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 (a))} 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 achieved¹. Paracetamol is chemically {N-(4-hydroxyphenyl) acetamide; Figure 1 (structure (b))}. It is used mainly as analgesic and antipyretic^{2, 3}. The combination offers faster as well as prolonged relief from pain and inflammation⁴. Literature survey reveals that many UV Spectrophotometric⁵, 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-MS²⁶ and capillary electro- phoresis²⁷ had been reported for determination of Paracetamol.

But no method is available for simultaneous estimation of Lornoxicam and Paracetamol in bulk and in synthetic mixture.

(a) Lornoxicam

(b) Paracetamol

Figure 1: Structure of Lornoxicam (a) and Paracetamol (b)

This paper describes simple, rapid, accurate, reproducible and economical RP-HPLC method for the simultaneous determination of Lornoxicam and Paracetamol in bulk and in synthetic mixture. The present RP-HPLC method was validated as per the ICH guidelines²⁸.

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 Merck 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 1:6.25 and used for the study.

Instrumentation

Youngling’s HPLC with UV-760 detector and Manual injector of 20 µl loop. The peaks were quantified by means of PC based Acme 9000 software.

Chromatographic condition

The column used for chromatographic separations was Varian microsorb mv C 18 (4.6mm i.d., 250mm 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 (40:30:30:0.3) pH adjusted to 7.3 with orthophosphoric acid which is filtered through 0.45µm filter (Millipore) and degassed in ultrasonic bath. Mobile phase was pumped in isocratic-mode at a flow rate of 1.5 ml/min at ambient temperature.

Preparation of standard and sample solution

The standard stock solutions of Lornoxicam (100 µg/ml) and Paracetamol (100 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 4 mg of LOR or 25 mg of PCM was weighed and dissolved in methanol. It was further diluted in order to get solution having concentration 4µg/ml of LOR and 25µg/ml of PCM.

RESULTS AND DISCUSSION:

Optimization of analytical condition

Different columns containing octyl, octadecyl, phenyl and base deactivated silane stationary phase were tried for separation and resolution. The Inertsil base deactivated silane 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.

Table 1: Results of system suitability study

Lornoxicam		Paracetamol
Retention time	3.44 min	Retention time	1.83 min
Theoretical plates	5738	Theoretical plates	4671
Assymetry factor	0.94	Assymetry factor	1.18
Resolution	13.17

The selected and optimized mobile phase was Water: Acetonitrile: Methanol: TEA (40:30:30:0.3) pH adjusted to 7.3 with orthophosphoric acid and conditions optimized were: flow rate was 1.5 ml/minute, detector wavelength was 293 nm and run time was 7 min. Here, the peaks were separated and showed better resolution, theoretical plate count and asymmetry was found as 1.18 & 0.94 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 3.44 min for Lornoxicam and 1.83 min for Paracetamol.

Figure 3: calibration curve of Lornoxicam

Figure 4: calibration curve of Paracetamol

Table 3: A- Results of intermediate precision for Lornoxicam

Conc. (μg/ml)	Intraday precision		Interday precision
Conc. (μg/ml)	Mean ± SD	RSD	Mean ± SD	RSD
2	149 ± 2	1.342	148.66 ± 2.014	1.355
4	302 ± 3	0.993	305.66 ± 2.677	0.876
6	468.33±3.511	0.749	464.22 ± 4.050	0.870

B-Results of intermediate precision for Paracetamol

Conc.. (μg/ml)	Intraday precision		Interday precision
Conc.. (μg/ml)	Mean ± SD	RSD	Mean ± SD	RSD
12.5	662 ± 3.605	0.544	659.44 ± 7.794	1.183
25	1288.66 ± 23.072	1.790	1278 ± 17.678	1.381
37.5	1914.66 ± 17.387	0.908	1907.55 ± 18.704	0.980

Table 4: Results summary of robustness study

Robustness of HPLC method for Lornoxicam
Sample		Flow Rate (ml/min)		Mobile Phase Composition (ACN : Water : TEA)
S.No.	Control*	1.4	1.6	67 :33 : 0.2	63: 37: 0.2
1	3.516	3.607	3.412	3.421	3.59
2	3.513	3.6	3.41	3.423	3.592
3	3.517	3.612	3.415	3.426	3.587
MEAN	3.515333	3.606333	3.412333	3.423333	3.589667
SD	0.002082	0.006028	0.002517	0.002517	0.002517
RSD	0.059217	0.167142	0.07375	0.073513	0.070107
Robustness of HPLC method for Paracetamol
Sample		Flow Rate (ml/min)		Mobile Phase Composition (ACN : Water : TEA)
S.No.	Control*	1.4	1.6	67 :33 : 0.2	63: 37: 0.2
1	1.716	1.749	1.652	1.758	1.674
2	1.714	1.745	1.65	1.758	1.671
3	1.716	1.748	1.655	1.759	1.676
MEAN	1.715333	1.747333	1.652333	1.758333	1.673667
SD	0.001155	0.002082	0.002517	0.000577	0.002517
RSD	0.067316	0.119134	0.152307	0.032835	0.150365

