Apr 15, 2026
Procedure to analyze cannabinoids in bovine urine using solid phase extraction & UPLC MS/MS
- 1Kansas State University
- Vet LIRN
Protocol Citation: Geraldine Magnin 2026. Procedure to analyze cannabinoids in bovine urine using solid phase extraction & UPLC MS/MS. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl495q8go5/v1
Manuscript citation:
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/
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: July 02, 2024
Last Modified: April 15, 2026
Protocol Integer ID: 102689
Keywords: cannabinoids in bovine urine, quantification of phytocannabinoid, cannabinoid, phytocannabinoid, bovine urine, list of analyte, using solid phase extraction, blinded method test, solid phase extraction, urine, bmt report, analyte, analysis by uplc
Funders Acknowledgements:
FDA Vet-LIRN
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 phytocannabinoids in bovine urine using solid phase extraction and analysis by UPLC-MS/MS.
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 Abbreviation
Table 1. List of cannabinoids and their abbreviation
| A | B | |
| CBC | Cannabichromene | |
| CBCA | Cannabichromenic acid | |
| CBD | Cannabidiol | |
| CBDA | Cannabidiolic acid | |
| CBD-7-acid | Cannabidiol-7-acid | |
| CBD-6-OH | 6-Hydroxy-cannabidiol | |
| CBD-7-OH | 7-Hydroxy-cannabidiol | |
| 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-11-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 Abbreviation
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
MATERIAL & EQUIPMENT
Material
- 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
- Vorter 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.
Reference standards/Chemicals/Reagents
Chemicals and 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 0.5% formic acid
- Water with 0.2% 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 | |
| Cannabinoid | P/N | Supplier | Concentration (mg/mL) | Solvent | |
| Cannabinoids Standards | |||||
| CBC | C-143 | Cerilliant Co | 1,000 | MeOH | |
| CBCA | 30879 | Cayman Chemical | 1,000 | ACN | |
| CBD | C-045 | Cerilliant Co | 1,000 | MeOH | |
| CBD-7-acid | C-181 | 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 | CAN | |
| 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-acid | T-018 | Cerilliant Co | 100 | MeOH | |
| THC-acid glu | T-038 | Cerilliant Co | 100 | MeOH | |
| THC-glu | S16 | ElSohly | 10 | MeOH | |
| THC-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 Chemical | 100 | MeOH | |
| 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-acid-d9 | T-007 | Cerilliant Co | 100 | MeOH | |
| THC-acid-glu-d3 | T-080 | Cerilliant Co | 100 | MeOH | |
| THC-OH-d3 | H-041 | Cerilliant Co | 100 | MeOH | |
Before start
Urine used for the validation was collected “in-house” in a plastic cup. Upon receiving, the urine was stored in 40-mL aliquots at -80 °C. Urine is let thawed on the bench for 20 min before analysis.
Preparation of solutions
Stock standards solutions in acetonitrile: From the commercially available solutions, prepare a stock solution containing the mixture of analytes at 10 mg/mL in ACN are prepared in acetonitrile and stored at -20 °C
In a 4-mL glass vial add the following components for a final volume of 2000 µL
Add20 µL of each of the following at 1000 mg/mL with a total volume of 320 µL
CBC
CBCA
CBD
CDB-7-acid
CBDA
CBDV
CBDVA
CBG
CBGA
CBLA
CBN
8-THC
9-THC
THCA-A
THCP,
THCV
Add 200 µL of each of the following at100 µg/mL
THC-acid
THC-acid-glu
THC-OH
Add40 µL of CBL at 500 µg/mL
Add 1040 µL of ACN
The 10 µg/mL stock standard solution is aliquoted in 0.5-mL portions stored at -20 °C .
Note
THC-glu will be added later while preparing the working standard because the commercial
solution is available only at a concentration of 10 mg/mL.
