The Synthetic Cannabinoid Chemical Arms Race and Its Effect on Pain Medication Monitoring

February 27, 2018
Figure 1: Chemical structures of recent synthetic cannabinoids.
Click to enlarge, Figure 1: Chemical structures of recent synthetic cannabinoids.
Figure 2
Click to enlarge, Figure 2: Total ion chromatography of 100 ng/mL calibrator in C18 and phenyl-hexyl columns with 2.5-min or 5-min methods.
Abstract / Synopsis: 

In recent years, synthetic cannabinoids (“K2” or “spice”) have experienced a boom in popularity. The negative health effects of these drugs coupled with their increasing popularity led to placement onto Schedule I by the Drug Enforcement Administration (DEA). In response, the chemists behind these illicit compounds frequently invent new compounds to circumvent the law. Thus, new classes and new examples within classes of “spice” continue to become available for illicit use. In this paper, we examine the use of two column chemistries (C18 and phenyl-hexyl) in an effort to definitively identify synthetic cannabinoid compounds in patient samples. Distinct synthetic cannabinoid compounds interact differently with specific stationary phases and the hope is that this extra dimension of data will help to rule out similar interferent compounds that would otherwise cause false-positive results.

Synthetic cannabinoids, commonly known as “K2,” “spice,” or “synthetic marijuana,” are often sprayed onto or mixed with dried plant materials and sold in convenience stores, gas stations, smoke shops, and on the internet. This ready availability causes confusion about their safety and legality (1). In recent years, synthetic cannabinoids have become increasingly popular among adolescents and young adults as one of several frequently abused substances. These synthetic drugs mimic delta-9-tetrahydrocannabinol (THC), but can be much more potent, which results in psychoactive doses less than 1 mg (2). In fact, synthetic cannabinoids, which have a similar psychoactive effect as cannabis, have strong addictive properties often coupled with unknown physiological impacts on users. A recent study indicates that the use of synthetic cannabinoids can be a cause of death (3).

Because of the high abuse potential and lack of medical knowledge or usage, these synthetic cannabinoids have been added to the Schedule I list by the United States Drug Enforcement Administration (DEA), as “necessary to avoid imminent hazard to the public safety” (4). In response, the chemists instigating this illegal proliferation have synthesized many new K2 analogs by slightly altering chemical structures (5). Therefore, compared with the relatively stagnant pool of other compounds, such as opiates, that most pain medication monitoring laboratories deal with, the number of agents on the list of synthetic cannabinoids has been and continues to be increasing (6). Testing for synthetic cannabinoids has become a routine demand among pain treatment clinics.

There are various types of synthetic cannabinoids with different modifications on the core structure. The first THC analogs, including HU-210 (7) and CP-47, 497 (8), were synthesized in the 1980s. Their inventions allowed the discovery of G protein-coupled receptors, CB1 and CB2 (9). Later on, a structurally different analog, WIN55, 212-2, was reported. Surprisingly, WIN55, 212-2 has higher affinity toward CB1 and CB2 than THC does (10). Subsequently, John W. Huffman developed a series of “JWH compounds” by simply replacing the aminoalkyl group in WIN55, 212-2 with simple alkyl chains (11). JWH-018 has become the prototypical JWH compound. Synthetic cannabinoids have also been developed by generating fluoro-derivatives of JWH compounds. For example, AM-2201 and MAM-2201 are fluoro-derivatives of JWH 018 and JWH 122, respectively (12). By replacing the ketone in the 3-indole position of JWH-018 with an ester linkage, PB-22 and BB-22 compounds have been synthesized (13). Furthermore, another class of synthetic cannabinoids contains the tetramethylcyclopropyl ketone indoles, such as UR-144 and its fluoro-derivative, XLR-11 (14). Both UR-144 and XLR-11 have cyclopropyl rings, and are therefore likely to exhibit similar retention times in liquid chromatography (LC). (See upper right for Figure 1, click to enlarge.)

