Elimination of caffeine interference in HPLC determination of urinary nicotine and cotinine

We report an analytical reversed-phase liquid-chromatographic procedure for quantifying nicotine and cotinine in urine, taking into account the presence of interfering caffeine frequently encountered in such specimens. These analytes are extracted from the alkalinized urine with chloroform. After evaporation of the chloroform, the residue is dissolved in methanol and injected into a chromatographic C18 column. Extraction recoveries averaged 80% to 97%. Chromatographic conditions were investigated to obviate caffeine interference. The proposed eluent mobile phase is a polar mixture of water, acetonitrile, methanol, and a pH 4 acetoacetate buffer (65/2/29/4 by vol) adjusted to pH 4.30 +/- 0.02 with triethylamine. High resolution and linearity were obtained for each analyte up to a concentration of 200 mg/L. The minimum detectable amount of each compound was 20 ng per injection, corresponding to 10 micrograms per liter of urine. Correlation with results of gas-liquid chromatography was excellent (r = 0.99). This simple, rapid procedure allows routine screening of tobacco exposure with acceptable precision: within- and between-run coefficients of variation were less than 2% and less than 5%, respectively.