Gas chromatography–mass spectrometry was used alongside quality control systems to analyze amino acids present in urine samples.
Dimitrios Tsikas, alongside fellow Hannover Medical School (Hannover, Germany) researchers, tested a quality control (QC) approach for analyzing amino acids in human urine and plasma using gas chromatography–mass spectrometry (GC–MS). Their findings were published in the Journal of Chromatography B (1).
Mass detector coupled with GC-MS - Gas Chromatography. Analytical laboratory. | Image Credit: © khaw - stock.adobe.com
Mass spectrometry (MS) underpins both targeted and untargeted metabolomics, allowing comprehensive analysis of metabolite classes in biological fluids such as plasma, serum, and urine. α-Amino acids remain a major focus because they are a crucial group of organic analytes explored with omics approaches. Robust quality-control (QC) procedures are therefore essential in analytical chemistry and, critically, in metabolomics workflows—including amino-acid profiling.
The team of researchers previously worked on the development and application of a QC system for the targeted metabolomics gas chromatography–mass spectrometry (GC–MS) analysis of amino acids, such as methyl ester pentafluoropropionyl (Me-PFP) derivatives, in human plasma. However, compared to serum and plasma, urine is a considerably less analyzed matrix.
In this study, the researchers adapted their QC strategy for quantitative (targeted) GC–MS analysis of amino acids in human urine. The method relies on the in-situ generation of d3-methyl esters of selected amino acids (d3Me-AAs) as internal standards, carried out alongside esterification of endogenous amino acids to their d0-methyl esters (d0Me-AAs) and their subsequent derivatization to pentafluoropropionyl (PFP) derivatives.
Endogenous (unlabeled) amino acids were analyzed in 10-μL aliquots of study urine samples and in QC urine samples as methyl ester pentafluoropropionyl derivatives (d0Me-PFP) using a mixture of in-situ prepared trideuteromethyl esters for use as internal standards, which were then converted into their PFP derivatives (such as d3Me-PFP). GC–MS analysis of 38 study urine samples and 8 QC urine samples was performed in the negative-ion chemical ionization (NICI) mode by selected-ion monitoring (SIM) of characteristic ions of d0Me-PFP and d3Me-PFP within a single run using an oven temperature program. For direct comparison, 35 study plasma samples and 8 QC plasma samples of the same study were performed.
The accuracy (recovery) and precision by which the analyzed amino acids were determined in the QC urine and plasma samples by GC–MS in the present work were, with minor exceptions, within generally acceptable ranges. This highlights the suitability of the overall analytical process for quantitative measurement of amino acids in human urine and plasma samples. The QC system was complemented with the determination of additional parameters, notably the retention times and the peak areas of the internal standards.
Peak area variability was not found to affect the accuracy of the GC–MS method for the analyzed amino acids in urine and plasma samples; however, it could have major implications. The researchers found that untargeted GC–MS metabolomics studies on amino acids in biological samples would be inappropriate because “targeted” GC–MS metabolomics is an improper substitute for the classical terminology “quantitative” GC–MS analysis, not only for amino acids, but in principle for every class of analytes, including amines and inorganic analytes, the researchers wrote. This could also apply to other classes of analytes and types of biological samples.
(1) Bollenbach, A.; Beckmann, B.; Tsikas, D. Quality Control in GC–MS Analysis of Amino Acids in Human Urine and Plasma: Possible Implications for Targeted and Untargeted Metabolomics. J. Chromatogr. B 2025, 1262, 124661. DOI: 10.1016/j.jchromb.2025.124661
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