Evaluation of a homogenous enzyme immunoassay for the detection of synthetic cannabinoids in urine.
BACKGROUND
The recent emergence and widespread availability of many new synthetic cannabinoids support the need for an accurate and high-throughput urine screen for these new designer drugs. We evaluated performance of the immunalysis homogeneous enzyme immunoassay (HEIA) to sensitively, selectively, and rapidly identify urinary synthetic cannabinoids.


METHODS
2443 authentic urine samples were analyzed with the HEIA that targets JWH-018 N-pentanoic acid, and a validated LC-MS/MS method for 29 synthetic cannabinoids and metabolites. Semi-quantitative HEIA results were obtained, permitting performance evaluation at and around three cutoffs (5, 10 and 20 μg/L), and diagnostic sensitivity, specificity and efficiency determination. Performance challenges at ±25 and ±50% of each cutoff level, cross-reactivity and interferences also were evaluated.


RESULTS
Sensitivity, specificity, and efficiency of the immunalysis HEIA K2 Spice kit with the manufacturer's recommended 10 μg/L cutoff were 75.6%, 99.6% and 96.8%, respectively, as compared to the reference LC-MS/MS method with limits of detection of 0.1-10 μg/L. Performance at 5 μg/L was 92.2%, 98.1% and 97.4%, and for the 20 μg/L cutoff were 62.9%, 99.7% and 95.4%. Semi-quantitative results for in-house prepared standards were obtained from 2.5-30 μg/L, and documented acceptable linearity from 5-25 μg/L, with inter-day imprecision <30% (n = 17). Thirteen of 74 synthetic cannabinoids evaluated were classified as highly cross-reactive (≥50% at 10 μg/L); 4 showed moderate cross-reactivity (10-50% at 10 μg/L), 30 low cross-reactivity (<10% at 500 μg/L), and 27 <1% cross-reactivity at 500 μg/L. There was no interference from 102 investigated compounds. Only a mixture containing 1000 μg/L each of buprenorphine/norbuprenorphine produced a positive result above our proposed cutoff (5 μg/L) but below the manufacturer's recommended cutoff concentration (10 μg/L).


CONCLUSIONS
The Immunalysis HEIA K2 Spice kit required no sample preparation, had a high-throughput, and acceptable sensitivity, specificity and efficiency, offering a viable method for screening synthetic cannabinoids in urine that cross-react with JWH-018 N-pentanoic acid antibodies.
Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD 21224, USA.
Allan J
AJ
Barnes
Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD 21224, USA.
Sheena
S
Young
Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD 21224, USA; School of Pharmacy, Fluminense Federal University, CAPES Foundation, BEX 1534/12-2, Rio de Janeiro, Brazil.
Eliani
E
Spinelli
Drug Testing and Program Policy, Office of the Secretary of Defense, Operational Readiness and Safety, Washington DC, USA.
Thomas M
TM
Martin
Drug Testing and Program Policy, Office of the Secretary of Defense, Operational Readiness and Safety, Washington DC, USA.
Kevin L
KL
Klette
Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD 21224, USA. Electronic address: mhuestis@intra.nida.nih.gov.
Marilyn A
MA
Huestis
D002186:Cannabinoids / Q000737:chemistry / Q000652:urine*; D015198:Designer Drugs / Q000032:analysis; D053593:Forensic Toxicology; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D015394:Molecular Structure; D012680:Sensitivity and Specificity
D002186:Cannabinoids; D015198:Designer Drugs
HEIA; Immunoassay; Synthetic cannabinoids