SwethaBhavani N, HimaBindu S, Venkateswara Rao A, Panikumar D Anumolu Simultaneous Quantification of Lornoxicam and Paracetamol Combination By Application of First Derivative UV- Spectroscopy SwethaBhavani N, HimaBindu S, Venkateswara Rao A, Panikumar D Anumolu

contents Introduction Objective References Drug profile 1 Introduction Objective Drug profile Results & discussions References Conclusion Materials & methods

DERIVATIVE SPECTROSCOPY 2 Derivatization of actual spectra with respect to wavelength Zero order First order Second order Zero, first and Second-order UV derivative spectrum

3 Advantages of First derivative spectroscopy: (1) Precise determination of the λmaxcan be obtained from the zero crossing of the first derivative. (2) Improved spectral resolution (3) Discrimination of broad bands Resolution enhancement in derivative spectroscopy

OBJECTIVES 4 Development and validation of UV derivative spectrophotometric method for simultaneous estimation of Lornoxicam and Paracetamol Application of developed method for simultaneous estimation of Lornoxicam and Paracetamol in marketed tablets

Drug profile of Lornoxicam 5 Category: NSAID Chemical name: 6-Chloro-4-hydroxy-2-methyl-N-2pyridinyl-2H-thieno[2,3-e]- 1,2thiazine-3-carboxamide1,1-dioxide Molecular formula:C13H10Cl N3O4S2 Molecular weight: 371.82 g/mol Melting point: 225-2300c Solubility: Slightly soluble in chloroform & 0.1mol/LNaOH, and very slightly soluble in methanol and acetonitrile, hardly soluble in water. pKa : 4.7 Partition coefficient : Log P (octanol/pH 7.4 buffer):1.8 ,octanol-water- 3.15(calc)

Drug profile of Paracetamol: 6 Category: Analgesic and antipyretic Chemical name: N-(4-Hydroxyphenyl)acetamide Molecular formula:C8H9NO2 Molecular weight:151.2 Melting point: 169.0° to 170.5° Solubility: Very slightly soluble in cold water, considerably more soluble in hot water; soluble in ethanol, methanol, dimethylformamide, ethylene dichloride, acetone, and ethyl acetate; very slightly soluble in chloroform; slightly soluble in ether; practically insoluble in petroleum ether, pentane, and benzene pKa : 9.5 (25°).Partition coefficient : Log P(octanol/water), 0.5. Appearance: White crystals or crystalline powder

Instrument : Shimadzu UV-1800 Spectrophotometer 7 Materials and methods Instrument : Shimadzu UV-1800 Spectrophotometer Chemicals Used –0.01N NaOH

Preparation of standard stock solutions Method development 8 Preparation of standard stock solutions LOR(10mg) and PAR (10mg) were weighed and transferred into two separate 10ml volumetric flasks Dissolved in 1ml 0.01N NaOH and volume was made up to 10ml These solutions have concentration 1000mcg/ml- stock A From stock A 5ml each of LOR and PAR are taken into two separate 50ml volumetric flasks and volume makeup was done with 0.01N NaOH 100mcg/ml stock B From stock B 10mcg/ml solutions of LOR and PAR were prepared with 0.01N NaOH and scanned in the range of 200-400nm using 0.01N NaOH as blank S.No.

9 A B S O R N C E WAVE LENGTH(nm) Zero-order UV overlaid spectrum of Lor(10mcg/ml) and Par(10mcg/ml) in 0.01N NaOH

10 Since the zero order spectra of two drugs are overlapping, zero order spectra were transformed into corresponding first derivative spectra גmax for LOR zcp for LOR dA/dג zcp for PAR גmax for PAR WAVELENGTH First Order UV Overlaid Spectrum of Lor (10mcg/ml) and Par (10mcg/ml) in 0.01N NaOH

Calibration curve for of LOR & PAR 11 Preparation of calibration curve for Lornoxicam From stock Solution - B (LOR) 0.2,0.6,1,1.4,1.8,2.2ml transferred into 10ml volumetric flasks and volume was adjusted to 10ml with 0.01N NaOH (2-22mcg/ml) First derivative spectra were recorded and derivative absorbance was measured at 347 nm Preparation of calibration curve for Paracetamol : From stock Solution-B (PAR) 0.1, 0.6, 1.2, 1.8, 2.4 , 3, 3.6ml transferred into 10ml volumetric flasks. (1-36mcg/ml) First derivative spectra were recorded and derivative absorbance measured at 272.5nm

Calibration plot of LOR & PAR 12 zcp for LOR גmax for LOR dA/dג zcp for PAR ג max for PAR WAVELENGTH (nm) UV first-derivative linearity range of LOR (2-22mcg/ml) and PAR (1-36mcg/ml) in 0.01N NaOH

Concentration (mcg/ml) Derivative absorbance (AM ± SD) (n=3) 13 Data for standard plot of LOR in 0.01N NaOH at 347nm Concentration(mcg/ml) S.No. Concentration (mcg/ml) Derivative absorbance (AM ± SD) (n=3) 1 2 0.00365 ± 0.00005 3 6 0.00945± 0.0013 4 10 0.01553± 0.00016 5 14 0.0221 ± 0.00005 18 0.02775 ± 0.00025 7 22 0.03475± 0.00025 Derivative absorbance Standard plot of LOR in 0.01N NaOH at 347 nm

