Journal:Leaner and greener analysis of cannabinoids - Revision history

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	==Abstract==		==Abstract==
	There is an explosion in the number of [[Laboratory\|labs]] analyzing [[wikipedia:Cannabinoid\|cannabinoids]] in marijuana ([[wikipedia:Cannabis\|''Cannabis sativa'' L.]], Cannabaceae); however, existing methods are inefficient, require expert analysts, and use large volumes of potentially environmentally damaging [[~~solvent~~]]s. 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 [[Chromatography detector\|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.		There is an explosion in the number of [[Laboratory\|labs]] analyzing [[wikipedia:Cannabinoid\|cannabinoids]] in marijuana ([[wikipedia:Cannabis\|''Cannabis sativa'' L.]], Cannabaceae); however, existing methods are inefficient, require expert analysts, and use large volumes of potentially environmentally damaging [[wikipedia:Solvent\|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 [[Chromatography detector\|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		'''Keywords''': green chemistry, single-laboratory validation, ''Cannabis'', cannabinoids, medical marijuana

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	==Abstract==		==Abstract==
	There is an explosion in the number of [[Laboratory\|labs]] analyzing [[~~cannabinoid~~]]s in marijuana ([[Cannabis\|''Cannabis sativa'' L.]], Cannabaceae); however, existing methods are inefficient, require expert analysts, and use large volumes of potentially environmentally damaging [[solvent]]s. 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 [[Chromatography detector\|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.		There is an explosion in the number of [[Laboratory\|labs]] analyzing [[wikipedia:Cannabinoid\|cannabinoids]] in marijuana ([[wikipedia:Cannabis\|''Cannabis sativa'' L.]], Cannabaceae); however, existing methods are inefficient, require expert analysts, and use large volumes of potentially environmentally damaging [[solvent]]s. 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 [[Chromatography detector\|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		'''Keywords''': green chemistry, single-laboratory validation, ''Cannabis'', cannabinoids, medical marijuana

