6 millimeter, five micron column. MP was having a gradient program. Sol-

A contains 75% of 10 mill molar CH3COONH4 buffer and 25% ACN mixture. Sol-B

contains 25% H2O and 75% ACN mixture). Both the solvent mixture was degassed before

use. The gradiant elution programe was sat as (Time (min)/% sol-B) 0/25, 12/35, 50/45,

60/80, 67/80, 69/25 and 75/25.The FR was kept as 1 milliliter per min. The sample was

injected as 20 micro liter. The column oven temp was set as 25 degree centigrade. +ve ESI

mode was used for analysis.

NMR and HRMS Conditions:

The proton and carbon Nuclear magnetic resonance (NMR) experiment were

performed for potential degradant in drug product. Deuterated methanol used as solvent.

Frequency for 1H and 13C was 500 megahertz and 125 megahertz correspondingly applied.

Bruker make INOVA was used for the spectrum.Tetramethylsilane is used as internal

standard which chemical shift considered as Zero ppm. High resolution mass spectroscopy

(HRMS) experiments were performed on Waters LCT premier XE (TOF).

Preparative LC Parameter:

Isolation and resolution of degradant was performed on Zodiac C18 (250 mm × 20

millimeter; 10 micron) preparative column.MP solvent A (75% of water pH 6.2 set with NH3

solution and 25% of ACN mixture) and B (25% of water and75 % of acetonitrile) at FR 18.0

milliliter per minute .The gradiant (Tim/%B) was programmed 0/25, 12/45, 20/100, 28/100,

30/25 and 32/25. The detection wavelength was 227 nanometer. The column oven temp was

set at 25°C.

Enrichment and Isolation of Impurity

During the drug product stability evaluation and specificity study, especially in

thermal degradation experiment 0.32 RRT impurity was found potential. In order to enhance

the impurity, about 500 mL of nicardipine injection was subjected for thermal degradation,

kept in oven at 105°C for 48 hrs. The impurity has been found to enhance up to 1.48%.The

isolation of the impurity was performed on prep chromatographic system. Combined isolated

portion of impurity was and evaporated by using rotavapour with 40 degree centigrade

vacuum. The resultant aqueous layer was lyophilized. The purity of the lyophilized impurity

Page 11 of 36

was checked in HPLC and found highly pure. Further it is utilized to identify the impurity by

advanced technique.

Structural Elucidation of Impurity

The positive electro-spray ionization mass spectrometer of potential degradant found

mass to charge ratio 290 amu [M+H]+ (Fig.4.11.a). Employ HRMS found C15H15NO5 as

elemental composition of the potential degradant (Tab.4.11.a).

Proton NMR, carbon NMR, two dimensional NMR (COSY, HSQC and HMBC) has

been performed to achieve structural elucidation degradation product. The 1H and 13C NMR

spectra (Fig.4.11.b & Fig.4.11.c) showed two sets of signals may be due to the presence of

diastreomeric mixture with few impurity peaks in the aliphatic region. The COSY spectrum

(Fig.4.11.d) showed the presence of two spin systems one in the aromatic and one in the

aliphatic regions. Three proton in the aromatic region at 8.22ppm (CH, 123.41), 7.75(CH,

135.09) and 7.60 ppm (CH, 131.24 ppm) confirms the presence of nitro phenyl moiety. The

aliphatic region showed correlation between 6.07(CH, 128.76), 2.00(CH3, 22.45), 3.89(CH,

45.04) and Ha2.82, Hb 2.66 (CH2, 22.45).One methoxy signal was observed at 3.59(CH3,

52.91).The corresponding 13C NMR chemical shifts were shown in the parenthesis from

HSQC (Fig.4.11.e) experiment.

The 13C NMR signal at 199.48 ppm is typical for ketone or aldehyde signal. The

absence of aldehyde proton shows that this corresponds to ketone group. The HMBC

correlations showed that this is next to CH2 group. The position at ester carbon at 173.38

ppm was confirmed by the HMBC (Fig.4.11.f.) correlate with 3.59(CH3) and 3.89(CH)

protons. The remaining quaternary carbons were confirmed by HMBC correlations.

