Modification of AOAC Official Method 2018.11 for Quantification of Cannabinoids in Plant Materials, Concentrates and Oils to Include Determination of Cannabinoids on a Dry-Weight Basis Lukas Vaclavik, Danielle Houston and Katerina Mastovska* Eurofins Food Integrity and Innovation, 3301 Kinsman Boulevard, Madison, WI, USA, *Corresponding author contact information: E-mail: KaterinaMastovska@eurofinsUS.com; Phone: +1-317-371-2968

Presented at the AOAC Annual Meeting and Exposition 2020

IntroductionUnder the current U.S. federal regulations, hemp plant material has to contain no more than 0.3% of ∆9-THC on dry weight basis in order to be considered legal.1 In addition to the psychoactive ∆9-THC, hemp plant may also contain ∆9-tetrahydrocannabinolic acid (∆9-THCA), which has no psychotropic activity, but may decarboxylate (e.g., upon heating) to form ∆9-THC. Recent testing guidelines for determination of ∆9-THC concentration in hemp published by the U.S. Department of Agriculture (USDA) require that the used testing methodology must consider the potential conversion of ∆9-THCA into ∆9-THC and the test result reflect the total available THC derived from the sum of the THC and THCA content.2

The AOAC Int. issued a call for methods for quantitation of cannabinoids in plant materials of hemp based on Standard Method Performance Requirements (SMPR) 2019.003 (see Table 1).3 The candidate method had to be able to report concentrations for individual cannabinoids and total THC (sum of ∆9-THC and ∆9-THCA expressed as ∆9-THC) on a dry-weight basis and include a detailed procedure for determination of plant material dry weight.

This poster summarizes the modifications made to the AOAC Official Method 2018.11 method to address the above requirements. Results of limited precision experiments obtained by the analysis of hemp plant material and calculated on “as is” and dry-weight basis are also presented.

Method

Cannabinoid Determination

• The AOAC 2018.11 First Action official method is based on analysis using ultra-high performance liquid chromatography coupled to diode array detection (UHPLC-DAD) and is also compatible with mass spectrometric (MS) detection.

• The method was thoroughly validated for 12 major cannabinoids in Cannabis dried plant materials (including hemp plants), concentrates and oils.4

• Method performance passes the requirements in AOAC SMPRs 2017.001 and 2017.002.

Table 1. AOAC SMPR 2019.003

Parameter Requirement for all analytes

Analyte scope (required analytes)

Cannabidiol (CBD), Cannabidiolic acid (CBDA), Cannabinol (CBN), ∆9-Tetrahydrocannabinol (∆9-THC),

∆9-Tetrahydrocannabinolic acid (∆9-THCA)

Analyte scope (desirable analytes)

Cannabichromene (CBC), Cannabichromenic acid (CBCA), Cannabidivarinic acid (CBVDA), Cannabigerol (CBG), Cannabigerolic acid

(CBGA), Cannabidivarin (CBDV), ∆8-Tetrahydrocannabinol (∆8-THC), Tetrahydrocannabivarin (THCV), Tetrahydrocannabivarinic acid (THCVA)

Limit of quantification ≤0.05 (%, w/w)a

Analytical range (CBD and CBDA)

0.05–35 (%, w/w)a

Analytical range (others)

0.05–5 (%, w/w)a

ParameterRange (%, w/w)a

0.05–0.5 >0.5–5 5–35b

Recovery (%) 85–118 90–111 95–105

RSDr (%) ≤5 ≤3 ≤2

RSDR (%) ≤10 ≤8 ≤6

a All calculated on dry weight basis. b Only applicable to CBD and CBDA. Figure 1. Example UHPLC-DAD chromatogram of mixed solvent standard.

Figure 2. Sample preparation scheme.