	==Introduction==		==Introduction==
	The modern [[Cannabis industry\|cannabis market]] is in a period of dramatic flux. In the United States, cannabis is classified as a [[Controlled Substances Act\|Schedule I]] drug<ref name="LamarineMari12">{{cite journal \|title=Marijuana: Modern medical chimaera \|journal=Journal of Drug Education \|author=Lamarine, R.J. \|volume=42 \|issue=1 \|pages=1–11 \|year=2012 \|doi=10.2190/DE.42.1.a \|pmid=22873011}}</ref>; however, eight U.S. states have legalized marijuana for recreational use, and 28 states have allowed [[Cannabis (drug)\|medical marijuana]] on the basis of evidence of anxiolytic, analgesic, sedative, anticancer, and appetite stimulation effects.<ref name="PorterReport13">{{cite journal \|title=Report of a parent survey of cannabidiol-enriched cannabis use in pediatric treatment-resistant epilepsy \|journal=Epilepsy & Behavior \|author=Porter, B.E.; Jacobson, C. \|volume=29 \|issue=3 \|pages=574–7 \|year=2013 \|doi=10.1016/j.yebeh.2013.08.037 \|pmid=24237632 \|pmc=PMC4157067}}</ref><ref name="TafelskiEfficacy16">{{cite journal \|title=Efficacy, tolerability, and safety of cannabinoids for chemotherapy-induced nausea and vomiting--a systematic review of systematic reviews \|journal=Schmerz \|author=Tafelski, S.; Häuser, W.; Schäfer, M. \|volume=30 \|issue=1 \|pages=14–24 \|year=2016 \|doi=10.1007/s00482-015-0092-3 \|pmid=26787227}}</ref><ref name="WhitingCanna15">{{cite journal \|title=Cannabinoids for Medical Use: A Systematic Review and Meta-analysis \|journal=JAMA \|author=Whiting, P.F.; Wolff, R.F.; Deshpande, S. et al. \|volume=313 \|issue=24 \|pages=2456–73 \|year=2015 \|doi=10.1001/jama.2015.6358 \|pmid=26103030}}</ref><ref name="BagshawMedical02">{{cite journal \|title=Medical efficacy of cannabinoids and marijuana: A comprehensive review of the literature \|journal=Journal of Palliative Care \|author=Bagshaw, S.M.; Hagen, N.A. \|volume=18 \|issue=2 \|pages=111–22 \|year=2002 \|doi=10.1177/082585970201800207 \|pmid=12164099}}</ref> 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.<ref name="GoCAccess16">{{cite web \|url=https://laws.justice.gc.ca/eng/regulations/sor-2016-230/ \|title=Access to Cannabis for Medical Purposes Regulations (SOR/2016-230) \|work=Justice Laws Website \|author=Government of Canada \|date=2016}}</ref> Canadian production of commercial products must take place in a facility using [[good manufacturing practice]]s, and products must be assayed for the presence and quantity of [[Tetrahydrocannabinol\|Δ9-tetrahydrocannabinol]] (Δ9-THC), [[Tetrahydrocannabinolic acid\|Δ9-tetrahydrocannabinolic acid]] (THCA), [[cannabidiol]] (CBD), and cannabidiolic acid (CBDA), using validated analytical methods.<ref name="GoCAccess16" /> In total, more than 100 cannabinoids in 11 subclasses have been characterized in cannabis and are concentrated in the glandular [[~~trichome~~]]s of the female inflorescences. Other cannabinoid classes include [[cannabigerol]] (CBG), [[cannabichromene]] (CBC), and [[cannabinol]] (CBN) (Fig. 1).<ref name="MahlbergAccum04">{{cite journal \|title=Accumulation of Cannabinoids in Glandular Trichomes of ''Cannabis'' (Cannabaceae) \|journal=Journal of Industrial Hemp \|author=Mahlberg, P.G.; Kim, E.S. \|volume=9 \|issue=1 \|pages=15–36 \|year=2004 \|doi=10.1300/J237v09n01_04}}</ref> 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]].<ref name="BrenneisenChem07">{{cite book \|chapter=Chapter 2: Chemistry and Analysis of Phytocannabinoids and Other Cannabis Constituents \|title=Marijuana and the Cannabinoids \|author=Brenneisen, R. \|editor=ElSohly, M.A. \|publisher=Humana Press \|pages=17–49 \|year=2007 \|isbn=9781592599479 \|doi=10.1007/978-1-59259-947-9}}</ref><ref name="SwiftAnalysis13">{{cite journal \|title=Analysis of cannabis seizures in NSW, Australia: Cannabis potency and cannabinoid profile \|journal=PLoS One \|author=Swift, W.; Wong, A.; Li, K.M. et al. \|volume=8 \|issue=7 \|pages=e70052 \|year=2013 \|doi=10.1371/journal.pone.0070052 \|pmid=23894589 \|pmc=PMC3722200}}</ref> 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.<ref name="SwiftAnalysis13" /><ref name="ZuardiAction82">{{cite journal \|title=Action of cannabidiol on the anxiety and other effects produced by delta 9-THC in normal subjects \|journal=Psychopharmacology \|author=Zuardi, A.W.; Shirakawa, I.; Finkelfarb, E. et al. \|volume=76 \|issue=3 \|pages=245–50 \|year=1982 \|pmid=6285406}}</ref>		The modern [[wikipedia:Cannabis industry\|cannabis market]] is in a period of dramatic flux. In the United States, cannabis is classified as a [[wikipedia:Controlled Substances Act\|Schedule I]] drug<ref name="LamarineMari12">{{cite journal \|title=Marijuana: Modern medical chimaera \|journal=Journal of Drug Education \|author=Lamarine, R.J. \|volume=42 \|issue=1 \|pages=1–11 \|year=2012 \|doi=10.2190/DE.42.1.a \|pmid=22873011}}</ref>; however, eight U.S. states have legalized marijuana for recreational use, and 28 states have allowed [[wikipedia:Cannabis (drug)\|medical marijuana]] on the basis of evidence of anxiolytic, analgesic, sedative, anticancer, and appetite stimulation effects.<ref name="PorterReport13">{{cite journal \|title=Report of a parent survey of cannabidiol-enriched cannabis use in pediatric treatment-resistant epilepsy \|journal=Epilepsy & Behavior \|author=Porter, B.E.; Jacobson, C. \|volume=29 \|issue=3 \|pages=574–7 \|year=2013 \|doi=10.1016/j.yebeh.2013.08.037 \|pmid=24237632 \|pmc=PMC4157067}}</ref><ref name="TafelskiEfficacy16">{{cite journal \|title=Efficacy, tolerability, and safety of cannabinoids for chemotherapy-induced nausea and vomiting--a systematic review of systematic reviews \|journal=Schmerz \|author=Tafelski, S.; Häuser, W.; Schäfer, M. \|volume=30 \|issue=1 \|pages=14–24 \|year=2016 \|doi=10.1007/s00482-015-0092-3 \|pmid=26787227}}</ref><ref name="WhitingCanna15">{{cite journal \|title=Cannabinoids for Medical Use: A Systematic Review and Meta-analysis \|journal=JAMA \|author=Whiting, P.F.; Wolff, R.F.; Deshpande, S. et al. \|volume=313 \|issue=24 \|pages=2456–73 \|year=2015 \|doi=10.1001/jama.2015.6358 \|pmid=26103030}}</ref><ref name="BagshawMedical02">{{cite journal \|title=Medical efficacy of cannabinoids and marijuana: A comprehensive review of the literature \|journal=Journal of Palliative Care \|author=Bagshaw, S.M.; Hagen, N.A. \|volume=18 \|issue=2 \|pages=111–22 \|year=2002 \|doi=10.1177/082585970201800207 \|pmid=12164099}}</ref> Regulations regarding ''[[wikipedia:Cannabis\|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.<ref name="GoCAccess16">{{cite web \|url=https://laws.justice.gc.ca/eng/regulations/sor-2016-230/ \|title=Access to Cannabis for Medical Purposes Regulations (SOR/2016-230) \|work=Justice Laws Website \|author=Government of Canada \|date=2016}}</ref> Canadian production of commercial products must take place in a facility using [[good manufacturing practice]]s, and products must be assayed for the presence and quantity of [[wikipedia:Tetrahydrocannabinol\|Δ9-tetrahydrocannabinol]] (Δ9-THC), [[wikipedia:Tetrahydrocannabinolic acid\|Δ9-tetrahydrocannabinolic acid]] (THCA), [[wikipedia:Cannabidiol\|cannabidiol]] (CBD), and cannabidiolic acid (CBDA), using validated analytical methods.<ref name="GoCAccess16" /> In total, more than 100 cannabinoids in 11 subclasses have been characterized in cannabis and are concentrated in the glandular [[wikipedia:Trichome\|trichomes]] of the female inflorescences. Other cannabinoid classes include [[wikipedia:Cannabigerol\|cannabigerol]] (CBG), [[wikipedia:Cannabichromene\|cannabichromene]] (CBC), and [[wikipedia:Cannabinol\|cannabinol]] (CBN) (Fig. 1).<ref name="MahlbergAccum04">{{cite journal \|title=Accumulation of Cannabinoids in Glandular Trichomes of ''Cannabis'' (Cannabaceae) \|journal=Journal of Industrial Hemp \|author=Mahlberg, P.G.; Kim, E.S. \|volume=9 \|issue=1 \|pages=15–36 \|year=2004 \|doi=10.1300/J237v09n01_04}}</ref> The cannabinoids occur primarily in acid form, with neutral cannabinoids formed during drying, storage, and [[wikipedia:Decarboxylation\|decarboxylation]] during smoking. Δ9-THC, the main psychoactive cannabinoid, can be over 20% by weight in specially bred [[wikipedia:Cannabis strains\|cannabis strains]].<ref name="BrenneisenChem07">{{cite book \|chapter=Chapter 2: Chemistry and Analysis of Phytocannabinoids and Other Cannabis Constituents \|title=Marijuana and the Cannabinoids \|author=Brenneisen, R. \|editor=ElSohly, M.A. \|publisher=Humana Press \|pages=17–49 \|year=2007 \|isbn=9781592599479 \|doi=10.1007/978-1-59259-947-9}}</ref><ref name="SwiftAnalysis13">{{cite journal \|title=Analysis of cannabis seizures in NSW, Australia: Cannabis potency and cannabinoid profile \|journal=PLoS One \|author=Swift, W.; Wong, A.; Li, K.M. et al. \|volume=8 \|issue=7 \|pages=e70052 \|year=2013 \|doi=10.1371/journal.pone.0070052 \|pmid=23894589 \|pmc=PMC3722200}}</ref> 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.<ref name="SwiftAnalysis13" /><ref name="ZuardiAction82">{{cite journal \|title=Action of cannabidiol on the anxiety and other effects produced by delta 9-THC in normal subjects \|journal=Psychopharmacology \|author=Zuardi, A.W.; Shirakawa, I.; Finkelfarb, E. et al. \|volume=76 \|issue=3 \|pages=245–50 \|year=1982 \|pmid=6285406}}</ref>


