OSAC 2022-S-0014 Building an Analytical 

Scheme for the Assessment of Tetrahydrocannabinol (THC)

in Suspected Marijuana Plant Material Samples

1

OSAC 2022-S-0014 Building an 

Analytical Scheme for the 

Assessment of 

Tetrahydrocannabinol (THC)

in Suspected Marijuana Plant 

Material Samples

Seized Drugs Subcommittee

Chemistry: Seized Drugs/Toxicology Scientific Area Committee

Organization of Scientific Area Committees (OSAC) for Forensic Science
OSAC 2022-S-0014 Building an Analytical 

Scheme for the Assessment of Tetrahydrocannabinol (THC)

in Suspected Marijuana Plant Material Samples

2

Draft OSAC Proposed Standard

OSAC 2022-S-0014 Building an 

Analytical Scheme for the Assessment 

of Tetrahydrocannabinol (THC)

in Suspected Marijuana

Plant Material Samples

Prepared by 

Seized Drugs Subcommittee

Version: 1.0

August 2022

Disclaimer:

This OSAC Proposed Standard was written by the Organization of Scientific Area Committees 

(OSAC) for Forensic Science following a process that includes an open comment period. This 

Proposed Standard will be submitted to a standards developing organization and is subject to 

change.

There may be references in an OSAC Proposed Standard to other publications under 

development by OSAC. The information in the Proposed Standard, and underlying concepts and 

methodologies, may be used by the forensic-science community before the completion of such 

companion publications.

Any identification of commercial equipment, instruments, or materials in the Proposed Standard 

is not a recommendation or endorsement by the U.S. Government and does not imply that the 

equipment, instruments, or materials are necessarily the best available for the purpose.
OSAC 2022-S-0014 Building an Analytical 

Scheme for the Assessment of Tetrahydrocannabinol (THC)

in Suspected Marijuana Plant Material Samples

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To be placed on the OSAC Registry, certain types of standards first must be reviewed by a 

Scientific and Technical Review Panel (STRP). The STRP process is vital to OSAC’s mission of 

generating and recognizing scientifically sound standards for producing and interpreting forensic 

science results. The STRP shall provide critical and knowledgeable reviews of draft standards or 

of proposed revisions of standards previously published by standards developing organizations 

(SDOs) to ensure that the published methods that practitioners employ are scientifically valid, 

and the resulting claims are trustworthy.

The STRP panel will consist of an independent and diverse panel, including subject matter 

experts, human factors scientists, quality assurance personnel, and legal experts, which will be 

tasked with evaluating the proposed standard based on a comprehensive list of science-based 

criteria.

For more information about this important process, please visit our website 

at: https://www.nist.gov/topics/organization-scientific-area-committees-forensicscience/scientific-technical-review-panels. 
OSAC 2022-S-0014 Building an Analytical 

Scheme for the Assessment of Tetrahydrocannabinol (THC)

in Suspected Marijuana Plant Material Samples

1

1 Rationale:

2 The Farm Bill of 2018 removed hemp from the Controlled Substance Act Schedule I and defines 

3 it as “...the plant Cannabis Sativa L. and any part of the plant, including the seeds thereof, all 

4 derivatives, extracts, cannabinoids, isomers, acids, salts, and salts of isomers, whether growing 

5 or not, with a delta-9-THC concentration of not more than 0.3% on a dry weight basis.” As such, 

6 the OSAC Seized Drugs Subcommittee has drafted this standard to assist forensic science service 

7 providers to analyze seized drug evidence submitted to their laboratories as suspected Marijuana.

8 Standard Practice for 

9 Building an Analytical Scheme for the Assessment of Tetrahydrocannabinol (THC) in 

10 Suspected Marijuana Plant Material Samples

11 1. Scope

12 1.1. This standard covers options for building an analytical scheme for the analysis and 

13 identification of suspected marijuana plant material in seized drugs.

14 1.2. This standard is intended for use by competent forensic science practitioners (FSPs) with 

15 the requisite formal education, discipline-specific training (see Practice E2917 and 

16 Practice E2326), and demonstrated proficiency to perform forensic casework.

17 1.3. This standard does not purport to address all of the safety concerns, if any, associated 

18 with its use. It is the responsibility of the user of this standard to establish appropriate 

19 safety and health practices and determine the applicability of regulatory limitations prior 

20 to use.

21

22 2. Referenced Documents 

2.1. ASTM Standards2 23

24 2.1.1. E1732 Terminology Relating to Forensic Science

25 2.1.2. E2326 Practice for Education and Training of Seized-Drug Analysts

26 2.1.3. E2548 Guide for Sampling Seized Drugs for Qualitative and Quantitative 

27 Analysis

28 2.1.4. E2549 Practice for Validation of Seized-Drugs Analytical Methods

29 2.1.5. E2917 Practice for Forensic Science Practitioner Training, Continuing Education, 

30 and Professional Development Programs

31

32 2.2. Other Documents

33 2.2.1. Establishment of a Domestic Hemp Production Program; Federal Register, vol. 