Based all above study and interpretation of spectral data confirms the identification of

unknown impurity as Methyl 3-methyl-3’-nitro-5-oxo-1,2,5,6-tetrahydro-[1,1’-biphenyl]-2-

carboxylate (imp-A).

Tab.4.11.a HRMS data for elemental composition

Experiment TOF(+ve) Elemental

composition TOF (-ve)

Elemental Composition

UPLC-TOF 307.1279 C15H19 N2O5

(M+NH4)+

288.0870 C15H14 NO5

(M-H)-

Fig. 4.11.a: Mass spectrum of potential degradant (I

Fig. 4.11.b.:

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Fig. 4.11.a: Mass spectrum of potential degradant (Impurity

Fig. 4.11.b.:1H NMR spectrum of potential degradant

mpurity-A)

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Fig. 4.11.c.:13C NMR spectrum of potential degradant

Fig. 4.11.d. COSY spectrum of potential degradant

Fig. 4.11.e. HSQCspectrum of potential degradant

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Fig. 4.11.f. HMBCspectrum of potential degradant

Pathway of Formation of Impurity

Nicardipine will undergo hydrolysis in presence of heat and forms an unstable

product, further rearrangement takes place. Subsequently hydroxyl group react with the

intermediate compound and gives hydroxy acid which further rearrange to keto acids and

open the ring. This compound undergoes decarboxylation as presence of keto group in β-

Position which gives keto amine compound and further reacts with hydroxyl group, forms

diketo compound. Supplementary dehydration takes place and form final product (impurity-

A).Complete stepwise mechanism on Fig.4.11.g is described.

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Fig. 4.11.g. Mechanism of degradation impurity

a. METHOD VALIDATION:

The method validetion of optimizd test procedure was performed as par ICH

guidlines[17,18]. The method was validated by executing the studies for the parameter are,

selectivity, linearity, recovery, precision, and robustness.

System Suitability

This parameter was verified by test procedure characteristic criteria before initiate the

analysis which determination has been done base on specification. The SST was assessed

based on retention time, the resolution between impurity C and nicardipine peak, ratio of

nicardipine peak areas from two standard injections and nicardipine analyte tailing from STD

preparation. The resolution between nicardipine and imp-C was observed 4.7 against

specification of NLT 2.0. USP tailing factor and area ratio of nicardipine peak observed 1.0

and 1.0, respectively. All SST parameter are complies to the limit.(Table-4.12.a).

Table 4.12.aSystem Suitability Data

Parameters Specification Result

Precision Inter. Precision

Resolutiona NLT 2.0 4.7 5.7

Peak response ratio Between 0.9

and1.1 1.0 1.0

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Tailing factor NLT 2.0 1.0 1.0

a Resolusion betwen nicardipine and Impu-C.

Specificity:

Specificity was performed to demonstrate the method capability to separate all

potential impurities from the main peak and from each other. Forced degradation was studied

on nicardipine HCl injection to prove the performance of test method related to its specificity.

Degradation was performed on different hassle condition of Photolytic degradation [Ultra

Violet light (200 watt hr per m2),], heat (60 hrs at 60°C ), acid (60°C with 0.1N HCl, 60 hrs),

base (60°C with 0.1N NaOH, 60 hrs), oxidation (1% H2O2 at 25°C, 60 hrs),hydrolytic

(40°C,55 hrs) and humidity (25°C/90%RH,72 hrs). The proposed method was assessed for its

capability to resolve nicardipine from its degradation product. In all stress samples peak

purity check performed for nicardipine peak by Photo Diode Array detector.

All degraded tests were assessed for Nicardipine peak purity and homogeneity by

photo diode array detector in high performance liquid chromatography. % Degradation is

summarized in table 4.12.b.

Table 4.12.b: FD Study Dat

Fig.4.12.a. Representative chromatograms of Placebo.

Page 17 of 36

Table 4.12.b: FD Study Data

Fig.4.12.a. Representative chromatograms of Placebo.

