Abstract

Identification and analysis of synthetic cannabinoids (SCs) in biological specimens remains an ongoing challenge for forensic toxicologists. Analytical method development is both resource and time consuming, and falls behind the illicit production of newer SCs. Distinguishing optimal metabolic targets and specific SC use is further complicated by metabolic pathway convergence between different SCs. Gaining further insight into the prevalence and psychopharmacologic role of these drugs in forensic cases, particularly in individuals suspected of driving impaired, is important. The prevalence of SC metabolites (SCMs) in suspects of impaired driving in Washington, DC between June 2012 and August 2013 was studied. A total of 526 urine samples were screened for 12 SCMs by liquid chromatography tandem mass spectrometry in separate duplicate analyses. Nineteen cases (3.6%) confirmed positive for the following SCMs: UR-144 N-pentanoic acid (n = 17;89%), JWH-073 butanoic acid (n = 3;16%), JWH-018 pentanoic acid (n = 3;16%), AM-2201 4-hydroxypentyl (n = 3;16%) and 5-fluoro PB22 3-carboxyindole (n = 1;5%). This study made use of existing analytical methodology to provide insight into the prevalence of synthetic cannabinoid use in DUID cases. Understanding the range and extent of use in these cases can provide valuable information to the forensic community.

Introduction

Cannabis is one of the most widely used recreational drugs within the USA (1). While marijuana's status as a legal drug is expanding statewide, the possession, distribution and use of the drug remains a federal offense within the USA. Many cannabinoid users have thus looked toward using alternative synthetic cannabinoid (SC) compounds, which can produce marijuana-like pharmacological effects. Sprayed onto herbal material, these SCs are sold under a variety of brand names as “herbal incense”, which is then smoked by the user. Marketed as “legal” alternatives to marijuana, it is unsurprising that SCs are attracting both naïve and frequent drug users.

SCs are chemically distinct compounds with pharmacological functionality similar to, or even greater than, delta-9-tetrahydrocannabinol (delta-9-THC), which is the primary psychoactive ingredient in Cannabis (2). Since the emergence of these designer drugs, referred to as “legal highs”, in the late 2000's in the US, their popularity has increased due to their affordability, accessibility and limited detection in most standard drug tests (3–6). The evolution and expansion of the illicit cannabimimetic agent market has thus occurred rapidly, particularly as existing SCs, and their salts and isomers, are scheduled (7).

The database of SC epidemiology, pharmacology and toxicology is gradually expanding, and clinical symptoms are widely reported (8–10). While there currently exists only few publications discussing the driving behavior of, and toxicological findings in, individuals suspected to drive under the influence of SCs, symptoms of slowed and slurred speech, marked lack of convergence, dilated pupils, confusion, instability and slowed movement have been reported in these cases (11, 12). It is reported that SC intoxication may lead to impaired psychomotor functionality similar to that caused by cannabis use (9, 11, 12), however, variations in symptom manifestation, such as incongruous speed and severe inattention in DUID cases, have been described (13). Conclusions reported in these studies indicate the potential of SC-intoxication to impair a driver's ability to operate a motor vehicle safely.

The forensic toxicologist is confronted with difficult and unique tasks associated with detecting and quantifying SCs and their metabolites in biological specimens. This is challenging due to the limited quality control in the synthesis of SCs, delay in obtaining reference standards, and the cost and time associated with developing and maintaining assays for these analyses. Nevertheless, methods for SC screening and confirmatory analyses have been described in whole blood (14, 15), serum (16, 17), oral fluid (17–20), hair (21–23) and urine (24–28). The use of tandem mass spectrometry and accurate mass techniques for greater sensitivity and selectivity, as well as simple method modification, alleviates the analytical challenges of such a rapidly changing market (29, 30).

Even amid evidence that SCs diminish sensory-motor performance, coordination and attention, and contribute to the impairment of individuals suspected of driving under the influence of drugs and/or alcohol (DUID) (11–13), there remains a dearth of information on the prevalence and degree of impairment associated with the use of these compounds. This study retrospectively evaluated the presence of SC metabolites in urine samples of individuals suspected of DUID in Washington, DC. While a positive urine result does not speak to the impairment of the drivers, it does increase our understanding of usage rate within this population.

