A novel validated procedure for the determination of nicotine, eight nicotine metabolites and two minor tobacco alkaloids in human plasma or urine by solid-phase extraction coupled with liquid chromatography–electrospray ionization–tandem mass spectrometry

Abstract

A novel validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) procedure was developed and fully validated for the simultaneous determination of nicotine-N-β-D-glucuronide, cotinine-N-oxide, trans-3-hydroxycotinine, norcotinine, trans-nicotine-1′-oxide, cotinine, nornicotine, nicotine, anatabine, anabasine and cotinine-N-β-D-glucuronide in human plasma or urine. Target analytes and corresponding deuterated internal standards were extracted by solid-phase extraction and analyzed by LC-MS/MS with electrospray ionization (ESI) using multiple reaction monitoring (MRM) data acquisition. Calibration curves were linear over the selected concentration ranges for each analyte, with calculated coefficients of determination (R2) of greater than 0.99. The total extraction recovery (%) was concentration dependent and ranged from 52–88 % in plasma and 51–118 % in urine. The limit of quantification for all analytes in plasma and urine were 1.0 ng/mL and 2.5 ng/mL respectively with the exception of cotinine-N-β-D-glucuronide which was 50 ng/mL. Intra-day and inter-day imprecision were ≤14 % and ≤17 % respectively. Matrix effect (%) was sufficiently minimized to ≤19 % for both matrices using the described sample preparation and extraction methods. The target analytes were stable in both matrices for at least 3 freeze thaw cycles, 24 hours at room temperature, 24 hours in the refrigerator (4 °C) and 1 week in the freezer (−20 °C). Reconstituted plasma and urine extracts were stable for at least 72 hours storage in the liquid chromatography autosampler at 4 °C. The plasma procedure has been successfully applied in the quantitative determination of selected analytes in samples collected from nicotine-abstinent human participants as part of a pharmacokinetic study investigating biomarkers of nicotine use in plasma following controlled low dose (7 mg) transdermal nicotine delivery. Nicotine, cotinine, trans-3-hydroxycotinine and trans-nicotine-1′-oxide were detected in the particular sample presented herein. The urine procedure has been used to facilitate the monitoring of unauthorized tobacco use by clinical study participants at the time of physical examination (before enrolment) and on the pharmacokinetic study day.

Determination of nicotine and three minor alkaloids in tobacco by hydrophilic interaction chromatography-tandem mass spectrometry

“…The most common methods include thin layer chromatography [3], capillary electrophoresis [4][5][6], gas chromatography [7][8][9][10], and high performance liquid chromatography (HPLC) [11][12][13][14][15][16][17]. Each of these methods has its advantages.…”

“…Each of these methods has its advantages. However, HPLC with its various separation modes can have the capability of being more specific and sensitive, especially if interfaced with tandem mass spectrometry (MS/MS) [13][14][15][16][17]. Nicotine related alkaloids belong to the group of very polar basic molecules, which in neutral and acidic solutions exist predominantly in the cationic form [4].…”

“…Nicotine related alkaloids belong to the group of very polar basic molecules, which in neutral and acidic solutions exist predominantly in the cationic form [4]. For this reason, HPLC separation of nicotine alkaloids can be carried out by ion pair [12], or ion chromatography [16], reversed-phase (RP) chromatography being the most common separation mode [11,[13][14][15]17].…”

Medicine students and exposure to environmental tobacco smoke

“…Moreover, Benowitz et al stated that the nicotine metabolites ratio could be used as a predictor of cigarette consumption [14]. The evaluation of xenobiotics exposure is often carried out by the immunochemical technique [15][16][17], however the chromatographic techniques are more popular due to their higher specificity [16,18]. Thin layer chromatography has been rarely used for this purpose (only to determine cotinine) [11], but the LC-MS/MS technique is frequently applied in this area [18].…”

“…The evaluation of xenobiotics exposure is often carried out by the immunochemical technique [15][16][17], however the chromatographic techniques are more popular due to their higher specificity [16,18]. Thin layer chromatography has been rarely used for this purpose (only to determine cotinine) [11], but the LC-MS/MS technique is frequently applied in this area [18]. The aim of the presented study was the assessment of exposure to chosen xenobiotic biomarkers in a group of young medical students from the Medical University of Silesia in Katowice, Faculty of Medicine in Zabrze.…”