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 of the following at100 µg/mL
THC-acid-glu-d3
THC-OH-d3
THC-acid-d9
9-THC-d3
CBD-d3
CBC-d9
THCA-A-d3
Working standards: Each day of the analysis, fresh standard solutions are prepared in acetonitrile at the following concentrations: 0.5 ng/mL ; 1 ng/mL ; 2.5 ng/mL ; 5 ng/mL ; 10 ng/mL ; 25 ng/mL ; 50 ng/mL and 100 ng/mL
In a 1.5-mL polypropylene microcentrifuge tube, mix the following:
1000 ng/mL
- 50 µL of cannabinoids working solution at 10 µg/mL in ACN
- 50 µL of THC-glu at 10 µg/mL
- 400 µL of ACN
In a 1.5-mL polypropylene microcentrifuge tube, mix the following:
500 ng/mL
- 100 µL of cannabinoids working solution at 1000 ng/mL in ACN
- 100 µL of solution at 1000 ng/mL in ACN
In a 1.5-mL polypropylene microcentrifuge tube, mix the following:
250 ng/mL
- 100 µL of cannabinoids mix at 500 ng/mL in ACN
- 100 µL of ACN
In a 1.5-mL polypropylene microcentrifuge tube, mix the following:
100 ng/mL
- 50 mL of cannabinoids mix at 1000 ng/mL in ACN
- 450 mL of ACN
In a 1.5-mL polypropylene microcentrifuge tube, mix the following:
50 ng/mL
- 50 mL of cannabinoids mix at 500 ng/mL in ACN
- 450 mL of ACN
In a 1.5-mL polypropylene microcentrifuge tube, mix the following:
25 ng/mL :
- 50 µL of cannabinoids mix at250 ng/mL in ACN
- 450 µL of ACN
In a 1.5-mL polypropylene microcentrifuge tube, mix the following:
10 ng/mL :
- 50 µL of cannabinoids mix at 100 ng/mL in ACN
- 450 µL of ACN
In a 1.5-mL polypropylene microcentrifuge tube, mix the following:
5 ng/mL :
- 50 µL of cannabinoids mix at 50 ng/mL in ACN
- 450 µL of ACN
In a 1.5-mL polypropylene microcentrifuge tube, mix the following:
1 ng/mL :
- 50 µL of mix 50 ng/mL
- 450 µL of ACN
0 mL Internal standards mixture. An IS working solution is prepared at 50 ng/mL in acetonitrile as follow:
In a 15-mL polypropylene tube add
- 75 µL of IS mixture at 10 µg/mL
- 14.9 mL of acetonitrile-0.1 % formic acid.
Working standards for the preparation of QCs
950 ng/mL:
- 47.5 µL of cannabinoids mix stock solution at 10 µg/mL in ACN
- 47.5 mL of THC-glu stock solution at 10 µg/mL in ACN
- 405 µL of ACN
475 ng/mL
- 100 µL of cannabinoids working solution at 950 ng/mL in ACN
- 100 µL of ACN
47.5 ng/mL
- 50 µL of cannabinoids working solution at 950 ng/mL in ACN
- 450 µL of ACN
Preparation of urine CTRL, standards and QCs before SPE
Negative CTRL: In 1.5-mL microcentrifuge tubes, add
- 90 µL of NEG CTRL urine
- 210 µL of ACN
- 400 µL of 18W water containing 0.5% formic acid
IS CTRL: In a 1.5-mL microcentrifuge tube, mix:
- 90 µL of NEG CTRL urine
- 10 µL of ACN
- 200 µL of IS 50 ng/mL in ACN- vortex
- 400 µL of 18W water containing 0.5% formic acid
Calibration standards: In a 1.5-mL microcentrifuge tube, mix:
- 90 µL of NEG CTRL urine
- 10 µL of calibration working standard in ACN. Vortex.
- 200 µL of IS 50 ng/mL in ACN
- 400 µL of 18W water containing 0.5% formic acid
QCs: In a 1.5-mL microcentrifuge tube, mix:
- 90 µL of urine (sample or QC)
- 10 µL of QC working standard in ACN
- 200 µL of IS 50 ng/mL in ACN. Vortex.
- 400 µL of 18W water containing 0.5% formic acid
Vortex briefly and centrifuge for 5 minutes at 13,000 g.
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 | M | |
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | ||
| A | |||||||||||||
| B | |||||||||||||
| C | |||||||||||||
| D | |||||||||||||
| E | |||||||||||||
| F | |||||||||||||
| G | |||||||||||||
| H |
Stack the HLB mElution 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 mElution plate on top of a clean collection plate.
