Development and validation of HPLC and GC methods for quantification of cannabinoids and terpenes extracted by ultrasound assisted extraction technique
DOI:
https://doi.org/10.22456/2527-2616.139666Keywords:
HPLC analysis, gas chromatography, cannabinoids, terpenes, quantificationAbstract
Cannabis exhibits a wide array of therapeutic effects due to its diverse and potent compounds. High-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) are the primary techniques used to quantify and qualify cannabinoids and terpenes in cannabis. This paper rigorously validates HPLC and GC-MS methods for quantifying cannabinoids and terpenes in cannabis plants. Cannabinoids and terpenes were extracted using an optimised ultrasound assisted extraction (UAE) method. The HPLC and GC-MS methods were validated by assessing linearity, limit of detection (LOD), limit of quantification (LOQ), recovery, precision (inter-day and intra-day), accuracy (inter-day and intra-day), and matrix effect for 12 cannabinoids and 30 terpenes using appropriate standards. The HPLC and GC results fell within acceptable ranges (for HPLC, recovery between 73-121%, precision, accuracy and matrix effect all were below 10%; and for GC, recovery between 79-91%, precision, accuracy and matrix effect all were below 10%) demonstrating the accuracy and reliability of the validated method. The findings of this study can be used effectively to measure these important compounds in cannabis samples, facilitating quality control and standardisation of cannabis-based products for therapeutic applications.
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References
Thakur, G.A., R.I. Duclos Jr, and A. Makriyannis, Natural cannabinoids: templates for drug discovery. Life sciences, 2005. 78(5): p. 454-466.
Andreae, M.H., et al., Inhaled cannabis for chronic neuropathic pain: a meta-analysis of individual patient data. The Journal of Pain, 2015. 16(12): p. 1221-1232.
Aizpurua-Olaizola, O., et al., Identification and quantification of cannabinoids in Cannabis sativa L. plants by high performance liquid chromatographymass spectrometry. Analytical and bioanalytical chemistry, 2014. 406: p. 7549-7560.
Hazekamp, A. and G. Pappas, Self-medication with Cannabis. Handbook of Cannabis, 2014. 319.
Citti, C., et al., Pharmaceutical and biomedical analysis of cannabinoids: A critical review. Journal of pharmaceutical and biomedical analysis, 2018. 147: p. 565-579.
Berman, P., et al., A new ESI-LC/MS approach for comprehensive metabolic profiling of phytocannabinoids in Cannabis. Scientific reports, 2018. 8(1): p. 14280.
Russo, E.B., Taming THC: potential cannabis synergy and phytocannabinoid‐terpenoid entourage effects. British journal of pharmacology, 2011. 163(7): p. 1344-1364.
Gertsch, J., et al., Beta-caryophyllene is a dietary cannabinoid. Proceedings of the National Academy of Sciences, 2008. 105(26): p. 9099-9104.
Lewis-Bakker, M.M., et al., Extractions of medical cannabis cultivars and the role of decarboxylation in optimal receptor responses. Cannabis and cannabinoid research, 2019. 4(3): p. 183-194.
Lazarjani, M.P., et al., Processing and extraction methods of medicinal cannabis: A narrative review. Journal of cannabis research, 2021. 3: p. 1-15.
Hazekamp, A., Y.H. Choi, and R. Verpoorte, Quantitative analysis of cannabinoids from Cannabis sativa using 1H-NMR. Chemical and pharmaceutical bulletin, 2004. 52(6): p. 718-721.
Lazarjani, M.P., et al., Processing and extraction methods of medicinal cannabis: A narrative review. Journal of cannabis research, 2021. 3(1): p. 1-15.
Pourseyed Lazarjani, M., et al., Methods for quantification of cannabinoids: A narrative review. Journal of Cannabis Research, 2020. 2(1): p. 1-10.
Hazekamp, A. and J. Fischedick, Cannabis‐from cultivar to chemovar. Drug testing and analysis, 2012. 4(7-8): p. 660-667.
Singh, P.K., et al., Steps to be considered during method development and validation for analysis of residual solvents by gas chromatography. International Research Journal of Pharmaceutical and Applied Sciences, 2013. 3(5): p. 74-80.
Den Boef, G. and A. Hulanicki, Recommendations for the usage of selective, selectivity and related terms in analytical chemistry. Pure and Applied Chemistry, 1983. 55(3): p. 553-556.
Stauffer, M., Calibration and Validation of Analytical Methods: A Sampling of Current Approaches. 2018: BoD–Books on Demand.