Materials and methods

Samples

At the Office of the Chief Medical Examiner (OCME) in the District of Columbia (DC), all the blood and/or urine samples collected from DUID suspects in Washington, DC are screened for the presence of drugs, including cannabinoids and alcohol. SCs, however, were not routinely screened. In this study, 526 urine samples collected from individuals suspected of impaired driving between June 2012 and August 2013 were analyzed for the presence of 12 pre-selected synthetic cannabinoid metabolites (SCMs). The SCMs added to the assay were chosen based on their reported prevalence at the time. This was determined by following the testing panels at other laboratories and monitoring other published accounts. These urine samples had sufficient volume for analysis and had all been stored at 4°C under temperature control prior to analysis.

Samples had previously undergone comprehensive screening for ethanol (cutoff of 0.01 g/100 mL), as well as methanol, acetone, isopropanol (cutoff of 0.005 g/100 mL) by headspace gas chromatography (HS-GC), and by ELISA for (cutoff in ng/mL): amphetamine (250), methamphetamine (250), barbiturates (Secobarbital) (500), benzodiazepines (Nordiazepam) (100), cocaine metabolites (100), phencyclidine (10), opiates (Morphine) (200), methadone (100), oxycodone (20), THCCOOH (50) and zolpidem (10). Presumptive-positive results for these screenings were confirmed by means of GC–MS, GC–MS/NPD, HPLC-MS and UPLC-MS/MS.

Chemicals, reagents and standards

All solvents used were LC–MS grade. Acetonitrile (ACN), methanol, formic acid and ammonium acetate were all obtained from Fisher Scientific (Fairlawn, New Jersey). Certified drug-free urine was obtained from UTAK laboratories (Valencia, CA). β-Glucuronidase (from Helix Pomatia, Type HP-2, aqueous solution, >1,000,000 units/mL, 121,715 units/mL β-glucuronidase) was purchased from Sigma-Aldrich (St. Louis, MO). Reference standards of the SCMs JWH-073 N-(4-butanoic acid), JWH-250 N-(5-hydroxypentyl), JWH-122 N-(5-hydroxypentyl), JWH-019 N-(6-hydroxyhexyl) and the internal standard (ISTD) JWH-073 (3-hydroxybutyl)-d5 were purchased from Cerilliant Corporation (Round Rock, TX) in ampoules as 100 μg/mL methanolic solutions. XLR-11 N-(4-hydroxypentyl), UR-144 N-pentanoic acid, MAM-2201 N-pentanoic acid metabolite, AM2201 N-(4-hydroxypentyl), JWH-018 N-(5-pentanoic acid), PB-22 N-pentanoic acid, BB-22 3-carboxyindole, 5-fluoro PB22 3-carboxyindole and the ISTD JWH-018 N-pentanoic acid-d4 were obtained from Cayman Chemicals (Ann Arbor, MI) as solids in screw-cap vials and dissolved in methanol to form 1 mg/mL solutions. The structures of all 12 metabolites are illustrated in Figure 1.

Figure 1.

Structures of the SCMs analyzed in this study.

Figure 1.

Structures of the SCMs analyzed in this study.

Preparation of reagents, standards and controls

Solutions of ammonium acetate and β-glucuronidase were prepared. A stock solution of the ISTDs JWH-073 3-hydroxybutyl-d5 and JWH-018 N-pentanoic acid-d4 was prepared at 0.001 mg/mL in ACN. From this solution, the ISTD working standard was prepared at a concentration of 1 ng/mL.

A stock calibrator solution of the metabolite standards was prepared by adding the following to a 5 mL flask: 200 μL of 1.0 mg/mL solutions of both 5-fluoro PB-22 3-carboxyindole and BB-22 3-carboxyindole (0.04 mg/mL), 5 μL of 1.0 mg/mL PB-22 N-pentanoic acid (0.001 mg/mL), 50 μL of each of the nine remaining 0.1 mg/mL metabolite standard solutions (0.001 mg/mL), and filling to quantity sufficient (QS) with ACN. A cutoff working standard was prepared by adding 10 μL of the stock solution to a 10 mL volumetric flask and filling to QS with ACN to produce final concentrations of 40 ng/mL for each of the 3-carboxyindole metabolites and 1 ng/mL for the remainder of the metabolites. The ISTD and cutoff standard solutions were stored in amber glass bottles at −20°C.

