Tuesday, 26 March 2013

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF TOLPERISONE HYDROCHLORIDE AND PARACETAMOL IN PHARMACEUTICAL FORMULATION



About Author:Monali G. Patel*, Ragin R. Shah, Krupa H. Shah, Bushra M. Malik
Department of Pharmacy, Pharmaceutical Quality Assurance Laboratory,
Arihant School of Pharmacy & BRI, Adalaj,
Gandhinagar-382421, Gujarat, India.
*monali_patel317@yahoo.in
ABSTRACT
A simple, novel, sensitive, precise and specific validated RP-HPLC method was developed for simultaneous determination of Tolperisone Hydrochloride (TOL) and Paracetamol (PCM) in Pharmaceutical Formulation (Tablet). The chromatographic separation was achieved on C18 Licrosphere column as a stationary phase usingMethanol: Tetrahydrofuran (90: 10 v/v, pH 8.2 with 0.1% v/v TEA) as mobile phase at detection wavelength 254 nm. The linearity range was 3-27 µg/ml for TOL and 10-90 µg/ml for PCM with Rt of 3.593 min and 5.023 min for PCM and TOL respectively. The correlation coefficient (r) values were found to be found to be 0.9994 and 0.9998 for TOL and PCM, respectively.Precision study showed % CV values less than 2% for both TOL and PCM, respectively in all selected concentrations. The % Recoveries of TOL and PCM are in the range of 99.96- 100.71 % and 99.73- 100.49 %, respectively. The assay results of TOL and PCM are 100.05 % and 99.98 %, respectively. This is comparable to labeled claim. The method was validated as per the International Conference on Harmonization (ICH) guidelines.The proposed validated method was successfully used for the quantitative analysis of commercially available dosage form.
INTRODUCTION
Tolperisone Hydrochloride (TOL), chemically (R, S) 2-methyl-1-(4 - methyl phenyl)-3- (1-piperidyl) propane -1 one is a piperidine derivative[1] [Fig. 1]. It is a centrally acting muscle relaxant which is used in the treatment of different pathological conditions like acute and chronic muscle spasm, electroconvulsive therapy, neurological conditions and orthopedic manipulation - multiocular sclerosis, myelopathy, encephalomyelitis, spondylosis, spondylarthrosis, cervical and lumbar syndrome, Arthrosis of the large joints obliterating artherosclerosis of the extremity vessels, Diabetical angiopathy, thromboangitis obliterans, raynauds syndrome[2, 3]. Tolperisone hydrochloride is official in Japanese pharmacopoeia[1]. Paracetamol is N- (4 - hydroxyphenyl) acetamide, a Para-aminophenol derivative [Fig. 2], has analgesic and antipyretic properties and weak anti-inflammatory activity. Paracetamol is official in Indian Pharmacopoeia[4], British Pharmacopoeia [5], United States Pharmacopoeia[6] and European Pharmacopoeia[7].  A combination of Tolperisone HCl with Paracetamol has been approved in India in the proportion of 150:500 mg proportion respectively. The literature survey revealed that there are some analytical methods reported for Tolperiosone Hydrochloride like Spectrophotometric [8-11], HPTLC [12], RP-HPLC [13-17] either individually or in combination with other drug and also reported on biological fluids. Many methods have been reported in literature for determination of paracetamol with other drugs[15-17]. TOL and PCM combination is not official in any pharmacopoeia. Hence, aim of present work is to develop simple, feasible, effective and economic validated analytical techniques for quantification of Tolperisone HCl & Paracetamol simultaneously in Pharmaceutical tablet formulation.
MATERIALS AND METHOD
Instrument:

