Assay of the acetyl-CoA probe acetyl-sulfamethoxazole and of sulfamethoxazole by gas chromatography-mass spectrometry.

We present gas chromatographic-mass spectrometric assays for (i) the concentration of sulfamethoxazole and (ii) the concentration and molar percentage enrichment of acetyl-sulfamethoxazole in biological fluids. The compounds are extracted with ethyl acetate, derivatized with either diazomethane or pentafluorobenzyl bromide, and analyzed by gas chromatography-mass spectrometry. Quantitation is achieved using internal standards, [2H4]sulfamethoxazole and acetyl-[2H4]sulfamethoxazole. Limits of detection are 200 nmol for the methyl derivatives and 2 nmol for the pentafluorobenzyl derivatives. The high sensitivity of the assay with the pentafluorobenzyl derivatives allows measuring in plasma and urine (i) the pharmacokinetics of sulfamethoxazole and acetyl-sulfamethoxazole and (ii) the stable isotope enrichment of the acetyl moiety of acetyl-sulfamethoxazole. The latter is used as a probe for the noninvasive chemical biopsy of liver extramitochondrial acetyl-CoA.