AOAC CASP SMPRs

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the asci which carry the ascospores. The asci are spread to the surrounding when the cleistothecium bursts. Cleistothecium is produced during the sexual reproduction stage of some Aspergillus species. Aleuriconidium is a type of conidium produced by lysis of the cell that supports it. The base is usually truncate and carries remnants of the lysed supporting cell. These remnants form annular frills at its base. Hulle cell is a large sterile cell bearing a small lumen. Similar to cleistothecium, it is associated with the sexual stage of some Aspergillus species . See Tables 2 and 3 for more macroscopic and microscopic information on Aspergillus species. Chen, S.C.A., Meyer, W., Sorrell, T.C., & Halliday, C.L. (2019) Manual of Clinical Microbiology , 12th Ed., Landry, M.L., McAdam, A.J., Patel, R., & Richter, S.S. (Eds) ASM Press, Washington, DC, USA, pp 2103–2131 Anaissie, E.J., McGinnis, M.R., & Pfaller, M.A. (2009) Clinical Mycology , 2nd Ed., Churchill Livingstone, New York, NY, USA, 687 pp Walsh, T.J., Hayden, R.T., & Larone, D.H. (2018) Larones Medically Important Fungi: A Guide to Identification , 6th Ed, ASM Press, Washington, DC, USA, 500 pp Candidate method .—Method submitted for validation [Appendix J: AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces, Official Methods of Analysis of AOAC INTERNATIONAL (2019) 21st Ed., AOAC INTERNATIONAL, Rockville, MD, USA] Candidate method confirmed result .—Final result obtained for a test portion after cultural confirmation of a candidate method. Candidate method presumptive result .—Preliminary result for a test portion produced by following a candidate method’s instructions for use. Cannabis .—Genus of flowering plants within the Cannabinaceae family that commonly contain 9-tetrahydrocannabinol (THC), cannabidiol (CBD), and other cannabinoids and terpenes. Cannabis includes, but is not limited to, high-THC and high-CBD cultivars. Cannabis concentrates .—Extracts (primarily composed of cannabinoids and/or terpenes) manufactured through the extraction and concentration of compounds derived from the cannabis plant or flower. Final products can be many forms, including oils, wax, or hash (Category II). Cannabis infused edibles .—Food and drinks containing extracts of cannabis and/or cannabis materials (Category III). Cannabis infused nonedibles .—Products containing extracts of cannabis and/or cannabis materials intended to be applied to the human body or any part thereof. Final products can be many forms, including creams, ointments, cosmetics, and therapeutic pads (Category IV). Cannabis plant and flower .—General terms for the structural and flowering unadulterated parts of the cannabis plant (Category I). Cannabis products .—Products (edible, and nonedible) extracted or infused with compounds derived from the cannabis plant, including, but not limited to, CBD and THC. Exclusivity .—Study involving pure nontarget strains, which are potentially cross-reactive, that shall be not detected or enumerated by the candidate method. See Table 4 for a list of recommended nontarget strains. [Appendix J: AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces, Official Methods of Analysis of AOAC INTERNATIONAL (2019) 21st Ed., AOAC INTERNATIONAL, Rockville, MD, USA]

AOAC SMPR ® 2019.001

Standard Method Performance Requirements (SMPRs ® ) for Detection of Aspergillus in Cannabis and Cannabis Products

