Validation of a Novel Immunoassay for the Detection o

Journal:

Journal of Analytical Toxicology

Article id:

bkt024

Article title:

Validation of a Novel Immunoassay for the Detection of Synthetic Cannabinoids and Metabolites in Urine Specimens

First Author:

Amanda Arntson

Corr. Author:

Barry Logan

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Journal of Analytical Toxicology 2013;00:1 –7 doi:10.1093/jat/bkt024 Advance Access publication Month 00, 0000

Article

Validation of a Novel Immunoassay for the Detection of Synthetic Cannabinoids and Metabolites in Urine Specimens Q1 5

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Amanda Arntson1, Bill Ofsa2, Denise Lancaster2, John R. Simon3, Matthew McMullin2 and Barry Logan1,2* 1

The Center for Forensic Science Research and Education, Willow Grove, PA, 2National Medical Services Laboratory, Willow Grove, PA, and 3Tulip Biolabs, Inc., West Point, PA 65

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*Author to whom correspondence should be addressed. Email: [email protected]

Synthetic cannabinoid drugs do not cross react on traditional marijuana immunoassay tests, preventing their use in large scale drug screening programs. This paper describes the validation and performance characteristics of two enzyme linked immunosorbent assays designed to detect the use of two common synthetic cannabinoids in urine, JWH-018 and JWH-250. The JWH-018 assay has significant cross-reactivity with several synthetic cannabinoids and their metabolites, whereas the JWH-250 assay has limited crossreactivity. The assays are calibrated at 5 ng/mL with the 5-OH metabolite of JWH-018 and the 4-OH metabolite of JWH-250. The method was validated with 114 urine samples for JHW-018 and 84 urine samples for JWH-250 and confirmed by using liquid chromatography tandem mass spectrometry, which tests for metabolites of JWH-018, JWH-019, JWH-073, JWH-250 and AM-2201. The accuracy was determined to be 98% with greater than 95% sensitivity and specificity for both assays. Introduction

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Synthetic cannabinoids, originally developed in university and laboratory research centers during the 1980s and 1990s, have been synthesized and assessed for their appetite stimulating properties and therapeutic potential for the treatment of as Multiple Sclerosis, Acquired Immunodeficiency Syndrome (AIDS) and nausea associated with chemotherapy (1–4). Initially, in 2004 in several European countries, clandestine laboratories began producing and distributing herbal or botanical products laced with synthetic cannabinoid chemicals as novelty “legal high” products, simultaneously promoting their legal status while labeling them as not for human consumption (5 –7). 1-Naphthyl-(1-pentylindol-3-yl)methanone (JWH-018) was one of the first psychoactive constituents identified in the herbal products. Another common synthetic cannabinoid found in the herbal products is 2-(2-methoxyphenyl)-1-(1-pentylindol-3-yl) ethanone (JWH-250), which emerged after regulations and restrictions were placed on the possession and distribution of JWH-018 and its C-4 homolog JWH-073 (5). Both JWH-018 and JWH-250 are classified as aminoalkylindoles (AAIs) and further divided into the subcategories of naphthoylindole (JWH-018) and phenylacetylindole (JWH-250) (Figures 1 and 2). Since 2008, synthetic cannabinoids, marketed as “legal alternatives to cannabis” or “legal highs” have dramatically increased in popularity among different drug user populations. The ability to purchase these products over the internet, or in gas stations, convenience stores or head or smoke shops, and the fact that drug testing programs have not been able to detect their use, adds to their overall appeal as substances of abuse.

