Identification of Potential Distinguishing Markers for the Use of Cannabis-Based Medicines or Street Cannabis in Serum Samples

2.2. Quantitative Study Sample Analysis

Within the study collective, THC was detected in 48 of 56 samples, ranging from approximately 0.048 (value below limit of quantification (LOQ)) to 49 ng/mL. THC-OH and THC-COOH concentrations ranged from approx. 0.20 to 26 ng/mL and approx. 1.0 to 29 ng/mL. Among minor cannabinoids, THCAA, CBN, CBC and THCVA were detected in most samples with maximum values ranging from 0.97 (CBN) to 9.3 ng/mL (THCAA). Other analytes, such as CBG or THCV, rarely occurred. CBDVA was detected in one sample only, and CBL and CBDV were not detected in any samples. Serum cannabinoid concentrations itemized by substance intake along with time intervals between last substance intake and blood sample collection are presented in Table 1.

2.3. Comparison to Forensic Serum Samples and Identification of Potential Distinguishing Markers

In order to identify potential distinguishing markers for the intake of cannabis-based medicines or use of street cannabis, all study subgroups, except for the CBD capsule group with n = 1, were compared to 55 forensic serum samples of different street cannabis users, that had been analyzed in a previous study [16]. Serum cannabinoid concentrations of forensic case samples (in the following referred to as street cannabis subgroup) are presented in Supplementary Table S2. For better comparability of different doses of cannabis or cannabis-based medicine applied as well as different time intervals between application, cannabinoid concentrations were standardized on THC by dividing them by the respective THC concentration of the sample. Resultant cannabinoid ratios of all samples are listed in Supplementary Table S3. For eight samples, no ratios could be calculated, as THC serum concentrations lay below limit of detection (LOD). Those samples were excluded from further analysis. If other cannabinoid concentrations lay below LOD, 0 (zero) was substituted as the respective ratio. CBDV was not detected in any samples and was therefore eliminated from further analyses. Boxplots of selected cannabinoid ratios are presented in Figure 1.

2.4. Application to Forensic Case Samples

Results of the presented study were used to analyze nine forensic case serum samples with self-reported intake of Sativex^® (n = 4, named Sat01 to Sat04), Dronabinol (n = 4, Dro01 to Dro04) and “THC medicine” (n = 1, THCM01). Cannabinoid concentrations of the samples were quantified as described above and are presented in Table 3. Cannabinoid concentrations standardized on THC are given in Supplementary Table S4. Standardized cannabinoid concentrations were analyzed in a PCA along with study and forensic samples (except for F03 and F21) described in 3.2. With a value of 0.606, the Kaiser–Meyer–Olkin measure of sampling adequacy was sufficient for this dataset, and Bartlett’s test of Sphericity was significant (p < 0.001) [18,19]. The resultant PCA scatter plot of all samples plotted on PC 1 and PC 2 is presented in Figure 3. Corresponding loading plots of PC 1 and PC 2 are displayed in Supplementary Figure S2.

Samples with reported Sativex^® intake exhibited different cannabinoid patterns than Sativex^® users from the study: Especially THC-OH/THC and CBD/THC ratios were much lower, ranging from 0.37 to 0.71 and 0.042 to 0.23, compared to ranges of 1.1 to 10 and 0.61 to 1.5 detected in Sativex^® user study samples. Minor cannabinoids, such as CBG, CBGA or THCV, on the other hand, occurred in higher quantities. As shown in Figure 3, cannabinoid patterns of forensic Sativex^® user samples were much more similar to medical or street cannabis users, suggesting use of cannabis or other cannabis-based products with notable quantities of minor cannabinoids. THC-OH/THC ratios <1 indicate inhalatory instead of oral application of THC. However, relatively high CBD and CBDA serum concentrations, compared to most street cannabis users, could be compatible with additional use of Sativex^®.

Forensic samples with self-reported Dronabinol intake mostly exhibited different cannabinoid patterns as well: THC-OH/THC ratios ranged from 0.23 to 0.42 in samples Dro01 to Dro03, compared to 1.1 to 10 in Dronabinol user study samples, indicating THC had been inhaled instead of oral application. Larger quantities of other minor cannabinoids, such as CBG or CBC, suggest the use of cannabis or other cannabis-based products with notable quantities of minor cannabinoids. Accordingly, samples Dro01, Dro02 and Dro03 were plotted among cannabis user samples in Figure 3. Sample Dro04, however, exhibited a THC-OH/THC ratio of almost 1.6 and only low concentrations of other cannabinoids. It was plotted among Dronabinol user study samples, indicating similar serum cannabinoid patterns that could indeed result from Dronabinol intake.

Sample THCM01 after self-reported intake of “THC medicine” featured a THC-OH/THC ratio of 0.24 and low concentrations of minor cannabinoids. It was plotted among cannabis user samples in Figure 3, suggesting inhalative use of cannabis or cannabis-based products.

