Feb 05, 2024
Procedure to analyze cannabinoids in bovine plasma using solid phase extraction & UPLC MS/MS
- Geraldine Magnin1
- 1Kansas State University
Protocol Citation: Geraldine Magnin 2024. Procedure to analyze cannabinoids in bovine plasma using solid phase extraction & UPLC MS/MS. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6927dlqe/v1
Manuscript citation:
Rapid quantification of cannabinoids in beef tissues and bodily fluids using direct-delivery electrospray ionization mass spectrometry. Shubhashis Chakrabarty, Eric M Serum, Thomas M Winders, Bryan Neville, Michael D Kleinhenz, Geraldine Magnin, Johann F Coetzee, Carl R Dahlen, Kendall C Swanson, David J Smith. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2022 Oct;39(10):1705-1717. doi: 10.1080/19440049.2022.2107711. Epub 2022 Aug 8.https://pubmed.ncbi.nlm.nih.gov/35939416/
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 01, 2022
Last Modified: February 05, 2024
Protocol Integer ID: 73400
Keywords: cannabinoids, animal diagnostic specimens, plasma, blood, serum, specimens, tissues, hemp, marijuana
Funders Acknowledgement:
FDA Vet-LIRN's
Grant ID: U18 FD006915-01
Disclaimer
Reference to any commercial materials, equipment, or process does not in any way constitute approval, endorsement, or recommendation by the Food and Drug Administration.
Abstract
This procedure described the quantification of 21 phytocannabinoids in 100uL bovine plasma. 200uL of acetonitrile is added to 100uL plasma extract, cleaned up by centrifugation and a 96-well plate solid phase extraction and analyzed by UPLC-MS/MS.Matrix-matched (not in solvent) calibration curve is used to quantify 21 cannabinoids at concentrations ranging 1-100 ng/mL (ppb). Internal Standards were applied for quantification some of the analytes.
Method validation/evaluation/verification:
The earlier version of the method (including in-house validation data) was published: https://pubmed.ncbi.nlm.nih.gov/35939416/
Then the method was extended (by Geraldine Magnin - lead) regarding the list of analytes (to include 21 cannabinoids in its 2nd version) and evaluated through Blinded Method Test (BMT) by Vet-LIRN. The BMT report summarizing method performance evaluation data is available upon request.
Materials
Cannabinoids abbreviations
Table 1. List of cannabinoids and their abbreviation.
| CBC | Cannabichromene | |
| CBCA | Cannabichromenic acid | |
| CBD | Cannabidiol | |
| CBDA | Cannabidiolic acid | |
| CBDV | Cannabidivarin | |
| CBDVA | Cannabidivarinic acid | |
| CBG | Cannabigerol | |
| CBGA | Cannabigerolic acid | |
| CBL | Cannabicyclol Tetrahydrocannabivarin | |
| CBLA | Cannabicyclolic acid | |
| CBN | Cannabinol | |
| 8-THC | D8-Tetrahydrocannabinol | |
| 9-THC | D9-Tetrahydrocannabinol | |
| THCA-A | D9-Tetrahydrocannabinolic acid A | |
| THC-acid | (±)-11-Nor-9-Carboxy-D9-tetrahydrocannabinol | |
| THC-acid glu | Δ9-11-Nor-9-Carboxy-D9-tetrahydrocannabinol glucuronide | |
| THC-glu | Δ9-Tetrahydrocannabinol glucuronide | |
| THC-OH | 11-Hydroxy-D9-tetrahydrocannabinol | |
| THCP | Tetrahydrocannabiphorol | |
| THCV | Tetrahydrocannabivarin | |
| CBC-d9 | Cannabichromene-d9 | |
| CBD-d3 | Cannabidiol-d3 | |
| 9-THC-d3 | D9-Tetrahydrocannabinol-d3 | |
| THCA-A-d3 | Δ9-Tetrahydrocannabinolic acid A-d3 | |
| THC-acid-d9 | (±)-11-nor-9-Carboxy-D9-Tetrahydrocannabinol-d9 | |
| THC-acid-glu-d3 | Δ9-11-Nor-9-Carboxy-D9-tetrahydrocannabinol glucuronide-d3 | |
| THC-OH-d3 | 11-Hydroxy-D9-Tetrahydrocannabinol-d3 |
Other abbrevations
NEG CTRL: Negative control
IS CTRL: Internal standard control
ACN: Acetonitrile
MeOH: Methanol
UPLC: Ultra high-pressure liquid chromatography
MRM: Multiple reaction monitoring
QC: Quality controls
Specimen
Plasma used for the validation was purchased through VWR International (P/N TDS- SBPUC35); the anticoagulant used is sodium citrate. Upon receiving, the plasma was stored in 50-mL aliquots at -80 °C. Plasma samples should be stored at -80 °C (to preserve the glucuronide metabolites which are not as stable as other cannabinoids) and let thawed on the bench for 20 min before analysis. The plasma is spun down at 4,500 g for 5 minutes before use.
