Clin Chem Lab Med 2017; aop

Letter to the Editor

Roberta Pacifici*, Emilia Marchei, Francesco Salvatore, Luca Guandalini, Francesco Paolo Busardò and Simona Pichini Evaluation of long-term stability of in standardized preparations of flowering tops and cannabis oil by ultra-high-performance liquid chromatography tandem mass spectrometry https://doi.org/10.1515/cclm-2017-0758 concentration and short-term (up to 14 days) stability in Received August 25, 2017; accepted September 21, 2017 standardized preparations of cannabis tea and cannabis oil [2]. We showed that the 10 different batches of flow- Keywords: ; stability; ultra-high-­ ering tops effectively contained a mean 5.8% THC and a performance liquid chromatography tandem mass mean 8.1% CBD with minimal variations between batches spectrometry (UHPLC-MS/MS). and that stability in aqueous solution was so short that extemporaneous tea preparation was rec- To the Editor, ommended, whereas in case of oil preparation, a loss of around 20% initial concentration was observed for all the From November 2015, Italy started autonomous cultiva- studied cannabinoids for the first 14 days of storage. tion and production of medical cannabis, authorized by a It has been shown that cannabinoid content in can- Decree of the Italian Ministry of Health [1]. From January nabis plant and in its products (e.g. resin and oil) 2017, the batches of Cannabis FM2, a cannabis with stand- fall as storage time increase in terms of months and years ardized amount of 6.5% (w/w) Δ-9- [3–6]. Nevertheless, up to now, no data are available for (THC) and 8% (w/w) (CBD) manufactured by long-term stability of cannabis flowering tops and medical the Military Pharmaceutical Chemical Works of Florence, cannabis oil. came into the market. Taking all this into account, the Italian National Insti- In order to guarantee the quality and the tempo- tute of Health, in agreement with the Health Commis- ral inalterability of cannabis preparations and conse- sion on Medical Cannabis and Military Pharmaceutical quently therapeutic continuity in individuals treated Chemical Works of Florence, aimed to evaluate the 1-year with medical cannabis, the Italian National Institute of stability of cannabinoids in standardized preparations Health, as indicated in the Ministerial Decree, was com- of cannabis flowering tops and cannabis oil by the ultra- mitted to evaluate cannabinoid concentration and stabil- high-performance liquid chromatography tandem mass ity in standardized preparations of cannabis flowering spectrometry (UHPLC-MS/MS) assay, developed and vali- tops, cannabis tea and oil. dated in the previous study [2]. In this concern, we recently evaluated cannabinoid We measured the content (% w/w) of THC, Δ9- content in the first 10 batches of FM2 and cannabinoid tetrahydrocannabinolic acid A (THCA-A), CBD, canna- bidiolic acid (CBDA), (CBN), (CBG), (CBC) in the 10 batches of FM2 *Corresponding author: Roberta Pacifici, National Centre on cannabis, previously used to study short-term stability Addiction and Doping, Istituto Superiore di Sanità, V.le Regina Elena [2], stored inside closed plastic containers in darkness at 299, 00161, Rome, Italy, Phone: +390649906544, Fax: +390649902016, E-mail: [email protected] room temperature (about 25 °C) and in six different repli- Emilia Marchei and Simona Pichini: National Centre on Addiction cates of cannabis oil, prepared as previously described [2] and doping, Istituto Superiore di Sanità, Rome, Italy and stored in darkness at 4 °C. In the specific case of oil, Francesco Salvatore and Luca Guandalini: Agenzia Industrie e no oven preheating was performed, which decarboxylated Difesa, Stabilimento Chimico Farmaceutico Militare; (Military THCA-A and CBDA to THC and CBD, respectively, prevent- Pharmaceutical Chemical Works of Florence), Florence, Italy Francesco Paolo Busardò: Unit of Forensic Toxicology (UoFT), ing the stability study of acidic compounds over the time. Department of Anatomical, Histological, Forensic and Orthopedic In case of FM2, the 1-year storage of cannabis at Sciences, Sapienza University of Rome, Rome, Italy ambient temperature caused only slight changes in the

Brought to you by | University of Sydney Authenticated Download Date | 11/27/17 8:49 PM 2 Pacifici et al.: Long-term stability of cannabinoids in cannabis flowering tops and cannabis oil content of THC and THCA-A, and a minimal decrease in flowering tops content of CBD and CBDA are likely to not case of CBG and CBC. Conversely, a significant increase be medically relevant. was observed for CBN, which in any case remained far Regarding the stability of cannabinoids in oil below 1% and for CBD with a simultaneous significant preparation after 1 year of storage in the darkness at decrease of CBDA content (Figure 1). In this latter case, it 4 °C, a significant decrease (p < 0.05) was observed in has to be underlined that both THC and CBD are present case of THC (14.6 ± 8.1%), THCA-A (24.7 ± 7.8%), CBD in the plant flowering tops together with their non-psy- (12.9 ± 4.9%), CBDA (9.9 ± 2.3%), CBN (22.1 ± 6.1%) and choactive carboxylated forms: THCA-A and CBDA, which CBG (36.2 ± 6.3%) and not in case of CBC (7.8 ± 5%) (Figure have recently been involved in the above-reported medical 2). However, the loss of cannabinoids mainly involved in benefits of cannabis, at least in an animal model [7, 8]. In the medical effect of cannabis (namely, THC, CBD and this sense, even significant variations in the percentage of CBDA were <20% and above 20% only in case of THCA-A)

