On-site monitoring of nandrolone in cattle farming samples by portable atmospheric pressure chemical ionization mass spectrometry with ambient sampling.

Nandrolone (NT) is a type of androgen anabolic steroid that is often illegally used in cattle farming, leading to unpredictable harm to human health via the food chain. In this study, a rapid detection method for NT in the samples of cattle farming was established using a portable mass spectrometer. The instrument parameters were optimized, including a thermal desorption temperature of 220 °C, a pump speed of 30 %, an APCI ionization voltage of 3900 v, and an injection volume of 6 µL. The samples of bovine urine, feed, sewage, and tissue were selected, and extracted using a solution of methanol:acetonitrile (1:1, v/v), followed by spiking a NT standard solution (1000 ng·mL-1) and ionization through the APCI ion source for detection. The results showed that NT could not be detected in beef and feed due to the complexity of the matrix, while clear signals of NT ions were observed in bovine urine and sewage samples, with LODs of 1000 and 100 ng·mL-1, respectively. Furthermore, quantitative analysis was attempted, and a good linear relationship (R2 = 0.9952) was observed for NT in sewage within the range of 100 to 1000 ng·mL-1. At spiked levels of 100, 500, 1000 and 2000 ng mL-1, the recovery rates ranged from 74.3 % to 92.8 %, with a relative standard deviation (n = 6) of less than 15 %. In conclusion, this detection method offers the advantages of simplicity, rapidity, strong timeliness, and specificity, making it suitable for on-site detection. It can be used for qualitative screening of nandrolone in bovine urine and quantitative analysis of nandrolone in sewage.

Trace Analysis of Five Androgens in Environmental Waters by Optimization of Enzymolysis and Solid-Phase Extraction Ultra-Performance Liquid Chromatography-Mass Spectrometry and its Risk Assessment.

Steroid hormones (SHs) have received widespread attention in recent years. However, current studies of SHs have primarily focused on estrogenic substances, with androgen-related studies being quite limited. We optimized the solid-phase extraction (SPE) pretreatment method, as well as the enzymolysis conditions of five androgens (androstenedione, boldenone, methandienone, nandrolone, and testosterone), to simultaneously determine their concentrations in the effluent from wastewater treatment plants and surface water samples. Then we evaluated the ecological risks of the five androgens in the effluent and Pearl River basin of Guangzhou (PR China) using the risk quotient method. The recovery rates of the targets were 90% to 99% in water samples when digested with ß-glucosidase for 90 min before solid-phase extraction, extracted with a Poly-Sery HLB column, and washed with 15% methanol aqueous solution and 2% ammonia. The established instrument's limit of detection was between 0.02 and 0.39 µg/L, and the limit of quantification was between 0.05 and 1.29 µg/L. Androstenedione, boldenone, methandienone, nandrolone, and testosterone were detected in all samples from the 2018 and 2022 wastewater influent and the 2018 surface water, with concentrations of 3.06 × 101 ng/L to 1.33 × 103 ng/L, 1.03-8.15 × 102 ng/L, and 0.93 × 101 ng/L to 5.50 × 102 ng/L, respectively. The ecological risks of androgens in wastewater influent and surface water were medium to high and low to medium, respectively. Moreover, the biotoxicity of androgens was predicted by the Ecological Structure Activity Relationships model, with methandienone and androstenedione having the highest and lowest acute and chronic toxicities, respectively. These results suggest that the risk of environmental androgens should not be ignored and that further research should be carried out. Environ Toxicol Chem 2024;43:915-925. © 2023 SETAC.

Novel nandrolone aptamer for rapid colorimetric detection of anabolic steroids.