Fig.4.12.b. Representative Chromatogram of unstressed Sample

Fig.4.12.c. Purity Plot of unstressed Sample

Fig.4.12.d. Representative Chromatograms of Acidstressed placebo

Page 18 of 36


Fig.4.12.c. Purity Plot of unstressed Sample




Fig.4.12.e. Representative Chromatograms of Acidstressed sample

Fig.4.12.f. Purity plot of Acidstressed sample

Fig.4.12.g. Representative Chromatograms of Alkalistressed Placebo

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Representative Chromatograms of Acidstressed sample

Fig.4.12.f. Purity plot of Acidstressed sample


Representative Chromatograms of Acidstressed sample


Fig.4.12.h. Representative chromatograms of Alkali stressed sample

Fig.4.12.i.

Fig.4.12.j. Representative Chromatograms of Peroxidestressed placebo

Page 20 of 36


Fig.4.12.i. Purity plot of Alkalistressed sample




Fig.4.12.k. Representative chromatograms of

Fig.4.12.l. Purity plot of Peroxidestressed sample

Fig.4.12.m. Representative chromatograms of

Page 21 of 36

Fig.4.12.k. Representative chromatograms of Peroxide stressed sample

Fig.4.12.l. Purity plot of Peroxidestressed sample

Fig.4.12.m. Representative chromatograms of Waterstressed placebo

stressed sample

stressed placebo

Fig.4.12.n. Representative Chromatograms of Waterstressed sample

Fig.4.12.o. Purity plot of Waterstressed sample

Fig.4.12.p. Representative Chromatograms of Photostressed placebo

Page 22 of 36


Fig.4.12.o. Purity plot of Waterstressed sample




Fig.4.12.q. Representative Chromatograms of Photostressed sample

Fig.4.12.r. Purity plot of Photostressed sample

Fig.4.12.s. Representative Chromatograms of Humiditystressed placebo

Page 23 of 36


Fig.4.12.r. Purity plot of Photostressed sample




Fig.4.12.t. Representative Chromatograms of Humiditystressed sample

Fig.4.12.u. Purity plot of Humiditystressed sample

Fig.4.12.v. Representative Chromatograms of Thermalstressed placebo

Page 24 of 36


Fig.4.12.u. Purity plot of Humiditystressed sample




Fig.4.12.w. Representative Chromatograms of Thermalstressed Sample

Fig.4.12.x. Purity plot of Thermalstressed Sample

Fig. 4.12.y. Representative chromatogrms of

impurities

Precission of the Method:

Page 25 of 36


Fig.4.12.x. Purity plot of Thermalstressed Sample

Fig. 4.12.y. Representative chromatogrms of Nicardipine spked test wi


ked test wiith its

Page 26 of 36

The method precission was confirmed with six sample injection. The nicardipine (0.5

mg/mL) solution spiked imp.-A, imp.-B at 0.2% and impu.-C, imp.-D 0.5% with regard to

nicardipine concentration. The RSD of peak response of impu.-A, impu.-B, impu.-C and

impu.-D was determined. In the same lab the intra-day precision was performed with

proposed method with different scientist and equipment. The % RSDs for the are nicardipine

impurity A, B, C and D in precission parameter was withein 2.7 % and interm. precission was

within 4.5%, conformiing excellent precission of the proposed test procedure. % RSDs data

are shown in Taable 4.12.c.