Table 2. UHPLC-DAD conditions

UHPLC Conditions

Analytical column Supelco Ascentis Express C18, 2.0 µm, 150 x 2.1 mm

Column temperature 25°C

Injection volume 3 µL

Mobile phase A 20 mM aqueous ammonium formate, pH 3.2

Mobile phase B Acetonitrile

Elution gradient

Time (min) A (%) B (%) Flow (mL/min)

0.00 40 60 0.400

12.00 5 95 0.400

12.01 5 95 0.600

14.00 5 95 0.600

14.01 40 60 0.400

16.00 40 60 0.400

DAD Conditions

UV absorbance spectra acq. range 200-400 nm

UV absorbance spectra acq. step 2 nm

Quantification wavelength 240 nm

Quantification bandwidth 10 nm

Acquisition rate 2.5 Hz

Reference wavelength 360 nm

Reference bandwidth 100 nm

Dry Weight Determination

• Dry weight determination for plant materials was added to the method to meet requirements in AOAC SMPR 2019.003.

• Plant material dry weight determined gravimetrically (on a separate sample aliquot) after drying of a sample aliquot in a vacuum oven at 100°C ± 2°C for 5 to 5.5 hours.

Calculations

Figure 3. Dry weight determination scheme.

Calculations

Extra figures:

Results • A dried hemp flower and leaves

composite sample was analyzed in six replicates to determine cannabinoid concentrations and in six replicates to determine % dry weight.

• To show the effect of variability of different combinations of cannabinoid concentration dry weight results, all possible combination sets were generated for each determined cannabinoid “as is” concentration result (n = 6) and dry weight results (n = 6), and cannabinoids concentrations on a dry-weight basis were calculated for each combination.

• Table 3 summarizes the results for each analyte present in the sample (≥LOQ) showing the mean values and RSD ranges obtained on dry weight basis side-by-side with values calculated on “as is” basis.

• No significant effect on the repeatability (RSD) of the final result expressed on a dry weight basis was observed.

• All the results met the AOAC SMPR 2019.003 repeatability requirements.

Conclusions• The AOAC Official Method 2018.11 First Action has been updated to include a procedure

for the determination of Cannabis plant material dry weight and calculation of cannabinoid concentrations on a dry-weight basis, including the total THC content.

• These method modifications were approved by an AOAC ERP to meet the AOAC SMPR 2019.003 requirements, which are in line with the USDA testing guidelines for total THC in hemp.

References1 Agriculture Improvement Act of 2018, Pub. L., 115-334 (2018 Farm Bill).

2 U.S Department of Agriculture: Testing Guidelines for Identifying Delta-9 Tetrahydrocannabinol (THC) Concentration in Hemp (available at https://www.ams.usda.gov/sites/default/files/media/TestingGuidelinesforHemp.pdf).

3 AOAC SMPR 2019.003 – Standard Method Performance Requirements (SMPRs) for Quantitation of Cannabinoids in Plant Materials of Hemp (Low THC Varieties Cannabis sp.).

4 Vaclavik, L., Benes, F., Fenclova, M., Hricko, J., Krmela, A., Svobodova, V., Hajslova, J., Mastovska, K. (2019) J. AOAC Int. 102, 1822–1833.

Table 3. Comparison of cannabinoid mean results (%, w/w) and RSDs (%) determined on “as is” and on a dry-weight basis (the RSD ranges for the dry weight determination were obtained

using all possible analyte-dry weight combinations)

Analyte

“As is” basis Dry-weight basis

Mean (%, w/w)

RSD (%)Mean

(%, w/w)RSD (%)

range

CBC 0.0683 4.01 0.0745 3.97 - 4.05

CBD 1.15 2.58 1.26 2.53 - 2.62

CBDA 0.541 3.69 0.590 3.65 - 3.72

CBDV 0.0311 4.68 0.0339 4.64 - 4.72

CBDVA 0.0099 3.17 0.0108 3.14 - 3.21

∆9-THC 0.0329 3.45 0.0359 3.41 - 3.50