Two immunoassays for the detection of 2C-B and related hallucinogenic phenethylamines.

INTRODUCTION: The use of new psychoactive substances as drugs of abuse has dramatically increased over the last years. Hallucinogenic phenethylamines gained particular popularity as they have both stimulating and psychedelic effects. Although generally perceived as safe, these illicit drugs pose a serious health risk; they have been linked to cases of severe poisoning or even deaths. Therefore, simple, cost-effective and reliable methods are needed for rapid determination of abused hallucinogens. METHODS: For this purpose, two haptens derived from 2C-H were designed, synthesized and subsequently attached to a carrier protein. Polyclonal antibodies obtained from a rabbit immunized with one of the prepared immunogens were used for the development of two immunoassays. RESULTS: In this study, a lateral flow immunoassay (LFIA) and an enzyme linked immunosorbent assay (ELISA) for the detection of 2C-B and related hallucinogenic phenethylamines in urine were developed. The presented LFIA is primarily suitable for on-site monitoring as it is simple and can provide a visual evidence of 2C-B presence within a few minutes. Its reasonable sensitivity (LODLFIA = 15 ±â€¯7 ng mL-1) allows detection of the drug presence in urine after acute exposure. For greater accuracy, highly sensitive ELISA (LODELISA = 6 ±â€¯3 pg mL-1) is proposed for toxicological quantitative analyses of positive samples captured by the LFIA. DISCUSSION: The comparison of the ELISA with the well-established UHPLC-MS-MS method shows excellent agreement of results, which confirms good potential of the ELISA to be used for routine analyses of 2C-B and related hallucinogenic phenethylamines of both main sub-families.

Sensitive enzyme immunoassay for screening methandienone in dietary supplements.

Methandienone is a synthetic exogenous steroid which, like other anabolic steroids, is strictly regulated in many countries. In recent years, increasing numbers have been detected of illegal additions into dietary supplements of methandienone and other anabolic androgenic steroids (AAS). In this work, a competitive indirect enzyme-linked immunosorbent assay (ELISA) has been constructed for the detection of methandienone using an antiserum against methandienone. Under optimal experimental conditions, the ELISA achieved a limit of detection of 0.04 ± 0.01 µg.g-1. The obtained intra- and inter-day coefficients of variation were less than 8%. The developed ELISA was applied in the analysis of real dietary supplement samples. To minimise the effect of the sample matrix, the sample extracts were simply diluted before addition into the immunoassay. The achieved recovery values were around 100%. Results obtained from the ELISA correlated well, both in terms of accuracy and precision, with those obtained by UHPLC-MS/MS (reference method). The presented ELISA could be successfully applied for the simple screening of dietary supplements.

Lateral flow immunoassay and enzyme linked immunosorbent assay as effective immunomethods for the detection of synthetic cannabinoid JWH-200 based on the newly synthesized hapten.

In recent years, the use of synthetic cannabinoids (SCs) as drugs of abuse has greatly increased. SCs are associated with a risk of severe poisoning or even death. Therefore, more rapid, cost effective and reliable methods are needed, especially for the screening of drivers after traffic accidents and for detailed toxicological analysis in forensic laboratories. In this study, we developed a lateral flow immunoassay (LFIA) and an enzyme linked immunosorbent assay (ELISA) for the detection of JWH-200 in oral fluids. For this purpose a new hapten was prepared using a ten-step synthetic route. The developed immuno methods are based on antibodies obtained from rabbit immunized with synthesized hapten conjugated to carrier protein. The proposed methods are highly sensitive (LODLFIA = 0.08 ±â€¯0.04 ng mL-1; LODELISA = 0.04 ±â€¯0.02 ng mL-1). They were applied to the quantification of JHW-200 in spiked oral fluids. The recoveries ranged from 82 to 134% for both methods. The results correlated excellently with results obtained using UHPLC-MS/MS (R2LFIA = 0.99; R2ELISA = 0.99). Our developed methods could be an important tool for analyses of JWH-200 in human oral fluids. The one-step LFIA is particularly suitable for roadside and on-site monitoring due to the rapid qualitative results it delivers, while the ELISA is especially useful for laboratory quantitative analyses of positive samples captured by LFIA.

Use of novel haptens in the production of antibodies for the detection of tryptamines.

Tryptamines are a group of hallucinogenic drugs whose detection in body fluids could be simplified by immunochemical assay kits. Antibodies for these assays are obtained by the immunization of laboratory animals with conjugates of a hapten similar to the target analyte and a suitable protein. Therefore we synthesized novel haptens derived from tryptamine-based drugs, with N,N-dimethyltryptamine (DMT), 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) and N,N-diisopropyltryptamine (DiPT) selected as the target analytes. Their structures were modified with a short linker ended with a carboxylic group. The haptens were conjugated with bovine serum albumin (BSA) and rabbits were immunized with the conjugates. The obtained polyclonal antibodies showed good reactivity and the LOD of the constructed ELISAs was in the range 0.006-0.254 ng mL-1. Thus, they are suitable for the development of immunochemical assay kits.