MSACL 2023 Abstract

∆8-THC by GC-MS: Unresolved Ion Ratio Failure Investigation Mark Girton; Jim Nicholson; Lindsay Bazydlo Department of Pathology, University of Virginia, Charlottesville, VA Mark Girton, M.D. (Presenter) University of Virginia >> POSTER (PDF)

Presenter Bio: Mark Girton is currently completing clinical chemistry and laboratory medicine fellowship at the University of Virginia. Before his current fellowship, he completed an AP/CP residency and hematopathology fellowship, also at University of Virginia. His clinical and research interests lie at the intersection of the clinical laboratory and hematologic disease diagnosis.

ABSTRACT SUMMARY:
In October 2022, our laboratory began GC-MS validation experiments to add the ∆8-THC metabolite 11-nor-9-carboxy-∆8-THC (∆8-THCCOOH) on to our existing ∆9-THC urine confirmation assay (metabolite 11-nor-9-carboxy-∆9-THC (∆9-THCCOOH)). Frequent ion ratio failures for ∆8-THCCOOH have become an obstacle. This was initially suspected to be due to a shared transition peak overlap at 488 in the context of high quantities of ∆9-THCCOOH. We were also seeing this issue more frequently on the Agilent 5975C than on the Agilent 5977A, but the latter instrument has been down for maintenance during the month of January 2023.

PROBLEM:
A clinical validation for urine ∆8-THCCOOH by GC-MS is stalled as there are frequent ion ratio failures. While we initially suspected interference from high ∆9-THCCOOH given the shared qualifier transition, further investigation demonstrated a more widespread issue, including both qualifier ions, that has no known systemic cause at this time.

METHOD INFORMATION:
Base hydrolysis:
• 1.0 mL patient urine 50 µL internal standard undergo alkaline hydrolysis with 200 µL 10 M KOH
• Vortex; incubate at 60°C for 20 minutes; cool 5-10 minutes at ambient temperature
• Add 1.5 mL glacial acetic acid and vortex
Extraction:
• Condition 10 mL UCT THC Clean Screen cartridges on positive pressure manifold
• Pour into column reservoir, slowly increase flow (1-2 mL/min)
• Add 3 mL DI H2O; 2 mL THC Wash Solution (100 mM HCl with 5% acetonitrile); air dry 5 min; 200 uL hexane
• Elute with 2 mL 1:1 hexane and ethyl acetate
• Evaporate under nitrogen gas (LABCONCO RapidVap Vertex Evaporator) at 12 psi and dry bath at 60°C for approximately 30 min
Derivatization:
• Add 100 μL BTSFA with 1% TMCS to the dry residue; cap
• Heat at 70 °C for 30 minutes; cool 5-10 min; transfer to vials with micro inserts, crimp seal
GC-MS Analysis:
• Confirmation analysis is using the Agilent MSD system with THC_ACQ.m program. The selected ion monitoring (SIM) mode analyzes for the following ions (m/z) for ∆9-THCCOOH: 371, 473, 488. For deuterated ∆9-THCCOOH: 374, 476, 491. For ∆8-THCCOOH: 303, 432, 488.
• 1 uL aliquots of derivatized samples are injected onto the column by the autosampler
• Agilent Technologies 15m HP-5MS, 0.25mm i.d., 0.25 μm film thickness fused silica capillary column
• GC oven temp. program: 175°C isothermal for 1 minute, then increase at 25°C /minute to 280°C, held at 280° for 2.5 minutes on the MSD 5975 and 175°C increasing at 25°C/min to 280°C, held at 280°C for 2.5 minutes on the MSD 5977
• Helium is used as the carrier gas
• Mass Spec Conditions: Positive Ion Electron Impact (EI).
• Transfer line temp. = 280°C on MSD 5975 and 310°C on MSD 5977.
• MS Source temp. = 230°C for MSD 5975 and 300°C for MSD 5977
• Tune file= Atune.u on MSD 5975 and etune.u on MSD 5977.
• Electron multiplier settings vary with instrument’s condition and design.

TROUBLESHOOTING STEPS:
Data analysis (n=304) is ongoing to identify result and parameter characteristics leading to ion ratio failure. Ion ratios are considered acceptable if within +/-15% of an appropriate reference material ion ratio. Overall, there were 117 specimens positive for ∆8-THCCOOH. 58 (49.6%) had acceptable ion ratios, 30 (25.6%) had failure at the 432 transition only, 14 (12.0%) had failure at the 488 transition only, and 15 (12.8%) failed at both transitions. For 20 specimens negative for ∆9-THCCOOH and positive for ∆8-THCCOOH, 14 (70%) have acceptable ∆8-THCCOOH ion ratios for both qualifiers. There are 70 cases positive for both analytes with 25 (37.1%) having ∆8-THCCOOH ion ratio failures. In some cases, high ∆8-THCCOOH results in ion ratio failure, and this is appreciated on chromatography review as broadened peaks with slightly delayed acquisition time. The majority of ion ratio failures do not demonstrate a clear pattern. Ion ratio failures are seen throughout the analytical measurement range for both ∆9-THCCOOH and ∆8-THCCOOH. Occasional cases have signal from unidentified substances which may cause interference, but this does not appear to account for the large majority of ion ratio failures.

OUTCOME:
The Agilent 5977A GC-MS is now operational as of February 1, 2023. This will allow us to perform instrument comparisons with the Agilent 5975C to attempt to resolve issues with unexplained ∆8-THCCOOH ion ratio failures. We will also continue to perform data analysis and chromatography review. Unfortunately, with an approximate 50% ion ratio failure rate, our validation study has been extended as troubleshooting continues.