Direct detection of boldenone sulfate and glucuronide conjugates in horse urine by ion trap liquid chromatography-mass spectrometry

A study of the equine phase II metabolism of the anabolic agent boldenone is reported. Boldenone sulfate, boldenone glucuronide and their C17-epimers were synthesised as reference standards in our lab and a method was developed for their detection in a horse urine matrix. Solid phase extraction was used to purify the analytes, which were then detected by ion trap LC/MS. Negative and positive ionisation mode MS(2) were used for the detection of sulfate and glucuronide conjugates, respectively. Boldenone sulfate and 17-epiboldenone glucuronide were detected as the major and minor phase II metabolites, respectively, in horse urine samples collected following the administration of boldenone undecylenate by intramuscular injection.     

Detection of endogenous boldenone in the entire male horses

Boldenone (1,2-dehydrotestosterone) is a common veterinary anabolic agent. Its structure is very similar to testosterone. Testosterone is endogenous in the horse, whereas there has been no report concerning the detection of endogenous boldenone. This paper reports the direct observation of sulphate conjugate of boldenone in equine urine from entires. The detection procedures involved solid-phase extraction, immunoaffinity column (IAC) purification, and then LC-MS-MS analysis on a Q-ToF instrument. The identification of boldenone sulphate has provided direct evidence for the endogenous nature of boldenone in entire male horses. Quantification data for the normal level of boldenone in Hong Kong racehorses will also be discussed.     

Criteria to distinguish between natural situations and illegal use of boldenone, boldenone esters and boldione in cattle 1. Metabolite profiles of boldenone, boldenone esters and boldione in cattle urine

Boldenone is an androgenic steroid that improves the growth and food conversion in food producing animals. In most countries worldwide, this anabolic steroid is forbidden for meat production. Until recently, the control of its illegal use was based either on 17beta-boldenone or 17alpha-boldenone (its main metabolite in cattle) identification in edible tissues, hair, faeces or urine. Recent observations and data tend to demonstrate the natural occurrence (but not ubiquitous) in cattle of these steroids, making the analytical strategy of the control more complicated. We investigated the metabolism of boldenone in cattle after intramuscular and oral treatment of boldenone, boldenone esters and boldione. The central objective was to elucidate the structures of the main metabolites (phase I and phase II) in urine, with main objective to be further in position to compare boldenone urinary profiles of treated and non-treated animals. Nine metabolites have been identified, only four were present whatever the treatment and the administered boldenone source. Nevertheless, all of them have been detected at least once in non-treated animals which did not permit us to use them as biomarkers of an illegal treatment. At last, but not at least, all metabolites were found mainly glucuro-conjugated, and rarely sulfo-conjugated, with the only exception of 17beta-boldenone. Current investigations are showing the absence of 17beta-boldenone sulfoconjugate in non-treated animals; that would permit to distinguish non-treated from treated animals with boldione, boldenone and boldenone esters.     

Excretion of norsteroids’ phase II metabolites of different origin in human

The urinary phase II metabolites of norsteroids, 19-norandrosterone, 19-noretiocholanolone and 19-norepiandrosterone glucuronide and sulphate, were analyzed in samples collected during the pregnancy, following the administration of norsteroids or the consumption of edible parts of non-castrated pig and in athletes’ samples in which they were found during routine controls. The level of the sulfo- and glucuroconjugated metabolites was precisely determined by GC/HRMS, after selective hydrolysis. The goal was to evaluate whether the fine analysis of the norsteroid conjugates produced and excreted in different conditions would show a pattern that could be linked to their origin. The delta ¹³C values of the metabolites formed following the ingestion of edible parts of non-castrated pig were measured by isotope ratio mass spectrometry. Our results indicated that it is not possible to determine the origin of the urinary metabolites based upon the sole evaluation of the different metabolites and conjugates. The GC/C/IRMS is the only method permitting to distinguish between the exogenous and endogenous origin of the metabolites.     

