Formation of ketoprofen methyl ester artifact in GC–MS analysis of basic drugs in horse urine using alkaline liquid-liquid extraction

Abstract

This study investigates the unexpected formation of ketoprofen methyl ester (KME) during the routine alkaline liquid–liquid extraction (LLE) process for analyzing basic drugs in horse urine samples using GC–MS analysis. An unidentified peak in the GC–MS chromatogram was observed in certain horse urine samples, identified as KME through mass spectral comparison with in situ synthesized KME. Since the KME is not a metabolite of ketoprofen present in urine, it is proposed that its formation occurs during the LLE process due to the reaction between ketoprofen contained in the urine and methanol used as the solvent of the spiked internal standard. Surprisingly, no artifact was detected when negative quality control horse urine samples (absence of ketoprofen) even when spiked with standard ketoprofen and methanol. Further investigation indicated that the presence of lipase enzymes from bacteria in specific urine samples is the key factor in the formation of the KME artifact. This hypothesis was confirmed when negative quality control horse urines were spiked with ketoprofen, methanol, and a lipase enzyme and the KME artifact was detected. Additionally, the formation of a methyl ester artifact was also detected for flunixin, a carboxylic acid NSAID drug, when negative quality control horse urines were spiked with the drug, methanol, and lipase enzyme. These findings will be valuable for scientists analyzing drugs in urine.