Pharmacologically, synthetic cannabinoids are designed with structural features that allow binding to the cannabinoid receptors (CB1 or CB2) in the human body to elicit effects similar to tetrahydrocannabinol (THC), the active ingredient in marijuana (6). The chemicals are sprayed onto inert plant material and subsequently smoked in the form of a cigarette or joint, or from a pipe, like marijuana. Once absorbed in the body, the effects of the cannabinoids are felt within minutes and vary in intensity and quality of effect between the different compounds (6, 9). The typical duration of action for JWH-018 has been documented to last a few hours following smoking (10). Unlike marijuana, synthetic cannabinoids are known to frequently elicit adverse effects such as hypertension, agitation, anxiety, hallucinations and recurring seizures (9 –13). Metabolism studies on AAIs have primarily been based on experiments with human liver microsomes and include a few involving the analysis of urine specimens of drug users known to have smoked products containing a specific synthetic cannabinoid. The metabolism of these aminoalkylindoles proceeds through hydroxylation of the alkyl side chain in the terminal (omega), or the omega minus one position (14 –17), as determined by analysis of the urine from pedigreed samples from subjects who smoked botanical products containing known synthetic cannabinoids. Further oxidation to the terminal carboxy group has been documented (17), and there is some evidence for additional hydroxylation on the indole ring for multiple hydroxylation (18). There is evidence that these hydroxylated and carboxylated metabolites are glucuronidated and may have some innate receptor binding activity themselves (11, 17, 19). In particular, in vivo and in vitro studies have shown that the ring- and side-chain hydroxylated derivatives of JWH-018 have greater pharmacological activity than D9-THC (11). Additionally, studies indicate that AM-2201, an analog of JWH-018 fluorinated on the terminal position of the side chain, is metabolized to the common JWH-018-omega hydroxyl metabolite (18). The hydroxylated metabolites retain significant affinity and activity at the CB1 receptor (11, 17, 20). Moreover, there is no indication from published work that the parent compounds are excreted in urine to any measurable extent (6, 16). Beginning in early 2009, several countries began placing restrictions on the primary cannabinoids found in herbal products, like JWH-018. In the United States, the Department of Justice Drug Enforcement Administration (DEA) restricted JWH-018 and similar compounds, temporarily classifying them Schedule I substances under the Controlled Substances Act, which went into effect in March of 2011 (20 –21). The initial

# The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]

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Figure 1. Chemical structures of JWH-018 and its common metabolites: JWH-018 (A); JWH-018-4-OH (B); JWH-018-5-OH (C); JWH-018 pentanoic acid (D).

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restrictions resulted in the emergence of new synthetic cannabinoid products in an attempt to avoid legal implications. As of July, 2012, in the United States, specific cannabimimetic agents (cannabinoid receptor type 1, CB1, agonists) including JWH-018, JWH-073, AM-2201, JWH-250 and other compounds were permanently scheduled as Schedule I (21 –22). In addition to the previously listed compounds, many others are either named directly in the schedule or may be considered as drug analogs under the statute or the terms of the US drug analog act. The most current widespread practice for screening for synthetic cannabinoid use in urine is through liquid chromatography tandem mass spectrometry (LC – MS-MS) (16, 18, 23, 24). This allows high sensitivity testing and distinction between various similar metabolites of related compounds. Considering instrument costs, sample preparation, consumables and supplies, LC–MS-MS is more time consuming and expensive than immunoassay testing. Furthermore, many hydroxylated metabolites of synthetic cannabinoids have identical transitions, requiring them to be distinguished chromatographically rather than through their multiple reaction monitoring (MRM) transitions, which lengthens run times. For this reason, LC–MS-MS is typically applied to confirmatory testing when a lower cost screening method exists with appropriate scope and sensitivity. Screening using immunoassays with antibodies specific to 11-nor-9-carboxy-D9-THC has yielded negative results in both blood and urine samples, indicating that compounds like JWH-018 and their metabolites cannot be detected without a 2 Arntson et al.

specialized assay (11, 23). To date, there are no published methodologies in peer-reviewed literature for the screening of synthetic cannabinoids using enzyme linked immunosorbent assays (ELISAs). Based on the recent permanent ban on cannabimimetic agents, it is anticipated that there will be additional demands for synthetic cannabinoid screening in workplace, transportation, safety sensitive and probation and parole populations, and the need to rapidly screen large volumes of specimens for synthetic cannabinoids will become more challenging. Additionally, the reports of adverse events, toxicity and fatalities associated with synthetic cannabinoids have increased, presenting a public health concern that adds to the need for a low cost screening procedure to assess patient samples (8– 13). This paper describes the development, validation and performance characteristics of ELISA technologies for the detection of the most prevalent members of the naphthoylindole and phenylacetylindole synthetic cannabinoid classes.

