An article published by Ganesh S. Moorthy et al at Children's Hospital of Philadelphia (CHOP) in the November 2019 issue of Journal of Chromatography B reported on a thorough validation of three common carotenoids found in medical cannabis. The research study was conducted using Mitra® microsampling devices based on VAMS® technology. The paper is entitled “A patient-centric liquid chromatography-tandem mass spectrometry microsampling assay for analysis of cannabinoids in human whole blood: Application to pediatric pharmacokinetic study.” It describes the development and validation of delta-9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN) extracted from dried blood samples on Mitra devices with VAMS® tips. Focusing on blood to plasma partitioning, the team was able to conclude that their findings were consistent with VAMS to plasma ratios for THC, CBD, and CBN. Their findings allowed them to demonstrate this as a proof-of-principal study for future patient-centric sampling projects involving cannabis.
Why Measure Cannabis?
The psychoactive effects of taking cannabis have long been known, but in recent years, cannabis has also been shown to have a wide number of pharmacological benefits in many diseases. For this reason, the medical community is applying cannabis to help manage certain conditions. For example, the National Health Service (NHS) in the United Kingdom can now prescribe medical cannabis for people with rare forms of epilepsy; for those experiencing nausea from chemotherapy medications; and those with muscle stiffness and spasms from Multiple Sclerosis (MS). However, it must be noted that cannabis is only considered when other treatments are not suitable or have not helped. Recently, medical cannabis has been shown to help with symptoms associated with autism spectrum disorders (ASD). A 2019 review published in the Nature Journal “Scientific Reports” stated that in the last three decades, there has been a threefold increase in ASD diagnoses. According to this review, “Cannabis in ASD patients appears to be a well-tolerated, safe and effective option to relieve symptoms associated with ASD.”
Object of the Pediatric Study
In 2017, a paper published by Protti et al, demonstrated the successful utilization of dried blood VAMS extracts for the analysis of natural and synthetic cannabinoids including THC. These findings led the research group at CHOP to look at the simultaneous quantitation of THC, CBD, and CBN from VAMS extracts for pediatric clinical trials involving both in-clinic and at-home sampling.
Sample Extraction Study Methods & Findings
An evaluation of optimal extraction conditions (from 20 µL Mitra devices) was evaluated and a final extraction method involving the reagents zinc sulphate, ammonium acetate, formic acid and acetonitrile involving both sonication and vortexing steps was used for validation. To reduce any matrix effects, a further purification of the VAMS extracts was conducted using RP SPE prior to LC-MS/MS analysis.
As discussed in a previous blog, analytes can sometimes undergo a temporal bias effect in terms of % recovery. Sometimes these can be solved by extraction chemistry. In the case of this study, it was observed that % recovery plateaued to around 70% after 60 hours drying and so drying time for the assay was set to 60 hours to allow for this natural plateauing.
The method was fully validated, including the following parameters: accuracy, precision, linearity dilution integrity, stability, carry-over, recovery, and matrix effect using stable labeled internal standards using human blood.
In terms of stability, the cannabinoids were stable on Mitra devices with VAMS tips for 2 weeks at RT and -4° C; 1 month at -20° C and 2 months at -78° C. Furthermore, post extracted samples were stable for at least 50 days at -78° C, and were also stable at 40 °C for 1 week.
Even though the % recoveries were at around 70% after drying for 60 hours, these were not significantly affected by the tested hematocrit (HCT) range (20.5–67.2%). The group reported that “The present assay employing Mitra® devices with VAMS® tips illustrates consistent recovery, matrix effect, and accurate quantitation of THC, CBD, and CBN, over a wide range of HCT values.”
In terms of blood to plasma partitioning, the results for WB and VAMS looked similar, but were lower in plasma due to the blood to plasma partitioning ratios of 0.544 to 0.775 for WB and 0.589 to 0.827 on VAMS for each of the analytes.
Study Authors’ Discussion and Conclusions
A robust method for measuring the three cannabinoids was validated from VAMS.
A wide range of drug concentrations were employed for validation 1 -500 ng/mL for THC and 0.5-500 ng/mL for both CBD & CBN. The range was necessary as one intended application of the method was to measure involuntary overdose in pediatrics.
The study demonstrated the importance of comparing literature PK to that of VAMS data and so calculated the blood to plasma partitioning ratio using the equation “([CVAMS/(1−HCT)]× fraction plasma protein binding=CPL)”. The study authors concluded that because plasma binding of CBD and THC has been reported to be >94% and >90%, respectively, and that the 1-HCT value was 0.58, then it made sense that the VAMS extracts gave values which were lower than plasma. They concluded that these findings were consistent with VAMS to plasma ratios for THC, CBD, and CBN.
The method was implemented for a pediatric PK study to measure WB CBD levels.
The group discussed a plan to expand the scope of the study to measure other cannabinoids and metabolites, such as 11-hydroxy-THC, from VAMS extracts.
Understanding blood to plasma partitioning is a critical step in developing assays from whole blood VAMS extracts using Mitra devices. By understanding these levels, plasma equivalent values can be estimated, allowing for comparisons against plasma reference ranges. Given that many current therapeutic drug monitoring (TDM) assays measure analytes from wet plasma, such bridging studies as demonstrated by Moorthy et al are critical to allow for a transition to whole blood measurements and increase the scope for more patient sampling TDM research studies.
This study paper was summarized for our readers by James Rudge, PhD, Neoteryx Technical Director. This is curated content. To learn more about the important research outlined in this review, visit the original article published in the Journal of Pharmaceutical and Biomedical Analysis.