This Is AuburnElectronic Theses and Dissertations

Development and validation of a UPLC-MS method for quantification of selected cannabinoids in canine plasma and its application to commercial cannabis products




Cruz-Espindola, Crisanta

Type of Degree

Master's Thesis


General Veterinary Medicine


Introduction. Cannabinoids are the important chemicals in cannabis plant with medicinal value [51]. However, effective and safe use is best based on studies that describe their behavior in the plasma of the species being treated. This requires a method for accurate and precise quantification of these closely chemically related compounds. Several LC-MS and GC-MS methods have been described in the literature that quantify cannabinoids in human plasma, rat urine, waste water, surface water, cannabis plant, and cannabis oil. However, the quantification of cannabinoids in canine plasma has not being described. This study describes the development and validation of a reverse phase ultra-performance liquid chromatographic (UPLC) method with mass spectrometry (MS) detection using solid phase extraction for the simultaneous determination of the major cannabinoids. (cannabidiol, tetrahydrocannabinol cannabigerol, cannabinol, and cannabichromene) in canine plasma. Methods. Based on the chemical structures, physical properties, sample type (canine plasma), and previously reported methods, an analytical method was developed and validated using solid phase extraction to clean up the sample, liquid chromatography for separation and tandem mass spectrometry (LC-MS/MS) for detection. Results. Cannabinoids were extracted from canine plasma by using Oasis HLB SPE cartridges. Cannabinoids detection, separation and quantification was accomplished using a C18 chromatographic column, a mobile phase consisting of formic acid in water and acetonitrile at a flow rate of 0.5 mL/min. LC-MS/MS with Electrospray ionization (ESI) in positive mode and multiple reaction monitoring (MRM) was used for quantification. The limit of detection (LOD) for the five major cannabinoids was 1.95 ng/mL. The lower limit of quantification (LLOQ) for cannabidiol, and tetrahydrocannabinol was 3.91 ng/mL. For cannabidiol the mean accuracy (% recovery) was 100% ± 18% with a 16% Precision. For tetrahydrocannabinol the mean accuracy (% recovery) was 105% ± 5% with a 5% Precision. Using this method, both cannabidiol and tetrahydrocannabinol were detected and quantified in the plasma of canine patients receiving commercial cannabis-based products. The analytical method for the analysis of cannabinoids in commercial products will require a future validation Conclusions. We have successfully validated a cannabinoid LC-MS/MS method for quantitation of cannabidiol and tetrahydrocannabinol in canine plasma. This assay will support clinical trials and pharmacokinetic studies necessary to demonstrate safety and efficacy of these promising agents. Identification of cannabigerol, cannabinol, and cannabichromene in canine plasma can be performed with this method, but validation is still pending.