Impact Factor: 0.3397/ICV: 4.10 59 ANALYTICAL METHOD DEVELOPMENT and VALIDATION of UV SPECTROSCOPIC METHOD for SIMULTANEOUS ESTI

Impact factor: 0.3397/ICV: 4.10 59

Pharma Science Monitor 5(3) Supl-1, Jul-Sep 2014

PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES Journal home page: http://www.pharmasm.com

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF UV SPECTROSCOPIC METHOD FOR SIMULTANEOUS ESTIMATION OF ZALTOPROFEN AND PARACETAMOL IN COMBINED DOSAGE FORM Chirag Dahyabhai Patel*, Ashim Kumar Sen, Dhanya B. Sen, Ujjwal Sahoo, A. K. Seth. Department of Pharmacy, Sumandeep Vidyapeeth, At & Po Piparia, Ta- Waghodia, Dist. Vadodara-391760. (Gujarat) India.

ABSTRACT A simple, accurate, precise & sensitive UV spectroscopic method was developed for the simultaneous determination of zaltoprofen (ZLT) & paracetamol (PCM) in combined tablet dosage form. For the simultaneous equation method, the wavelength range of both the drugs zaltoprofen & paracetamol were selected as 227 nm and 243 nm. The linearity was found in the concentration range of 1 to 5 µg/ml and 4.062 to 20.31 µg/ml for zaltoprofen and paracetamol, respectively. The slope, intercept, correlation coefficient values of zaltoprofen & paracetamol at 227 nm & 243 nm were found to be 0.032, 0.047, 0.999 (227 nm); 0.029, 0.043, 0.998 (243 nm) & 0.041, 0.073, 0.999 (227 nm); 0.059, 0.095, 0.999 (243 nm). The % recovery values were found to be 98.66 % - 99.09 % for zaltoprofen and 98.40 % - 98.86% for paracetamol, stated that the method was accurate. The LOD and LOQ values of both the drugs zaltoprofen & paracetamol were found to be 0.216 µg/ml, 0.656 µg/ml (227 nm); 0.238 µg/ml, 0.724 µg/ml (243 nm) & 0.225 µg/ml, 0.682 µg/ml (227 nm); 0.117 µg/ml, 0.355 µg/ml (243 nm). This method was successfully applied to the determination of zaltoprofen & paracetamol content in marketed formulation. The method was validated statistically as per ICH guideline. The results proved that the method can be employed for the routine analysis of zaltoprofen & paracetamol in the combined formulations. KEYWORDS: Simultaneous equation method, UV-Visible spectrophotometer, Zaltoprofen, Paracetamol.

INTRODUCTION Zaltoprofen (ZLT), 10, 11-Dihydro-alpha-methyl-10-oxo-dibenzo [b,f]thiepin-2-acetic acid is a nonsteroidal anti-inflammatory drug (NSAIDs) with powerful analgesic action on inflammatory pain (Figure: 1).[1,2] Zaltoprofen has also been reported to inhibit bradykinin induced nociceptive responses by blocking the activation of protein kinase C. Zaltoprofen is a preferential COX-2 inhibitor and selectively inhibits prostaglandin E(PGE2) production at inflammatory sites.[3] Paracetamol (PCM), N-(4-hydroxyphenyl) acetamide is an analgesic and antipyretic drug (Figure: 2). [4] It is used for the relief of mild to moderate pain & fever. Paracetamol reduces the synthesis of prostaglandins which are responsible for the mediation of pain and fever. The main

Chirag et al. / Pharma Science Monitor 5(3) Supl-1, Jul-Sep 2014, 59-70 Impact factor: 0.3397/ICV: 4.10 60 mechanism proposed is the inhibition of cyclooxygenase (COX) and recent findings suggest that it is highly selective for COX-2. [5]

O NH CH3 OH O S CH 3 HO

Zaltoprofen Paracetamol

Figure: 1 & 2 - Chemical structures of Zaltoprofen and Paracetamol.

Literature survey reveals various analytical methods for the estimation of zaltoprofen and paracetamol individually by UV spectroscopic method [6-11], HPLC [12-14] and HPTLC [15]. However, no analytical method has been reported for their simultaneous estimation from combined dosage form. Hence, the present work aims at developing newer analytical method that is simple, accurate, rapid, precise, sensitive and reliable. Development & validation of method is an important requirement in analytic purpose. For this, the United States Pharmacopoeia (USP) [16] and International Conference on Harmonization (ICH) [17] provide guidelines for performing these validations. The present work describes new methods for simultaneous determination of zaltoprofen and paracetamol using UV- Visible spectroscopic method. The proposed method is simple, easy and suitable for determination of the two drugs in combined formulations simultaneously. MATERIALS AND METHODS Instrument: A Shimadzu UV-Visible spectrophotometer (UV - 1800, Shimadzu, Japan) was used for all absorbance measurements with matched quartz cells; Digital balance: Ohaus - 0 to 250 gm; Sonicator: Ultra sonica cleaner - Toshcon Pvt. Ltd. Chemicals: All the chemicals and reagents used were of analytical grade. Zaltoprofen and Paracetamol Active Pharmaceutical Ingredient (API) were received as gift sample from IPCA laboretories, Mumbai (India) & Osaka Pharmaceutical Pvt. Ltd, Vadodara (Gujarat, India). Zaltoprofen & Paracetamol film coated tablet (Label claim 80/325 mg) was purchased from market.

