EFFECTS OF PH AND AMOUNT OF ACETONITRILE ON THE SEPARATION OF CANNABINOIDS
Keywords:Reversed-phase high-performance liquid chromatography, pH, Acetonitrile, Delta 9 tetrahydrocannabinol, Cannabidiol, Cannabinol
Objective: During reversed-phase high-performance liquid chromatography (HPLC) analyses, optimization of separation can be achieved by selecting appropriate chromatographic conditions. The retention time, peak shape, and peak size of chromatographic peaks are dependent on amount of organic modifier in the mobile phase and buffer pH. The aim of this study was to investigate the effects of varying pH, acetonitrile composition and flow rate of the mobile phase, and temperature of the stationary phase and wavelength in the development of a method to separate Δ9 tetrahydrocannabinol, cannabidiol, and cannabinol.
Methods: Mobile phases with different buffer pHs and acetonitrile composition were used with ultraviolet (UV) detection wavelength of 220 nm and 228 nm. The AUPs and retention times were observed using different mobile phase flow rates and stationary phase temperatures.
Results: The best results were obtained when using a mobile phase composition of 20% phosphate buffer pH 2.5 or pH 3 and 80% acetonitrile v/v at a flow rate of 2 mL/min at 220 nm.
Conclusion: This rapid and easy-to-use HPLC method describes the effect of changing important chromatographic parameters on separation and retention time of cannabinoids and can be effectively applied for high throughput analysis.
Nahar L, Onder A, Sarker SD. A review on the recent advances in HPLC, UHPLC and UPLC analyses of naturally occurring cannabinoids (2010-2019). Phytochem Anal 2020;31:413-57.
Nikolin B, Imamović B, Medanhodzić S, Sober M. High performance liquid chromatography in pharmaceutical analyses. Bosn J Basic Med Sci 2004;4:5-9.
Rafferty JL, Siepmann JI, Schure MR. Mobile phase effects in reversed-phase liquid chromatography: A comparison of acetonitrile/ water and methanol/water solvents as studied by molecular stimulation. J Chromatogr A 2011;1218:2203-13.
Wilson RE, Groskreutz SR, Weber SG. Improving the sensitivity, resolution and peak capacity of gradient elution in capillary liquid chromatography with large-volume injections by using temperature-assisted on-column solute focusing. Anal Chem 2016;88:5112-21.
Locatelli M, Melucci MD, Carlucci G, Locatelli C. Recent HPLC strategies to improve sensitivity and selectivity for the analysis of complex matrices. Instrum Sci Technol 2012;40:112-37.
Vella J, Busuttil F, Bartolo NS, Sammut C, Ferrito V, Serracino- Inglott A, et al. A simple HPLC-UV method for the determination of ciprofloxacin in human plasma. J Chromatogr B Analyt Technol Biomed Life Sci 2015;989:80-5.
Venn RF. Principles and Practice of Bio-Analysis. 2nd ed. London: Taylor and Francis; 2005.
Muchohi SM, Thuo N, Karisa J, Muturi A, Kokwaro GO, Maitland K. Determination of ciprofloxacin in human plasma using high-performance liquid chromatography coupled with fluorescence detection: Application to a population pharmacokinetics study in children with severe malnutrition. J Chromatogr B 2011;879:146-52.
Yang Y. High-Temperature Liquid Chromatography, LcGC North America; 2008. Available from: https://www.researchgate.net/ publication/269874595_high-temperature_liquid_chromatography. [Last accessed on 2020 Oct 05].
Wenclawiak BW, Sascha G, Tuetenberg T. High-temperature liquid chromatography. Anal Lett 2008;41:1097-105.
Hendriks G. Theoretical models in LC based bioanalytical method development. J Pharm Biomed Anal 2009;49:1-10.
Marques RM, Schoenmakers PJ. Modelling retention in reversed-phase liquid chromatography as a function of pH and solvent composition. J Chromatogr A 1992;592:157-82.
Galaon T, David V. The influence of mobile phase pH on the retention and selectivity of related basic compounds in reversed-phase liquid chromatography. Rev Roum Chim 2012;57:131-40.
Mifsud-Buhagiar L, Scorpiniti M, Bartolo NS, Szijj JV, Ferrito V, Serracino-Inglott A, et al. Implications of mobile phase composition and pH of the chromatographic separation of amitriptyline and its metabolite nortriptyline. Int J Pharm Pharm Sci 2018;10:132-8.
Vella J, Mifsud M, Bartolo NS, Ferrito V, Serracino-Inglott A, Azzopardi LM, et al. The combined effects of pH and acetonitrile composition on the separation of two lincosamide antibiotics. Asian J Pharm Clin Res 2014;7:96-100.
Ahuja A, Rasmussen H. HPLC Method Development for Pharmaceuticals. Amsterdam: Elsevier, Academic Press; 2007.
Sanli S, Sanli N, Alsancak G. Spectrophotometric determination of acidity constants of some macrolides in acetonitrile-water binary mixtures. Acta Chim Slov 2010;57:980-7.
Deidda R, Avohou TH, Orlandin S, Baronti R, Pasquini B, Furlanetto S, et al. Analytical quality by design: Development and control strategy for a RP-HPLC method implemented in routine to evaluate the cannabinoids content in Cannabis olive oil extracts. J Pharm Biomed Anal 2019;166:326-35.
de Petrocellis L, Ligresti A, Moriello AS, Allarà M, Bisogno T, Petrosino S, et al. Effects of cannabinoids and cannabinoid-enriched Cannabis extracts on TRP channels and endocannabinoid metabolic enzymes. Br J Pharmacol 2011;163:1479-94.
