MSACL 2025 Abstract

LC-MS/MS Separation of 11-Nor-9-Carboxy-Δ9-THC and 11-Nor-9-Carboxy-Δ8-THC in Urine Using the SCIEX Citrine System

Mahesheema Ali (1,2), Michael Breckenridge (1)
(1) The MetroHealth System, Cleveland, Ohio, (2) Case Western Reserve University School of Medicine, Cleveland, Ohio.

Mahesheema Ali, PhD, NRCC (Presenter) The MetroHealth Medical Center

Presenter Bio: Dr. Mahesheema Ali is the Medical Director of the Core laboratory, Toxicology& POCT at MetroHealth Systems and an Associate Professor of Laboratory Medicine at Case Western Reserve University School of Medicine. She is board-certified by the American Board of Clinical Chemistry and is a fellow of the ADLM Academy. Dr. Ali's academic interests focus on implementing innovative laboratory tests for in vitro diagnostic testing in areas such as automated chemistry, toxicology, mass spectrometry, and point-of-care testing (POCT). She is also committed to promoting excellence and enhancing the quality of laboratory testing.

Relevant Financial Disclosures (within past 24 months, reported on Mar 25, 2025) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION
11-Nor-9-carboxy-Δ8-tetrahydrocannabinol (Carboxy-Δ8-THC) is a metabolite of Delta-8-tetrahydrocannabinol (Δ8-THC), a cannabinoid found in cannabis, while 11-Nor-9-carboxy-Δ9-tetrahydrocannabinol (Carboxy-Δ9-THC) is the primary metabolite of Delta-9-tetrahydrocannabinol (Δ9-THC). Due to their structural similarities with other cannabinoids and metabolites, their separation and analysis present significant analytical challenges. Accurate quantification and differentiation of Carboxy-Δ8-THC and Carboxy-Δ9-THC are critical for regulatory compliance, forensic toxicology, and clinical research.

OBJECTIVE
To develop and optimize an LC-MS/MS method for the separation, quantification, and reporting of Carboxy-Δ8-THC and Carboxy-Δ9-THC in urine.

LC-MS/MS METHODOLOGY and MATERIALS
Standards:
(-)-11-nor-9-Carboxy-Δ8-THC (Item No. T-010)
(±)-11-nor-9-Carboxy-Δ9-THC (Racemic mixture; not for immunoassay, Item No. T-170)
Deuterated internal standards from Cerilliant
Preparation of Internal Standard
Add 100 µL of Δ8-THC-D3 and 100 µL of Δ9-THC-D3 to a test tube.
Add 12.3 mL of methanol.
Mix thoroughly and vortex.
Preparation of Top Stock Solution
Transfer 15 µL of Δ8-THC and 15 µL of Δ9-THC into a 10 mL volumetric flask.
Adjust the volume to 10 mL using methanol.
Mix thoroughly.
Follow the Standard Operating Procedure (SOP) for further steps.
Instrumentation and Conditions
System: SCIEX® Citrine/6500 Triple Quadrupole
Column: Phenyl-hexyl (Kinetex®)
Mobile Phase:
(A) 0.1% formic acid in water
(B) 0.1% formic acid in methanol
Gradient: 2–75% over 9 minutes
Mass Spectrometry (MS/MS) Conditions:
Ionization Mode: Negative
Optimized Parameters:
Ion source: Turbo Spray Ion Drive
Polarity: Negative
Curtain gas (CUR): 30
Collision gas (CAD): 8
Ion spray voltage (IS): -4500V
Temperature: 650°C
Ion source gas 1 (GS1): 60
Ion source gas 2 (GS2): 50
Declustering potential (DP): -110V
Entrance potential (EP): -10V
Collision energy (CE): -28V
Collision cell exit potential (CXP): -11V
Liquid Chromatography (LC) Conditions
Column Oven Temperature: 50°C
Injection Volume: 2 µL
Flow Rate: 0.8 mL/min
Detection: MS/MS (SCIEX® 6500 Triple Quad)

RESULTS
The method demonstrated robust analytical performance, ensuring optimized separation, increased throughput, and reproducible quantification across relevant analyte concentration ranges. It exhibited excellent repeatability, with calibration curves showing good linearity. The complete resolution of Carboxy-Δ8-THC and Carboxy-Δ9-THC stereoisomers was achieved, allowing for both qualitative and quantitative analysis. The respective retention times for Carboxy-Δ8-THC and Carboxy-Δ9-THC were determined to be 7.45 and 7.70 minutes.

CONCLUSION
The developed LC-MS/MS method effectively differentiates Carboxy-Δ8-THC and Carboxy-Δ9-THC metabolites. The separation of these compounds was accomplished within approximately 9 minutes using a phenyl-hexyl column and 0.1% formic acid in methanol as the mobile phase under optimized conditions on the SCIEX® Triple Quad system. This method provides a reliable approach for regulatory, forensic, and clinical applications.