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

Abstract

On-line automation of two different liquid chromatographic procedures, a pre-column derivatization system and a pre- and post-column system, in order to generate chemiluminescence is reported. Dansyl chloride (Dns-Cl) was used as a pre-column reagent to form fluorophores and bis(2,4,6-trichlorophenyl) 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 C₁₈ material inserted in a multi-dimensional assembly for sample clean-up and derivatization with Dns-Cl. The dansyl derivatives formed are transferred and separated in a LiChrospher 100 RP₁₈ analytical column (125 × 4 mm id, 5 µm film thickness) using acetonitrile–imidazole buffer (pH 6.8) (70 + 30) as eluent. The separated derivatives were transferred to the detector for fluorescence detection or to the post-column system where the chemiluminescence response was generated by using TCPO–H₂O₂ and the products were detected by chemiluminescence. The procedure was optimised for amphetamine and related compounds. A comparison between the on-line pre-column and pre- and post-column systems was performed. The results show that the sensitivity of chemiluminescence detection can be higher than that of fluorescence detection. The recoveries obtained ranged from 98 ± 8 up to 108 ± 8% for amphetamine and methamphetamine, respectively. The accuracy and precision of these methods were evaluated.