Pre-column Derivatization High Performance Liquid Chromatography Tandem Mass Spectrometric Determination of Trace Level of Amino Acids in Rat Serum

A simple and sensitive method based on the derivatization of 20 amino acids with 1,2-benzo-3,4-dihydrocarbazole-9- ethyl chloroformate (BCEOC) as derivatization reagent on a reversed-phase Hypersil BDS C18 column (4.6 mm × 200 mm, 5 μm) with a gradient elution followed by high performance liquid chromatography (HPLC) via fluorescence detection at 390 nm (excitation wavelength of 333 nm) and tandem mass spectrometric identification has been developed. Optimum derivatization, giving the corresponding stable fluorescent derivatives, was obtained by reacting amino acids with BCEOC at 40 ° for 10 min in borate buffer (pH 9.0) with four times excess of molar reagent. The linear range of 20 amino acids were 51.6 fmol–105.6 pmol, all correlation coefficients >0.9995, detection limits were 6.3–177.6 fmol (at signal to noise 3:1, S/N=3:1). The identification of amino acid derivatives was carried out by post-column tandem mass spectrometry with electrospray ion (ESI) source, and the MS/MS cleavage mode of representative tyrosine derivative was analyzed. Under all the above optimum experimental conditions, the contents of 20 amino acids in rat serum of 3 groups (A: Quiet, B: At exercising exhaust, C: 12 h after exercising exhaust) were determined. It was indicated that the contents of 20 amino acids in group B were obviously higher than those in group A, and the contents of amino acids of group C and group A were almost the same. The established method exhibited high sensitivity and excellent reproducibility, and provided a new technology for the determination of amino acids in rat serum.