Materials
UPLC column: Eclipse Plus C18, Agilent Technologies (Santa Clara, CA) 100 x 2.1 mm, 1.8 m.
1.5-mL microcentrifuge tubes
Oasis PRIME HLB 96-well µElution Plate, 3 mg Sorbent per well, 1/pk (Waters, P/N 186008052)
Cap-mat 96 well 7 mm round plug pre-slit silicone/PTFE, 5/pk (Waters, P/N 186006332)
Pipette, single channel, variable, 0.5-5 mL (Eppendorf North America or equivalent)
Pipet, multichannel 25-300 mL
Equipment
Vortex mixer
Refrigerated microcentrifuge
96-well Sample Collection Plate, 2mL Square well 50/pkg (Waters, P/N 186002482)
Cap-mat 96 well 7 mm round plug pre-slit silicone/PTFE, 5/pkg (Waters, P/N 186006332)
Reservoir, White, 50mL, Non-Sterile, Fisher Scientific (Hampton, NH), P/N 50143700.
Positive Pressure Manifold Spacer, Waters Co. 9Milford MA), P/N 186007987.
UPLC system, Waters Acquity H equipped with a degasser, a column heater, a refrigerated autosampler, a quaternary pump. The UPLC is interfaced with a Waters triple quadrupole spectrometer Xevo TQ-S.
Chemicals/Reagents
Methanol, LC-MS grade
Acetonitrile, LC-MS grade
Formic acid, LC-MS grade
Ultrapure 18W water is obtained in-house with a Millipore Synergy UV-R system.
Methanol-water (25:75)
Acetonitrile-Methanol (90:10)
Water with 0.2% formic acid
Acetonitrile-0.1% formic acid
Reference Standards
All standards are purchased in solutions from the following suppliers (Table 2)
Table 2. List of cannabinoids standards, suppliers and parts numbers
| A | B | C | D | E | |
| Cannabinoids standards | P/N | Supplier | Concentration (ug/mL) | Solvent | |
| CBC | C-143 | Cerilliant Co | 1,000 | MeOH | |
| CBCA | 30879 | Cayman Chemical | 1,000 | ACN | |
| CBD | C-045 | Cerilliant Co | 1,000 | MeOH | |
| CBDA | 18090 | Cayman Chemical | 1,000 | ACN | |
| CBDV | C-140 | Cerilliant Co | 1,000 | MeOH | |
| CBDVA | C-152 | Cerilliant Co | 1,000 | ACN | |
| CBG | C-141 | Cerilliant Co | 1,000 | MeOH | |
| CBGA | 20019 | Cayman Chemical | 1,000 | ACN | |
| CBL | 22036 | Cayman Chemical | 1,000 | ACN | |
| CBLA | C-171 | Cerilliant Co | 500 | ACN | |
| CBN | C-046 | Cerilliant Co | 1,000 | MeOH | |
| 8-THC | T-032 | Cerilliant Co | 1,000 | MeOH | |
| 9-THC | T-005 | Cerilliant Co | 1,000 | MeOH | |
| THCA-A | ISO60175 | Cayman Chemical | 1,000 | ACN | |
| THC-11-acid | T-018 | Cerilliant Co | 100 | MeOH | |
| THC-11-acid glu | T-038 | Cerilliant Co | 100 | MeOH | |
| THC-glu | S16 | ElSohly | 10 | MeOH | |
| THC-11-OH | H-026 | Cerilliant Co | 100 | MeOH | |
| THCP | 30171 | Cayman Chemical | 1,000 | ACN | |
| THCV | T-094 | Cerilliant Co | 1,0000 | MeOH | |
| Internal standards | |||||
| CBC-d9 | 21294 | Cayman Chemicals | 100 | ||
