DEVELOPMENT AND VALIDATION OF RP-HPLC …ibdr.in/Dacuments/fwresearchpaperforistissueofibdr6/3.pdf · AND PARACETAMOL IN THEIR PHARMACEUTICAL DOSAGE FORM ... Email: [email protected]

International Bulletin of Drug Research., 4(7): 41-52, 2014

41

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD

FOR SIMULTANEOUS ESTIMATION OF CINNARIZINE

AND PARACETAMOL IN THEIR PHARMACEUTICAL

DOSAGE FORM

RUCHITA J. CHAUHAN*1, T.Y. PASHA

2, STAVAN M. MASTER

3,

KHUSHBU A. THAKOR4, PARTH U. PATEL

5

___________________________________________________________________________

ABSTRACT

In the present work, sensitive RP-HPLC method been developed for the quantitative

estimation of Cinnarizine (CINN) and Paracetamol (PCM) in their pharmaceutical dosage

form. RP-HPLC method for estimation of CINN and PCM was carried on Enable C18

column (5 μm, 250 mm x 4.6 mm i.d) using a mobile phase consisting of Acetonitrile: Water

(70: 30 V/V), pH 2.4 adjusted with 0.1% Ortho phosphoric acid. The mobile phase was

pumped at flow rate of 0.8 ml/min and the detection was carried out at 229 nm. The linearity

was found to be in the range of 2-12 µg/ml and 50-300 µg/ml with (r2=0.9989, and

r2=0.9976) for CINN and PCM respectively. The peaks obtained were sharp having clear

baseline separation with a retention time of 3.209 ± 0.01 and 4.719 ± 0.01 min for PCM and

CINN respectively. The % recoveries of both the drugs CINN and PCM (in sample

preparation) were found to be 99.18-99.88 % and 99.78-99.93 % respectively. The developed

method was validated as per ICH guidelines, for its accuracy, precision, LOD & LOQ,

robustness and the results were found to be satisfactory, thus the method is accurate, precise,

rapid and simple with good sensitivity for estimation of CINN and PCM and was successfully

used for the quantitative analysis of commercially available dosage form.

KEYWORDS

RP-HPLC, Cinnarizine, Paracetamol, Drug analysis, Validation.

AUTHOR’S AFFILIATION

*Author for correspondence

Department of Quality Assurance, Parul Institute of Pharmacy and Research, Limda,

Vadodara- 391760, Gujarat, India, Email: [email protected].


42

INTRODUCTION

Cinnarizine (CINN) is chemically (1-(diphenylmethyl)-4-(3-phenylprop-2-en-1-yl)

piperazine). It is well known Calcium channel blocker. It is used for the symptomatic

treatment of nausea and vertigo caused by Meniere disease and other vestibular disorders and

for the treatment of motion sickness. It is official in IP, BP and EP. IP[1]

, BP[3]

, and EP[4]

describe Potentiometric Titration and Liquid Chromatography method for its estimation.

Literature survey reveals that Colorimetry, Potentiometry titrationand Simple

Spectrophotometry methods[5]

has been carried out for determination of CINN in

pharmaceutical dosage forms as well as in biological fluids. Literature survey also reveals

that RP-HPLC[6]

and HPTLC methods[7]

for determination of CINN with other drugs in

combination have been reported.

Paracetamol (PCM) chemically, N- (4-hydroxyphenyl) acetamide is an analgesic-antipyretic

agent. It is effective in treating mild to moderate pain such as headache, neuralgia and pain of

musculo-skeletal origin. Paracetamol acts predominantly by inhibiting prostaglandin

synthesis in the central nervous system (CNS). Paracetamol probably produces antipyresis by

acting centrally on the hypothalamic heat-regulating center to produce peripheral vasodilation

resulting in increased blood flow through the skin, sweating, and heat loss. The central action

probably involves inhibition of prostaglandin synthesis in the hypothalamus. Paracetamol

