Determination of DCA Concentration in the Plasma

The DCA levels in the blood samples were measured after derivatization and extraction, as previously described (Yan et al., 1997). Plasma samples (200 µl) and an internal standard of 2-ketohexanoic acid (10 µg) were mixed with 500 µl of boron trifluoride–methanol complex in a glass tube and heated for 15 minutes at 110°C. Upon cooling, 1 ml of methylene chloride and 1 ml of water were added to the reaction mixture, then vortexed for 5 minutes and allowed to stand for 10 minutes. After centrifugation at 3000 rpm, the methylene chloride layer was transferred to a glass vial for gas chromatography–mass spectrometry analysis.