HPLC System (Thermo LC), with UV-2000, UV detector, AS 3000, Auto samplerSN 4000, Software with CromQuest 4.1 System Controllerand Licrosphere C18 Column (250 × 4.6 mm, 5 µm).
Ultrasonicator (Soltec-Sonica Ultrasonic Cleaner, Spincotech Pvt. Ltd.) was used for degassing purpose.
Chemicals:
Tolperisone Hydrochloride reference standard was kindly provided by Zydus Cadila, Ahmedabad. The purity of reference standard was 98.9%w/w. Paracetamol was provided by Lincoln Pharmaceuticals Ltd. Marketed Tablet formulation containing Tolperisone Hydrochloride 150 mg and Paracetamol 500 mg (MYO-MR PLUS) manufactured by Amanath Pharmaceuticals and marketed by Grandix Pharmaceuticals, A Division of Strides Arcolab Ltd, Bangalore, India were used for estimation.
Methanol, Water, Triethylamine and Tetrahydrofuran of HPLC grade (RANKEM, RFCL chemicals Ltd.) were used as solvents.
ChromatographicConditions:
Preparation of Mobile Phase
Mix Methanol and Tetrahydrofuran in the proportion of 90:10 v/v (pH 8.2 using 0.1% v/v TEA). Filter it through 0.45µm membrane filter. Sonicate the mobile phase for 20 min to degas it.
Preparation of Standard Stock Solution:
Tolperisone hydrochloride (15.0 mg) and Paracetamol (50.0 mg) were accurately weighed, transferred into 25 ml volumetric flask and dissolved in 10 ml methanol. The flask was shaken and diluted up to the mark with methanol (600 µg/ml TOL and 2000 µg/ml PCM respectively i.e. Solution M1). An aliquot (2.5 ml) was withdrawn from solution Minto 25 ml volumetric flask and diluted up to 25 ml with methanol (60 µg/ml TOL and 200 µg/ml respectively i.e. Standard stock solution M). This solution was used to prepare diluted solutions for calibration curve. Aliquots of 0.5, 1.5, 2.5, 3.5 and 4.5 ml were taken from the combined Stock Solution Min a series of 10 ml volumetric flask and diluted up to the mark with methanol giving the final diluted concentration 3, 9, 15, 21 and 27 µg/ml TOL and 10, 30, 50, 70 and 90 µg/ml PCM respectively. The diluted solutions were filtered through 0.45 µm membrane filter.
Preparation of Sample Solution:
Twenty tablets were weighed accurately; the average weight was calculated and finely powdered. Tablet powder equivalent to 150 mg of TOL and 500 mg of PCM were weighed and transferred into 25 ml of volumetric flask, sonicate it for 15 minutes and diluted up to mark with methanol. The solution was filtered using Whatman filter paper no.41 and first few drops of filtrate were discarded. (TS1:6000 μg/ml of TOL and 20,000 μg/ml of PCM). An aliquot (1 ml) of TS1H solution was withdrawn in 10 ml volumetric flask and diluted up to mark with methanol (TS2:600 μg/ml of TOL and 2000 μg/ml of PCM). An aliquot (1 ml) of TSsolution was withdrawn in 10 ml volumetric flask and diluted up to mark with methanol (TS3:60 μg/ml of TOL and 200 μg/ml of PCM). An aliquot (2.5 ml) of TSsolution was withdrawn in 10 ml volumetric flask and diluted up to mark with methanol (Final TS: 15 μg/ml of TOL and 50 μg/ml of PCM). The final test solution was filtered through 0.45 µm membrane filter.
Validation of the Method
The developed method was validated for assay of TOL and PCM in combination in accordance with ICH guidelines [18].
Linearity:
The linearity of measurement was evaluated by analyzing five different concentration of the standard solution of TOL (3-27 μg /ml) and PCM (10-90 μg /ml) (Fig. 3). A linear relationship was observed between Peak Area Vs Concentration for mentioned concentration range. This range was selected as a linear range for estimation of TOL and PCM The linearity data was shown in Table 1 and regression analysis data was shown in Table 2.
Precision:
The reproducibility of the proposed method was determined by performing the assay for the same day (intraday assay precision) and on three different days (interday assay precision). Precision studies were performed by preparing nine determinations covering the specified range for the procedure (3 x 3 replicates for each concentration). Low %RSD shows that the method has good precision. The results of repeatability, intraday and inter day precision were expressed in % RSD was tabulated in Table 3 and Table 4.
Specificity
Specificity study is required to show that the method is unaffected by the presence of impurities and/ or excipients. Specificity was checked by comparison of chromatogram of standard TOL (27 μg/ml) and PCM (90 μg/ml), test sample (15 μg/ml TOL and 50 μg/ml PCM) and blank (Fig. 4, Fig. 5 and Fig. 6).
Accuracy
In order to ensure the suitability and reliability of proposed method, recovery studies were carried out. The accuracy study was carried out by the analysis of standard additions at three levels that is multi-level recovery studies. To a fixed equivalent quantity of formulation powder as well as synthetic mixture, a known quantity of standard TOL and PCM added at 50%, 100% and 150% level and the contents were re-analyzed by the proposed method. The % recovery and %RSD were calculated (Table 5).
Limit of Detection and Limit of Quantitation:
The LOD and LOQ was separately determined (table 8) based on the standard calibration curve. The residual standard deviation of y- intercept of regression lines may be used to calculate LOD and LOQ. LOD=3.3*D/S and LOQ=10*D/S where , D is the standard deviation of the intercept of regression line and S is the slope of the calibration curve (Table 6).
RESULT AND DISCUSSION:
Mobile Phase for the simultaneous estimation of TOL and PCM using isocratic RP- HPLC was chosen after several trials with different proportion of methanol along with other organic solvents. The best separation was obtained using the mobile phase consisted of a mixture of Methanol: THF (90:10 v/v, pH 8.2 using 0.1% v/v TEA) at detection wavelength 254 nm.A flow rate of 1 ml/min gave an optimal signal to noise ratio with excellent separation time. Standard calibration curves for TOL and PCM were linear with Correlation coefficients (r) values in the range of 0.9994 and 0.9998 respectively at 254 nm and the values were average of five readings. LOD and LOQ were found to be 0.289 µg/ml and 0.875 µg/ml for TOL and 0.827 µg/ml and 2.505 µg/ml for PCM.Precision study showed co-efficient of variation (%CV) values less than 2% for both TOL and PCM respectivelyin all selected concentrations. The accuracy of the method was confirmed by recovery studies from tablet at three different levels of 50 %, 100 %, 150 % recovery in the range of 99.96-100.71 % for TOL and 99.73-100.49% for PCMjustifies the accuracy of method. The results obtained from the recoveries of both drugs showed excellent accuracy. The developed RP-HPLC method was found to be specific as it was unaffected by the presence of impurities and/ or excipients. The assay results obtained showed good agreement with the labelled claim.
CONCLUSION:
The proposed work provides a precise, specific and accurate method for simultaneous analysis of Tolperisone hydrochloride and Paracetamol in tablet formulation using isocratic RP-HPLC method. The proposed method can be applied for routine analysis in laboratory.
ACKNOWLEDGEMENT:
The authors are thankful to Zydus Cadila Ltd, Ahmedabad and Lincoln Pharmaceuticals Ltd. for providing standard sample of drugs and also to the Arians School of Pharmacy & BRI, Adalaj, Gandhinagar for providing facilities to carry out research work.
TABLE 1: PEAK AREA DATA FOR TOLPERISONE HYDROCHLORIDE AND PARACETAMOL at 254 nm (n=5)
Sr.
No.