| CBD-d3 | C-084 | Cerilliant Co | 100 | MeOH | |
| 9-THC-d3 | T-003 | Cerilliant Co | 100 | MeOH | |
| THCA-A-d3 | T-145 | Cerilliant Co | 100 | ACN | |
| THC-11-acid-d9 | T-007 | Cerilliant Co | 100 | MeOH | |
| THC-11-acid-glu-d3 | T-080 | Cerilliant Co | 100 | MeOH | |
| THC-11-OH-d3 | H-041 | Cerilliant Co | 100 | MeOH |
Before start
Bovine plasma. Upon receiving, plasma samples should be stored at -80 °C (to preserve the glucuronide metabolites which are not as stable as other cannabinoids). Before use, the plasma is thawed on the bench for 20 min mixed well with a vortex mixer and spun down at 4,500 g for 5 minutes.
Preparation of solutions
Preparation of solutions
Standards stock mixture. From the commercially available solutions, a stock solution containing the mixture of analytes at 10 µg/mL in ACN is prepared and stored at -20 °C . In a 4-mL glass vial,
add 20 µL of CBC, CBCA, CBD, CBD-7-acid, CBDA, CBDV, CBDVA, CBG, CBGA, CBLA, CBN,
8-THC, 9-THC, THCA-A, THCP, THCV each at 10000 µg/mL
add200 µL of THC-11-acid, and THC-11-OH at 100 µg/mL
add 40 µL of CBL at 500 µg/mL
add 1240 µL of ACN
The stock standard solution is aliquoted in 0.5-mL portions stored at -20 °C .
Note
THC-11-acid-glu and THC-glu are added to the working solution on the day of the analysis because they are not stable at -20 °C (they should be stored at -80 °C ).
Internal standards mixture. A mixture of internal standards is prepared at 10 µg/mL in ACN and stored at -20 °C. In a 4-mL glass vial.
add 200 µL of each IS (THC-11-acid-glu-d3, THC-11-OH-d3, THC-11-acid-d9, 9-THC-d3,
CBD-d3, CBC-d9, THCA-A-d3)at 100 µg/mL
add 600 µL of ACN.
The stock IS solution is aliquoted in 0.5-mL portions stored at -20 °C .
Standards mixture. Each day of the analysis, fresh standard solutions are prepared in ACN-0.1% formic acid, at the following concentrations: 0.5; 1; 2.5; 5; 10; 25; 50 and 100 ng/mL. In a 1.5-mL polypropylene microcentrifuge tube, mix the following:
For 1,000 ng/mL:
- 100 µL of cannabinoids working solution at 10 µg/mL in ACN
- 10 µL of THC-11-acid-glu at 100 µg/mL
- 100 µL of THC-glu at 10 µg/mL
- 790 µL of ACN
For 100 ng/mL
- 100 µL of cannabinoids working solution at 1,000 ng/mL in ACN
- 900 µL of ACN
For 50 ng/mL
- 50 µL of cannabinoids working solution at 1,000 ng/mL in ACN
- 950 µL of ACN
For 25 ng/mL
- 25 µL of cannabinoids working solution at 1,000 ng/mL in ACN.
- 975 µL of ACN.
For 10 ng/mL
- 100 µL of cannabinoids working solution at 100 ng/mL in ACN.
- 900 µL of ACN.
For 5 ng/mL:
- 100 µL of cannabinoids working solution at 50 ng/mL in ACN.
- 900 µL of ACN.
For 2.5 ng/mL
- 100 µL of cannabinoids working solution at 25 ng/mL in ACN.
- 900 µL of ACN.