Data for standard plot of PAR in 0.01N NaOH at 272.5 nm 14 S.No. Concentration (mcg/ml) Derivative absorbance (AM ± SD) (n=3) 1 2 -0.00517±0.00007 3 6 -0.0194±0.0146 4 12 -0.04026 ± 0.0002 5 18 -0.0598± 0.00041 24 -0.07856 ± 0.0002 7 30 -0.09755 ± 0.0012 8 36 -0.1157±0.00107 Derivative absorbance Concentration(mcg/ml) Standard plot of PAR in 0.01N NaOH at 272.5 nm

Precision Lornoxicam Intra-day precision Inter-day precision 15 Lornoxicam Concentration (mcg/ml) Intra-day precision Inter-day precision Concentration estimated (mcg/ml) (AM ± SD) (n=3) % RSD %RSD 2 2.06 ± 0.004 0.22 1.98± 0.010 0.54 14  14.54 ± 0.065 0.45 14.02 ± 0.096 0.69 32 22.56 ± 0.22 0.98 22.34± 0.205 0.92 Paracetamol Concentration estimated (mcg/ml) (AM ± SD) (n=3) 1  1.22± 0.003 0.25  1.12± 0.004 0.357 18 18.14 ± 0.116 0.64 18.02 ± 0.129 0.72 36 36.32± 0.272 0.75 36.24± 0.358 0.99

Theoretical content (mg) Amount found (mg) (AM ± SD) (n=3) Recovery studies 16 Formulation Recovery level (%) Recovery of analyte Theoretical content (mg) Amount found (mg) (AM ± SD) (n=3) Recovery (%) % RSD Lornoxi-P LOR 2 2.1± 0.015 105 0.714 PAR 125 125.1 ± 0.145 100.08 0.115 80 3.6 3.5± 0.045 97.2 1.285 225 224.5 ± 0.34 99.77 0.151 100 4 4.2 ± 0.009 0.214 250 250.24 ± 0.985 100.09 0.393 120 4.4 4.4 ± 0.012 0.272 275 275.12± 1.256 100.04 0.456 Lornasafe -plus 2.2 ± 0.012 110 0.56 125.04 ± 0.252 100.03 0.201 3.4 ± 0.008 94.4 0.24 225.05 ± 0.556 100.02 0.247 3.7 ± 0.024 92.5 0.67 250.12 ± 0.357 0.142 4.3 ± 0.014 97.7 0.34 274.9± 0.852 99.9 0.309

System Suitability Parameters 17 System Suitability Parameters S.No Parameter Lornoxicam Paracetamol 1 Absorption maxima (nm) 347 272.5 2 Beer's Law Limit (mcg/ml) 2-22 1- 36 3 slope 0.001 -0.003 4 Intercept 5 Correlation coefficient 0.999 0.995 6 Regression equation y = 0.001x +0.0 y =-0.003x +0.0000 7 LOD (mcg/ml) 0.1643 0.0745 8 LOQ (mcg/ml) 0.5477 0.248

Amount found (mg) (AM ± SD) (n=3) Assay 18 Formulation Lornoxicam Paracetamol Label claim (mg) Amount found (mg) (AM ± SD) (n=3) % Recovery % RSD Lornoxi-p 8 8.4 ± 0.011 105 0.13 500 500.16±0.252 100.032 0.05 Lornasafe-plus 8.2 ± 0.102 102 1.243 500.04 ±0.562 100 0.112

ADVANTAGES OF PROPOSED METHOD 19 Simple spectra and less interference. High sensitivity and wide linearity range. Rapid, economical. Eco-friendly, accurate and precise method. High spectral discrimination. Single analytical method for quantification of LOR and PAR in combined tablet dosage forms.

conclusion 20 It is a simple, novel, more economic, reliable, precise and accurate method for simultaneous estimation of Lornoxicam and Paracetamol in bulk and formulations. So this method can be successfully applied to routine analysis of studied drugs in their tablet dosage forms.

References: 21 ICH Harmonized Tripartite guideline validation of analytical procedures: text and methodology, Q2(R1) Current step 4 version, 27 october 1994. Mark, H. and Workman, J. Derivatives in spectroscopy. Spectroscopy. 2003 ,18 (4): 32-37 Beckett AH and Stenlake JB. Practical pharmaceutical chemistry; 4th edition, the athlone press. 2007, 269-299.

22 Lakshmi Sivasubramanian*, Lakshmi K.S. and TinTu.T. PB Khedikar and G Pawnikar, Absorption Correction Method for Simultaneous Estimation of lornoxicam and paracetamol tablet dosage form by absorbance ratio method , International Journal of pharmacy and pharmaceutical sciences, 2010,vol 2,Issue 4 . DharaJ.Patel*Vivek Patel., Simultaneous determination of paracetamol and lornoxicam in tablets by thin layer chromatography combined with densitometry, An International Journal of chem tech research, pp 1929-1932,Vol-2 no.4. T.Raja*,A.LakshmanaRao,Validated High performance Thin layer liquid chromatography method for simultaneous quantitation of paracetamol and lornoxicam in bulk drug and pharmaceutical formulation , International Journal Pharm Biomed Res 2012,3(3),162- 166 .

23 Thanq…