34 86, No, 11 January 19, 2021

35 2.2.2. SWGDRUG Recommendations Version 8.0, 2019 

36 (https://www.swgdrug.org/Documents/SWGDRUG%20Recommendations%20V

37 ersion%208_FINAL_ForPosting_092919.pdf)

38 2.2.3. United Nations Office on Drugs and Crime (UNODC) Recommended methods 

39 for the identification and analysis of Cannabis and Cannabis products, 2022. 
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40 (https://www.unodc.org/documents/scientific/Recommended_methods_for_the_i

41 dentification_and_analysis_of_cannabis_and_cannabis_products.pdf)

42

43

44 3. Terminology

45 3.1. Definitions:

46 3.1.1. For definitions of terms used in this practice, refer to Terminology E1732. 

47 3.2. Definitions of terms specific to this standard:

48 3.2.1. Cannabis, n - a genus of flowering plants in the family Cannabaceae of which 

49 Cannabis sativa is a species, and Cannabis indica and Cannabis ruderalis are 

50 subspecies thereof. Cannabis refers to any form of the plant where the total 

51 delta-9 tetrahydrocannabinol concentration on a dry weight basis has not yet been 

52 determined. (DOA 7 CFR Part 990 Establishment of a Domestic Hemp 

53 Production Program)

54 3.2.1.1. Discussion - “The chemical and morphological distinctions by which 

55 Cannabis has been split into these subspecies are often not readily 

56 discernible, appear to be environmentally modifiable, and vary in a 

57 continuous fashion. For most purposes, it will suffice to apply the name 

58 Cannabis sativa to all Cannabis plants encountered.” (United Nations 

59 Office on Drugs and Crime (UNODC) Recommended methods for the 

60 identification and analysis of Cannabis and Cannabis products, 2022.)

61 3.2.2. decarboxylation, n - the removal or elimination of a carboxyl group from a 

62 molecule or organic compound. (DOA 7 CFR Part 990 Establishment of a 

63 Domestic Hemp Production Program)

64 3.2.3. decision point, n - an administratively defined cutoff or concentration that is at or 

65 above the method’s limit of detection or limit of quantitation and is used to 

66 discriminate between positive and negative results. (Scientific Working Group 

67 for Forensic Toxicology (SWGTOX), “Scientific Working Group for Forensic 

68 Toxicology (SWGTOX) Standard Practices for Method Validation in Forensic 

69 Toxicology.” Journal of Analytical Toxicology, 37:7, 452-474, 2013.)

70 3.2.4. dry weight basis, n - a basis for expressing the percentage of a chemical in a 

71 substance after removing the moisture from the substance. Percentage of THC 

72 on a dry weight basis means the percentage of THC, by weight, in a Cannabis 

73 item, after excluding moisture from the item. (DOA 7 CFR Part 990 

74 Establishment of a Domestic Hemp Production Program)

75 3.2.5. hemp, n - the plant species Cannabis sativa L., and any part of that plant, 

76 including the seeds thereof and all derivatives, extracts, cannabinoids, isomers, 

77 acids, salts, and salts of isomers, whether growing or not, with a total delta-9 

78 tetrahydrocannabinol concentration of not more than 0.3 percent on a dry weight 

79 basis. (DOA 7 CFR Part 990 Establishment of a Domestic Hemp Production 

80 Program) 

81 3.2.6. inconclusive results, n - results that do not meet criteria for reporting, or were 

82 unsuitable due to analytical interferences or condition of the sample. 

83 (ANSI/ASB Standard 053)
OSAC 2022-S-0014 Building an Analytical 

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84 3.2.7. internal standard, n - a compound of known concentration added to a sample to 

85 facilitate the qualitative identification and/or quantitative determination of the 

86 sample components (ISO 20752) 

87 3.2.8. marijuana, n - or “marihuana” as defined in the Federal Controlled Substances 

88 Act (CSA) means all parts of the plant Cannabis sativa L., whether growing or 

89 not; the seeds thereof; the resin extracted from any part of such plant; and every 

90 compound, manufacture, salt, derivative, mixture, or preparation of such plant, its 

91 seeds or resin. The term “marihuana” does not include hemp and does not 

92 include the mature stalks of such plant, fiber produced from such stalks, oil or 

93 cake made from the seeds of such plant, any other compound, manufacture, salt, 

94 derivative, mixture, or preparation of such mature stalks (except the resin 

95 extracted therefrom), fiber, oil, or cake, or the sterilized seed of such plant which 

96 is incapable of germination. “Marihuana” means all Cannabis that tests as 

97 having a THC concentration level of higher than 0.3 percent on a dry weight 

98 basis. (DOA 7 CFR Part 990 Establishment of a Domestic Hemp Production 

99 Program)

100 3.2.9. total THC, n - the value determined after the process of decarboxylation, or the 

101 application of a conversion factor if the testing methodology does not include 

102 decarboxylation, that expresses the potential total delta-9 tetrahydrocannabinol 

103 content derived from the sum of the THC and THCA content and reported on a 

104 dry weight basis. (DOA 7 CFR Part 990 Establishment of a Domestic Hemp 

105 Production Program)

106 3.2.9.1. Discussion - delta-9 THCA is a component of Cannabis that 

107 decarboxylates to delta-9 THC when heated. Also known as delta-9 

108 Tetrahydrocannabinolic Acid or delta-9 THC Carboxylic Acid.

109 3.2.10. THC, n - for the purpose of this standard, refers primarily to delta-9 THC, but can 

110 include other THC isomers (e.g., delta-8-THC) depending on jurisdictional 

111 requirements. 

112 3.2.11. trichome, n - hair-like projections from a plant epidermal cell. (United Nations 

113 Office on Drugs and Crime (UNODC) Recommended methods for the 

114 identification and analysis of Cannabis and Cannabis products, 2022.)