Table 4.12.c. Nicardipine impurities method precision data

Sample Imprity-A Imprity -B Imprity-C Imprity-D

RRT % RRT % RRT % RRT %

1 0.32 0.472 0.49 0.192 1.20 0.520 2.12 0.212

2 0.32 0.470 0.49 0.192 1.20 0.505 2.12 0.217

3 0.32 0.469 0.49 0.190 1.20 0.481 2.12 0.218

4 0.32 0.474 0.49 0.193 1.20 0.491 2.12 0.216

5 0.32 0.471 0.50 0.192 1.20 0.494 2.12 0.220

6 0.32 0.478 0.50 0.194 1.20 0.496 2.12 0.214

MEAN 0.32 0.472 0.49 0.192 1.20 0.498 2.12 0.216

(%) RSD NA 0.7 NA 0.7 NA 2.7 NA 1.3

Table 4.12.d.: Nicardipine impurities intermediate precision data

Sample Impurity-A Impurity -B Impurity-C Impurity-D

RRT % RRT % RRT % RRT %

1 0.33 0.508 0.50 0.212 1.21 0.511 2.10 0.238

2 0.33 0.514 0.50 0.212 1.20 0.509 2.10 0.238

3 0.32 0.508 0.49 0.210 1.20 0.532 2.12 0.242

4 0.32 0.505 0.48 0.209 1.20 0.550 2.13 0.236

5 0.31 0.503 0.48 0.209 1.19 0.560 2.14 0.241

6 0.31 0.502 0.47 0.209 1.19 0.564 2.15 0.240

Mean 0.32 0.507 0.49 0.210 1.20 0.538 2.13 0.239

(%) RSD NA 0.9 NA 0.7 NA 4.5 NA 0.9

Limits of Detection and Quantification of Method

Page 27 of 36

The LOD and LOQ imprity-A, B, C and D ware established with S/N ratio

correspondingly 3:1 and 10:1. The S/N ratio was established based on performing injection of

known impurity at different concentrations. The precision also performed at the Limit of

quantification level. It was done with six test solution injection all the impurities. The peak

area % RSD was calculated.

The determination values of LOD and LOQ of all the impurities namely nicardipine

impurit-A, B, C and D are tabularized in Table 4.12.e. The precision at the LOQ level for

nicardipine impurity -A, impurity -B, impurity -C and impurity -D was below 3.2 %.

Table 4.12.e: LOQ Precision and Linearity data

Parameter Impurty -A Impurty-B Impurty-C Impurty-D

LOD (µg/mL) 0.005 0.005 0.025 0.035

LOQ (µg/mL) 0.015 0.015 0.06 0.085

Precision at LOQ (%RSD)

0.0 3.2 2.0 0.0

Linearity of the Method

To perform this parameter of the proposed test procedure 6 different parts per million

solutions were prepared. The series of level covers the LOQ to 200% of specification level.

The slope, correlation coefficient and Y-intercept were calculated.

Based on outcome of the linearity study it was found that the concentration and

analyte area correlation is satisfactory. The calibration plot was found linear for all the

concentration of nicardipine and its impurity, i.e. Limit of quantification to 200 % for impu.-

A, impu.-B, impu.-C, and impu.-D. The R gained was more than 0.999 (Fig. 4.12.z-zc).

Page 28 of 36

Fig. 4.12.z: Linearity Plot of Impurity-A

Fig. 4.12.za: Linearity Plot of Impurity-B

y = 69807x - 6089.

R² = 0.999

0

100000

200000

300000

400000

500000

600000

700000

800000

0 2 4 6 8 10 12

Are

a

Concentration (µg/mL)

Impurity-A

y = 10543x - 6.704

R² = 0.999

0

50000

100000

150000

200000

250000

0 0.5 1 1.5 2 2.5

Are

a


Impurity-B

Page 29 of 36

Fig. 4.12.zb: Linearity Plot of Impurity-C

Fig. 4.12.zc: Linearity Plot of Impurity-D

Accuracy

The recovery parameter of impurity was performed in three sample preparation at

Limit of quantification to 50 percent, 75percent, 100percent, 125percent and 150percent of

targeted concentration level. All the impurities % accuracy was calculated. The % accuracy

of impurities of nicardipine tests varies from 87.3 to 103.1 % at limit of quantification, 50%,

75%, 100%, 125% and 150% levels of test concentration. The Liquid chromatography

chromatogram is shown in figure 4.12.y. with spiked test at target concentration of all

y = 40944x - 3052.

R² = 0.999

0

200000

400000

600000

800000

1000000

1200000

1400000

0 5 10 15 20 25 30 35

Are

a


Impurity-C

y = 47963x - 1249.

R² = 0.999

0

20000

40000

60000

80000

100000

120000

0 0.5 1 1.5 2 2.5

Are

a


Impurity-D

Page 30 of 36

impurities in nicardipine. Individual % accuracy results for impurities are tabulated in Table-

4.12.f-i.

