Liquid chromatography/electrospray ionization tandem mass spectrometry assay for determination of nicotine and metabolites, caffeine and arecoline in breast milk

A procedure based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) is described for the determination of nicotine and its principal metabolites cotinine, trans ‐3‐hydroxycotinine and cotinine‐ N ‐oxide, caffeine and arecoline in breast milk, using N ‐ethylnorcotinine as internal standard. Liquid/liquid extraction with chloroform/isopropanol (95:5, v/v) was used for nicotine, cotinine, trans ‐3‐hydroxycotinine, cotinine‐ N ‐oxide and caffeine under neutral conditions and for arecoline under basic conditions. Chromatography was performed on a C 8 reversed‐phase column using a gradient of 50 mM ammonium formate, pH 5.0, and acetonitrile as a mobile phase at a flow rate of 0.5 mL/min. Separated analytes were determined by electrospray ionization tandem mass spectrometry in the positive ion mode using multiple reaction monitoring. Limits of quantification were 5 µg/L for nicotine, cotinine, trans ‐3‐hydroxycotinine, cotinine‐ N ‐oxide and caffeine, and 50 µg/L for arecoline using 1 mL human milk per assay. Calibration curves were linear over the calibration ranges for all the substances under investigation, with a minimum r 2  > 0.998. At three concentrations spanning the linear dynamic range of the assay, mean recoveries from breast milk ranged between 71.8 and 77.4% for different analytes. This method was applied to the analysis of analytes in human milk to assess substance exposure in breast‐fed infants in relation to eventual clinical outcomes. This LC/MS/MS assay provides adequate sensitivity and performance characteristics for the simultaneous quantification of biomarkers of three of the drugs most commonly used worldwide (tobacco, caffeine and areca nut). Copyright © 2007 John Wiley & Sons, Ltd.