Positive urine controls, 10 times the concentration of each drug's respective cutoff, were prepared in certified drug-free urine (final concentrations of 100 ng/mL for the 3-carboxyindole metabolites and 2.5 ng/mL for the other metabolites). Two hundred and fifty aliquots of the urine control solution were placed in micro-centrifuge tubes and stored at −20°C.

Sample preparation and extraction procedure

The samples in this study were prepared and extracted by means of a rapid, room temperature glucuronide hydrolysis and salting out-assisted liquid–liquid extraction (24).

Aliquots of 100 μL of certified blank drug-free urine (for cutoff, blank and negative samples); positive urine controls stored at −20°C, 4°C and RT (for stability monitoring over the period of the study); and case urine samples were transferred to labeled micro-centrifuge tubes. Aliquots of 25 μL of the ISTD were added to all samples except for a blank, and 25 μL of cutoff standard was also added to the cutoff tube. All samples were hydrolyzed with 25 μL of β-glucuronidase solution (12.3 units/μL), vortexed and left to stand at room temperature for 10 min. To each sample, 200 μL ACN and 50 μL of 10 M ammonium acetate were added, and samples were vortexed and then centrifuged at 10,000 rpm for 3 min. The upper organic layer (100 μL) was removed and added to labeled LC–MS/MS Snap Top vials diluted with 100 μL of 0.1% formic acid in DI water, mixed using clean pipette tips and loaded onto the instrument.

Instrumental analysis

The specimens were analyzed using Agilent's 1260 LC and 6460 ESI-MS/MS in positive ESI mode. Chromatographic separation was performed using an Agilent Poroshell 120 SB-C18 HPLC column (2.7 μm × 2.1 mm × 100 mm) attached to a Poroshell 120 SB-C18 HPLC guard column (2.7 μm × 3.0 mm × 5.0 mm) operating in gradient mode at 50°C. The mobile phases were made up of 0.1% formic acid in DI water (Solvent A) and 0.1% formic acid in ACN (Solvent B), with a flow rate of 0.5 mL/min and a total run time of 9 min for each sample, with an additional 1.5 min for re-equilibration. Injections of 50 μL of sample were programmed with 30-s needle washes with MeOH to eliminate carryover. The gradient elution system proceeded as follows: %B increased from 40 to 60% (0–7 min); %B increased from 60 to 95% (7.0–7.5 min); %B held at 95% (7.5–8.5 min); %B decreased from 95 to 40% (8.5–9 min).

The ESI source auxiliary gas flow and temperature were set at 5 L/min and 320°C, respectively, while sheath gas flow and temperature were set at 12 L/min and 360°C, respectively. The applied capillary voltage was set at +3.5 kV. The nebulizer pressure was maintained at 45 psi with nitrogen used as the ion trap collision gas. The triple quadrupole parameters were set in dynamic multiple reaction-monitoring mode (DMRM), and mass spectral data were collected within a start and stop time of 2.0 and 8.5 min, respectively. Two MRM parent to product ion transitions were monitored for each synthetic metabolite and ISTD, with the fragmentor voltage and collision energy being optimized for each individual analyte (Table I).

Table I.