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Experimental Apparatus and reagents Two 96-well ELISA assay kits were developed in-house for the screening procedure. The JWH-018 Direct ELISA assay kit was designed to test for the presence of JWH-018 metabolites and was calibrated with urine standards containing a JWH-018-5-OH metabolite (Cayman Chemicals, Ann Arbor, MI). The JWH-250 kit was developed to test for JWH-250 metabolites and was

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Figure 2. Chemical structures of JWH-250 and its common metabolites: JWH-250 (A); JWH-250-4-OH (B); JWH-250-5-OH (C); JWH-250-5-carboxypentyl (D).

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calibrated with JWH-250-4-OH metabolites (Cayman Chemicals). The assays were run and measured on a Freedom Evo instrument and microplate reader (Tecan, San Jose, CA). Standards of other drugs and metabolites for interference and cross-reactivity experiments were purchased from Cayman Chemical and Cerilliant (Dallas, TX).

Principle of the assay Both kits were developed by immunizing rabbits with their respective parent compound conjugated to a carrier. Sera were screened by competitive ELISA by using the respective hydroxy metabolites of the target compounds in buffer to ascertain sensitivity. Urine from control and known JWH-018 or JWH-250 users was also tested to screen the sera and optimize the final assay conditions to best detect users, irrespective of the identity of the metabolites present. Both kits were designed to be suitable for the analysis of whole blood, serum and urine. The principle of both ELISA kits was based on the competitive binding of the analytes and the JWH-018 or JWH-250 peroxidase conjugate to the antibody coated on the 96 well microplate. Twenty microliters of test sample were applied to the well simultaneously with 100 mL of JWH-018 or JWH-250 peroxidase conjugate and incubated for 60 min. This was followed by a total of six washes, each using 300 mL of deionized water, followed by the addition

of a buffer chromogenic substrate solution containing tetramethylbenzidine and hydrogen peroxide for 30 minu. The reaction was stopped with the addition of a dilute acid. Optical density (OD) was subsequently measured at 450 nm on a microplate reader.

Stability, precision and drift The long-term stability of the JWH-018 and JWH-250 kits was evaluated in-house at 48C. Kits were assayed immediately after assembly, then stored refrigerated at 48C and assayed. The OD of phosphate buffered saline (PBS) and 100 ng/mL of the respective 4-OH and 5-OH metabolites in PBS were assayed and compared both at the time of manufacture and some months later. Intra-plate and inter-plate precision were assessed by preparing three 100 mL pools containing both JWH-5-OH and JWH-4-OH at concentrations of 3.75, 5.0 and 6.25 ng/mL in PBS. The pools were aliquoted in duplicate into 75 100 mm tubes, along with negative PBS to serve as a blank. The order of analysis was blank, blank, 3.75, 3.75, 5.0, 5.0, 6.25 and 6.25. The duplicates were reiterated six times occupying a total of 48 tubes. The tubes were placed in three 16 tube racks on the Tecan. The racks were sampled twice to completely utilize a 96 well plate. This procedure was repeated for a total of three replicates. The raw absorbance data acquired were normalized to the blank and subsequent calculations were made using the normalized data.

Validation of a Novel Immunoassay for the Detection of Synthetic Cannabinoids and Metabolites in Urine Specimens 3

