TITLE: Procedure to analyze cannabinoids in bovine plasma using solid phase extraction &amp; UPLC MS/MS
AUTHORS: Geraldine Magnin

[DESCRIPTION]
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]
SECTION: Preparation of solutions
1. 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,

SECTION: Preparation of solutions
1.1. 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
SECTION: Preparation of solutions
1.2. add200 µL of THC-11-acid, and THC-11-OH at 100 µg/mL
SECTION: Preparation of solutions
1.3. add 40 µL of CBL at 500 µg/mL
SECTION: Preparation of solutions
1.4. add 1240 µL of ACN
SECTION: Preparation of solutions
1.5. The stock standard solution is aliquoted in 0.5-mL portions stored at -20 °C.
 


SECTION: Preparation of solutions
2. 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.
SECTION: Preparation of solutions
2.1. 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
SECTION: Preparation of solutions
2.2.  add 600 µL of ACN.
SECTION: Preparation of solutions
2.3. The stock IS solution is aliquoted in 0.5-mL portions stored at -20 °C.

SECTION: Preparation of solutions
3. 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:
SECTION: Preparation of solutions
3.1. 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
SECTION: Preparation of solutions
3.2. For 100 ng/mL
100 µL of cannabinoids working solution at 1,000 ng/mL in ACN
900 µL of ACN
SECTION: Preparation of solutions
3.3. For 50 ng/mL
50 µL of cannabinoids working solution at 1,000 ng/mL in ACN
950 µLof ACN

SECTION: Preparation of solutions
3.4. For 25 ng/mL
25 µL of cannabinoids working solution at 1,000 ng/mL in ACN.
975 µL of ACN.

SECTION: Preparation of solutions
3.5. For 10 ng/mL
100 µL of cannabinoids working solution at 100 ng/mL in ACN.
900 µLof ACN.

SECTION: Preparation of solutions
3.6. For 5 ng/mL:
100 µL of cannabinoids working solution at 50 ng/mL in ACN.
900 µL of ACN.

SECTION: Preparation of solutions
3.7. For 2.5 ng/mL
100 µLof cannabinoids working solution at 25 ng/mL in ACN.
900 µLof ACN.

SECTION: Preparation of solutions
3.8. For 1.0 ng/mL
100 µLof cannabinoids working solution at 10 ng/mL in ACN
900 µLof ACN

SECTION: Preparation of solutions
4. Working solution of internal standards mixture. An IS working solution is prepared at 100 ng/mL in ACN-0.1% formic acid as follow:
SECTION: Preparation of solutions
4.1. In a 15-mL polypropylene tube,
SECTION: Preparation of solutions
4.2. add 100 µLof IS mixture at 10 ng/mL
SECTION: Preparation of solutions
4.3. add 9.9 mLof ACN-0.1 % formic acid.
SECTION: Preparation of solutions
5. Working quality control standards solutions
SECTION: Preparation of solutions
5.1. For 950 ng/mL
475 µL of cannabinoids working solution at 1,000 ng/mL in ACN
25 µL of ACN

SECTION: Preparation of solutions
5.2. For 475 ng/mL
100 µL of cannabinoids working solution at 950 ng/mL in ACN
100 µLof ACN

SECTION: Preparation of solutions
5.3. For 47.5 ng/mL
50 µLof cannabinoids working solution at 475 µg/mL in ACN
450 µLof ACN


SECTION: Preparation of QCs
6. 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:
SECTION: Preparation of QCs
6.1. QC3 (95 ng/mL)- Add:
10 µL of mix 950 ng/mL 
990 µL of NEG CTRL bovine plasma
SECTION: Preparation of QCs
6.2. QC2 (47.5 ng/mL)- Add:
10 µL of mix 475 ng/mL 
990 µLof NEG CTRL bovine plasm
SECTION: Preparation of QCs
6.3. QC1 (4.75 ng/mL)- Add:
10 µL of mix 47.5 ng/mL 
990 µL of NEG CTRL bovine plasma

SECTION: Protein precipitation
7. Negative control: In a 1.5-mL microcentrifuge tube, mix: 
SECTION: Protein precipitation
7.1. 100 µL of NEG CTRL bovine plasma

SECTION: Protein precipitation
7.2. 100 µL of ACN

SECTION: Protein precipitation
7.3. 100 µL of acetonitrile-0.1% formic acid

SECTION: Protein precipitation
8. IS control: In a 1.5-mL microcentrifuge tube, mix:
SECTION: Protein precipitation
8.1. 100 µL of NEG CTRL bovine plasma
SECTION: Protein precipitation
8.2. 100 µL of ACN
SECTION: Protein precipitation
8.3. 100 µL of IS mixture at 100 ng/mL in ACN-formic acid 0.1%

SECTION: Protein precipitation
9. Standard: In a 1.5-mL microcentrifuge tube, mix:
SECTION: Protein precipitation
9.1. 100 µL of NEG CTRL bovine plasma

SECTION: Protein precipitation
9.2. 100 µL of working standard in ACN

SECTION: Protein precipitation
9.3. 100 µL of IS working mixture at 100 ng/mL in ACN-formic acid 0.1%.