Elute the cannabinoids with 2 x 25 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
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
The chromatographic separation is performed with a UPLC column Eclipse Plus C18 from Agilent Technologies (Santa Clara, CA) 100 x 2.1 mm, 1.8 m. The following parameters are used:
- Column temperature: 55 °C
- Autosampler compartment: 8 °
- Flow rate: 0.5 mL/min
- Injection volume: 5 µL
- The total run time is 10 minutes.
Table 3. 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 |
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 4.
Table 4. Source parameters
| A | B | |
| Source temperature | 150 °C | |
| Desolvation temperature | 550 °C | |
| Desolvation gas flow | 1,000 L/hour | |
| Cone gas flow | 150 L/hour | |
| Nebulizer gas flow | 7.0 bar | |
| Capillary voltage | 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. The LC flow is diverted to waste from 0 to 0.70 min and from 8.00 to 10 min.
Table 5. MRM parameters for each cannabinoid (Quantifiers product ions are in bold; other product ions
are used as qualifiers)
| A | B | C | D | E | F | G | H | I | |
| Cannabinoid | RT (min) | Acquisition time (min) | Precursor (m/z) | Cone (V) | Product (m/z) | CE (V) | Mode | IS | |
| CBD-7-acid | 1.08 | 0.9-1.2 | 345.34 | 74 | 119.14 193.17 299.24 | 26 28 18 | ES+ | THC-acid-d9 | |
| CBD-6-OH | 1.15 | 0.8-1.4 | 331.507 | 8 | 201.20 259.34 271.36 | 24 16 18 | ES+ | THC-OH-d3 | |
| CBD-7-OH | 1.25 | 1.0-1.5 | 331.51 | 6 | 193.27 201.20 313.43 | 24 24 12 | ES+ | THC-OH-d3 | |
| THC-acid-glu | 1.00 | 0.90-1.20 | 521.47 | 44 | 299.39 327.35 345.36 | 32 24 14 | ES+ | THC-acid-glu-d3 | |
| THC-acid-glu-d3 | 1.00 | 0.90-1.20 | 524.49 | 2 | 348.33 | 14 | ES+ | n/a | |
| THC-glu | 1.48 | 1.30-1.70 | 491.30 | 2 | 123.03 193.07 315.11 | 52 36 16 | ES+ | THC-acid-glu-d3 | |
| CBVDA | 2.01 | 1.85-2.15 | 329.30 | 2 | 151.10 217.11 283.14 311.18 | 22 24 20 20 | ES- | THC-acid-d9 | |
| THC-acid | 2.18 | 1.8-2.40 | 345.41 | 60 | 193.20 299.29 327.33 | 28 20 14 | ES+ | THC-acid-d9 | |
| THC-acid-d9 | 2.14 | 1.80-2.40 | 354.47 | 28 | 196.56 | 26 | ES+ | n/a | |
| THC-acid-d9 | 2.14 | 1.80-2.40 | 352.47 | 68 | 308.44 | 20 | ES- | n/a | |
| THC-OH | 2.17 | 1.95-2.30 | 331.40 | 36 | 193.19 201.19 313.38 | 26 24 14 | ES+ | THC-OH-d3 | |
| THC-OH-d3 | 2.17 | 1.95-2.30 | 334.44 | 18 | 196.31 | 26 | ES+ | n/a | |
| CBDV | 2.24 | 1.90-2.40 | 287.24 | 66 | 123.07 135.10 165.09 231.10 | 32 18 26 18 | ES+ | CBD-d3 | |
| CBDA | 2.91 | 2.80-3.15 | 357.40 | 4 | 179.20 245.35 339.39 | 26 28 20 | ES- | THC-acid-d9 | |
| CBGA | 3.07 | 2.90-3.35 | 359.47 | 32 | 191.29 315.45 341.39 | 34 20 20 | ES- | THC-acid-d9 | |
| THCV | 3.25 | 3.15-3.45 | 287.37 | 40 | 123.10 135.17 165.15 231.23 | 30 16 20 16 | ES+ | CBD-d3 | |
| CBG | 3.28 | 3.10-3.55 | 317.40 | 50 | 95.10 123.13 193.24 | 38 30 18 | ES+ | CBD-d3 | |
| CBD | 3.36 | 3.20-3.55 | 315.21 | 14 | 122.86 134.95 193.01 259.06 | 32 20 22 20 | ES+ | CBD-d3 | |
