Journal:Effects of the storage conditions on the stability of natural and synthetic cannabis in biological matrices for forensic toxicology analysis: An update from the literature
| Full article title | Effects of the storage conditions on the stability of natural and synthetic cannabis in biological matrices for forensic toxicology analysis: An update from the literature |
|---|---|
| Journal | Metabolites |
| Author(s) | Djilali, Elias; Pappalardo, Lucia; Posadino, Anna M.; Giordo, Roberta; Pintus, Gianfranco |
| Author affiliation(s) | American University of Sharjah, University of Sassari, Mohammed Bin Rashid University of Medicine and Health Sciences |
| Primary contact | Email: lpappalardo at aus dot edu |
| Year published | 2022 |
| Volume and issue | 12(9) |
| Article # | 801 |
| DOI | 10.3390/metabo12090801 |
| ISSN | 2218-1989 |
| Distribution license | Creative Commons Attribution 4.0 International |
| Website | https://www.mdpi.com/2218-1989/12/9/801 |
| Download | https://www.mdpi.com/2218-1989/12/9/801/pdf (PDF) |
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This article should be considered a work in progress and incomplete. Consider this article incomplete until this notice is removed. |
Abstract
The use and abuse of cannabis, be it for medicinal or recreational purposes, is widely spread among the population. Consequently, a market for more potent and consequently more toxic synthetic cannabinoids has flourished, and with it, the need for accurate testing of these substances in intoxicated people. In this regard, one of the critical factors in forensic toxicology is the stability of these drugs in different biological matrices due to different storage conditions. This review aims to present the most updated and relevant literature of studies performed on the effects of different storage conditions on the stability of cannabis compounds present in various biological matrices, such as blood and plasma, urine, and oral fluids, as well as in alternative matrices such as breath, bile fluid, hair, sweat, cerumen, and dried blood spots.
Keywords: cannabinoids, stability, urine, plasma, oral fluids, hair, dried blood spots
Introduction
As of 2020, cannabis has become the most frequently used drug worldwide. Its use is associated with the impairment of the assuming individual’s cognitive and psychomotor abilities. [1] However, authentic marijuana is not the sole cause of concern, as synthetic cannabinoids also exist. These drugs were created to mimic the binding of delta-9 tetrahydrocannabinol (Δ9-THC) to cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2). However, it was later discovered that the binding potential possessed by these synthetic drugs is far stronger than that of their natural counterparts, causing them to have a greater chance of resulting in toxic effects. [2] Most of the abused synthetic cannabinoids legally available on the market appear to be CB1 receptor agonists showing an affinity greater than THC. [3] Due to their stronger cannabimimetic effects, a greater incidence of cognitive and psychomotor impairment, seizures, psychosis, tissue injury, and death associated with these drugs’ intake has been observed. [4] Data have shown that accidents, sometimes resulting in fatalities, have grown in number due to the increased use of these drugs. [1]
The primary psychoactive components of cannabis are THC and its metabolites, primarily THC-COOH. As a consequence, given the increment of both the use and abuse of such psychoactive substances, it is imperative for forensic laboratories to properly understand their stability within the biological matrices of collection. Indeed, their degradation is one of the most significant causes of concern during forensic cases. [5] These compounds are, in fact, subject to numerous processes that lead to the eventual degradation of or decrease in the cannabinoids from the sample. Such processes include but are not limited to conjugate formation, adsorption to surface containers, microbial action, thermal decomposition, and sample handling errors. [6,7] Therefore, sample storage conditions are critical for forensic toxicology analysis.
This review will provide insights into the overall stability of cannabinoids within different conventional and alternative biological matrices—namely blood, plasma, urine, oral fluids, breath, bile fluid, hair, sweat, cerumen, and dried blood spots—and gather the currently published literature about the ideal sample storage conditions for forensic toxicology analysis.