8.0 * 7.0 * 6.0

5.0

4.0

3.0

2.0 Cannabinoid concentration, % w/w 1.0 *

0.0 %THC %THCA-A %CBD %CBDA %CBN %CBG %CBC July-2016 3.37 ± 0.66 2.82 ± 0.77 2.66 ± 0.52 6.18 ± 0.66 0.09 ± 0.05 0.06 ± 0.01 0.12 ± 0.03 July-2017 3.71 ± 0.48 2.86 ± 0.80 4.81 ± 0.92 3.86 ± 0.60 0.16 ± 0.04 0.05 ± 0.01 0.08 ± 0.02

Figure 1: Effect of 1-year storage in darkeness at ambient temperature on cannabinoid content in FM2 cannabis flowering tops. *Student’s t-test, p < 0.05.

% THC % THCA-A % CBD % CBDA % CBN % CBG % CBC

100

75

50

% Initial concentration 25

0 014 365

Time, days

Figure 2: Stability of cannabinoids in cannabis oil after 1-year storage in darkeness and at 4 °C.

Brought to you by | University of Sydney Authenticated Download Date | 11/27/17 8:49 PM Pacifici et al.: Long-term stability of cannabinoids in cannabis flowering tops and cannabis oil 3 was higher than that observed after 14 days of storage, Competing interests: The funding organization(s) played as previously shown [2]. Hence, after an initial cannabi- no role in the study design; in the collection, analysis, and noid loss of around 15%–20% is taken into consideration interpretation of data; in the writing of the report; or in the when prescribing the product for medical purposes, then decision to submit the report for publication. up to 1 year, the active compounds in oil preparation remain quite stable. The minimal changes in the content of THC and References THCA-A present in the flowering tops and in oil prepara- tion are in accordance with the study of Trofin et al. [4] 1. Decreto 9 novembre 2015: Funzioni di Organismo statale per la and Taschwer and Schmid [5], whereas concerning CBN cannabis previsto dagli articoli 23 e 28 della convenzione unica increase, some studies reported a time-related slight sugli stupefacenti del 1961, come modificata nel 1972. Available ­progressive conversion of THC into CBN in aged material at: http://www.gazzettaufficiale.it/eli/id/2015/11/30/15A08888/ sg;jsessionid=p1rnwNujUKlqQ5azhAQ95A__.ntc-as3-guri2a. [3, 9]. Accessed: 24 Aug 2017. From the obtained results, we can conclude that can- 2. Pacifici R, Marchei E, Salvatore F, Guandalini L, Busardò FP, nabinoid content in Italian FM2 cannabis flowering tops Pichini S. Evaluation of cannabinoids concentration and stability grown at fixed temperature and light-dark cycles by Mili- in standardized preparations of cannabis tea and cannabis oil tary Pharmaceutical Chemical Works and in oil prepared by ultra-high performance liquid chromatography tandem mass with a standardized procedure undergo slight changes, spectrometry. Clin Chem Lab Med 2017;55:1555–63. [Epub ahead of print]. which do not affect therapeutic continuity in treated indi- 3. Trofin IG, Dabija G, Váireanu DI, Filipescu L. The influence of long- viduals and assure a homogeneous product of defined sta- term storage conditions on the stability of cannabinoids derived bility. This long-term study complements the information from cannabis resin. Rev Chim 2012;63:422–7. on short-term stability of medical cannabis provided to 4. Trofin IG, Dabija G, Váireanu DI, Filipescu L. Long term storage the physicians, who need to know the precise composition and cannabis oil stability. Rev Chim 2012;63:293–7. 5. Taschwer M, Schmid MG. Determination of the relative percent- of a prescribed preparation and stability of a prescribed age distribution of THCA and Δ(9)-THC in herbal cannabis seized preparation. in Austria – Impact of different storage temperatures on stability. Forensic Sci Int 2015;254:167–71. Acknowledgments: The authors thank Stefano Gentili, 6. Lindholst C. Long term stability of cannabis resin and cannabis Michele Sciotti, Simonetta di Carlo, Antonella Bacosi and extracts. Australian J Forensic Sci 2010;42:181–90. Laura Martucci for their technical assistance. 7. Rock EM, Limebeer CL, Parker LA. Effect of combined doses of Δ(9)-tetrahydrocannabinol (THC) and cannabidiolic acid (CBDA) on All the authors have accepted Author contributions: acute and anticipatory nausea using rat (Sprague-Dawley) models responsibility for the entire content of this submitted of conditioned gaping. Psychopharmacology 2015;232:4445–4. manuscript and approved submission. 8. Brierley DI, Samuels J, Duncan M, Whalley BJ, Williams CM. Research funding: Funded by the project: “Implemen- ­Neuromotor tolerability and behavioural characterisation tazione dei Sistemi di Sorveglianza sul disturbo da gioco of cannabidiolic acid, a phytocannabinoid with therapeu- tic potential for anticipatory nausea. Psychopharmacology d’azzardo e sull’uso medico della cannabis sul territorio 2016;233:243–54. Nazionale” by the Ministry of Health. 9. Carbone M, Castelluccio F, Daniele A, Sutton A, Ligresti A, Employment or leadership: None declared. Di Marzo V, et al. Chemical characterisation of oxidative Honorarium: None declared. ­degradation products of Δ9-THC. Tetrahedron 2010;66:9497–501.

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