| CBD-d3 | 3.36 | 3.20-3.55 | 318.45 | 34 | 196.24 | 24 | ES+ | n/a | |
| CBN | 4.19 | 3.90-4.40 | 311.37 | 72 | 195.40 208.21 223.17 241.25 | 28 28 20 18 | ES+ | CBD-d3 | |
| 9-THC | 4.78 | 4.60-5.20 | 315.40 | 46 | 123.14 135.17 193.19 | 34 20 22 | ES+ | 9-THC-d3 | |
| 9-THC-d3 | 5.78 | 4.60-5.20 | 318.45 | 36 | 196.24 | 24 | ES+ | n/a | |
| 8-THC | 4.89 | 4.60-5.20 | 315.40 | 24 | 123.14 135.17 193.19 | 30 18 22 | ES+ | 9-THC-d3 | |
| CBL | 5.25 | 5.00-5.45 | 315.35 | 42 | 122.97 165.01 235.07 | 28 26 18 | ES+ | CBC-d9 | |
| THCA-A | 5.42 | 5.20-5.80 | 359.34 | 30 | 219.20 261.28 341.33 | 32 16 | ES+ | THCA-A-d3 | |
| THCA-A-d3 | 5.43 | 5.20-5.80 | 362.33 | 26 | 264.10 | 26 | ES+ | n/a | |
| CBC | 5.67 | 5.50-6.00 | 315.43 | 16 | 123.14 193.20 259.29 | 30 18 16 | ES+ | CBC-d9 | |
| CBC-d9 | 5.67 | 5.10-5.60 | 324.40 | 40 | 268.33 | 14 | ES+ | n/a | |
| CBLA | 5.83 | 5.60-6.10 | 359.28 | 52 | 177.02 219.00 261.04 | 36 32 26 | ES- | THCA-A-d3 | |
| CBCA | 5.99 | 5.50-6.20 | 357.44 | 70 | 179.19 313.42 339.43 | 24 22 22 | ES- | THCA-A-d3 | |
| THCP | 6.43 | 6.10-6.80 | 343.36 | 48 | 123.01 135.06 221.09 | 22 22 22 | ES+ | CBC-d9 |
Thedwell time is adjusted automatically in order to have 20 data points across each peak.
Data processing- using the Waters TargetLynx software 4.2.
Signal to noise determination
S/N calculations are performed on unsmoothed data obtained at low cannabinoids concentration: 0.5, 1.0 and 2.5 ng/mL. An example is represented in Figure 1 below.
Noise. A peak-free section in the current chromatogram is used to determine the signal noise (classified as the difference between the largest and the smallest point of the baseline). To calculate the noise, right-click and drag the mouse across the noise region.
Signal. To measure the signal, right-click and drag the mouse across the peak of interest.
Signal over noise (S/N) is displayed as peak-to-peak values, the greatest height of the signal range above the mean noise value being divided by the variance. Cannabinoids S/N ratio at concentrations 0.5 ng/mL, 1.0 ng/mL and 2.5 ng/mL are listed in Appendix Table 6.
Figure 1. Example of S/N calculation for THC-acid at 0.5, 1.0 and 2.5 ng/mL
Smoothing and Integration
Smoothing is done using the mean method with the following parameters:
- Smoothing iterations: 5
- Smoothing width: 3
Note
Peak integration is done using the Apex Track method because it improves the
integration of all the peak in the chromatogram and reduced the use of manual
integration to correct for errors.
Acceptance criteria for each compound
Chromatograms are presented in Appendix Figures 2A to Figures 22A.
Each compound is characterized by the following parameters:
The transition from precursor ion to product ion in positive or negative ionization. A quantifier ion and 2 qualifier ions are used for each compound. Transitions are listed in Table 5.