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	===Calibration standards===		===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.		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 [[wikipedia:Tetrahydrocannabivarin\|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===		===Test materials===

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	\|download = [https://link.springer.com/content/pdf/10.1007%2Fs00216-017-0256-3.pdf https://link.springer.com/content/pdf/10.1007%2Fs00216-017-0256-3.pdf] (PDF)		\|download = [https://link.springer.com/content/pdf/10.1007%2Fs00216-017-0256-3.pdf https://link.springer.com/content/pdf/10.1007%2Fs00216-017-0256-3.pdf] (PDF)
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	==Abstract==		==Abstract==
	There is an explosion in the number of [[Laboratory\|labs]] analyzing [[cannabinoid]]s in marijuana ([[Cannabis\|''Cannabis sativa'' L.]], Cannabaceae); however, existing methods are inefficient, require expert analysts, and use large volumes of potentially environmentally damaging [[solvent]]s. 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 [[Chromatography detector\|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.		There is an explosion in the number of [[Laboratory\|labs]] analyzing [[cannabinoid]]s in marijuana ([[Cannabis\|''Cannabis sativa'' L.]], Cannabaceae); however, existing methods are inefficient, require expert analysts, and use large volumes of potentially environmentally damaging [[solvent]]s. 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 [[Chromatography detector\|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.
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	The demand for cost-efficient quantitative methods for cannabinoids is growing. Many laboratories engaged in this work lack the expertise for advanced analytical instrumentation, such as mass spectrometric detectors. In this case, these detectors would impose a significant cost increase in infrastructure and expertise. [[Mass spectrometry]] (MS) would allow for improved detection limits, selectivity, and sensitivity, but given the performance of this method with UV absorbance detection and the high concentrations of the major cannabinoids, the use of MS detection does not improve the method fitness. Care was taken to ensure that the mobile phases used for this method are compatible with mass spectrometry for those who with the instrument capabilities require improved sensitivity and putative identification of additional minor cannabinoids.		The demand for cost-efficient quantitative methods for cannabinoids is growing. Many laboratories engaged in this work lack the expertise for advanced analytical instrumentation, such as mass spectrometric detectors. In this case, these detectors would impose a significant cost increase in infrastructure and expertise. [[Mass spectrometry]] (MS) would allow for improved detection limits, selectivity, and sensitivity, but given the performance of this method with UV absorbance detection and the high concentrations of the major cannabinoids, the use of MS detection does not improve the method fitness. Care was taken to ensure that the mobile phases used for this method are compatible with mass spectrometry for those who with the instrument capabilities require improved sensitivity and putative identification of additional minor cannabinoids.