Baron EP. Comprehensive review of medicinal marijuana, cannabinoids, and therapeutic implications in medicine and headache: What a long strange trip it’s been. Headache 2015;55:885-916.
Pellati F, Borgonetti V, Bighenti V, Biagi M, Benvenuti S, Corsi S. Cannabis sativa L. and nonpsychoactive cannabinoids: Their chemistry and role against oxidative stress, inflammation, and cancer. Biomed Res Int 2018;2018:1691428.
Zuardi AW. History of Cannabis as a medicine: A review. Braz J Psychiatry 2006;28:153-7.
Eliott J, Deirdre D, Clifford T, Coyle D, Potter B, Skidmore B, et al. Cannabis for pediatric epilepsy: Protocol for a living systematic review. Syst Rev 2018;7:95.
Klumpers LE, Thacker DL. A brief background on Cannabis: From plant to medical indications. J AOAC Int 2019;12:412-20.
Lafaye G, Karila L, Blecha L, Benyamina A. Cannabis, cannabinoids and health. Dialogues Clin Neurosci 2017;19:309-16.
Zgair A, Wong JC, Sabri A, Fischer PM, Barrett DA, Constantinescu CS, et al. Development of a simple and sensitive HPLC-UV method for the simultaneous determination of cannabidiol and Δ9-tetrahydrocannabinol in rat plasma. J Pharm Biomed Anal 2015;114:145-51.
Gallo-Molina AC, Castro-Vargas HI, Garzón-Méndez WF, Ramirez JA, Monro ZJ, King JW, et al. Extraction, isolation and purification of tetrahydrocannabinol from the Cannabis sativa L. plant using supercritical fluid extraction and solid phase extraction. J Supercrit Fluids 2019;146:208-16.
Jamwal R, Topletz AR, Ramratnam B, Akhlaghi F. Ultra-high performance liquid chromatography tandem mass-spectrometry for simple and simultaneous quantification of cannabinoids. J Chromatogr B Analyt Technol Biomed Life Sci 2017;1048:10-8.
Mazina J, Spiljova A, Vaher M, Kaljurand M, Kulp M. A rapid capillary electrophoresis method with LED-native fluorescence detection for analysis of cannabinoids in oral fluid. Anal Methods 2015;7:7741.
ChEMBL; 2018. Available from: https://www.ebi.ac.uk/chembl/ compound_report_card/chembl74415. [Last accessed on 2020 Oct 06].
Cas MD, Casagni E, Saccardo A, Arnoldi S, Young C, Scotti S, et al. The Italian panorama of Cannabis light preparation: Determination of cannabinoids by LC-UV. Forensic Sci Int 2020;307:110-3.
Elkins AC, Deseo MA, Rochfort S, Ezenieks V, Spangenberg G. Development of a validated method for the qualitative and quantitative analysis of cannabinoids in plant biomass and medicinal Cannabis resin extracts obtained by super-critical fluid extraction. J Chromatogr B Analyt Technol Biomed Life Sci 2019;1109:76-83.
Burnier C, Esseiva P, Rouseel C. Quantification of THC in Cannabis plants by fast-HPLC-DAD: A promising method for routine analyses. Talanta 2019;192:135-41.
Mandrioli M, Tura M, Scotti S, Gallina T, Toschi T. Fast detection of 10 cannabinoids by RP-HPLC-UV method in Cannabis sativa L. Molecules 2019;24:2113.
Bettiol A, Lombardi N, Crescioli G, Maggioni V, Gallo E, Mugelli A, et al. Galenic preparations of therapeutic Cannabis sativa differ in cannabinoids concentration: A quantitative analysis of variability and possible clinical implications. Front Pharmacol 2018;9:1543.
Citti C, Linciano P, Forni F, Vandelli MA, Gigli G, Laganà A, et al. Analysis of impurities of cannabidiol from hemp. Isolation, characterization and synthesis of cannabidibutol, the novel cannabidiol butyl analog. J Pharm Biomed Anal 2019;175:112752.
Križman M. A simplified approach for isocratic HPLC analysis of cannabinoids by fine tuning chromatographic selectivity. Eur Food Res Technol 2020;2:315-22.
Namdar D, Mazuz M, Ion A, Koltai H. Variation in the compositions of cannabinoid and terpenoids in Cannabis sativa derived from inflorescence position along the stem and extraction methods. Ind Crops Prod 2018;113:376-82.
Hazekamp A, Peltenburg A, Verpoorte R, Giroud C. Chromatographic and spectroscopic data of cannabinoids from Cannabis sativa L. J Liq Chromatogr Relat Technol 2005;28:2361-82.
United Nations Office on Drugs and Crime. WHO Scheduling Recommendations on Cannabis and Cannabis-Related Substances; 2020. Available from: https://www.unodc.org/unodc/en/commissions/ cnd/mandate_functions/current-scheduling-recommendations.html. [Last accessed on 2020 Oct 26].
How to Cite
The publication is licensed under CC By and is open access. Copyright is with author and allowed to retain publishing rights without restrictions.