Anabasine analysis in human plasma using liquid chromatography coupled to tandem mass spectrometry to Verify tobacco use: empirical challenges and limitations

Anabasine is an alkaloid frequently quantified by LC–MS/MS to differentiate tobacco use from nicotine replacement therapy. While urine provides reliable measurement, plasma remains a poorly validated and analytically challenging matrix. This study assessed widely used sample preparation strategies for anabasine determination in human plasma. Across all methods, recovery in undiluted plasma was highly variable and largely outside acceptable analytical ranges, with marked ion suppression and poor reproducibility. Matrix dilution increased apparent signal but substantially worsened variability. Experiments in albumin-enriched saline excluded protein binding as the main determinant of analyte loss, indicating broader plasma-related matrix effects. In human plasma, including time-course sampling during and after smoking, anabasine remained consistently below quantifiable levels. None of the tested workflows met the robustness criteria required for quantitative LC–MS/MS analysis, indicating that current preparation approaches do not enable reliable anabasine measurement in plasma, a critical limitation for studies using anabasine as a biomarker of tobacco exposure. • Plasma anabasine recovery was inconsistent across all tested LC–MS/MS methods. • Matrix dilution raised recovery but added high variability, reducing reproducibility. • Plasma anabasine quantification proved unreliable and is not suitable for clinical use.