Automated pre-column derivatization of amines in biological samples with dansyl chloride and with or without post-column chemiluminescence formation by using TCPO-H2O2

On-line automation of two different liquid chromatographic procedures, a pr e-column derivatization system and a pre- and post-column system, in order to generate chemiluminescence is reported. Dansyl chloride (Dns-Cl) was use d as a pre-column reagent to form fluorophores and bis(2,4,6-trichloropheny l) oxalate (TCPO) and hydrogen peroxide (H2O2) as a post-column reagent to generate chemiluminescence. This procedure is based on the employment of a primary column packed with C-18 material inserted in a multi-dimensional as sembly for sample clean-up and derivatization with Dns-Cl. The dansyl deriv atives formed are transferred and separated in a LiChrospher 100 RP18 analy tical column (125 x 4 mm id, 5 mu m film thickness) using acetonitrile-imid azole buffer (pH 6.8) (70 + 30) as eluent. The separated derivatives were t ransferred to the detector for fluorescence detection or to the post-column system where the chemiluminescence response was generated by using TCPO-H2 O2 and the products were detected by chemiluminescence. The procedure was o ptimised for amphetamine and related compounds. A comparison between the on -line pre-column and pre- and post-column systems was performed. The result s show that the sensitivity of chemiluminescence detection can be higher th an that of fluorescence detection. The recoveries obtained ranged from 98 /- 8 up to 108 +/- 8% for amphetamine and methamphetamine, respectively. Th e accuracy and precision of these methods were evaluated.