Table 4.12.f: Accuracy result of Nicardipine imp-A

Sample No.

level Spiked

‘microgram/ milliliter’ added

‘microgram/ milliliter’ found

(recovered)

% recovery

Mean % recovery

1. 50% 2.4936 2.1850 87.6

87.3 2. 50% 2.4936 2.1750 87.2

3. 50% 2.4936 2.1750 87.2

1. 75% 3.7403 3.3200 88.8

88.6 2. 75% 3.7403 3.3100 88.5

3. 75% 3.7403 3.3100 88.5

1. 100% 4.9871 4.3300 86.8

87.7 2. 100% 4.9871 4.400 88.2

3. 100% 4.9871 4.3950 88.1

1. 125% 6.2339 5.4450 87.3

88.0 2. 125% 6.2339 5.5400 88.9

3. 125% 6.2339 5.4750 87.8

1. 150% 7.4807 6.6100 88.4

88.3 2. 150%

7.4807 6.6650 87.8

3. 150%

7.4807 6.6450 88.8

Page 31 of 36

Table 4.12.g: Accuracy result of Nicardipine imp-B

Sample No.

level Spiked



(recovered)

% recovery

Mean % recovery

1. 50% 0.5117 0.5300 103.6

101.9 2. 50% 0.5117 0.5150 100.6

3. 50% 0.5117 0.5200 101.6

1. 75% 0.7676 0.7800 101.6

102.3 2. 75% 0.7676 0.7900 102.9

3. 75% 0.7676 0.7850 102.3

1. 100% 1.0234 1.0250 100.2

99.9 2. 100% 1.0234 1.0250 100.2

3. 100% 1.0234 1.0150 99.2

1. 125% 1.2793 1.3050 102.0

101.6 2. 125% 1.2793 1.3000 101.6

3. 125% 1.2793 1.2950 101.2

1. 150% 1.5351 1.3650 88.9

88.8 2. 150% 1.5351 1.3550 88.3

3. 150% 1.5351 1.3700 89.2

Table 4.12.h: Accuracy result of Nicardipine imp-C

Sample No.

level Spiked



(recovered)

% recovery

Mean % recovery

1. 50% 7.711 7.100

92.1

91.9 2. 50% 7.711 7.0700 91.7

3. 50% 7.711 7.0800 91.8

1. 75% 11.5667 10.7950 93.3 93.1

Page 32 of 36

2. 75% 11.5667 10.7800 93.2

3. 75% 11.5667 10.7350 92.8

1. 100% 15.4222 14.0600 91.2

92.1 2. 100% 15.4222 14.2750 92.6

3. 100% 15.4222 14.2650 92.5

1. 125% 19.2778 17.6300 91.5

92.2 2. 125% 19.2778 17.9200 93.0

3. 125% 19.2778 17.7300 92.0

1. 150% 23.1334 21.4750 92.8

92.8 2. 150% 23.1334 21.3050 92.1

3. 150% 23.1334 21.6000 93.4

Table 4.12.i: Accuracy result of Nicardipine imp-D

Sample No.

level Spiked



(recovered)

% recovery

Mean % recovery

1. 50% 0.5021 0.4700 93.6

98.3 2. 50% 0.5021 0.4950 98.6

3. 50% 0.5021 0.5150 102.6

1. 75% 0.7532 0.7100 94.3

94.0 2. 75% 0.7532 0.6850 90.9

3. 75% 0.7532 0.7300 96.9

1. 100% 0.9941 0.9500 95.6

96.3 2. 100% 0.9941 0.9500 95.6

3. 100% 0.9941 0.9700 97.6

1. 125%

1.2553 1.1400 90.8 93.1

Page 33 of 36

2. 125%

1.2553 1.1850 94.4

3. 125%

1.2553 1.1800 94.0

1. 150%

1.4911 1.3800 92.5

92.8 2. 150%

1.4911 1.3750 92.2

3. 150%

1.4911 1.3950 93.6

Robustness

The proposed method robustness was established by deliberated change in the method

and the separation between nicardipine and imp-C, the peak area ratio of nicardipine peak

from two injections of standard, and USP tailing factor of nicardipine peak in standard

solution were calculated. The mobile phase FR was kept 2.0 milliliter per min. The flow rate

was altered to 1.8 and 2.2 mL per minute to ensure the performance of the test procedure on

separation by FR change. The check consequence of the column temp on separation was

checked at 25°C and 35° C in place of 30° C. The outcome of MP buffer pH was evaluated at

5.8 and 6.1 instead of 6.0 .The organic phase proportion change effect was estimated by

altering acetonitrile ration in solvent A & B by ±10%.