Conventional biological matrices
Blood and plasma
Analyte stability is among the essential parameters in forensic toxicology. [8] In blood, THC concentration reaches its highest point approximately 10 minutes after smoking cannabis and is then quickly distributed throughout the body due to its lipophilic nature. THC's metabolite THC-COOH, on the other hand, can persist within the body for up to a month. [1] Therefore, studies of these two metabolites have become more prominent in the past decade, as they may provide a practical guideline to properly detect the abuse of cannabinoids in forensic cases. To better understand the stability of these cannabinoids, different storage temperatures (i.e., room-temperature, refrigerated, and frozen) over time were carefully examined, since the concentration of both THC and THC-COOH is time-dependent. [9] Where the temperature is concerned, storing blood samples containing cannabinoids in a frozen condition, or refrigerated at the very least, appears to be the most effective way to ensure the greatest stability for the longest period of time. [10] At room temperature, cannabinoid concentrations tend to significantly decrease after a time ranging between two weeks and two months, regardless of the container material. [10] Storing whole blood containing cannabinoids in Venoject tubes with rubber stoppers for six months at room temperature decreased their concentrations by approximately 90%. Johnson et al. highlight the possibility of a THC concentration loss to the rubber stoppers used for the containers, but no further data is provided. [10]
Furthermore, other variables to consider are the properties of the containers in which the matrices are being stored. Because of the cannabinoids’ lipophilic nature, studies have highlighted the possibility of a drug adsorptive loss onto the container, which is made of similarly lipophilic plastic. [11] Experimental studies comparing the efficacy of polystyrene plastic and glass vials on THC-containing whole blood samples stored at −20 °C for 4–24 weeks showed a loss of THC concentration of 60 to 100% in the samples stored in plastic containers, while a loss of 30 to 50% was observed in the samples stored in glass vials. [11]
Whole-blood-contained cannabinoids stored in green-top sodium heparin vacutainers were found to remain stable for three to four months when stored under refrigerated conditions, whereas when stored under frozen conditions, they remained stable for up to six months. [12] The same tests were executed on plasma samples stored in grey-top sodium fluoride tubes, with results showing that cannabinoids would remain stable for up to 12 months at −20 °C. [13] However, it is worth mentioning that the same results were not observed in all the THC metabolites. Toennes and Kauert reported that, in plasma, the THC-COOH ester glucuronide metabolite, called THCCOOH-glucoronide (THC-COOH-glu), tends to significantly degrade. The study concluded that the susceptibility of the metabolite to the esterase enzymes naturally present in the blood might be at the base of the observed phenomenon. [14] Fort et al. performed a similar experiment on synthetic cannabinoids, namely XLR-11, UR-144, AB-Pinaca, and AB-Fubinaca, obtaining similar results. [2] The concentration of the synthetic cannabinoids was stable for the entire period of the experiment (12 weeks) when the blood samples were kept frozen. In contrast, under the other two conditions (refrigerated and room-temperature), there was a significant loss of the samples spiked with XLR-11, while the concentrations of UR-144, AB-Pinaca, and AB-Fubinaca remained stable at all three different temperatures for the entire experiment duration (t = 12 weeks). [2] Similarly to THC-COOH-glu, AB-Pinaca and AB-Fubinaca were found to be susceptible to degradation by carboxylesterase enzymes. [4] WIN 55,212-2 is another synthetic cannabinoid that was observed to be metabolized by the hepatic microsomes at the same rates as the previously mentioned synthetic cannabinoids. Its metabolites may be extracted for detection purposes from bio-matrices, although further research is required to fully confirm this aspect. [15]
Using whole blood samples collected in glass vials, Meneses and Mata repeated similar experiments on a variety of cannabis compounds, namely 11-nor-9-carboxy-THC (i.e., THC-COOH), cannabinol (CBN), and cannabidiol (CBD) under refrigerated and frozen conditions. The study results showed that the cannabinoids remained stable for approximately six months, losing about 20% of their initial concentration. While working with samples suspected of containing cannabinoids, the authors concluded that it would be ideal to analyze the samples as rapidly as possible, as it would provide the most accurate results. Should that not be possible, storage under frozen conditions is recommended. [16] Hess et al. analyzed the freeze/thaw stability of several synthetic cannabinoids in glass tubes, concluding that, while not advisable, continuously freezing and thawing a serum sample containing synthetic cannabinoids does not significantly decrease the initial drugs’ concentration. [17] On the other hand, another study performed on whole blood stored at −20 °C in plastic vacuette containers observed a significant difference between samples that had undergone freeze/thaw multiple times and samples that remained frozen uninterruptedly. This study, however, showed that the decrease in stability and concentration over time can be avoided using antioxidants as preserving agents. Indeed, applying a mixture of fluoride oxalate (FX) and ascorbic acid (ASC) to the samples resulted in no significant cannabinoid loss after five months, even when storage was interrupted by six freeze/thaw cycles. [18] A summary of the reported data is presented in Table 1 and Table 2.
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References
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.
