A simple and mild derivatization method for hydroxyl compounds using condensation agent carbazole-9-N-(2-methyl)-acetyl-benzene-disulfonate and its application for the determination of volatile alcohols from plasma by HPLC with fluorescence detection

A simple and sensitive HPLC method for the determination of hydroxyl compou nds including volatile alcohols from spiked plasma, using a fluorescent con densation agent, namely carbazole-9-N-(2-methyl)-acetyl -benzene-disulfonat e (CMABS), has been developed. A mixture of alcohols and pyridine in dichlo romethane was treated with CMABS to give a quantitative yield of esters. Th e maximum fluorescence emissions for the derivatized alcohols are at 365 nm (lambda(ex) 335 nm). Studies on derivatization conditions indicate that al cohols react very fast with CMABS in presence of pyridine in dichloromethan e to give the corresponding fluorescent derivatives. The method, in conjunc tion with a multi-step gradient, offers a baseline resolution of the common alcohol derivatives on a reversed-phase C-18 column, which is more conveni ent and more efficient than previous methods which require the prior conver sion of the carboxylic acids to the acyl chlorides. The application of this method to the analysis of volatile alcohols in plasma was also investigate d. The LC separation shows good selectivity and reproducibility for alcohol derivatives. The relative standard deviations (n = 5) for 100 pmol of each alcohol are < 4%. The detection limits are at the fmol level for C-1-C-3 a lcohols and pmol level for C-4-C-9 alcohols.