Measurement of cotinine in urine by liquid chromatography tandem mass spectrometry

Abstract

Background: Cotinine is the major metabolite of nicotine. It is also a specific biomarker for nicotine exposure in cigarette smokers. The measurement of urine cotinine concentration will enable: (1) the assessment of the smoking status of lung transplant patients and (2) tobacco abstinence to be studied in patients during treatment under smoking cessation programmes. Methods: We have developed and validated a method for the measurement of urinary cotinine using reversed phase high-performance liquid chromatography (HPLC) coupled to tandem mass spectrometry (LC-MS/MS). This technique utilizes online ion exchange coupled with an analytical column to eliminate ion suppression effects. The chromatography was performed using a WatersTM 2795 Alliance HT LC system. Results: Cotinine and d3-cotinine had a retention time of 2.5 min and the cycle time from injection to injection was 4 min. The transition identified for cotinine was m/z 177.1>79.6 and for d3-cotinine m/z 180.2>79.6. This method was linear up to 1000 μg/L. Mean recovery of the assay was 112% with a range of 107-117% ( n=9). The limit of quantitation for this assay was 2.5 µg/L and the limit of detection was 0.156 µg/L. The intra- and inter-assay imprecision was <12% and <10% respectively over a concentration range of 22-660 μg/L. Conclusions: We have developed a robust and rapid assay for measuring and analysing urine cotinine by LC-MS/MS, by utilizing a technique, which has reduced ion suppression effects. Ultimately, the method will facilitate the assessment of lung transplant patients' smoking status.