Primary and Secondary Ion Transitions for the SCMs and ISTDs

Analyte Composition ([M] m/zQ1 mass m/z [M + H] Q2 and Q3 mass m/z RT (min) Fragmentor voltage (V) Collision energy 
UR-144 N-pentanoic acid C21H27NO3 (341.2) 342.2 125.1a 5.0 120 20 
342.2 97.1  120 32 
XLR-11 N-(4-hydroxypentyl) C21H28FNO2 (345.2) 346.2 248.1a 4.6 115 20 
346.2 144  115 36 
++JWH-073 3-hydroxybutyl-d5 C23H16D5NO2 (348.5) 349.2 155.1a 3.8 125 24 
349.2 127.1  125 52 
JWH-250 5-hydroxypentyl C22H25NO3 (351.2) 352.2 121.1a 3.3 135 20 
352.2 91.1  135 56 
JWH-073 4-butanoic acid C23H19NO3 (357.2) 358.2 155a 3.2 155 24 
358.2 127  155 56 
JWH-019 6-hydroxyhexyl C25H25NO2 (371.2) 372.2 155.1a 4.9 135 24 
372.2 127.1  135 56 
JWH-018 5-pentanoic acid C24H21NO3 (371.2) 372.2 155a 3.7 140 24 
372.2 127  140 60 
JWH-122 5-hydroxypentyl C25H25NO2 (371.2) 372.2 169.1a 5.0 115 24 
372.2 141.1  115 52 
+JWH-018 N-pentanoic acid-d4 C24H17D4NO3 (375.5) 376.2 155a 3.7 130 24 
376.2 127.1  130 56 
AM2201 N-pentanoic acid C24H22FNO2 (375.2) 376.2 155a 3.7 145 24 
376.2 127  145 50 
MAM2201 N-pentanoic acid C25H23NO3 (385.2) 386.2 169.1a 4.5 160 24 
386.2 141.1  160 48 
5-Fluoro PB-22 3-carboxyindole C14H16FNO2 (249.1) 250.1 206.2a 2.3 115 12 
250.1 118  115 24 
BB-22 3-carboxyindole C16H19NO2 (257.1) 258.2 132.1a 4.6 120 16 
258.2 118  120 24 
PB-22 N-pentanoic acid C23H20N2O4 (388.1) 389.2 244.1a 2.8 100 
389.2 144  100 40 
Analyte Composition ([M] m/zQ1 mass m/z [M + H] Q2 and Q3 mass m/z RT (min) Fragmentor voltage (V) Collision energy 
UR-144 N-pentanoic acid C21H27NO3 (341.2) 342.2 125.1a 5.0 120 20 
342.2 97.1  120 32 
XLR-11 N-(4-hydroxypentyl) C21H28FNO2 (345.2) 346.2 248.1a 4.6 115 20 
346.2 144  115 36 
++JWH-073 3-hydroxybutyl-d5 C23H16D5NO2 (348.5) 349.2 155.1a 3.8 125 24 
349.2 127.1  125 52 
JWH-250 5-hydroxypentyl C22H25NO3 (351.2) 352.2 121.1a 3.3 135 20 
352.2 91.1  135 56 
JWH-073 4-butanoic acid C23H19NO3 (357.2) 358.2 155a 3.2 155 24 
358.2 127  155 56 
JWH-019 6-hydroxyhexyl C25H25NO2 (371.2) 372.2 155.1a 4.9 135 24 
372.2 127.1  135 56 
JWH-018 5-pentanoic acid C24H21NO3 (371.2) 372.2 155a 3.7 140 24 
372.2 127  140 60 
JWH-122 5-hydroxypentyl C25H25NO2 (371.2) 372.2 169.1a 5.0 115 24 
372.2 141.1  115 52 
+JWH-018 N-pentanoic acid-d4 C24H17D4NO3 (375.5) 376.2 155a 3.7 130 24 
376.2 127.1  130 56 
AM2201 N-pentanoic acid C24H22FNO2 (375.2) 376.2 155a 3.7 145 24 
376.2 127  145 50 
MAM2201 N-pentanoic acid C25H23NO3 (385.2) 386.2 169.1a 4.5 160 24 
386.2 141.1  160 48 
5-Fluoro PB-22 3-carboxyindole C14H16FNO2 (249.1) 250.1 206.2a 2.3 115 12 
250.1 118  115 24 
BB-22 3-carboxyindole C16H19NO2 (257.1) 258.2 132.1a 4.6 120 16 
258.2 118  120 24 
PB-22 N-pentanoic acid C23H20N2O4 (388.1) 389.2 244.1a 2.8 100 
389.2 144  100 40 

aDenotes the quantification transition.