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Intra-plate and inter-plate precision were calculated for each concentration on each plate. The average intra-plate precision was calculated by taking the average of the three sets of intraplate precision results. The precision of the assays was determined acceptable if the coefficient of variation (CV) was equal to or less than 15%. The percent plate drift was calculated for each concentration by using the averaged normalized absorbance of the duplicate aliquots of Rows 1 and 12 on the plate. Carryover Carryover was assessed at two levels, 1,000 and 5,000 ng/mL, by spiking the calibration analytes JWH-018-5-OH and JWH-250-4-OH into PBS. The order of analysis was designed to separately challenge each tip of the Tecan. The assays were calibrated per protocol with additional aliquots of the precision pools at 3.75, 5.0 and 6.25 ng/mL to complete a set of eight calibration samples. This was followed by eight blank samples, eight 1,000 ng/mL samples, 16 blank samples, eight 5,000 ng/mL samples and 16 blank samples. Carryover was considered significant if any of the first eight blanks following the spiked samples were interpreted as positive per protocol. LC –MS-MS confirmation Confirmatory analyses were performed by a validated LC– MS-MS procedure that qualitatively detected the alkyl side chain monohydroxy metabolites of five synthetic cannabinoids: JWH-018, JWH-019, JWH-073, JWH-250 and AM-2201. The scope of analysis included JWH-018-4-OH, JWH-018-5-OH, JWH-019-5-OH, JWH-073-3-OH, JWH-073- 4-OH, JWH-250-4-OH and AM-22014-OH, each at a cutoff concentration of 0.10 ng/mL. In summary, two internal standards (JWH-018-4-OH-d5 and JWH-073-3OH-d5) were added to 1.0 mL urine aliquots that were enzymatically hydrolyzed using type H-2 Helix Pomatia at pH 5.5. Samples were made strongly basic with NaOH and extracted into methyl t-butyl ether. Following evaporation and reconstitution with a mixture of water and mobile phase, samples were analyzed using an Acquity Ultra Performance Liquid Chromatograph (Waters, Milford, MA) and a tandem mass spectrometer (TQD, Waters) using positive ion electrospray ionization. The analytical column was a Restek 1.9 micron Pinnacle DB Biphenyl, 2.1 100 mm. A reversed-phase gradient elution was used with increasing amounts of acetonitrile mixed with 0.1% formic acid in water. Two transitions were monitored for each analyte and internal standard (Table I). Duplicate cutoff calibrators at

Table I LC –MS-MS MRM Transitions for Synthetic Cannabinoids 390

Analyte

Quant ion

Ratio ion

395

JWH-018-4-OH JWH-018-5-OH JWH-018-4-OH-D5 JWH-018-5-OH-D5 JWH-019-5-OH JWH-019-6-OH JWH-073-3-OH JWH-073-4-OH JWH-073-3-OH-D5 JWH-073-4-OH-D5 JWH-250-4-OH AM-2201-4-OH

358.0.155.0 358.0.155.0 363.0.155.0 363.0.155.0 372.4.155.0 372.4.155.0 344.0.155.0 344.0.155.0 349.0.155.0 349.0.155.0 352.2.121.1 376.4.154.7

344.0.127.0 358.0.127.0 363.0.127.0 363.0.127.0 372.4.127.0 372.4.127.0 351.0.127.0 344.0.127.0 349.0.127.0 349.0.127.0 352.2.186.6 352.2.127.0

4 Arntson et al.

0.1 ng/mL in urine were run in each batch with positive controls at 0.15 ng/mL, in addition to a hydrolysis control, which was a pool of positive urine samples. Positivity was based on a response greater than the cutoff calibrators, in addition to correlating retention time and transition ion ratios within defined limits. Sensitivity and specificity were 100% for all analytes in the scope of the confirmatory method. Peaks were required to have a relative retention time within +2% of that observed in the calibrators. Calibrators were required to back-calculate to within +20% of the expected concentration. Ion intensity ratios were within +30% of ion intensity ratios obtained from calibrators in the same analytical run for both analyte and internal standard.

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Results and Discussion Sensitivity The ELISA assays were validated to determine their performance at the defined cutoff concentration of 5 ng/mL. A cutoff concentration of 5 ng/mL was selected based on LC–MS-MS results of incurred authentic positive urine samples from subjects known to have ingested specific synthetic cannabinoid compounds, in addition to JWH-018 and JWH-250 metabolite concentrations determined in authentic samples from large scale screening populations. Limits of performance below the 5 ng/mL cutoff were not evaluated.

