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-beta-D-glucuronide, cotinine-N-oxide, trans-3-hydroxycotinine, norcotinine, trans-nicotine-1'-oxide, cotinine, nornicotine, nicotine, anatabine, anabasine and cotinine-N-beta-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 (R(2)) of greater than 0.99. The total extraction recovery (%) was concentration dependent and ranged between 52-88% in plasma and 51-118% in urine. The limits 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-beta-D-glucuronide, which was 50 ng/mL. Intra-day and inter-day imprecision were < or = 14% and < or = 17%, respectively. Matrix effect (%) was sufficiently minimized to < or = 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 h at room temperature, 24 h in the refrigerator (4 degrees C) and 1 week in the freezer (-20 degrees C). Reconstituted plasma and urine extracts were stable for at least 72 h storage in the liquid chromatography autosampler at 4 degrees 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 enrollment) and on the pharmacokinetic study day.

Figure 1

Nicotine Metabolic Pathway⁸

Figure 2

Figure 2a Chromatogram for low concentration quality control in human plasma for nicotine, eight nicotine metabolites and two minor tobacco alkaloids with corresponding deuterated internal standards* Figure 2b Chromatogram of analytes detected in participant’s plasma sample 1 h after patch removal Figure 2c Chromatogram for analyte-free human plasma fortified with deuterated internal standard only (corresponding to analytes detected in participant sample in Figure 2b)

Figure 2

Figure 2a Chromatogram for low concentration quality control in human plasma for nicotine, eight nicotine metabolites and two minor tobacco alkaloids with corresponding deuterated internal standards* Figure 2b Chromatogram of analytes detected in participant’s plasma sample 1 h after patch removal Figure 2c Chromatogram for analyte-free human plasma fortified with deuterated internal standard only (corresponding to analytes detected in participant sample in Figure 2b)

Figure 2

Figure 2a Chromatogram for low concentration quality control in human plasma for nicotine, eight nicotine metabolites and two minor tobacco alkaloids with corresponding deuterated internal standards* Figure 2b Chromatogram of analytes detected in participant’s plasma sample 1 h after patch removal Figure 2c Chromatogram for analyte-free human plasma fortified with deuterated internal standard only (corresponding to analytes detected in participant sample in Figure 2b)

Figure 2

Figure 2a Chromatogram for low concentration quality control in human plasma for nicotine, eight nicotine metabolites and two minor tobacco alkaloids with corresponding deuterated internal standards* Figure 2b Chromatogram of analytes detected in participant’s plasma sample 1 h after patch removal Figure 2c Chromatogram for analyte-free human plasma fortified with deuterated internal standard only (corresponding to analytes detected in participant sample in Figure 2b)

Figure 3

Figure 3a Chromatogram of lowest urine calibrator for cotinine (2.5 ng/mL) Figure 3b Chromatogram of a participant’s urine sample collected on day of physical examination before enrolment into the study Figure 3c Chromatogram of cotinine and cotinine d3 transitions for urine fortified with deuterated internal standard only

Figure 3

Figure 3a Chromatogram of lowest urine calibrator for cotinine (2.5 ng/mL) Figure 3b Chromatogram of a participant’s urine sample collected on day of physical examination before enrolment into the study Figure 3c Chromatogram of cotinine and cotinine d3 transitions for urine fortified with deuterated internal standard only

Figure 3

Figure 3a Chromatogram of lowest urine calibrator for cotinine (2.5 ng/mL) Figure 3b Chromatogram of a participant’s urine sample collected on day of physical examination before enrolment into the study Figure 3c Chromatogram of cotinine and cotinine d3 transitions for urine fortified with deuterated internal standard only