+JWH-018 N-pentanoic acid-d4 used as ISTD for all R-COOH analytes.

++JWH-073 3-hydroxybutyl-d5 used as ISTD for all R-OH analytes.

Method validation

The LC–MS/MS method for SCM screening was validated according to SWGTOX guidelines using fortified drug-free urine (31).

Cutoff concentration

The laboratory selected a cutoff concentration using a one-point calibration standard. LC–MS/MS chromatographs were analyzed over spiked ranges for quality and response. The cutoff was the lowest concentration using this method where quality chromatography was observed (clear baseline resolution and Gaussian and symmetrical chromatographic peaks) and where the ion ratios and retention times passed.

Within-run and between-run control precision

Within-day precision was measured by analyzing control samples (n = 5), stored at −20°C, within a single run (% CV = 4.8–9.7). Within-day control values were used to determine the range within which the controls pass. Between-run precision was analyzed for all the controls over 10 consecutive runs (% CV = 4.3–17.7).

Stability

Positive controls made in certified drug-free urine were evaluated at RT, 4°C and −20°C for the duration of the study, or ∼216 consecutive days. All metabolites in the positive controls maintained stability at −20 and 4°C for that period of time, with fluctuation in concentration being observed for controls stored at RT. This was also performed to distinguish optimal control conditions for future work.

Criteria for acceptance

Data were processed using Agilent MassHunter® Workstation software. For the analytes 5-fluoro PB-22 3-carboxyindole and BB-22 3-carboxyindole, a positive result was defined as an analyte concentration above that of the established 10.0 ng/mL cutoff standard, with a retention time (Rt) ± 2% of the standard, and ion ratios that fell within 20% of the assigned range. For the remainder of the synthetic metabolites, a positive result was defined as an analyte concentration above that of the established 0.25 ng/mL cutoff standard, with the same Rt and ion ratio requirements stated above.

Results

Figure 2 illustrates the ion chromatogram results of an extracted urine sample of the working calibrator at the cutoff concentration. The SCMs and ISTDs were identified by chromatographic retention time, mass resolution and ion transitions.

Figure 2.

Overlaying qualitative ion chromatogram of the extracted cutoff urine sample illustrating the retention times of the working calibrator and ISTDs. Reponses are not scaled.

Figure 2.

Overlaying qualitative ion chromatogram of the extracted cutoff urine sample illustrating the retention times of the working calibrator and ISTDs. Reponses are not scaled.

A total of 526 urine samples from suspected DUID cases that were obtained over approximately a year, were analyzed for 12 pre-selected SCMs. A total of 19 cases (3.6%) screened positive for one or more SCMs (Table II) by means of separate duplicate extractions and analyses.

Table II.

Demographic and Toxicological Data for 19 Synthetic Cannabinoid Metabolite-Positive Cases