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Stability, precision and drift With respect to long term stability, the JWH-018 and JWH-250 assays performed acceptably (relative change in OD/OD0 , 25%) at 8.3 and 7.6 months, respectively. With respect to intraplate and inter-plate precision, both the JWH-018 and JWH-250 kits were precise, with a CV less than 15% for both assays. Drift from Rows 1 to 12 was 4.4% or better for the JWH-250 kit and 21% or better for the JWH-018 kit across a range of concentrations up to 6.25 ng/mL. Although this was larger than optimum, each control was spiked with only one cross-reactive species, whereas actual human samples have multiple reactive species present. As reflected in the sensitivity/specificity data, this drift did not negatively impact the assay performance with actual human samples.

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Carryover The carryover experiment showed no significant carryover for either assay at 1,000 ng/mL. At 5,000 ng/mL, both assays exhibited some carryover. However, 1,000 ng/mL is 200 times and 5,000 ng/mL is 1,000 times the cutoff concentration, 5 ng/mL. The cumulative effect of multiple metabolites and cross-reacting species that are likely to have similar carryover characteristics make the carryover results worth noting at 5,000 ng/mL.

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Cross reactivity Cross-reactivity data for the assays are shown in Tables II– V. Several naphthoylindoles and their hydroxylated metabolites gave cross-reactivity above 1%, including JWH-018-4-OH-pentyl, JWH-018-5-OH-pentyl, JWH-081, JWH-081-4-OH-pentyl, JWH081-5-OH-pentyl, JWH-122, JWH-122-5-OH-pentyl, AM-2201 and

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Table II Cross-Reactivity on JWH-018 Direct ELISA of Synthetic Cannabinoids or Metabolites Relative to JWH-018-5-OH (5 ng/mL)

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Drug

Cross-reactivity (%)

Drug


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JWH-122-4-OH-pentyl metab JWH-019-6-OH-hexyl metab JWH-073-N-4-OH-butyl metab JWH-081-O-desmethyl-4-OH-pentyl metab JWH-018-5-OH (calibrator) AM-2201-N-4-OH-pentyl metab JWH-022-4-keto JWH-073-N-3-OH-butyl metab JWH-081-O-desmethyl-5-OH-pentyl metab JWH-398-5-OH-pentyl metab JWH-018-N-4-OH-pentyl metab JWH-018-N-pentanoic acid JWH-019-5-OH-hexyl metab JWH-200 JWH-122-5-OH-pentyl metab JWH-022-3-OH JWH-398 AM-2201 JWH-073 N-butanoic acid metab JWH-210-4-OH-pentyl metab JWH-210-5-OH-pentyl metab AM-1220 JWH-018 5-OH glucuronide JWH-018 JWH-019 JWH-073 WIN 55,212-2 JWH-018-N-5-Cl-pentyl analog JWH-022

200 125 125 125 100 50 50 50 50 50 25 25 25 25 17 10 10 10 8 7 7 6 6 5 5 5 5 3 3

AM-694 JWH-073 6-OH-indole metab AM-2233 JWH-007 JWH-015 JWH-018 adamantyl JWH-018-4-OH-indole JWH-018-5-OH-indole JWH-018-6-OH-indole metab JWH-018-7-OH-indole metab JWH-020 JWH-073-4-OH-indole JWH-073-5-OH-indole metab JWH-073-7-OH-indole metab JWH-081 JWH-098 JWH-122 JWH-175 JWH-210 JWH-250-4-OH JWH-250-5-OH JWH-250 5-carboxypentyl metab JWH-302 JWH-307 RCS-4 N-5-OH-pentyl metab RCS-8 4-methoxy isomer UR-144 UR-144-5-OH UR-144-4-OH

1 1 ,1 ,1 ,1 ,1 ,1 ,1 ,1 ,1 ,1 ,1 ,1 ,1 ,1 ,1 ,1 ,1 ,1 ,1 ,1 ,1 ,1 ,1 ,1 ,1 ,1 ,1 ,1

485

Table III Drugs Showing No Cross-Reactivity with the JWH-018 Direct ELISA at 1,000 ng/mL or As Indicated

Table IV Cross-Reactivity on JWH-250 Direct ELISA of Synthetic Cannabinoids or Metabolites Relative to JWH-250-4-OH (5 ng/mL)

