INTRODUCTION:

Lornoxicam, {6-chloro-4-hydroxy-2-methyl-N-2-pyridyl-2H-thieno-[2, 3-e]-1, 2-thiazine-3-carboxamide-1, 1-dioxide; Figure 1 (structure 1)} is a nonsteroidal anti-inflammatory drug (NSAID) of oxicam class. Lornoxicam is used to treat acute mild to moderate pain, and to treat pain and inflammation of the joints caused by certain types of rheumatic diseases. Lornoxicam inhibits both isoforms in the same concentration range i.e., COX1/COX2 = 1. Thus, a perfectly balanced inhibition of COX-1 and COX-2 is achieved.^[1] Paracetamol is chemically {N-(4-hydroxyphenyl) acetamide; Figure 1 (structure 2)}. It is used mainly as analgesic and antipyretic.^{[2, 4]} The combination offers faster as well as prolonged relief from pain and inflammation. Literature survey reveals that many UV Spectrophotometric,^[5] RP-HPLC^[6-7]and Polarographic methods^[8] have been reported for the determination of Lornoxicam in human plasma and in tablet dosage form. While spectrophometry, ^[9-14] HPLC, ^[15-25] LC-MS ^[26] and capillary electrophoresis ^[27] had been reported for determination of PARA.

But no method is available for simultaneous estimation of Lornoxicam and Paracetamol in bulk and in tablet dosage form.

Structure 1: Lornoxicam

Structure 2: Paracetamol

Figure 1: Structure of Lornoxicam and Paracetamol ^{[1, 2]}

This paper describes simple, rapid, accurate, reproducible and economical RP-HPLC method for the simultaneous determination of Lornoxicam and Paracetamol in bulk and in tablet dosage form. The present RP-HPLC method was validated following ICH guideline. ^[28]

EXPERIMENTAL:

CHEMICALS AND REAGENTS USED:

The reference standard of Lornoxicam and Paracetamol were obtained as gift samples from Shree Pramukh laboratory. All chemicals used were of HPLC grade of Merck. Triethylamine and Ortho-phosphoric acid, as having HPLC grade of Merk Limited were used for chromatographic procedure. Water for HPLC was used to prepare mobile phase. Synthetic mixture containing common tablet additives were prepared in ratio of 1:62.5 and used for the study.

INSTRUMENTATION:

Youngling’s HPLC with UV-760 detector and Manual injector of 20µl loop. The peaks were quantified by means of PC based Autochrom 3000 software.

CHROMATOGRAPHIC CONDITIONS:

The column used for chromatographic separations was Varian microsorb mv C 18 (4.6mm i.d., 250 mm length, 5 µm particle size). The analytical wavelength was set at 293 nm and samples 20 µl were manually injected. The chromatographic separations were accomplished using mobile phase, consisting of Water: Acetonitrile: Methanol: TEA (55:30:15:0.3) and pH adjusted to 7.25 pH with orthophosphoric acid which is filtered through 0.45µm filter (Millipore) and degassed in ultrasonic bath. Mobile phase was pumped in isocratic-mode at a flow rate of 2.0 ml/min at ambient temperature.

PREPARATION OF STANDARD AND SAMPLE SOLUTION:

The standard stock solutions of Lornoxicam (20 µg/ml) and Paracetamol (1250 µg/ml) were prepared by dissolving appropriate amounts of respective compounds in methanol. Whereas in the preparation of sample solution, quantity of powdered API and common tablet additives equivalent to 2 mg of LOR or 125 mg of PARA was weighed and dissolved in methanol. It was further diluted with mobile phase in order to get solution having final concentration 2 µg/ml of LOR and 125 µg/ml of PCM.

RESULTS AND DISCUSSION:

OPTIMIZATION OF ANALYTICAL CONDITIONS:

Different columns containing octyl, octadecyl, phenyl and base deactivated silane stationary phase were tried for separation and resolution. The Varian microsorb mv column became more advantageous over the other columns. Individual drug solution was injected into column, both elution pattern and resolution parameters studied as a function of pH, as a function of mobile phase component and their ratio. To develop a suitable LC method for estimation of lornoxicam and paracetamol in bulk forms, different mobile phases were employed to achieve the best separation. The selected and optimized mobile phase was Water: Acetonitrile: Methanol: TEA (55:30:15:0.3) and pH adjusted to 7.25 pH with orthophosphoric acid and conditions optimized were: flow rate (2.0 ml/min), detector wavelength (293 nm). Run time was 8 min. Here the peaks were separated and showed better resolution, theoretical plate count and asymmetry was found as 1.27 and 1.33 respectively for lornoxicam and paracetamol. The proposed chromatographic conditions were found appropriate for the quantitative determination of the drugs. The typical chromatogram of two drugs assayed is shown in Figure 2.