The increasing number of sophisticated reversed-phase LC separations has led to the need for optimized stationary phases to offer improved selectivity and efficiency (15). In the present work, we investigate C18 and phenyl-hexyl column chemistries for definitively identifying 13 synthetic cannabinoid metabolites in standards and patient samples. (See upper right for Figure 2, click to enlarge. Caption: Figure 2: Total ion chromatography of 100 ng/mL calibrator in C18 and phenyl-hexyl columns with 2.5-min or 5-min methods. Red, blue, and green peaks represent XLR11 N-(4-hydroxypentyl), UR-144 N-pentanoic acid, and UR-144 N-(5-hydroxypentyl), respectively. Blue dashed lines indicate solvent gradients.)

Materials and Methods


Reference standards of AKB48 5-hydroxypentyl metabolite, AKB48 pentanoic acid metabolite, AM2201 4-hydroxypentyl metabolite, BB-22 3-carboxyindole metabolite, JWH-018 pentanoic acid metabolite, JWH-073 butanoic acid metabolite, JWH-122 5-hydroxypentyl metabolite, MAM-2201 4-hydroxypentyl metabolite, PB-22 3-carboxyindole metabolite, PB-22 pentanoic acid metabolite, UR-144 5-hydroxypentyl metabolite, UR-144 pentanoic acid metabolite, and XLR11 4-hydroxypentyl metabolite were purchased from Cayman Chemical Company. Reference standards of 11-nor-9-Carboxy-Δ9-THC (THCA), THCA glucuronide, and THCA-D9 were purchased from Cerilliant Corporation. Solvents including methanol (optima grade), acetonitrile (optima grade), and formic acid (88%) were purchased from VWR. Dimethylsulfoxide (DMSO) (HPLC grade), ethyl acetate (optima grade), and ammonium hydroxide (A.C.S. Plus) were purchased from Fisher Scientific. Recombinant β-glucuronidase enzyme was purchased from IMCS. Drug-free normal human urine (NHU) was purchased from UTAK Laboratories, Inc. Deionized (DI) water was obtained in-house from a Thermo Scientific Barnstead Nanopure water purification system.

Sample Preparation

Reference standards not already in solution were dissolved in DMSO. Solutions of reference standards were aliquoted, dried, and reconstituted with NHU to make a low calibrator concentration at 1 ng/mL for all analytes except BB-22 3-carboxyindole metabolite and THCA with low calibrator levels at 5 ng/mL and 10 ng/mL, respectively. A high calibrator concentration of 100 ng/mL in NHU was used for all analytes. An 18.5-ng/mL THCA glucuronide hydrolysis–negative control (HNEG) and a 20-ng/mL positive control (20CON) were similarly prepared in NHU. This protocol uses THCA glucuronide as a hydrolysis control. Accordingly, every curve and patient batch has a hydrolysis control that contains 18.5 ng/mL of THCA glucuronide. For this control to be considered passing, it must return the expected THCA (parent) concentration within 30%.

Into 13 mm x 10 mm borosilicate glass tubes, 800 µL of calibrators, controls, and samples were each aliquoted and combined with 200 µL of THCA-D9 (2.5 µg/mL)/recombinant β-glucuronidase (1000 enzyme units/mL) solution in 25:25:50 methanol–DI water–pH 7.5 phosphate buffer. All samples were vortexed, transferred to SPEware CEREX PSAX 3 mL/35 mg extraction columns in sample racks by SPEware, and heated in a VWR Symphony oven for 15 min at 60 °C. Samples were cooled for 5 min and placed on an automated liquid dispensing-II (ALD-II) system for extraction. A light positive pressure was applied to push the samples onto the solid-phase extraction (SPE) packing. The ALD-II system then washed columns with 85:14:1 DI water–acetonitrile–ammonium hydroxide, washed with 30:70 DI water–methanol, and finally eluted samples into 1800-μL amber autosampler vials using 98:2 ethyl acetate–formic acid. Samples were dried under nitrogen for ~35 min at 25 °C in a SPEware Cerex sample concentrator, then each reconstituted with 400 μL of 50:50 DI water–methanol. Samples were capped, vortexed for 20 s, and spun for 5 min at 4000 rpm on a Sorvall ST 40 centrifuge.

Patient Sample Collection

Patient urine specimens were collected at clinics and shipped to Ameritox Ltd. These de-identified patient samples were treated similarly to standards, that is, they were diluted, extracted, and subjected to liquid chromatography–tandem mass spectrometry (LC–MS-MS). Patient samples were selected for this study that were deemed positive by the current method’s criteria, but were then deemed negative upon closer manual inspection.

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