	Our method is a significant improvement over previous methods that can be used in a variety of settings and has the potential to be expanded for inclusion of new cannabinoids as required. To date, many jurisdictions only require the quantity of total THC and total CBD in the products<ref name="CSoS1CC1212-2">{{cite web \|url=https://www.sos.state.co.us/CCR/DisplayRule.do?action=ruleinfo&ruleId=3175&deptID=19&agencyID=185&deptName=200%20Revenue&agencyName=212%A0Marijuana%20Enforcement%20Division&seriesNum=1%20CCR%20212-2		Our method is a significant improvement over previous methods that can be used in a variety of settings and has the potential to be expanded for inclusion of new cannabinoids as required. To date, many jurisdictions only require the quantity of total THC and total CBD in the products<ref name="CSoS1CC1212-2">{{cite web \|url=https://www.sos.state.co.us/CCR/DisplayRule.do?action=ruleinfo&ruleId=3175&deptID=19&agencyID=185&deptName=200%20Revenue&agencyName=212%A0Marijuana%20Enforcement%20Division&seriesNum=1%20CCR%20212-2 \|title=1 CCR 212-2 Retail Marijuana Rules \|work=Code of Colorado Regulations \|publisher=Colorado Secretary of State}}</ref><ref name="WSoSInitiative11">{{cite web \|url=https://sos.wa.gov/_assets/elections/initiatives/i502.pdf \|format=PDF \|title=Initiative Measure No. 502 \|publisher=Washington Secretary of State \|date=08 July 2011}}</ref>, while with improvements in analytical instrumentation, columns, and detection techniques, the ability to expand the regulations to acids, neutral forms, and minor cannabinoids is straightforward. There is a significant amount of concern around the use of [[gas chromatography]] (GC) for quantitation of cannabinoids using in-injector decarboxylation because of conversion issues, which can reduce the accuracy and precision.<ref name="UNODCRecomm09" /> This issue is no longer a concern when quantifying cannabinolic acids separately from neutral cannabinoids. The information on acid content is also important for those that do not smoke ''Cannabis'' as the pharmacology of acids varies compared to neutral cannabinoids.<ref name="BursteinTheCanna99">{{cite journal \|title=The Cannabinoid Acids: Nonpsychoactive Derivatives with Therapeutic Potential \|journal=Pharmacology & Therapeutics \|author=Burstein, S.H. \|volume=82 \|issue=1 \|pages=87–96 \|year=1999 \|doi=10.1016/S0163-7258(98)00069-2 \|pmid=10341359}}</ref> Understanding the cannabinoid profiles of different ''Cannabis'' strains will allow additional information for clinical researchers to understand the complex composition of these plants and their roles in pain regulation and treatment of a variety of other illnesses.


	~~[25, 26]~~, while with improvements in analytical instrumentation, columns, and detection techniques, the ability to expand the regulations to acids, neutral forms, and minor cannabinoids is straightforward. There is a significant amount of concern around the use of [[gas chromatography]] (GC) for quantitation of cannabinoids using in-injector decarboxylation because of conversion issues, which can reduce the accuracy and precision ~~[24]~~. This issue is no longer a concern when quantifying cannabinolic acids separately from neutral cannabinoids. The information on acid content is also important for those that do not smoke ''Cannabis'' as the pharmacology of acids varies compared to neutral cannabinoids ~~[27]~~. Understanding the cannabinoid profiles of different ''Cannabis'' strains will allow additional information for clinical researchers to understand the complex composition of these plants and their roles in pain regulation and treatment of a variety of other illnesses.

			==Conclusions==
			We developed an optimized HPLC-DAD method with reduced extraction time and greener solvents for adoption into ''Cannabis'' testing laboratories. Sample turnaround is significantly reduced, while method validation confirmed that the method produces repeatable, accurate results. The sample preparation eliminates the use of chloroform, which has been routinely used in cannabinoid analysis, reducing material costs, promoting greener solvents, and improving laboratory safety for personnel. This method can be used in a variety of settings from clinical studies, research, quality control, and regulatory evaluation of this growing industry.

	==Acknowledgements==		==Acknowledgements==
			We would like to acknowledge partial financial support from Northern Vine Canada Ltd (Langley, BC) in the development and validation of an analytical method suitable for the intended purpose of conducting routine analysis. This research was undertaken, in part, thanks to funding from the Canada Research Chairs program.

	~~===Author contributions===~~


	~~===Funding===~~


	===Conflicts of interest===		===Conflicts of interest===
			The authors declare that they have no conflict of interest.

			==Supplementary material==
			[https://static-content.springer.com/esm/art%3A10.1007%2Fs00216-017-0256-3/MediaObjects/216_2017_256_MOESM1_ESM.pdf 216_2017_256_MOESM1_ESM.pdf] (PDF, 390 kb)

	==References==		==References==
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	==Notes==		==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.		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. The original had the incorrect title for the final citation; it has been corrected for this version.

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Shawndouglas at 17:52, 2 July 2019

2019-07-02T17:52:26Z

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	==Discussion==		==Discussion==
			With the rapid expansion of labs analyzing ''Cannabis'', it is essential to have robust, versatile analytical methods. The currently available methods have several limitations. For example, the resolution of the minor cannabinoids in some cases has been achieved only by selecting a less sensitive UV wavelength to achieve baseline, while this reduction in sensitivity would impact the quantitation of low-level compounds found in many products.<ref name="SwiftAnalysis13" /> Some methods fail to provide sufficient method development information to explain extraction solvent selection, extraction times, potential losses, degradation, or inefficiencies.<ref name="SwiftAnalysis13" /><ref name="DeBackerInno09" /><ref name="MehmedicPotency10" /> Many methods use chlorinated solvents with potential negative health and environmental impacts. Other methods use high pH mobile phases pH >5.0 which is above the p''K''<sub>a</sub> of the cannabinolic acids.<ref name="DeBackerInno09" /> We found that this elution system caused the peaks to tail significantly, and early eluting peaks were asymmetrical.

