MSACL 2025 Abstract

Distorted Oxymorphone Peak in Urine Drug Testing Panel

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

Jaime Noguez, PhD (Presenter) University Hospitals Cleveland Medical Center/Case Western Reserve University

Relevant Financial Disclosures (within past 24 months, reported on Aug 14, 2025) Other Potential Conflicts Siemens Healthineers USA / Speaker / Ended Conflict mitigation NOT REQUIRED.

1. Problem
Distorted peak shapes were observed in the oxymorphone quantification, qualification, and internal standard transition chromatograms on two LC-MS/MS systems, compromising the accuracy of quantitation.

2. Method Information
- A 100 µL urine sample was 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 A, 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

3. Troubleshooting Steps
Oxymorphone was identified as an early-eluting compound within the panel, exhibiting consistent distortion on the left side of the peak. This observation suggested that the sample solvent might be the contributing factor.

Step 1: Further diluting the extracted sample (x2) with the mobile phase prior to injection resulted in improved peak shape.

Step 2: Reducing the injection volume also (3uL or 1uL) enhanced peak symmetry, whereas increasing the injection volume exacerbated peak distortion.

These steps were intended to minimize the concentration of organic solvent prior to injection or before the analytes reached the column, aligning conditions more closely with the initial LC gradient. The findings confirmed that the peak distortion was due to a high proportion of organic solvent in the extracted sample.

4. Outcome
Three approaches were identified to improve peak shape in this scenario:
- Replacing the tube connecting the autosampler and column with a longer, larger tube.
- Reducing the injection volume.
- Diluting the extracted sample with the mobile phase before injection or modifying the extraction procedure to lower the organic solvent content.