Determination of methylmalonic acid and glutaric acid in urine by aqueous-phase derivatization followed by headspace solid-phase microextraction and gas chromatography-mass spectrometry.

In this work, a novel technique of aqueous-phase derivatization followed by headspace solid-phase microextraction and gas chromatography-mass spectrometry was developed for the determination of organic acids in urine. The analytical procedure involves derivatization of organic acids to their ethyl esters with diethyl sulfate, headspace sampling, and GC/MS analysis. The proposed method was applied to the determination of methylmalonic acid and glutaric acid in urine. The experimental parameters and method validation were studied. Optimal conditions were obtained: PDMS fiber, extraction temperature 55 degrees C, extraction time 30 min, and 60 microL of diethyl sulfate as derivatization reagent with 2 mg of the ion pairing agent tetrabutylammonium hydrogensulfate. The method was linear over three orders of magnitude, and detection limits were 21 nM for methylmalonic acid and 34 nM for glutaric acid, respectively. Consequently, in-situ derivatization/HS-SPME/GC/MS is an alternative and powerful method for determination of organic acids as biomarkers in biological fluids.