Intended Use: Consensus-Based Reference Method 1 Purpose AOAC SMPRs describe the minimum recommended performance characteristics to be used during the evaluation of a method. The evaluation may be an on-site verification, a single- laboratory validation, or a multi-site collaborative study. SMPRs are written and adopted by AOAC composed of representatives from industry, regulatory organizations, contract laboratories, test kit manufacturers, and academic institutions. AOAC SMPRs are used by AOAC expert review panels in their evaluation of validation study data for methods being considered for Performance Tested Methods SM or AOAC Official Methods of Analysis SM and can be used as acceptance criteria for verification at user laboratories. [Refer to Appendix F: Guidelines for Standard Method Performance Requirements , Official Methods of Analysis of AOAC INTERNATIONAL (2019) 21st Ed., AOAC INTERNATIONAL, Rockville, MD, USA.] 2 Applicability Candidate methods used to detect Aspergillus ( Aspergillus niger , Aspergillus fumigatus , Aspergillus flavus , and Aspergillus terreus ) in cannabis (plants/flowers) and/or cannabis products (concentrates, infused edibles, and infused nonedibles). Candidate methods may be validated for specific matrices, categories or broader claims. See Table 1 for matrix/category claim acceptance criteria. 3 Analytical Technique Any analytical technique that meets the method performance requirements is acceptable. 4 Definitions Aspergillus .—Filamentous, cosmopolitan, and ubiquitous fungus found in nature producing colonies typically of 1–9 cm in size (select species produce 0.5–1 cm colonies). Colonies are powdery in texture and color varies based on species. Reverse color is typically uncolored to pale yellow. Growth is typical at 20–30°C. Aspergillus fumigatus is thermotolerant and can grow at a temperature range of 20 to 50°C. For all species, hyphae are septate and hyaline. The conidiophores originate from the basal foot cell located on the supporting hyphae and terminate in a vesicle at the apex. Vesicle is the typical formation for the genus Aspergillus . The morphology and color of the conidiophore vary from one species to another. Covering the surface of the vesicle entirely (“radiate” head) or partially only at the upper surface (“columnar” head) are the flask-shaped phialides, which are either uniseriate and attached to the vesicle directly or are biseriate and attached to the vesicle via a supporting cell, metula. Over the phialides are the round conidia (2–5 µm in diameter) forming radial chains. Other microscopic structures include sclerotia, cleistothecia, aleuriconidia, and Hulle cells are of key importance in identification of some Aspergillus species. Cleistothecium is a round, closed structure enclosing

Fractional positive .—Validation criterion that is satisfied when an unknown sample yields both positive and negative responses within a set of replicate analyses. The proportion of positive responses should fall within 25 and 75% and should ideally approximate 50% of the total number of replicates in the set. A set of replicate analyses are those replicates analyzed by one method. Only one set of replicates per matrix is required to satisfy this criterion. Inclusivity .—Study involving pure target strains that shall be detected or enumerated by the candidate method. See Table 5 for a list of recommended target strains. [Appendix J: AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces, Official Methods of Analysis of AOAC INTERNATIONAL (2019) 21st Ed., AOAC INTERNATIONAL, Rockville, MD, USA] Laboratory probability of detection (LPOD) .—POD value obtained from combining all valid collaborator data sets for a method for a given matrix at a given analyte level or concentration. [Appendix H: Probability of Detection (POD) as a Statistical Model for the Validation of Qualitative Methods, Official Methods of Analysis of AOAC INTERNATIONAL (2019) 21st Ed., AOAC INTERNATIONAL, Rockville, MD, USA] LCL.— Lower confidence limit . Probability of detection (POD) .—Portion of positive analytical outcomes for a qualitative method for a given matrix at a given analyte level or concentration. The difference in POD values between presumptive and confirmed results is termed dPOD CP . Test portion .—Sample size used in most validation studies. For cannabis flower/plant and cannabis infused nonedible products, a 10 g test portion is used. For cannabis concentrates, a 5 g test portion is used. For cannabis infused edibles, a 25 g test portion is used. A larger test portion can be used in validation studies when appropriate. See Table 6 for minimum test portion requirements. Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests (61), USP 40, United States Pharmacopeia, Rockville, MD, USA Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms (62), USP 40, United States Pharmacopeia, Rockville, MD, USA Feng, P., Weagant, S.D., Grant, M.A., & Burkhardt, W. (2017) Bacteriological Analytical Manual , Ch. 4 Enumeration of Escherichia coli and the Coliform Bacteria, https://www.fda.gov/ Food/FoodScienceResearch/LaboratoryMethods/ucm064948.htm Andrews, W. H., Wang, H., Jacobson, A., & Hammack, T. (2018) Bacteriological Analytical Manual, Ch. 5 Salmonella, https:// www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ ucm070149.htm UCL.— Upper confidence limit . 5 System Suitability Tests and/or Analytical Quality Control Positive and negative controls shall be embedded in assays as appropriate. Inhibition controls should be used for method verification for each new matrix. Manufacturer must provide written justification if controls are not appropriate to an assay. 6 Reference Material(s) Use of live cultures and/or fungal spores (liquid stressed/ nonstressed, lyophilized) is required for inclusivity and exclusivity testing and for inoculation of test matrices during the matrix studies. Extracted DNA is not suitable for use in validating methods against this SMPR but may be used to develop supplemental information.