			The demand for cost-efficient quantitative methods for cannabinoids is growing. Many laboratories engaged in this work lack the expertise for advanced analytical instrumentation, such as mass spectrometric detectors. In this case, these detectors would impose a significant cost increase in infrastructure and expertise. [[Mass spectrometry]] (MS) would allow for improved detection limits, selectivity, and sensitivity, but given the performance of this method with UV absorbance detection and the high concentrations of the major cannabinoids, the use of MS detection does not improve the method fitness. Care was taken to ensure that the mobile phases used for this method are compatible with mass spectrometry for those who with the instrument capabilities require improved sensitivity and putative identification of additional minor cannabinoids.

			Our method is a significant improvement over previous methods that can be used in a variety of settings and has the potential to be expanded for inclusion of new cannabinoids as required. To date, many jurisdictions only require the quantity of total THC and total CBD in the products<ref name="CSoS1CC1212-2">{{cite web \|url=https://www.sos.state.co.us/CCR/DisplayRule.do?action=ruleinfo&ruleId=3175&deptID=19&agencyID=185&deptName=200%20Revenue&agencyName=212%A0Marijuana%20Enforcement%20Division&seriesNum=1%20CCR%20212-2


			[25, 26], while with improvements in analytical instrumentation, columns, and detection techniques, the ability to expand the regulations to acids, neutral forms, and minor cannabinoids is straightforward. There is a significant amount of concern around the use of [[gas chromatography]] (GC) for quantitation of cannabinoids using in-injector decarboxylation because of conversion issues, which can reduce the accuracy and precision [24]. This issue is no longer a concern when quantifying cannabinolic acids separately from neutral cannabinoids. The information on acid content is also important for those that do not smoke ''Cannabis'' as the pharmacology of acids varies compared to neutral cannabinoids [27]. Understanding the cannabinoid profiles of different ''Cannabis'' strains will allow additional information for clinical researchers to understand the complex composition of these plants and their roles in pain regulation and treatment of a variety of other illnesses.