Tolperisone hydrochloride
Paracetamol
Conc. (µg/ml)

Mean Peak area ± SD
%CV
Conc.  (µg/ml)

Mean Peak area ± SD
%CV
1.
3
175815.0 ± 1787.33

1.02
10
1471361.4 ± 9630.94

0.65
2.
9
496282.4 ± 4351.58

0.88
30
3646275.0 ± 31715.63

0.87
3.
15
895012.6 ± 5135.91

0.57
50
5850892.4 ± 39151.97

0.67
4.
21
1282749.6 ± 8009.72

0.62
70
8146941.6 ± 89537.72

1.01
5.
27
1622201.6 ± 15529.63

0.95
90
10531325.0 ± 90639.27
0.86
TABLE 2: DATA OF REGRESSION ANALYSIS
Drug
Regression Equation
Correlation Coefficient (r)
Tolperisone hydrochloride
y = 61,320.67x - 25,397.86
0.9994
Paracetamol
y = 113,102.96x + 274,210.63
0.9998
TABLE 3: REPEATABILITY DATA OF TOLPERISONE HYDROCHLORIDE AND PARACETAMOL(n=6)
Tolperisone hydrochloride
Paracetamol
Conc. (µg/ml)
Peak Area
Conc.  (µg/ml)
Peak Area



     
15
892035




50
5851792
896697
5855698
895482
5857123
894998
5863321
896124
5852456
895115
5841132
Mean Peak Area ± SD
895075.2 ± 1621.48
Mean Peak Area ± SD
5853587 ± 7366.66
% CV
0.18
% CV
0.13
TABLE 4: INTRA AND INTER DAY PRECISION DATA OF TOLPERISONEHYDROCHLORIDE AND PARACETAMOL(n=3)
Drug
Conc. (µg/ml)
Intra Day Variation
Inter Day Variation
Mean Peak Area ± SD
% CV
Mean Peak Area ± SD
% CV