7 Validation Guidance Appendix J: AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces [Official Methods of Analysis of AOAC INTERNATIONAL (2019) 21st Ed., AOAC INTERNATIONAL, Rockville, MD, USA]; or ISO 16140-2:2016 At the time of the publication, no national reference method exists for the confirmation of Aspergillus from cannabis products. Until a suitable reference method is established, the following is recommended for method developers: ( 1 ) To screen samples for the presence or absence of the target analyte, two methods that employ different technologies (agar plate, PCR, ELISA) must be used. ( 2 ) To ensure the viability of the inoculating organism (both confirming presumptive results or determining false-negative results), an extended primary enrichment (up to at least 48 total hours) followed by plating of the sample to a minimum of two types of agar plates [examples: Dichloran rose bengal chloramphenicol (DRBC), Sabouraud dextrose (SAB-DEX), potato dextrose agar (PDA), Czapek’s] is required. Final confirmation can be achieved via matrix-assisted laser desorption/ionization time-of-flight (MALDI- TOF) mass spectroscopy, sequencing, or other suitable confirmatory procedures (microscopic examination, biochemical analysis, etc). ( 3 ) When performing the validation, bulk inoculation of test material is required. In certain instances (for example, therapeutic patches), individual item inoculation may be required. ( 4 ) For the single-laboratory validation (SLV) with artificial contamination, matrix naturally contaminated with nontarget organisms (when available) shall be used. For at least one matrix evaluated during the SLV, competing nontarget microflora must be at least 10x the level of the target microorganism. If the concentration of competing microflora does not exceed 10x the target organism for any matrix, artificial contamination of one matrix with nontarget organism (s) is required. ( 5 ) Aminimum three-level most probable number (MPN) study should be performed to determine the concentration of the target organism used in the validation. If possible, the use of test portions included in the matrix study should be included as a level in the MPN study. See Appendix J guidelines for details on performing the MPN study. 8 Method Performance Requirements See Table 7 for acceptance criteria for validation. See Table 6 for category test portion requirement. See Table 1 for matrix claims acceptance criteria. See Table 8 for descriptions of MPN analysis. See Table 9 for condition of inoculating culture and stabilization of matrix for inoculation. See Table 10 for inclusivity and exclusivity guidance. Approved by attending stakeholders of the AOAC Cannabis Analytical Science Program (CASP) meeting on September 7, 2019. Final Version Date: October 3, 2019. Posted: October 9, 2019

Table 1. Acceptable matrix claims a

Table 2. Colony color in various Aspergillus species Species Surface Reverse A. clavatus Blue-green White, brownish with age A. flavus Yellow-green Goldish to red brown A. fumigatus Blue-green to gray White to tan A. glaucus group Green with yellow areas Yellowish to brown A. nidulans Green, buff to yellow Purplish red to olive A. niger Black White to yellow A. terreus Cinnamon to brown White to brown A. versicolor

Criteria

Minimum No. categories

Matrix claim

No. matrices

Broad range of cannabis and cannabis products Variety of cannabis and cannabis products Select cannabis products

15 (minimum 3 matrices/category) ≥10 (minimum 2 matrices/category)

≥5 ≥5 ≥1

2 1

Specific category Specific matrix(es)

1 a Reference: AOAC Technical Bulletin: TB02MAY2016: Acceptable Validation Claims for Proprietary/Commercial Microbiology Methods for Foods and Environmental Surfaces .