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← Older revision		Revision as of 16:27, 2 July 2019
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	==Introduction==		==Introduction==
	The modern [[Cannabis industry\|cannabis market]] is in a period of dramatic flux. In the United States, cannabis is classified as a [[Controlled Substances Act\|Schedule I]] drug<ref name="LamarineMari12">{{cite journal \|title=Marijuana: Modern medical chimaera \|journal=Journal of Drug Education \|author=Lamarine, R.J. \|volume=42 \|issue=1 \|pages=1–11 \|year=2012 \|doi=10.2190/DE.42.1.a \|pmid=22873011}}</ref>; however, eight U.S. states have legalized marijuana for recreational use, and 28 states have allowed [[Cannabis (drug)\|medical marijuana]] on the basis of evidence of anxiolytic, analgesic, sedative, anticancer, and appetite stimulation effects.<ref name="PorterReport13">{{cite journal \|title=Report of a parent survey of cannabidiol-enriched cannabis use in pediatric treatment-resistant epilepsy \|journal=Epilepsy & Behavior \|author=Porter, B.E.; Jacobson, C. \|volume=29 \|issue=3 \|pages=574–7 \|year=2013 \|doi=10.1016/j.yebeh.2013.08.037 \|pmid=24237632 \|pmc=PMC4157067}}</ref><ref name="TafelskiEfficacy16">{{cite journal \|title=Efficacy, tolerability, and safety of cannabinoids for chemotherapy-induced nausea and vomiting--a systematic review of systematic reviews \|journal=Schmerz \|author=Tafelski, S.; Häuser, W.; Schäfer, M. \|volume=30 \|issue=1 \|pages=14–24 \|year=2016 \|doi=10.1007/s00482-015-0092-3 \|pmid=26787227}}</ref><ref name="WhitingCanna15">{{cite journal \|title=Cannabinoids for Medical Use: A Systematic Review and Meta-analysis \|journal=JAMA \|author=Whiting, P.F.; Wolff, R.F.; Deshpande, S. et al. \|volume=313 \|issue=24 \|pages=2456–73 \|year=2015 \|doi=10.1001/jama.2015.6358 \|pmid=26103030}}</ref><ref name="BagshawMedical02">{{cite journal \|title=Medical efficacy of cannabinoids and marijuana: A comprehensive review of the literature \|journal=Journal of Palliative Care \|author=Bagshaw, S.M.; Hagen, N.A. \|volume=18 \|issue=2 \|pages=111–22 \|year=2002 \|doi=10.1177/082585970201800207 \|pmid=12164099}}</ref> 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.<ref name="GoCAccess16">{{cite web \|url=https://laws.justice.gc.ca/eng/regulations/sor-2016-230/ \|title=Access to Cannabis for Medical Purposes Regulations (SOR/2016-230) \|work=Justice Laws Website \|author=Government of Canada \|date=2016}}</ref> Canadian production of commercial products must take place in a facility using [[good manufacturing practice]]s, and products must be assayed for the presence and quantity of [[Tetrahydrocannabinol\|Δ9-tetrahydrocannabinol]] (Δ9-THC), [[Tetrahydrocannabinolic acid\|Δ9-tetrahydrocannabinolic acid]] (THCA), [[cannabidiol]] (CBD), and cannabidiolic acid (CBDA), using validated analytical methods.<ref name="GoCAccess16" /> In total, more than 100 cannabinoids in 11 subclasses have been characterized in cannabis and are concentrated in the glandular [[trichome]]s of the female inflorescences. Other cannabinoid classes include [[cannabigerol]] (CBG), [[cannabichromene]] (CBC), and [[cannabinol]] (CBN) (Fig. 1).<ref name="MahlbergAccum04">{{cite journal \|title=Accumulation of Cannabinoids in Glandular Trichomes of ''Cannabis'' (Cannabaceae) \|journal=Journal of Industrial Hemp \|author=Mahlberg, P.G.; Kim, E.S. \|volume=9 \|issue=1 \|pages=15–36 \|year=2004 \|doi=10.1300/J237v09n01_04}}</ref> 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]].<ref name="BrenneisenChem07">{{cite book \|chapter=Chapter 2: Chemistry and Analysis of Phytocannabinoids and Other Cannabis Constituents \|title=Marijuana and the Cannabinoids \|author=Brenneisen, R. \|editor=ElSohly, M.A. \|publisher=Humana Press \|pages=17–49 \|year=2007 \|isbn=9781592599479 \|doi=10.1007/978-1-59259-947-9}}</ref><ref name="SwiftAnalysis13">{{cite journal \|title=Analysis of cannabis seizures in NSW, Australia: Cannabis potency and cannabinoid profile \|journal=PLoS One \|author=Swift, W.; Wong, A.; Li, K.M. et al. \|volume=8 \|issue=7 \|pages=e70052 \|year=2013 \|doi=10.1371/journal.pone.0070052 \|pmid=23894589 \|pmc=PMC3722200}}</ref> 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.<ref name="SwiftAnalysis13" /><ref name="ZuardiAction82">{{cite journal \|title=Action of cannabidiol on the anxiety and other effects produced by delta 9-THC in normal subjects \|journal=Psychopharmacology \|author=Zuardi, A.W.; Shirakawa, I.; Finkelfarb, E. et al. \|volume=76 \|issue=3 \|pages=245–50 \|year=1982 \|pmid=6285406}}</ref>		The modern [[Cannabis industry\|cannabis market]] is in a period of dramatic flux. In the United States, cannabis is classified as a [[Controlled Substances Act\|Schedule I]] drug<ref name="LamarineMari12">{{cite journal \|title=Marijuana: Modern medical chimaera \|journal=Journal of Drug Education \|author=Lamarine, R.J. \|volume=42 \|issue=1 \|pages=1–11 \|year=2012 \|doi=10.2190/DE.42.1.a \|pmid=22873011}}</ref>; however, eight U.S. states have legalized marijuana for recreational use, and 28 states have allowed [[Cannabis (drug)\|medical marijuana]] on the basis of evidence of anxiolytic, analgesic, sedative, anticancer, and appetite stimulation effects.<ref name="PorterReport13">{{cite journal \|title=Report of a parent survey of cannabidiol-enriched cannabis use in pediatric treatment-resistant epilepsy \|journal=Epilepsy & Behavior \|author=Porter, B.E.; Jacobson, C. \|volume=29 \|issue=3 \|pages=574–7 \|year=2013 \|doi=10.1016/j.yebeh.2013.08.037 \|pmid=24237632 \|pmc=PMC4157067}}</ref><ref name="TafelskiEfficacy16">{{cite journal \|title=Efficacy, tolerability, and safety of cannabinoids for chemotherapy-induced nausea and vomiting--a systematic review of systematic reviews \|journal=Schmerz \|author=Tafelski, S.; Häuser, W.; Schäfer, M. \|volume=30 \|issue=1 \|pages=14–24 \|year=2016 \|doi=10.1007/s00482-015-0092-3 \|pmid=26787227}}</ref><ref name="WhitingCanna15">{{cite journal \|title=Cannabinoids for Medical Use: A Systematic Review and Meta-analysis \|journal=JAMA \|author=Whiting, P.F.; Wolff, R.F.; Deshpande, S. et al. \|volume=313 \|issue=24 \|pages=2456–73 \|year=2015 \|doi=10.1001/jama.2015.6358 \|pmid=26103030}}</ref><ref name="BagshawMedical02">{{cite journal \|title=Medical efficacy of cannabinoids and marijuana: A comprehensive review of the literature \|journal=Journal of Palliative Care \|author=Bagshaw, S.M.; Hagen, N.A. \|volume=18 \|issue=2 \|pages=111–22 \|year=2002 \|doi=10.1177/082585970201800207 \|pmid=12164099}}</ref> 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.<ref name="GoCAccess16">{{cite web \|url=https://laws.justice.gc.ca/eng/regulations/sor-2016-230/ \|title=Access to Cannabis for Medical Purposes Regulations (SOR/2016-230) \|work=Justice Laws Website \|author=Government of Canada \|date=2016}}</ref> Canadian production of commercial products must take place in a facility using [[good manufacturing practice]]s, and products must be assayed for the presence and quantity of [[Tetrahydrocannabinol\|Δ9-tetrahydrocannabinol]] (Δ9-THC), [[Tetrahydrocannabinolic acid\|Δ9-tetrahydrocannabinolic acid]] (THCA), [[cannabidiol]] (CBD), and cannabidiolic acid (CBDA), using validated analytical methods.<ref name="GoCAccess16" /> In total, more than 100 cannabinoids in 11 subclasses have been characterized in cannabis and are concentrated in the glandular [[trichome]]s of the female inflorescences. Other cannabinoid classes include [[cannabigerol]] (CBG), [[cannabichromene]] (CBC), and [[cannabinol]] (CBN) (Fig. 1).<ref name="MahlbergAccum04">{{cite journal \|title=Accumulation of Cannabinoids in Glandular Trichomes of ''Cannabis'' (Cannabaceae) \|journal=Journal of Industrial Hemp \|author=Mahlberg, P.G.; Kim, E.S. \|volume=9 \|issue=1 \|pages=15–36 \|year=2004 \|doi=10.1300/J237v09n01_04}}</ref> 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]].<ref name="BrenneisenChem07">{{cite book \|chapter=Chapter 2: Chemistry and Analysis of Phytocannabinoids and Other Cannabis Constituents \|title=Marijuana and the Cannabinoids \|author=Brenneisen, R. \|editor=ElSohly, M.A. \|publisher=Humana Press \|pages=17–49 \|year=2007 \|isbn=9781592599479 \|doi=10.1007/978-1-59259-947-9}}</ref><ref name="SwiftAnalysis13">{{cite journal \|title=Analysis of cannabis seizures in NSW, Australia: Cannabis potency and cannabinoid profile \|journal=PLoS One \|author=Swift, W.; Wong, A.; Li, K.M. et al. \|volume=8 \|issue=7 \|pages=e70052 \|year=2013 \|doi=10.1371/journal.pone.0070052 \|pmid=23894589 \|pmc=PMC3722200}}</ref> 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.<ref name="SwiftAnalysis13" /><ref name="ZuardiAction82">{{cite journal \|title=Action of cannabidiol on the anxiety and other effects produced by delta 9-THC in normal subjects \|journal=Psychopharmacology \|author=Zuardi, A.W.; Shirakawa, I.; Finkelfarb, E. et al. \|volume=76 \|issue=3 \|pages=245–50 \|year=1982 \|pmid=6285406}}</ref>


