Journal:Leaner and greener analysis of cannabinoids

Full article title	Leaner and greener analysis of cannabinoids
Journal	Analytical and Bioanalytical Chemistry
Author(s)	Mudge, Elizabeth M.; Murch, Susan J.; Brown, Paula N.
Author affiliation(s)	British Columbia Institute of Technology, University of British Columbia
Primary contact	Email: Paula underscore brown at bcit dot ca
Year published	2017
Volume and issue	409(12)
Page(s)	3153–63
DOI	10.1007/s00216-017-0256-3
ISSN	1618-2650
Distribution license	Creative Commons Attribution 4.0 International
Website	https://link.springer.com/article/10.1007%2Fs00216-017-0256-3
Download	https://link.springer.com/content/pdf/10.1007%2Fs00216-017-0256-3.pdf (PDF)

Abstract

There is an explosion in the number of labs analyzing cannabinoids in marijuana (Cannabis sativa L., Cannabaceae); however, existing methods are inefficient, require expert analysts, and use large volumes of potentially environmentally damaging solvents. The objective of this work was to develop and validate an accurate method for analyzing cannabinoids in cannabis raw materials and finished products that is more efficient and uses fewer toxic solvents. A method using high-performance liquid chromatography (HPLC) with diode-array detection (DAD) was developed for eight cannabinoids in Cannabis flowers and oils using a statistically guided optimization plan based on the principles of green chemistry. A single-laboratory validation determined the linearity, selectivity, accuracy, repeatability, intermediate precision, limit of detection, and limit of quantitation of the method. Amounts of individual cannabinoids above the limit of quantitation in the flowers ranged from 0.02 to 14.9% concentration (w/w), with repeatability ranging from 0.78 to 10.08% relative standard deviation. The intermediate precision determined using Horwitz ratios (HorRat) ranged from 0.3 to 2.0. The limits of quantitation (LoQs) for individual cannabinoids in flowers ranged from 0.02 to 0.17% w/w. This is a significant improvement over previous methods and is suitable for a wide range of applications, including regulatory compliance, clinical studies, direct patient medical services, and commercial suppliers.

Keywords: green chemistry, single-laboratory validation, Cannabis, cannabinoids, medical marijuana

Introduction

The modern cannabis market is in a period of dramatic flux. In the United States, cannabis is classified as a Schedule I drug; however, eight U.S. states have legalized marijuana for recreational use, and 28 states have allowed medical marijuana on the basis of evidence of anxiolytic, analgesic, sedative, anticancer, and appetite stimulation effects. Regulations regarding Cannabis spp. vary globally. The Netherlands, Uruguay, and Portugal have decriminalized possession. In Canada, cannabis is a Schedule II controlled substance, but regulations have allowed production for medical purposes through licensed producers and personal production licenses. Canadian production of commercial products must take place in a facility using good manufacturing practices, and products must be assayed for the presence and quantity of Δ9-tetrahydrocannabinol (Δ9-THC), Δ9-tetrahydrocannabinolic acid (THCA), cannabidiol (CBD), and cannabidiolic acid (CBDA), using validated analytical methods. In total, more than 100 cannabinoids in 11 subclasses have been characterized in cannabis and are concentrated in the glandular trichomes of the female inflorescences. Other cannabinoid classes include cannabigerol (CBG), cannabichromene (CBC), and cannabinol (CBN) (Fig. 1). The cannabinoids occur primarily in acid form, with neutral cannabinoids formed during drying, storage, and decarboxylation during smoking. Δ9-THC, the main psychoactive cannabinoid, can be over 20% by weight in specially bred cannabis strains. CBD, known for its anti-inflammatory activity and antagonism of Δ9-THC-induced anxiety, can range from below 0.5% up to 6.5% by weight.

Fig. 1 Structures for the main neutral cannabinoids found in Cannabis flowers

There are a significant number of analytical methods to quantify cannabinoids available, many of which do not provide sufficient validation data to establish the method performance and reliability. Without this information, there is a possibility that the methods are not fit for purpose. The solvent composition, mass-to-solvent ratio, extraction technique, and time vary considerably between methods. Separations of cannabinoids use different mobile phases, columns, and gradients, and given the number of minor cannabinoids present in authentic materials, there is a possibility for coelution of peaks and inaccurate quantitative results. Rigorous validation procedures are necessary to ensure that the results of any analytical method are reliable. Without this data on method performance, the final method may not meet the needs of the users who adopt it for routine use, therefore producing inaccurate information pertaining to the products that people are using for the treatment of medical conditions. The speed with which regulations have changed and the nature of the rapidly expanding cannabis marketplace have created increased pressure for fast, safe, simple, and accurate analysis of phytochemicals to meet the demands of high-throughput laboratories and rapid release of finished products.

