Quality Control in Targeted GC-MS for Amino Acid-OMICS

Abstract

Gas chromatography-mass spectrometry (GC-MS) is suitable for the analysis of non-polar analytes. Free amino acids (AA) are polar, zwitterionic, non-volatile and thermally labile analytes. Chemical derivatization of AA is indispensable for their measurement by GC-MS. Specific conversion of AA to their unlabeled methyl esters (d₀Me) using 2 M HCl in methanol (CH₃OH) is a suitable derivatization procedure (60 min, 80 °C). Performance of this reaction in 2 M HCl in tetradeutero-methanol (CD₃OD) generates deuterated methyl esters (d₃Me) of AA, which can be used as internal standards in GC-MS. d₀Me-AA and d₃Me-AA require subsequent conversion to their pentafluoropropionyl (PFP) derivatives for GC-MS analysis using pentafluoropropionic anhydride (PFPA) in ethyl acetate (30 min, 65 °C). d₀Me-AA-PFP and d₃Me-AA-PFP derivatives of AA are readily extractable into water-immiscible, GC-compatible organic solvents such as toluene. d₀Me-AA-PFP and d₃Me-AA-PFP derivatives are stable in toluene extracts for several weeks, thus enabling high throughput quantitative measurement of biological AA by GC-MS using in situ prepared d₃Me-AA as internal standards in OMICS format. Here, we describe the development of a novel OMICS-compatible QC system and demonstrate its utility for the quality control of quantitative analysis of 21 free AA and metabolites in human plasma samples by GC-MS as Me-PFP derivatives. The QC system involves cross-standardization of the concentrations of the AA in their aqueous solutions at four concentration levels and a quantitative control of AA at the same four concentration levels in pooled human plasma samples. The retention time (t_R)-based isotope effects (IE) and the difference (δ(H/D) of the retention times of the d₀Me-AA-PFP derivatives (t_R(H)) and the d₃Me-AA-PFP derivatives (t_R(D)) were determined in study human plasma samples of a nutritional study (n = 353) and in co-processed QC human plasma samples (n = 64). In total, more than 400 plasma samples were measured in eight runs in seven working days performed by a single person. The proposed QC system provides information about the quantitative performance of the GC-MS analysis of AA in human plasma. IE, δ(H/D) and a massive drop of the peak area values of the d₃Me-AA-PFP derivatives may be suitable as additional parameters of qualitative analysis in targeted GC-MS amino acid-OMICS.

Keywords: OMICS; amino acids; metabolites; plasma; quality control; sample preparation.