Analysis Of Cannabinoids In Hemp Seed Oils By HPLC
A P P L I C AT I O N N O T ELiquid ChromatographyAuthors:Catharine LaytonWilhad M. ReuterPerkinElmer, Inc.Shelton, CTAnalysis of Cannabinoidsin Hemp Seed Oils byHPLC Using PDA DetectionIntroductionCannabis sativa, from which hemp andmarijuana are derived, has been a sourceof medicinal, industrial, and recreationalcommodities for centuries. The term“hemp” refers primarily to cannabis grown as an agricultural crop and is characterized by cannabisplants that are low in delta-9 tetrahydrocannabinol (THC), the main psychoactive ingredient inmarijuana. Although more than 30 nations worldwide grow hemp as an agricultural commodity,in the United States, production is strictly controlled under existing drug enforcement laws.1As of January 2015, twenty states have passed legislation favorable to hemp cultivation. The mainobstacles facing the potential U.S. market are the government drug policies and Drug EnforcementAdministration (DEA) concerns that the commercial cultivation could increase the likelihood ofcovert production of high-THC cannabis varieties or inadvertent cross pollination, complicatingDEA surveillance.In recent years, scientific knowledge regarding the composition and health benefits of edible hempproducts has significantly increased. Hemp seed oil has been promoted as a good source ofnutritious omega-6 and omega-3 polyunsaturated acids, and may be a cleaner, more sustainablealternative to fish oil.3 While hemp seed oil has been shown to have high nutritive value, it isgenerally believed that it may also afford other beneficial qualities.
Naturally occurring cannabinoids, the main biologically activecomponent of the cannabis plant, form a complex group ofclosely related compounds, of which 70 are known and welldescribed. Although no cannabinoids are metabolically producedby the hemp seed itself, contamination with resin from cannabisflowers and leaves during hemp seed oil processing can occur.Impurity reports have been primarily focused on THC for itspharmacological and toxicological relevance, upon which strictlegal limits have been enforced.4 However, other cannabinoidsprovide unique biological activities. For example, cannabidiol(CBD), typically present at higher levels than THC, showsevidence of providing anticonvulsive, anti-epileptic, andantimicrobial properties.2,3,4In this application, we describe a technique for the monitoringof six cannabinoids, including THC and CBD, in hemp seed oil byHPLC with PDA detection. Figure 1 shows the chemical structuresfor the six cannabinoids analyzed in this application.ExperimentalHardware/SoftwareA PerkinElmer HPLC system was used, including the UV detector(PerkinElmer, Shelton, CT, USA). A PDA (photodiode array) detectorwas also used for spectral confirmation. PerkinElmer BrownleeAnalytical C18, 3.0 μm, 4.6 x 150-mm (Part# N9303508) columnwas used for all analyses (PerkinElmer, Shelton, CT, USA). Allinstrument control, analysis, and data processing was performed viaassociated Chromatography Data Software (CDS).Method ParametersThe LC method parameters are shown in Table 1.Solvents, Standards and SamplesAll solvents and diluents used were HPLC-grade and filtered via0.45-µm filters.Figure 1. Chemical structure of six cannabinoids analyzed in this study.2Table 1. LC Method Parameters.PerkinElmer Brownlee Analytical C18, 3.0 µm, 4.6 x 150-mmColumn:(Part# N9303508)Solvent A: 0.1% formic acid in waterSolvent B: 0.1% formic acid in acetonitrileSolvent program:Mobile Phase:Analysis Time:Flow Rate:Pressure:Oven Temp.:PDA Detection:Injection Volume:Sampling (Data) Rate:Diluent:Sampling (Data) Rate:1Time(min)Flow 1.11.020.080.048.11.05.095.058.61.050.050.08.0 min.; wash/equilibration time 7.0 min.1.0 mL/min. 2500 psi/172 bar maximum50 ºCWavelength: 210 nm25 µL10 pts./secIsopropyl alcohol (IPA)5 pts./sec1 mg/ml each of the following cannabinoids was obtained fromRestek Corporation (Bellefonte, PA): cannabidiol (CBD),cannabigerol (CBG), cannabinol (CBN), cannabichromene (CBC)and tetrahydrocannabinolic acid (THC-A). 1 mg/mL of delta-9tetrahydrocannabinol (THC) was obtained from Sigma-Aldrich ,Inc (Allentown, PA).A 14-ppm mixture of the six standards was prepared by adding25 µL of each standard to 1.650 mL of isopropyl alcohol (IPA).For hemp seed oil quantitation, a standard solution containing THCand CBD was prepared by adding 25.0 µL of each into 1.750 mLof IPA, to equal a final concentration of 14 ppm.
