A simple high performance liquid chromatographic method for the quantification of total cotinine, total 3'-hydroxycotinine and caffeine in the plasma of smokers

J Pharm Biomed Anal. 2000 Aug 15;23(2-3):543-9. doi: 10.1016/s0731-7085(00)00339-3.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Caffeine / blood*
  • Chromatography, High Pressure Liquid / methods*
  • Cotinine / analogs & derivatives
  • Cotinine / blood*
  • Humans
  • Reference Standards
  • Reproducibility of Results
  • Smoking / blood*
  • Spectrophotometry, Ultraviolet


  • hydroxycotinine
  • Caffeine
  • Cotinine