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Biochip array technology immunoassay performance and quantitative confirmation of designer piperazines for urine workplace drug testing

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Abstract

Designer piperazines are emerging novel psychoactive substances (NPS) with few high-throughput screening methods for their identification. We evaluated a biochip array technology (BAT) immunoassay for phenylpiperazines (PNP) and benzylpiperazines (BZP) and analyzed 20,017 randomly collected urine workplace specimens. Immunoassay performance at recommended cutoffs was evaluated for PNPI (5 μg/L), PNPII (7.5 μg/L), and BZP (5 μg/L) antibodies. Eight hundred forty positive and 206 randomly selected presumptive negative specimens were confirmed by liquid chromatography high-resolution mass spectrometry (LC-HRMS). Assay limits of detection for PNPI, PNPII, and BZP were 2.9, 6.3, and 2.1 μg/L, respectively. Calibration curves were linear (R 2 > 0.99) with upper limits of 42 μg/L for PNPI/PNII and 100 μg/L for BZP. Quality control samples demonstrated imprecision <19.3 %CV and accuracies 86.0–94.5 % of target. There were no interferences from 106 non-piperazine substances. Seventy-eight of 840 presumptive positive specimens (9.3 %) were LC-HRMS positive, with 72 positive for 1-(3-chlorophenyl)piperazine (mCPP), a designer piperazine and antidepressant trazodone metabolite. Of 206 presumptive negative specimens, one confirmed positive for mCPP (3.3 μg/L) and one for BZP (3.6 μg/L). BAT specificity (21.1 to 91.4 %) and efficiency (27.0 to 91.6 %) increased, and sensitivity slightly decreased (97.5 to 93.8 %) with optimized cutoffs of 25 μg/L PNPI, 42 μg/L PNPI, and 100 μg/L BZP. A high-throughput screening method is needed to identify piperazine NPS. We evaluated performance of the Randox BAT immunoassay to identify urinary piperazines and documented improved performance when antibody cutoffs were raised. In addition, in randomized workplace urine specimens, all but two positive specimens contained mCPP and/or trazodone, most likely from legitimate medical prescriptions.

Biochip array technology (BAT) immunoassay for designer piperazines detection in urine. In chemiluminescent immunoassay, the labeled-drug (antigen) competes with the drug in the urine. In the absence of drug, the labeled-drug binds to the antibody releasing an enzyme (horseradish peroxidase) to react with the substrate and producing chemiluminescence. The higher the drug concentration in urine, the weaker the chemiluminescent signal is produced. All presumptive positive specimens and randomly selected presumptive negative specimens were analyzed and confirmed by a liquid chromatography high-resolution mass spectrometry with limit of quantification of 2.5 or 5 μg/L

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Acknowledgments

An Interagency Agreement (Y3-DA-1116-01) between DOD Counter Narcotics Program and Intramural Research Program of the National Institute on Drug Abuse, NIH helped fund this research. Assistance and support of the Forensic Toxicology Drug Testing Laboratory, Fort Meade, MD and Chemistry and Drug Metabolism staff in the conduct of this study are greatly appreciated.

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The opinions or assertions herein are those of the authors and do not necessarily reflect the views of the National Institutes of Health and Department of Defense.

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Authors and Affiliations

  1. Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, 251 Bayview Blvd., Baltimore, MD, 21224, USA

    Marisol S. Castaneto, Allan J. Barnes, Marta Concheiro & Marilyn A. Huestis

  2. Program in Toxicology, University of Maryland Baltimore School of Medicine, Baltimore, MD, 20742, USA

    Marisol S. Castaneto

  3. Drug Testing and Program Policy, Office of the Under Secretary of Defense (Personnel and Readiness), Personnel Risk Reduction, Washington, DC, USA

    Kevin L. Klette & Thomas A. Martin

  4. Department of Sciences, John Jay College of Criminal Justice, City University of New York, New York, NY, USA

    Marta Concheiro

Authors
  1. Marisol S. Castaneto
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  2. Allan J. Barnes
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  3. Marta Concheiro
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  4. Kevin L. Klette
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  5. Thomas A. Martin
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  6. Marilyn A. Huestis
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Correspondence to Marilyn A. Huestis.

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Castaneto, M.S., Barnes, A.J., Concheiro, M. et al. Biochip array technology immunoassay performance and quantitative confirmation of designer piperazines for urine workplace drug testing. Anal Bioanal Chem 407, 4639–4648 (2015). https://doi.org/10.1007/s00216-015-8660-z

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  • DOI: https://doi.org/10.1007/s00216-015-8660-z

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