Sensitive and rapid method for the determination of urinary cotinine in non-smokers: an application for studies assessing exposures to second hand smoke (SHS)

We describe a sensitive and rapid method to assay urinary cotinine levels among non-smokers using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) and its application in studies assessing exposures to second hand smoke (SHS). Cotinine was initially extracted from 1 ml of urine with methylene chloride by using a liquid-liquid extraction Chem Elut™ column. The extracted sample was further separated by using a BetaBasic C18 column (1 mm × 150 mm, 3 μm) with isocratic elution (60:40 acetonitrile and 5 mM ammonium acetate at pH 5), and then examined using a triple quadrupole mass spectrometer with an electrospray ionization (ESI) source in multiple-reaction-monitoring (MRM) mode. The elution of cotinine from the LC column took approximately 2.3 min and the detection of cotinine by ESI/MS/MS provided a limit of detection (LOD) of 0.3 ng/ml. The ESI/MS/MS detection was able to easily distinguish between cotinine and nicotine. This method, validated using a cotinine concentration range from 0.8 to 102.4 ng/ml, was successfully applied in a cross-sectional study examining differences in levels and sources of second hand smoke (SHS) exposure among non-smokers. Self-reported measures of SHS exposure were significantly associated with urinary cotinine levels. This urinary cotinine assay using LC-ESI/MS/MS provides a robust, high throughput and very sensitive method for the evaluation of SHS exposure for use in epidemiologic and clinical research studies.