Topic: Tox / TDM / Endocrine
Podium Presentation in Room 1 on Wednesday at 11:40 (Chair: Jen Colby / Chris Koch)
Authors: Raymond Suhandynata, Melissa Hoffman, Thomas Marcotte and Robert Fitzgerald
Background: The discovery of cannabinoid (CB) receptors and the growing popularity of cannabis use has sparked interest in understanding the biological function of CB receptors’ endogenous ligands, or endocannabinoids (eCBs). Most attention has focused on the physiological role of two eCBs, anandamide (AEA) and 2-arachidonoyl glycerol (2-AG). THC and CBD are capable of functioning as CB1 and CB2 agonists and, therefore, impact important physiological processes. Widespread recreational and medical use of cannabis and CBD products underscores the importance of understanding how exogenous cannabinoids effect the endocannabinoid system. To address this, liquid chromatography – multiple reaction monitoring mass spectrometry (LC-MRM) assays have been developed to quantify AEA and 2-AG in patient plasma.
Methods: Detection of the eCBs was optimized by direct infusion ESI-MS/MS (Waters, TQ-S micro) of pure standard compounds (Cayman). Both compounds performed best in positive ion mode and at least two precursor and product ion pairs (quantifier and qualifier ions) were selected to monitor each compound. Cone voltage and collision energy optimization was performed for ion transitions monitored. Separation of analytes was achieved using reversed-phase liquid chromatography on a Waters Acquity UPLC equipped with a BEH C18 column (2.1 ID x 50 mm, 1.7 um particles) and mobile phases A and B consisting of 5 mM ammonium formate in water with 0.1% formic acid and acetonitrile with 0.1% formic, respectively. A 3.5 min gradient was used with a total run time of 5 min per sample.
Results: Collection tube type was evaluated and did not grossly affect the measured concentrations of AEA. However, the measured concentrations of 2-AG varied up to 5-fold depending on collection tube type. Based on stability of 2-AG, K-EDTA was selected as the collection tube of choice. Assay linearity was determined for each analyte using a seven point calibration curve. The linear ranges for AEA and 2-AG quantification in plasma are 0.05 to 25 ng/mL and 0.25 to 25 ng/mL, respectively. High, mid, and low concentration samples (4.0 ng/mL, 1.0 and 0.6 ng/mL) were analyzed to assess accuracy and precision. The observable bias for the high, mid, and low spiked samples were measured. In addition, stability in the auto-sampler was assessed at 24, 48, and 96 hours. Results demonstrate that AEA and 2-AG are stable in the LC auto-sampler for up to 24 hours. Sample carryover was evaluated and none was observed following injection of the highest calibrator (25 ng/mL). Within run accuracy and precision was determined in stripped plasma, indicating that calibrator and QC bias was < 20% and coefficient of variations (%CV) were <10%. Matrix effect studies revealed no matrix effects for AEA but significant interpatient matrix effects for 2-AG.
Conclusion: This AEA LC-MRM assay will be applied to patient plasma in a clinical trial assessing the therapeutic efficacy and safety of CBD therapy.
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