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 |
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| 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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Urine
Urine, among the other biological matrices used for illicit drug detection, is considered the most popular. Indeed, urine sapling requires noninvasive collection techniques and allows for a fairly wide detection window for most psychoactive drugs and their metabolites. [19] Due to its easy application, urine drug testing is often used in workplaces to test all workers to create a "drug-free work environment." [20] Thus, similarly to blood, a thorough understanding of the drug of abuse's stability in urine matrices is essential. In urine, the stability of a drug depends on the sample pH, storage temperature, bacterial contamination, and the container material used. [21] In this context, Ciuti et al. tested the effects of temperature (−20 °C, 4 °C, and 25 °C), over 20 weeks on THC-containing urine samples using both glass and polyethylene vials. The data indicated a recovery of approximately 85% of the original content in samples stored at −20 °C (frozen conditions), thus indicating the analytes’ relative stability. Conversely, this was not observed in samples stored at 4 °C and at 25 °C, where the recovery was 37 and 33%, respectively. [21] These findings align with another study’s conclusions, whereby frozen conditions allowed for greater cannabinoid stability within the urine matrix. This experimental study spanned over three years and showed a maximum loss in cannabinoid (THC-COOH) concentration of 19.6 ± 6.7% when samples were stored at −20 °C in polypropylene containers. [22] Desrosiers et al. replicated a similar experimental design and were also able to observe better cannabinoid stability when samples were stored under frozen conditions for up to six months. In their experiment, polypropylene vials were utilized instead of polyethylene ones, as they seemingly cause less adsorptive loss. [23] The authors also stated that glass vials are less preferred to store biological matrices due to the easy possibility of breaking. [23] Frozen conditions appear to be the most favorable for another THC conjugate, THC-COOH glucuronide, a THCCOOH metabolite. Unlike in blood, however, the THCCOOH-Glu-degrading esterase enzymes are not present in urine, allowing this molecule to remain present within the solution, and therefore making it a viable marker for the detection of cannabis use. [7]
Further insight on the cannabinoids’ stability has been provided by studies focusing on the containers utilized to store the drugs of abuse. Jamerson et al. showed the effects of container composition, pH, and temperature on the cannabinoids’ adsorptive loss. Tests performed using polypropylene plastic containers and borosilicate glass containers showed that the adsorptive loss was highly present in polypropylene containers compared to the borosilicate ones, and that it appeared to be relatively absent in urine solutions near neutral or basic pH. [24] Although glass vials show no cannabinoid adsorptive loss, their usage is not the preferred one when it comes to the storage of biological matrices due to easy breakability. [23] In light of such conclusions, researchers have tried to observe whether the type of plastic container employed may cause lower, or higher, cannabinoid metabolite adsorptive loss. [25] In this regard, the effect of both polypropylene and polyethylene containers on cannabinoid stability was tested at both 4 °C and 25 °C in the same study. A rapid cannabinoid loss was observed for both containers at 4 °C, while at 25 °C only a small loss was observed for polypropylene containers, and no significant loss was observed in polyethylene containers. The authors mentioned that the observed effects could be related to the cannabinoid’s lower solubility in water at lower temperatures. In addition, as the overall loss appeared to stabilize after approximately one hour, the researchers concluded that the observed loss was due to a surface phenomenon and not to an absorption effect into the container plastic matrix. [25] Similarly, it was determined that a solution of urine spiked with THC could be stored in (Nalgene) high-density polyethylene plastic containers for up to 40 days. The study illustrated that, at 2–8 °C, the analyte concentration remained constant for 42 days and showed a minimal decrease following day 42. The analyte concentration decreased from 72.44 ng/mL to 65.71 ng/mL on day 72. [26]
