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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	==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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