Case Sex Age Positive synthetic metabolite Other confirmed drugs Concentrations 
Male 28 UR-144 N-pentanoic acid PCP 245.43 ng/mL 
Male Unknown UR-144 N-pentanoic acid THCCOOH 3,652.62 ng/mL 
Female 33 UR-144 N-pentanoic acid EtOH 0.26 g/100 mL 
Female 30 UR-144 N-pentanoic acid Acetone 0.016 g/100 mL 
    PCP 345.16 ng/mL 
Male 37 UR-144 N-pentanoic acid EtOH 0.23 g/100 mL 
    THCCOOH 582.37 ng/mL 
Male 32 UR-144 N-pentanoic acid None – 
Male 36 UR-144 N-pentanoic acid PCP 1,133.18 ng/mL 
Female 35 UR-144 N-pentanoic acid EtOH 0.09 g/100 mL 
    PCP 85.4 ng/mL 
Male 53 5-Fluoro PB-22 3-carboxyindole EtOH 0.07 g/100 mL 
   UR-144 N-pentanoic acid THCCOOH 98.23 ng/mL 
10 Male 45 UR-144 N-pentanoic acid EtOH 0.27 g/100 mL 
11 Male 28 UR-144 N-pentanoic acid THCCOOH 31.31 ng/mL 
    EtOH 0.19 g/100 mL 
12 Male 22 UR-144 N-pentanoic acid THCCOOH 14.73 ng/mL 
    EtOH 0.14 g/100 mL 
13 Male 35 UR-144 N-pentanoic acid THCCOOH >ULOQ 
    EtOH 0.11 g/100 mL 
14 Female 33 UR-144 N-pentanoic acid None – 
15 Male Unknown JWH-073 4-butanoic acid EtOH 0.02 g/100 mL 
   JWH-018 5-pentanoic acid   
   AM2201 N-pentanoic acid   
16 Male 22 UR-144 N-pentanoic acid EtOH 0.2 g/100 mL 
    THCCOOH 138.61 ng/mL 
17 Male 35 UR-144 N-pentanoic acid EtOH 0.03 g/100 mL 
   JWH-073 4-butanoic acid   
   JWH-018 5-pentanoic acid   
   AM2201 N-pentanoic acid   
18 Male 29 UR-144 N-pentanoic acid EtOH 0.22 g/100 mL 
19 Male 33 JWH-073 4-butanoic acid EtOH 0.33 g/100 mL 
   JWH-018 5-pentanoic acid THCCOOH 74.79 ng/mL 
   AM2201 N-pentanoic acid   
Case Sex Age Positive synthetic metabolite Other confirmed drugs Concentrations 
Male 28 UR-144 N-pentanoic acid PCP 245.43 ng/mL 
Male Unknown UR-144 N-pentanoic acid THCCOOH 3,652.62 ng/mL 
Female 33 UR-144 N-pentanoic acid EtOH 0.26 g/100 mL 
Female 30 UR-144 N-pentanoic acid Acetone 0.016 g/100 mL 
    PCP 345.16 ng/mL 
Male 37 UR-144 N-pentanoic acid EtOH 0.23 g/100 mL 
    THCCOOH 582.37 ng/mL 
Male 32 UR-144 N-pentanoic acid None – 
Male 36 UR-144 N-pentanoic acid PCP 1,133.18 ng/mL 
Female 35 UR-144 N-pentanoic acid EtOH 0.09 g/100 mL 
    PCP 85.4 ng/mL 
Male 53 5-Fluoro PB-22 3-carboxyindole EtOH 0.07 g/100 mL 
   UR-144 N-pentanoic acid THCCOOH 98.23 ng/mL 
10 Male 45 UR-144 N-pentanoic acid EtOH 0.27 g/100 mL 
11 Male 28 UR-144 N-pentanoic acid THCCOOH 31.31 ng/mL 
    EtOH 0.19 g/100 mL 
12 Male 22 UR-144 N-pentanoic acid THCCOOH 14.73 ng/mL 
    EtOH 0.14 g/100 mL 
13 Male 35 UR-144 N-pentanoic acid THCCOOH >ULOQ 
    EtOH 0.11 g/100 mL 
14 Female 33 UR-144 N-pentanoic acid None – 
15 Male Unknown JWH-073 4-butanoic acid EtOH 0.02 g/100 mL 
   JWH-018 5-pentanoic acid   
   AM2201 N-pentanoic acid   
16 Male 22 UR-144 N-pentanoic acid EtOH 0.2 g/100 mL 
    THCCOOH 138.61 ng/mL 
17 Male 35 UR-144 N-pentanoic acid EtOH 0.03 g/100 mL 
   JWH-073 4-butanoic acid   
   JWH-018 5-pentanoic acid   
   AM2201 N-pentanoic acid   
18 Male 29 UR-144 N-pentanoic acid EtOH 0.22 g/100 mL 
19 Male 33 JWH-073 4-butanoic acid EtOH 0.33 g/100 mL 
   JWH-018 5-pentanoic acid THCCOOH 74.79 ng/mL 
   AM2201 N-pentanoic acid   

EtOH, ethanol; THCCOOH, 11-nor-9-carboxy-delta9-tetrahydrocannabinol; PCP, phencyclidine.

