牛血清白蛋白中氨基酸的高效液相色谱荧光测定及质谱鉴定

Determination of Amino Acids From Hydrolyzed Bovine Serum Albumin by High-Performance Liquid Chromatography-Mass Spectrometry with Fluorescence and Electrospray Ionization Detection

利用新型荧光试剂1,2-苯并-3,4-二氢咔唑-9-乙基氯甲酸酯（BCEOC）作为柱前衍生化试剂,在Hypersil BDS C18 （4.6 mm×200 mm, 5 μm）反相色谱柱上,采用梯度洗脱对氨基酸衍生物进行了分离检测.检测的激发和发射波长分别为λex=333 nm,λem=390 nm.采用电喷雾离子源（ESI Source）,实现了牛血清白蛋白中氨基酸的定性测定.荧光定量检测的线性回归系数均大于0.999;检出限为6.3～117.6 fmol.

On a reversed-phase Hypersil BDS C18 （4.6 mm × 200 mm, 5 μm） column, 20 amino acids derivatized using 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate（BCEOC） as pre-column derivatization reagent were separated in conjunction with a gradient elution. Optimum derivatization, giving the corresponding stable fluorescent derivatives, was obtained the reaction of amino acids with BCEOC at room temperature for 10 min in the presence of sodium borate catalyst in acetonitrile solvent. The fluorescence excitation and emission wavelengths were 333 nm and 390 nm. The identification of amino acid derivatives from hydrolyzed bovine serum albumin was obtained on the basis of electrospray ionization detection at positive ion mode. Linear correlation coefficients for the determination amino acid derivatives are over 0. 999, and detection limits （at signal-to-noise of 3：1 ） are 6.3 - 117.6 fmol for the labeled amino acids.