White at the beginning, turns to yellow, tan, pale green or pink

White to yellow or purplish red

Table 3. Microscopic features of various Aspergillus species Species Conidiophore Phialides

Vesicle

S a

C a

HC a

A a

Long, smooth

Uniseriate Huge, clavate-shaped

–

– –

A. clavatus

Colorless, rough Uni-/biseriate Round, radiate head + (in some strains, brown) –

A. flavus

Short (<300 µm), smooth, colorless or greenish

Uniseriate Round, columnar head

–

A. fumigatus

A. glaucus group Variable length, smooth, colorless

Uniseriate Round, radiate to very loosely columnar head

–

+ (yellow -orange)

–

Short (<250 µm), smooth, brown

Biseriate, short

Round, columnar head

–

+ (red)

–

A. nidulans

Long, smooth, colorless or brown

Biseriate Round, radiate head

–

A. niger

Short (<250 µm), smooth, colorless

Biseriate

Round, compactly columnar head

–

+ (solitary, round, produced directly on hyphae)

A. terreus

Long, smooth, colorless Biseriate

Round, loosely radiate head

–

+ (in some strains)

–

A. versicolor

a S = Sclerotia; C = Cleistothecia; HC = Hulle cells; A = Aleuriconidia.

Table 4. Aspergillus exclusivity panel a

Table 5. Aspergillus inclusivity panel a Aspergillus spp.

Reference ID (where applicable)

Minimum No. strains

Organism

Aspergillus niger Aspergillus flavus

10 10 10

Acinetobacter baumanii Alternia alternata Aspergillus aculeatus Aspergillus alabamensis Aspergillus brasiliensis Varga et al. Aspergillus carneus Aspergillus clavatus Aspergillus deflectus Aspergillus fijiensis Varga et al. Aspergillus fischeri Aspergillus glaucus Aspergillus janponicus Aspergillus nidulans Aspergillus oryzae (Ahlburg) Cohn Aspergillus parasiticus Speare Aspergillus ustus Aspergillus versicolor Botrytis cinerea Persoon Candida albicans Cryptococcus laurentii Cryptococcus neoformans Fusarium proliferatum Fusarium oxysporum Fusarium solani Golovinomyces cichoracearum Mucor circinelloides Mucor hiemalis Penicillium chrysogenum Penicillium rubens Penicillium venetum Pseudomonas aeruginosa Rhizopus nigrans Rhizopus stolonifer Scopulariopsis acremonium Yarrowia lipolytica Aspergillus caesiellus Aspergillus carbonarius Aspergillus pseudoterreus Peterson et al. Aspergillus steynii Aspergillus tamarii Aspergillus tubingensis (Schober) Mosseray Aspergillus tubingensis (Schober) Mosseray

Aspergillus fumigatus Aspergillus terreus

10 a Required species method developers must use to validate their methods. A minimum of 50 total strains, including 10 strains of each of the species are required for AOAC adoption. Strains utilized should be well-characterized and information provided must include source, strain, numbers and origin (if available).

ATCC 9642 b

Table 6. Category test portion requirements

ATCC 20611 b

Minimum test portion size, g a

Category

Plants and flowers

Low 20 x 10 a High 5 x 10 a

3 x 5 3 x 5

3 x 1 3 x 1

Concentrates Concentrates

Low 20 x 5 3 x 2.5 3 x 1 High 5 x 5 a 3 x 2.5 3 x 1

Infused edibles

Low 20 x 25 a High 5 x 25 a Low 20 x 10 a High 5 x 10 a

3 x 10 3 x 5 3 x 10 3 x 5

Infused nonedibles

3 x 5 3 x 5

3 x 1 3 x 1

culture (if shelf stable product is processed, cells must be heat stressed)

a Test portions from matrix study.

Table 10. Inclusivity/exclusivity performance requirements

Parameter requirements

Final test concn, CFU/mL 10–100 x limit of detection of the candidate method Overnight growth undiluted

Minimum acceptable results

Parameter

Inclusivity Single-laboratory validation (SLV) study: At least 10 strains per required Aspergillus spp. (Table 5) cultured by the candidate method enrichment procedure. A minimum of 50 strains is required. Exclusivity SLV study: At least 30 nontarget organisms (including those required in Table 4), cultured under optimal conditions for growth b

100% positive results a

100% negative results a

a 100% correct analyses are expected. All unexpected results are to be retested following internationally recognized guidelines (ISO 16140, AOAC OMA Appendix J, and Compendium of Analytical Methods of Health Canada). Some unexpected results may be acceptable if the unexpected results are investigated, and acceptable explanations can be determined and communicated to method users. b In instances where an exclusivity culture produces a positive result by the candidate method, the culture may be reanalyzed after culture following the candidate method enrichment procedure. Both results (optimal growth conditions and candidate method enrichment) must be reported.

