title: Liquid chromatography/electrospray ionization tandem mass spectrometry assay for determination of nicotine and metabolites, caffeine and arecoline in breast milk.
abstract: 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 microg/L for nicotine, cotinine, trans-3-hydroxycotinine, cotinine-N-oxide and caffeine, and 50 microg/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).
authors.affiliation: Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanitá, Rome, Italy.
authors.forename: Manuela
authors.initials: M
authors.lastname: Pellegrini
authors.forename: Emilia
authors.initials: E
authors.lastname: Marchei
authors.forename: Silvia
authors.initials: S
authors.lastname: Rossi
authors.forename: Federica
authors.initials: F
authors.lastname: Vagnarelli
authors.forename: Abhilasha
authors.initials: A
authors.lastname: Durgbanshi
authors.forename: Oscar
authors.initials: O
authors.lastname: García-Algar
authors.forename: Oriol
authors.initials: O
authors.lastname: Vall
authors.forename: Simona
authors.initials: S
authors.lastname: Pichini
mesh_terms: D001115:Arecoline / Q000032:analysis*; D002110:Caffeine / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D008895:Milk, Human / Q000737:chemistry*; D009538:Nicotine / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D002110:Caffeine; D001115:Arecoline; D009538:Nicotine