박성현 (한국화학연구원)

조성희 (한국화학연구원)

박성현 (한국화학연구원)

이장재 (한국화학연구원)

김윤지 (한국화학연구원)

이정미 (성균관대학교)

조성희 (한국화학연구원)

Liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based acylcarnitine profiling has become the standard analytical approach for newborn screening. However, accurate differentiation of clinically important C4 and C5 acylcarnitine isomers remains a major analytical challenge. In particular, isovalerylcarnitine (C5-I), a diagnostic biomarker of isovaleric acidemia (IVA), cannot be readily distinguished from the exogenous metabolite pivaloylcarnitine (C5-P), frequently leading to false-positive IVA screening results. In this study, we developed and validated an LC–MS/MS method capable of achieving baseline separation of C4 and C5 acylcarnitine isomers while simultaneously quantifying 32 acylcarnitines. Optimized mobile-phase conditions (acetonitrile, 0.1% formic acid, and 2 mM ammonium formate) enabled effective isomer separation and enhanced MS response. Among protein precipitation (PP), liquid–liquid extraction (LLE), and solid-phase extraction (SPE), SPE showed the highest MS response. The optimized method improved analytical sensitivity without derivatization, enabling simultaneous quantification within 10 min. Method validation demonstrated excellent linearity (r² > 0.990), together with accuracy and precision that met the acceptance criteria of the U.S. Food and Drug Administration (FDA) Bioanalytical Method Validation Guidance. The proposed method can provide an effective analytical platform for newborn screening.

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