Figure 2: Typical chromatogram showing retention time of 3.334 for Lornoxicam and 1.483 min for Paracetamol

METHOD VALIDATION:^[33]

SYSTEM SUITABILITY:

The system suitability of method was studied to determine reproducibility of chromatographic system and column performance was acceptable for intended analytical application. Four parameters i.e. retention time of eluted drugs, number of theoretical plates, asymmetry factor and resolution between two peak of analyte were evaluated. The results are shown in Table 1.

Table 1: RESULTS OF SYSTEM SUITABILITY STUDY

Lornoxicam		Paracetamol
Retention time	1.483 min	Retention time	3.334 min
Theoretical plates	5781	Theoreticalplates	3025
Assymetry factor	1.27	Assymetry factor	1.33
Resolution	12.35

LINEARITY:

The Linearity of analytical method is its ability to obtain test results, which are directly proportional to the concentration of analyte in the test sample .The linearity of the assay method for synthetic mixture, was established by injecting test samples in the range of 1-5 µg/ml for lornoxicam and 62.5-387.5 µg/ml for paracetamol. The regression analysis was carried out from graph of peak area Vs concentration; correlation co-efficient and Y- Intercept of plot was also evaluated. Calibration curves of LOR and PCM are shown in Figures 3 and 4.

Figure 3: Calibration curve of Lornoxicam

Figure 4: Calibration curve of Paracetamol

ACCURACY:

The accuracy study was assured by spiking the previously analyzed sample with 50%, 100%, and 150 % of target concentration. The resulting mixtures were assayed, and the results obtained for both drugs were compared to those expected. The good recoveries with the spiking method prove the good accuracy of the proposed methods. The recovery samples were prepared in triplicate at each level. The precision of recovery at each level was determined by computing the relative standard deviation of triplicate recovery results. The result for accuracy is shown in Table 2, indicating good accuracy of the method for simultaneous determination of two drugs.

PRECISION:

Precision was determined by two ways; by System precision and Intermediate precision. System precision was demonstrated by making six replicate injections of standard solution. The peak area of analyte for replicate injections was recorded. The %RSD for the analyte peak area of these replicate injections was evaluated. The results of System precision is indicating that an acceptable precision was achieved for simultaneous determination of lornoxicam and paracetamol, as revealed by RSD < 2.0%. The intermediate precision of test method was demonstrated by carrying out precision study at three concentration level as 50 %, 100%, 150% (i.e 1, 2, 3 µg/ml and 62.5, 125, 187.5 µg/ml ) for LOR and PCM respectively. Intermediate precision study includes intra-day and inter-day analysis. The result summary of intermediate precision is shown in Table 3A and 3B.

ROBUSTNESS:

Robustness of the test method was demonstrated by carrying out system suitability under normal conditions and each of the altered conditions as follows.

Flow rate was changed by -10% and +10%; Organic phase ratio of mobile phase was changed by -5% and +5% absolute; the result summary of robustness study are summarized in Table 4, result indicates that the method is robust for simultaneous determination of lornoxicam and paracetamol.

LIMIT OF DETECTION AND LIMIT OF QUANTITATION:

Limit of detection and limit of quantitation was established based on the residual standard deviation method. The limit of detection (LOD) of LOR and PCM was found to be 0.039 μg/ml and 0.617 μg/ml respectively. The limit of quantification (LOQ) of LOR and PCM was found to be 0.121 μg/ml and 1.871 µg/ml respectively.

SPECIFICITY:

Specificity was determined by observing interference between binary mixture containing LOR and PCM and binary mixture of LOR and PCM spiked with tablet additives. No peaks were found at retention of LOR and PCM. Specificity studies indicating that excipients did not interfere with analysis. Chromatogram of binary mixture containing LOR and PCM is shown in Figure 5 and Chromatogram of binary mixture spiked with tablet additive is shown in Figure 6.

