INTRODUCTION:

Lornoxicam, (6-chloro-4-hydroxy-2-methyl-N-2-pyridinyl-2H-thieno[2,3-e]-1,2-thiazine-3-carboxamide1,1-dioxide (Figure 1) is a non-steroidal anti-inflammatory drug with analgesic and antipyretic properties that belongs to the class of oxicams¹. It acts by nonselective inhibition of cyclo-oxygenase-1 and -2. It is prescribed for osteoarthritis, rheumatoid arthritis, acute lumbar-sciatica conditions and postoperative pain management. In the literature, a voltammetric², polarographic³, UV spectrophotometric⁴, LC-MS⁵and HPLC⁶methods were reported for the analysis of lornoxicam. The present study was aimed to develop a simple, rapid, precise, accurate and selective chromatographic method for the estimation of lornoxicam in bulk and tablet dosage forms with the use of buffer in the mobile phase in short duration.

MATERIALS AND METHODS:

The analysis of the drug was carried out on a Waters HPLC system equipped with a reverse phase Xterra C₁₈ column (100mmx4.6mm; 5 μm), a 2695 binary pump, a 20 μl injection loop and a 2487 dual absorbance detector and running on Waters Empower2 software.

Chemicals and solvents:

The reference sample of lornoxicam was supplied by Glenmark Pharmaceuticals Ltd, Mumbai. HPLC grade water and methanol were purchased from E. Merck (India) Ltd., Mumbai. Potassium dihydrogen phosphate and orthophosphoric acid of AR grade were obtained from S.D. Fine Chemicals Ltd., Mumbai.

Preparation of phosphate buffer (pH 3.0):

Seven grams of KH₂PO₄was weighed into a 1000 ml beaker, dissolved and diluted to 1000 ml with HPLC water. 2 ml of triethyl amine was added and pH adjusted to 3.0 with orthophosporic acid.

Preparation of mobile phase and diluents:

650 ml of the phosphate buffer was mixed with 350 ml of methanol. The solution was degassed in an ultrasonic water bath for 5 minutes and filtered through 0.45 µ filter under vacuum.

Procedure:

A mixture of buffer and methanol in the ratio of 65:35 v/v was found to be the most suitable mobile phase for ideal separation of lornoxicam. The solvent mixture was filtered through a 0.45μ membrane filter and sonicated before use. It was pumped through the column at a flow rate of 1.5 ml/min. The column was maintained at ambient temperature. The column was equilibrated by pumping the mobile phase through the column for at least 30 min prior to the injection of the drug solution. The detection of the drug was monitored at 289 nm. The run time was set at 10 min. Under these optimized chromatographic conditions the retention time obtained for the drug was 7.166 min. A typical chromatogram showing the separation of the drug is given in Fig. 2.

Figure 1: Chemical structure of lornoxicam

Figure 2: Typical chromatogram of lornoxicam

Calibration plot:

About 25 mg of lornoxicam was weighed accurately, transferred into a 100 ml volumetric flask and dissolved in 25 ml of a 65:35 v/v mixture of phosphate buffer and methanol. The solution was sonicated for 15 min and the volume made up to the mark with a further quantity of the diluent to get a 100 µg/ml solution. From this, a working standard solution of the drug (40µg/ml) was prepared by diluting 2 ml of the above solution to 10 ml in a volumetric flask. Further dilutions ranging from 30-150 µg/ml were prepared from the solution in 10 ml volumetric flasks using the above diluent. 20 µl of each dilution was injected six times into the column at a flow rate of 0.6 ml/min and the corresponding chromatograms were obtained. From these chromatograms, the average area under the peak of each dilution was computed. The calibration graph constructed by plotting concentration of the drug against peak area was found to be linear in the concentration range of 30-150 µg/ml of the drug. The relevant data are furnished in Table-1. The regression equation of this curve was computed. This regression equation was later used to estimate the amount of lornoxicam in tablet dosage forms.

Validation of the proposed method:

The specificity, linearity, precision, accuracy, limit of detection, limit of quantitation, robustness and system suitability parameters were studied systematically to validate the proposed HPLC method for the determination of lornoxicam. Solution containing 50 µg/ml of lornoxicam was subjected to the proposed HPLC analysis to check intra-day and inter-day variation of the method and the results are furnished in Table-2. The accuracy of the HPLC method was assessed by analyzing solutions of lornoxicam at 50, 100 and 130 % concentrated levels by the proposed method. The results are furnished in Table-3. The system suitability parameters are given in Table-4.

