Development of a Dried Blood Spot Assay for Detecting Prenatal Cannabis Exposure in Newborns Stephanie Vuong1, Deborah Michel1, Richard Huntsman2, Andrew W Lyon3, and Jane Alcorn1 1College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK, Canada, 2Pediatrics,3Pathology and Laboratory Medicine, College of Medicine, University of Saskatchewan, Saskatoon, SK, Canada

INTRODUCTION LC-MS/MS CONDITIONS RESULTS • Significant concerns about potential increase of Liquid Chromatography (Agilent 1290 Infinity LC System) pregnant women using Cannabis since the legalization of recreational Cannabis in October Column: Agilent Zorbax Eclipse XDB-C18 Narrow Injection volume: 5 µL/min 2018. Bore with Zorbax Eclipse XDB-C8 Narrow Flow Rate: 700 µL/min

Bore Guard Column Ratio Area • Women reportedly use Cannabis to alleviate symptoms of pregnancy, including nausea and Mobile phase A: 100 mM formic acid + 0.1 mM Time (min) Figure 5: Chromatogram showing the Lowest Limit of Quantification (LLOQ) of morning sickness. ammonium formate in LCMS CBD, with a concentration of 1.28 ng/mL and signal-to-noise ratio of 10. grade water • Cannabinoids are highly lipophilic compounds- having the ability to cross the blood-placental Mobile phase B: 100 mM formic acid + 0.1 mM

barrier, exposing the fetus to Cannabis, and ammonium formate in LCMS MobilePhase Composition (%) Area Ratio Area potentially leading to long-term neurological grade methanol Time (min) impairments. Figure 3: Gradient method of mobile phase A (blue) and B (red) during a Concentration (ng/mL) a) b) c) run time of 9.0 min. Figure 6: Standard curve for CBD ranging from 1.28-217.20 ng/mL, with a R2 Mass Spectrometry (SCIEX 6500 Hybrid Triple Quadrupole with TurboIonSpray) value of 0.9912, representing good linearity. CONCLUSION Figure 1: Chemical structures of Cannabidiol (a); ∆9-Tetrahydrocannabinol (b); Curtain gas (CUR): 50.0 Ion Spray (IS) Voltage: 4500.0 V • LC-MS/MS method displayed great efficiency 9 and 11-nor-9-Carboxy- ∆ -Tetrahydrocannabinol (c). Collision Activated Dissociation (CAD): 10.0 Temperature: 600.0°C and sensitivity for the quantification of CBD, Gas Source 1 (GS1): 70.0 Gas Source 2 (GS2): 60.0 OBJECTIVE THC, and THC-COOH in human dried blood Table 1: Mass spectrometry parameters for multiple reaction monitoring (MRM) of CBD, THC, THC-COOH and their corresponding internal standards spots, achieving a lowest limit of quantification Develop an efficient liquid chromatography- (precursor ion➝ product ion) of 1.28 ng/mL. tandem mass spectrometry assay to detect MRM Transition Dwell Time Declustering Potential Entrance Potential Collision Energy Exit Potential cannabinoids in dried blood spots from newborns. (m/z precursor ion ➝ product ion) (msec) (DP) (EP) (CE) (CXP) • Our sample extraction procedures provided CBD Quantifier 315.1 ➝ 193.1 50 61 10 25 12 great effectiveness in removal of potentially METHOD CBD Qualifier 315.1 ➝ 259.2 50 61 10 19 14 interfering endogenous components found in • Blank human blood was obtained from the CBD-d3 318.1 ➝ 196.1 50 56 10 29 12 THC Quantifier 50 91 10 31 12 human blood, contributing to better sensitivity in Royal University Hospital. 315.1 ➝ 193.1 THC Qualifier 315.1 ➝ 259.2 50 91 10 27 14 method. THC-d3 318.1 ➝ 196.1 50 96 10 31 10 FUTURE WORK • Cannabinoids were mixed with blood, applied to THC-COOH Quantifier 345.1 ➝ 299.2 50 111 10 27 16 collection cards, and dried (Human ethics- THC-COOH Qualifier 345.1 ➝ 193.2 50 111 10 35 12 • Validate the LC-MS/MS method using FDA approved). THC-COOH-d3 348.1 ➝ 302.2 50 121 10 27 16 Bioanalytical Method Validation Guidelines. RESULTS • Sample preparation techniques were used for • Analyze archived newborn dried blood spots to efficient removal of proteins, phospholipids, and CBN determine prevalence rate of prenatal Cannabis red blood cells prior to LC-MS/MS analyses exposure before and after legalization of using deuterated internal standards. recreational Cannabis. ACKNOWLEDGMENTS • This project was funded by the College of Medicine Research and Development (CoMRAD) program. Area ratio Area

CBD THC THC-COOH CBC 11-OH-THC

Time (min) Figure 2: Sample preparation procedure displaying the extraction of plasma Figure 4: Chromatogram displaying the retention times of 11-OH-THC (2.12 min), THC-COOH (2.54 min), CBD (2.63 min), CBN (4.24 min), THC (4.73 min), and CBC protein, red blood cell, and phospholipid. (5.36 min).

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