A simple isocratic HPLC procedure has been developed for the quantification of caffeine and the nicotine metabolites cotinine, 3'-hydroxycotinine, cotinine glucuronide and 3'-hydroxycotinine glucuronide in the plasma of smokers. The glucuronide conjugates were determined indirectly via initial basic hydrolysis of the analyte sample followed by quantification of the resulting deconjugation product. Plasma was basified, extracted with dichloromethane, evaporated, the residue dissolved water and an aliquot part was analyzed by HPLC. The method utilized a Partisil-10 SCX cation-exchange column and an isocratic mobile phase of sodium phosphate buffer: methanol (92:8 v/v, 0.1 M, adjusted to pH 4.8 with triethylamine) at a flow rate of 1.5 ml/min. UV detection was at 254 nm. All solutes were separated with good resolution, and quantification was determined using an internal standard of N,N-diethylnicotinamide. The retention times were: caffeine 5.1 min, 3'-hydroxycotinine 7.2 min, N,N-diethylnicotinamide 9.5 min, and cotinine 15.5 min. Detection limits for caffeine, 3'-hydroxycotinine, cotinine, and total cotinine were 10 ng/ml; the detection limit for total 3'-hydroxycotinine was 20 ng/ml. The inter-day and intra-day variations for all analytes were between 1 and 8%. This analytical method is suitable for the determination of caffeine and nicotine metabolite levels in large numbers of clinical samples. Copyright (C) 2000 Elsevier Science B.V.
|Number of pages||7|
|Journal||Journal of Pharmaceutical and Biomedical Analysis|
|State||Published - Aug 15 2000|
Bibliographical noteFunding Information:
This work was supported by two NARSAD Young Investigator Awards (JdL and TR), NIH grant DA 08656 (LPD and PAC) and a grant from the Tobacco and Health Research Institute, Lexington, KY (LPD and PAC).
- 3'-Hydroxycotinine glucuronide
- Cationic exchange chromatography
- Cotinine glucuronide
ASJC Scopus subject areas
- Analytical Chemistry
- Pharmaceutical Science
- Drug Discovery
- Clinical Biochemistry