De Backer, B., et al., Innovative development and validation of an HPLC/DAD method for the qualitative and quantitative determination of major cannabinoids in cannabis plant material. Journal of Chromatography B, 2009. 877(32): p. 4115-4124.
Ibrahim, E.A., et al., Analysis of terpenes in Cannabis sativa L. using GC/MS: method development, validation, and application. Planta medica, 2019. 85(05): p. 431-438.
Araujo, P., Key aspects of analytical method validation and linearity evaluation. Journal of chromatography B, 2009. 877(23): p. 2224-2234.
Rozet, E., et al., Analysis of recent pharmaceutical regulatory documents on analytical method validation. Journal of Chromatography A, 2007. 1158(1-2): p. 111-125.
Giese, M.W., M.A. Lewis, and L. Giese, Reliable and robust method for the analysis of cannabinoids and terpenes in cannabis. 2019, Google Patents.
Le, T.T., et al., Development and validation of a simple LC-MS/MS method for the simultaneous quantitative determination of trimethylamine-Noxide and branched chain amino acids in human serum. Analytical and bioanalytical chemistry, 2019. 411: p. 1019-1028.
Pugliese, J., et al., Dispersive SPE, an alternative to traditional SPE for extraction of 43 doping peptides from equine urine prior to LC–MS screening. Forensic Toxicology, 2020. 38: p. 365-377.
Weitzel, M.J., The estimation and use of measurement uncertainty for a drug substance test procedure validated according to USP< 1225>. Accreditation and Quality Assurance, 2012. 17(2): p. 139-146.
Nielsen, S.D., et al., Development and application of a multiple reaction monitoring mass spectrometry method for absolute quantification of lysinoalanine and lanthionine in dairy products. International Dairy Journal, 2020. 105: p. 104693.
Jin, D., et al., Classification of Cannabis cultivars marketed in Canada for medical purposes by quantification of cannabinoids and terpenes using HPLC-DAD and GC-MS. J. Anal. Bioanal. Tech, 2017. 8(2).
Wu, J., et al., Rapid simultaneous determination of 11 synthetic cannabinoids in urine by liquid chromatography-triple quadrupole mass spectrometry. Separations, 2023. 10(3): p. 203.
Chu, M.H.C. and O.H. Drummer, Determination of Δ9-THC in whole blood using gas chromatographymass spectrometry. Journal of analytical toxicology, 2002. 26(8): p. 575-581.
Brighenti, V., et al., Development of a new extraction technique and HPLC method for the analysis of non-psychoactive cannabinoids in fibretype Cannabis sativa L.(hemp). Journal of Pharmaceutical and Biomedical Analysis, 2017. 143: p. 228-236.
Patel, B., D. Wene, and Z.T. Fan, Qualitative and quantitative measurement of cannabinoids in cannabis using modified HPLC/DAD method. Journal of Pharmaceutical and Biomedical Analysis, 2017. 146: p. 15-23.
Zivovinovic, S., et al., Determination of cannabinoids in Cannabis sativa L. samples for recreational, medical, and forensic purposes by reversed-phase liquid chromatography-ultraviolet detection. Journal of Analytical Science and Technology, 2018. 9(1): p. 1-10.
Ciolino, L.A., T.L. Ranieri, and A.M. Taylor, Commercial cannabis consumer products part 2: HPLC-DAD quantitative analysis of cannabis cannabinoids. Forensic science international, 2018. 289: p. 438-447.
Myers, C., et al., Accelerated solvent extraction of terpenes in Cannabis coupled with various injection techniques for GC-MS analysis. Frontiers in Chemistry, 2021. 9: p. 619770.
Fernández, N., et al., Development and validation of a GC-MS method for quantification of terpenes in cannabis oil (Cannabis sativa L.). Application to commercially available preparations in Argentina. Journal of Applied Research on Medicinal and Aromatic Plants, 2023. 35: p. 100466.
Premier, I., M.J.M. Aznar, and S.P. Minister, FOOD AND AGRICULTURE ORGANISATION OF THE UNITED NATIONS (FAO). 2015.
Little, T., Establishing acceptance criteria for analytical methods. BioPharm International, 2016. 29(10).
38.Steiner, D., et al., Evaluation of matrix effects and extraction efficiencies of LC–MS/MS methods as the essential part for proper validation of multiclass contaminants in complex feed. Journal of agricultural and food chemistry, 2020. 68(12): p. 3868-3880.
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