The retention time (RT) in minutes. The variation should not exceed 0.25%. The retention times of the standards from 0.5 to50 ng/mL are averaged. The retention times deviation for the QCs is calculated (N=3). Retention times deviations are presented in Appendix Table 7.
The ratio Quantifiers/Qualifiers in the QCs should not exceed 20% of the average ratios in the standards.
Calibration curve
Linear regression with a weighing factor of 1/x is used for the calibration curve. The squared coefficient of variation is accepted if ≥ 0.99. The calibration curves are presented in Appendix figures 2B-22B.
The calculated concentration shouldn’t deviate more than 15% of the theoretical value (20% for the LOQ). Deviation from the expected concentrations are presented in Appendix, Table 8.
LOD and LOQ
By convention:
- The Limit of Detection (LOD) is the concentration at which the signal level of the substance reaches at least 3 times the signal noise of the baseline.
- The Limit of Quantitation (LOQ) is the concentration at which the signal level of the substance reaches at least 10 times the signal noise of the baseline.
In addition, the following acceptance criteria needs to be met to determine the LOQ:
- Intra-day and inter-day precision < 20%
- Deviation of the calculated concentration < 20%
- Accuracy of QC at LLOQ should be 80-120%
Intra-day precision and accuracy at LOQ are presented in Appendix Tables 9A-28B
Inter-day precision and accuracy at LOQ are presented in Appendix Tables 9B-28C
Table 24. LOQ and linear range of cannabinoids in bovine urine
| A | B | C | |
| Cannabinoids | LOQ (ng/mL) | Linear range ng/mL | |
| CBD-7-acid | 1.0 | 1.0-100 | |
| THC-acid-glu | 0.25 | 0.25-100 | |
| CBD-6-OH | 2.5 | 2.5-100 | |
| CBD-7-OH | 1.0 | 1.0-100 | |
| THC-glu | 0.1 | 0.1-100 | |
| CBDVA | 1.0 | 1-100 | |
| THC-11-acid | 0.25 | 0.25-100 | |
| THC-11-OH | 0.25 | 0.25-100 | |
| CBDV | 0.25 | 0.25-100 | |
| CBDA | 1.0 | 1-100 | |
| CBGA | 1.0 | 1-100 | |
| THCV | 0.1 | 0.1-100 | |
| CBG | 0.25 | 0.25-100 | |
| CBD | 1.0 | 0.25-100 | |
| CBN | 0.1 | 0.1-100 | |
| 9-THC | 0.1 | 0.1-100 | |
| 8-THC | 0.25 | 0.25-100 | |
| CBL | 2.5 | 2.5-100 | |
| CBC | 0.25 | 0.25-100 | |
| THCA-A | 0.25 | 0.25-100 | |
| CBCA | 2.5 | 2.5-100 | |
| CBLA | 1.0 | 1.0-100 | |
| THCP | 0.1 | 0.1-100 |
APPENDIX
Table 6. Signal over noise ratio at 0.1, 0.25, 0.5, 1.0 and 2.5 ng/mL
| Signal over noise ratio (S/N) | ||||||
| Expected ng/mL | 0.10 | 0.25 | 0.50 | 1.0 | 2.5 | |
| CBD-7-acid | 163 | 286 | 852 | 1,461 | 3,920 |
Figure 2A. Chromatogram at 2.5 ng/mL
Figure 22A. Chromatogram at 2.5 ng/mL
Table 7. Deviation from the expected concentration and precision (inter-day data, N=6)
| A | B | C | D | E | F | |
| CBDVA standard | ||||||
| Expected ng/mL | 0.10 | 0.25 | 0.50 | 1.0 | 2.5 | |
| Deviation % | -15.0 | -14.7 | 27.2 | 14.7 | -1.0 | |
| RSD or precision % | 30.9 | 20.2 | 51.2 | 12.1 | 8.2 | |
Table 8. Deviation from the expected concentration and precision (inter-day data, N=6)
| THC-11-OH standard | ||||||
| Expected ng/mL | 0.10 | 0.25 | 0.50 | 1.0 | 2.5 | |
| Deviation % | -7.0 | -2.1 | 1.8 | 4.1 | -7.0 | |
| RSD or precision % | 29.4 | 9.1 | 3.8 | 4.4 | 4.8 |