Fluorescence properties of carbazole- N-(2-methyl)acetyl chloride and determination of amino compounds via high-performance liquid chromatography with pre-column fluorescence derivatization

Abstract

A sensitive LC method for the determination of amino compounds with fluorescence detection has been developed. The reaction of fluorescent tagging reagent, namely carbazole- N-(2-methyl)acetyl chloride (CMA-Cl), with amino acids is reported. Emission maximum for the CMA derivatives of amino acids is 360 nm ( λ ex=335 nm). In all cases, the derivatives exhibit strong fluorescence, whereas the reagent itself also exhibits fluorescence. It is found that the labelled derivatives using CMA-Cl are very stable; <4% decomposition occurs after heating at 40°C for 24 h in neutral solution. Fluorescence intensity of amino acid derivatives is higher in neutral and alkaline than in acidic solutions. This method, in conjunction with a gradient elution, offers baseline resolution of 18 amino acids including Orn from a linear acetonitrile gradient (here, Asn, Gln and Trp are not tested, the principal reasons: Gln and Asn, in a real sample hydrolysed, have been changed into Glu and Asp; Trp gives as much as 60% loss of its monosubstituted derivative under proposed derivatization conditions.). Separation of derivatives is carried out on a reversed-phase C 18 column with good reproducibility. Derivatization and chromatographic conditions are optimized. The relative standard deviations ( n=6) for 50 pmol of each amino acid derivative are <4.5%. The detection limits, calculated by the corresponding peak heights (in cm) by injecting successively lower concentrations until a signal-to-noise of 3:1, are 10–65 fmol for the labelled amino acids.