TOL
3
177783.0 ±
1255.83
0.71
177524 ±
1453.49338
0.82
15
890261.0 ±
5579.82
0.63
851057 ±
6824.06418
0.80
27
1627225.0 ±13412.16
0.82
1644867 ±17409.7897
1.06


PCM
10
1473690.0 ±9279.21
0.63
1467243 ±
12062.086
0.82
50
5848332.0 ±48334.16
0.83
5873348 ±
61762.622
1.05
90
10597609.0 ±48682.39
0.46
10593003 ±59203.268
0.56
TABLE 5: RECOVERY DATA FOR TOLPERISONE HYDROCHLORIDE AND PARACETAMOL (n=3)

Drug
Amt. of taken (mg)
Amt. of added (mg)
Total Amt. found (mg) ± SD
Mean %Recovery ± SD
% CV








TOL
150
0
150.33 ± 0.76376
-
0.51
 
 
 
150
75
224.95 ± 0.70614
99.98 ± 0.31384
0.31
 
 
 
150
150
299.87 ± 1.09509
99.96 ± 0.36502
0.37
 
 
 
150
225
377.68 ± 0.78009

100.71 ± 0.20802
0.21
 
 
 







PCM
500
0
500.72 ±
1.11509
-
0.22
 
 
 
500
250
748.72 ± 0.80033
99.83 ± 0.10671
0.11
 
 
 
500
500
1004.93 ± 1.59919
100.49 ± 0.15992
0.16
 
 
 
500
750
1246.63 ± 1.85595
99.73 ± 0.14839
0.15
 
 
 
TABLE 6: LIMIT OF DETECTION (LOD) AND LIMIT OF QUANTITATION (LOQ)

Parameters
Tolperisone Hydrochloride
Paracetamol
LOD
0.289 μg/ml
0.827 μg/ml
LOQ
0.875 μg/ml
2.505 μg/ml