The most commonly used extraction solvent for cannabinoid analysis is 9:1 methanol/chloroform (% v/v), with some exceptions. It was originally selected to dissolve the internal standard di-n-octyl phthalate, which is no longer necessary with commercially available reference standards. There is an increasing desire to find greener methods to reduce use of chlorinated solvents, which can be toxic, expensive to dispose of, and hazardous to transport and store. Long-term, chronic exposure to chloroform is associated with liver and kidney damage, where the occupational exposure limit is 2 ppm in air. While laboratory safety procedures reduce exposure significantly, the risks of spills and inhalation of vapors are increased with chloroform use, and there is a diversity of safety equipment used in the labs engaged in this analysis. Removal of chloroform from the extraction solvent will improve laboratory safety and reduce reagent and disposal costs, while improving the environmental impact associated with chlorinated solvent usage.

The objective of the current work was to develop a fully validated, simplified, green chemistry method for labs to implement that may not have high levels of expertise or capacity for method development or validation. We developed the method using statistically guided method development protocols for the quantitation of eight cannabinoids in Cannabis flowers and oils. Nine authentic Cannabis flower materials and one cannabis oil with a wide range of cannabinoid contents were obtained and used as test articles for the validation of the method of the AOAC International guidelines. This method does not use chlorinated solvents, reduces sample preparation time, and ensures precise and accurate determination of cannabinoids.

Materials and methods

Reagents

Methanol and acetonitrile suitable for high-performance liquid chromatography (HPLC) were purchased from VWR International (Mississauga, ON, Canada). Chloroform suitable for the American Chemical Society (ACS) was obtained from VWR International. Water was purified to 18 MΩ using a Barnstead Smart2Pure nanopure system (Thermo Scientific, Waltham, MA). Ammonium formate for HPLC (>99.0%) was purchased from Sigma Aldrich (Oakville, ON, Canada), and formic acid (98% pure) was purchased from Fisher Scientific (Ottawa, ON, Canada).

Calibration standards

Certified reference materials (CRMs) were purchased from Cerilliant Corp. (Round Rock, TX) for nine cannabinoids: Δ9-THC, THCA, Δ8-THC, CBD, CBDA, CBG, CBN, CBC, and tetrahydrocannabivarin (THCV). The individual cannabinoids were provided in solution at 1.0 mg/mL concentration, certified by the supplier. The acidic cannabinoids were provided in acetonitrile and neutral cannabinoids in methanol. Fresh ampules were used for the validation study to ensure accurate quantitation of the individual constituents.

Test materials

Dried medical marijuana samples were purchased from several licensed producers within Canada. Nine products were selected for a variety of cannabinoid concentrations ranging from 0.2% to 17% total THC and 0.3% to 9% total CBD. As a result of the legal restrictions pertaining to these products, voucher specimens were not possible, but the samples were purchased directly from the source to ensure authenticity. A dried ethanol extract was dissolved in oil at a 1:10 dilution.

HPLC analysis

An Agilent 1200 RRLC system equipped with a temperature-controlled autosampler, binary pump, and diode-array detector (Agilent Technologies, Mississauga, ON, Canada) was used to separate the cannabinoids. The separation was achieved on a Kinetex C18, 1.7 μm, 100 × 3.0 mm i.d. column (Phenomenex, Torrance, CA). Mobile phase compositions were (A) 10 mM ammonium formate, pH 3.6 and (B) acetonitrile using gradient conditions at 0.6 mL/min. The separation was achieved according to the following gradient: 0–8 min, 52–66%B; 8–8.5 min, 66–70%B; 8.5–13 min, 70–80%B; 13–15 min, 80%B. A 7-min column equilibration was performed after each run. The injection volume was 5 μL and detection was at 220 nm. The autosampler was maintained at 4 °C.

Preparation of test materials

Plant tissues

A minimum of five grams of dried flowers was ground together from each test sample to ensure sample homogeneity. Ground flowers were extracted by weighing 200.0 mg into a 50-mL amber centrifuge tube. Then 25.00 mL of 80% methanol was added and vortexed for 30 seconds. Extraction took place using a sonicating bath for 15 minutes, where samples were vortexed every five minutes. Extracts were filtered with a 0.22-μm Teflon filter; diluted at 1:2, 1:5, or 1:10 using the extraction solvent into amber HPLC vials; and stored at 4 °C until analysis.