(PCM) is official in IP[1]

, USP[2]

and BP[3]

. The pharmacopeial methods include

potentiometric titraton and LC. Literature servey revels that HPLC[8],[9],[10],[11]

, UV

Spectrophotometry [12], [13], [14]

and HPTLC method for the estimation of PCM alone in bulk

drug and in combination with other drugs has been reported. To the best of our knowledge,

there is no official pharmacopeial as well as non-official method reported for simultaneous

determination of CINN and PCM in pharmaceutical formulation dosage form. The present

manuscript describes simple, sensitive, rapid, accurate, precise and cost effective RP-HPLC

method for simultaneous estimation of both drugs in their combined dosage form.

Figure 1: (A) Cinnarizine. Figure 1: (B) Paracetamol.

MATERIALS AND METHODS

Materials & Chemicals

TheCINN and PCM pure powders were procured as gratis samples from Meridian Medicare

Limited (Himachal Pradesh, India) and Unicure Remedies pvt ltd, (Vadodara, India) with

99.95 % and 99.94 % purity, respectively. Tablet formulation, NEUROZINE (Ultramark

Healthcare Ltd.), was obtained commercially with the labeled amounts of 20 mg CINN and

500 mg PCM. Methanol HPLC Grade (Rankem), HPLC Water, Acetonitrile HPLC Grade

(Rankem), Ortho- phosphoric acid (Analytical Reagent grade) were used for HPLC Method.


43

Instruments

A HPLC instrument (isocratic mode, Shimadzu) equipped with UV detector, Rheodyne

injector and Enable C-18 column. A Shimadzu 1800 (Japan) double beam UV/Visible

spectrophotometer with spectral width of 2 nm, wavelength accuracy of 0.5 nm and a pair of

10 mm matched quartz cell was used to detect isoabsorbtivity of both the drugs. A Digital

analytical balance BL 220H (Japan), an ultrasonic bath (Frontline FS 4, India) was used in

the study.

Methodology (RP-HPLC METHOD)

Selection of detection wavelength

In the present study individual drug solutions of 10 µg/ml CINN and 10 µg/ml PCM were

prepared in Methanol.These drug solutions were than scanned in the UV region of 200-400

nm and overlaid spectrum was recorded. Results showed that at 229 nm both the drug possess

good absorbance. So it was selected as detection wavelength for both drugs.

Selection and Preparation of Mobile Phase

Selection of Mobile Phase

Various mobile phase system containing Methanol : Water and Acetonitrile : Water were

tried. When Methanol : Water (different ratios) at different flow rates were tried for

chromatographic separation, the resolution was not satisfactory. Hence, various trails having

different ratio of Acetonitrile : Water at different flow-rates and at different pH had been

taken. Finally, the optimal composition of the mobile phase was determined to be

Acetonitrile : Water (70:30 v/v), pH 2.4 adjusted with OPA at flow rate (0.8 ml/min).

Preparation of mobile phase

A mixture of 70 ml Acetonitrile and 30 ml of HPLC grade water filtered through 0.45 μm

filter paper, sonicated for 10 minutes to degas the mixture and used as mobile phase. Adjust

the pH to 2.4 by using O-phosphoric acid.

Chromatographic Conditions

Enable C-18 column (250 mm x 4.6 mm i.d., 5 µm) was used as stationary phase. The mobile

phase composed of Acetonitrile: Water (70:30 v/v), pH adjusted to 2.4 with OPA; at flow rate

of 1.0 ml/min with run time of 10 min. The detection of both the drugs was carried out at 270

nm.

Preparation of Standard Solutions

CINN Standard stock solution (200 μg/mL):

A 20 mg of standard CINN was weighed and transferred to a 100 ml volumetric flask and

dissolved in 25 ml mobile phase. The flask was shaken and volume was made up to the mark

with mobile phase to give a solution containing 200 μg/mL CINN.


