Stanazolol derived ELISA as a sensitive forensic tool for the detection of multiple 17α-methylated anabolics

Huml, Lukáš; Havlová, Dominika; Longin, Ondřej; Staňková, Eliška; Holubová, Barbora; Kuchař, Martin; Prokudina, E.A.; Rottnerová, Zdeňka; Zimmermann, Tomáš; Drašar, Pavel; Lapčı́k, Oldřich; Jurášek, Michal

doi:10.1016/j.steroids.2019.108550

Cited by 8 publications

(20 citation statements)

References 26 publications

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“…Conjugated hapten to rabbit serum albumin (RSA) was simply marked RSA/ST-3 (Supplementary Figure S1). As the result of this work [7], we found that the RAb 212 antibody had strong cross-reactivity (CR) for 17α-methylated AAS in an indirect competitive ELISA setup (Figure 1). The applicability of this group-selective method was further demonstrated in a series of police-intercepted preparations containing AAS.…”

Section: Introductionmentioning

confidence: 64%

“…The basic methods for the detection of AAS include, in particular, gas or liquid chromatography combined with mass detection (GC-MS, HPLC-MS, respectively). Other commonly used methods include enzyme-linked immunosorbent assay (ELISA) [6][7][8][9]. In our recent work by Huml et al [7], we have described the development of a group-selective immunoassay method based on stanazolol (ST)-derived polyclonal rabbit antibodies (RAb).…”

Section: Introductionmentioning

confidence: 99%

“…Other commonly used methods include enzyme-linked immunosorbent assay (ELISA) [6][7][8][9]. In our recent work by Huml et al [7], we have described the development of a group-selective immunoassay method based on stanazolol (ST)-derived polyclonal rabbit antibodies (RAb). In the latter paper [7], hapten ST-3 obtained by alkylation of the pyrazole ring showed the best performance in convenient ELISA assays.…”

Section: Introductionmentioning

confidence: 99%

“…In our recent work by Huml et al [7], we have described the development of a group-selective immunoassay method based on stanazolol (ST)-derived polyclonal rabbit antibodies (RAb). In the latter paper [7], hapten ST-3 obtained by alkylation of the pyrazole ring showed the best performance in convenient ELISA assays. Conjugated hapten to rabbit serum albumin (RSA) was simply marked RSA/ST-3 (Supplementary Figure S1).…”

Section: Introductionmentioning

confidence: 99%

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Tailor-Made Immunochromatographic Test for the Detection of Multiple 17α-Methylated Anabolics in Dietary Supplements

Holubová

Kubešová

Huml

et al. 2021

Foods

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In recent years, the undeclared presence of various anabolic androgenic steroids (AAS) in commercial supplements has been confirmed. This fact can be a potential threat to all athletes using these supplements, and therefore, there is of increased interest in the implementation of rapid methods for the detection of AAS. The presented study describes the development of an immunostrip test for the detection of multiple 17α-methylated AAS based on direct and indirect competitive principle using gold nanoparticles as a label. As a capture reagent on test lines conjugated stanazolol to rabbit serum albumin (RSA/ST-3) was used, the intensity of color formed in the test line of the AAS-positive sample was visually distinguishable from that of negative sample within 10 min. The optimized closed direct and indirect format of the test provided a similar visual detection limit (0.7 and 0.9 ng/mL, respectively). The most commonly orally abused AAS (17α-methyltestosterone, methandienone, methyldihydrotestosterone, oxandrolone and oxymetholone) showed a strong cross-reaction. Developed immunostrips were successfully applied to analysis of artificially contaminated dietary supplements with 17α-methylated AASs. The developed immunostrips offer potential as a useful user-friendly method for capturing suspicious dietary supplement samples with different contents of AAS at levels far below the usually used concentrations of AAS.

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Section: Introductionmentioning

confidence: 64%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Tailor-Made Immunochromatographic Test for the Detection of Multiple 17α-Methylated Anabolics in Dietary Supplements

Holubová

Kubešová

Huml

et al. 2021

Foods

Self Cite

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“…Instead, ELISA (enzyme-linked immunosorbent assay) is a commonly used analytical assay, as it enables parallel analysis of multiple samples in a short time, utilizing a simple procedure, and appears to be a useful cost-effective screening tool for the preselection of suspect samples for further instrumental approaches, which are, however, irreplaceable in identification and quantification of particular steroids [ 28 ]. As previously reported, a carrier protein with bound molecules of the target steroid via a defined spacer is used to elicit anti-histone antibodies [ 29 ]. This is also the case for Stz , where a specific derivative, with an appropriate spacer, is necessary to build up an efficient antibody for the preparation of an ELISA kit for Stz and its metabolites analysis.…”

Section: Introductionmentioning

confidence: 99%

Simple Synthesis of 17-β-O-hemisuccinate of Stanozolol for Immunoanalytical Methods

2020

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The use of doping in sports is a global problem that affects athletes around the world. Among the different methods developed to detect doping agents in biological samples, there are antibody-based methods that need an appropriate hapten design. Steroids with a hydroxyl group can be converted to the corresponding hemisuccinates. A novel approach to the synthesis of 17β-O-hemisuccinate of the common doping agent stanozolol is described here. Acylation of stanozolol with methyl 4-chloro-4-oxobutyrate/4-dimethylaminopyridine, followed by mild alkaline hydrolysis with methanolic sodium hydroxide at room temperature, gave the simultaneous protection and deprotection of pyrazole-nitrogen atoms. The proposed new synthetic method allows the desired hemisuccinate derivative to be obtained in only two steps, and with a good total yield starting from stanozolol.

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Screening method for the characterization of anabolic steroids seized in Brazil using paper spray mass spectrometry and chemometric tools

et al. 2022

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This paper reports the use of paper spray mass spectrometry (PS‐MS) combined with chemometric models to analyze seized samples of anabolic steroids. Because many forensic laboratories typically demand high‐throughput analysis for this type of sample, we developed a quicker and simpler alternative analytical method for routine analysis with minimal sample preparation. Oily samples (n = 39) resulting from seizures carried out by Brazilian Federal and State Police units were selected for this study. These samples were analyzed by PS‐MS in the positive ion mode and full scan (50–1000 m/z), providing spectra containing patterns of the respective active ingredients present in each product. A principal component analysis (PCA) model was built, which discriminated samples mainly according to their active ingredients and allowed to detect and characterize some cases of product counterfeiting. The variable selection method ordered predictors selection was employed jointly with PCA to improve sample cluster separation and to provide model simplification. The final PCA model was built with three principal components and using only 28 spectral variables. This model accounted for 69.82% of the variance and discriminated samples according to their specific active ingredients.

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Stanazolol derived ELISA as a sensitive forensic tool for the detection of multiple 17α-methylated anabolics

Cited by 8 publications

References 26 publications

Tailor-Made Immunochromatographic Test for the Detection of Multiple 17α-Methylated Anabolics in Dietary Supplements

Tailor-Made Immunochromatographic Test for the Detection of Multiple 17α-Methylated Anabolics in Dietary Supplements

Simple Synthesis of 17-β-O-hemisuccinate of Stanozolol for Immunoanalytical Methods

Screening method for the characterization of anabolic steroids seized in Brazil using paper spray mass spectrometry and chemometric tools

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