Oil

Cannabis oil was mixed by inversion prior to sample preparation. Then 50.0 mg of oil was weighed into a 50-mL amber centrifuge tube, to which 10.00 mL of methanol was added and vortexed for 30 seconds. Extracts were sonicated for 15 minutes, with vortexing every five minutes. Samples were filtered with 0.22-μm Teflon filters into amber HPLC vials and stored at 4 °C until analysis.

Method optimization

Analyte stability

Mixed calibration standards were stored at −20 °C, 4 °C, and 22 °C in the dark and tested at regular intervals to assess cannabinoid stability in solutions. Sample extracts were stored at 4 °C and 22 °C in light and dark conditions. A sample with greater than 5% loss from time zero was considered unstable.

Fractional factorial

The partial factorial design for method optimization and data analysis was completed using Minitab 16 (Minitab 16, State College, PA). Individual cannabinoids were quantified as percentage weight for weight in Cannabis flowers and milligrams per gram in oil. Microsoft Excel (Richmond, WA) was used for quantitative calculations and statistical analysis of validation data.

Single-laboratory validation parameters

The optimized method was subjected to a single-laboratory validation according to AOAC International guidelines for dietary supplements. Δ8-THC was not observed in any of the samples and therefore was not considered in the method validation.

Preparation of calibration solutions

Individual cannabinoid CRMs were used to prepare seven-point standard calibration curves for eight cannabinoids in concentrations ranging from 0.5 to 250 μg/mL. Dilutions of the CRMs were performed using the extraction solvent composed of 80% methanol. Concentration ranges were modified for each cannabinoid as summarized in Table 1. The calibration curves were plotted, and the slope and y intercept for each cannabinoid were used for linear regression analysis. Calibration curves were visually inspected and correlation coefficients were determined. An r² of at least 0.995 was deemed suitable for quantitation. Mixed standards were stored at 4 °C and were stable for up to three days.

Table 1. Concentration of cannabinoids used in the calibration standards for the method validation and resolution of analytes in chromatographic separation Cannabinoid Approximate Concentration (μg mL−1) Average correlation coefficients (r2) Resolution (Between component of interest and closest eluting peak) Lin 1 Lin 2 Lin 3 Lin 4 Lin 5 Lin 6 Lin 7 CBDA 250 200 100 50 25 10 5 0.9990 6.34 THCV 25 20 10 5 2.5 1 0.5 0.9993 1.75 CBD 50 40 20 10 5 2 1 0.9982 1.64 CBG 25 20 10 5 2.5 1 0.5 0.9995 1.85 CBN 25 20 10 5 2.5 1 0.5 0.9992 3.18 THCA 250 200 100 50 25 10 5 0.9992 1.95 THC 50 40 20 10 5 2 1 0.9983 2.89 CBC 25 20 10 5 2.5 1 0.5 0.9991 2.29

Selectivity

Selectivity was demonstrated by injecting the reference materials and raw flower extracts to evaluate the resolution between closely eluting peaks and potential interferences at 220 nm. Resolution of greater than 1.5 is deemed acceptable by AOAC guidelines. Peak purity was verified for all cannabinoids of interest.

Repeatability and intermediate precision

Quadruplicate samples of each test material were prepared on a single day to evaluate the repeatability as relative standard deviation (% RSD) for the individual cannabinoids. Intermediate precision was determined by repeating the repeatability studies on three separate days. The within-day, between-day, and total standard deviations were calculated for each cannabinoid in each test material. HorRat values were calculated to assess the overall precision of the method.

Recovery

Recovery was determined at three concentration levels of the major cannabinoids: CBDA, CBD, THCA, and THC. Ground stinging nettle, used as the negative recovery material, was spiked with individual cannabinoids and prepared according to the sample preparation protocol.

Limits of detection and quantitation

The limits of detection and quantitation were determined using the U.S. Environmental Protection Agency (EPA) method detection limit (MDL) protocol. The MDL is defined as the minimum concentration of substance that can be measured and reported with 99% confidence that the analyte concentration is greater than zero. Extract solutions containing low concentrations of the cannabinoids were used to evaluate the method limits. Seven replicates were injected, and the calculation for MDL was determined as the standard deviation of the calculated concentration between the seven replicates multiplied by the t statistic at 99% confidence interval. LOQ was determined as 10 times the standard deviation for the replicates to determine the MDL.

Notes

This presentation is faithful to the original, with only a few minor changes to presentation. Some grammar and punctuation was cleaned up to improve readability. In some cases important information was missing from the references, and that information was added. In the original, reference 22 cites Appendix D of 40 CFR 136, but the topic cited was on the method detection limit, which is covered in Appendix B; presumably the authors meant Appendix B, which is cited here.