Procedure to analyze cannabinoids in bovine plasma using solid phase extraction & UPLC MS/MS
Geraldine Magnin
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.
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.
Steps
Preparation of solutions
Standards stock mixture. From the commercially available solutions, a stock solution containing the mixture of analytes at 10µg/mL in ACN is prepared and stored at -20°C. In a 4-mL glass vial,
add 20µL of CBC, CBCA, CBD, CBD-7-acid, CBDA, CBDV, CBDVA, CBG, CBGA, CBLA, CBN, 8-THC, 9-THC, THCA-A, THCP, THCV each at 10000µg/mL
add200µL of THC-11-acid, and THC-11-OH at 100µg/mL
add 40µL of CBL at 500µg/mL
add 1240µL of ACN
The stock standard solution is aliquoted in 0.5-mL portions stored at -20°C.
Internal standards mixture . A mixture of internal standards is prepared at 10µg/mL in ACN and stored at -20 °C. In a 4-mL glass vial.
add 200µL 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 100µg/mL
add 600µL 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:
100µLof cannabinoids working solution at10µg/mLin ACN10µLof THC-11-acid-glu at100µg/mL100µLof THC-glu at10µg/mL790µLof ACN
For 100 ng/mL
100µLof cannabinoids working solution at 1,000 ng/mL in ACN900µLof ACN
For 50 ng/mL
50µLof cannabinoids working solution at 1,000 ng/mL in ACN950µLof ACN
For 25 ng/mL
25µLof cannabinoids working solution at 1,000 ng/mL in ACN.975µLof ACN.
For 10 ng/mL
100µLof cannabinoids working solution at 100 ng/mL in ACN.900µLof ACN.
For 5 ng/mL:
100µLof cannabinoids working solution at 50 ng/mL in ACN.900µLof ACN.
For 2.5 ng/mL
100µLof cannabinoids working solution at 25 ng/mL in ACN.900µLof ACN.
For 1.0 ng/mL
100µLof cannabinoids working solution at 10 ng/mL in ACN900µLof 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 100µLof IS mixture at 10ng/mL
add 9.9mLof ACN-0.1 % formic acid.
Working quality control standards solutions
For 950 ng/mL
475µLof cannabinoids working solution at 1,000 ng/mL in ACN25µLof ACN
For 475 ng/mL
100µLof cannabinoids working solution at 950 ng/mL in ACN100µLof ACN
For 47.5 ng/mL
50µLof cannabinoids working solution at475µg/mLin ACN450µLof ACN
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:
10µLof mix 950 ng/mL990µLof NEG CTRL bovine plasma
QC2 (47.5 ng/mL)- Add:
10µLof mix 475 ng/mL990µLof NEG CTRL bovine plasm
QC1 (4.75 ng/mL)- Add:
10µLof mix 47.5 ng/mL990µLof NEG CTRL bovine plasma
Protein precipitation
Negative control : In a 1.5-mL microcentrifuge tube, mix:
100µL of NEG CTRL bovine plasma
100µL of ACN
100µL of acetonitrile-0.1% formic acid
IS control: In a 1.5-mL microcentrifuge tube, mix:
100µL of NEG CTRL bovine plasma
100µL of ACN
100µL of IS mixture at 100 ng/mL in ACN-formic acid 0.1%
Standard: In a 1.5-mL microcentrifuge tube, mix:
100µL of NEG CTRL bovine plasma
100µL of working standard in ACN
100µL 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:
100µL of plasma (samples or QCs)
100µL of ACN
100µL 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 0.4mL of water before clean-up.
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 250µLof MeOH-water (25:75).
Stack the HLB µElution plate on top of a clean collection plate.
Elute the cannabinoids with 2 x25µL aliquots of ACN-MeOH (90:10).
Add 50µL 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
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: 5µL
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 |