Figure 5: Chromatogram of binary mixture containing Lornoxicam and Paracetamol

Figure 6: Chromatogram of binary mixture spiked with tablet additives

TABLE 2: RESULTS OF ACCURACY STUDY

Drug	Amount taken (µg/ml)	Amount added (µg/ml)	Amount found (µg/ml)	% Recovery ± S.D (n=3)
LOR	2.0048	1	3.0240	100.63 ± 0.656
	2.0048	2	4.0427	100.95 ± 0.510
	2.0048	3	4.9905	99.71 ± 1.166
PCM	124.98	62.5	187.2562	99.880 ± 0.090
	124.98	125	251.5249	100.618 ± 0.197
	124.98	187.5	312.7674	100.092 ± 0.444

TABLE 3: A- RESULTS OF INTERMEDIATE PRECISION FOR LORNOXICAM

Concentration ofLOR (μg/ml)	Intraday precision		Inter day precision
Concentration ofLOR (μg/ml)	Mean ± SD	%RSD	Mean ± SD	% RSD
1	27.993 ± 0.518	1.851	27.286 ± 0.390	1.430
2	54.393 ± 0.905	1.664	55.602 ± 0.619	1.113
3	85.380 ± 1.004	1.176	86.217 ± 1.093	1.276

B- RESULTS OF INTERMEDIATE PRECISION FOR PARACETMOL

Concentration of PCM (μg/ml)	Intraday precision		Inter day precision
Concentration of PCM (μg/ml)	Mean ± SD	%RSD	Mean ± SD	% RSD
62.5	245.63 ± 3.662	1.491	244.475 ± 2.363	0.966
125	680.36 ± 7.352	1.080	678.238 ± 7.352	1.055
187.5	1117.636 ± 7.920	0.708	1128.778 ± 16.84	1.492

TABLE 4: RESULT SUMMARY OF ROBUSTNESS STUDY

Robustness of HPLC method for LOR
Sample		Flow Rate		Mobile Phase Composition (Water : ACN : MeOH : TEA)
Sr. No.	Control*	1.9 ml/min	2.1 ml/min	57: 29: 14: 0.3	53: 31:16: 0.3
1	3.334	3.336	3.331	3.336	3.329
2	3.335	3.338	3.33	3.337	3.329
3	3.334	3.337	3.329	3.339	3.33
MEAN	3.3343	3.337	3.33	3.3373	3.3293
SD	0.0005	0.001	0.001	0.0015	0.0005
RSD	0.0173	0.0299	0.030	0.0457	0.0173
Robustness of HPLC method for PCM
Sample		Flow Rate		Mobile Phase Composition (Water : ACN : MeOH : TEA)
Sr. No.	Control	1.9 ml/min	2.1 ml/min	57: 29: 14: 0.3	53: 31:16: 0.3
1	1.483	1.485	1.482	1.48	1.489
2	1.483	1.486	1.481	1.479	1.49
3	1.482	1.486	1.48	1.479	1.487
MEAN	1.4826	1.4856	1.481	1.4793	1.4886
SD	0.0005	0.0005	0.001	0.0005	0.0015
RSD	0.0389	0.0388	0.0675	0.0390	0.1026

*Control condition: mobile phase composition (Water: ACN: MeOH: TEA) 55:30:15:0.3 and flow rate 2.0 ml/min at working temperature of laboratory.

TABLE 5: APPLICATION OF METHOD TO TABLET DOSAGE FORM

Formulations	Label claim (mg)		Amount found (mg)		% Assay* ± SD*
Formulations	LOR	PCM	LOR	PCM	LOR	PCM
ABDOMAX tab.	8.0	500.0	7.989	496.37	99.86 ± 1.25	99.27 ± 0.869

* Average of five determination

APPLICATION OF PROPOSED METHOD TO MARKETED FORMULATION:

Synthetic mixture were prepared by weighing accurately bulk powdered drug and common tablet additives. Powder equivalent to 2 mg of LOR and 125 mg of PCM into 100 ml volumetric flask and diluted to 100 ml with methanol. This solution is sonicated for 20 minutes. The solution was filtered through Whatman filter paper No. 41. Transfer 5 ml of solution into 50 ml volumetric flask and dilute to the mark with mobile phase to get a final concentration 2 µg/ml of LOR and 125 µg/ml of PCM.

CONCLUSION:

The data demonstrate that the new RP-HPLC method we have developed showed acceptable linearity, specificity, accuracy, precision and robustness in the concentration range of 1-5 µg/ml for Lornoxicam and 62.5-387.5 µg/ml for paracetamol, as per the requirement of ICH guidelines. The method described is rapid since chromatographic run time is 8 min. In conclusion, the proposed method could be routinely used for the analysis of lornoxicam and paracetamol in API and tablet dosage form.

ACKNOWLEDGEMENT:

The authors are grateful to Shree Pramukh Laboratory for providing gift samples of Lornoxicam and paracetamol, respectively. Also thanks to Jodhpur National University, Jodhpur.

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Received on 07.05.2012 Modified on 22.05.2012

Asian J. Research Chem. 5(6): June, 2012; Page 774-778