The illicit use of anabolic androgenic steroids (AAS) as performance-enhancing drugs remains a global issue threatening not only the credibility of competitive sports but also public health due to the well-documented adverse effects they elicit. AAS abuse is not restricted only to professional sports, but also extends to recreational athletes and adolescents as well as in livestock production as growth-promoting agents. Testosterone and nandrolone are among the AAS most frequently exploited. Gas chromatography-mass spectrometry is the reference method for AAS detection, but it is strictly laboratory-based and cannot be performed on-site. The great potential of aptamers in bioanalytical applications and specifically for the development of simple analytical tools suitable for on-site analysis has been extensively documented. In this report, we describe the selection and identification of aptamers binding nandrolone, exhibiting affinity dissociation constants in the low nanomolar range. A label-free colorimetric assay based on gold nanoparticles was developed using one of these novel aptamers for the detection of nandrolone and/or its metabolites. The assay could be deployed for the rapid, on-site, facile and cost-effective screening of samples and provide qualitative visual results with a red to purple/blue color change being indicative of a positive result.

Detection of 18-methyl steroids: Case report on a forensic urine sample and corresponding dietary supplements.

The detection of a putative 18-methyl-19-nortestosterone metabolite in a forensic bodybuilder's urine sample collected as part of a criminal proceeding has triggered a follow-up investigation. Four different dietary supplements in the possession of the suspect were examined with regard to possible precursor steroids. This led to the detection of the declared ingredient methoxydienone, which was confirmed by both, GC-MSMS and LC-HRMSMS. As neither 18-methyl-testosterone, nor 18-methyl-19-nortestosterone were detectable in the supplements, the possibility that the metabolite originates from methoxydienone was investigated. For this purpose, the metabolic fate of methoxydienone was studied in vitro using human HepG2 cells and in vivo by a single oral administration. While the 18-methyl-19-nortestosterone metabolite was not generated by HepG2 cells incubated with methoxydienone, it was observed in the urine samples collected at 2, 6, 10 and 24 h after methoxydienone administration. Moreover, the potential binding of methoxydienone as ligand to the human androgen receptor was modelled in silico in comparison with 18-methylnandrolone, for which androgen receptor activation had been shown in an in vitro approach before. In conclusion, we could ascribe the presence of the 18-methyl-19-nortestosterone metabolite in a forensic urine sample to originate from methoxydienone present in dietary supplements. Methoxydienone was observed to slowly degrade by demethylation of the methoxy substituent in liquid solutions. While no compound-specific intermediates were identified that allowed differentiation from other 18-methyl steroids, the 18-methyl-19-nortestosterone metabolite proved to be a suitable marker for reliable detection in doping analysis.

Nandrolone and estradiol biomarkers identification in bovine urine applying a liquid chromatography high-resolution mass spectrometry metabolomics approach.

With the aim of specifically investigating patterns associated with three steroid treatments (17ß-nandrolone, 17ß-estradiol, and 17ß-nandrolone + 17ß-estradiol) in bovine, an reversed phase liquid chromatography (RPLC)-electrospray ionization (ESI)(+/-)-high-resolution mass spectrometry (HRMS) study was conducted to characterize the urinary profiles of involved animals. Although specific fingerprints with strong differences could be highlighted between urinary metabolite profiles within urine samples collected on control and treated animals, it appeared further that significant discriminations could also be observed between steroid treatments, evidencing thus specific patterns and candidate biomarkers associated to each treatment. An MS-2 structural elucidation step enabled level-1 identification of two biomarkers mainly involved in energy pathways, in relation to skeletal muscle functioning. These results make it possible to envisage a global strategy for the detection of anabolic practices involving steroids, while at the same time providing clues as to the compounds used, which would facilitate the confirmation stage to follow.

5α-reductase inhibitors: Evaluation of their potential confounding effect on GC-C-IRMS doping analysis.