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Method:- Method Development: Selection of solvent: Solubility of the drugs were checked in different solvents (methanol, acetonitrile, water) and UV-spectra of zaltoprofen & paracetamol (10 μg/ml) were recorded. Absorbances of both drugs were higher and both the drugs exhibited distinct λmax in water. So, water was selected as solvent for further studies & shown in Figure: 3. Selection of analytical wavelength: Appropriate dilutions were made from the standard stock solution A (ZLT) and B (PCM). The solutions were scanned in the wavelength range of 200-400 nm. Drugs showed optimum absorbance at 227 nm & 243 nm. Overlain spectra of Zaltoprofen (3 µg/ml) and Paracetamol (12.19 µg/ml) shown in Figure: 3. Preparation of standard stock solution of Zaltoprofen (ZLT): Accurately weighed 10 mg of ZLT was dissolved in a 5 ml of methanol in a 100 ml volumetric flask and volume was made up to the mark with distilled water to get a solution of 100 μg/ml (Stock solution A). Preparation of standard stock solution of Paracetamol (PCM): Accurately weighed 40.62 mg of PCM was dissolved in a 5 ml of methanol in 100 ml volumetric flask and volume was made up to the mark with distilled water to get a solution of 406.2 μg/ml (Stock solution B).

Figure: 3 – Overlain UV spectra of Zaltoprofen (3 µg/ml) and Paracetamol (12.19 µg/ml) at 227 nm and 243 nm.

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RESULT AND DISCUSSION Method Validation:- Linearity & Range: From the standard stock solution A and B, appropriate aliquots were pipetted out in to a series of 10 ml volumetric flasks and dilutions were made up to the mark with water to obtain working standard solutions, concentrations ranging from 1-5 g/ml for ZLT and 4.062 - 20.31 µg/ml for PCM, respectively. Absorbances of these solutions were measured at 227 nm and 243 nm against water as blank for ZLT and PCM. The calibration curve was plotted of concentration v/s absorbance as shown in Figure: 4, 5. Linear regression data are shown in Table: 1 & 2. Table: 1 – Absorbances and Absorptivities values of Zaltoprofen at 227 nm & 243 nm. Zaltoprofen Sr. Conc. 227 nm 243 nm No (µg/ml Average Average Absorban Absorptivi Absorban Absorptivi . ) Absorptivi Absorptivi ce ty ce ty ty ty 1 1 0.081 0.0810 0.075 0.0750 2 2 0.110 0.0550 0.099 0.0495 0.0538 0.0494 3 3 0.143 0.0476 0.132 0.0440 (ax1) (ax2) 4 4 0.175 0.0437 0.162 0.0405 5 5 0.209 0.0418 0.191 0.0382

Table: 2 – Absorbances and Absorptivities values of Paracetamol at 227 nm & 2437 nm. Paracetamol Sr. Conc. 227 nm 243 nm No (µg/ml Average Average Absorban Absorptivi Absorban Absorptivi . ) Absorptivi Absorptivi ce ty ce ty ty ty 1 4.062 0.240 0.0600 0.332 0.0817 2 8.12 0.403 0.0500 0.572 0.0704 0.0498 0.0697 3 12.19 0.583 0.0480 0.831 0.0681 (ay1) (ay2) 4 16.25 0.739 0.0460 1.055 0.0649 5 20.31 0.906 0.0450 1.294 0.0637

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Figure: 4 - Calibration graph of Zaltoprofen at 227 nm and 243 nm.

