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Deterrence of synthetic cathinone abuse is hampered by the lack of a high-throughput immunoassay screen. The Randox Drugs of Abuse V (DOA-V) Biochip Array Technology contains two synthetic cathinone antibodies: Bath Salt I (BSI) targets mephedrone/methcathinone and Bath Salt II (BSII) targets 3',4'-methylenedioxypyrovalerone (MDPV)/3',4'-methylenedioxy-α-pyrrolidinobutiophenone (MDPBP). We evaluated DOA-V synthetic cathinones performance and conducted a full validation on the original assay with calibrators reconstituted in water, and the new assay with calibrators prepared in lyophilized urine; both utilized the same antibodies and were run on the fully automated Evidence® Analyzer. We screened 20 017 authentic military urine specimens and confirmed positives by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for 28 synthetic cathinones. Limits of detection (LOD) for the original and new assays were 0.35 and 0.18 (BSI), and 8.5 and 9.2 µg/L (BSII), respectively. Linearity was acceptable (R(2)  >0.98); however, a large negative bias was observed with in-house prepared calibrators. Intra-assay imprecision was <20% BSI-II, while inter-assay imprecision was 18-42% BSI and <22% BSII. Precision was acceptable for Randox controls. Cross-reactivities of many additional synthetic cathinones were determined. Authentic drug-free negative urine pH <4 produced false positive results for BSI (6.3 µg/L) and BSII (473 µg/L). Oxidizing agents reduced BSI and increased BSII results. Sensitivity, specificity, and efficiency of 100%, 52.1%, and 53.0% were obtained at manufacturer's proposed cut-offs (BSI 5 µg/L, BSII 30 µg/L). Performance improved if cut-off concentrations increased (BSI 7.5 µg/L, BSII 40 µg/L); however, there were limited confirmed positive specimens. Currently, this is the first and only fully validated immunoassay for preliminary detection of synthetic cathinones in urine. Published 2014. This article is a U.S. Government work and is in the public domain in the USA., (Published 2014. This article is a U.S. Government work and is in the public domain in the USA.)

Synthetic cathinones are novel stimulants derived from cathinone, with amphetamines or cocaine-like effects, often labeled "not for human consumption" and considered "legal highs". Emergence of these new designer drugs complicate interpretation of forensic and clinical cases, with introduction of many new analogs designed to circumvent legislation and vary effects and potencies. We developed a method for the simultaneous quantification of 28 synthetic cathinones, including four metabolites, in urine by liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS). These cathinones include cathinone, methcathinone, and synthetic cathinones position-3'-substituted, N-alkyl-substituted, ring-substituted, methylenedioxy-substituted, and pyrrolidinyl-substituted. One mL phosphate buffer pH 6 and 25 μL IStd solution were combined with 0.25 mL urine, and subjected to solid phase cation exchange extraction (SOLA SCX). The chromatographic reverse-phase separation was achieved with a gradient mobile phase of 0.1 % formic acid in water and in acetonitrile in 20 min. We employed a Q Exactive high resolution mass spectrometer, with compounds identified and quantified by target-MSMS experiments. The assay was linear from 0.5-1 to 100 μg/L, with limits of detection of 0.25-1 μg/L. Imprecision (n = 20) was <15.9 % and accuracy (n = 20) 85.2-118.1 %. Extraction efficiency was 78.9-116.7 % (CV 1.4-16.7 %, n = 5), process efficiency 57.7-104.9 %, and matrix effects from -29.5 % to 1.5 % (CV 1.9-13.1 %, n = 10). Most synthetic cathinones were stable at 4 °C for 72 h (n = 27) and after 3 freeze-thaw cycles (n = 26), but many (n = 19) were not stable at room temperature for 24 h (losses up to -67.6 %). The method was applied to authentic urine specimens from synthetic cathinone users. This method provides a comprehensive confirmation method for 28 synthetic cathinones in urine, with good selectivity and specificity.