DEVELOPMENT AND VALIDATION OF A HIGHLY SENSITIVE HIGH PERFORMANCE LIQUID CHRMOATOGRAPHY (HPLC) METHOD FOR THE ESTIMATION OF METHOTREXATE (MTX) PURE DRUG AND MARKETED FORMULATION IN SPIKED RAT PLASMA
Objective: Methotrexate (MTX) is used as an anti-tumor agent, and its clinical pharmacokinetics requires an accurate method for estimation of its plasma concentration. We describe a rapid, selective and sensitive HPLC method coupled with UV detection for determination of MTX in rat plasma.
Methods: Internal standard (IS; caffeine) was added to plasma aliquots prior to protein precipitation with acetonitrile. MTX and IS were eluted on a Phenomenex C18 column (250 mm Ã— 4.6 mm, 5 Î¼m) protected by a guard column (4 mm Ã— 3 mm Ã—5 Î¼m) (Torrance, CA, USA). The mobile phase comprised a mixture of aqueous 0.01 M phosphate buffer (pH 3.9)/acetonitrile (89/11%, v/v). To further improve the applicability of the method, a marketed formulation of high dose MTX (Folitrax 10 mg, IPCA) was spiked in rat plasma and the developed method was applied for the detection of MTX.
Results: Adequate specificity, precision, and accuracy of the proposed method were demonstrated over the concentration range of 10 to 1000 ng/ml.
Conclusion: This developed method was more sensitive and specific than any reported methods for the estimation of MTX and hence was successfully applied to measure plasma MTX concentrations in rat plasma which can be useful to elucidate the pharmacokinetic data of MTX.
Keywords: Methotrexate, Folitrax, Protein precipitation, Rat plasma, Caffeine
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