MSACL 2025 Abstract

Investigating Intermittent Retention Time Shifts

Darcy Dochterman (1), Anna Merrill (2)
U.S. Department of Veterans Affairs

Darcy Dochterman, Bachelor of Science: Medical Laboratory Science (Presenter) U.S. Department of Veterans Affairs

Relevant Financial Disclosures (within past 24 months, reported on Mar 14, 2025) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

1. PROBLEM
The isobaric cannabinoid metabolites delta-8-carboxy-tetrahydrocannabinol (THC) and delta-9-carboxy-THC have significant shifts to earlier retention times (RT) of 4.55 RT versus a normal RT of 4.77, and impaired chromatographic separation causing unreliable quantification of the assay target, delta-9-carboxy-THC. This is an intermittent problem affecting both patient and QC samples that was not observed during method development or validation.
2. METHOD INFORMATION
• 200ul urine treated with glucuronidase and extracted with solid phase extraction (SPE) using HLB uElution plate
• Waters Acquity LC with Column Manager
o Column heat: 45⁰C
o Injection volume: 10uL
o 2.1 x 50mm C18 1.7 um column with a 2.1 x 5mm 1.7 um C18 pre-column
o A 7.5 min method with a 4.0 minute isocratic flow of 0.3mL/min 29% aqueous 0.1% formic acid/0.5% ammonium acetate mobile phase A and 71% methanol mobile phase B
• Waters Xevo TQD
o MRM acquisition

3. TROUBLESHOOTING STEPS
The intermittent shift of retention time (RT) was seen across multiple sample preparations using multiple lots of reagents and the same method. Thus, a system problem was suspected. We eliminated the possibility of incorrectly prepared mobile phases by remaking them prior to sample injection. A new column and pre-column were installed. We noticed that when shutting off the binary solvent manager (BSM) and turning it back on, the RT did not shift from the normal 4.77 RT to 4.55 RT or earlier. Then upon investigation of the pressure trace files, a drop in psi was noted during the isocratic phase of the method. Constant 0.3mL/min flow of 29% mobile phase A and 71% mobile phase B2 should hold a steady psi. We then suspected the system leak was present, contaminating the mobile phases. The column manager contains two separate columns and separate mobile phases for each. The phenyl column uses mobile phase B1, acetonitrile/0.1% formic acid. The C18 column, what is used for the cannabinoid testing, uses mobile phase B2, methanol. Reversed-phase chromatography methods that use acetonitrile as the organic mobile phase have earlier RT since acetonitrile has higher elution strength. The accumulator and vent valve were checked for leaks, the mixer was changed and finally the solvent select valve was investigated. If the solvent select valve was leaking acetonitrile into the methanol mobile phase, the chromatographic peak of interest would shift earlier causing the decrease separation of delta-8-carboxy-THC and delta-9-carboxy-THC. When the peak tubing was placed together into mobile phase B2, primed, and a sample with known delta-8-carboxy-THC and delta-9-carboxy-THC standards was injected, the RT of the delta-9-carboxy-THC was 4.77 and separation between the isobaric compounds was optimal. Separation of the peak tubing into the respected mobile phase B1 and B2, priming, and injection of the known sample, shifted the RT earlier to 4.55. This proves that the acetonitrile B1 was leaking in the solvent select valve and contaminating the methanol mobile phase B2.
4. OUTCOME
The methanol mobile phase B2 was no longer contaminated by the acetonitrile mobile phase B1 once the select valve was replaced in the binary solvent manager of the LC/MS-MS. The RT was no longer shifted and the separation of chromatography between delta-8-carboxy-THC and delta-9-carboxy-THC was optimal for quantification of delta-9-carboxy-THC.