A mass spectrometric method for measuring glycerol levels and enrichments in plasma using 13C and 2H stable isotopic tracers

Abstract

The stable isotope tracer [1,1,2,3,3,-2H5]glycerol has been commonly used as a tracer to measure glycerol kinetics and lipolysis in vivo. The method for measuring samples using the trimethylsilyl derivative and electron impact gas chromatograph-mass spectrometry retains only three of the five deuteriums, resulting in the possibility of incorrectly identifying the whole glycerol tracer molecule. This reports preparation of glycerol as the heptafluorobutyrl derivative and measurement by negative ion chemical ionization gas chromatography-mass spectrometry to produce a derivative with an intense molecular ion that retains all five deuterium labels. Thus the heptafluorobutyrl derivative analyzed by negative ion mass spectrometry overcomes the problems associated with fragmentation and loss of the isotopic label. Glycerol concentration using a labeled internal standard can be determined in plasma with a precision of 3%. Nanomole amounts of glycerol can be analyzed for 13C or 2H enrichments with a precision of +/- 0.14 mol% excess isotope. This simple, sensitive method for measuring glycerol levels and stable isotopic enrichment in plasma uses a simple extraction procedure and requires a minimal volume of plasma (less than 300 microliters).