	[[File:Fig1 Mudge AnalBioChem2017 409-12.gif\|450px]]		[[File:Fig1 Mudge AnalBioChem2017 409-12.gif\|450px]]
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	===Preparation of test materials===		===Preparation of test materials===
	====Plant tissues====		====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.		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 5 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====		====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.		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 5 minutes. Samples were filtered with 0.22-μm Teflon filters into amber HPLC vials and stored at 4 °C until analysis.

	===Method optimization===		===Method optimization===
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	==Results==		==Results==
			===Method optimization===
			A statistically guided optimization plan was developed using a partial factorial design to determine the impact of four parameters used in cannabinoid extractions from dried flowers.

			====Extraction of tissues====
			Two levels were selected for each factor: solvent composition (80% methanol, 9:1 methanol/chloroform), extraction technique (sonication, wrist action shaking), extraction time (15 minutes, one hour), and solvent volume (10 mL, 25 mL). An initial prescreening of solvents indicated that the extraction efficiency of methanol/chloroform mixture was not different from 80% methanol, allowing further studies to use the greener option. The statistical analysis of these data indicated that solvent volume was the most significant factor, followed by solvent composition (Fig. 2). Extraction technique and time did not affect the extraction of cannabinoids. Further studies evaluating the solvent volume to mass and solvent composition using 25 mL extraction solvent with 100 and 200 mg of sample confirmed that 200 mg was equivalent to 100 mg, without saturating the extraction solvent (Fig. 3a). The mass to volume ratios used previously range considerably. In some cases, the mass of sample was as high as 100 mg in 1 mL, up to 500 mg in 100 mL.<ref name="LehmannHigh95" /><ref name="StolkerDeterm04">{{cite journal \|title=Determination of cannabinoids in cannabis products using liquid chromatography-ion trap mass spectrometry \|journal=Journal of Chromatography A \|author=Stolker, A.A.; van Schoonhoven, J.; de Vries, A.J. et al. \|volume=1058 \|issue=1–2 \|pages=143–51 \|year=2004 \|doi=10.1016/j.chroma.2004.08.089 \|pmid=15595662}}</ref> Although extraction time did not significantly impact the resulting cannabinoid content, it was optimized to increase sample throughput. The factorial design showed slightly lower total cannabinoids at 60 minutes in comparison with 15 minutes, potentially indicating some degradation during long extractions. Three time points were assessed: 15, 30, and 60 minutes. The level of cannabinoids was not significantly different between the time points (Fig. 3b). It was then verified if extraction time could be reduced by evaluating 5, 10, and 15 minutes in comparison with 15 minutes with vortexing every 5 minutes. Again, no significant differences were observed between all four extraction times, while 15 minutes with vortexing was significantly higher than 5 minutes (Fig. 3c). These data were used to formulate an optimized standard operating protocol (see Supplementary Material at the end) using 200 mg of dried flowers with 25 mL of 80% aqueous methanol for 15 minutes by sonication with vortexing every 5 minutes.


