DEVELOPMENT AND VALIDATION OF HPLC/UV METHOD FOR DETERMINATION OF MELOXICAM IN HUMAN PLASMA AND APPLICATION IN PHARMACOKINETIC STUDIES
Keywords:
Meloxicam, HPLCUV, Human plasma, PharmacokineticsAbstract
Objective: To develop and validate a modified isocratic reversed-phase high performance liquid chromatographic/ultraviolet (RP-HPLC/UV) method for determination of Meloxicam in human plasma to be used for pharmacokinetic studies.
Methods: The drug was extracted from plasma samples by direct protein precipitation technique using perchloric acid: acetonitrile (1:1). Piroxicam was used as internal standard (IS). Samples were analyzed on phenomenex C18 column(150 x 4.6 mm, 5 µm), applying sodium acetate buffer (0.17M): acetonitrile, at a ratio of 62:38 v/v in isocratic mode as a mobile phase at a flow rate of 1 ml/min to attain adequate resolution. Using a spectra autosampler, separations were performed at room temperature and monitored at a wavelength of 353 nm after injection a 100μl samples into the HPLC system. The analytical method was validated according to FDA bioanalytical method validation guidance. The method was applied for pharmacokinetic study of Meloxicam tablets (Mobic®,Boehringer Ingelheim, Germany). Mobic®15mg tablets were administered as a single dose to 12 healthy male adult volunteers under fasting condition. Twenty blood samples were withdrawn from each volunteer over 72 hours periods. From the plasma concentration-time data of each individual, the pharmacokinetic parameters; Cmax, Tmax, AUC0-t, AUC0-¥, Cmax/AUC0-¥, β and t0.5 were calculated.
Results: A peak area was obtained for piroxicam and Meloxicam at 6.1 and 10.3 min retention time, respectively. Linearity was established at a concentration range of 50– 1500ng/ml with R2 = 0.999 as the regression coefficient. The lower limit of quantitation (LLOQ) was identifiable and reproducible at 50ng/ml with a precision of 1.012%. The coefficients of variation(% CV) of the intra-day and inter-day precision at 150, 750 and 1200ng /ml levels were found to be 2.906%, 1.139%, 2.938%; and 4.347%, 4.985%, 3.556%, respectively, which are lower than the accepted criteria limits (15-20 %). The relative recovery (%) of Meloxicam at 150, 750, and 1200ng/ml was found to be 100.706%, 102.638% and 100.292%, respectively. Stability at different conditions and in autosampler was also established. The mean pharmacokinetic parameters; Cmax, Tmax, AUC0-t, AUC0-¥, Cmax/AUC0-¥, β and t0.5 were; 1262.2 ng/ml, 4.8 hr, 40905.2 ng. hr/ml, 45460.5 ng. hr/ml, 0.029 hr-1, 0.035 hr-1, 19.9 hr, respectively.
Conclusion: The present analytical method was found to be specific, sensitive, accurate and precise for quantification of Meloxicam in human plasma. It can be successively applied for pharmacokinetics, bioavailability and bioequivalence studies.
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