Rapid liquid chromatography-tandem mass spectrometry to determine very-long-chain fatty acids in human and to establish reference intervals for the Chinese population

Abstract

Very-long-chain fatty acids (VLCFAs), including hexacosanoic, tetracosanoic, and docosanoic acids, are peroxisomal disease markers, whose abnormal accumulation warrants prompt detection for timely, effective treatment. This study aimed to establish and validate a robust liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method to simultaneously quantify VLCFAs and provide reference intervals among Chinese individuals, quantify VLCFAs in pregnancy, and explore potential associations between plasma and amniotic fluid. Analytes were extracted via water-bath incubation with HCl and liquid-liquid extraction. Method linearity, limit of detection/quantitation, precision, carryover, and recovery were evaluated according to Clinical and Laboratory Standard Institute (CLSI) guidelines. VLCFAs showed good reproducibility based on low within-run coefficient variations (CVs) and total CVs, and correlation coefficients of linearity were > 0.99. The reference interval of C22:0, C24:0, and C26:0 were 32.0-73.4 μmol/L, 30.3-72.0 μmol/L, and 0.20-0.71 μmol/L, respectively; C24:0/C22:0 and C26:0/C22:0 ratios were 0.75-1.28 and 0.005-0.0139, respectively. Plasma and amniotic fluid of the same pregnant women displayed no significant correlation in the second trimester. This study presents the simple, efficient, accurate, and robust LC-MS/MS method to simultaneously detect C22:0, C24:0, and C26:0 without derivatization; it can be used to establish reference intervals among Chinese individuals and has diagnostic and other clinical applications.

Keywords: Ammonic fluid; Liquid chromatography-tandem mass spectrometry; Very-long-chain fatty acid; X-linked adrenoleukodystrophy.