Different greenness assessment perspectives for stability-indicating RP-HPLC method used for the assay of isoxsuprine hydrochloride and four nephrotoxic and hepatotoxic photothermal degradation products

Abstract

Nowadays, the analytical community focuses on applying principles of green analytical chemistry (GAC) to develop new analytical methods to reduce harmful effects on the environment and human beings. In this work, we developed, validated, and assessed the greenness profile of a RP-HPLC method for stability and purity assay of isoxsuprine hydrochloride (ISX) and four of its nephrotoxic/hepatotoxic photothermal degradation products; p-hydroxyacetophenone (AC), 1-methyl-3-phenylpropylamine (AM), and p-hydroxybenzaldehyde (HB) in pure form, laboratory-prepared mixtures, and in the pharmaceutical dosage form. A C18 Waters XTerra (3.5 μm, 4.6 × 100 mm) separated the five analytes using a mobile phase composed of 20 mM potassium dihydrogen phosphate (adjusted to pH 2.3 with ortho-phosphoric acid) and methanol. We performed gradient elution at a constant flow rate of 0.8 mL/min while monitoring the effluent absorbance at 216 nm. The method shows high sensitivity with limits of quantifications equal to 0.557, 0.304, 5.791, 0.446, and 0.497 μg/mL for ISX, AC, AM, BZ, and HB, moreover, ISX low limit of detection (0.184 µg/mL) enabled its detection in spiked human plasma at its therapeutic level of concentration (0.4 µg/mL). We used four greenness assessment tools to assess the method greenness, including the “National Environmental Method Index” (NEMI), the “Eco-Scale Assessment” (ESA), the “Green Analytical Procedure Index” (GAPI), and Analytical Greenness metric (AGREE); additionally, we used the tools as a comparative greenness study between the proposed and the reported HPLC methods. The comparison proved that the proposed method was the best regarding the principles and guidelines of GAC. Our method proved superior eco-friendly qualitative and quantitative scores relative to the reported methods, where the method achieved more green NEMI and GAPI quadrants, scored the higher ESA score (85) than reported (81,81, and 77), and the higher AGREE score (0.71) than reported (0.53,0.56,0.58).