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 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).
Pellegrini
Manuela
M
Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanitá, Rome, Italy.
Marchei
Emilia
E
Rossi
Silvia
S
Vagnarelli
Federica
F
Durgbanshi
Abhilasha
A
García-Algar
Oscar
O
Vall
Oriol
O
Pichini
Simona
S
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*
D002110:Caffeine; D001115:Arecoline; D009538:Nicotine