Determination of tetrahydro-beta-carbolines in urine by high-performance liquid chromatography with suppression of artefact formation.
A high-performance liquid chromatographic method has been developed for the determination of urinary tetrahydro-beta-carbolines. When standing tryptamine with formaldehyde and acetaldehyde under extraction conditions, the significant amounts of artefact 1,2,3,4-tetrahydro-beta-carboline (TBC) and 1-methyl-1,2,3,4-tetrahydro-beta-carboline (MTBC) were formed in a short time. Urine samples added with 2-ethyl-1,2,3,4-tetrahydro-beta-carboline (an internal standard) were treated with fluorescamine, and then with glycine, followed by serial solvent extractions. Such a pretreatment using two-step reactions removed a precursor (trypamine) by extracting its fluorescamine derivative, and enhanced the detection response by consuming excess fluorescamine. It solved the analytical problem that artefact TBC and MTBC are formed during analysis. Reversed-phase ion-pair chromatography using a C8-column and trifluoroacetic acid as a counter ion completed a base-line separation of three analytes within 10 min. The calibration graphs showed a good linearity in the range 0.1-50.0 ng ml-1 of urine samples spiked with standard TBC and MTBC. In the spike experiment, the recovery and relative standard deviation were almost 100% and less than 3.0%, respectively, for both TBC and MTBC. The proposed method enables the determination of the genuine urinary concentrations of TBC and MTBC without involving their artefacts.
Tsuchiya
H
H
Department of Dental Pharmacology, Asahi University School of Dentistry, Gifu, Japan.
Ohtani
S
S
Yamada
K
K
Takagi
N
N
Hayashi
T
T
D000079:Acetaldehyde / Q000737:chemistry; D000328:Adult; D000596:Amino Acids; D002243:Carbolines / Q000737:chemistry / Q000652:urine*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D005450:Fluorescamine; D005557:Formaldehyde / Q000737:chemistry; D006801:Humans; D008297:Male; D013050:Spectrometry, Fluorescence / Q000379:methods; D014363:Tryptamines / Q000737:chemistry
D000596:Amino Acids; D002243:Carbolines; D014363:Tryptamines; C009777:2-methyltryptoline; D005557:Formaldehyde; D005450:Fluorescamine; C009804:tryptoline; D000079:Acetaldehyde