Validation of the only commercially available immunoassay for synthetic cathinones in urine: Randox Drugs of Abuse V Biochip Array Technology

Abstract

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.

Keywords: MDPV; Randox; immunoassay; mephedrone; synthetic cathinones.

Figure 1

Mean %bias of the Randox DOA-V synthetic cathinone biochip A) BSI and B) BSII assays across their linear ranges using DOA-V original and new assays. Randox proposed cutoffs were 5 and 30µg/L for BSI and BSII. The vertical line (····) represents the manufacturer’s proposed cutoffs for original and new assays and (—) highest calibrators (grey: original assay, black: new assay). Horizontal lines represent (····) ±20% acceptance criteria.

Figure 2

Randox and Chemistry and Drug Metabolism (CDM)-evaluated synthetic cathinone cross-reactivity with A) BSI (mephedrone) and B) BSII (MDPV). (*) represent no Randox cross-reactivity reported and (+) represent % cross-reactivity <1%.

Figure 3

Sensitivity, specificity, and efficiency of Randox DOA-V for the detection of synthetic cathinones in authentic confirmed urine specimens (N=198) evaluated with different cutoffs: A) BSI (5µg/L), B^*) BSI (7.5µg/L), C) BSI (10µg/L), D) BSII (30µg/L), E^*) BSII (40µg/L), F) BSII (50µg/L), G) BSI (5µg/L) & BSII (30µg/L) (Randox proposed cutoffs), H^*) BSI (7.5µg/L) & BSII (40µg/L), I) BSI (10µg/L) & BSII (50µg/L). Only four specimens were confirmed positive via LC-HRMS. ^* Represents optimal cutoff concentrations for BSI only (B), BSII only (E), and BSI & BSII (H).

Figure 4

Performance around cutoff concentrations of Randox synthetic cathinone BSI biochip for original and new DOA-V kits. Mephedrone fortified negative urine samples at cutoff A) 2.5, B) 5, C) 7.5, and D) 10µg/L, and ±25% and ±50% of cutoffs. Error bars represent ±2SD from mean observed concentration.

Figure 5

Performance around cutoff concentrations of Randox synthetic cathinone BSII biochip for original and new DOA-V assays. MDPV fortified negative urine samples at A) 20, B) 30, and C) 40µg/L cutoffs, and ±25% and ±50% of these cutoffs. Error bars represent ±2SD from the mean observed concentration.