Extraordinary long detection window of a synthetic cannabinoid metabolite in human urine - potential impact on therapeutic decisions.

Synthetic cannabinoids (SCs) have become established drugs of abuse. They play an increasing role in drug therapy, where abstinence control testing is required. Differentiation between recent drug uptake and uptake in the distant past is important for drug therapy. This study aimed at evaluating the detection window of a metabolite commonly used as a consumption marker for AB-FUBINACA and AMB-FUBINACA (synonym: FUB-AMB) in urine analysis. The acidic hydrolysis metabolite was quantified in urine samples of a drug user applying a validated analytical method. The concentration profile of the metabolite was correlated with usage data of the subject. Pharmacokinetic properties of AB-FUBINACA were collected by analysis of serum and urine samples from a controlled administration study (single oral ingestion of AB-FUBINACA). Thirteen urine samples were taken without advance notice over 2 years. The metabolite was detected in the first urine sample at 0.77 ng per mg creatinine and subsequently in concentrations ranging from 0.06 to 0.29 ng per mg creatinine. Usage data showed credible abstinence from SCs during this period. The pharmacokinetic properties observed within the controlled self-administration study supported the hypothesis of distribution in deeper compartments and long-lasting elimination (serum concentration-time curve showing biphasic kinetics). An elimination phase of over one year after the last drug uptake seems plausible in cases of extensive consumption. To avoid misinterpretation of positive findings, we recommend testing patients with known SC use at the beginning of abstinence program and to re-test continuously in short time intervals. These data enable correct interpretation of analytical findings.


| INTRODUCTION
New psychoactive substances (NPS) have gained high popularity over the past decade and have become established drugs of abuse. 1 With about 180 different compounds currently monitored by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA), synthetic cannabinoids (SCs) represent the largest group of NPS. 2 Synthetic cannabinoid receptor agonists induce cannabis-like effects and are commonly consumed by smoking. Products containing SCs include ready-to-smoke herbal mixtures ("incense blends") and liquids for e-cigarettes ("cannabinoid liquids") but also highly pure substances in powder form ("research chemicals"). Such drug preparations are easily available via the Internet and are often believed to be legal, harmless, and non-detectable by drug tests. For these reasons SCs are increasingly being used as cannabis alternatives among populations undergoing drug abstinence control testing and among people with restricted access to classical drugs of abuse. In particular, the (ab)use of SCs has become a serious problem in penitentiaries and forensicpsychiatric clinics and probably also play a role in institutions incorporating drug withdrawal therapy. Besides the acute toxicity of these compounds, 3 there are increasing concerns about the dependence potential and other psychiatric conditions (e.g. psychosis 4 ) associated with the consumption of SCs, making this class of drugs relevant for drug therapeutic casework in general. 5,6 Since undetected consumption of SCs may hamper the therapeutic process, abstinence control testing can be very important. Urine is typically the preferred sample material for abstinence control testing as sampling is non-invasive and urine usually provides a wider detection window for most drugs of abuse when compared with other body fluids (e.g. blood or oral fluid). In contrast to hair or oral fluid testingwhere contamination can be a problema positive urine test result unambiguously proves drug uptake.
We received an increasing number of urine samples to be tested for SCs and their metabolites mainly from psychiatric institutions over the past few years that reflected the increasing relevance of these drugs within the abstinence control environment. Positive test results may indicate therapeutic non-compliance and may have serious consequences for the patient (e.g. cessation of therapy and dismissal from clinic, or prison for forensic patients). Hence, correct interpretation of analytical findings is of utmost importance.
A positive finding can typically raise questions about the time point of drug consumption to differentiate between recent and more distant substance use. Positive findings detected in consecutive samples with a time distance of several weeks and relatively low concentrations can be challenging in this context, in particular when the question arises as to whether the respective substance was consumed again between two sampling points. Another issue is the possibility of passive exposure to side stream smoke as a cause of positive test results. Unfortunately, patients' statements regarding their substance use are often not reliable and the scientific data that could support a valid interpretation regarding the pharmacokinetic/toxicokinetic behavior of these drugs is scarce. During the past few years we encountered several exemplary cases with long elimination times over several months involving SCs and/or their metabolites after cessation of consumption. This observation indicates pronounced distribution into deeper compartments (e.g. fatty tissue) similar to other lipophilic drugs such as Δ 9 -tetrahydrocannabinol (THC) particularly following frequent and extensive use. 7 In the following we illustrate the difficulties in the differentiation between immediate drug intake and uptake in the distant past using This acidic hydrolysis metabolite is an important analytical target for urine screening as the parent compounds themselves are usually not excreted in urine to a relevant extent ( Figure 1). 8,9 In order to verify the hypothesis of long terminal elimination, the pharmacokinetic properties of AB-FUBINACA were collected by a controlled low-dose administration study. This study was conducted in order to provide reliable data for correct interpretation of positive metabolite findings in abstinence control.

| Quantification of N-{[1-(4-fluorobenzyl)-1Hindazol-3-yl]carbonyl}valine in urine samples
An analytical method was developed and fully validated for the detec- Method validation was performed according to the guidelines of the Starting condition of mobile phase B was 30%, linearly increased to 40% in 2.5 min, further increased to 70% in 2.0 min, further increased to 90% in 0.5 min, held for 2.0 min, decreased to starting conditions of 30% in 0.5 min, and held for 1.5 min for re-equilibration. The flow rate was set to 0.5 mL/min. Autosampler and column oven temperature were set to 10 C and 40 C, respectively. The injection volume was 10 μL. The mass spectrometer was operated in positive electrospray ionization mode. Multiple reaction monitoring (MRM) scan mode was applied for analysis and the respective potentials for the monitored ion transitions were carefully optimized (Table 1). Analyst software 1.6.2 (Sciex, Darmstadt, Germany) and Valistat software 2.0 (Arvecon GmbH, Germany) were used for data evaluation.

| Urine samples from abstinence control
Thirteen urine samples were collected from a drug user without advance notice over a time period of 2 years (from June 2017 to June 2019) together with self-reported drug consumption behavior.

| Preparation of authentic urine samples
After the addition of 0.5 mL phosphate buffer (pH 6) and 30 μL β-  The concentration of the acidic hydrolysis metabolite of AB-FUBINACA and AMB-FUBINACA was determined in all urine samples obtained from the authentic case and the administration study with the previously mentioned fully validated method.

| Method validation
The Metabolite concentration in urine was normalized to creatinine concentration for better comparability (ng/mg creatinine).

| User data
The

| Metabolite quantification in urine samples (abstinence control)
The 10 days for AM-2201) of the main metabolites in urine after single dosing. 13,15 From this experience, we recommend testing patients with known SC use at the very beginning of the abstinence program and to re-test continuously in short intervals in order to ensure reliable detection of a new event of consumption and to avoid misinterpretation of positive findings. Regarding the often stated "passive exposure defense": an exposure to the side stream smoke of cigarettes containing SCs is not necessarily perceivable by the exposed person due to the absence of typical odors. In a situation where, for example, a roommate shows urine concentrations of more than an order of magnitude lower than other patients, passive exposure should be taken into consideration as a cause of the finding. Again, repeated testing is recommended after stopping the exposure.

ACKNOWLEDGEMENT
The test person gave his informed consent about publication of the presented case. Open access funding enabled and organized by