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Abstract Abstract Summary:
Ghost peaks appeared in our plasma catecholamine assay during all injections containing more than 10% organic solvent. We hypothesized that the LC system was contaminated, leading us to replace the needle, seals, and various tubing components. We also hypothesized the mobile phases or column were at fault, but replacing them did not eliminate the peaks. Since increasing the injection volume did not change the peak intensity, we hypothesized the issue was related to the solvent strength. We have temporarily resolved the issue by resuspending samples in 90% water, which suppresses the contaminants while maintaining acceptable chromatographic quality. Investigation into the source of the contamination is ongoing.
(1) Problem
Ghost peaks have appeared in several transitions of our catecholamines assay, most notably in the quantifier ion of epinephrine (166.1>107.1), when injecting >10% organic solvent.
(2) Method Information
• 500μL Plasma (+ sodium heparin) extracted with 30mg sorbent SPE plate on ice
• SCIEX QTRAP 6500+
• Shimadzu ExionLC™ AD
• MP-A: 0.1% FA in water
• MP-B: 0.1% FA in methanol
• 3 minute LC gradient, 0.900 mL/min flow rate
• Column: 50 x 4.6 mm, 3μm Luna Omega Polar C18
• Column oven: 50°C
• Injection volume 10 μL, Resuspension solvent: 75/25* water/ACN
• MRM acquisition, 3 analytes + 3 deuterated internal standards, 11 transitions
(3) Troubleshooting Steps
Injecting a 100% organic wash produced ghost peaks across nearly all transitions at approximately 0.8 minutes, aligning with the elution time of epinephrine. Most peaks disappeared with higher aqueous content, with only the >107.1 transition of epinephrine remaining with injections of 25% ACN. No ghost peaks were observed with a 100% water injection.
Contamination of the LC system was suspected. Several components were cleaned or replaced, including the needle and needle port, needle seals, rotor seals, sample loop, and peek tubing before/after the column. A column of similar chemistry was also tested; however, the contaminant peaks persisted. Further testing with fresh solvent bottles and mobile phases, and increased injection volumes (5, 10, 20 μL) showed no changes in peak intensity.
(4) Outcome
Samples are resuspended in 90/10 water/ACN to avoid eluting the contaminants, while maintaining acceptable peak shape and retention for all analytes. The source of the ghost peaks is still under investigation.
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= Discovery stage. (57.21%, 2026)
= Translation stage. (23.38%, 2026)
= Clinically available. (19.40%, 2026)