AOAC SMPR ® 2019.002

Repeatability.— Variation arising when all efforts are made to keep conditions constant by using the same instrument and operator and repeated on individually prepared sample/test portions. Expressed as the repeatability standard deviation (SD r ); or % repeatability relative standard deviation (%RSD r ). Reproducibility.— Standard deviation or relative standard deviation calculated from among-laboratory data. Expressed as the reproducibility standard deviation (SD R ); or % reproducibility relative standard deviation (% RSD R ). 5 Method Performance Requirements See Table 2. 6 System Suitability Tests and/or Analytical Quality Control Suitable methods will include blank check samples, and check standards at the lowest point and midrange point of the analytical range. 7 Reference Material(s) Refer to Annex F: Development and Use of In-House Reference Materials in Appendix F: Guidelines for Standard Method Performance Requirements , Official Methods of Analysis of AOAC INTERNATIONAL (2019) 21st Ed., AOAC INTERNATIONAL, Rockville, MD, USA. Available at: http://www.eoma.aoac.org/ app_f.pdf 8 Validation Guidance <467> Residual Solvents, USP Dietary Supplements (2016) International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use. Impurities: Guideline for Residual Solvents Q3C (R6) (2016). Available at www.ich.org Appendix D: Guidelines for Collaborative Study Procedures to Validate Characteristics of a Method of Analysis, Official Methods of Analysis of AOAC INTERNATIONAL (2019) 21st Ed., AOAC INTERNATIONAL, Rockville, MD, USA. Available at: http:// www.eoma.aoac.org/app_d.pdf Appendix F: Guidelines for Standard Method Performance Requirements, Official Methods of Analysis of AOAC INTERNATIONAL (2019) 21st Ed., AOAC INTERNATIONAL, Rockville, MD, USA. Available at: http://www.eoma.aoac.org/ app_f.pdf AppendixK: Guidelines for Dietary Supplements and Botanicals, Official Methods of Analysis of AOAC INTERNATIONAL (2019) 21st Ed., AOAC INTERNATIONAL, Rockville, MD, USA. Available on line at: http://www.eoma.aoac.org/app_k.pdf U.S. Food and Drug Administration (May 2018) Bioanalytical Method Validation Guidance for Industry 9 Maximum Time-to-Result None Approved by attending stakeholders of the AOAC Cannabis Analytical Science Program (CASP) meeting on September 7, 2019. Final Version Date: October 3, 2019. Posted: October 9, 2019

Standard Method Performance Requirements (SMPRs ® ) for Identification and Quantitation of Selected Residual Solvents in Cannabis-Derived Materials

Intended Use: Consensus-Based Reference Method 1 Purpose AOAC SMPRs describe the minimum recommended performance characteristics to be used during the evaluation of a method. The evaluation may be an on-site verification, a single- laboratory validation, or a multi-site collaborative study. SMPRs are written and adopted by AOAC composed of representatives from the industry, regulatory organizations, contract laboratories, test kit manufacturers, and academic institutions. AOAC SMPRs are used by AOAC expert review panels in their evaluation of validation study data for method being considered for Performance Tested Methods SM certification or AOAC Official Methods of Analysis SM adoption and can be used as acceptance criteria for verification at user laboratories. 2 Applicability Method, or a suite of methods, to identify and quantify selected residual solvents (Table 1) in cannabis derivatives. 3 Analytical Technique Any analytical technique(s) that measures the analytes of interest and meets the following method performance requirements is/are acceptable. More than one analytical technique may be needed. 4 Definitions Cannabis plant material.— Plant material from Cannabis spp. and its chemical varieties or “chemovars.” Cannabis derivatives.— Products or extracts derived from cannabis plant material. Limit of detection (LOD).— Minimum concentration or mass of analyte in a given matrix that can be detected. A minimum 3 to 1 signal to background noise ratio (S/N). Limit of quantitation (LOQ).— Minimum concentration or mass of analyte in a given matrix that can be reported as a quantitative result. A minimum 10 to 1 S/N. Parts per million (ppm).— mg of analyte per kg of cannabis derivatives. Quantitative method.— Method of analysis where response is the amount of the analyte measured either directly (enumeration in a mass or a volume), or indirectly (color, absorbance, impedance, etc.) in a certain amount of sample. Recovery.— Fraction or percentage of spiked analyte that is recovered when the test sample is analyzed using the entire method.