115 3.2.12. isomers, n - Compounds that have the same elemental formula, but have different 

116 structural configurations, and different physical and/or chemical properties.

117 (Retrieved January 26, 2022 from OSAC lexicon, 

118 https://lexicon.forensicosac.org/) 

119

120 4. Significance and Use

121 4.1. An analytical scheme is created to generate results for the assessment of THC in the 

122 analysis of suspected marijuana in seized-drug evidence. An analytical scheme is a 

123 combination of selected techniques used to reach a result, and is comprised of validated 

124 analytical methods that are appropriate for the analyte(s) or properties of interest. The 

125 combination of techniques chosen should aim to minimize false positives and false 

126 negatives.

127 NOTE 1 – This standard provides information that could assist in the differentiation 

128 between marijuana and hemp. 
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129 4.1.1. Identification only analytical schemes that do not include a decision point 

130 analysis or quantitative analysis cannot differentiate hemp from marijuana. They 

131 only provide information for the identification of Cannabis. 

132 4.2. This Practice applies to plant material only, and does not cover derivatives, mixtures, or 

133 preparations such as concentrates, oils, or edibles.

134 4.3. These techniques cannot determine subspecies.

135

136 5. Sampling and Storage

137 5.1. If sampling in the field, follow DOA 7 CFR Part 990 Establishment of a Domestic Hemp 

138 Production Program.

139 5.2. Random sampling should be conducted (see Guide E2548) to address variations of THC 

140 content. 

141 5.2.1. If one unit is received, sample portions from different areas within the unit.

142 5.2.2. If multiple units are received, do not combine. Use a sampling plan (e.g., 

143 hypergeometric approach, sample selection, sampling to penalty) to determine 

144 the number of units to sample individually.

145 5.2.3. Stems, stalks, and seeds should be excluded from sampling for qualitative and 

146 quantitative analysis.

147 5.3. Packaging/Storage - Fresh plant material should be packaged to allow the samples to dry 

148 (e.g., paper bags or perforated cardboard boxes), minimizing the amount of moisture and 

149 deterioration of the plant material. 

150 5.3.1. THC is sensitive to air and UV light, therefore storage in a dark and cool place is 

151 recommended. (UNODC Recommended methods for the identification and 

152 analysis of Cannabis and Cannabis products, 2022).

153 5.3.2. The laboratory can establish additional procedures to refrigerate plant material 

154 samples.

155 5.3.3. If samples are received in a deteriorating state, the samples can still be analyzed. 

156 Document the state of the evidence in the case file.

157

158 6. Building an Analytical Scheme

159 6.1. The combination of analyses can be selected based on the information required (i.e.

160 quantitation vs. decision point). Figure 1 illustrates the combination of testing that can be 

161 performed to identify marijuana. The individual tests are described in detail in the 

162 subsequent sections.

163 6.2. Minimum test requirements 

164 6.2.1. Morphological Assessment

165 6.2.1.1. If a negative result is observed, this standard is no longer applicable.

166 6.2.2. An analysis that provides structural data to confirm the presence of THC

167 6.2.2.1. This can be combined with the decision point analysis or full quantitation 

168 if those tests provide structural data.

169 6.2.3. An analysis to assess the amount of THC present in the item (decision point or 

170 full quantitation, or both).

171 6.3. The analytical scheme provides a scientifically supported conclusion when each 

172 technique achieves the level of selectivity required and the positive test results 

173 corroborate each other. (SWGDRUG Recommendations Version 8.0, SWGDRUG, 2019)
OSAC 2022-S-0014 Building an Analytical 

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174 6.4. Additional testing can be completed as described in Section 8.

175

176

177

178
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179

180 Figure 1: Suspected marijuana analysis scheme flowcharts

181

182 7. Morphological Assessment

183 7.1. Macroscopic Examination differentiates between plant material and non-plant material 

184 exhibits. Additional macroscopic observations can include documentation of color, 

185 stems/fruiting stalks, form (e.g., loose, compressed, ground), presence of seeds, palmate 

186 leaves with 3-11 leaflets, individual leaves with ellipsoid blade and serrated edges.

187 7.2. Microscopic Examination is conducted using a microscope with magnification of at least 

188 10 times (e.g., 10-40x). Document observed features of the plant material; a picture can 

189 be captured to document the observations. These features can include:

190 7.2.1. Unicellular cystolithic trichomes found on the upper surface of the leaves with a 

191 characteristic bear-claw shape. See Figure 2 for an illustration.

192 7.2.1.1. Unicellular cystolithic trichomes contain a crystal of calcium carbonate 

193 at the base. Addition of dilute acid to the plant material surface, and 

194 observation of the resulting effervescence of the carbon dioxide formed 

195 as a result of the chemical reaction, can aid in distinguishing these hairs 

196 from other unicellular covering trichomes but is not required.

197 7.2.2. Multicellular glandular trichomes are found on the upper and lower surfaces of 

198 the leaves and have a shiny appearance. See Figure 2 for an illustration.

199
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200

201 Figure 2: Figure 2 depicts cystolithic hairs and multicellular glandular trichomes 

202 of the Cannabis plant material at a magnification of 50X (source: DuPage county 

203 Sheriff’s Office).

204 7.2.3. Observation of cystolithic hairs alone is not sufficient to report marijuana. The 

205 simultaneous presence of cystolithic trichomes on the upper surface of the leaves, 

206 along with the presence of non-cystolithic trichomes on the lower surface of the 

207 leaves must be observed (UNODC Recommended methods for the identification 

208 and analysis of Cannabis and Cannabis products, 2022). 

