MSACL 2026 Abstract

Characterization of Sertraline Metabolites Responsible for Benzodiazepine Immunoassay Cross‑Reactivity Using LC‑HRMS and Targeted Spiking Studies

Olumuyiwa Adesoye (1), Hannah Lusk (2), Kara Lynch (2), Xander Van Wijk (2), Joyce Liao (1)
(1) Department of Laboratory Medicine and Pathology, University of Washington, Seattle. (2) Department of Laboratory Medicine, University of California San Francisco, California

Olumuyiwa Adesoye, PhD, PMP (Presenter) University of Washington

Presenter Bio: Dr. Adesoye is a clinical chemist and organic chemist whose expertise bridges advanced medicinal chemistry with clinical laboratory science. With over nine years of experience in drug synthesis and public health, he possesses a deep technical command of mass spectrometry and nuclear magnetic resonance spectroscopy. Following his Ph.D. at The Ohio State University, Dr. Adesoye held several academic appointments before transitioning to a military career, where he served as the director for congressionally funded initiatives and as a public health sustainment expert. He is currently completing clinical chemistry training at the University of Washington and is a certified Project Management Professional (PMP).

Relevant Financial Disclosures (within past 24 months, reported on Apr 21, 2026) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION:
Sertraline is a widely prescribed selective serotonin reuptake inhibitor (SSRI). Several reports have shown that sertraline exposure may produce presumptive false positive benzodiazepine results in urine immunoassays, but the specific metabolite(s) responsible for this cross reactivity remain unclear. Identifying the causative metabolite is important for improving interpretation of benzodiazepine screening results and guiding confirmatory testing strategies in clinical toxicology laboratories.

OBJECTIVES:
To identify sertraline metabolites present in urine specimens associated with false positive benzodiazepine immunoassay results and determine which metabolites can generate immunoassay responses exceeding the assay detection threshold.

METHODS:
Using a two-step forward and reverse approach, urine specimens producing presumptive benzodiazepine immunoassay positives in patients prescribed sertraline were first analyzed using high resolution quadrupole time of flight liquid chromatography mass spectrometry (LC HRMS) to identify sertraline and related metabolites.

To evaluate immunoassay cross reactivity, commercially available standards of sertraline and six metabolites were spiked into drug free urine at 0.1, 1.0, 10, 25, 50, and 100 µg/mL and analyzed using a benzodiazepine immunoassay on the Beckman Coulter AU680. The assay generates unitless instrument response values proportional to enzymatic activity relative to the assay calibrator; a response ≥100 represents the cutoff for presumptive benzodiazepine positivity.

RESULTS:
LC-HRMS analysis confirmed the presence of multiple sertraline metabolites in urine specimens associated with presumptive benzodiazepine immunoassay positives, identified by exact mass measurements, including sertraline (305.0738 Da), norsertraline (291.0582 Da), sertraline ketone (290.0265 Da), α hydroxy sertraline ketone (306.0214 Da), α hydroxy sertraline ketone glucuronide (462.1081 Da), sertraline carbamoyl glucuronide (525.0957 Da), N hydroxy sertraline (321.0687 Da), N hydroxy sertraline glucuronide (497.1008 Da), and sertraline oxime (305.0374 Da).

Subsequent spiking experiments demonstrated concentration dependent immunoassay responses. Sertraline ketone produced the strongest cross reactivity, exceeding the assay cutoff beginning at 25 µg/mL (responses 107 and 96) and increasing further at higher concentrations (up to 131 at 100 µg/mL). Sertraline carbamoyl glucuronide exceeded the cutoff at ≥50 µg/mL (responses 108–122). N hydroxy sertraline and α hydroxy sertraline ketone exceeded the cutoff only at 100 µg/mL (responses approximately 110–112 and 101, respectively). Parent sertraline reached the cutoff only at the highest concentration tested (103 at 100 µg/mL), while norsertraline and sertraline oxime did not exceed the cutoff at any concentration evaluated (maximum responses 77 and 80, respectively).

CONCLUSION:
LC-HRMS identified several sertraline metabolites present in urine specimens associated with benzodiazepine immunoassay false positive results. Reverse confirmation using spiked standards demonstrated that multiple metabolites can produce immunoassay cross reactivity, with sertraline ketone showing the strongest effect and exceeding the benzodiazepine immunoassay cutoff at relatively lower concentrations. These findings suggest that sertraline metabolite profiles, rather than parent drug alone, may contribute to presumptive benzodiazepine positivity. Characterizing these cross reactivity patterns may improve interpretation of benzodiazepine immunoassay results and support appropriate use of confirmatory mass spectrometry in clinical toxicology testing.