Green evaluation of human plasma levels of metformin, linagliptin, and empagliflozin using HPLC and HPTLC methods: a pharmacokinetic study
Abstract
Two simple, rapid, cost-effective, and environmentally friendly chromatographic methods were developed and validated for the simultaneous determination of metformin (MEF), linagliptin (LIN), and empagliflozin (EMP) in human plasma, with successful application to pharmacokinetic study. Plasma sample preparation was performed using a straightforward protein precipitation technique employing acetonitrile: methanol: trichloroacetic acid (50:49:1, by volume), which provided high extraction recovery and minimal matrix interference. The first method was based on high-performance liquid chromatography with diode array detection (HPLC-DAD) using an ODS Hypersil C18 column and isocratic elution with a mobile phase consisting of acetonitrile, methanol, and phosphate buffer (pH 3) in a ratio of (40:40:20, by volume), at a flow rate of 1.3 mL/min, with detection at 230 nm. The second method employed high-performance thin-layer chromatography (HPTLC) with densitometric detection at 225 nm, using silica gel 60 F254 plates and n-hexane: methanol: glacial acetic acid (6:3:1, by volume) as the developing system. Excellent linearity was achieved over concentration ranges of 85-1650 ng/mL for MEF, 50-1100 ng/mL for EMP, and 45-950 ng/mL for LIN using the HPLC method, and 500-2800, 100-800, and 50-550 ng/band, respectively, using the HPTLC method, with correlation coefficients exceeding 0.998. The lower limits of quantitation for the HPLC method were 85, 50, and 45 ng/mL for MEF, EMP, and LIN,…
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