Figure: 5 - Calibration graph of Paracetamol at 227 nm and 243 nm. Table: 3 Regression Analysis Data and Summary of the Calibration Curves. Parameters Zaltoprofen Paracetamol Detection wavelength (nm) 227 243 227 243 Beer’s law limits (µg/ml) 1-5 4.062 - 20.31

Regression y = 0.032x + y = 0.029x + 0.043 y = 0.041x + y = 0.059x + equation (y*) 0.047 0.073 0.094 Slope (m) 0.032 0.029 0.041 0.059 Intercept (c) 0.047 0.043 0.073 0.095 Correlation- coefficient (r2) 0.999 0.998 0.999 0.999 LOD (µg/ml) 0.216 0.238 0.225 0.117 LOQ (µg/ml) 0.656 0.724 0.682 0.355

Sensitivity: The sensitivity of the proposed method for measurement of ZLT and PCM was estimated in terms of limit of detection [LOD] and limit of quantification [LOQ]. The LOD and LOQ were calculated by using the value of slope and SD of response (intercept). The mean slope value and

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SD of response were obtained after plotting six calibration curves. The LOD and LOQ obtained are reported in Table: 3. Precision: The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision of the method was determined in terms of repeatability, intraday and interday precisions. Repeatability studies were done by six times consequently taking absorbances of the standard solution containing 3 μg/ml of ZLT and 12.19 μg/ml PCM & the % RSD of the replicate absorbances was calculated. Values of repeatability in terms of %RSD was found to be < 2.0 %. Table: 4 -Repeatability for Zaltoprofen and Paracetamol

REPEATABILITY Zaltoprofen Sr. Conc. Absorbance Mean* % Absorbance Mean* % No. (µg/ml) (227 nm) ± SD RSD (243 nm) ± SD RSD 1 0.140 0.130 2 0.141 0.135 3 0.138 0.140 0.131 3 ± 1.42 0.131 1.52 4 0.142 0.0020 0.129 ± 5 0.144 0.128 0.0026 6 0.140 0.133 Paracetamol Sr. Conc. Absorbance Mean* % Absorbance Mean* % No. (µg/ml) (227 nm) ± SD RSD (243 nm) ± SD RSD 1 0.583 0.833 2 0.584 0.837 0.831 3 0.582 0.584 0.830 12.19 0.513 ± 0.625 ± 0.0030 4 0.589 0.828 0.0052 5 0.588 0.824 6 0.582 0.829 *Average of six determinations.

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Intra day precision studies were done by three times in a day taking absorbances of the standard solutions containing 2 μg/ml, 3 μg/ml of ZLT & 8.12 μg/ml, 12.19 μg/ml of PCM and the % RSD of the replicate absorbances was calculated. Values of Intra day precision in terms of %RSD were found to be < 2.0 %. Table: 5 -Intraday Precision for Zaltoprofen and Paracetamol

INTRADAY PRECISION Zaltoprofen Sr. Conc. Absorbance Mean* % Absorbance Mean* % No. (µg/ml) (227 nm) ± SD RSD (243 nm) ± SD RSD 1 0.108 0.108 0.096 0.096 2 0.111 ± 1.37 0.097 ± 1.04 2 3 0.107 0.0015 0.095 0.0010 4 0.138 0.139 0.133 0.131 5 3 0.142 ± 0.131 ± 1.07 1.52 6 0.141 0.0015 0.129 0.0020

Paracetamol Sr. Conc. Absorbance Mean* % Absorbance Mean* % No. (µg/ml) (227 nm) ± SD RSD (243 nm) ± SD RSD 1 0.402 0.402 0.571 0.571 2 0.405 ± 0.62 0.574 ± 0.35 8.12 3 0.400 0.0025 0.570 0.0020 4 0.583 0.584 0.834 0.829 5 12.19 0.581 ± 0.70 0.829 ± 0.48 6 0.589 0.0041 0.826 0.0040 *Average of three determinations.

Inter day precision studies were done by once in three different day taking absorbances of the standard solution containing 2 μg/ml, 3 μg/ml of ZLT and 8.12 μg/ml, 12.19 μg/ml of PCM the % RSD of the replicate absorbances was calculated. Values of Inter day precision in terms of %RSD were found to be <2.0 %.

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Table: 6 -Interday Precision for Zaltoprofen and Paracetamol

INTER DAY PRECISION Zaltoprofen Absorbance Absorbance Mean* Sr. Conc. (227 nm) % (243 nm) Mean* % ± No. (µg/ml) RSD ± RSD DAY SD DAY 1 2 3 1 2 3 SD 0.109 0.096 1 2 0.109 0.111 0.108 ± 1.37 0.095 0.097 0.098 ± 1.56 0.0015 0.0015 0.140 0.130 2 3 0.138 0.143 0.140 ± 1.78 0.130 0.128 0.133 ± 1.92 0.0025 0.0025

Paracetamol Absorbance Absorbance Sr. Conc. Mean* (243) Mean* (227) % % No. (µg/ml) ± ± DAY RSD DAY RSD SD SD 1 2 3 1 2 3 0.404 0.573 1 8.12 0.401 0.404 0.407 ± 0.62 0.570 0.575 0.574 ± 0.45 0.003 0.0026 0.584 0.833 2 12.19 0.582 0.589 0.581 ± 0.73 ± 0.66 0.827 0.838 0.834 0.0043 0.0055 *Average of three determinations.

