MSACL 2024 Abstract

Implementation and Validation of an Ultra-High Performance LC-MS/MS Method for Nicotine Metabolites and Anabasine Quantification in Urine Specimens

Ahram Yi (1), Jun Hyung Lee (1), Hye Jin Lim (1), Jungsun Han (1), Sung-Eun Cho (1), Sang Gon Lee (2), Eun Hee Lee (2)
(1) Endocrine Substance Analysis Center (ESAC), Department of Laboratory Medicine, GC Labs, Yongin, Republic of Korea (2) Department of Laboratory Medicine, GC Labs, Yongin, Republic of Korea

Ahram Yi, M.D., M.Sc. (Presenter) GC Labs

Relevant Financial Disclosures (within past 24 months, reported on Jan 28, 2024) Salary GC Labs Conflict mitigation NOT REQUIRED.

Background: Reliable quantification of urine nicotine metabolites is essential for identifying smoke exposure. Further, detection of minor tobacco alkaloids, such as anabasine, may aid in determining smoking status or compliance with nicotine-replacement therapy. We implemented and validated an ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method for quantifying 3-OH cotinine, nornicotine, cotinine, anabasine, and nicotine in urine specimens.

Methods: The UHPLC–MS/MS method used a Triple Quad 5500+ QTRAP Ready (SCIEX, USA) with 1 μg/mL 3-OH cotinine-d3 (3-OH cotinine-IS), 1 μg/mL nornicotine-d4 (norcotinine-IS), 1 μg/mL cotinine-d3 (cotinine-IS), 1 μg/mL nicotine-d4 (nicotine-IS), and 20 μg/mL anabasine-d4 (anabasine-IS) as internal standards. The method consists of precipitation extraction with methanol and dilution with distilled water followed by the analysis of the prepared sample by UHPLC-MS/MS in multiple reaction monitoring (MRM) using electrospray ionization positive (ESI+) mode. The mass spectrometer was operated through transitions from the precursor to the product ions (m/z 193.1 to 134.0 for 3-OH cotinine, m/z 196.2 to 134.0 for 3-OH cotinine-IS, m/z 149.1 to 130.1 for norcotinine, m/z 153.2 to 134.0 for norcotinine-IS, m/z 177.1 to 98.0 for cotinine, m/z 180.2 to 101.1 for cotinine-IS, m/z 163.1 to 94.0 for anabasine, m/z 167.2 to 150.2 for anabasine-IS, m/z 163.1 to 84.1 for nicotine, and m/z 167.2 to 134.1 for nicotine-IS). The total run time was 6 min. We conducted a gradient of mobile phase A of 30 mM ammonium bicarbonate and mobile phase B of 100% MeOH at a flow rate of 0.40 mL/min on a Kinetex EVO C18 column (2.1 mm × 150 mm, 5 μm; Phenomenex, USA) in an ExionLC™ system (Sciex, USA). Linearity, recovery, precision, carry-over, and matrix effect were evaluated to validate the method.

Results: Linear dynamic ranges were 4.98–23406.90 ng/mL for 3-OH cotinine, 1.02–9381.70 ng/mL for nornicotine, 1.04–8592.06 ng/mL for cotinine, 1.00–9106.58 ng/mL for anabasine, and 0.99–9252.99 ng/mL for nicotine (R² ≥ 0.9988). The lower limits of quantification were 4.98 ng/mL for 3-OH cotinine, 1.02 ng/mL for nornicotine, 1.04 ng/mL for cotinine, 1.00 ng/mL for anabasine, and 0.99 ng/mL for nicotine. The recovery of 3-OH cotinine, nornicotine, cotinine, anabasine, and nicotine UHPLC–MS/MS measurements were within ±9% of the targeted values. The intra- and inter-day coefficients of variation were all acceptable (≤5% for all tests). Carry-over was not found for all compounds. Ion suppression or enhancement was not observed in the blank and six patient samples for all compounds.

Conclusions: The UHPLC–MS/MS nicotine metabolite and anabasine assay showed an adequate recovery, precision, sensitivity, and AMR, making it suitable for measuring urine nicotine metabolite and anabasine concentrations.