Table 1. Residual solvents and targeted LOQs

Table 1. ( continued )

Analytical range Lower (≤ ppm) Upper (ppm)

Proposed target LOQ, ppm

Residual solvents a

Class 1

Ethyl acetate

5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000 5000

2500 2500 2500 2500 2500 2500 2500 2500 2500 2500 2500 2500 2500 2500 2500 2500

150000 150000 150000 150000 150000 150000 150000 150000 150000 150000 150000 150000 150000 150000 150000 150000

Benzene

2 4 5 8

1 2

Ethyl ether

Carbon tetrachloride 1,2-Dichloroethane 1,1-Dichloroethene 1,1,1-Trichloroethane

120 150 240

Ethyl formate Formic acid

2.5

Heptane

1500

750

45000

Isobutyl acetate Isopropyl acetate Methyl acetate 3-Methyl-1-butanol Methylethylketone Methylisobutylketone 2-Methyl-1-propanol

Class 2

Acetonitrile

180

Chlorobenzene

360

180

10800

Chloroform

60 70

30 35

1800 2100

Cumene

Cyclohexane

3880 1870

1940

116400 56100

1,2-Dichloroethene 1,2-Dimethoxyethane N,N -dimethylacetamide N,N -dimethylformamide

935

Pentane

100

3000

1-Pentanol 1-Propanol 2-Propanol

1090

545 440 190 310 110 145 80

32700 26400 11400

880 380 160 620 220 290

1,4-Dioxane

Isopropanol (2-propanol) Propyl acetate

2-Ethoxyethanol Ethylene glycol

4800

5000

2500

150000

18600

Additional

Formamide

6600 8700

Butane (sum of n - and iso-)

5000

2500

150000

Hexane

Propane

360

n -Hexane Methanol

2,2-Dimethylbutane 2,3-Dimethylbutane

290 290

145 145

8700 8700

400

200

12000

2-Methoxyethanol Methylbutylketone Methylcyclohexane Methylene chloride N -Methylpyrrolidone

50 50

25 25

1500 1500

2-Butanone

5000 5000

2500 2500

150000 150000

2-Methylbutane 2-Methylpentane 3-Methylpentane Ethylbenzene Ethylene oxide

1180

590 300 265

35400 18000 15900

290 290

145 145

8700 8700

600 530

2170

1085

65100

Nitromethane

1500 6000 4800

50 12 12

1500

Pyridine Sulfolane

200 160 720 100

100

Isobutane (methyl propane)

6 6

360 360

n -Butane n -Heptane n -Pentane Nitrogen o -Xylene

Tetrahydrofuran

360

21600

1000 1000

500 500

30000 30000

Tetralin Toluene

50 15 40

3000

30 80

900

0 5

Trichloroethylene

2400

300

Xylene

2170

1085

65100

Triethylamine

5000

2500

150000

m,p -Xylenes

a USP Guidance (467) Residual Solvents.

