Developing a reference measurement procedure for free glycerol in human serum by two-step gas chromatography–isotope dilution mass spectrometry

Abstract

BackgroundFree glycerol in human serum is measured in clinical laboratories using enzymatic methods, which can be affected by interferences from biological samples. These methods are not applicable when stable isotopic tracers are used to determine lipid kinetics. Hence, a reference measurement procedure for free glycerol in human serum is needed. MethodsA reference measurement procedure based on two-step gas chromatography–isotope dilution mass spectrometry (GC–IDMS) was developed for the measurement of free glycerol in human serum. This procedure involved spiking with 13C3-glycerol, protein precipitation and cation exchange SPE, followed by two-step derivatization with 1-butylboronic acid and N-methyl-N-trimethylsilyltrifluoroacetamide. Tripalmitin certified reference material (CRM) was used as the calibration standard to ensure metrological traceability. ResultsGood precision and accuracy were obtained as demonstrated by relative standard deviation (RSD) of 1.51%–3.33%, with average recoveries over 98%. The relative measurement uncertainty was below 3% with major contributions from the concentration of glycerol calibration solution, choice of ion pair, linear regression, and measurement precision. ConclusionsWith good accuracy and precision, as well as clear metrological traceability, the developed GC–IDMS procedure is useful in producing traceable and accurate measurement of free glycerol in human serum.