| Summary: | Unknown <i>N</i>-acylated amino acid (<i>N</i>-AAA) conjugates have been detected in maple syrup urine disease (MSUD) and other inborn errors of metabolism (IEMs). This study aimed to elucidate the mechanism behind the formation of urinary <i>N</i>-AAA conjugates. Liquid–liquid extraction was employed to determine the enantiomeric composition of <i>N</i>-AAA conjugates, followed by liberation of conjugated amino acids through acid hydrolysis. Gas chromatography–mass spectrometry (GC–MS) was used to separate amino acid enantiomers. In vitro experiments were conducted to test the non-enzymatic formation of <i>N</i>-AAA conjugates from 2-keto acids and ammonia, with molecular modelling used to assess possible reaction mechanisms. Adequate amounts of <i>N</i>-AAA conjugates were obtained via organic acid extraction without concurrent extraction of native amino acids, and hydrolysis was complete without significant racemisation. GC–MS analysis successfully distinguished amino acid enantiomers, with some limitations observed for L-isoleucine and D-alloisoleucine. Furthermore, investigation of racemic <i>N</i>-AAA conjugates from an MSUD case confirmed its non-enzymatic origin. These findings highlight the value of employing chiral strategy and molecular modelling to investigate the origin of unknown constituents in biological samples. Additionally, these conjugates warrant further investigation as potential factors contributing to MSUD and other IEMs.
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