Accuracy: To study the accuracy of the method, recovery studies were carried out by addition of standard drug solution to preanalyzed sample at three different levels: 50, 100 and 150%. The resulting solutions were then reanalyzed by the proposed method. The % recovery and %RSD were calculated Values of % recovery in terms of %RSD were found to be < 2.0 %.

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Table: 7 Accuracy Data (% Recovery)

Amount of Amount Total Final Amount % Mean Level sample of drug Amoun Conc. Recoverd Recover * %RS (Formulation added t (µg/ml) (µg/ml) y ± D ) (mg) (API) (mg) SD (mg) ZALTOPROFEN 2.95 98.33 98.77 50% 10 05 15 3 2.98 99.33 ± 0.5158 2.96 98.66 0.5095 2.96 98.66 98.66 100% 10 10 20 3 2.97 99.00 ± 0.3406 2.95 98.33 0.3350 2.94 98.00 99.09 150% 10 15 25 3 2.98 99.33 ± 0.7011 2.97 99.00 0.5163 PARACETAMOL

12.08 99.09 98.80 50% 40.62 20.31 60.93 12.19 11.95 98.03 ± 0.692 12.11 99.34 0.6846 11.97 98.00 98.40 100% 40.62 40.62 81.24 12.19 12.05 98.85 ± 0.4341 11.99 98.35 0.4272 12.12 99.42 98.86 150% 40.62 60.93 101.55 12.19 11.94 98.00 ± 0.7686 12.09 99.17 0.7580 *Average of three determinations.

Analysis of formulation: Twenty tablets of marketed formulation, Reducin A (J. B. Chemicals & Pharmaceuticals Ltd. Mumbai, India) containing Zaltoprofen 80 mg and Paracetamol 325 mg were weighed and powdered. An accurately weighed quantity of the powder equivalent to 10 mg ZLT & 40.62 mg PCM was dissolved in 5 ml methanol and sonicated for 20 min and volume was made up to 100 ml with distilled water (100 µg/ml ZLT & 406.2 µg/ml PCM). The solution was filtered through Whatman filter paper No. 41 and aliquot portion of the filtrate was diluted to produce solution having final concentration of 3 μg/ml of ZLT and 12.19 μg/ml of PCM. The absorbance of sample solution was measured at selected wavelengths and the concentrations of the two drugs were estimated & shown in Table: 8.

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Table: 8 - Analysis of Formulation

Formulation : REDUCIN A

Drug Label claim (mg/tab) Amount found (mg/tab) % Drug found

ZLT 80 79.45 99.31

PCM 325 324.62 99.88

CONCLUSION: The proposed UV-Visible spectroscopic method is simple, precise, accurate and rapid for the simultaneous determination of zaltoprofen & paracetamol from tablets. Hence, it can be easily & conveniently employed for the routine quality control analysis of these drugs in combined formulation. ACKNOWLEDGEMENTS: Authors are thankful to Principal and Management, Department of Pharmacy, Sumandeep Vidyapeeth, Vadodara, Gujarat for providing the facilities to conduct this research work. REFERENCES 1. Kameyama T, Nabeshima T, Yamada S, Sato M. Analgesic and antiinflammatory effects of 2-(10,11-dihydro-10-oxo-dibenzo[b,f]thiepin-2-yl)propionic acid in rat and mouse. Drug Res 1987;37(1):19-26. 2. Tsurumi K, Kyuki K, Niwa M, Kokuba S, Fujimura H. Pharmacological investigations of the new antiinflammatory agent 2-(10,11-dihydro-10-oxodibenzo[b,f]thiepin-2- yl)propionic acid. 1986;36(12):1796-1800. 3. Kawai S, Nishida S, Kato M, Furumaya Y, Okamoto R, Koshino T, Mizushima Y. Comparison of cyclooxygenase-1 and -2 inhibitory activities of various nonsteroidal antiinflammatory drugs using human platelets and synovial cells. Eur J Pharmacol 1998;347:87-94. 4. Ministry of Health & Family Welfare. Indian Pharmacopoeia, Indian Pharmacopoeia Commission.Goverment of India, Ghaziabad 2007;3:1544. 5. Hinz B, Cheremina O, Brune K, Acetaminophen (paracetamol) is a selective cyclooxygenase-2 inhibitor in man. The FASEB journal: official publication of the Federation of American Societies for Experimental Biology 2008;22(2):383-90.

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For Correspondence Chirag Patel Email: [email protected]

Chirag et al. / Pharma Science Monitor 5(3) Supl-1, Jul-Sep 2014, 59-70