Profiling of urinary formestane and confirmation by isotope ratio mass spectrometry

Formestane (F, androst-4-en-4-ol-3,17-dione) is an irreversible aromatase inhibitor with the ability to suppress the estrogen production from anabolic steroids. Consequently, F is mentioned on the World Anti-Doping Agency (WADA) prohibited list and because studies have shown that F is produced endogenously in small amounts, a threshold for urinary excreted F of 150 ng/mL was introduced. Lower concentrations could be due to endogenous production and need further investigation to prove the exact origin through determination of the carbon isotope ratio.However, because the current screening methods are a lot more sensitive, F is detected in practically every urine sample. A strict implementation of this WADA rule would imply that almost every urine sample needs additional investigation to verify an exogenous or endogenous origin. The main aim of this study was to propose and introduce a lower concentration limit of 25 ng/mL beneath which the detected F is considered as being endogenous and no further investigation is needed. The data presented in this paper suggests that this threshold provides a good balance between a sufficiently large detection window and not having to perform isotope ratio mass spectrometry (IRMS) analyses on negative urine samples.     

A new sulphate metabolite as a long-term marker of metandienone misuse

Metandienone is one of the most frequently detected anabolic androgenic steroids in sports drug testing. Metandienone misuse is commonly detected by monitoring different metabolites excreted free or conjugated with glucuronic acid using gas chromatography mass spectrometry (GC–MS) and liquid chromatography tandem mass spectrometry (LC–MS/MS) after hydrolysis with β-glucuronidase and liquid–liquid extraction. It is known that several metabolites are the result of the formation of sulphate conjugates in C17, which are converted to their 17-epimers in urine. Therefore, sulphation is an important phase II metabolic pathway of metandienone that has not been comprehensively studied. The aim of this work was to evaluate the sulphate fraction of metandienone metabolism by LC–MS/MS. Seven sulphate metabolites were detected after the analysis of excretion study samples by applying different neutral loss scan, precursor ion scan and SRM methods. One of the metabolites (M1) was identified and characterised by GC–MS/MS and LC–MS/MS as 18-nor-17β-hydroxymethyl-17α-methylandrost-1,4,13-triene-3-one sulphate. M1 could be detected up to 26 days after the administration of a single dose of metandienone (5 mg), thus improving the period in which the misuse can be reported with respect to the last long-term metandienone metabolite described (18-nor-17β-hydroxymethyl-17α-methylandrost-1,4,13-triene-3-one excreted in the glucuronide fraction).     

Integrated analytical approach in veal calves administered the anabolic androgenic steroids boldenone and boldione: urine and plasma kinetic profile and changes in plasma protein expression

Surveillance of illegal use of steroids hormones in cattle breeding is a key issue to preserve human health. To this purpose, an integrated approach has been developed for the analysis of plasma and urine from calves treated orally with a single dose of a combination of the androgenic steroids boldenone and boldione. A quantitative estimation of steroid hormones was obtained by LC-APCI-Q-MS/MS analysis of plasma and urine samples obtained at various times up to 36 and 24 h after treatment, respectively. These experiments demonstrated that boldione was never found, while boldenone alpha- and beta-epimers were detected in plasma and urine only within 2 and 24 h after drug administration, respectively. Parallel proteomic analysis of plasma samples was obtained by combined 2-DE, MALDI-TOF-MS and muLC-ESI-IT-MS/MS procedures. A specific protein, poorly represented in normal plasma samples collected before treatment, was found upregulated even 36 h after hormone treatment. Extensive mass mapping experiments proved this component as an N-terminal truncated form of apolipoprotein A1 (ApoA1), a protein involved in cholesterol transport. The expression profile of ApoA1 analysed by Western blot analysis confirmed a significant and time dependent increase of this ApoA1 fragment. Then, provided that further experiments performed with a growth-promoting schedule will confirm these preliminary findings, truncated ApoA1 may be proposed as a candidate biomarker for steroid boldenone and possibly other anabolic androgens misuse in cattle veal calves, when no traces of hormones are detectable in plasma or urine.     

Criteria to distinguish between natural situations and illegal use of boldenone, boldenone esters and boldione in cattle: 2. Direct measurement of 17β-boldenone sulpho-conjugate in calf urine by liquid chromatography–high resolution and tandem mass spectrometry

