MSACL 2026 Abstract

Reverse Charging, the Symptom You’ve Never Heard Of

Katharina Iacob (1), Julie A Ray (1), Tatiana Yuzyuk (1,2)
(1) ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT (2) University of Utah, Salt Lake City, Utah

Kat Iacob, Dr (Presenter) ARUP Institute for Clinical and Experimental Pathology

Presenter Bio: Kat is a scientist II in R&D at ARUP. Before joining ARUP, she was a Botany professor and plant-insect ecologist. What she loves about her job: nerding out over the perfect separation of analytes. Making a difference.

Relevant Financial Disclosures (within past 24 months, reported on Apr 22, 2026) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

PROBLEM:
Bile acids are a group of amphiphilic endogenous metabolites that are involved in a wide range of physiological activities. They are commonly analyzed in panels, and the number and chemistry of these metabolites can be challenging. In development of an updated LC-MS/MS method for our bile acid panel, we faced a range of chromatography and mass-spec-related complications related to reproducibility, calibration curve failures, linearity, peak deterioration, sensitivity, retention time stability, and unexplained increase in peak areas. While these problems occurred intermittently, reproducible quantitation was challenging to achieve.

METHOD INFORMATION:
• 50 µL serum, with methanolic protein crash as sample clean-up
• LC-MS/MS in negative MRM mode
• MP A: 5mM ammonium acetate in water
• MP B: MeOH-ACN 1:1
• 10 min gradient, starting at 40% MP B
• Column: 100 x 2.1mm, C18
• Column oven: 50 °C
• Injection volume: 10 µL

TROUBLESHOOTING STEPS:
• Daily cleaning of curtain plate.
• Extensive scrutiny of analytical method with gradient changes, changes in additives, buffering components and concentrations.
• Remaking calibrators several times and in different solvents. Extraction of the same samples performed by various hands.
• Evaluation of reagent quality.
• Repeat injections of the same samples, both from the same and from repeated extractions.
• Adjusting injection volumes to yield reproducible LLOQ area counts and peak quality. Critical LC components that could affect chromatography were exchanged.
• Various needle wash solutions and needle wash regimens were tested. Cleaning of the source. Extensive technical discussions and powwows with engineers, vendor staff, and scientists.
• Ultimately, instrument testing revealed measurable ‘reverse charging’- within allowable limits.
• Cleaning the front-end of the source proved to be the definitive remedy, even though it had to be repeated every few weeks to maintain linearity.

OUTCOME:
Eventually a technician discovered an oily residue on the source which turned out to be the reason for the observed problems. This oily residue came from a failed air compressor which operated using oil and only got serviced once a year. Therefore, the failure was not uncovered until after the contamination had wreaked havoc on the instrument. Decontamination took several weeks and required the replacement of an increasing number of parts until, finally, we had to admit that the MS was catastrophically contaminated.

LESSONS LEARNED: Make sure that all parts of the system supporting the mass spec are adequate. In negative mode, reverse charging/increasing peak sizes for calibrators is not a happy coincidence that improves instrument sensitivity. It is a critical diagnostic indicator of system contamination that needs to be addressed immediately.