5α-reductase inhibitors (5-ARIs) are considered by the World Anti-doping Agency as potential confounding factors in evaluating the athlete steroid profile, since they may interfere with the urinary excretion of several diagnostic compounds. We herein investigated 5α-reductase inhibitors from a different perspective, by verifying their influence on the carbon isotopic composition of 5α- and 5ß-reduced testosterone and nandrolone metabolites. The GC-C-IRMS analysis was performed on a set of urine samples collected from three male Caucasian volunteers after the acute and chronic administration of finasteride in combination with the intake of 19-norandrostenedione, a nandrolone precursor. The excretion and the isotopic profile of androsterone (A), etiocholanolone (Etio) 5α-androstane-3α,17ß-diol (5αAdiol), and 5ß-androstane-3α,17ß-diol (5ßAdiol) were determined as well as those of 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE). Pregnanediol (PD) and pregnanetriol (PT) were also measured as endogenous reference compounds to define the individual endogenous isotopic profile. Our results confirmed the impact of finasteride, especially if chronically administered, on the enzymatic pathway of testosterone and nandrolone, and pointed out the influence of 5-ARIs on δ13 C values of the selected target compounds determined in the IRMS confirmation analysis.

Nanobody-Based Indirect Competitive ELISA for Sensitive Detection of 19-Nortestosterone in Animal Urine.

Nanobody (Nb), a new type of biorecognition element generally from Camelidae, has the characteristics of small molecular weight, high stability, great solubility and high expression level in E. coli. In this study, with 19-nortestosterone (19-NT), an anabolic androgenic steroid as target drug, three specific Nbs against 19-NT were selected from camel immune library by phage display technology. The obtained Nbs showed excellent thermostability and organic solvent tolerance. The nanobody Nb2F7 with the best performance was used to develop a sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for 19-NT detection. Under optimized conditions, the standard curve of ic-ELISA was fitted with a half-maximal inhibitory concentration (IC50) of 1.03 ng/mL and a detection limit (LOD) of 0.10 ng/mL for 19-NT. Meanwhile, the developed assay had low cross- reactivity with analogs and the recoveries of 19-NT ranged from 82.61% to 99.24% in spiked samples. The correlation coefficient between ic-ELISA and the ultra-performance liquid chromatography/mass spectrometry (UPLC-MS/MS) method was 0.9975, which indicated that the nanobody-based ic-ELISA could be a useful tool for a rapid analysis of 19-NT in animal urine samples.

Classification and detection of testosterone propionate and nandrolone residues in duck meat using surface-enhanced Raman spectroscopy coupled with multivariate analysis.

There is a critical need for a rapid and simple method of qualitative and quantitative analysis of testosterone propionate (TP) and nandrolone (NT) residues in duck meat. In this study, we applied surface-enhanced Raman spectroscopy (SERS) coupled multivariate analysis for the classification and detection of TP and NT residues in duck meat. A total of 294 duck meat extract samples were obtained from duck breast meats based on a LC-MS/MS sample preparation method with slight modification including 102 duck meat extract samples without TP and NT, 43 duck meat samples containing TP, 47 duck meat extract samples containing NT, and 102 duck meat extract samples containing TP and NT. Raw Raman spectra were pretreated by using adaptive iteratively reweighted penalized least squares (airPLS), normalization and first derivative, and then the score values of first 10 principal components were selected as the inputs of the developed models. A particle swarm optimization-support vector classification (PSO-SVC) model was created to classify all the duck meat samples into the 4 groups (i.e., control group, TP group, NT group, and TP combined with NT group) with the classification accuracies of 99.49 and 100% for training set and test set, respectively. Furthermore, 2 least squares support vector regression (LS-SVR) models were developed to predict the TP values in samples with a determination coefficient (R2) value of 0.9316, root mean square error (RMSE) value of 2.1739, and ratio of prediction to deviation (RPD) value of 3.2189 for the test set, and NT values in samples with an R2 value of 0.9038, RMSE value of 2.2914, and RPD value of 2.9701 for the test set. Surface-enhanced Raman spectroscopy technology, in combination with multivariate analysis, has the potential to become the qualitative and quantitative analysis tool for TP and NT residues in duck meat extract.

The determination of common anabolic steroid and stimulants in nutritional supplements by HPLC-DAD and LC-MS.

