DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR SIMULTANEOUS DETERMINATION OF METHOTREXATE, DEXAMETHASONE AND INDOMETHACIN
Keywords:RP-HPLC, Methotrexate, Dexamethasone, Indomethacin, LOD, LOQ
Objective: To develop a reverse-phase high performance liquid chromatographic method for simultaneous estimation of methotrexate (MTX), dexamethasone (DXM) and indomethacin (IND).
Methods: A simple, isocratic reverse-phase high performance liquid chromatographic (RP-HPLC) method has been developed for simultaneous estimation of methotrexate, dexamethasone and indomethacin using ibuprofen (IBP) as internal standard with photodiode array detection at 254 nm. The process was carried out on C18 column (5 Î¼m, 250 mm X 4.6 mm) using methanol: orthophosphoric acid (1.67 % v/v) in 70: 30 ratio at a flow rate of 1.5 ml per minute.
Results: The retention time for MTX, DXM, IND and IBP were found to be 1.47, 4.05, 11.29 and 12.69 minutes, respectively. Calibration curves of the drugs were linear in the concentration range 1-500 Î¼g/ml. The limit of detection and limit of quantitation was 3.3 ng and 10.9 ng for MTX, 0.3 and 0.9 ng for DXM and 2.1 and 6.7 ng for IND respectively. The intra-day precision varied from 0.2 to 1.6 % and inter-day precision ranged from 0.2 to 1.8 %. The intra-day accuracy ranged from 98.3 to 101.13 % while inter-day accuracy varied from 98.6 to 101.4 %.
Conclusion: The developed method is ideally suited for simultaneous estimation of the drugs.
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