The ultra-trace determination of nicotine and its 4 major metabolites (cotinine, nornicotine, norcotinine and anabasine) from rabbit plasma was achieved by a newly developed solid phase microextraction-liquid chromatography-tandem mass spectrometry method. Extraction of the target analytes was performed with hydrophilic/lipophilic balance-polyacrylonitrile SPME fibers. Dual fiber extraction was necessary to guarantee improved recovery at parts-per-trillion levels. Liquid chromatographic analysis was achieved in a 6-min run using a C18 (1.9 µm C18, 50 mm x 2.1 mm) column with a mobile phase flow rate of 0.4 mL/min. Tandem mass spectrometry was used for detection and quantification in positive electrospray ionization (ESI+) mode for all the targeted analytes. Two stable isotope-labeled internal standards were used for signal correction and accurate quantification. The mass spectrometer with laminar flow ion flux transport, guaranteed improved signal stability, minimal contamination of the ion guide and reproducibility into the first quadrupole analyzer. The method was validated in line with the Food and Drug Administration (FDA) guidelines for bioanalytical method validation. The results met the acceptance criteria as proposed by the FDA: accuracy was tested at 0.35, 10 and 75 µg L - 1 and ranged between 98.3-112.2% for nicotine, 94.1-101.9% for cotinine, 94.7-107.0% for nornicotine, 81.1-107.2% for norcotinine and 94.3-115.2% for anabasine, with precision up to 14.2%. Stability tests indicated that all the targeted analytes were stable in the desorption solution for at least 1 week. LOQs ranged from 0.05 to 1 µg L-1. The method was successfully applied to analyze plasma samples obtained from rabbits following transdermal application of a smoking cessation formulation loaded with solid lipid nanoparticles containing a nicotine-stearic acid conjugate.
Keywords: Biocompatible SPME; Liquid chromatography-tandem mass spectrometry; Nicotine; Rabbit plasma; Smoking cessation.
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