Table 1. Calibration data of the method

Concentration (µg/ml)	Mean peak area (n=5)
30	618407
60	1161835
90	1724787
120	2337682
150	2968750

Table 2. Precision of the proposed HPLC method

Concentration of Lornoxicam (µg/ml)	Measured concentration of lornoxicam (µg/ml)
	Intra-day		Inter-day
	Mean (n=5)	% RSD	Mean (n=5)	% RSD
90	1725608	0.05	1727586	0.11

Table 3. Accuracy studies

Conc.	Area	Amount added (mg)	Amount found (mg)	% Recovery	% Mean recovery
50 %	1161 033	14.9	15.0	99.8 %	99.8
100 %	1727 214	29.9	29.03	100.2 %
150 %	2339 108	40.2	40.4	99.5 %

Table 4. System suitability parameters

Parameter	Result
Linearity (mg/ml)	30-150
Correlation coefficient	0.999
Theoretical plates (N)	2387
Tailing factor	0.9
LOD (mg/ml)	0.01
LOQ (mg/ml)	0.04

Table 5. Assay studies

Formulation	Label claim (mg)	Amount found (mg)	% Amount found
Lorsin	8	7.98	99.67

Estimation of lornoxicam in tablet dosage forms:

Commercial brand of tablets were chosen for testing the suitability of the proposed method to estimate lornoxicam in tablet formulations. Twenty tablets were weighed and powdered. An accurately weighed portion of this powder equivalent to 25 mg of lornoxicam was transferred into a 100 ml volumetric flask and dissolved in 25 ml of a 65:35 v/v mixture of phosphate buffer and methanol. The contents of the flask were sonicated for 15 min and a further 25 ml of the diluent was added, the flask was shaken continuously for 15 min to ensure complete solubility of the drug. The volume was made up with the diluent and the solution was filtered through a 0.45 µ membrane filter. This solution was injected into the column six times. The average peak area of the drug was computed from the chromatograms and the amount of the drug present in the tablet dosage form was calculated by using the regression equation obtained for the pure drug. The relevant results are furnished in Table-5.

RESULTS AND DISCUSSION:

In the proposed method, the retention time of lornoxicam was found to be 7.193 min. Quantification was linear in the concentration range of 30-150 µg/ml. The regression equation of the linearity plot of concentration of lornoxicam over its peak area was found to be Y=19582+38432.49X (r²=0.999), where X is the concentration of lornoxicam (µg/ml) and Y is the corresponding peak area. The number of theoretical plates calculated was 2387, which indicates efficient performance of the column. The limit of detection and limit of quantitation were found to be 0.01 μg/ml and 0.04 μg/ml respectively, which indicate the sensitivity of the method. The use of phosphate buffer and methanol in the ratio of 65:35 v/v resulted in peak with good shape and resolution. The high percentage of recovery indicates that the proposed method is highly accurate. No interfering peaks were found in the chromatogram of the formulation within the run time indicating that excipients used in tablet formulations did not interfere with the estimation of the drug by the proposed HPLC method.

CONCLUSION:

The proposed HPLC method is rapid, sensitive, precise and accurate for the determination of lornoxicam and can be reliably adopted for routine quality control analysis of lornoxicam in its tablet dosage forms.

ACKNOWLEDGEMENTS:

The authors are thankful to M/s Glenmark Pharmaceuticals Ltd, Mumbai for providing a reference sample of lornoxicam.

REFERENCES:

1. www.drugsupdate.com

2. B. Bozal and B. Uslu. Combinatorial Chemistry and High Throughput Screening, 2010, 13(7), 598-609.

3. I. Cetin, N. Kocak and S. Aycan. C.B.U. Journal of Science, 2009, 5(1), 11-18.

4. E. Venumadhav, T. Neeha, P. Bhargavi, Amreen Nishat, A. Swetha and G. Devala Rao. International Journal of Applied Biology and Pharmaceutical Technology, 2011, 2(1), 23-26.

5. Y.L. Zeng, X.Y. Chen, Y.F. Zhang and D.F. Zhong. Yao Xue Xue Bao, 2004, 39(2), 132-135.

6. A. Mahesh. Journal of Basic and Clinical Pharmacy, 2010, 1(2), 115-120.

Received on 10.04.2011 Modified on 02.05.2011

Asian J. Research Chem. 4(7): July, 2011; Page 1081-1083

Table 2. Precision of the proposed HPLC method

Intra-day

Inter-day

Table 4. System suitability parameters