Class 3

Acetic acid Acetone Anisole 1-Butanol 2-Butanol Butyl acetate

5000 1000 5000 5000 5000 5000 5000 5000 1000

2500

150000 30000 150000 150000 150000 150000 150000 150000 30000

500

2500 2500 2500 2500 2500 2500

Table 2. Method performance requirements for residual solvents in Table 1

Analytical range, ppm

Parameter

1–100

100–500 90–107

500–1000

>1000 97–103

tert -Butylmethyl ether Dimethyl sulfoxide

Recovery, % 60–120

95–105

RSD r RSD

, %

≤20 ≤30

≤5 ≤8

≤4 ≤6

≤3 ≤4

Ethanol

500

, %

AOAC SMPR ® 2019.003

Reproducibility .—Standard deviation or relative standard deviation calculated from among-laboratory data. Expressed as the reproducibility standard deviation (SD R ); or % reproducibility relative standard deviation (%RSD R ). Total THC .—Maximum potential percentage w/w delta-9- tetrahydrocannabinol that the test sample could yield on a dry weight basis, including delta-9-THC and delta-9-THCA. 5 Method Performance Requirements See Tables 3 and 4. 6 System Suitability Tests and/or Analytical Quality Control Suitable methods will include blank check samples, and check standards at the lowest point and midrange point of the analytical range. A detailed description of the method’s dry weight procedures and calculations must be included. 7 Reference Material(s) See Tables 1 and 2 for sources of reference materials. Refer to Annex F: Development and Use of In-House Reference Materials in Appendix F: Guidelines for Standard Method Performance Requirements , Official Methods of Analysis of AOAC INTERNATIONAL (2019) 21st Ed., AOAC INTERNATIONAL, Rockville, MD, USA. Available at: http://www.eoma.aoac.org/ app_f.pdf 8 Validation Guidance An initial and important stage in cannabis testing is preparation of a homogeneous sample of the plant material. Detailed and complete procedures for reproducible preparation of test samples from the plant material must be addressed during method validation and those data must be included in the method validation submission. A detailed description of the method’s dry weight procedures and calculations must be included in the submission. Appendix D: Guidelines for Collaborative Study Procedures to Validate Characteristics of a Method of Analysis, Official Methods of Analysis of AOAC INTERNATIONAL (2019) 21st Ed., AOAC INTERNATIONAL, Rockville, MD, USA. Available at: http:// www.eoma.aoac.org/app_d.pdf Appendix F: Guidelines for Standard Method Performance Requirements, Official Methods of Analysis of AOAC INTERNATIONAL (2019) 21st Ed., AOAC INTERNATIONAL, Rockville, MD, USA. Available at: http://www.eoma.aoac.org/ app_f.pdf AppendixK: Guidelines for Dietary Supplements and Botanicals, Official Methods of Analysis of AOAC INTERNATIONAL (2019) 21st Ed., AOAC INTERNATIONAL, Rockville, MD, USA. Available at: http://www.eoma.aoac.org/app_k.pdf 9 Maximum Time-to-Result None Approved by attending stakeholders of the AOAC Cannabis Analytical Science Program (CASP) meeting on September 7, 2019. Final Version Date: October 3, 2019. Posted: October 9, 2019

Standard Method Performance Requirements (SMPRs ® ) for Quantitation of Cannabinoids in Plant Materials of Hemp (Low THC Varieties Cannabis sp.)

Intended Use: Consensus-Based Reference Method 1 Purpose AOAC SMPRs describe the minimum recommended performance characteristics to be used during the evaluation of a method. The evaluation may be an on-site verification, a single- laboratory validation, or a multi-site collaborative study. SMPRs are written and adopted by AOAC composed of representatives from the industry, regulatory organizations, contract laboratories, test kit manufacturers, and academic institutions. AOAC SMPRs are used by AOAC expert review panels in their evaluation of validation study data for methods being considered for Performance Tested Methods SM certification or AOAC Official Methods of Analysis SM adoption and can be used as acceptance criteria for verification at user laboratories. 2 Applicability The method will be able to identify and quantify individual cannabinoids (as listed in Tables 1 and 2) in plant materials expressed on a dry weight basis. The method must be able to report total THC (as defined in this SMPR), regardless of how it is measured. 3 Analytical Technique Any analytical technique(s) that measures the analytes of interest and meets the following method performance requirements is/are acceptable. 4 Definitions Hemp plant materials .—Fresh or dried, whole or milled plant material of low THC cultivars of Cannabis spp. Limit of quantitation (LOQ) .—Minimum concentration or mass of analyte in a given matrix that can be reported as a quantitative result. Quantitative method .—Method of analysis which response is the amount of the analyte measured either directly (enumeration in a mass or a volume), or indirectly (color, absorbance, impedance, etc.) in a certain amount of sample. Recovery .—Fraction or percentage of spiked analyte that is recovered when the test sample is analyzed using the entire method. Repeatability .—Variation arising when all efforts are made to keep conditions constant by using the same instrument and operator and repeating during a short time period. Expressed as the repeatability standard deviation (SD r ); or % repeatability relative standard deviation (%RSD r ).

Table 1. Required cannabinoids

Reference material Restek Cerilliant

Common name

Abbreviation

IUPAC name

CAS No.