*Control condition: mobile phase comp+osition (ACN: Water: TEA) 65:35:0.2 and flow rate 1.5 ml/min at working temperature of laboratory that is 25 °C.

Accuracy

The accuracy study was performed by spiking placebo with known quantity of API. The accuracy of test method was demonstrated by preparing recovery samples at the level of 50%, 100%, and 150% of target concentration. The recovery samples were prepared in triplicate at each level. The above samples were injected and the percentage recovery for amount added, were estimated. 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

The precision was determined by two ways; (i) system precision and (ii) intermediate precision. the system precision was demonstrated by making five 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 was demonstrated by carrying out precision study at three concentration level as 50 %, 100% and 150% (i.e 2, 4, 6 µg/ml and 12.5, 25, 37.5 µg/ml) for LOR and PCM respectively. The intermediate precision study includes intra-day and inter-day analysis. The result summary of intermediate precision is shown in Table 3A and 3B.

Robustness

The robustness was demonstrated by carrying out system suitability under normal conditions and each of the altered conditions as follows: The flow rate was changed by -10% and +10%; the organic phase ratio of mobile phase was changed by -5% and +5% absolute; the result summary of robustness study is summarized in Table 4. The result indicates that method is robust for simultaneous determination of Lornoxicam and Paracetamol.

Limit of detection and Limit of Quantitation

The limit of detection and the limit of quantitation were established based on the residual standard deviation method. LOD and LOQ for Lornoxicam were 0.032 and 0.293 µg/ml, respectively, and for Paracetamol 0.099 and 1.09 µg/ml, respectively.

Specificity

The specificity was carried as interference from placebo. The placebo was injected first, then, the synthetic mixture containing placebo was injected and finally, API’s as tablet ratio was injected. The result shows that the method is specific.

Proposed method to synthetic mixture:

The synthetic mixture was prepared by weighing accurately bulk powder drug and common tablet additives. The drug powder equivalent to 4 mg of Lornoxicam and 25 mg of Paracetamol into 100 ml volumetric flask and diluted to 100 ml with methanol. This solution was sonicated for 20 minutes. The solution was filtered through Whatman filter paper No. 41, then after 5 ml of solution was taken into 50 ml of volumetric flask and dilute to the mark with mobile phase to get a final concentration 4 µg/ml of Lornoxicam and 25 µg/ml of Paracetamol.

Table 5: Application of Method to synthetic mixture

Lornoxicam
Amount labeled (mg)	Amount found (mg)	% Amount Found S.D. (n=3)
80	80.320	100.401±1.435
Paracetamol
Amount labeled (mg)	Amount found (mg)	% Amount Found S.D. (n=3)
500	500.515	100.103±1.720

CONCLUSION:

We have developed the new RP-HPLC method. The method fits with the requirement of ICH guidelines. The data shows acceptable linearity, specificity, accuracy, precision and robustness in the concentration range of 2-30 µg/ml for Lornoxicam and 2-100 µg/ml for Paracetamol. The method is rapid since chromatographic run time is 7 minutes. The present method can be used for the analysis of Lornoxicam and Paracetamol in API and synthetic mixture containing common tablet additives.

ACKNOWLEDGEMENT:

The authors are grateful to Shree Pramukh Laboratory for providing gift samples of Lornoxicam and Paracetamol, respectively.

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Received on 13.07.2012 Modified on 11.08.2012

Asian J. Research Chem. 5(8): August, 2012; Page 1042-1046

Drug	Amount taken conc. (μg/ml)	Amount spiked* (μg/ml)	Amount recovered* (μg/ml)	% recovery ± S.D *	RSD
LOR	4	2	6.0598	100.998 ± 1.392	1.19
	4	4	8.0476	100.595 ± 0.729
	4	6	9.9886	99.886 ± 1.476
PCM	25	12.5	37.5470	101.125 ± 0.928	1.09
	25	25	50.2050	100.410 ± 1.021
	25	37.5	62.3893	99.822 ± 1.344