Boldenone is banned in the European Union (Directive 96/22/EC) as growth promoter for meat producing animals. Boldione (ADD), boldenone and boldenone esters (mainly the undecylenate form) are commercially available as anabolic preparations, either to the destination of human, horse or cattle. Since the late 90s, the natural occurrence of boldenone metabolites has been reported in cattle. According to EU regulation, the unambiguous demonstration of boldenone administration in bovine urine should be provided on the basis of boldenone identification in the corresponding conjugate fraction. An analytical method has been developed and validated according to current standards with main concern to the measurement of intact 17β-boldenone-sulphate. The analytical procedure included direct extraction–purification of target analyte on octadecylsilyl cartridges and direct detection of phase II metabolite by liquid chromatography (negative electrospray), tandem mass spectrometry (QqQ) or high resolution mass spectrometry (Orbitrap™). Decision limit (CCα) and detection capability (CCβ) were respectively 0.2 μg L⁻¹ and 0.4 μg L⁻¹ on triple quadrupole and 0.1 μg L⁻¹ and 0.2 μg L⁻¹ on hybrid system. The method was successfully applied to the analysis of incurred samples collected in different experiments. 17β-Boldenone-sulphate was measurable up to 36 h after oral administration of boldione, and 30 days after 17β-boldenone undecylenate intra-muscular injection. This conjugate form was never detected in non-treated animals, confirming its status of definitive candidate marker for boldenone administration in calf.     

Criteria to distinguish between natural situations and illegal use of boldenone,boldenone esters and boldione in cattle: 2. Direct measurement of 17β-boldenone sulpho-conjugate in calf urine by liquid chromatography–high resolution and tandem mass spectrometry

Boldenone is banned in the European Union (Directive 96/22/EC) as growth promoter for meat producing animals. Boldione (ADD), boldenone and boldenone esters (mainly the undecylenate form) are commercially available as anabolic preparations, either to the destination of human, horse or cattle. Since the late 90s, the natural occurrence of boldenone metabolites has been reported in cattle. According to EU regulation, the unambiguous demonstration of boldenone administration in bovine urine should be provided on the basis of boldenone identification in the corresponding conjugate fraction. An analytical method has been developed and validated according to current standards with main concern to the measurement of intact 17β-boldenone-sulphate. The analytical procedure included direct extraction–purification of target analyte on octadecylsilyl cartridges and direct detection of phase II metabolite by liquid chromatography (negative electrospray), tandem mass spectrometry (QqQ) or high resolution mass spectrometry (Orbitrap?). Decision limit (CCα) and detection capability (CCβ) were respectively 0.2 μg L^?1 and 0.4 μg L^?1 on triple quadrupole and 0.1 μg L^?1 and 0.2 μg L^?1 on hybrid system. The method was successfully applied to the analysis of incurred samples collected in different experiments. 17β-Boldenone-sulphate was measurable up to 36 h after oral administration of boldione, and 30 days after 17β-boldenone undecylenate intra-muscular injection. This conjugate form was never detected in non-treated animals, confirming its status of definitive candidate marker for boldenone administration in calf.     

Phenol sulphotransferase: purification and characterization of the rat kidney and stomach enzymes

3'-Phosphoadenosine-5'-phosphosulphate-dependent enzymes that catalyse sulphation of p-nitrophenol have been purified from rat kidney and stomach mucosa by affinity chromatography on the p-hydroxyphenylacetic acid-agarose conjugate, by chromatography on DEAE-cellulose and Sephadex G-100. The phenol sulphotransferase (PST) from rat kidney had Mr of 69 000 and that of the stomach enzyme was 32 000. With p-nitrophenol as the sulphate acceptor, the pH optima were 6.4 for the stomach PST and 5.4 and 6.6 for the kidney enzyme. Both enzymes were inhibited by 2,6-dichloro-4-nitrophenol and phenylglyoxal, an arginine specific modifying reagent. Both enzymes readily sulphated p-nitrophenol, 2-naphthol, 1-naphthol and salicylamide and did not act on biogenic amines (e.g. epinephrine, norepinephrine, dopamine, serotonin), acid metabolites of catecholamines (e.g. 3,4-dihydroxyphenylacetic acid, homovanillic acid), and O-methylated metabolites of catecholamines. Only the stomach enzyme sulphated such catecholamine metabolites as homovanillic alcohol and 3-methoxy-4-hydroxyphenylglycol. In contrast to the brain enzyme, but similarly to the liver enzyme, the kidney and stomach phenol sulphotransferases appear to sulphate exogenous phenolic substrates in preference to potential endogenous substrates.     