For 1.0 ng/mL
- 100 µL of cannabinoids working solution at 10 ng/mL in ACN
- 900 µL of ACN
Working solution of internal standards mixture. An IS working solution is prepared at 100 ng/mL in ACN-0.1% formic acid as follow:
In a 15-mL polypropylene tube,
add 100 µL of IS mixture at 10 ng/mL
add 9.9 mL of ACN-0.1 % formic acid.
Working quality control standards solutions
For 950 ng/mL
- 475 µL of cannabinoids working solution at 1,000 ng/mL in ACN
- 25 µL of ACN
For 475 ng/mL
- 100 µL of cannabinoids working solution at 950 ng/mL in ACN
- 100 µL of ACN
For 47.5 ng/mL
- 50 µL of cannabinoids working solution at 475 µg/mL in ACN
- 450 µL of ACN
Preparation of QCs
Preparation of QCs
Quality controls are prepared each day of the analysis in negative control bovine plasma containing a mixture of the cannabinoids at the following concentrations: 4.75, 47.5, 95 ng/mL. In 1.5-mL microcentrifuge tubes:
QC3 (95 ng/mL)- Add:
- 10 µL of mix 950 ng/mL
- 990 µL of NEG CTRL bovine plasma
QC2 (47.5 ng/mL)- Add:
- 10 µL of mix 475 ng/mL
- 990 µL of NEG CTRL bovine plasm
QC1 (4.75 ng/mL)- Add:
- 10 µL of mix 47.5 ng/mL
- 990 µL of NEG CTRL bovine plasma
Protein precipitation
Protein precipitation
Negative control: In a 1.5-mL microcentrifuge tube, mix:
100 µL of NEG CTRL bovine plasma
100 µL of ACN
100 µL of acetonitrile-0.1% formic acid
IS control: In a 1.5-mL microcentrifuge tube, mix:
100 µL of NEG CTRL bovine plasma
100 µL of ACN
100 µL of IS mixture at 100 ng/mL in ACN-formic acid 0.1%
Standard: In a 1.5-mL microcentrifuge tube, mix:
100 µL of NEG CTRL bovine plasma
100 µL of working standard in ACN
100 µL of IS working mixture at 100 ng/mL in ACN-formic acid 0.1%.
Samples and QCs: In a 1.5-mL microcentrifuge tube, mix:
100 µL of plasma (samples or QCs)
100 µL of ACN
100 µL of internal standard mixture at 100 ng/mL in ACN-0.1% formic acid
Vortex each mixture for 5 seconds and centrifuge for 5 minutes at 13,000 g. The supernatant is then transferred to another 1.5-mL microcentrifuge tube and diluted with 0.4 mL of water before clean-up.
Solid-phase extraction
Solid-phase extraction
Fill out the plate template below before added each solution (NEG CTRL, IS CTRL, standards, QCs or sample to the wells.
| A | B | C | D | E | F | G | H | I | J | K | L | |
| NEG | IS | 1.0 | 2.5 | 5 | 10 | 25 | 50 | 100 | QC1 | QC2 | QC3 | |
Stack the HLB µElution plate on top of a waste collection plate
Load each diluted supernatant into a well using positive pressure with nitrogen.
Wash each well twice with 250 µL of MeOH-water (25:75).
Stack the HLB µElution plate on top of a clean collection plate.
Elute the cannabinoids with 2 x25 µL aliquots of ACN-MeOH (90:10).
Add 50 µL of water with 0.2% formic acid in each well before analysis.
Stack a cap mat on top of the plate.
Mix gently.
E. Analytical parameters
E. Analytical parameters
The analysis is performed with a system from Waters Corporation (Milford, MA) including an Acquity H UPLC and a TQ-S triple quadrupole mass spectrometer. The software used to control the UPLC and the mass spectrometer is MassLynx 4.2.
Chromatographic separation
UPLC column: Eclipse Plus C18 from Agilent Technologies (Santa Clara, CA) 100 x 2.1 mm, 1.8 µ.
