Development and Validation of Derivative Spectrophotometric Method for Simultaneous Estimation of Lornoxicam and Eperisone in their Synthetic Mixture

 

Jawed Akhtar¹*, Jatin Prajapati², ShamimAhmad¹, Mohammad Mujahid¹,

Gamal Osman Elhassan³

¹Translam Institute of Pharmaceutical Education and Research, Meerut, Uttar Pradesh-250001

²Astra Life Care Pvt. Ltd, Ahmedabad.

³Faculty of Pharmacy, Omdruman Islamic University, Khartoum, Sudan

*Corresponding Author E-mail: jawed.pharmacy@gmail.com

A simple, precise, accurate and reproducible spectrophotometric method has been developed for simultaneous estimation of Lornoxicam (LXM) and Eperisone (EPE) by employing first order derivative zero crossing method in methanol. The first order derivative absorption at 264.5 nm (zero cross point of LXM) was used for quantification of EPE and 254 nm (zero cross point of EPE) for quantification of LXM. The linearity was established over the concentration range of 2-24 µg/ml with correlation coefficient 0.9987 and 0.997, respectively. The mean % recoveries were found to be in the range of 99.69 and 99.13 for LXM and EPE respectively. The developed method has been validated as per ICH guidelines and successfully applied to the estimation of LXM and EPE in their Synthetic Mixture.

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INTRODUCTION:

Lornoxicam (LXM) is chemically (3E)-6-chloro-3-[hydroxyl (pyridin-2-ylamino) methylene]-2 methyl-2,3-dihydro - 4H- thieno [2,3-e] [1,2] thiazin-4-one 1, 1-dioxide.^[1] Lornoxicam is an NSAID of the oxicam class with analgesic, anti-inflammatory and antipyretic properties. It inhibits prostaglandin synthesis by inhibiting both cyclo-oxygenase enzyme (COX-1 and COX-2).  EPE is chemically (2RS)-1-(4-ethylphenyl)-2-methyl-3-(1-piperidyl) propane-1-one.^[2] EPE is an antispasmodic drug. It acts by relaxing both skeletal muscles and vascular smooth muscles, and demonstrates a variety of effects such as reduction of myotonia, improvement of circulation, and suppression of the pain reflex. The review of literature revealed that various involving spectrophotometry have been reported for LXM in single form and in combination with other drugs.

 

Lornoxicam is estimated by UV and HPLC method.^[3-17] According to literature review, Eperisone is estimated by UV, HPLC, LC-EI-MS methods.^[18] There is no any method reported for Simultaneous estimation of Lornoxicam and Eperisone in a combination by UV and HPLC, but individually available for each drug and in combination with other drug. The present work describes the development of a simple, precise, accurate and reproducible spectrophotometric method for the simultaneous estimation of LXM and EPE in synthetic mixture. The developed method was validated in accordance with ICH Guidelines and successfully employed for the assay of LXM and EPE in synthetic mixture.

 

Structure of Eperisone

 

Structure of Lornoxicam

 

MATERIALS AND METHODS:

Reagents and Chemicals:

Analytically pure LXM and EPE were kindly provided by Cirex Pharmaceuticals Limited. Analytical grade methanol was purchased from Merck Pvt. Ltd., India.

 

Instrument and Apparatus:

Shimadzu-1800 UV‐Visible Spectrophotometer was used for spectral measurements with spectral band width 1 nm, wavelength accuracy is 0.5 nm and 1 cm matched quartz cells. Software used was UV Probe (version 2.34). Glassware used in each procedure were soaked overnight in a mixture of chromic acid and sulphuric acid rinsed thoroughly with double distilled water and dried in hot air oven.

 

Spectrophotometric Condition:

All zero order spectrums (D0) were converted to first derivative spectrum (D1) using delta lambda 1.

 

Preparation Standard Stock Solutions:

Accurately weighed 10mg of LXM and EPE standard were transferred to separate 100 ml volumetric flask and dissolved in 50 ml 0.1 N methanolic NaOH. The flasks were shaken and volume was made up to the mark with the same solvent to give solutions containing 100 μg/ml LXM and 100 μg/ml EPE.