Development of a GC/C/IRMS method - Confirmation of a novel steroid profiling approach in doping control

In doping control, an athlete can only be convicted with the misuse with endogenous steroids like testosterone (T), if abnormal values of steroid metabolites and steroid ratios are observed and if the subsequent analysis with isotope ratios mass spectrometry (IRMS) confirms the presence of exogenously administered androgens. In this work, we compare the results of a novel steroid profiling approach with the performance an in-house developed IRMS method. The developed IRMS has the advantage over other methods to be relatively short in time and with target compounds androsterone, etiocholanolone, 5β-androstane 3α,17β-diol and 5α-androstane 3α,17β-diol. Pregnanediol was used as an endogenous reference compound (ERC). Reference limits for the IRMS values were established and applied as decision limits for the evaluation of excretion urine from administration with oral T, T-gel, dihydrotestosterone (DHT) - gel and dehydroepiandrosterone (DHEA). Results indicated the importance of both androstanediols as important IRMS markers where relative values compared to an ERC (Δδ(13)C) yielded better detection accuracy than absolute δ(13)C-values. The detection times of all administered endogenous steroids were evaluated using the proposed thresholds. The results of traditional steroid profiling and a new approach based upon minor steroid metabolites monitoring introduced in a longitudinal framework were evaluated with IRMS. With traditional steroid profiling methods, 95% of the atypical samples could be confirmed whereas an additional 74% of IRMS confirmed was provided by a new biomarkers strategy. These results prove that the other steroid profiling strategies can improve the efficiency in detection of misuse with endogenous steroids.     

GC/MS analysis of the rat urine for metabonomic research

In this paper, an optimized protocol was established and validated for the metabonomic profiling in rat urine using GC/MS. The urine samples were extracted by methanol after treatment with urease to remove excessive urea, then the resulted supernatant was dried, methoximated, trimethylsilylated, and analyzed by GC/MS. Forty-nine endogenous metabolites were separated and identified in GC/MS chromatogram, of which 26 identified compounds were selected for quantitative analysis to evaluate the linearity, precision, and sensitivity of the method. It showed good linearity between mass spectrometry responses and relative concentrations of the 26 endogenous compounds over the range from 0.063 to 1.000 (v/v, urine/urine+water) and satisfactory reproducibility with intra-day and inter-days precision values all below 15%. The metabonomic profiling method based on GC/MS was successfully applied to urine samples from hyperlipidemia model rats. Obviously, separated clustering of model rats and the control rats were shown by principal components analysis (PCA); time-dependent metabonomic modification was detected as well. It was suggested that metabonomic profiling based on GC/MS be a robust method for urine samples.     

Excretion of endogenous boldione in human urine: Influence of phytosterol consumption

Boldenone (17-hydroxy-androsta-1,4-diene-3-one, Bol) and boldione (androst-1,4-diene-3,17-dione, ADD), are currently listed as exogenous anabolic steroids by the World Anti-Doping Agency. However, it has been reported that these analytes can be produced endogenously. Interestingly, only for Bol a comment is included in the list on its potential endogenous origin. In this study, the endogenous origin of ADD in human urine was investigated, and the potential influence of phytosterol consumption was evaluated.We carried out a 5-week in vivo trial with both men (n = 6) and women (n = 6) and measured α-boldenone, β-boldenone, boldione, androstenedione, β-testosterone and α-testosterone in their urine using gas chromatography coupled to multiple mass spectrometry (GC–MS–MS). The results demonstrate that endogenous ADD is sporadically produced at concentrations ranging from 0.751 ng mL⁻¹ to 1.73 ng mL⁻¹, whereas endogenous Bol could not be proven. We also tested the effect of the daily consumption of a commercially available phytosterol-enriched yogurt drink on the presence of these analytes in human urine. Results from this study could not indicate a relation of ADD-excretion with the consumption of phytosterols at the recommended dose. The correlations between ADD and other steroids were consistently stronger for volunteers consuming phytosterols (test) than for those refraining from phytosterol consumption (control). Excretion of AED, bT and aT did not appear to be dependent on the consumption of phytosterols.This preliminary in vivo trial indicates the endogenous origin of boldione or ADD in human urine, independent on the presence of any structural related analytes such as phytosterols.     

Normalization strategies for metabonomic analysis of urine samples

Unlike plasma and most biological fluids which have solute concentrations that are tightly controlled, urine volume can vary widely based upon water consumption and other physiological factors. As a result, the concentrations of endogenous metabolites in urine vary widely and normalizing for these effects is necessary. Normalization approaches that utilized urine volume, osmolality, creatinine concentration, and components that are common to all samples (“total useful MS signal”) were compared in order to determine which strategies could be successfully used to differentiate between dose groups based upon the complete endogenous metabolite profile. Variability observed in LC/MS results obtained from targeted and non-targeted metabonomic analyses was highly dependent on the strategy used for normalization. We therefore recommend the use of two different normalization techniques in order to facilitate detection of statistically significant changes in the endogenous metabolite profile when working with urine samples.     