SECTION: Protein precipitation
10. Samples and QCs: In a 1.5-mL microcentrifuge tube, mix:

SECTION: Protein precipitation
10.1. 100 µL of plasma (samples or QCs)

SECTION: Protein precipitation
10.2. 100 µL of ACN

SECTION: Protein precipitation
10.3. 100 µL of internal standard mixture at 100 ng/mL in ACN-0.1% formic acid

SECTION: Protein precipitation
11. 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.

SECTION: Solid-phase extraction
12. Fill out the plate template below before added each solution (NEG CTRL, IS CTRL, standards, QCs or sample to the wells.

            1      2      3      4      5      6      7      8      9      10      11      12        A                                                                                            B                                                                                            C                                                                                            D                                                                                            E                                                                                            F                                                                                            G                                                                                            H                                                                                       


SECTION: Solid-phase extraction
13. Stack the HLB µElution plate on top of a waste collection plate
SECTION: Solid-phase extraction
14. Load each diluted supernatant into a well using positive pressure with nitrogen.
SECTION: Solid-phase extraction
15. Wash each well twice with 250 µLof MeOH-water (25:75).

SECTION: Solid-phase extraction
16. Stack the HLB µElution plate on top of a clean collection plate.
SECTION: Solid-phase extraction
17. Elute the cannabinoids with 2 x25 µL aliquots of ACN-MeOH (90:10).

SECTION: Solid-phase extraction
18. Add 50 µL of water with 0.2% formic acid in each well before analysis.

SECTION: Solid-phase extraction
19. Stack a cap mat on top of the plate.
SECTION: Solid-phase extraction
20. Mix gently.
SECTION: E.	Analytical parameters
21. 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.
SECTION: E.	Analytical parameters
22. Chromatographic separation 

SECTION: E.	Analytical parameters
22.1. UPLC column: Eclipse Plus C18 from Agilent Technologies (Santa Clara, CA) 100 x 2.1 mm, 1.8 µ.
SECTION: E.	Analytical parameters
22.2. 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
       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.
 
 
 

SECTION: E.	Analytical parameters
23. 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
     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   
 
SECTION: E.	Analytical parameters
24. 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.
     Cannabinoid      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  327.4  345.4      32  24  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  193.2  299.2      26  28  18      ES+      THC-11-acid-d9        THC-glu      1.48      491.3      2      123.0  193.1  315.1      52  36  16      ES+      THC-11-acid-glu-d3        CBVDA      2.01      329.3      2      217.1  283.1  311.2      24  20  20      ES-      THC-11-acid-d9        THC-11-acid      2.18      345.4      60      193.2  299.3  327.3      28  20  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  201.2  313.4      26  24  14      ES+      THC-11-OH-d3        THC-OH-d3      2.17      334.4      18      196.31      26      ES+      n/a        CBDV      2.24      287.2      66      135.1  165.1  231.1      18  26  18      ES+      CBD-d3        CBDA      2.91      357.4      4      179.20  245.35  339.39      26  28  20      ES-      THC-acid-d9        CBGA      3.07      359.5      32      191.29  315.45  341.39      34  20  20      ES-      THC-acid-d9        THCV      3.25      287.5      40      135.17  165.15  231.23      16  20  16      ES+      CBD-d3        CBG      3.28      317.4      50      95.1  123.1  193.2      38  30  18      ES+      CBD-d3        CBD      3.36      315.2      14      135.0  193.0  259.1      20  22  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  223.2  241.3      28  20  18      ES+      CBD-d3        9-THC      4.78      315.4      46      123.1  135.2  193.2      34  20  22      ES+      9-THC-d3        9-THC-d3      5.78      318.5      36      196.24      24      ES+      n/a        8-THC      4.89      315.4      24      123.1  135.2  193.2      30  18  22      ES+      9-THC-d3        CBL      5.25      315.4      42      123.0  165.0  235.1      28  26  18      ES+      CBC-d9        THCA-A      5.42      359.4      30      219.2  261.3  341.3      32  16      ES+      THCA-A-d3        THCA-A      5.42      357.4      8      191.2  245.4  313.4      34  30  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  193.2  259.3      30  18  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  219.0  261.0      36  32  26      ES-      THCA-A-d3        CBCA      5.99      357.4      70      179.2  313.4  339.4      24  22  22      ES-      THCA-A-d3        THCP      6.43      343.4      48      123.0  135.1  221.1      22  22  22      ES+      CBC-d9   
 

 Table 6: Linear range and limit of quantitation
     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