Column temperature: 55 °C
Autosampler compartment: 8 °C
Flow rate: 0.5 mL/min
Injection volume: 5 µL
Table 1. Gradient used for the chromatographic separation of cannabinoids
| A | B | C | |
| Time (min) | Acetonitrile % | Aqueous formic acid 0.1% | |
| 0.00 | 40 | 60 | |
| 6.50 | 14 | 86 | |
| 6.51 | 0 | 100 | |
| 7.50 | 0 | 100 | |
| 7.51 | 40 | 60 | |
| 10.00 | 40 | 60 |
The total run time is 10 minutes.
Mass spectrometer parameters
Data acquisition is performed by Electrospray Ionization (ESI) in positive (ES+) and negative mode (ES-). The source parameters are described in Table 2.
Table 2. Source parameters
| A | B | |
| Temperature | ||
| Source | 150 °C | |
| Desolvation | 550 °C | |
| Gas Flow | ||
| Desolvation | 1,000 L/hour | |
| Cone | 150 L/hour | |
| Nebulizer | 7.0 bar | |
| Voltages | ||
| Capillary | 3.0 kV |
Multiple reaction monitoring (MRM) mode is used to detect quantify each cannabinoid. The precursor ion, the products ions, the quantifier ion (bold), the qualifiers ions, the cone voltage, the collision energy (CE) as well as the ionization mode (ES+ or ES-) are indicated in Table 5.
Table 5. MRM parameters for each cannabinoid. (Quantifiers product ions are in bold; other product ions are used as qualifiers). Note: The dwell time is adjusted automatically in order to have 20 data points across each peak.
| A | B | C | D | E | F | G | H | |
| Cannabinoids | RT (min) | Precursor (m/z) (Cone, V) | Product (m/z)(CE, V) | Mode | IS | |||
| THC-11-acid-glu | 1.00 | 521.5 (44) | 299.4 (32) | 327.4 (24) | 345.4 (14) | ES+ | THC-11-acid-glu-d3 | |
| THC-11-acid-glu-d3 | 1.00 | 524.5 (2) | 348.3 (14) | ES+ | n/a | |||
| CBD-7-acid | 1.08 | 524.5 (74) | 119.1 (26) | 193.3 (28) | 299.2 (18) | ES+ | THC-11-acid-d9 | |
| THC-glu | 1.48 | 491.3 (2) | 123.0 (52) | 193.1 (36) | 315.1 (16) | ES+ | THC-11-acid-glu-d3 | |
| CBVDA | 2.01 | 329.3 (2) | 217.1 (24) | 283.1 (20) | 311.2 (20) | ES- | THC-11-acid-d9 | |
| THC-11-acid | 2.18 | 345.4 (60) | 193.2 (28) | 299.3 (20) | 327.3 (14) | ES+ | THC-11-acid-d9) | |
| THC-11-acid-d9 | 2.14 | 354.5 (28) | 196.6 (26) | ES+ | n/a | |||
| THC-11-acid-d9 | 2.14 | 352.5 (68) | 308.4 (20) | ES- | n/a | |||
| THC-11-OH | 2.17 | 331.4 (36) | 193.2 (26) | 201.2 (24) | 313.4 (14) | ES+ | THC-11-OH-d3 | |
| THC-11-OH-d3 | 2.17 | 334.4 (18) | 196.3 (26) | ES+ | n/a | |||
| CBDV | 2.24 | 287.2 (66) | 135.1 (18) | 165.1 (26) | 231.1 (18) | ES+ | CBD-d3 | |
| CBDA | 2.91 | 357.4 (4) | 179.2 (26) | 245.4 (28) | 339.4 (20) | ES- | THC-acid-d9 | |
| CBGA | 3.07 | 359.5 (32) | 191.3 (34) | 315.5 (20) | 341.4 (20) | ES- | THC-acid-d9 | |
