title: A fully validated LC-MS/MS method for simultaneous determination of nicotine and its metabolite cotinine in human serum and its application to a pharmacokinetic study after using nicotine transdermal delivery systems with standard heat application in adult smokers.
abstract: A sensitive and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous determination of nicotine and its main metabolite cotinine in human serum samples. Liquid-liquid extraction using ethyl acetate was employed for serum sample extractions. Chromatographic separation was achieved on Phenomenex Luna(®) HILIC column (150 mm x 3.0mm, 5 μm). Isocratic elution was performed using acetonitrile:100mM ammonium formate buffer (pH=3.2) (90:10, v/v) as the mobile phase, at a flow rate of 0.4 mL/min. Tandem mass spectrometric detection was employed at positive electrospray ionization in MRM mode for the determination of both nicotine and cotinine and their stable isotope labeled internal standards. Analysis was carried out in 8 min over a concentration range of 0.26-52.5 ng/mL and 7.0-1500 ng/mL for nicotine and cotinine, respectively. The assay was validated according to FDA guidelines for bioanalytical method validation and satisfactory results were obtained; the accuracy ranged between 93.39% and 105.73% for nicotine and between 93.04% and 107.26% for cotinine. No significant matrix effect was observed. Stability assays indicated both nicotine and cotinine were stable during sample storage, preparation and analytical procedures. The method was successfully applied to biological samples obtained from a pharmacokinetic study conducted in adult smokers to investigate heat effect on nicotine and cotinine serum levels after nicotine transdermal delivery system (TDS) application.
authors.affiliation: Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD, United States; Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Misr International University, Cairo, Egypt.
authors.forename: Inas A
authors.initials: IA
authors.lastname: Abdallah
authors.affiliation: Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD, United States.
authors.forename: Dana C
authors.initials: DC
authors.lastname: Hammell
authors.affiliation: Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD, United States. Electronic address: astinchc@rx.umaryland.edu.
authors.forename: Audra L
authors.initials: AL
authors.lastname: Stinchcomb
authors.affiliation: Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD, United States; Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Helwan University, Cairo, Egypt. Electronic address: hhassan@rx.umaryland.edu.
authors.forename: Hazem E
authors.initials: HE
authors.lastname: Hassan
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D003367:Cotinine / Q000097:blood* / Q000737:chemistry / Q000378:metabolism; D004355:Drug Stability; D006801:Humans; D006979:Hyperthermia, Induced; D016014:Linear Models; D009538:Nicotine / Q000097:blood* / Q000737:chemistry / Q000493:pharmacokinetics / Q000627:therapeutic use; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016540:Smoking Cessation; D053719:Tandem Mass Spectrometry / Q000379:methods*; D061485:Tobacco Use Cessation Devices; D014029:Tobacco Use Disorder / Q000628:therapy
chemical_list: D009538:Nicotine; D003367:Cotinine
keywords: Cotinine; HILIC; LC–MS/MS; Nicotine; TDS