TABLE 7: SYSTEM SUITABILITY PARAMETERS
Sr. No.
Parameters
Tolperisone hydrochloride
Paracetamol
1
Retention time
5.023 min
3.593 min
2
Theoretical Plate
3831
5816
3
Asymmetry
0.85
0.46
4
Resolution
5.241
TABLE 8: ASSAY STUDY OF TOL AND PCM TABLET FORMULATION
Tolperisone hydrochloride
Paracetamol
Amount of TOL taken in mg
Amount of TOL Found in mg
% Purity
Amount of PCM taken in mg
Amount of PCM Found in mg
% Purity
150
150.27
100.18
500
499.78
99.96
150.50
100.33
500.05
100.01
149.73
99.82
499.21
99.84
150.02
100.01
500.65
100.13
149.82
99.88
499.86
99.97
Mean Amt. ± SD
150.07 ± 0.3183
Mean Amt. ± SD
499.91 ± 0.5188
Mean % Purity ± SD
100.05 ± 0.21226
Mean % Purity ± SD
99.98 ± 0.10376
% CV
0.21
% CV
0.10
Fig. 3: Chromatogram of standard solution (TOL 3-27 μg/ml and PCM 10-90 μg/ml)
Fig. 4: Chromatogram of standard solution (TOL 27 μg/ml and PCM 90 μg/ml)
Fig. 5: Chromatogram of test solution (TOL 15 μg/ml: PCM 50 μg/ml)
Fig. 6: Chromatogram of blank
REFERENCES:
1.    Japanese Pharmacopoeia, The ministry of Health, Labour and Welfare, Prefectural office in Japan, 15th Edn; 2006, pp 1190-1.
2.    Gayraud M., Raynaud’s phenomenon. Joint, bone and spine, 74(1), el-8 Jan- (2007)
3.    “Muscle Relaxants Introduction”, Nov 2011, en.wikipedia.org/wiki/Muscle_ relaxants
4.    Indian Pharmacopoeia, Government of India Ministry of Health & Family Welfare, 2007, Vol.III, pp 900, 901,903.
5.    British Pharmacopoeia, Government of British, 2011, Vol.-I and II, pp 840-854.
6.    The United States pharmacopoeia 32, National Formulary-27, Asian Edition, The United States Pharmacopoeia Convention, Rockville, 2009, Vol.-III, pp 1266-91.
7.    European Pharmacopoeia 5.0, pp 2184.
8.    Raghavi K, Shaiba M, Jagathi V, Sindhura M and Prashanth R, “Assay of Tolperisone by extractive spectrophotometry.” Asian J. Res. Chem. 2011, 4 (2).
9.    Sai PP, Anupama B, Jagathi V and Rao DG, “Spectrophotometric determination of Tolperisone using 2, 4-dinitrophenylhydrazine reagent.” Int. J. Res. Pharm. Sci. 2010, 1 (3), 317-320.
10.    Carolin NI, Balan P, Chiranjeevi N, Maheswari UV and Rajasekar S, “Method development and statistical validation of UV spectrophotometric method.” J. Pharm. Research. 2011, 4 (5), 1356-1357.
11.    Badmanaban R, Patel MJ and Patel CN, “Simultaneous analysis of Tolperisone hydrochloride and Etodolac in combined fixed oral dosage formulation by spectrophotometry.” Res. J. Pharm. Techno. 2011, 4 (7).
12.    Saisunee L and Boonsom L, “Simultaneous determination of Tolperisone and Lidocaine by HPTLC.” J. Pharma. And Bio. Ana. 1999, 20, 401–404.
13.    Patel MJ, Badmanaban R and Patel CN, “RP-HPLC method for simultaneous analysis of Tolperisone hydrochloride and Etodolac in combined fixed oral dosage formulation.” Pharmaceutica Methods-A Pharma. J. Pharm Assoc. 2011, 2 (2), 124-129.
14.    Bae JW, Park YS, Sohn UD, Myung CS, Ryu BK, Jang CG and Lee SY, “HPLC determination of Tolperisone in human plasma.” Arch Pharm. Res. 2006, 29 (4), 339-42.
15.    Carolin NI, Balan P, Chiranjeevi N, Maheswari UV and Rajasekar S, “Method development, validation and forced degradation studies of Tolperisone hydrochloride by RP-HPLC method in bulk and tablet dosage form.” J. Pharm. Res. 2011, 4 (5), 1356-1357.
16.    Liawruangrath S, Liawruangrath B and Pibool P, “Simultaneous determination of Tolperisone and Lidocaine by high performance liquid chromatography.” J. Pharm.And Biomed. Anal. 2001, 26 (5-6), 865-72.
17.    Murali. M. and Satyanarayana. PV, “Simple validated isocratic RP –HPLC method for estimation of Tolperisone in bulk and pharmaceutical dosage form.” Der Pharma. Chemica. 2011, 3 (5), 13-19.
18.    Gharge D and Dhabale P, “Spectrophotometric estimation of Paracetamol from tablet formulations.” Int. J. Chem. And Ana. Sci. 2010, 1 (1), 3-5.
19.    Narayan S, Pradeep Kumar, Sindhu RK, Tiwari A and Ghosh M, “Simultaneous analysis of Paracetamol and Tramadol – Analytical method development & validation.” Der Pharma. Chemica. 2009, 1 (2), 72-78.
20.    Jain NA, Dudane NP and Lohiya RT, “Simultaneous determination of Metaclopramide and Paracetamol by area under curve spectrophotometric method in combined tablet dosage form.” J. Pharma. Bio. Chem. Sci. 2011, 2 (2), 250.
21.    Attimarad M et al, “Simultaneous determination of Paracetamol and Lornoxicam by RP-HPLC in bulk and tablet formulation.” Pharma. Methods. 2011, 1(4).
22.    Gharge D and Dhabale P, “Simultaneous estimation of Aceclofenac and Paracetamol in solid dosage form by RP-HPLC method.” Int. J. ChemTech. Res. 2010, 2 (2), 942-946.
23.    Ebru CD, Mehmet G and Hale C, “Validation of method for   simultaneous determination of Paracetamol and Phenylephrine in pharmaceutical formulation by reversed phase liquid chromatography.” Int. J. Comprehensive Pharm.2011, 6.
24.    Shukla R, Sivakumar R, Vijayanand PR, Nallasivan P and Venkatnarayanan R, “RP-HPLC method for simultaneous estimation of Tramadol hydrochloride and Paracetamol bulk drug and its combined dosage form.” Asian J. Res. Chem. 2010, 3 (3).
25.    ICH Q2 (R1) Validation of Analytical Procedure: Text and Methodology, Geneva, International Conference on Harmonisation, 2005.




1 comment:

  1. Thanks so much for sharing all of this information! I have always wanted to learn more about HPLC analysis because I have a friend who just got into it in college. I now know what he'll be talking about whenever he discusses it with me!

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