Protocol - Biochemical Validation of Cannabis Consumption

Description

The assay from Klawitter et al. (2017) provides details for purchasing analytes, internal standards, and reagents performing quality control and calibration curves extracting, processing and storing samples and analyzing samples using high-performance liquid chromatography (HPLC) and tandem mass spectrometry.

Specific Instructions

The PhenX Substance Use and Recovery Working Group (WG) recommends research studies record the biospecimen type and the name of the laboratory performing the assay (if applicable).

Availability

This protocol is freely available; permission not required for use.

Protocol

Biochemical Validation of Cannabis Consumption in Plasma and Urine by Liquid Chromatography–Tandem Mass Spectrometry (LC-MS/MS)

The following is a summary of the protocol from Klawitter et al. (2017). The Substance Use and Recovery WG encourages interested investigators to identify laboratories that offer this assay and to ask about any specific requirements for biospecimen collection and transport. Investigators are encouraged to review the manuscript for more details.

Reagents

See Klawitter et al. (2017) for a description of the 11 cannabis analytes, internal standards, buffers, and where they were purchased.

Calibration Curves

For both urine and plasma, calibration standards for all 11 analytes were prepared by diluting 20 uL of 10X stock solutions into 180 uL of matrix resulting in following concentrations:

0.39 ng/mL
0.78 ng/mL
1.56 ng/mL
3.13 ng/mL
6.25 ng/mL
12.5 ng/mL
25 ng/mL
50 ng/mL
100 ng/mL
200 ng/mL
400 ng/mL

The authors note that the calibration curves for THC-C-gluc needs to be 5x higher to account for its higher concentration in urine

Sample Extraction

Aliquots are transferred to 1.5 mL low-binding polypropylene vials, vortexed, centrifuged and added to high-performance liquid chromatography (HPLC) autosampler vials.

Analysis

Investigators are encouraged to review Klawitter et al. (2017) for the details concerning the detection of the 11 cannabis analytes according to HPLC–tandem mass spectrometry. The Substance Use and Recovery WG notes that over time, additional analytes may become recognized and available.

The Substance Use and Recovery WG notes that there are other HPLC tandem mass spectrometry procedures and instruments that are appropriate for the measurement of cannabis analytes in urine and plasma. Once an assay is chosen for a particular study, the WG recommends that no changes in the protocol be made over the course of the study. To aid comparability, the WG recommends that the investigator record the make and manufacturer of equipment used and the repeatability and coefficients of variation for the assay. If using a commercial provider, then that provider should be used for the duration of the study, whenever possible.

Results

Δ9-tetrahydrocannabinol (THC): ______ ng/mL
11-hydroxy-Δ9-tetrahydrocannabinol (11OH-THC): ______ ng/mL
11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid glucuronide (THC-C-gluc): ______ ng/mL
cannabinol (CBN): ______ ng/mL
cannabidivarin (CBDV): ______ ng/mL
Cannabidiol (CBD): ______ ng/mL
cannabichromene (CBC): ______ ng/mL
cannabigerol (CBG): ______ ng/mL
Δ9-tetrahydrocannabivarin (THCV): ______ ng/mL
11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH): ______ ng/mL
11-nor-9-carboxy-Δ9-tetrahydrocannabivarin (THCV-COOH): ______ ng/mL

Personnel and Training Required

Expertise with liquid chromatography and tandem mass spectrometry is required (unless sending samples to a separate laboratory for testing).

Equipment Needs

This method requires high-performance liquid chromatography and tandem mass spectrometry on a triple quadrupole mass spectrometer.

Requirements

Requirement Category	Required
Major equipment	Yes
Specialized training	Yes
Specialized requirements for biospecimen collection	No
Average time of greater than 15 minutes in an unaffected individual	No

Mode of Administration

Bioassay

Lifestage

Adult, Senior

Participants

Adults, ages 18 and older

Selection Rationale

Liquid chromatography–tandem mass spectrometry (LC-MS/MS) is the preferred method for determination of cannabis analytes in human plasma or urine. The protocol from Klawitter et al., 2017 was selected as an example of an established, validated protocol for the biochemical validation of cannabis consumption using plasma or urine.

