Quantitative GC-NICI-MS and GC-NICI-MS/MS analysis by using isotopologs as internal standards: A tutorial review

The use of analytes labelled with stable-isotopes of ²H, ¹³C, ¹⁵N, ¹⁷O and/or ¹⁸O, i.e., the isotopologs, as internal standards is unique and considered the Golden Standard is quantitative analyses based on mass spectrometry. However, the handling with isotopologs deserves a great extent of care and attentiveness from the very begin of the analytical process. Many issues need to be considered in order to create an analytical method that generates close-to-reality concentrations of a certain analyte or of group of analytes of the same or different chemical classes in complex biological samples. They including isotopic purity, stability through the entire analytical process including sampling, derivatization, ionization and collision-induced dissociation (CID). The present work deals specifically with the use of isotopologs as internal standards for the quantitative analysis of endogenous and exogenous substances in plasma, serum and urine samples. It focuses on GC–MS and GC–MS/MS, negative-ion chemical ionization (NICI) with methane as the reagent gas, and selected-ion monitoring (SIM) or selected-reaction monitoring (SRM) using argon as the collision gas. Special attention has been paid to purity of isotopologs, cross-contribution of analyte-internal standard, and stability of isotopologs during NICI and CID, to linearity of analyte-isotope detector response upon the analyte concentration. This tutorial review re-examines and discusses exemplarily previously reported validated GC–MS and GC–MS/MS methods, and gives recommendations regarding the handling with stable-isotope labelled analogs in quantitative analyses.