Review of HPLC–MS methods for the analysis of nicotine and its active metabolite cotinine in various biological matrices

In recent years, tobacco smoking is a risk factor for a series of diseases, including cardiovascular diseases, cerebrovascular diseases, and cancers. Nicotine, the primary component of tobacco smoke, is mainly transformed to its active metabolite cotinine, which is often used as a biomarker for tobacco exposure for its higher blood concentration and longer residence time than nicotine. Various analytical methods have been developed for the determination of nicotine and cotinine in biological matrices. This article reviewed the HPLC-MS based methods for nicotine and/or cotinine analysis in various biological matrices. The sample preparation, mass and chromatographic conditions, and method validation results of these methods have been summarized and analyzed. The sample was mainly pretreated by protein precipitation and/or extraction. Separation was achieved using methanol and/or acetonitrile:water (with or without ammonium acetate) on C18 columns and acetonitrile:water (with formic acid, ammonium acetate/formate) on HILIC columns. Nicotine-d3, nicotine-d4, and cotinine-d3 were commonly used internal standards (ISs). Other non-deuterated ISs such as ritonavir, N-ethylnorcotinine, and milrinone were also used. For both nicotine and cotinine, the calibration range was 0.005-35,000 ng/mL, the matrix effect was 75.96-126.8%, and the recovery was 53-124.5%. The two analytes were stable at room temperature for 1-10 days, at -80°C for up to 6 months, and after three to six freeze-thaw cycles. Comedications did not affect nicotine and cotinine analyses.