A simple, sensitive method for the simultaneous determination of cotinine and trans-3´-hydroxycotinine in human serum by LC-MS/MS

Abstract A simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of cotinine and its metabolite, trans -3´-hydroxycotinine, in human serum. Liquid–liquid extraction (LLE) using dichloromethane (DCM) and ethyl acetate (ETAC) (4:1 v/v) was employed for serum sample extractions. Chromatographic separation was achieved on an Agilent Eclipse Plus Phenyl-Hexyl column (4.6 mm × 100 mm, 5 µm) with a gradient elution of 0.05% (v/v) formic acid/5 mM ammonium formate (1:1) in water and methanol at an initial flow rate of 1 mL/min. Tandem mass spectrometry detection used positive electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode for the determination of cotinine, trans -3´-hydroxycotinine, and their deuterated internal standards. The limits of detection (LOD) for cotinine and trans -3´-hydroxycotinine were 0.005 and 0.006 ng/mL, respectively. The limit of quantification (LOQ) was 0.025 ng/mL for both analytes. The accuracy ranged from 96.1 to 104.9% for cotinine and 98.9 to 102.9% for trans -3´-hydroxycotinine. The linear range for both metabolites was 0.025–10 ng/mL. Extraction recovery ranged from 100.0 to 106.4% for cotinine and 89.6 to 102.5% for trans -3´-hydroxycotinine, respectively. The intra- and interday precision (% CV) ranged from 0.9 to 8.0% for cotinine and 1 to 9.6% for trans -3´-hydroxycotinine. Neither carryover nor endogenous or exogenous interference was observed. The method was successfully applied to pooled serum samples from a biomonitoring study of pregnant women in Alberta, Canada.