A high-performance liquid chromatography method employing a diode-array detector and mass spectrometry detector was developed, validated and implemented for determining Synephrine, Caffeine, Clenbuterol, Nandrolone, Testosterone and Methylhexaneamine in Nutritional supplements. The use of Nutritional supplements is widespread. Hazards relating to concentration, composition, individual contaminants, supplements interactions as well as positive doping results among athletes present increasing concerns regarding nutritional supplement consuming. The proposed method was validated according to the International Conference on the Harmonization of the Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) standards. The proposed method observed to be accurate, linear, precise, sensitive, required minimal sample preparation and uncomplicated mobile phase. The implementation of the proposed method on nine commercial supplements shows that inaccurate labeling for some supplements regarding the concentration of the ingredients.

IRMS delta values (13 C) of nandrolone and testosterone products available in the UK: Implications for anti-doping.

Anabolic androgenic steroids (AAS) are the most widely abused class of drugs by athletes and thus represent a significant problem to the anti-doping community. Confirmation of a doping violation for AAS cannot always be based on their presence alone due to the endogenous production of some steroids. Both testosterone (and its metabolites) and the major diagnostic metabolite of nandrolone (19-norandrosterone) are produced endogenously. Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is used in such cases to differentiate between the administration of a synthetic preparation and endogenous steroid production by measurement of their differing carbon isotope (13 C/12 C) ratio. The purpose of this study was to investigate the availability of steroid preparations in the UK with a 13 C content analytically indistinguishable from that of endogenous steroids. Fourteen preparations containing nandrolone (n = 9) and testosterone (n = 5) were analyzed. The δ13 C values were determined using GC-C-IRMS and the identity of the steroid preparations was confirmed using gas chromatography-mass spectrometry (GC-MS). Ten steroid preparations displayed δ13 C values within the range expected for synthetic steroids (less than -27‰). However, four nandrolone preparations displayed δ13 C values that overlap with the values considered to be endogenous in origin (range: -26 to -16‰). Misuse of these preparations could prevent the confirmation of nandrolone administration using GC-C-IRMS in anti-doping cases.

Evidence of boldenone, nandrolone, 5(10)-estrene-3ß-17α-diol and 4-estrene-3,17-dione as minor metabolites of testosterone in equine.

The detection of boldenone, nandrolone, 5(10)-estrene-3ß,17α-diol, and 4-estrene-3,17-dione in a urine sample collected from a gelding having been treated with testosterone (500 mg 'Testosterone Suspension 100', single dose, injected intramuscularly) in 2009 led the authors' laboratory to suspect that these 'testicular' steroids could be minor metabolites of testosterone in geldings. Administration trials on six castrated horses with Testosterone Suspension 100 confirmed that low levels of boldenone, nandrolone, 5(10)-estrene-3ß,17α-diol, and 4-estrene-3,17-dione could indeed be detected and confirmed in the early post-administration urine samples from all six geldings. Although boldenone has been reported to be present in urine after testosterone administration, there has been no direct evidence reported that boldenone, nandrolone, 5(10)-estrene-3ß,17α-diol, and 4-estrene-3,17-dione are metabolites of testosterone in geldings. Subsequent in vitro experiments involving the incubation of testosterone with horse liver microsomes, liver, adipose and muscle tissues, and adrenal cortex homogenates failed to provide evidence that these four substances are minor metabolites of testosterone. An administration trial using 'Testosterone Suspension 100' supplemented with 13 C-labelled testosterone (500 mg, 1:1 ratio, injected intramuscularly) was performed. The similarities of the excretion curves of 12 C-testosterone and 13 C-testosterone in urine suggest that there was minimal kinetic isotope effect. 13 C-Labelled boldenone, nandrolone and 4-estrene-3,17-dione were detected but not 5(10)-estrene-3ß,17α-diol and its 13 C-counterpart. This is the first unequivocal evidence of boldenone, nandrolone and 4-estrene-3,17-dione being metabolites of testosterone in geldings. In view of these results, caution should be exercised when interpreting findings of boldenone, nandrolone and/or 4-estrene-3,17-dione together with a relatively high level of testosterone in gelding urine. Copyright © 2017 John Wiley & Sons, Ltd.

