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 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 x 4 mm id, 5 microns 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.
Molins-Legua
C
C
Departament de Química Analítica, Facultad de Química, Universitat de Valencia, Burjassot, Spain.
Campíns-Falcó
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P
Sevillano-Cabeza
A
A
D000588:Amines / Q000652:urine*; D000661:Amphetamine / Q000652:urine; D002853:Chromatography, Liquid / Q000379:methods*; D003619:Dansyl Compounds; D006801:Humans; D007202:Indicators and Reagents; D008163:Luminescent Measurements; D008694:Methamphetamine / Q000652:urine; D010070:Oxalates
D000588:Amines; D003619:Dansyl Compounds; D007202:Indicators and Reagents; D010070:Oxalates; C036521:bis(2,4,6-trichlorophenyl)oxalate; D008694:Methamphetamine; D000661:Amphetamine; C029038:dansyl chloride