209 7.2.4. It should be noted, however, that very immature seedlings and stems with no 

210 leaves attached cannot be definitively identified as Cannabis by botanical 

211 examination. (UNODC Recommended methods for the identification and 

212 analysis of Cannabis and Cannabis products, 2022).

213

214 8. Qualitative Analysis

215 8.1. Color Tests

216 8.1.1. Duquenois-Levine - Place a small amount of plant material (30 mg - 100 mg) in a 

217 test tube or other container. Cover with petroleum ether (or other organic solvent) 

218 to extract the cannabinoids into the solvent, filter or decant the solution to 

219 remove the residual plant material, evaporate to dryness, and add a small amount 

220 of Duquenois reagent and an equal amount of concentrated hydrochloric acid. A 

221 blue to purple color should develop within a few minutes. Add a small amount 

222 of chloroform or methylene chloride, shake, and let the layers separate. A violet 

223 to purple color in the organic layer indicates a positive test for cannabinoids.

224 8.1.1.1. Alternatively, the Duquenois reagent and concentrated hydrochloric acid 

225 can be added directly to a small amount of plant material.

226 8.1.1.2. Duquenois reagent consists of 0.5 mL acetaldehyde and 0.4 g vanillin in 

227 20 mL of ethanol (e.g., 95%, 200 proof). Store the solution in a cool dark 

228 place and discard if it assumes a deep yellow color. (UNODC 

229 Recommended methods for the identification and analysis of Cannabis 

230 and Cannabis products, 2022) The reagent preparation can be scaled up 

231 or down as needed.
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232

233 Figure 3A Figure 3B Figure 3C

234 Figures 3A-3C: Figure 3A depicts resulting observations when the Duquenois reagent is 

235 added to samples of cannabidiol (CBD), THC, and suspected marijuana plant material. 

236 Figure 3B depicts the resulting observations when an equal volume of concentrated 

237 hydrochloric acid is added. Figure 3C depicts the resulting observations when 

238 chloroform or methylene chloride is added. (source DEA)

239 8.1.2. 4-Aminophenol (4-AP) - A small amount of material (5 mg) can be placed in a 

240 test tube or spot plate and covered with the 4-AP Reagent A Solution. Add 2-4 

241 drops of the Reagent B solution and wait 1-2 minutes. A blue color is indicative 

242 of the THC concentration being greater than the cannabidiol (CBD) 

243 concentration in the sample. A pink color is indicative of the THC concentration 

244 being less than the CBD concentration in the sample. If the concentration of 

245 THC is equivalent to the concentration of CBD, the test will be inconclusive.

246 8.1.2.1. Note: 4-AP Reagent A consists of 300 mg of 4-aminophenol, 5 mL of 

247 2N HCl, and 995 mL of ethanol (e.g., 95%, 200 proof). Reagent B 

248 consists of 30 g sodium hydroxide, 300 mL of water, and 700 mL of 

249 ethanol. The reagent preparation can be scaled up or down as needed.

250

251

252 Figure 4A Figure 4B

253 Figures 4A and 4B: Figure 4A depicts resulting observations when the 4-AP test Reagent 

254 A added to samples of CBD, THC, and suspected marijuana plant material. Figure 4B 

255 depicts resulting observations when the 4-AP test Reagent B is added to the same 

256 samples of CBD, THC, and suspected marijuana plant material. The color blue will 

257 appear for a sample where THC was in greater concentration than CBD. (source: DEA)

258 8.2. Thin Layer Chromatography (TLC) - TLC can be used to compare the retention factor of 

259 the cannabinoid to that of a reference material. Possible TLC plates include silica gel G 

260 250 micron. Possible solvent systems include 4:1 Petroleum Ether:Diethyl ether or 4:1 
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261 Hexane:Diethyl Ether. Visualization reagents that can be utilized are Fast Blue B, Fast 

262 Blue 2B spray and iodine (vapor).

263 8.2.1. Use method validation data to determine the acceptance criteria for retention 

264 factor comparisons. For example, the retention factor of the analyte can be 

265 within 5% of the retention factor of the reference material).

266 8.2.2. Alternatively, visually compare the sample spots to the reference spots at the 

267 greatest density in position and color. Some visualization reagents also allow for 

268 color differences between the substances present.

269 8.2.3. A picture of the TLC plate can be captured to document the observations.

270

271

272

273

274

275

276

277

278

279

280 Figure 5: TLC plate developed using 4:1 petroleum ether:diethyl ether solvent system, 

281 silica gel G 250 micron plate, and visualized using iodine (vapor). Source: GBI DOFS

282
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283

284 Figure 6: TLC plate developed using 4:1 hexane:diethyl ether solvent system and 

285 visualized using Fast Blue B spray. Source: NMS Labs

286 8.3. Gas Chromatography/Mass Spectrometry (GC/MS) - GCMS can be used for the 

287 identification of individual cannabinoids by comparing the retention time, mass spectrum, 

288 or both, to reference materials. 100% dimethylpolysiloxane, 5% (phenyl)-

289 methylpolysiloxane, or 35% phenyl 65% dimethylpolysiloxane stationary phases are 

290 routinely utilized, with a 1μL injection volume and inlet temperature of at least 250 °C. 