Highly sensitive avidin-biotin ELISA for detection of nandrolone and testosterone in dietary supplements.

Avidin-biotin technology was used for the implementation of an enzyme-linked immunosorbent assay (AB-ELISA) as a sensitive method for the detection of anabolic androgenic steroids (AAS) present in dietary supplements. Using click chemistry, novel haptens (linker-optimized biotinylated nandrolone (NT) and testosterone (T) at positions C-3 and C-17, respectively) were designed and synthesized to be then applied as four different immobilized competitors in a proposed set of four indirect competitive AB-ELISAs. Four rabbit polyclonal antibodies of various specificities were prepared using four different immunogens synthesized from C-3 and C-17 carboxymethyloxime and hemisuccinate derivatives of NT and T, respectively. Assembled AB-ELISAs were characterized to establish method parameters such as a half-maximum inhibition concentration (0.18-12.99 ng/mL), limit of detection (0.004-0.032 ng/mL) and linear working range (the best with 0.02-1.38 ng/mL). The stability of the set simulating storage in different conditions was demonstrated. Cross reactivity (CR) was tested for 59 steroids including both endogenous and synthetic analogues in four assembled AB-systems. The focus was placed on the practical use of the method in detection of various AAS in 49 samples of counterfeit dietary supplements. The concordance between ultra high performance liquid chromatography-mass spectrometry (UHPLC-MS) and the CR corrected data from AB-ELISA indicated the potential of this method even to quantification of T propionate, NT phenyl propionate, and NT decanoate in such a complex matter. Copyright © 2016 John Wiley & Sons, Ltd.

Bovine teeth as a novel matrix for the control of the food chain: liquid chromatography-tandem mass spectrometry detection of treatments with prednisolone, dexamethasone, estradiol, nandrolone and seven ß2-agonists.

Veterinary drugs usually have rapid clearance rates in the liver and kidney, hampering their detection in conventional matrices such as the liver or urine. Pharmacological principles such as esterification may be applied to facilitate the administration of veterinary drugs and increase drug half-life. Prednisolone, whose therapeutic administration is regulated for food producing animals in the EU, is available in its acetate form as well as nandrolone, a banned anabolic steroid, which may be obtained as nandrolone phenylpropionate and estradiol as a benzoyl ester. While the distribution and accumulation of lipophilic and hydrophilic substances in human teeth have been well documented, studies on residues in bovine teeth are lacking. We hypothesised that analysis of bovine teeth could be used to detect both regulated and banned veterinary drugs. Steroids may be illegally used as growth promoters in food producing animals, alone or combined with ß2-agonists; therefore, we developed, and validated, in accordance with the Commission Decision 2002/657/EC, two analytical confirmatory LC-MS/MS methods to detect these classes of compounds following a unique liquid extraction procedure. Finally, we analysed teeth from three male Friesian veal calves treated with intramuscular estradiol benzoate, oral prednisolone acetate or intramuscular nandrolone phenylpropionate in combination with oral ractopamine, respectively, and from seven bovines from the food chain. Teeth from treated animals were positive for their respective drugs, with the exception of nandrolone phenylpropionate. One sample from a food chain bovine was positive for isoxsuprine, one of the seven ß2-agonists studied. Non-esterified forms of the steroids were not found. These results demonstrate that bovine teeth are a suitable matrix for the determination of pseudoendogenous substances or illicit administration of veterinary drugs.

Development of a rapid method for the analysis of trenbolone, nortestosterone, and zeranol in bovine liver using liquid chromatography tandem mass spectrometry.