44

PCM standard stock solution (5000 μg/mL)

A 500 mg of standard PCM was accurately weighed and transferred to a 100 ml volumetric

flask and dissolved in 25 ml mobile phase. The flask was shaken and volume was made up

tothe mark with mobile phase to give a solution containing 5000 μg/mL PCM.

Calibration curve for CINN and PCM

Calibration curve for the CINN (2-12 μg/mL) and PCM (50-300 μg/mL)

5 ml of stock solution was transferred to a 50 ml volumetric flask and volume was made up to

the mark with mobile phase to give a solution containing 20 μg/mL CINN and 500 μg/mL

PCM. Appropriate volume was transferred to different volumetric flasks of 10 ml capacity,

adjusted to the mark with the mobile phase to obtain 2, 4, 6, 8, 10 and 12 μg/mL CINN and

50, 100, 150, 200, 250 and 300 μg/mL PCM. The mixed standard solution was

chromatographed for 10 minutes using mobile phase at a flow rate of 0.8 ml/min. The graphs

were plotted for Concentration. vs Peak area for both the drugs.

Method Validation

The proposed method was validated according to International Conference on Harmonization

(ICH) guidelines.

Linearity and Range (Calibration Curve)

The linearity response was determined by analyzing 6 independent levels of calibration curve

in the range of 2-14 µg/ml for CINN and 50-300 µg/ml for PCM. The plot of peak area

against concentration was plotted. Correlation coefficient and regression line equations for

CINN and PCM were calculated.

Accuracy (Recovery Study)

Accuracy is the closeness of the test results obtained by the method to the true value.The

accuracy of the method was determined by calculating the recoveries of CINN and PCM by

standard addition method. Known amounts of standard solutions of CINN and PCM were

added at 50, 100 and 150 % level to prequantified sample solutions of CINN and PCM. The

amounts of CINN and PCM were estimated by applying obtained values to the respective

regression line equations.

Precision

The precision of an analytical method is the degree of agreement among individual test

results when the method is applied repeatedly to multiple samplings of homogenous samples.

It provides an indication of random error results and was expressed as coefficient of variation

(CV).

Repeatability

6 replicates of standard mixture solution having CINN (4 μg/mL) and PCM (100 μg/mL)

were prepared and chromatograms were recorded and % RSD was calculated.


45

Intraday precision

Standard solutions containing 4, 6 and 8 μg/mL CINN and 50, 100 and 150 μg/mL PCM

were analyzed 3 times on the same day as per the procedure. Chromatogram of each sample

was taken. SD and % RSD were calculated.

Interday precision

Standard solutions containing 4, 6 and 8 μg/mL CINN and 50, 100 and 150 μg/mL PCM

were analyzed on three consecutive days as per the procedure. Chromatogram of each sample

was taken. SD and % RSD were calculated.

Limit of Detection

Calibration curve was constructed for 6 times and the SD of the intercepts was calculated and

LOD was calculated using the following formula,

LOD= (3.3*SD)/Slope

Where, SD = the standard deviation of Y- intercept of 6 calibration curves.

Slope = the mean slope of the 6 calibration curves.

Limit of Quantitation

Calibration curve was constructed for 6 times and the SD of the intercepts was calculated and

LOQ was calculated using the following formula,

LOQ= (10*SD)/Slope

Where, SD = the standard deviation of Y- intercept of 6 calibration curves.

Slope = the mean slope of the 6 calibration curves.

System suitability testing

System suitability testing is an integral part of many analytical procedures. The tests are

based on the concept that the equipment, electronics, analytical operations and samples to be

analyzed constitute an integral system that can be evaluated as such. System suitability test

parameters to be established for a particular procedure depend on the type of procedure being

validated.

Robustness

Change in Flow rate

One conc. of mixture CINN (4 μg/mL) and PCM (100 μg/mL) was analyzed 3 times at 3

different flow rate and record the peak area, Rs and T and % RSD is calculated.