291 The oven temperature program can vary. Individual method set points including inlet 

292 temperature, temperature program, flow rates, and column chemistry are evaluated during 

293 method development and tested during method validation.

294 8.3.1. Example oven temperature program: initial temperature 220°C, ramping at 

295 1°C/minute, to a final temperature of 250°C, holding for 2 minutes.

296

297 9. Qualitative Analysis Using Decision Point Thresholds

298 9.1. The qualitative analysis of Cannabis samples using a decision point threshold can be 

299 performed in a number of ways. However, procedures typically follow the same basic 

300 steps.

301 NOTE 2 – Decision point thresholds are above those jurisdictionally defined as legal 

302 thresholds. The measurement uncertainty around the decision point should not encompass 

303 the legal threshold.

304 9.2. Sample Preparation

305 9.2.1. Drying (optional) - Dry plant material in an oven to obtain a sample dry enough 

306 to be homogenized (e.g., 1 hour at 60 °C).

307 9.2.2. Homogenization (optional) - Grind plant material with a device such as a 

308 disposable hand-held herb grinder or mortar and pestle. The material sampled for 

309 homogenization should exclude stalks, stems, roots, and seeds.

310 9.2.3. Extraction - Weigh a sample of plant material. The amount of sample to weigh is 

311 determined during validation of the method. If the material was not previously 

312 homogenized, crumble the weighed plant material (approximately 50 mg) and 

313 place into a test tube. Add extraction solution. Allow the plant material to extract 

314 for approximately 10-15 minutes or a period of time as determined during 
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315 method development or optimization. Vortex during this time. After extraction 

316 the solution can be filtered or centrifuged and the supernatant collected to remove 

317 particulates.

318 9.2.4. Use an Internal Standard – An internal standard solution can be added as part of 

319 the extraction solvent or added post extraction. Common internal standards 

320 utilized are 4-Androstene-3,17-dione, tribenzylamine (TBA), testosterone, and 

321 deuterated delta-9-THC. The concentration of the internal standard is typically 

322 the cutoff value for the defined decision point.

323 9.2.5. Aliquot Samples for Instrumental Analysis - Pipette a set volume of extract into a 

324 test tube and add internal standard if internal standard is not part of the extraction 

325 solvent. Vortex and transfer to an autosampler vial. If samples are believed to be 

326 high concentration, a dilution can be performed prior to analysis. 

327 9.2.6. Derivatization (optional) - Derivatization can be conducted to obtain 

328 identification of THC and THCA separately as opposed to the total THC. Add 

329 the derivatizing agent (e.g., BSTFA-TMCS) to the extracted solution containing 

330 the internal standard or the dried residue from the extracted solution. The time 

331 and temperature at which the sample is derivatized as well as appropriate 

332 volumes of sample and derivatizing agent are determined during method 

333 validation.

334 9.3. Preparation of Calibrators and Controls 

335 9.3.1. Decision Point with One-point Comparison

336 9.3.1.1. THC Calibrator at Decision Point - Prepare a THC standard by diluting a 

337 certified reference material (CRM) to an appropriate concentration in 

338 solvent. Then add a set volume of the standard to a test tube and add 

339 internal standard. Vortex and transfer to an autosampler vial.

340 9.3.1.2. CBD Conversion Control - Because of potential conversion of CBD to 

341 THC in the GC injection port when the sample is analyzed underivatized, 

342 procedures that use GC with no derivitzation should include a CBD 

343 conversion control. This can be prepared at a high concentration to 

344 demonstrate no conversion to THC or used to determine a cut-off 

345 concentration above which the THC result cannot be used. The FSSP 

346 should assess the conversion of CBD to THC during method 

347 development and validation. Frequency of injection port maintenance, 

348 consumables used in the injection port, and amount of CBD present in 

349 samples can affect the magnitude of conversion. This should be taken 

350 into account in validation experiments. Prepare a CBD standard by 

351 diluting a CRM to an appropriate concentration in solvent. Add a set 

352 volume of standard to a test tube and add internal standard. Vortex and 

353 transfer to an autosampler vial. The CBD conversion control should be 

354 analyzed throughout the run to monitor conversion.

355 9.3.1.3. THC Control at Decision Point - Prepare a second THC standard from a 

356 different CRM to an appropriate concentration in solvent. This can be 

357 done using a different lot of CRM, or a different manufacturer. Add a set 

358 volume of standard to a test tube and add internal standard. Vortex and 

359 transfer to an autosampler vial.

360 9.3.2. Decision Point with Internal Standard

361 9.3.2.1. THC control above decision point - prepare a THC standard by 

362 dissolving a reference material to an appropriate concentration (above 
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363 the decision point) in internal standard solution. Vortex and transfer to an 

364 autosampler vial.

365 9.3.2.2. THC control below decision point - prepare a second THC standard by 

366 dissolving a reference material to an appropriate concentration (below 

367 the decision point) in internal standard solution. Vortex and transfer to an 

368 autosampler vial.

369 9.3.2.3. Alternatively, controls can be prepared by extracting well-characterized 

370 plant reference materials that are above and below the decision point, 

371 using the same preparation procedure as sample(s) (see 8.2 above).

