Determination of amino acids in biological fluids by capillary gas chromatography with nitrogen-phosphorus selective detection

Abstract

A selective and sensitive method for the determination of protein and non-protein amino acids in biological fluids by capillary gas chromatography (GC) has been developed. The amino acids in the samples were directly converted into their N( O, S)-isobutoxycarbonyl methyl ester derivatives and measured by GC with nitrogen-phosphorus selective detection (NPD) using a DB-17ht capillary column. Using this method, the derivatives of the 21 protein amino acids and the 25 non-protein amino acids provided excellent NPD responses and were quantitatively and reproducibly resolved within 28 min. The lower detection limits of these amino acids, at a signal-to-noise ratio of 3, were ca. 6–150 pg injected. The calibration curves for each amino acid in the range of 0.02–2 μg were linear and sufficiently reproducible for quantitative analysis. This method was successfully applied to small urine and serum samples without prior clean-up; there was no evidence of interference from coexisting substances. Overall recoveries of amino acids added to urine and serum samples were 83–112%. The intra-assay and inter-assay R.S.D. of amino acids in these samples were 0.3–8.9% ( n = 3) and 1.9–15.8% ( n = 3) respectively.