Determination of 27 bovine plasma amino acids and metabolites using zwitterionic-hydrophilic interaction liquid chromatography coupled with isotope dilution electrospray ionization triple quadrupole liquid chromatography-mass spectrometry and the effect of deproteinization timing

Abstract

The use of zwitterionic-hydrophilic interaction liquid chromatography (Z-HILIC) columns for analysis of underivatized analytes has allowed simpler sample preparation of bovine plasma for sensitive and selective analysis, when coupled with mass spectrometry (MS). The objective of this study was to evaluate and validate this analytical technique to measure AA and metabolites in bovine plasma at 2 deproteinization times. A robust method using Z-HILIC coupled to a triple quadrupole MS (TQMS) was evaluated and validated to quantitatively analyze 19 AA using isotope dilution and 8 AA metabolites qualitatively in bovine deproteinized plasma. The timing of deproteinization was investigated to determine if plasma should be deproteinized upon collection (on-site) or immediately before analysis (in-lab). Analytes were separated using a Z-HILIC column in a 21 min run and analyzed with a TQMS in positive electrospray ionization for identification and quantification. The method was validated for standard curve linearity, limits of detection (LOD) and quantification (LOQ), intra- and interday precision (% coefficient of variation; CV), recovery (%), and freeze-thaw stability (% CV) after 1 mo. Coefficients of determination (R²) were over 0.993, and LOD and LOQ were below measured values for all AA. The CV for the intraday and interday precision were below 18%, except for cystine (Cys2) and Orn in-lab. Recoveries on-site and in-lab ranged from 75% to 120% for all analytes except Cys2 in-lab. Most analytes were stable after 1 mo of freezing regardless of deproteinization timing, CV <25%, except for hydroxyproline (Hyp). The concentration of Cys2 was affected by deproteinization in-lab compared with on-site, and even though Glu and Hyp were different between the 2 deproteinization timings, the concentrations between the 2 timings were within the standard deviation.