A rapid liquid chromatography tandem mass spectrometry method has been developed and validated for the determination of α-trenbolone, ß-trenbolone, α-nortestosterone, ß-nortestosterone, zeranol, and taleranol in bovine liver. The impact of liquid-liquid extraction with methyl tert-butyl ether and optimized solid phase extraction on silica cartridges significantly reduced effort and time of sample preparation. Electrospray ionization gives a significant signal increase compared with atmospheric pressure chemical ionization and atmospheric pressure photoionization. The HPLC gradient was optimized to separate isobaric analytes and matrix constituents from the hormone molecules. The optimized time and temperature of enzymatic hydrolysis of conjugated trenbolone was 4 h at 52 °C. The method validated in the range of 0.5-30 µg kg(-1) for α-trenbolone, ß-trenbolone, zeranol, taleranol, and 2-30 µg kg(-1) for α-nortestosterone, ß-nortestosterone. Combined uncertainty of measurements was in the range of 4%-23%. The matrix effect was negligible (1%-5%) for all analytes except of α-nortestosterone (19%). The developed method with changes concerning sample size and hydrolysis was also applied for the analysis of meat, serum, and urine samples. Graphical Abstract Determination of trenbolone, nortestosterone and zeranol in bovine liver.

Carbon isotope ratios of nandrolone, boldenone, and testosterone preparations seized in Norway compared to those of endogenously produced steroids in a Nordic reference population.

Determining the origin of anabolic androgenic steroids (AAS) that also are produced endogenously in the human body is a major issue in doping control. In some cases, the presence of nandrolone and boldenone metabolites might result from endogenous production. The GC-C-IRMS technique (gas chromatography-combustion-isotope ratio mass spectrometry) enables the carbon isotopic ratio (CIR) to be measured to determine the origin of these metabolites. The aim of this study was to use GC-C-IRMS to determine the δ(13) CVPDB values of seized boldenone and nandrolone preparations to decide if the steroids themselves were depleted in (13) C, compared to what is normally seen in endogenously produced steroids. In addition, several testosterone preparations were analyzed. A total of 69 seized preparations were analyzed. The nandrolone preparations showed δ(13) CVPDB values in the range of -31.5 ‰ to -26.7 ‰. The boldenone preparations showed δ(13) CVPDB values in the range of -32.0 ‰ to -27.8 ‰, and for comparison the testosterone preparations showed δ(13) CVPDB values of -31.0 ‰ to -24.2 ‰. The results showed that the values measured in the nandrolone and boldenone preparations were in the same range as those measured in the testosterone preparations. The study also included measurements of CIR of endogenously produced steroids in a Norwegian/Danish reference population. The δ(13) CVPDB values measured for the endogenous steroids in this population were in the range of -21.7 to -26.8. In general, most of the preparations investigated in this study show (13) C-depleted delta values compared to endogenously produced steroids reflecting a northern European diet.

Statistical discrimination of steroid profiles in doping control with support vector machines.

Due to their performance enhancing properties, use of anabolic steroids (e.g. testosterone, nandrolone, etc.) is banned in elite sports. Therefore, doping control laboratories accredited by the World Anti-Doping Agency (WADA) screen among others for these prohibited substances in urine. It is particularly challenging to detect misuse with naturally occurring anabolic steroids such as testosterone (T), which is a popular ergogenic agent in sports and society. To screen for misuse with these compounds, drug testing laboratories monitor the urinary concentrations of endogenous steroid metabolites and their ratios, which constitute the steroid profile and compare them with reference ranges to detect unnaturally high values. However, the interpretation of the steroid profile is difficult due to large inter-individual variances, various confounding factors and different endogenous steroids marketed that influence the steroid profile in various ways. A support vector machine (SVM) algorithm was developed to statistically evaluate urinary steroid profiles composed of an extended range of steroid profile metabolites. This model makes the interpretation of the analytical data in the quest for deviating steroid profiles feasible and shows its versatility towards different kinds of misused endogenous steroids. The SVM model outperforms the current biomarkers with respect to detection sensitivity and accuracy, particularly when it is coupled to individual data as stored in the Athlete Biological Passport.

