|CHAKRABARTY, SHUBHASHIS - North Dakota State University|
|WINDERS, THOMAS - North Dakota State University|
|SWANSON, KENDAL - North Dakota State University|
|DAHLEN, CARL - North Dakota State University|
|KLEINHENZ, MICHAEL - Kansas State University|
|COETZEE, JOHANN - Kansas State University|
|MAGNIN, GERALDINE - Kansas State University|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 8/10/2021
Publication Date: 10/31/2021
Citation: Chakrabarty, S., Courneya, I.T., Winders, T.M., Serum, E.M., Swanson, K.C., Dahlen, C.R., Kleinhenz, M.D., Coetzee, J.F., Magnin, G., Smith, D.J. 2021. Rapid quantification of cannabinoids in bovine plasma using electrospray ionization mass spectrometry sans LC column. 2022 Reciprocal Meat Conference. Abstract.
Technical Abstract: Introduction. Hempseed cake has potential use as a ruminant feed ingredient, but it is not known whether hemp-derived cannabinoids are absorbed in animals fed hempseed cake. Mass spectrometry (MS), in conjunction with gas or liquid chromatography is a sensitive technique for accurate quantification of contaminants in food animal products. However, sample preparation and analysis can be time consuming, labor intensive, and costly. In this work, we demonstrated the quantitative capability of electrospray ionization rapid screening (RS-ESI-MS), a modification of ESI-MS, where LC-based separation and lengthy sample preparation were eliminated. Using RS-ESI-MS, we quantified 10 cannabinoids in low ppb levels in plasma from six cattle fed hemp-seed cake over a 112-day feeding period and in six cattle fed control rations. Method. Quadruplicate aliquots (5 µL) of plasma (300 µL) ethyl acetate extracts (300 µL) were injected (Shimadzu-Nexera LC) onto a Sciex 5600+ MS consisting a dual-spray source with an isocratic acetonitrile:water (1:1; 5 mM NH4HCO2) mobile phase (200 µL/min). Cannabinol (CBN), cannabidiol (CBD), cannabinolic acid (CBNA), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabichromenic acid (CBCA), cannabidivarin (CBDV), cannabidivarinic acid (CBDVA), tetrahydrocannabinol (THC) and tetrahydrocannabinolic acid (THCA) were analyzed in negative electrospray ionization mode. The spray voltage was 4,500 V, collision energy 30 – 50 V, declustering potential 25 V, and source temperature was 400 °C. Desolvation and nebulization gases were 40 psi, and the curtain gas was 25 psi. For quantification, four internal standards, cannabinol-d3, cannabidiol-d3, THC-d3 and cannabigerol-d3 were used. Preliminary Data. The rapid screening MS method (1 min/injection) was validated by evaluating matrix effect, linearity, repeatability, sensitivity and percentage recovery of each analyte. Ion suppression was observed for all compounds in bovine plasma (7.7 to 35.6%) relative to standards dissolved in acetonitrile. Regression coefficients of matrix-matched standard curves across all compounds ranged from 0.9995 to 0.9999. Average percentage recoveries across all compounds ranged from 90.2 ± 15.5 to 108.7 ± 18.7. Limits of detection and quantification ranged from 0.06 to 1.56 ng/mL and 0.22 to 5.20 ng/mL, respectively, across analytes. The inter-day relative standard deviation (RSDs) of all analytes were in the range of 9.1 to 12.7%. Incurred plasma samples (n = 72) were blinded to analysts with respect to animal treatment. No false positives were returned by RS-ESI-MS for any cannabinoid in plasma from control animals (n = 36 plasma samples; 10 analytes). In treated animals, CBNA, CBDA/THCA, CBGA, CBCA and CBDVA were detected at concentrations of 0.1 to 9.0 ng/mL, while CBN, CBD/THC and CBDV were not detected in control or test animals. Low levels of measured cannabinoids in plasma are commensurate with the relatively low cannabinoid content of hempseed cake. To validate RS-ESI-MS results against LC-MS/MS, previously analyzed plasma samples (n = 32) from cattle dosed with ground hemp (Kansas State University) were analyzed by RS-ESI-MS. Across quantifiable cannabinoids and animal plasma means for each time point as measured by RS-ESI-MS and LC-MS/MS were well correlated (r2 = 0.963). Analysis of variance indicated that major variables affecting mean concentrations of cannabinoids in cattle plasma were sample collection time (P < 0.001), and animal (P < 0.001), but not analysis method (P > 0.05). Novel Aspect. RS-ESI-MS method sans LC provided rapid, sensitive, and accurate analyses of cannabinoids from cow plasma with little sample preparation.