13C-labelled yeast as internal standard for LC–MS/MS and LC high resolution MS based amino acid quantification in human plasma

Abstract

Extracts from isotopically labelled organisms can be a versatile source for isotopically labelled chemical compounds providing ideal internal standards in mass spectrometry based assays. In this work, the application of 13C enriched yeast (Pichia pastoris) for accurate absolute metabolite quantification in human samples was investigated. >99% 13C enriched Pichia pastoris was produced via fermentation and extracted employing established protocols. Quantitative assays based on LC-triple quadrupole mass spectrometry (QQQ-MS) and LC-high resolution mass spectrometry (HRMS) were validated using the Standard Reference Material, SRM 1950 – metabolites in frozen human plasma. 14 amino acids (as given in the certificate) were quantified using separations by reversed phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC). The latter chromatographic separation provided retention and selectivity for the amino acid panel, while the studied approaches employing RPLC relied on the selectivity of the MS detection. Cross-validation using the different MS platforms showed that in all cases the application of in-vivo labelled standards resulted in a significant improvement of trueness and precision. LODs and LOQs ranged, regardless of the detection system and addition of internal standards, in the same order of magnitude. The linear dynamic range of the employed detection systems was enhanced at least for one order of magnitude for several analytes when the internal standards were applied.