Establishment and optimization of monoclonal antibody-based heterologous dcELISA for 19-nortestosterone residue in bovine edible tissue.

This paper presents the generation of monoclonal antibodies (mAbs) with high specificity against 19-nortestosterone (NT) through cell fusion procedures, and the development of mAb-based heterologous direct competitive enzyme-linked immunoabsorbent assay (dcELISA) methods to detect NT residue using one of these hybridomas (clone 3B8-E6). Under optimal experimental conditions, this assay exhibited a working range of 0.004 to 19 ng/mL with IC50 and limit of detection values of 0.28 and 0.002 ng/mL, respectively, when it was run in 0.01M phosphate-buffered saline (pH 7.4). Except for minor cross-reactivity with ß-boldenone (6.9%) and trenbolone (1.2%), other interference to the assay was negligible (<0.05%). No significant differences (P > 0.05) were found for IC50 values when the pH of the assay buffer ranged from 6 to 8 and phosphate ion concentration was less than 20 mM. The dcELISA can tolerate higher concentrations of methanol than other organic solvents tested. When applied to bovine sample, the correlation coefficients (R) of the dcELISA and GC-MS data were 0.9918 in muscle, 0.9834 in liver, and 0.9976 in kidney. Therefore, this assay has the potential to be incorporated into a quantitative monitoring program for the rapid screening of NT residue in food.

Illegal treatment of barrows with nandrolone ester: effect on growth, histology and residue levels in urine and hair.

The effect of 17ß-19-nortestosterone (17ßNT) treatment of barrows on residue levels and growth was evaluated. Five barrows were treated three times during the fattening period with 17ßNT phenylpropionate (Nandrosol, nandrolone phenylpropionate 50 mg/ml,1 mg/kg body weight). Another five barrows were untreated and five boars (untreated) were kept as positive control. Boars and treated barrows showed a 13 and 9% improvement in growth compared to untreated barrows, with mean final body weights of 121.6, 117.8 and 109.0 kg, respectively. The bulbourethral glands of the treated barrows were three times heavier than untreated barrows. The histology of the prostate and bulbourethral gland of the treated barrows was comparable to the boars, whereas the control barrows showed atrophic glands. Levels of 17ßNT ester in hair from treated barrows were high, whereas boars and untreated barrows did not show levels above LLQ. It is concluded that analysis of hair can detect illegal treatment with 17ßNT ester in barrows. The size of the bulbourethral gland can also be used for screening in the slaughterhouse.

Monoclonal antibody-based ELISA and colloidal gold immunoassay for detecting 19-nortestosterone residue in animal tissues.

This article presents the generation of monoclonal antibodies (mAbs) with high specificity against 19-nortestosterone (NT) through cell fusion techniques and the development of a mAb-based indirect competitive ELISA (icELISA) method and colloidal gold-based immuno-chromatographic assay to detect NT residues in beef and pork samples. A modified carbodiimide method was employed to synthesize the artificial antigen, and BALB/c mice were used to produce anti-NT mAbs. On the basis of the checkerboard titration, an indirect competitive ELISA standard curve was established. This assay was sensitive and had a linear range from 0.03 to 38 ng/mL in phosphate buffered saline (PBS), with IC(50) and LOD values of 0.52 ng/mL and 0.01 ng/mL, respectively. Of all the competitive analogues, the produced mAb exhibited a high cross-reactivity to 17α-nortestosterone (83.6%), the main metabolite of NT in animal tissues. Except for moderate cross-reactivities with trenbolone (22.6%) and ß-boldenone (13.8%), the other interference to the assay was negligible (<0.05%). In contrast, the strip test had a visual detection limit of 1 ng/mL in PBS, 2 µg/kg in beef, and 2 µg/kg in pork, respectively, and the results can be judged within 10 min. The ELISA and GC-MS results showed close correlation in beef (R2=0.9945) and in pork (R2=0.9977). Therefore, the combination of two immunoassays provides a useful screening method for quantitative or qualitative detection of NT residues in animal-origin products.