Language

English

Standards

Standard	Name	ID	Source

Derived Variables

None

Process and Review

Not Applicable

Protocol Name from Source

Biochemical Validation of Cannabis Consumption

Source

Klawitter, J., Sempio, C., Mörlein, S., De Bloois, E., Klepacki, J., Henthorn, T., Leehey, M. A., Hoffenberg, E. J., Knupp, K., Wang, G. S., Hopfer, C., Kinney, G., Bowler, R., Foreman, N., Galinkin, J., Christians, U., & Klawitter, J. (2017). An atmospheric pressure chemical ionization MS/MS assay using online extraction for the analysis of 11 cannabinoids and metabolites in human plasma and urine. Therapeutic Drug Monitoring, 39(5), 556–564.
https://doi.org/10.1097/FTD.0000000000000427

General References

Aizpurua-Olaizola, O., Zarandona, I., Ortiz, L., Navarro, P., Etxebarria, N., & Usobiaga, A. (2017). Simultaneous quantification of major cannabinoids and metabolites in human urine and plasma by HPLC-MS/MS and enzyme-alkaline hydrolysis. Drug Testing and Analysis, 9(4), 626–633.
https://doi.org/10.1002/dta.1998

Hubbard, J. A., Smith, B. E., Sobolesky, P. M., Kim, S., Hoffman, M. A., Stone, J., Huestis, M. A., Grelotti, D. J., Grant, I., Marcotte, T. D., & Fitzgerald, R. L. (2020). Validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to detect cannabinoids in whole blood and breath. Clinical Chemistry and Laboratory Medicine, 58(5), 673–681.
https://doi.org/10.1515/cclm-2019-0600

Klawitter, J., Sempio, C., Mörlein, S., De Bloois, E., Klepacki, J., Henthorn, T., Leehey, M. A., Hoffenberg, E. J., Knupp, K., Wang, G. S., Hopfer, C., Kinney, G., Bowler, R., Foreman, N., Galinkin, J., Christians, W., & Klawitter, J. (2017). An atmospheric pressure chemical ionization MS/MS assay using online extraction for the analysis of 11 cannabinoids and metabolites in human plasma and urine. Therapeutic Drug Monitoring, 39(5), 556–564

Protocol ID

511401

Variables

Export Variables

Variable Name	Variable ID	Variable Description	dbGaP Mapping

Assessment of Substance Use and Substance Use Disorders

Measure Name

Biochemical Validation of Cannabis Components in Human Tissue

Release Date

January 30, 2025

Definition

An assay to quantify cannabis components from human samples.

Purpose

Cannabis is a plant that contains many active or potentially active ingredients, including tetrahydrocannabinol (THC), cannabidiol (CBD), minor cannabinoids, terpenes, flavonoids. Many cannabis studies only measure frequency of cannabis use, making it challenging to estimate the pharmacological exposure of THC and other cannabis components. Lab-based assays of cannabis exposure can confirm the presence or absence of THC, other components of cannabis, and various metabolites that have varying half-lives. From the legal and/or regulatory perspective, lab-based assays can potentially be used to ascertain levels of THC in the blood for determining possible impairment. From the medical perspective, the pharmacologic effects of cannabis products have been linked to the THC and CBD content, the THC/CBD ratio, minor cannabinoids, and potentially cannabis-derived terpenes and flavonoids.

Keywords

Liquid chromatography–tandem mass spectrometry, LC-MS/MS, cannabis, tetrahydrocannabinol, THC, cannabidiol, CBD, cannabinoids, substance use

Measure Protocols

Protocol ID	Protocol Name
511401	Biochemical Validation of Cannabis Consumption