In above every the deliberately change in LC parameter like FR of mobile phase, pH

of buffer, column oven temp and solvent proportion, the separation between important

compound i.e. nicardipine and imp-C, and system suitability criteria such as area ratio of

nicardipine peak from two standard injection, and USP tailing for nicardipine peak were

found satisfactory which demonstrate the robustness of the method (Table-4.12.j).

Tab.4.12.j: HPLC Method Robustness Results

Stability of STD/Sample and Mobile Phase

The nicardipine and its impurity STD and test solution stability was conducted on

proposed method. Stability of sample was studied on nicardipine spiked solution. Solution

were made as per proposed method and kept

estimation for all impurity was performed on every day and calculated against fresh standard

preparation. The stability of mobile phase also monitored on bench top at every say up to 6

days.

The outcome of this study was there was changes are within acceptable range for all

the impurity, standard and mobile phase. The study confirms, standard preparation, the test

preparations and mobile phases found stable for 6 days.

Page 34 of 36

Tab.4.12.j: HPLC Method Robustness Results

Stability of STD/Sample and Mobile Phase



were made as per proposed method and kept on bench top for six days. The related substance



this study was there was changes are within acceptable range for all


preparations and mobile phases found stable for 6 days.



on bench top for six days. The related substance



this study was there was changes are within acceptable range for all


The results (precision, spcificity, li

present study indicate that the developed and validated methods are reproducible, rugged and

stability indicating.

Formal stability Data and Method Application

To check the application of the method and

time, formal stability study has been performed. Nicardipine injection finished product was

tested at different stability intervals. Sample were kept at refrigerator condition (2

term condition (25°C/60% RH )and acceler

samples were analyzed as per aforementioned method and results are summarized in Tab

4.12.k.The data indicates, imp

proportionally increasing with temperature.

Tab.4.12.k. Stability data of finished product

The present study illustrated the identification, isolation and structural elucidation of major

degradant of nicardipine injection pharmaceutical dosage form. Degradant was characterized

by advanced spectroscopic technique namely NMR, LC/MS, 2D

elemental composition by HRMS. The formation and mechanism of the impurity also

discussed. The stability indicating RP

impurity profile was as well discussed in detail. The propos

selective, liner, accurate, rugged and robust for nicardipine and its impurities in finished

Page 35 of 36

The results (precision, spcificity, linerity, accurcy, robustnes and ruggednes) of the


Formal stability Data and Method Application

To check the application of the method and finished product stability over a period of


tested at different stability intervals. Sample were kept at refrigerator condition (2

0% RH )and accelerted condision (40°C/75% RH) for 3 month .The


4.12.k.The data indicates, imp-A was increased in formal stability study which is

with temperature.

Tab.4.12.k. Stability data of finished product



by advanced spectroscopic technique namely NMR, LC/MS, 2D-NMR


discussed. The stability indicating RP-LC method development followed by validation for

file was as well discussed in detail. The proposd method fou


cy, robustnes and ruggednes) of the


finished product stability over a period of


tested at different stability intervals. Sample were kept at refrigerator condition (2-8°C), long

ion (40°C/75% RH) for 3 month .The


A was increased in formal stability study which is



NMR, TOF-MS and


LC method development followed by validation for

d method foud to be precisse,


Page 36 of 36

product .The proposed method is found as selective and specific. The proposed method can

be appliaed for routin analysi in QC laboratores for commercial batch sample release and

stability analysis of nicardipine.

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14 Chapter-IV NICARDIPINE INJECTION METHOD ...shodhganga.inflibnet.ac.in/bitstream/10603/19305/14/14...METHOD VALIDATION This chapter express about Identification followed by characterization