Simultaneous determination of acylcarnitines, fatty acids, and amino acids by 3-nitrophenylhydrazine derivatization and ultra-high performance liquid chromatography/tandem mass spectrometry

• Method for simultaneous quantification of acylcarnitines, amino acids and fatty acids. • Method covers key metabolite classes involved in metabolic diseases. • 3-nitrophenylhydrazine derivatization resolved amino acid and fatty acid isomers. • Low detection limits achieved with high precision and acceptable recoveries. Acylcarnitines (ACs) play a pivotal role in metabolism, most notably by facilitating the transport of fatty acids (FAs) into mitochondria for β-oxidation, a key step in cellular energy production. Dysregulation of AC, FA, and amino acid (AA) levels has been linked to various metabolic disorders, including cardiovascular diseases, neurodegenerative conditions, and cancer. Consequently, monitoring these metabolites in blood samples provides valuable insights into metabolic health and disease progression. In this study, we developed a method for the quantification of carnitine, seven ACs, fifteen FAs, and thirteen AAs in human serum using reversed-phase ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). By employing 3-nitrophenylhydrazine (3-NPH) derivatization, we achieved high detectability for ACs, with limits of detection (LODs) ranging from 0.01-0.27 ng/mL for ACs, 0.22-1.76 ng/mL for FAs and 0.17-18.25 ng/mL for AAs. Recovery rates ranged from 92-126% for ACs, 56-116% for FAs and 86-115% for AAs. Inter- and intra-day precision were below 20% for all metabolites except two FAs. This method provides a reliable and sensitive tool for the simultaneous analysis of ACs, FAs, and AAs in serum, with potential applications in clinical diagnostics and metabolic research.