Synthetic cannabinoid related drugs

Other

Drug


A796-260 AKB 48 AM-1241 AM-1248 CB-13 CB-25 CB-52 CP47,497 C8 analog CP55,940 HU-210 HU-308 HU-331 JP-104 JWH-201 JWH-203 JWH-250 RCS-4 RCS-8 URB 447 URB 597 URB 602 URB 754 URB 937 UR-144 pentanoic acid XLR-11 XLR-11-4-OH

Benzoylecgonine* Cannabidiol* Cocaine* Codeine* Dextromethorphan* EDDP* Methamphetamine* Methadone* Morphine* PCP* THC* THC-COOH* THC-OH*

JWH-250-4-OH (calibrator) JWH-250-5-OH JWH-250-5-carboxypentyl metab AKB 48 AM-1220 JWH-018-N-5-OH-pentyl metab JWH-018-5-OH glucuronide JWH-018-6-OH-indole metab JWH-018-7-OH-indole metab JWH-022 C4 keto JWH-201 JWH-203 JWH-250 JWH-302 RCS-8 RCS-8 4-methoxy isomer

100 50 50 ,1 ,1 ,1 ,1 ,1 ,1 ,1 ,1 ,1 ,1 ,1 ,1 ,1

460

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540

490

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505

*No cross-reactivity at 20,000 ng/mL.

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AM-2201-4-OH-pentyl. JWH-018 itself cross-reacted at 5%. Hydroxylation of the alkyl side chain enhanced cross-reactivity, and C-4 through C-7 homologs of JWH-018 resulted in considerable cross-reactivity. The additions of methyl, ethyl and methoxy substituents on the naphthyl ring did not interfere with the

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cross-reactivity of the compounds. Replacement of the naphthoyl group with a phenyl resulted in a loss of reactivity. Only JWH-250-5-OH-pentyl and JWH-250-5-carboxypentyl 560 metabolites had cross-reactivity of greater than 1% on the Direct ELISA relative to the JWH-250-4-OH calibrator. The assay did not significantly cross-react with the parent compound, JWH-250. Commonly used drugs of abuse and therapeutic drugs were evaluated to determine potential interference with both assays. 565 At a concentration of 20,000 ng/mL, the following analytes produced negative results on both ELISA tests: benzoylecgonine, cocaine, codeine, dextromethorphan, EDDP, methamphetamine, Q2 methadone, morphine and phencyclidine (PCP). Previous research has shown that synthetic cannabinoids yield negative 570


Table V Drugs Showing No Cross-Reactivity with the JWH-250 Direct ELISA at 1,000 ng/mL or As Indicated

Table VI Authentic Subject Urine Specimens 630

LC –MS-MS 575

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585

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Other

A796-260 AM-694 AM-1241 AM-1248 AM-2201 AM-2201-4-OH metab AM-2201-N-4-OH-pentyl metab AM-2233

JWH-073-4-OH-indole metab JWH-073-5-OH-indole metab JWH-073-6-OH-indole metab JWH-073-7-OH-indole metab JWH-073 butanoic acid metab JWH-081 JWH-081-O-desmethyl-4-OH-pentyl metab JWH-081-O-desmethyl-5-OH-pentyl metab JWH-098 JWH-122 JWH-122-4-OH-pentyl metab JWH-122-5-OH-pentyl metab JWH-175 JWH-200 JWH-210 JWH-210-4-OH-pentyl metab JWH-210-5-OH-pentyl metab JWH-307 JWH-398 JWH-398-5-OH-pentyl metab RCS-4 RCS-4-N-5-OH-pentyl metab URB-447 URB-597 URB-602

Benzoylecgonine* Cannabidiol* Cocaine* Codeine* Dextromethorphan* EDDP* Methamphetamine*

CB-13 CB-25 CB-52 CP55,940 CP47,497 C8 analog HU-210 HU-308 HU-331 JP-104 JWH-007 JWH-015 JWH-018 JWH-018-N-5-Cl-pentyl JWH-018-4-OH-indole metab JWH-018-4-OH-pentyl metab JWH-018-5-OH-indole metab JWH-018-N-pentanoic acid metab JWH-018 adamantyl JWH-019 JWH-019-N-5-OH-hexyl metab JWH-019-N-6-OH-hexyl metab JWH-020 JWH-022 JWH-022-3-OH JWH-073 JWH-073-N-3-OH-butyl metab JWH-073-N-4-OH-butyl metab

