Simultaneous analysis of plasma free fatty acids and their 3-hydroxy analogs in fatty acid beta-oxidation disorders.

We present a new derivatization procedure for the simultaneous gas chromatographic-mass spectrometric analysis of free fatty acids and 3-hydroxyfatty acids in plasma. Derivatization of target compounds involved trifluoroacetylation of hydroxyl groups and tert-butyldimethylsilylation of the carboxyl groups. This new derivatization procedure had the advantage of allowing the complete baseline separation of free fatty acids and 3-hydroxyfatty acids while the superior gas chromatographic and mass spectrometric properties of tert-butyldimethylsilyl derivatives remained unchanged, permitting a sensitive analysis of the target compounds. Thirty-nine plasma samples from control subjects and patients with known defects of mitochondrial fatty acid beta-oxidation were analyzed. A characteristic increase of long-chain 3-hydroxyfatty acids was observed for all of the long-chain 3-hydroxyacyl-CoA dehydrogenase-deficient and mitochondrial trifunctional protein-deficient plasma samples. For medium-chain acyl-CoA dehydrogenase deficiency and very-long-chain acyl-CoA dehydrogenase deficiency, decenoic and tetradecenoic acids, respectively, were the main abnormal fatty acids, whereas the multiple acyl-CoA dehydrogenase-deficient patients showed variable increases of these unusual intermediates. The results showed that this selective and sensitive method is a powerful tool in the diagnosis and monitoring of mitochondrial fatty acid beta-oxidation disorders.