 

Selection of Analytical Wavelength:

2-12 μg/ml solutions of both LXM and EPE were prepared in methanol by appropriate dilution and spectrum was recorded between 200-500 nm and first derivative spectrums were obtained using above condition. The overlain first derivative spectrums of LXM and EPE at different concentration were recorded. The zero crossing point (ZCP) of LXM was found to be 264.5 nm and ZCP of EPE was found to be 254 nm.

 

METHOD VALIDATION: ^[19-20]

The proposed method has been extensively validated in terms of specificity, linearity, accuracy, precision, limits of detection (LOD) and quantification (LOQ), robustness and reproducibility. The accuracy was expressed in terms of percent recovery of the known amount of the standard drugs added to the known amount of the pharmaceutical dosage forms. The precision (Coefficient of Variation - C.V.) was expressed with respect to the repeatability, intra-day and inter-day variation in the expected drug concentrations. After validation, the developed methods have been applied to pharmaceutical dosage form.

 

Specificity:

Commonly used excipients (starch, microcrystalline cellulose and magnesium stearate) were spiked into a pre weighed quantity of drugs. The D1 spectrum was recorded by appropriate dilutions and the quantities of drugs were determined.

 

Linearity:

Appropriate volume of aliquot from LXM and EPE standard stock solution was transferred to volumetric flask of 10 ml capacity. The volume was adjusted to the mark with methanol to give a solutions containing 2-16 μg/ml of both LXM and EPE All D1 Spectrum were recorded using above spectrophotometric condition. D1absorbance at 254 nm and 264.5 nm were recorded for LXM and EPE, respectively (n=6). Calibration curves were constructed by plotting average absorbance versus concentrations for both drugs. Straight line equations were obtained from these calibration curves.

 

Accuracy:

Accuracy was assessed by determination of the recovery of the method by addition of standard drug to the pre-quantified placebo preparation at 3 different concentration levels 80, 100 and 120 %, taking into consideration percentage purity of added bulk drug samples. Each concentration was analyzed 3 times and average recoveries were measured.

 

Precision:

The repeatability was evaluated by assaying 6 times of sample solution prepared for assay determination. The intraday and interday precision study of LXM and EPE was carried out by estimating different concentrations of LXM (0.16, 0.48, 0.96 μg/ml) and EPE(2, 6, 12 μg/ml), 3 times on the same day and on 3 different days (first, second, third) and the results are reported in terms of C.V.

 

Detection limit and Quantitation limit:

ICH guideline describes several approaches to determine the detection and quantitation limits. These include visual evaluation, signal-to-noise ratio and the use of standard deviation of the response and the slope of the calibration curve. In the present study, the LOD and LOQ were based on the third approach and were calculated according to the 3.3σ/S and 10σ/S criterions, respectively; where σ is the standard deviation of y-intercepts of regression lines and s is the slope of the calibration curve.

 

Robustness:

The sample solution was prepared and then analyzed with change in the typical analytical conditions like stability of analytical solution.

 

Reproducibility:

The absorbance readings were measured at different laboratory for sample solution using another spectrophotometer by analyst and the values obtained were evaluated using t- test to verify their reproducibility.

 

Determination of Lornoxicam and Eperisone in their synthetic mixture Sample preparation:

A powder quantity equivalent to 0.8 mg LXM and 10 mg EPE was accurately weighed and transferred to volumetric flask of 100 ml capacity. 60 ml of 0.1 N methanolic NaOH was transferred to this volumetric flask and sonicated for 15 min. The flask was shaken and volume was made up to the mark with the same solvent. From this solution 2 ml was transferred to volumetric flask of 100 ml capacity. Volume was made up to the mark to give a solution containing 0.16 μg/ml of LXM and 2 μg/ml of EPE. The resulting solution was analyzed by proposed method. The quantitation was carried out by keeping these values to the straight line equation of calibration curve.

 

RESULTS AND DISCUSSION:

First order derivative spectrophotometric method was developed for determination of LXM and EPE The proposed method has been extensively validated as per ICH guidelines. Summary of validation parameters for proposed method was given in Table 1.

