Measurement of stable isotopic enrichment of underivatized acetate by gas chromatography/mass spectrometry: application to in vivo estimation of acetate production.

In order to assess endogenous and colonic production of acetate, we have developed an assay for determining the isotopic enrichment of plasma acetate using gas chromatography/mass spectrometry (GC/MS). Acidified, deproteinized plasma (200 microliters) was extracted into ethyl ether, and the ether phase was then injected into a gas chromatograph/mass spectrometer fitted with a 30 m x 0.252 mm i.d. capillary column (temperature program 50-245 degrees C at 10 degrees C min-1). Using electron impact GC/MS and selected ion monitoring, peak areas of ions with m/z 60 and 61 (M + 1) were determined. Triplicate extractions of enriched plasma samples (mol.% excess 1.38-1.5%) resulted in a coefficient of variation of 1.6-5.9%. Unenriched plasma samples were found to have an enrichment close to theoretical natural abundance, and analysis of our (1-13C)acetate tracer (99 at.% excess) revealed an ion at m/z 61 and no ion at m/z 60. To verify accuracy, we conducted an in vivo isotope dilution study. In a 1-month-old piglet, fasted for 24 h, changing the rate of a 4 h infusion (mmol h-1) of (1-13C)acetate from 0.141 to 0.282 doubled the isotope enrichment (2.08 x) of the second plateau. The rate of appearance of acetate was 26.0 mumols kg-1 min-1, which is comparable to that reported in fasting sheep.