Determination of nicotine, cotinine, and related alkaloids in human urine and saliva by automated in-tube solid-phase microextraction coupled with liquid chromatography–mass spectrometry

A simple, rapid and sensitive method for the determination of nicotine, cotinine, nornicotine, anabasine, and anatabine in human urine and saliva was developed. These compounds were analyzed by on-line in-tube solid-phase microextraction (SPME) coupled with liquid chromatography–mass spectrometry (LC–MS). Nicotine, cotinine and related alkaloids were separated within 7 min by high performance liquid chromatography (HPLC) using a Synergi 4u POLAR-RP 80A column and 5 mM ammonium formate/methanol (55/45, v/v) as a mobile phase at a flow-rate of 0.8 mL/min. Electrospray ionization conditions in the positive ion mode were optimized for MS detection of these compounds. The optimum in-tube SPME conditions were 25 draw/eject cycles with a sample size of 40 μL using a CP-Pora PLOT amine capillary column as the extraction device. The extracted compounds could be desorbed easily from the capillary by passage of the mobile phase, and no carryover was observed. Using the in-tube SPME LC–MS method, the calibration curves were linear in the concentration range of 0.5–20 ng/mL of nicotine, cotinine and related compounds in urine and saliva, and the detection limits (S/N = 3) were 15–40 pg/mL. The method described here showed 20–46-fold higher sensitivity than the direct injection method (5 μL injection). The within-run and between-day precision (relative standard deviations) were below 4.7% and 11.3% (n = 5), respectively. This method was applied successfully to analysis of urine and saliva samples without interference peaks. The recoveries of nicotine, cotinine and related compounds spiked into urine and saliva samples were above 83%, and the relative standard deviations were below 7.1%. This method was used to analyze urinary and salivary levels of these compounds in nicotine intake and smoking.