 

The overlain D1 spectrum of LXM and EPE at different concentrations revealed that at 254 nm (ZCP of EPE) TRA possesses significant D1 absorbance and at 264.5 nm (ZCP of LXM) EPE possesses significant D1 absorbance. Considering above facts, wavelength 254 nm and 264.5 nm were selected for the estimation of LXM and EPE, respectively (figure 2). Linearity was assessed for LXM and EPE by plotting calibration curves of the D1 absorbance versus the concentration over the concentration range 2-18 μg/ml for both drugs. The correlation coefficients (r2) for LXM and EPE were found to be 0.9986 and 0.997, respectively (Table 2). The following equations for straight line were obtained for LXM and EPE. Linear equation for LXM, y = 0.0014x + 0.0004 Linear equation for EPE, y = 0.0041x + 0.0032. The % recoveries were found to be in the range of 99.69 % for LXM and 99.13 for EPE (Table 3).

 

The precision of method was determined by repeatability, intraday and interday precision and was expressed as the C.V. (Table 1), which indicate good method precision. The Limit of detection for LXM and EPE was found to be 0.144μg/ml and 1.60μg/ml respectively. Limit of quantification for LXM and EPEwas found to be 0.43μg/ml and 4.86μg/ml at 254 nm and at 264.5 nm respectively (Table 1). The method was also found to be specific, as there was no interference observed when the drugs were estimated in presence of excipients and robust, as there was no significant change in absorbance up to 24 hours of preparation of solution in methanol. The proposed spectrophotometric method was successfully applied to LXM and EPE synthetic mixture and its combined dosage form. The results are shown in Table 6.

Derivative Spectra

 

Table 1 - Specificity study for synthetic mixture

Mixture	Conc. µg/ml	Max (nm.)	Before addition of excipients		After addition of  excipients		% Interference
			Absorbance	Conc. µg /ml	Absorbance	Conc. µg/ml
1	0.64L	254	0.0012	0.57	0.00113	0.52	8.75
	8.0E	264.5	0.0331	7.29	0.03291	7.24	0.63
2	0.8 L	254	0.0014	0.71	0.00131	0.65	9.0
	10.0E	264.5	0.0419	9.43	0.04165	9.37	0.64
3	0.96L	254	0.0015	0.78	0.0014	0.71	9.09
	12.0E	264.5	0.0507	11.58	0.0504	11.51	0.63
Mean	LXM	8.947 ± 0.17		% RSD 1.96
	EPE	0.63 ± 0.0074		% RSD 1.16

 

Table 2 -Linearity

 

Eperisone

 

Lornoxicam

 

Table 3: Accuracy - Recovery study for the synthetic mixture

Mixture (LXM:EPE)	Conc. (µg/ml)	Wavelength (nm)	Absorbance	Conc. before spiking (µg/ml)	Reference standard added (µg/ml)	Conc. after spiking (µg/ml)	% Recovery	Mean ± SD	% RSD
1.	LXM (0.8) + EPE (10.0)	254	0.0015	0.78	80 % ( 0.64 L+ 8.0 E)	1.42	100.44	LXM 99.61 ± 1.22	LXM 1.22
		264.5	0.0418	9.41		17.29	98.47
2.		254	0.0014	0.71	100% (0.8 L + 10.0 E)	1.50	98.21
		264.5	0.0417	9.39		19.36	99.75	EPE 99.13 ± 0.64	EPE 0.64
3.		254	0.0014	0.71	120% (0.96L + 12.0 E)	1.67	100.44
		264.5	0.0419	9.43		21.34	99.18

 

 

Table 4: Intraday precision study for the synthetic mixture

Conc. (µg/ml)		Wavelength (nm)	Absorbance			Mean	S.D.	RSD
LXM	EPE		A	B	C
0.16	2.0	254	0.00062	0.00061	0.00062	0.000616	5.7×10-6	0.936
		264.5	0.0104	0.0105	0.0105	0.01046	5.7×10-6	0.551
0.48	6.0	254	0.00098	0.001	0.00098	0.00098	1.1×10-5	1.17
		264.5	0.0264	0.0263	0.0265	0.0264	1×10-4	0.378
0.96	12.0	254	0.00167	0.00168	0.00164	0.00166	2.1×10-5	1.25
		264.5	0.0509	0.0508	0.0508	0.05083	5.7×10-5	0.113

 

 

