Quantification of cotinine in plasma and saliva by liquid chromatography

Measurement of cotinine, a nicotine metabolite, has been studied as a method for monitoring smoking behavior and determining smoking status. We describe a specific, sensitive method for quantifying it in plasma and saliva by reversed-phase paired-ion liquid chromatography and detection by absorbance at 257 nm. The cotinine is extracted with methylene chloride, and 2-phenylimidazole is the internal standard. Cotinine peak heights are linearly related to the amount on the column from 0 to 500 ng. The mean (+/- SD) concentration of cotinine in plasma of 31 passively exposed nonsmokers was 2.1 +/- 1.6 micrograms/L (range, 0-7.9 micrograms/L). The regression of saliva cotinine concentration (y) on plasma cotinine concentration (x) at 0, 24, and 48 h in 10 smokers who refrained from smoking for 48 h was y (micrograms/L) = 1.155x (micrograms/L) + 0.245 (r = 0.986). The efficiency of cotinine as a biological marker was determined at 0, 24, and 48 h of smoking abstinence. Within-run CVs were 3.5% (n = 5) and day-to-day CVs 4.4% (n = 6) at 150 micrograms/L.