Liquid Chromatographic analysis of Methotrexate and Minocycline- Relevance to the determination in Plasma/Nanoparticulate formulations

Abstract

To develop an RP-HPLC method for the simultaneous estimation of methotrexate (MTX) and minocycline (MNC). Different HPLC methods were reported for the estimation of MTX/MNC individually, but there is no report for the simultaneous estimation of both MTX and MNC in a simple method. The objective of the developed method is to utilize the method for the estimation of MTX/MNC in different pharmaceutical formulations and in biological fluids. An HPLC method for the estimation of methotrexate (MTX) and minocycline (MNC) relevance to the evaluation of nanoparticulate formulations has been developed and validated. Chromatographic estimation was achieved using the mobile phase composition of sodium acetate buffer and acetonitrile (70:30% v/v) at pH 4.0 at a flow rate of 0.2 mL/min at 307 nm. The calibration curve for MTX and MNC was found to be linear at nanogram (5 to 25 ng.mL-1) and microgram (5 to 25 μg.mL-1) levels at a correlation coefficient range of 0.98 to 0.99 for both MTX/MNC. The lower limit of detection and limit of quantification were found to be 0.026 ng.mL-1 and 0.079 ng.mL-1 for MTX and MNC, respectively. The percentage relative standard deviation for validation parameters of both drugs was found to be less than 6.5%. The amount of MTX and MNC present within the nanoparticles was found to be MTX (0.84 mg/mL) and MNC (0.61 mg/mL). The in vitro release showed an immediate release pattern for MTX (64.95±2.08%) and MNC (90.90±1.78%) within 12 h. The developed analytical method for the simultaneous estimation of MTX and MNC was found to be simple, affordable, dynamic, low cost, rapid and easy to perform with good repeatability. This method is also time consuming, since the peaks were obtained within a moderate analysis time.