RP-HPLC method development and validation for simultaneous estimation of methotrexate and naringenin in pharmaceutical dosage form

Methotrexate is a folic acid inhibitor that works by inhibiting the enzyme dihydrofolate reductase and naringenin is an active phytochemical compound found in citrus fruits as a glycoside conjugate and has a number of therapeutic attributes, including inhibitory and anti-inflammatory effect on chemokine production, which is linked to the pathophysiology of psoriasis. The present investigation provides a simple, efficient, and predictable reverse phase high performance liquid chromatography (RP-HPLC) technique for estimating methotrexate and naringenin in pharmaceutical dose form. Inertsil ODS-3 (15 cm × 4.6 mm, 5μm) was the stationary phase, phosphate buffer pH 6.0 was the mobile phase A and acetonitrile was used as mobile phase B. The flow rate of the gradient was 1.0mL/min, and the eluents were detected through a UV detector at 302 nm. Methotrexate and naringenin retention times were reported to be 6.980 and 21.317 minutes, respectively. The devised technique was verified in accordance with the ICH guidelines Q2 (R1) was determined to be linear with a range of 50–150 ppm for methotrexate and 100-300 ppm for naringenin. The devised approach was effectively employed for methotrexate and naringenin testing in a combination dose form.