Change in Mobile Phase Ratio


different mobile phase ratio and record the peak area, Rs and T and % RSD is calculated.


46

Change in Wavelength


different wavelength and record the peak area, Rs and T and % RSD is calculated.

Determination of CINN and PCM from its tablet dosage form

Sample preparation

Twenty tablets were weighed and finely powered. Powder equivalent to 20 mg CINN and

500 mg PCM was accurately weighed and transferred to volumetric flask of 100 ml capacity.

50 ml of mobile phase was transferred to this volumetric flask and sonicated for 20 min and

volume was made up to the mark with mobile phase. The above solution was filtered through

whatman filter paper No. 41 (0.45µ) to give a solution containing 200 µg/ml CINN and 5000

µg/ml PCM (solution A). 5 ml of solution A was transferred to a 50 ml volumetric flask and

volume was made up to the mark with mobile phase to give a solution containing 20 μg/mL

CINN and 500 μg/mL PCM (solution B). 2 ml of solution B was transferred to a 10 ml

volumetric flask and volume was made up to the mark with mobile phase to give a solution

containing 4 μg/mL CINN and 100 μg/mL PCM.

RESULTS AND DISCUSSION

The RP-HPLC Method showed adequate separation of CINN and PCM in their tablet dosage

form. The separation was achieved on a Enable C18 (250mm X 4.6 mm i.d., 5 μm particle

size) with a isocratic system of (Acetonitrile : Water ) in the ratio of (70:30 v/v), pH adjusted

2.4 with Ortho-Phosphoric acid. The mobile phase at a flow rate of 0.8 ml/min, Injection

volume 20 μl and wavelength of detection used was 229 nm. The retention time for PCM and

CINN was obtained as 3.209 ± 0.1 min and 4.719 ± 0.1 min, respectively. The linearity of the

proposed method was investigated in the range of 2-12 μg/ml and 50-300 μg/ml for CINN

and PCM respectively. Correlation coefficient was 0.9989 and 0.9976 for CINN and PCM,

respectively. The developed method was validated as per ICH guideline, for its accuracy,

precision, LOD & LOQ, robustness and the results were found to be satisfactory, thus the

method is specific, rapid and simple with good sensitivity for estimation of CINN and PCM.

These analytical methods are also applicable in ordinary laboratories. It can also be adopted

for quality control tests for these drugs in tablets. The % recoveries of both the drugs CINN

and PCM (in sample preparation) were found to be 99.18-99.88 % and 99.78-99.93 %

respectively.

Figure 2: Selection of Detection Wavelength.

PCM

CINN


47

Figure 3: Optimized Chromatogram of binary mixture of 100 µg/ml of PCM and 04

µg/ml of CINN.

Table 1: Optimized Parameters for Chromatographic conditions.

S.

No. PARAMETERS CONDITIONS

1. Mobile Phase Acetonitrile : Water (70:30 v/v)

(pH 2.4 adjusted by 0.1 % Orthophosphoric acid)

2. Pump Mode Isocratic

3. Stationary Phase Enable C18 (250mm X 4.6 mm i.d., 5 μmparticle size)

4. Diluent Mobile Phase

5. Flow Rate (mL/min) 0.8

6. Run Time (min) 10

7. Injection volume 20 μL

8. Detection Wavelength 229 nm UV Detector

9.

Retention Time (mins)

- Paracetamol

- Cinnarizine

3.209

4.719

Figure 4: Chromatograms of Paracetamol and Cinnarizine for Linearity.

PCM

CINN


48

Table 2: Linearity Data of CINN. Table 3: Linearity Data of PCM.

Concentration

(µg/ml)

Peak Area of

Cinnarizine

Concentration

(µg/ml)

Peak area of

Paracetamol

2 6748 50 145166

4 26649 100 488466

6 43736 150 969469

8 60184 200 1279084

10 78231 250 1661692

12 93786 300 2018634

Figure 5: Calibration curve of Cinnarizine.