Of these 19 SC-positive cases, 15 of the drivers were male (79%), and the mean reported age was 33.3 years (range: 22–53 years). A total of 15 (79%) cases were positive for one SCM, while 4 cases (21%) were positive for more than one metabolite. UR-144 N-pentanoic acid was confirmed positive in 17 cases (89%), one of which was also positive for 5-fluoro PB-22 3-carboxyindole. JWH-018 5-pentanoic acid, JWH-073 4-butanoic acid and AM-2201 4-hydroxypentyl were all detected in three cases (16%), one sample of which was also positive for UR-144 N-pentanoic acid.

In addition, 18 SCM-positive cases tested positive for alcohol or illicit drugs (Table II). Ethanol was the most frequently detected substance in the SCM-positive cases (n = 13; 68%). THCCOOH (n = 8; 42%) and phencyclidine (PCP) (n = 4; 21%) were also confirmed. No other drugs were detected in one case (5%).

Discussion

An increase in structural variability and recreational use of SCs has become a challenge for forensic toxicologists. Reports on the prevalence rates of SCs in forensic cases are limited, and implementing standard screening panels for these compounds in forensic toxicology laboratories remains a costly, timeous and challenging endeavor, particularly in cases where common drugs of abuse are already detected.

In this study, 526 urine samples collected from DUID suspects in Washington, DC over the period of approximately a year were analyzed. Samples were subjected to a rapid room temperature glucuronide hydrolysis and salting out-assisted liquid–liquid extraction (24). This was followed by positive ESI-LC–MS/MS analysis with the identification of two MRM transitions for each analyte. This provided a rapid and easy workflow for routine sample analysis of SCMs in urine, with simple and cost-effective sample preparation.

Stability of the SCMs in spiked urine controls was established over the 216-day period of analyses. Any calculated deviations were within the accepted criteria (±20%) for all the analytes, based upon a single calibrator used throughout the study. This study was limited to a 12-targeted SCM assay, which was later expanded to include more SCMs as well as SC parent compounds.

Nineteen cases were confirmed positive for SCMs, with an estimated 3.6% prevalence. Similar prevalence estimates have been reported in other traffic offense publications. A total of 422 serum samples in traffic and criminal offense cases in Germany in 2010 were analyzed for 18 SCs, with 12 cases (2.8%) confirming positive (32). Similarly, 726 blood samples of Norwegian drivers collected between 2011 and 2012 were analyzed for 18 SCs, of which 16 samples (2.2%) were confirmed positive (33). The slightly higher prevalence determined in this study's cohort may be partially due to the analyses of the urine matrix, instead of blood or serum. Metabolites are typically found in higher concentrations for a longer period of time in urine, than the parent compounds in blood. A higher SC detection rate in males than females was found in these studies, which also correlates with our findings of 79% of the SC-positive drivers being male.

In this study, UR-144 N-pentanoic acid was the most prevalent metabolite detected in the positive cases (n = 17; 89%). Metabolism of cyclopropylindole SCs, such as UR-144 and XLR-11, produce this metabolite (34). It was therefore not possible to unambiguously distinguish between their administrations in these cases. While detection of JWH-073 butanoic acid, JWH-018 pentanoic acid, and AM2201 4-hydroxypentyl metabolites have been reported elsewhere (35–37), all three metabolites were detected in three separate cases in this study. This may be indicative of the composition of a particular brand of SC product. This is the first study however, that the authors were aware of at the time of writing, to report the detection of 5-fluoro PB-22 3-carboxyindole in human urine.

Upon analysis of the analytical data, the authors observed instances in which the requirements for acceptance in the form of peak shape, retention times and ion ratios passed but the analyte's concentrations fell below cutoff. This suggests that a lower LOD for some of the analytes (such as the UR-144 metabolite) is possible, and indicates the need to re-evaluate the cutoff upon the addition of new analytes to the assay in future.

In the study of German drivers, JWH-015, JWH-016, JWH-018, JWH-122, JWH-210 and JWH-250 SCs were detected (32), while JWH-081, JWH-250, JWH-018, AM-2201, RSC-4 and JWH-122 were confirmed in Norwegian drivers (33). It is not surprising that these results vary from our study due to the rapid production of novel SCs, variation in international SC drug scheduling, and the targeting of newer UR-144 and XLR-11 metabolites. This is supported by Louis et al.(13) who report the detection of synthetic cannabinoids XLR-11 and UR-144 in 18 driving cases in the states of Washington and Alaska.

