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)

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.

Table 1. Duration of storage stability for cannabinoids in blood based on temperature and collection container. RT: Room temperature; ID, inconclusive data (only one or two participant pools exceeded assay limit of quantification during baseline analysis). Matrix T (°C) Container Stability Note Reference Blood −20 °C Polystyrene plastic vials 60–100% loss between 4–24 weeks Losses observed were 30–50% lower when stored in glass vials. [11] Blood RT Venoject tubes with rubber stoppers 2–8 weeks At RT, THC concentrations significantly decreased after 2–8 weeks. Losses >90% after 6 months at RT. [10] Blood RT Green-top (Sodium heparin) Stable for up to 1 week THC-glu: ID THC: 1 week THC-COOH-glu: < 1 week THC-COOH < 1 week 11-OH-THC: < 1 week CBN: 1 week CBD: 1 week [12] Blood 4 °C Green-top (Sodium heparin) Stable for up to 6 months THC-glu: ID THC: 3 months THC-COOH-glu: 1 month THC-COOH: 1 month 11-OH-THC: 3 months CBN: 6 months CBD: ID [12] Blood −20 °C Green-top (Sodium heparin) Stable for up to 6 months THC-glu: ID THC: 3 months THC-COOH-glu: 3 months THC-COOH: 6 months 11-OH-THC: 6 months CBN: 3 months CBD: ID [12] Blood RT Gray-top tubes (Sodium fluoride tubes) Stable for up to 1 week THC: 1 week 11-OH-THC: 1 week THC-COOH-glu: < 1 week THC-COOH: 1 week [13] Blood 4 °C Gray-top tubes (Sodium fluoride tubes) Stable for up to 6 months THC: 6 months 11-OH-THC: 6 months THC-COOH-glu: 1 week THC-COOH: 6 months [13] Blood −20 °C Gray-top tubes (Sodium fluoride tubes) Stable for up to 1 year THC: 6 months 11-OH-THC: 1 year THC-COOH-glu: 3 months THC-COOH: 1 year [13] Blood RT Green-top tubes (Sodium heparin) Stable for up to 1 week THC: 1 week 11-OH-THC: 1 week THC-COOH-glu: < 1 week THC-COOH: < 1 week [13] Blood 4 °C Green-top tubes (Sodium heparin) Stable for up to 3 months THC: 3 months 11-OH-THC: 3 months THC-COOH-glu: 1 month THC-COOH: 1 month [13] Blood −20 °C Green-top tubes (Sodium heparin) Stable for up to 6 months THC: 3 months 11-OH-THC: 6 months THC-COOH-glu: 3 months THC-COOH: 6 months [13] Blood (synthetic cannabinoids) −20 °C Glass vials Stable for up to 12 weeks AB-Fubinaca, AB-Pinaca, UR-144 and XLR-11 remained stable for 12 weeks. [2] Blood (synthetic cannabinoids) 4 °C and RT Glass vials Stable for up to 12 weeks AB-Fubinaca, AB-Pinaca, and UR-144 remained stable for 12 weeks. XLR-11 significantly degraded by 31–73% after 3 weeks, and by 70–90% after 12 weeks. [2] Blood −20 °C and 4 °C Glass vials Stable for approximately 6 months Loss of approximately 20% of their initial concentration. [16]

Table 2. Duration of storage stability for cannabinoids in plasma based on temperature and collection container. RT: Room temperature. Matrix T (°C) Container Stability Note Reference Plasma RT Green-top (Sodium heparin) Stable up to 1 week THC-glu: 1 week THC: 1 week THC-COOH-glu: < 1 week THC-COOH < 1 week 11-OH-THC: < 1 week CBN: 1 week CBD: 1 week [12] Plasma 4 °C Green-top (Sodium heparin) Stable for up to 6 months THC-glu: 6 months THC: 6 months THC-COOH-glu: 2 weeks THC-COOH: 2 weeks 11-OH-THC: 6 months CBN: 3 months CBD: 6 months [12] Plasma −20 °C Green-top (Sodium heparin) Stable for up to 1 year THC-glu: 1 year THC: 1 year THC-COOH-glu: 6 months THC-COOH: 6 months 11-OH-THC: 1 year CBN: 1 year CBD: 1 year [12] Plasma RT Gray-top tubes (Sodium fluoride tubes) Stable for up to 1 week THC: 1 week 11-OH-THC: 1 week THC-COOH-glu: < 1 week THC-COOH: < 1 week [13] Plasma 4 °C Gray-top tubes (Sodium fluoride tubes) Stable for up to 6 months THC: 3 months 11-OH-THC: 6 months THC-COOH-glu: 1 week THC-COOH: 1 month [13] Plasma −20 °C Gray-top tubes (Sodium fluoride tubes) Stable for up to 1 year THC: 1 year 11-OH-THC: 1 year THC-COOH-glu: 3 months THC-COOH: 1 year [13] Plasma RT Green-top tubes (Sodium heparin) Stable for up to 1 week THC: 1 week 11-OH-THC: < 1 week THC-COOH-glu: < 1 week THC-COOH: < 1 week [13] Plasma 4 °C Green-top tubes (Sodium heparin) Stable for up to 6 months THC: 6 months 11-OH-THC: 6 months THC-COOH-glu: 2 weeks THC-COOH: 2 weeks [13] Plasma −20 °C Green-top tubes (Sodium heparin) Stable for up to 1 year THC: 1 year 11-OH-THC: 1 year THC-COOH-glu: 6 months THC-COOH: 6 months [13]

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.

Table 3. Duration of storage stability for cannabinoids in urine based on temperature and collection container. RT: Room temperature. Matrix T (°C) Container Stability Note Reference Urine −20 °C Glass vials and polyethylene plastic vials Stable for approximately 20 weeks 85% recovery. [21] Urine 4 °C and RT Glass vials and polyethylene plastic vials Not stable At 4 °C and RT, in glass vials, recoveries were approximately 37% and 33%, respectively. In plastic vials, losses were 17% and 5% higher respectively. [21] Urine −20 °C Polypropylene container Stable for 3 years Maximum loss of 19.6 +/− 6.7% over a maximum time of 3 years. [22] Urine −20 °C Polypropylene container Stable for 6 months Remained stable for the whole duration of the experiments. [23] Urine 4 °C Polyethylene and polypropylene plastic n/a Rapid loss was observed for both containers. [25] Urine 25 °C Polyethylene plastic Stable No significant loss observed. [25] Urine 25 °C Polypropylene plastic Stable Small loss (approximately 5%) was observed. [25] Urine 2–8 °C High-density polyethylene nalgene containers Minimal decrease in concentration over a 79-day period Approximately 11% loss. [26] Urine Doxtech bottles with external barcodes Loss of approximately 14% compared to the initial solution. [28] Urine Doxtech bottles with internal barcodes Loss of approximately 50% compared to the initial solution. [28] Urine −20 °C Pyrex bottles Losses < 20% after 49 weeks Solution treated with the surfactant Tergitol. [29] Urine −20 °C Pyrex bottles Losses > 20% after 21 weeks Untreated solution. [29]

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.