Measurement of 13C Enrichment of Plasma Lactate by Gas Chromatography/Isotope Ratio Mass Spectrometry

Abstract

An application of a gas chromatography/isotope ratio mass spectrometry (GC/IRMS) method for stable carbon isotope analysis of blood plasma lactic acid is presented. The method involves a simple extraction procedure followed by derivatization with diazomethane. It is shown that derivatization is by single methylation, thus minimizing the dilution of the derivative's 13C content, yet still ensuring good chromatographic behaviour on a polar capillary column. This ensures a high sensitivity of the isotopic analysis. Repeatability, expressed by the coefficient of variation, varied from 0.3% to 19%, depending on sample enrichment. Reproducibility was 2.3% over a 10 day period. The detection limit, defined as 2SD, was about 0.0004 atom % excess (APE), equivalent to 0.001 mol % excess, when based on a measured precision of about 0.2/1000 in delta notation. A comparison is made between enrichments obtained using a calibration curve and those obtained using a correction for the added methyl carbon. The two methods agreed well, with a relative difference (delta APE/APE x 100%) of less than 0.5% for samples enriched with between 0.004 and 1.28 APE. It is concluded that the method provides simple and precise isotope analysis of picomole quantities of blood lactate.