In this study, all cases but one tested positive for other psychoactive drugs. These included ethanol, THCCOOH, and PCP. While poly-drug use has been reported in previous retrospective studies (32, 33), psychophysical impairment has been attributed to the detection of synthetic cannabinoids alone or in combination (11–13). Detection of drugs that produce psychophysical impairment may confound the interpretation as to whether SC-induced impairment was contributory to the traffic offense. Furthermore, poly-drug detection in these cases may suggest the use of SCs for their availability and affordability, and not necessarily for their limited detection on standard drug screens.

Details of driving behaviors of the subjects in our study were not available and, as we were analyzing urine samples, may only have been informative in cases where no other drugs were detected. However, other cases of suspected impaired driving in which the drivers tested positive for SCs alone have been reported. One or more SCs were detected in the blood of 12 DUID suspects who subsequently tested negative for other drugs and alcohol (11). AM-2201 was most frequently detected, with JWH-018, JWH-210, JWH-250, JWH-081 and JWH-122 also being confirmed. The drivers displayed a marked lack of ocular convergence, slow and slurred speech, but variable signs of impairment in cognition and field sobriety test performance. In 10 of these cases, the Drug Recognition Expert (DRE) assigned their impairment to the DRE cannabis category.

In another report, the serum of seven subjects suspected of DUID tested positive for multiple SCs including JWH-018, AM-2201, JWH-210, JWH-307 and JWH-122 (12). One subject tested positive for all these SCs as well as MAM2201 and UR-144. The subjects exhibited impairment and psychophysical effects including disorientation, slurred speech, mydriasis, confusion, irritability, impaired coordination and sluggish movement. Louis et al. (13) report the finding of similar symptoms with XLR-11 and/or UR-144 use in drivers. These studies suggest that SCs may produce impairment similar to that of cannabis, which is incompatible with safe driving.

This study revealed possible changing trends in SC use, moving away from AM and JWH SCs to the newer UR-144 and/or XLR-11 compounds. Following this study, the SC urinary assay was expanded to include a greater number of more recently reported metabolites and parent SCs, such as the PINACA and FUBINACA compounds. It is important to monitor the prevalence of SCs in various population cohorts, so as to provide further information on usage patterns in such a rapidly changing drug market. This expanded urinary assay, together with future method development and validation of the analysis of these compounds in blood, will provide more robust and broader analyses of forensic cases in Washington, DC in which SCs may be involved.

Conclusion

SC identification, detection and quantitation in forensic toxicology laboratories is challenging due to the evolution and expansion of the designer drug market. When using a targeted LC–MS/MS method, continuous analytical modifications are required. While this technology allows for relatively rapid and simple method validation, a non-targeted instrumental screen would be ideal for detecting these rapidly changing compounds.

While this study's prevalence of SCMs in urine of individuals suspected of driving impaired was low (3.6%), it is relatively higher than DUID case studies of SCs in serum and blood. The study indicates that over this timeframe there was a movement toward newer SC such as UR-144, XLR-11 and 5F PB-22 and they tend to be mixed with other drugs. This again illustrates that the availability of these drugs may outweigh the risk associated with detection. While adding these compounds to a laboratory's routine drug screening assays is of benefit, it needs to be based on the availability of resources. In addition, controlled studies on the elimination kinetics and pharmacodynamics in humans need to be performed to better understand the pharmacology of these compounds and their metabolites.

Funding

This work was supported by the Forensic Toxicology Laboratory of the Office of the Chief Medical Examiner in Washington, DC.

Conflict of interest

None declared.

Acknowledgments

This research was supported by the Forensic Toxicology Laboratory of the Office of the Chief Medical Examiner in Washington, DC. The authors acknowledge the contributions of Jerome Robinson, as well as the support and assistance of Professors Nicholas Lappas and Walter Rowe.

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