372 9.4. Instrumental Analysis

373 9.4.1. Gas Chromatography/Mass Spectrometry (GC/MS) - Acquire data by analyzing 

374 samples, calibrators, and controls (as applicable) on an appropriate, validated 

375 method. A 5% (phenyl)-methylpolysiloxane stationary phase is routinely utilized, 

376 along with a 1μL injection volume and an inlet temperature of 250 °C. The oven 

377 temperature program can vary. Data acquisition can be performed using full scan, 

378 SIM or SIM/scan. Individual method set points including inlet temperature, 

379 temperature program, flow rates, and column chemistry are evaluated during 

380 method development and tested during method validation.

381 9.4.2. Gas Chromatography/Flame Ionization Detection (GC/FID) - A second aliquot of 

382 the extracted sample is often analyzed by GC/FID. A 100% 

383 dimethylpolysiloxane stationary phase is often utilized. Instrument parameters 

384 are usually similar or the same as those used during the GC/MS analysis.

385 9.4.3. The method should be able to resolve delta-6a,10a-THC, delta-7-THC, delta-8-

386 THC, delta-9-THC, delta-10-THC, and other THC isomers present in Cannabis. 

387 Method validation should include an assessment of interference to ensure the 

388 presence of multiple cannabinoids will not prevent the accurate determination of 

389 analytes of interest.

390 9.5. Data Analysis

391 9.5.1. If analysis is performed using a one-point threshold, create a one-point 

392 comparison and calculate the amount of THC in case samples and control 

393 samples. 

394 9.5.2. If analysis is performed using a decision point with internal standard, calculate 

395 the ratio of sample THC to internal standard, using either peak area or peak 

396 height.

397

398 10. Quantitative Analysis 

399 10.1. Drying 

400 10.1.1. Quantitation is performed on plant material on a dry weight basis. During method 

401 validation, determine a drying time and temperature that renders the majority of 

402 samples seen in casework sufficiently dry for quantitative analysis. In order to 

403 dry the sample, one of the following procedures should be performed: 

404 10.1.1.1. Moisture balance - The moisture balance will dry and weigh a sample. 

405 The loss on drying can be calculated and applied as a correction to the 

406 quantitative value obtained. The sample weighed on the moisture balance 

407 should not be the same sample used to perform the quantitation.

408 10.1.1.2. Obtain a weight of the sample. Dry plant material in an oven for 

409 approximately 1 hour at 60 ͒C. Re-weigh the sample. If the change in 
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410 weight is greater than the acceptance criteria established in validation, 

411 place the sample back in the oven for further drying. Dry until the change 

412 in weight meets validation acceptance criteria. 

413 10.1.1.3. Dry to constant weight - Continue drying until two consecutive 

414 weighings do not differ by more than 0.50 mg per g of substance taken. 

415 (USP-NF General Notices and Requirements, 6.40.20, GUID-6E790F63-

416 0496-4C20-AF21-E7C283E3343E_6_en-US)

417 10.2. Homogenization

418 10.2.1. Grind plant material with a device, such as mortar and pestle. The material 

419 sampled for homogenization should exclude stalks, stems, roots, and seeds.

420 10.3. Weigh Quantitation Sample(s)

421 10.3.1. Weigh an aliquot of the homogenized material for extraction. The aliquot weight 

422 should be determined in method validation. 

423 10.4. Extraction

424 10.4.1. Add extraction solution and internal standard solution (if applicable) and 

425 vortex/rotate . Sonicate/rotate the plant material to facilitate extraction of the 

426 cannabinoids. Sonicate/rotate the samples for approximately 10-15 minutes or a 

427 set period of time that is determined during validation of the method. 

428 Centrifuge/filter the samples and collect the supernatant to exclude particulates.

429 10.5. Dilution (Optional) 

430 10.5.1. Pipette a set volume of supernatant into a test tube. Dilute with solvent or mobile 

431 phase at an appropriate volume so quantitated samples will fall within the 

432 calibration curve.

433 10.6. Additional Sample Preparation (Optional)

434 10.6.1. Perform a liquid/liquid extraction or solid phase extraction on a specified volume 

435 of supernatant. This step can be used for additional sample clean up prior to 

436 instrumental analysis if required.

437 10.7. Derivatization (Optional)

438 10.7.1. To prevent conversion of THCA to THC, the sample can be derivatized using 

439 BSTFA-TCMS. This will allow for quantitation of the components separately if 

440 analysis is performed on a platform such as GC/MS where decarboxylation will 

441 occur during analysis. The time and temperature at which the sample is 

442 derivatized as well as appropriate volumes of sample and derivatizing agent 

443 should be determined during method validation.

444 10.8. Preparation of Calibrators and Control

445 10.8.1. Prepare THC calibration standards by diluting a CRM to appropriate 

446 concentrations in the extraction/dilution solvent and internal standard (if 

447 applicable). Vortex/rotate and transfer to an autosampler vial.

448 10.8.2. Prepare THC control samples by diluting a different lot (or vendor) of CRM to 

449 appropriate concentrations in the extraction/dilution solvent and internal standard 

450 (if applicable). Vortex/rotate and transfer to an autosampler vial.

451 10.8.3. Prepare CBD conversion control if performing quantitative analysis using a 

452 heated method without derivatization by diluting a CRM to appropriate 

453 concentrations in the extraction/dilution solvent and internal standard (if 

454 applicable). Vortex/rotate and transfer to an autosampler vial. 

