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

A sensitive LC method for the determination of amino compounds with fluores cence detection has been developed. The reaction of fluorescent tagging rea gent, namely carbazole-N-(2-methyl)acetyl chloride (CMA-Cl), with amino aci ds is reported. Emission maximum for the CMA derivatives of amino acids is 360 nm (lambda(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% decom position occurs after heating at 40 degrees 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 gradi ent elution, offers baseline resolution of 18 amino acids including Om 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 cha nged into Glu and Asp; Trp gives as much as 60% loss of its monosubstituted derivative under proposed derivatization conditions.). Separation of deriv atives is carried out on a reversed-phase C-18 column with good reproducibi lity. Derivatization and chromatographic conditions are optimized. The rela tive standard deviations (n=6) for 50 pmol of each amino acid derivative ar e <4.5%. The detection limits, calculated by the corresponding peak heights (in cm) by injecting successively lower concentrations until a signal-to-n oise of 3:1, are 10-65 fmol for the labelled amino acids. (C) 1999 Elsevier Science B.V. All rights reserved.