Two brands of hemp seed oils, labelled "Brand A" and "Brand B",were obtained from a local health food store. In order to reducethe viscosity of the hemp seed oil prior to analysis, each samplewas diluted nine fold in IPA by adding 0.2 mL hemp seed oil to 1.6mL of IPA and mixing by manual shaking. A 14-ppm spikedsolution of each hemp seed oil sample was prepared by adding25.0 µL THC standard, 25.0 µL CBD standard, 0.2 mL hemp seedoil, and 1.55 mL IPA to a 2-mL sample vial and then mixed bymanual shaking. Considering the nine fold dilution of sample, thefinal cannabinoid concentration corresponded to 125-ppm inundiluted hemp seed oil.Results and DiscussionFigure 2 shows the chromatogram of a standard mixture containingthe six cannabinoids, all separated in under eight minutes.As shown in Figure 3, chromatographic repeatability wasconfirmed via 10 injections of the 14-ppm standard mixture,demonstrating good hromene (CBC)THC-A1.10Cannabinol (CBN)Cannabigerol (CBG)Cannabidiol 2.00MinutesFigure 2. HPLC chromatogram showing separation of six cannabinoids in the 14-ppm standard mixture; wavelength: 210 509.00MinutesFigure 3. Overlay of 10 replicates of the 14-ppm standard mixture.3
Linearity was determined for all six cannabinoids. Representativelinearity plots for CBD and THC, respectively, are shown in Figure 4and Figure 5. The linearity coefficients of determination for all sixcannabinoids are listed in Table 2.Table 2. Linearity coefficient of determination for six cannabinoids between 1 –14 ppmdetected by PDA at 210 nm.CannabinoidCBD2.5x10 62.0x10 60.99951Cannabidiol (CBD)Cannabinol (CBN)Delta-9 tetrahydrocannabinol (THC)Cannabichromene (CBC)Delta-9-tetrahydrocannabinolic acid (THC-A)0.999940.999960.999250.999580.99857As listed in Table 3, LOD (limit of detection) and LOQ (limit ofquantitation) levels were established for each cannabinoid, basedupon undiluted hemp seed oil and a s/n of 3/1 for LOD and 10/1 for LOQ. The LOD and LOQ values were less than 200 ppband 600 ppb, respectively, for all cannabinoids.1.5x10 6AreaLinearity R2Cannabigerol (CBG)1.0x10 65.0x10 50.0Table 3. LOQ and LOD of six cannabinoids in undiluted Hemp Seed Oil.R2 0.99994-5.0x10 50.002.004.006.008.0010.0012.0014.00Figure 4. Linearity plot of CBD; concentration range: 1-14 ppm in IPA.4.0x10 6THC3.5x10 6LOD via PDA(ppb)LOQ via PDA(ppb)Cannabigerol (CBG)31102Cannabidiol (CBD)Cannabinol (CBN)Delta-9-tetrahydrocannabinol (THC)Cannabichromene (CBC)Delta-9-tetrahydrocannabinolic acid x10 62.5x10 6Figures 6 and 7 show the chromatograms of un-spiked Brand Aand Brand B hemp seed oils, respectively, overlaid with standardmix. CBD was present in both of the un-spiked hemp seed oils,eluting at approximately 5.1 minutes and confirmed by PDAdetection (data not shown). THC was detected at very low levels(below LOQ) in both samples, while the other five analytes werenot detected in either of the analyzed oils.Area2.0x10 61.5x10 61.0x10 65.0x10 50.0R2 0.99925-5.0x10 50.002.004.006.008.0010.0012.0014.00For comparison, an overlay of THC/CBD-spiked and un-spikedBrand B hemp seed oil is shown in Figure 8.ppmFigure 5. Linearity plot of THC; concentration range: 1-14 ppm in IPA.1.40AU0.800.70THC-A0.90Cannabichromene (CBC)1.00THC1.10Cannabinol (CBN)1.20Cannabidiol (CBD)Cannabigerol .506.006.50MINUTESFigure 6. Overlay of a mixture of six cannabinoids (black) and Brand A of the un-spiked hemp seed oil (blue).47.007.508.008.50
1.40AU0.800.700.60THC-A0.90Cannabichromene (CBC)1.00THC1.10Cannabinol (CBN)1.20Cannabidiol (CBD)Cannabigerol re 7. Overlay of a mixture of six cannabinoids (black) and Brand B of the un-spiked hemp seed oil (blue).1.401.201.101.000.90THCCannabidiol 04.505.005.506.00MINUTES6.50Figure 8. Overlaid chromatograms of a blank (red), un-spiked Brand B hemp seed oil (blue) and Brand B hemp seed oil spiked with 125-ppm THC and CBD (black).The recovery of the spiked THC and CBD analytes was between91-110%, as shown in Table 4. As CBD was observed in bothbrands of the un-spiked hemp seed oil, the recovery of spiked CBDwas calculated by subtracting the un-spiked CBD concentrationfrom the overall recovery.Table 4. Recovery results for hemp seed oil spiked with THC and CBD at 125 ppm (n 2).Hemp Seed OilCBD (%)THC (%)Brand A spiked91.096.6Brand B spiked95.6110.25