Molecular structure

Cannabidiol

CBD

2-[(1 R ,6 R )-6-isopropenyl-3- methylcyclohex-2-en-1-yl]-5- pentylbenzene-1,3-diol

13956-29-1

Sigma-Aldrich API Standards Echo Pharm Lipomed AG Cerilliant USP Restek Lipomed AG Echo Pharmaceutical

Cannabidiolic acid

CBDA 2,4-Dihydroxy-3-[(1 R ,6 R )-3-methyl-6- prop-1-en-2-ylcyclohex-2-en-1-yl]-6- pentylbenzoic acid

1244-58-2

Cannabinol

CBN 6,6,9-Trimethyl-3-pentyl-benzo[ c ] chromen-1-ol

521-35-7

Cerilliant Restek

Tetrahydro-cannabinol

THC (−)-(6a R ,10a R )-6,6,9-trimethyl-3- pentyl-6a,7,8,10a-tetrahydro-6 H - benzo[ c ]chromen-1-ol

1972-08-3

Cerilliant USP Echo Pharmaceuticals Cerilliant USP Echo Pharmaceuticals

Tetrahydro-cannabinolic acid THCA (6a R ,10a R )-1-hydroxy-6,6,9-trimethyl- 3-pentyl-6a,7,8,10a-tetrahydro-6h- benzo[c]chromene-2-carboxylic acid

23978-85-0

Table 2. Additional, desirable cannabinoids Common name Abbreviation

IUPAC name

CAS No.

Molecular structure Reference material

Cannabichromene

CBC 2-Methyl-2-(4-methylpent-3- enyl)-7-pentyl-5-chromenol

20675-51-8

Cerilliant Sigma Aldrich Echo Pharmaceuticals

Cannabichromenic acid

CBCA 5-Hydroxy-2-methyl-2-(4- methyl-3-penten-1-yl)-7-pentyl- 2 H -chromene-6-carboxylic acid

20408-52-0

Cerilliant

Cannabidivarinic acid

CBDVA 2,4-Dihydroxy-3-[(1 R ,6 R )- 3-methyl-6-prop-1-en-2- ylcyclohex-2-en-1-yl]-6- propylbenzoic acid

31932-13-5

Cerilliant

Cannabigerol

CBG 2-[(2 E )-3,7-dimethylocta-2,6- dienyl]-5-pentyl-benzene-1,3- diol NIST: 1,3-Benzenediol,

25654-31-3

Cerilliant Lipomed AG Echo

Pharmaceuticals SPEX Certiprep Tocris (UK)

NIST: 2808-33-5

2-(3,7-dimethyl-2,6- octadienyl)-5-pentyl-

Cannabigerolic acid

CBGA 3-[(2 E )-3,7-dimethylocta- 2,6-dienyl]-2,4-dihydroxy-6- pentylbenzoic acid

25555-57-1

Cerilliant Echo

Pharmaceuticals SPEX Certiprep

Cannabidivarin

CBDV 2-((1 S ,6 S )-3-methyl-6-(prop- 1-en-2-yl) cyclohex-2-enyl)-5- propylbenzene-1,3-diol

24274-48-4

Cerilliant SPEX Certiprep

Δ8 Tetrahydro-cannabinol

Δ8 THC

6,6,9-Trimethyl-3- pentyl-6a,7,10,10a- tetrahydrobenzo[c]chromen- 1-ol

5957-75-5

Cerilliant SPEX Certiprep

Tetrahydro-cannabivarin

THCV

6,6,9-Trimethyl-3-propyl- 6a,7,8,10a-tetrahydro-6H- benzo[c]chromen-1-ol

28172-17-0

Cerilliant USP

Tetrahydrocannabivaric acid THCVA

39986-26-0

Cerilliant

Table 3. Method performance requirements (part 1) for cannabinoids Parameter Requirement a Limit of quantitation (LOQ), % ≤0.05 Analytical range (CBD and CBDA), % 0.05–35 Analytical range (others), % 0.05–5 a All calculated on dry weight basis.

Table 4. Method performance requirements (part 2) for cannabinoids

Analytical range, % a

Parameter

0.05–0.5 85–118

>0.5–5 90–111

5–35 b

Recovery, %

95–105

RSD r RSD

, %

≤5

≤3 ≤8

≤2

R ≤6 a All calculated on dry weight basis; observed values to be compared to indicated limits for acceptability. b Only applicable to CBD and CBDA. , % ≤10

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