3.2. Study Design

In the multi-centered pilot study, a blood sample was collected from the participants, every time they had a medical appointment at one of the study centers involved. For blood sampling, 9 mL S-Monovette^® neutral blood tubes (Sarstedt AG und Co., Nümbrecht, Germany) were used. Information about gender, age, body height and weight, cannabis-based medicine received, dosage regimen, further intake of cannabis or cannabis-based products in the last seven days and time between last application of the medicine and blood draw was collected in a questionnaire after every sample collection. The study was carried out over a period of approximately 12 months. Following study centers were involved:

Department of Neurology—Movement Disorder and Neurostimulation Outpatient Clinic of the University Medical Center of the Johannes Gutenberg University Mainz

Department of Anesthesiology of the University Medical Center of the Johannes Gutenberg University Mainz

Department of Internal Medicine III of the University Medical Center of the Johannes Gutenberg University Mainz

Pain and Palliative Center Rhine-Main in Wiesbaden

Medical practice Dr. Löwenstein in Mainz

The study was approved by the ethics committees of the State Medical Associations of Rhineland-Palatinate and Hessen (application number 2018-13337).

3.3. Chemicals and Reagents

Reference standards for THC-d3, THC-OH, THC-OH-d3, THC-COOH-d9, CBD, CBD-d3, CBDA, CBN-d3, CBNA, CBG, CBGA, CBC, CBCA, CBL, THCV, THCVA, CBDV and CBDVA were purchased from Cerilliant (Round Rock, TX, USA), CBG-d9 and CBC-d9 from Cayman Chemical Co. (Ann Arbor, MI, USA) via LGC Standards (Wesel, Germany), CBN, THC and THC-COOH from LGC Standards and THCAA from THC Pharm (Frankfurt a. M., Germany). All standards were certified reference material and had a purity of at least 99.0%, except for THC-OH-d3, THCAA, CBC, CBC-d9, CBCA, CBL, THCVA, CBDV and CBDVA (97.7%), THC-d3 (96.7%), and CBG-d9 (95.0%). All chemicals were obtained from Carl Roth (Karlsruhe, Germany), except for methanol (from Fisher Scientific (Loughborough, UK)) and acetonitrile (from AppliChem (Darmstadt, Germany)). Chemicals used in LC-MS/MS measurement processes were LC-MS grade. All other chemicals were HPLC grade, except for acetone and dichloromethane, which had a purity of 99.8% and 99.5%, respectively. Blank human serum used for calibration was obtained from voluntary healthy blood donors via blood donation service and was tested to be drug-free.

3.4. Sample Preparation

Serum samples were prepared as described previously [16]. 500 µL serum, 50 µL ISTD (consisting of 0.2 ng/µL of THC-COOH-d9 and 0.02 ng/µL of each THC-d3, THC-OH-d3, CBD-d3, CBC-d9, CBN-d3 and CBG-d9) and 1.5 mL acetonitrile were mixed. The supernatant was diluted with 6 mL phosphate buffer (0.1 M, pH 6). A fully automated solid phase extraction (SPE) was performed subsequently, using a Gilson Aspec GX 271 Workstation (Gilson International B.V. Germany, Limburg an der Lahn, Germany). After the SPE column (Bond Elut C18 column; 300 mg, 3 mL, Agilent, Santa Clara, CA, USA) was conditioned with 6 mL methanol and 2 mL water at a flow rate of 1 mL/min, the sample mixture was applied. The column was flushed with 4 mL water and 4 mL water/methanol (80:20 v/v) and subsequently rinsed with 1 mL 0.1 M acetic acid. After drying the column for 10 min, the cannabinoid compounds were eluted with 3 mL acetone/dichloromethane (50:50 v/v) [29]. The extract was evaporated to dryness at 40 °C and transferred into glass vials with 200 µL ethyl acetate. After evaporation at 30 °C, the samples were reconstituted in 50 µL of an acetonitrile/methanol/water mixture (3:3:2). For both evaporation steps, nitrogen was used.

3.5. LC-MS/MS Analysis

Samples were analyzed, using a previously described validated analysis method [16]. Analysis was performed with an UHPLC 1290 Infinity from Agilent Technologies (Waldbronn, Germany) and an Agilent Technologies 6490 Triple Quadrupole mass spectrometer, using a C18 column (ZORBAX Eclipse Plus C18 Rapid Resolution HD 2.1 × 100 mm 1.8-µ, Agilent) at 30 °C with 5.00 µL sample injection volume at a flow rate of 0.6 mL/min. Mobile phase A was 5 mM ammonium formate in water, mobile phase B consisted of 0.1% (v/v) formic acid in acetonitrile. Analytes were separated using the following elution: 65% mobile phase B for the first 4.5 min, changed to 80% B and increased to 80.9% B at 7.50 min, changed to 100% B till 9.50 min. The column was re-equilibrated with 65% B for 3.5 min. Total run time was 13 min. Analytes were measured using electrospray ionization (ESI) in positive and negative reaction mode with a gas temperature of 250 °C, gas flow of 15 L/min, nebulizer pressure of 20 psi, sheath gas heater temperature of 400 °C, sheath gas flow of 12 L/min, capillary voltage of 4000 V and a nozzle voltage of 1000 V. For evaluation of the data, the Agilent Mass Hunter software B 09.00 was used. Analytes were calibrated in blank human serum.

The authors would like to express their special gratitude to R. Laufenberg-Feldmann and M. Ferner, V. Fleischer, U. Reinholz, T. Schmitt, J. Spielberger and E. Wagner-Drouet for their kind cooperation and support in study conduction. The authors also thank O. Löwenstein, J. Meier and the staffs of the study centers involved for supply of study samples. The Institute for medical biometry, epidemiology and informatics (IMBEI) is kindly acknowledged for support with statistical analysis.