Methadone* Morphine* PCP* THC* THC-COOH* THC-OH*

URB-754 URB-937 UR-144 UR 144-4-OH UR 144-5-OH UR-144 pentanoic acid XLR-11 XLR-11-4-OH WIN 55,212-2

*No cross-reactivity at 20,000 ng/mL. 605

results on assays targeted to THC metabolites (9). Similarly, THC, 11-nor-9-carboxy-THC, 11-hydroxy-THC and cannabidiol gave negative results on both direct ELISA kits at concentrations of 20,000 ng/mL. 610

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Authentic subject urine samples Authentic subject urine samples testing positive for JWH-018-5-OH-pentyl and JWH-018-4-OH-pentyl metabolite by LC–MS-MS with a cutoff concentration of 0.1 ng/mL were assessed using the JWH-018 Direct ELISA kit. Of the 63 samples confirmed positive by LC–MS-MS, 61 also tested positive with the JWH-018 Direct ELISA kit. Negative urine samples were collected from 51 volunteer self-identified non-drug users and analyzed by using the kit. All 51 samples yielded negative results. The sensitivity, specificity and accuracy for the JWH-018 Direct ELISA kit were calculated to be 96, 100 and 98%, respectively (Table VI). Additionally, 33 samples testing positive for JWH-250-4-OH pentyl metabolite using LC –MS-MS with a cutoff concentration of 0.5 ng/mL were assessed by using the JWH-250 Direct ELISA kit. Thirty-two of the 33 samples tested positive with the ELISA kit. The 51 pedigreed negative urine samples were also screened with this kit and all yielded negative results, 6 Arntson et al.

JWH-018 kit Positive Negative

Positive

Negative

61 2

0 51 635

Table VII Authentic Subject Urine Specimens LC –MS-MS

JWH-250 kit Positive Negative

640

Positive

Negative

32 1

0 51 645

leading to 96% sensitivity, 100% specificity and 98% accuracy (Table VII). The assay was placed into production for monitoring synthetic cannabinoid use in various at-risk populations. Over a period of six months between January and June of 2012, a total of 23,875 specimens were screened using both ELISA kits. Overall, 2,887 specimens (12.1%) tested positive on one or both ELISA plates. All samples screening positive were subsequently analyzed by using the previously described LC–MS-MS method. The confirmation rate on the ELISA screened positives was 75.7% for one or more metabolites of the target compounds. Given the rapidly changing dynamics of the illicit synthetic cannabinoid market, it is likely that many of these ELISA screened positive samples contained synthetic cannabinoid analogs outside of the scope of the LC–MS-MS method. As determined from the previously described cross-reactivity experiments, several of the closely related synthetic cannabinoid analogs did demonstrate cross-reactivity. Additionally, each drug is known to be metabolized to multiple metabolites, hydroxylated in one or more positions and further oxidized to carboxylic acid metabolites, which may collectively have been sufficient to cause an ELISA positive result, but may individually have been below the cutoff of the confirmatory method.

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Conclusions This paper describes the validation of a novel and highly effective technique using ELISA for the detection of metabolites of JWH-018 and JWH-250 in urine. Both the JWH-018 and JWH-250 Direct ELISA Assay Kits have a recommended cutoff concentration of 5 ng/mL. The JWH-018 ELISA has significant crossreactivity with several related synthetic cannabinoid drugs in the naphthoylindole family, which increases its utility. Confirmatory methods will need to be continually updated to ensure that popular cross-reacting drugs and their metabolites are included in the scope of confirmation, because this will determine the positivity rate. The specificity of the tests for synthetic cannabinoids is demonstrated by the limited cross-reactivity with common substances of abuse, including THC and its metabolites. Both kits, coupled with the described confirmation method,

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have yielded a method with a high degree of sensitivity and specificity, making the Direct ELISA Assay Kits suitable for screening synthetic cannabinoids in urine.

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