455 10.8.4. Any calibrators and controls should be prepared using the same process as the 

456 samples. For instance, if sample preparation requires derivatization, the 

457 calibrators and controls should also be derivatized. 

458 10.9. Instrumental Analysis
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459 10.9.1. Analyze the THC calibration standards, THC controls, and samples on a 

460 validated method. The method should be able to resolve delta-6a,10a-THC, delta461 7-THC, delta-8-THC, delta-9-THC, delta-10-THC, and other THC isomers 

462 present in Cannabis. Method validation should include an assessment of 

463 interference to ensure the presence of multiple cannabinoids will not prevent the 

464 accurate quantitation of analytes of interest.

465 10.9.2. Multi-point calibration curves are preferable to calculate the amount of THC in 

466 case samples.

467 10.9.2.1. The recommended minimum is three calibrators, not including the origin.

468 10.9.2.2. A single-point calibration can be valid for quantitation as long as method 

469 validation includes assessing linearity, and the y-intercept is shown to be 

470 negligible.

471 10.9.3. Establish acceptance criteria for any control samples analyzed. These criteria 

472 must be met for the data to be considered acceptable and results reported for case 

473 samples

474 10.9.4. Instrumental analysis can be performed on a variety of platforms. Example 

475 instrument parameters are listed below. Alternatives are acceptable as long as the 

476 method is validated. 

477 10.9.4.1. LC:

478 ● C18 columns are routinely used.

479 ● Routine mobile phases can include 0.1% v/v formic acid in water:0.05% 

480 v/v formic acid in methanol or 0.1% TFA in water:0.1% TFA in 

481 acetonitrile.

482 10.9.4.2. GC

483 ● Routine stationary phases can include 100% dimethylpolysiloxane, 5% 

484 (phenyl)-methylpolysiloxane, or 35% phenyl 65% dimethylpolysiloxane. 

485

486 11. Uncertainty of Measurement

487 11.1. Calculate the uncertainty of measurement for quantitative analysis and qualitative 

488 decision point analyses at the threshold value. 

489 11.1.1. Measurement uncertainty is reported with quantitative results and for statements 

490 of conformity (see ISO/IEC 17025).

491 11.1.2. The uncertainty for the method should be calculated at the decision point 

492 threshold. As a quantitative value is not reported, a specific uncertainty value is 

493 not reported for samples.

494 11.2. There are a variety of approaches that can be used for the determination of measurement 

495 uncertainty. At a minimum, uncertainty from sampling and the method of analysis should 

496 be included when determining uncertainty of measurement for quantitative analysis 

497 (Supplemental Document SD-4: Measurement Uncertainty for Quantitative 

498 Determinations in Seized Drug Analysis, SWGDRUG, 2013). The following are 

499 examples of components that can be assessed when calculating measurement uncertainty:

500 11.2.1. Calibration material

501 11.2.2. Balances used to weigh aliquots

502 11.2.3. Uncertainty associated with volumetric glassware and pipettes

503 11.2.4. Measurement reproducibility/imprecision

504 11.2.5. Method bias
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505

506 12. Reporting Language

507 12.1. THC is not identified in analysis

508 12.1.1. Samples can be reported as THC not present/detected or marijuana negative/not 

509 detected

510 12.2. Qualitative Analysis Using Decision Point Threshold 

511 12.2.1. Above Decision Point Threshold

512 12.2.1.1. If the testing scheme includes morphological examination to identify the 

513 Cannabis plant and the amount of THC or total THC is greater than the 

514 decision point, the sample can be reported as “marijuana” or the term 

515 defined in the respective State law. 

516 12.2.1.2. The THC content can be reported as greater than the decision point 

517 threshold. 

518 12.2.2. Below Decision Point Threshold

519 12.2.2.1. If the testing scheme includes an analysis to identify the Cannabis plant 

520 and the amount of THC or total THC is less than the decision point, the 

521 sample can be reported as “inconclusive for marijuana” or the THC 

522 content as less than the decision point threshold.

523 12.2.3. Alternatively reports can contain a statement with an explanation of the results if 

524 greater than/less than the decision point is not indicated for each item. See 

525 examples below:

526 12.2.3.1. This item was tested using Gas Chromatography-Mass Spectrometry 

527 (GC-MS), macroscopic examination, microscopic examination, and color 

528 test(s). The <FSSP Name> uses a decision point threshold of <## %> 

529 delta-9-tetrahydrocannabinol (THC) content in plant material, without 

530 decarboxylation of tetrahydrocannabinolic acid (THCA), to conclusively 

531 identify marijuana. Items above <decision point threshold value> are 

532 reported as “marijuana” and items below <decision point threshold 

533 value> are reported as “Inconclusive, not able to differentiate between 

534 marijuana or hemp.” Quantitative (purity) analysis was not performed.

535 12.2.3.2. Inconclusive - A determination of inconclusive indicates that the plant 

536 material was unable to be identified as marijuana or hemp based on the 

537 analytical results obtained from the analytical scheme.

538 12.3. Quantitative Analysis

539 12.3.1. Purity values with associated uncertainty are reported when quantitative analysis 

540 is performed on samples. The report can contain a result pertaining to 

541 “marijuana” or the term defined in the respective State law as appropriate.

542 12.3.1.1. Quantitative result is above 0.3% THC: Marijuana can be reported in 

543 addition to the purity.

