= Emerging. More than 5 years before clinical availability. (19.79%)
= Expected to be clinically available in 1 to 4 years. (37.97%)
= Clinically available now. (42.25%)
MSACL 2022 : Jensen

MSACL 2022 Abstract

Self-Classified Topic Area(s): Troubleshooting

Poster Presentation
Poster #27a
Attended on Tuesday at 20:30

Nonspecific Adsorption and Loss of 11-Nor-9-carboxy-Δ9-tetrahydrocannabinol. Dude, where’s my THC?

Triniti L. Jensen1, Kiumars Shahrokh1, Gwendolyn A. McMillin1,2
1ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108, 2Department of Pathology, University of Utah, Salt Lake City, UT 84132

Triniti Jensen (Presenter)


1. Problem

Here, we present the troubleshooting and lessons learned during the redevelopment of an assay to quantitate 11-Nor-9-carboxy-Δ9-tetrahydrocannabinol (THCA) in human urine by liquid chromatography tandem mass spectroscopy (LC-MS/MS). The initial development of the test following a traditional assay development process by a single scientist and the subsequent implementation into production to be performed by multiple staff members proved unsuccessful. The redevelopment of the test was then performed in close collaboration with the production lab. The second development was successful and produced a test with improved analytical performance, robustness and additional high throughput automated solutions to meet the increasing demand of this test.

2. Final Method

• Calibrators (CAL) and controls (QC) are prepared in authentic urine using a glass syringe
• 200 µL of CAL, QC, and patient samples are transferred to a Phenomenex glass-lined 96 well plate
• 400 µL of 5% KOH is added to samples for hydrolysis at 55°C for 30 min
• 250 µL 17% glacial acidic acid in methanol is added to quench
• A SPEware automated liquid dispenser (ALD) is used to automate sample clean-up coupled with a Phenomenex Strata-X-Drug B solid phase extraction (SPE) plate
• Final eluates are collected in a glass lined 96 well plate and diluted to LC starting conditions in 50:50 H2O:ACN
• Separation is performed with a Phenomenex Kinetex®2.6 µm biphenyl 100 Å, 30 x 2.1 mm column using 0.1% acetic acid in H2O and ACN with a total run time of 3.5 minutes on an Agilent LC system with a CTC-PAL autosampler.
• MS analysis is conducted on an AB SCIEX 5500 in negative mode

3. Troubleshooting Steps

• Recovery of THCA in synthetic and authentic urine:
o Average synthetic urine recovered 61% vs. 85% in authentic urine

Strategy 1: prepare CAL and QC in authentic urine
• Stability and solubility of THCA prepared in synthetic urine and stored at -80°C:
o After 1 and 5 months there was a loss of 33% and 55% of THCA, respectively

Strategy 2: CAL and QC cannot be stored in synthetic urine

Strategy 3: CAL and QC should be made fresh daily
• Solubility of THCA in extracted samples:
o Samples in 50:50 H2O:MeOH took more than 24 hours to equilibrate when stored at 4°C (constant increase in THCA area counts over time)
o Samples in 50:50 H2O:ACN were stable for 3 days stored at 4°C

Strategy 4: extracted samples need to be in 50:50 H2O:ACN
• Adsorption effects of plastic vs glass lined collection plates:
o Area counts of THCA-d3 were on average 50% lower when prepared in plastic vs glass plates
o Average accuracy of proficiency/spiked samples increased from 66% to 94%

Strategy 5: glass lined plates should be used during hydrolysis and collection steps
• Accuracy of measuring free THCA vs THCA-glucuronide spiked into authentic urine:
o Average accuracy of THCA (N=116) vs THCA-glucuronide (N=114) spiked into authentic urine was 90% vs 94%, respectively

Strategy 6: THCA-glucuronide spikes should be used to determine accuracy and precision
• Implementation of a less traditional “DevOps” approach during development with close collaboration between R&D and production staff coupled with the implementation of automation increased the robustness of the method:
o A combination of manual vs automated extractions was compared
o Six different bench staff preformed the assay to show it was reproducible

Strategy 7: When redevelopment is performed in collaboration with the production lab staff using lab resources assay robustness increases

4. Outcome

After the implementation of the above 7 strategies, a reproducible, robust, precise, and accurate assay was developed and successfully implemented into the production lab involving multiple staff members and shifts. Four quality control (QC) samples were monitored pre and post-test implementation. Imprecision of QC pre implementation was ±15% (N=141) versus ±5% (N=141) after implementation of the final method.

Financial Disclosure

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