While trying to understand the mechanism of cannabinoid concentration loss in urine matrix, research studies have shown that loss could be divided into loss during equilibrium conditions, that is, during storage, and loss during kinetic conditions, indicating losses that occur while transporting, manipulating, and testing urine samples. [27] The study’s conclusion showed that equilibrium losses are affected by the solvent, the container material, and the exposed surface area. In contrast, kinetic losses are affected mainly by temperature. Furthermore, Roth et al. advised the usage of glass containers for storage and glass pipettes for sample handling. Conversely, the poorest results were observed when using high-density polyethylene containers. [27] Lastly, using containers possessing internal bar code labels is not advised, as test results showed a significant reduction in THC-COOH levels when urine samples were stored in Doxtech bottles with an internal bar code. Instead, losses were relatively insignificant when urine samples were stored in the same containers but with an external barcode. This phenomenon appears to be due to the internal ID itself being made of waterproof polypropylene materials. [28]
In a study by Welsh et al., the authors reported that the adsorptive loss issue during the sample’s storing and handling might be bypassed if the cannabinoid-containing urine solution is treated with a non-ionic surfactant such as Tergitol. Their results showed a significantly higher THC recovery from the surfactant-treated samples. [29] Additionally, fungal and bacterial growth appear to be factors involved in significantly decreasing cannabinoid concentration in urine samples. However, this decrease appears to occur only when the storage temperature is above a threshold (near room temperature) that would allow for bacterial and fungal growth in the first place. [7] However, it is yet to be determined whether bacteria and fungi possess the ability to specifically degrade cannabinoids or otherwise. [5]
A summary of the reported data is presented in Table 3.
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Oral fluids
When it comes to psychoactive impairment caused by cannabinoid drugs, oral fluids have become increasingly studied biological matrices for the early detection of drugs of abuse. The reasons for their increase in popularity are several, including noninvasive collection methods, no requirement for trained medical professionals, and the possibility of multiple sample collection allowing for early detection in workplaces. [30] Reports show that oral fluids can also be utilized for the detection of new psychoactive drugs (NPS), such as new synthetic cannabinoids that continuously appear on the market. [31]
Just like it has been done for blood and urine, to further understand cannabinoids’ stability in oral fluids, researchers have studied the effects of storage temperature and sample container material on the psychoactive drug in this biological matrix. When stored at −20 °C, 4 °C, and 21 °C in polypropylene plastic containers for six weeks, THC losses were reported to be 21%, 87%, and 86%, respectively. [32] Similar tests were repeated using expectorated oral fluids stored for dix days in polypropylene tubes and glass tubes at 4 °C and room temperature. Results showed a loss of <10% for the samples stored in glass vials at both temperature conditions and >20% when stored in polypropylene tubes. [33] Kneisel et al. compared the effects of glass and plastic containers on 11 synthetic cannabinoids. [34] After 24 hours of storage in polypropylene tubes at room temperature, the authors observed recoveries ranging from 29 to 65%, while at 4 °C, recoveries ranged between 83 and 103% (RSD ≤ 13%). After 72 hours of storage in plastic containers, recoveries dropped to a range between 9 and 54% at room temperature and 75–79% at 4 °C. When using RapidEASE borosilicate glass tubes, on the other hand, recoveries ranged between 84 and 114% (RSD ≤ 12%) for the entire duration of the experiment (72 hours) and at all temperature conditions. [34] Likewise, to confirm that adsorptive loss is indeed the main storage-associated issue of cannabinoid-containing oral fluids, Molnar et al. observed a 23–30% THC adsorptive loss in polypropylene containers within a six-day storage period. [35] The study determined that lower oral fluid volumes led to a more significant adsorptive loss of the cannabinoid to the tube’s surfaces. Concerning temperature, the oral fluid samples were stored at both 4 °C and room temperature for four weeks, and a total cannabinoid concentration loss of 40–50% was observed in both temperature conditions. [35] Among the various suggestions, Moore et al. indicated the Quantisal collection device as an efficient THC extraction method from oral fluid samples, as long as certain conditions are satisfied. [36] The conditions specified by the authors are in-line with the other previously discussed findings and include that the samples must not be in contact with plastic surfaces, must be frozen or refrigerated, and must be stored in the dark. [36]
A summary of the reported data is presented in Table 4.
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Alternative matrices
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.