Table 5: Interday precision study for the synthetic mixture

Conc. (µg/ml)		Wavelength (nm)	Absorbance			Mean	S.D.	RSD
LXM	EPE		A	B	C
0.16	2.0	254	0.00062	0.00061	0.00062	0.000616	5.7×10-6	0.936
		264.5	0.0107	0.0108	0.0107	0.01046	5.7×10-5	0.537
0.48	6.0	254	0.001	0.001	0.00098	0.000993	1.5×10-5	1.16
		264.5	0.0262	0.0261	0.0262	0.02616	5.7×10-5	0.22
0.96	12.0	254	0.00167	0.00168	0.00164	0.00166	2.1×10-5	1.25
		264.5	0.0507	0.0506	0.0505	0.0506	1×10-4	0.19

 

 

 

Table 6.1: Robustness study for the synthetic mixture at 254 nm

Mixture LXM:EPE	Conc. (µg/ ml)	Absorbance at wavelength in (nm)
		At 253	Conc. obtained	At 254	Conc. obtained	At 255	Conc. obtained
1	0.32	0.00089	0.28	0.0009	0.35	0.00091	0.42
2	0.64	0.00119	0.56	0.0012	0.57	0.00121	0.58
3	0.8	0.0011	0.71	0.0014	0.71	0.0016	0.73
Mean			0.35		0.57		0.716
S.D.			0.0057		1.01		0.0115
R.S.D.			1.63		1.75		1.61

 

 

Table 6.2: Robustness study for the synthetic mixture at 264.5 nm

Mixture LXM:EPE	Conc. (µg/ml)	Absorbance at wavelength in (nm)
		At 263.5	Conc. obtained	At.264.5	Conc. obtained	At 265.5	Conc. obtained
1	4	0.0183	3.68	0.0182	3.65	0.0181	3.63
2	8	0.0332	7.31	0.0331	7.29	0.0329	7.24
3	10	0.0421	9.48	0.0419	9.43	0.0418	9.41
Mean			3.653		7.28		9.41
S.D.			0.025		0.036		0.036
R.S.D.			0.68		0.49		0.38

 

 

Table 7: Reproducibility study for the synthetic mixture

Conc.(µg/ml)		Wavelength (nm)	Absorbance			Mean	S.D.	RSD
LXM	EPE		A	B	C
0.16	2.0	254	0.00062	0.00061	0.00062	0.000616	5.7×10-6	0.94
		264.5	0.0104	0.0105	0.0105	0.01046	5.7×10-6	0.55
0.48	6.0	254	0.00098	0.001	0.00098	0.00098	1.15×10-5	1.17
		264.5	0.0264	0.0263	0.0265	0.0264	1×10-4	0.38
0.96	12.0	254	0.00167	0.00168	0.00164	0.00166	2.1×10-5	1.25
		264.5	0.0509	0.0508	0.0508	0.05083	5.7×10-5	0.11

 

 

Table 8: Assay

Sr. No.	Conc. (µg/ml)	Wavelength(nm)	Abs.	Conc.	% Assay	Mean % Assay
1	LXM (0.8) + EPE (10.0)	254	0.0015	0.78	98.21	LXM 98.51
		264.5	0.0434	9.80	98.04
2		254	0.00151	0.79	99.107
		264.5	0.0436	9.85	98.53	EPE 98.29
3		254	0.0015	0.78	98.21
		264.5	0.0435	9.82	98.29

 

 

Table 9: Result

 

 

CONCLUSION:

The proposed first order derivative method provides simple, specific, precise, accurate and reproducible quantitative analysis for simultaneous determination of LXM and EPE in synthetic mixture. The method was validated as per ICH guidelines in terms of specificity, linearity, accuracy, precision, limits of  detection  (LOD) and quantification (LOQ), robustness and reproducibility. The proposed method can be used for routine analysis and quality control assay of LXM and EPE in combined dosage form.

 

ACKNOWLEDGEMENT:

Authors are thankful to Cirex Pharmaceuticals Limited, Hyderabad for providing gratis sample.

 

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Received on 06.05.2015         Modified on 17.06.2015

Accepted on 04.07.2015         © AJRC All right reserved

Asian J. Research Chem. 8(7): July- 2015 ; Page 465-471