544 12.3.2. Quantitative result is below 0.3% THC or the uncertainty of measurement 

545 encompasses 0.3% THC: Samples can be reported as Cannabis, hemp, or 

546 marijuana negative/not detected along with the purity.

547 12.3.3. Quantitative result for THC is below the limit of quantitation (LOQ): In this 

548 situation a purity value is not reported. Samples can be reported as not present 

549 above the reporting limit, below the value of the low calibrator (##), or below the 

550 LOQ (##). In regards to the marijuana result, samples can be reported as 

551 Cannabis, hemp, or marijuana negative/not detected.
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552

553 13. Quality Assurance

554 13.1. Quality control samples will be analyzed with each instrumental analytical run. Establish 

555 acceptance criteria for control samples. These criteria must be met for the results obtained 

556 for unknown samples to be reported. 

557 13.1.1. Negative controls:

558 13.1.1.1. Solvent blanks are used to determine that the instrument is free from 

559 contamination/carryover. 

560 13.1.1.2. Method or procedural blanks (e.g., internal standard blank)/reagent 

561 blanks are quality control samples used to assess the process. They 

562 ensure that the reagents used to prepare the samples are free from 

563 contamination.

564 13.1.2. Positive controls:

565 13.1.2.1. Positive controls are samples of known concentration analyzed on the 

566 same method as casework samples. They ensure the method is producing 

567 acceptable results. Positive control concentrations are chosen so they 

568 encompass the analytical measurement range. In analyses where there is 

569 a legal threshold, controls can be prepared above and below the legal 

570 limit.

571 13.2. Validation

572 13.2.1. Method validations should be to conducted to evaluate each method for the 

573 following when applicable (see Practice E2549): sensitivity, specificity, 

574 selectivity, detection limits, accuracy, precision, effects of decarboxylation, and 

575 any interferences from other cannabinoids (e.g., in situ production) or other 

576 commonly seen substances.

577 13.3. Limitations 

578 13.3.1. Limitations associated with instrumental analysis

579 13.3.1.1. Cannabinoid acids decarboxylate in a GC injection port if samples are 

580 not derivatized. If analysis is performed by GC/MS or GC/FID without 

581 derivatization, the delta-9-THC result will include free delta-9-THC and 

582 decarboxylated THCA. CBDA will also decarboxylate to CBD.

583 13.3.1.2. It is possible for cannabinoids to interconvert to some extent under 

584 different conditions. The potential for degradation and conversion should 

585 be evaluated during method development and validation and monitored 

586 when necessary during analysis of casework. 

587 13.3.2. Limitations associated with color tests

588 13.3.2.1. Color tests are presumptive tests. Other cannabinoids and non589 cannabinoid compounds with similar structural features can result in the 

590 same color changes as the analyte of interest.

591 13.3.3. Limitations associated with TLC

592 13.3.3.1. Thin layer chromatography is a comparison technique. More than one 

593 compound can have the same retention factor. Potential interferences 

594 should be assessed and documented during validation.

595

596 14. Keywords

597 14.1. Cannabis; Marijuana; Tetrahydrocannabinol; Seized Drugs
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598

599 Appendices

600 XI. Table of Summary of Analytical Tests

Technique Qualitative Decision Point 

Threshold

Quantitative

Morphological 

Assessment

X

Duquenois Levine X

FBBB X

4-AP X

TLC X

GC/FID X X X

GC/MS X X X

LC-UV X X X

LC/MS X X X

601

602

603 XII. Examples of Analytical Schemes

604

605 Example Scenario 1: Determine if the sample is Marijuana using a full mass spectral scan decision point 

606 threshold.

Technique Result Test Type

Morphological Assessment cystolithic hairs Qualitative

GC/MS THC over the decision point threshold Decision Point Threshold

607 Reporting: The sample is Marijuana.

608

609 Example Scenario 2: Plant material submitted to determine if the sample is Marijuana using a decision 

610 point threshold.
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Technique Result Test Type

Morphological Assessment cystolithic hairs Qualitative

GC/MS Delta-9-THC Qualitative

GC/FID THC over the decision point threshold Decision Point Threshold

611 Reporting: The sample is Marijuana.

612

613 Example Scenario 3: Plant material with THC content more than CBD. Determine if the sample is 

614 Marijuana using a decision point threshold.

Technique Result Test Type

Morphological Assessment cystolithic hairs Qualitative

4-AP Blue Qualitative

GC/MS THC over the decision point threshold Decision Point Threshold

615 Reporting: The sample is Marijuana.

616

617 Example Scenario 4: Plant material with THC less than the decision point and delta-8-THC is present. 

618 Determine if the sample is Marijuana using a decision point threshold.

Technique Result Test Type

Morphological Assessment cystolithic hairs Qualitative

GC/MS (full scan) Delta-9-THC below the decision point 

threshold and contains delta-8-THC

Decision Point Threshold

619 Reporting: The sample is inconclusive for Marijuana and contains delta-8-THC.

620

621 Example Scenario 5: Plant material with THC content less than CBD, but contains high concentrations of 

622 CBN (a known false positive on the 4-AP test). Determine if the sample is Marijuana using a decision 

623 point threshold.

Technique Result Test Type
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Morphological Assessment cystolithic hairs Qualitative

4-AP Blue (false positive) Qualitative

GC/MS (full scan) CBD > THC and THC over the decision 

point threshold. High CBN observed.

Decision Point Threshold

624 Reporting: The results are inconclusive.