Figure 6: Calibration curve of Paracetamol.


49

Table 4: System Suitability Parameters for PCM and CINN.

Parameter PCM CINN

Retention Time

(min ± SD) (n=6) 3.209 ± 0.0108 4.719 ± 0.0321

Tailing factor (T) 1.246 1.275

Theoretical plate (N) 8186.163 9933.094

Resolution (R) 6.798

Table 5: Summary of Validation Parameters.

PARAMETER CINNARIZINE PARACETAMOL

Detection Wavelength 229 nm

Beer’s Range 2 – 12 μg/mL 50 – 300 μg/mL

Equation Y= 8662.6 X +

9082.7 Y= 7540.9 X – 225911

r2 0.9989 0.9976

Repeatability

(% RSD) (n=6) 0.743 0.533

Intraday Precision

(% RSD) 0.385 – 0.715 0.365 – 0.591

Interday Precision

(% RSD) 0.48 – 1.16 0.495 – 1.05

LOD 0.275 0.617

LOQ 0.835 1.869

% Recovery (n=3)

50 99.88 ± 0.280 99.78 ± 0.13

100 99.18 ± 0.261 99.93 ± 0.14

150 99.79 ± 0.236 99.87 ± 0.24


50

System Suitability

Retention

time 4.718 ± 0.1 min 3.209 ± 0.1 min

Resolution 6.587 –

Tailing

Factor 1.237 1.219

Theoretical

Plates 9933.094 8186.163

Robustness Change

in Flow Rate

(mL/min)

(% RSD)

0.7 0.671 0.481

0.8 0.384 0.250

0.9 0.904 0.272

Robustness Change

in Mobile Phase

Ratio (ACN : Water,

V/V)

(% RSD)

68:32 0.977 0.251

70:30 0.420 0.490

72:28 0.491 0.360

Robustness Change

in Wavelength (nm)

(% RSD)

228 0.849 0.320

229 0.651 0.493

230 1.086 0.463

Analysis of Pharmaceutical Preparation

%Assay of Paracetamol (100μg/ml) and Cinnarizine (04 μg/ml) in their combined dosage form.

Figure 7: Chromatograph of 100 μg/ml of Paracetamol and 04μg/ml of

Cinnarizinefrom tablet (NEUROZINE).


51

Table 6: % Assay of Cinnarizine and Paracetamol.

Drug Content

Conc. taken

for % assay

(µg/ml)

Peak Area of

Sample

Amount

found

(mg)

% Assay Mean %

Assay ± SD

CINN (20 mg)

4 25149 19.758 98.79 98.73

±

0.35

4 24998 19.671 98.35

4 25239 19.810 99.05

PCM (500 mg)

100 518953 493.88 98.77 99.13

±

0.39

100 521328 495.45 99.09

100 524842 497.78 99.55

CONCLUSION

The proposed RP-HPLC method for simultaneous estimation of Cinnarizine and Paracetamol

in combined dosage form was found to be simple, sensitive, accurate, precise and rapid. The

method utilizes easily available and cheap solvent for the analysis of CINN and PCM and

hence it provides clear evidence that the proposed method can be successfully used for

simultaneous estimation of both the drugs from marketed formulations.

ACKNOWLEDGEMENT

We are grateful to Meridian Medicare Limited (Himachal Pradesh, India) and Unicure

Remedies pvt.ltd, (Vadodara, India) for the gratis samples of pure CINN and PCM,

respectively. We also thank to Dr. T.Y. Pasha, Principal, Parul Institute of Pharmacy and

Research, Limda, Vadodara for providing necessary facilities to carry out research work.

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Documents

DEVELOPMENT AND VALIDATION OF RP-HPLC …ibdr.in/Dacuments/fwresearchpaperforistissueofibdr6/3.pdf · AND PARACETAMOL IN THEIR PHARMACEUTICAL DOSAGE FORM ... Email: [email protected]