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

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 1020; pp. 67 - 77
• Main Authors: Abdallah, Inas A., Hammell, Dana C., Stinchcomb, Audra L., Hassan, Hazem E.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.05.2016
• Subjects: Adults; blood serum; Chromatography; Chromatography, Liquid - methods; Cotinine; Cotinine - blood; Cotinine - chemistry; Cotinine - metabolism; Delivery systems; Drug Stability; ethyl acetate; formates; guidelines; heat; HILIC; Humans; hydrophilic interaction chromatography; Hyperthermia, Induced; ionization; LC–MS/MS; Linear Models; liquid-liquid extraction; Metabolites; Nicotine; Nicotine - blood; Nicotine - chemistry; Nicotine - pharmacokinetics; Nicotine - therapeutic use; pharmacokinetics; Pharmacology; Reproducibility of Results; Sensitivity and Specificity; Serums; Smoking Cessation; stable isotopes; tandem mass spectrometry; Tandem Mass Spectrometry - methods; TDS; Tobacco Use Cessation Products; Tobacco Use Disorder - therapy; TDS; Cotinine; LC–MS/MS; HILIC; Nicotine
• ISSN: 1570-0232; 1873-376X; 1873-376X
• DOI: 10.1016/j.jchromb.2016.03.020

•The new LC–MS/MS method allows the simultaneous determination of nicotine and its metabolite cotinine in human serum samples.•The method is highly sensitive in comparison to other reported methods.•It can be employed to analyze nicotine and cotinine samples following pharmacokinetic studies on smokers or non-smokers subjects administering nicotine products.•The method was applied for analysis of samples obtained from a clinical study involving the use of nicotine TDS.•It can be used in future clinical studies for evaluating different nicotine products. 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 (150mm x 3.0mm, 5μm). 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 0.4mL/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 8min over a concentration range of 0.26–52.5ng/mL and 7.0–1500ng/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.