| THCV | 3.25 | 287.5 (40) | 135.2 (16) | 165.2 (20) | 231.2 (16) | ES+ | CBD-d3 | |
| CBG | 3.28 | 317.4 (50) | 123.1 (30) | 193.0 (18) | ES+ | CBD-d3 | ||
| CBD | 3.36 | 315.2 (14) | 135.0 (20) | 193.0 (22) | 259.1 (20) | ES+ | CBD-d3 | |
| CBD-d3 | 3.36 | 318.5 (34) | 196.2 (24) | ES+ | n/a | |||
| CBN | 4.19 | 311.4 (72) | 208.2 (28) | 223.2 (20) | 241.3 (18) | ES+ | CBD-d3 | |
| 9-THC | 4.78 | 315.4 (46) | 123.1 (34) | 135.2 (20) | 193.2 (22) | ES+ | 9-THC-d3 | |
| 9-THC-d3 | 5.78 | 318.5 (36) | 196.2 (24) | ES+ | n/a | |||
| 8-THC | 4.89 | 315.4 (24) | 123.1 (30) | 135.2 (18) | 193.2 (22) | ES+ | 9-THC-d3 | |
| CBL | 5.25 | 315.4 (42) | 123.0 (280 | 165.0 (26) | 235.1 (18) | ES+ | CBC-d9 | |
| THCA-A | 5.42 | 359.4 (30) | 219.2 (32) | 261.3 (24) | 341.3 (16) | ES+ | THCA-A-d3 | |
| THCA-A | 5.42 | 357.4 (8) | 191.2 (34) | 245.4 (30) | 313.4 (24) | ES- | THCA-A-d3 | |
| THCA-A-d3 | 5.43 | 362.3 (26) | 264.1 (26) | ES+ | n/a | |||
| THCA-A-d3 | 5.43 | 360.3 (76) | 316.6 (28) | ES- | n/a | |||
| CBC | 315.4 (16) | 123.1 (30) | 193.2 (18) | 259.3 (16) | ES+ | CBC-d9 | ||
| CBC-d9 | 5.38 | 324.4 (40) | 268.3 (14) | ES+ | n/a | |||
| CBLA | 5.83 | 359.3 (52) | 177.0 (36) | 219.0 (32) | 261.0 (26) | ES- | THCA-A-d3 | |
| CBCA | 5.99 | 357.4 (70) | 179.2 (24) | 313.4 (22) | 339.4 (22) | ES- | THCA-A-d3 | |
| THCP | 6.43 | 343.4 (48) | 123.0 (22) | 135.1 (22) | 221.1 (22) | ES+ | CBC-d9 | |
Table 6: Linear range and limit of quantitation
| A | B | C | |
| Cannabinoid | LOQ ng/mL | Linear range (ng/mL) | |
| THC-11-acid glu | 1.0 | 1-100 | |
| THC-11-glu | 1.0 | 1-100 | |
| CBD-7-acid | 1.0 | 1-100 | |
| CBDVA | 2.5 | 2.5-100 | |
| THC-acid | 1.0 | 1-100 | |
| THC-11-OH | 1.0 | 1-100 | |
| CBDV | 1.0 | 1-100 | |
| CBDA | 1.0 | 1-100 | |
| CBGA | 2.5 | 2.5-100 | |
| THCV | 1.0 | 1-100 | |
| CBG | 1.0 | 1-100 | |
| CBD | 1.0 | 1-100 | |
| CBN | 1.0 | 1-100 | |
| 9-THC | 1.0 | 1-100 | |
| 8-THC | 1.0 | 1-100 | |
| CBL | 1.0 | 1-100 | |
| CBC | 1.0 | 1-100 | |
| THCA-A | 1.0 | 1-100 | |
| CBCA | 2.5 | 2.5-100 | |
| CBLA | 1.0 | 1-100 | |
| THCP | 1.0 | 1-100 |
Protocol references
Rapid quantification of cannabinoids in beef tissues and bodily fluids using direct-delivery electrospray ionization mass spectrometry. Shubhashis Chakrabarty 1 2, Eric M Serum 2, Thomas M Winders 1, Bryan Neville 3, Michael D Kleinhenz 4, Geraldine Magnin 5, Johann F Coetzee 5, Carl R Dahlen 1, Kendall C Swanson 1, David J Smith 2. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2022 Oct;39(10):1705-1717. doi: 10.1080/19440049.2022.2107711. Epub 2022 Aug 8.https://pubmed.ncbi.nlm.nih.gov/35939416/