MSACL 2025 Abstract

Investigation of Noroxycodone Interference in LC-MS/MS Urine Drug Testing

Jacob Rininger (1), Wenbo Li (1), Jaime H Noguez (1,2)
(1) University Hospitals Cleveland Medical Center, Cleveland, OH 44106 (2) Case Western Reserve University School of Medicine, Cleveland, OH 44106

Jacob Rininger, B.S. Chemistry (Presenter) John Carroll University

Relevant Financial Disclosures (within past 24 months, reported on Aug 15, 2025) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

1. Problem
Routine LC-MS/MS urine drug testing identified multiple patient specimens as positive for noroxycodone with undetectable levels of the parent drug oxycodone and its secondary metabolite, oxymorphone. The mass spectrometry middleware program flagged the specimens for review based on a rule requiring at least two of the three compounds be detectable. Upon chart review, the positive noroxycodone results were inconsistent with the patients' controlled substance prescriptions and clinical histories. Further analysis by an external laboratory using an alternative analytical method yielded negative noroxycodone results for all specimens. This discrepancy prompted an investigation into a potential interference affecting noroxycodone quantification.

2. Method Information
• Urine specimens (100 µL) were hydrolyzed with 200 µL of Kura BGTurbo Enzyme solution (including the internal standard), followed by termination of the reaction with 300 µL of cold methanol. After centrifugation, 200 µL of the supernatant was combined with 300 µL of mobile phase, preparing the sample for injection.
• Waters Acquity UPLC-TQD Mass Spectrometer
• Mobile Phase A: 0.1% formic acid in water
• Mobile Phase B: 0.1% formic acid in acetonitrile (ACN)
• 4.5 minutes gradient LC program, with a flow rate of 0.6 mL/min
• LC gradient starts at 98:2 Mobile Phase A: Mobile Phase B
• Column: Waters Acquity UPLC BEH C18 1.7 µm, 2.1 x 100 mm
• Column oven temperature: 40°C
• Injection volume: 7.5 µL
• Quantitative MRM acquisition
• Noroxycodone transitions: Quantifier 302.1384 -> 186.9814; Qualifier 302.1384 -> 226.9733

3. Troubleshooting Steps
The investigation identified an interfering compound with the same ion mass and retention time as noroxycodone, which did not trigger ion ratio flags. To enhance specificity, a new qualifier transition (302.1384 → 284.1000) was evaluated and found to be exclusively detected in specimens truly positive for noroxycodone, effectively distinguishing authentic signals from interference.

4. Outcomes
Specimens confirmed to contain noroxycodone exhibited detectable peaks across all three monitored transitions, whereas those affected by interference lacked a peak for the third transition. Despite rigorous method validation, this rare interfering substance was only identified during clinical testing and was flagged by a rules-based algorithm in the middleware. These findings suggest that an unidentified compound contributed to a possible false-positive noroxycodone result being reported and emphasize the utility of enhanced data review using AI-based tools for improved performance of LC-MS/MS drug analysis.