			[[File:Fig2 Mudge AnalBioChem2017 409-12.gif\|450px]]
			{{clear}}
			{\|
			\| STYLE="vertical-align:top;"\|
			{\| border="0" cellpadding="5" cellspacing="0" width="450px"
			\|-
			\| style="background-color:white; padding-left:10px; padding-right:10px;"\| <blockquote>'''Fig. 2''' Variation of total cannabinoids determined using a two-level partial factorial design to optimize the sample preparation</blockquote>
			\|-
			\|}
			\|}


			[[File:Fig3 Mudge AnalBioChem2017 409-12.gif\|550px]]
			{{clear}}
			{\|
			\| STYLE="vertical-align:top;"\|
			{\| border="0" cellpadding="5" cellspacing="0" width="550px"
			\|-
			\| style="background-color:white; padding-left:10px; padding-right:10px;"\| <blockquote>'''Fig. 3''' Optimization of a green chemistry extraction protocol. The figure shows concentration of the four major cannabinoids comparing ('''a''') solvent composition and volume to mass ratio; ('''b''') extraction times of 15, 30, and 60 minutes, and ('''c''') short extraction times of 5, 10, and 15 minutes, as well as 15 minutes with vortexing every 5 minutes (''n'' = 3).</blockquote>
			\|-
			\|}
			\|}

			====Extraction of oils====
			The extraction method was also optimized for cannabis oil comparing (a) 9:1 methanol/chloroform as used in the United Nations Office on Drugs and Crime (UNODC) method for cannabis products<ref name="UNODCRecomm09">{{cite book \|url=https://www.unodc.org/unodc/en/scientists/recommended-methods-for-the-identification-and-analysis-of-cannabis-and-cannabis-products.html \|title=Recommended Methods for the Identification and Analysis of Cannabis and Cannabis Products (Revised and Updated) \|author=United Nations Office on Drugs and Crime \|publisher=United Nations \|pages=50 \|year=2009 \|isbn=9789211482423}}</ref>, (b) 80% methanol, and (c) 100% methanol. These data show that 80% methanol was not sufficient for extracting the cannabinoids in an oil matrix, but 100% methanol was equivalent to the methanol/chloroform mixture. The final optimized standard operating protocol (see Supplementary Material at the end) for cannabis oil samples was 50 mg of oil extracted with 10 mL of methanol using the same extraction parameters as the dried flowers.

			====Stability====
			The stability of cannabinoids was assessed to determine whether losses occur prior to analysis that may significantly affect the quantitative data. Mixed calibration standards prepared in 80% methanol were stored at −20 °C, 4 °C, and 22 °C in the dark. Variations of less than 5% were considered stable and acceptable. Significant changes in the cannabinoids were found after 30 hours at room temperature, with THCA/CBDA contents decreasing by 6.3 and 9.6% respectively, while mixed standards stored at −20 °C were degraded after 48 hours with THCA and CBDA contents reduced by 8.1 and 10.6% respectively. The standards were stable at 4 °C for the duration of the 72-hour study. Sample extracts were prepared with 80% methanol and stored at 22 °C in light and dark conditions. Under these conditions, THCA and CBDA were considered unstable by 24 hours into the study, with reductions of 6.7% for both, resulting in 8–10% increases in the neutral forms of these cannabinoids. Reductions of 11–23% of acidic cannabinoids occurred under light conditions within 24 hours. On the basis of these findings, an additional study was performed to evaluate extract stability in 80% methanol and 9:1 methanol/chloroform stored in the dark at 22 °C and 4 °C. The 9:1 methanol/chloroform extracts were found to be stable at 22 °C and 4 °C for the duration of the study, 36 and 48 hours respectively, while 80% methanol at 4 ° C was stable for 48 hours. The extracts in 80% methanol were not stable at room temperature for 24 hours, similar to the previous study. As the elimination of chloroform from the extraction solvent improves analyst safety, reduces solvent costs, and maintains cannabinoid contents, 80% methanol proves to be a viable alternative extraction solvent for analytical quantitative analysis with the use of a dark, temperature-controlled autosampler.

			====Optimized chromatography====
			Several HPLC columns, phases, dimensions, mobile phases, and gradients were compared in preliminary experiments to determine a method for baseline separation of as many cannabinoids as possible while maintaining a short separation time. The optimal column used for separation of cannabinoids was a Phenomenex Kinetex core shell C18 column with sub-2.0-micron particle size. This required an HPLC system capable of pumping above 400 bar, but a 600-bar instrument was sufficient. Mobile phase pH appeared to be a significant factor in cannabinoid separation; as pH decreases, retention of cannabinolic acids increases, while at higher pH the retention decreases with poor peak shape. By optimizing the pH of the aqueous mobile phase for the separation to 3.6, it was possible to separate THCA from other cannabinoids, while maintaining adequate peak shapes. The final methodology was a 15-minute analytical run time with 10 mM ammonium formate pH 3.6 and acetonitrile as the mobile phase as shown in Fig. 4. Resolutions of major cannabinoids were greater than 2.0, while minor components were greater than 1.70 using the mixed calibration standards. Sample extracts were used to confirm resolution during the validation study.


			[[File:Fig4 Mudge AnalBioChem2017 409-12.gif\|520px]]
			{{clear}}
			{\|
			\| STYLE="vertical-align:top;"\|
			{\| border="0" cellpadding="5" cellspacing="0" width="520px"
			\|-
			\| style="background-color:white; padding-left:10px; padding-right:10px;"\| <blockquote>'''Fig. 4''' HPLC separation of ('''a''') standard mixture of cannabinoid standards and ('''b''') ''Cannabis'' flower extract at 220 nm</blockquote>
			\|-
			\|}
			\|}

			===Method validation===