ConclusionReferencesThis work has demonstrated the effective chromatographicseparation and quantitation of six cannabinoids, including THCand CBD, in hemp seed oils using the PerkinElmer HPLC Systemwith UV and PDA detectors. The results exhibited exceptionallinearity for each of the six cannabinoids over the concentrationrange tested.1. Johnson, Renee. “Hemp as an Agricultural Commodity:Congressional Research Service”, CRS Report, 2015.2. Hazekamp, Arno. “Cannabis Review”, Department ofPlant Metobolomics, Leiden University. 2008-2009.3. Leizer, Cary et. al. "The Composition of Hemp Seed Oil and ItsPotential as an Important Source of Nutrition", Journal ofNeutraceuticals, Functional and Medicinal Food. 2000.4. "Cannabis oil vs hemp seed oil; Cannabis oil, CDB Oil, MedicalMarijuana”. nElmer, Inc.940 Winter StreetWaltham, MA 02451 USAP: (800) 762-4000 or( 1) 203-925-4602www.perkinelmer.comFor a complete listing of our global offices, visit www.perkinelmer.com/ContactUsCopyright 2015-2018, PerkinElmer, Inc. All rights reserved. PerkinElmer is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners.012317B 01PKI
the viscosity of the hemp seed oil prior to analysis, each sample was diluted nine fold in IPA by adding 0.2 mL hemp seed oil to 1.6 mL of IPA and mixing by manual shaking. A 14-ppm spiked solution of each hemp seed oil sample was prepared by adding 25.0 µL THC standard, 25.0 µL CBD standard, 0.2 mL hemp seed
Products containing CBD, other cannabinoids, or any part of the Hemp plant are considered "Hemp Extract" and will be subject to Rule 5K-4.034, FAC. 2. Is CBD the same as Hemp? They are both varieties of the plant Cannabis sativa L., however, Hemp has a total THC level that does not exceed 0.3% on a dry weight basis.
i. Hemp extract or CBD in milk and dairy products j. Restaurants / Bars / Cosmetics k. Report a problem with a hemp product for human consumption l. Hemp and CBD smoking and vaping 7. ANIMAL FOOD AND FEED (DOG TREATS) 8. HEMP SEED 9. ADDITIONAL INFORMATION LINKS . SECTION IV: LAW ENFORCEMENT ISSUES . 10. PERSONAL POSSESSION OF HEMP PRODUCTS 11.
Testing for Cannabinoids Standardized Method of Cannabinoid Analysis AOAC International announced the approval of Official Method 2018.11 for detecting and measuring cannabinoids in hemp in April 2020 Initially created and validated for dry weight cannabis flower Additional validation and approval to be used to evaluate all plant materials of hemp
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Hemp Disease and Pest Management 5 Hemp rust Hemp rust is a fungal disease caused by Uredo kriegeriana (Figure 5). Little is known about this pathogen’s life cycle and mechanisms for overwintering. Hemp rust was first confirmed on samples collected in East Tennessee on August 26, 2019, at the UT Soil, Plant and Pest Center.
o No hemp production or processing is allowed in medical cannabis production centers Dispensary licensees can also be hemp processors, but hemp processing facility must be separate o Chapter 11-37 compliant hemp products may be used as an ingredient in a manufactured cannabis product o Chapter 11-37 compliant hemp products may be sold in .
ACCOUNTING 0452/22 Paper 2 May/June 2019 1 hour 45 minutes Candidates answer on the Question Paper. No Additional Materials are required. READ THESE INSTRUCTIONS FIRST Write your Centre number, candidate number and name on all the work you hand in. Write in dark blue or black pen. You may use an HB pencil for any diagrams or graphs. Do not use staples, paper clips, glue or correction fluid. DO .