Gas chromatography–combustion–isotope ratio mass spectrometry analysis of 19-norsteroids: application to the detection of a nandrolone metabolite in urine

Determination of whether the major metabolite of nandrolone in urine, 19-norandrosterone (19-NA), is exogenous or endogenous in origin is one of the most exciting challenges for antidoping laboratories. Gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) can be used to differentiate these two origins by carbon isotopic ratio analysis. A complete method for purification of 19-NA in urine has been established. Acetylated ketosteroids, and in particular 19-NA, are isolated from the urine matrix before analysis after hydrolysis and purification of urine by reversed-phase and normal solid-phase extraction. The limit of detection for 19-NA was about 60 ng with recoveries of 54–60%. Evidence of exogenous administration of 19-NA may be established from isotope ratio determination from the ¹³C/¹²C ratios of several synthetic 19-norsteroids compared to those obtained for endogenous steroids.     

Capillary electrophoresis with UV detection and mass spectrometry in method development for profiling metabolites of steroid hormone metabolism

The aim of this study was to develop a method for comprehensive profiling of metabolites involved in mammalian steroid metabolism. The study was performed using the partial filling micellar electrokinetic chromatography (PF-MEKC) technique for determination of endogenous low-hydrophilic steroids. The detection techniques in capillary electrophoresis were UV absorption and electrospray mass spectrometry (ESI-MS). Thirteen steroids were included in the method development, and the selected were metabolites involved in major pathways of steroid biosynthesis. Although only eight of them could be separated and detected with UV, they could be identified by ESI-MS using selected ion monitoring (SIM) technique. Tandem MS spectra were also collected. UV detection was more sensitive than MS due to better separation of compounds and the selective signal sensitivity. The lowest limits of detection were 10-100ng/mL for cortisone, corticosterone, hydrocortisone and testosterone. The other steroids could be detected at 500-1000ng/mL. The identification of cortisone, corticosterone, hydrocortisone, estrogen and testosterone were made in patient urine samples and their concentrations were 1-40mug/L.     

Drug testing data from the 2007 Pan American Games: δ C values of urinary androsterone, etiocholanolone and androstanediols determined by GC/C/IRMS

The main purpose of this article is to show the application of the CG/C/IRMS in real time during competition in the steroid confirmation analysis. For this reason, this paper summarizes the results obtained from the doping control analysis during the period of the 2007 Pan American Games held in Rio de Janeiro, Brazil. Approximately 5600 athletes from 42 different countries competed in the games. Testing was performed in accordance to World Anti-Doping Agency (WADA) technical note for prohibited substances. This paper reports data where abnormal urinary steroid profiles, have been found with the screening procedures. One 8 mL urine sample was used for the analysis of five steroid metabolites with two separate analyses by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Urine samples were submitted to GC/C/IRMS for confirmation analysis to determine the ¹³C/¹²C ratio of selected steroids. Fifty-seven urine samples were analyzed by GC/C/IRMS and the δ ¹³C values (‰) of androsterone, etiocholanolone, 5β-androstane-3α, 17β-diol (5β-diol), 5α-androstane-3α, 17β-diol (5α-diol) and 5β-pregnane-3α, 20α-diol (5β-pdiol), the endogenous reference compound are presented. One urine sample with a testosterone/epitestosterone (T/E) ratio of 4.7 was confirmed to be positive of doping by GC/C/IRMS analysis. The δ values of 5β-diol and 5α-diol were 3.8 and 10.8, respectively, compared to the endogenous reference compound 5β-pdiol, which exceeded the WADA limit of 3‰. The results obtained by CG/C/IRMS confirmation analyses, in suspicious samples, were conclusive in deciding whether or not a doping steroid violation had occurred.     

The metabonomics of aging and development in the rat: an investigation into the effect of age on the profile of endogenous metabolites in the urine of male rats using 1H NMR and HPLC-TOF MS

The effect of aging and development in male Wistar-derived rats on the profile of endogenous metabolites excreted in the urine was investigated using both (1)H NMR spectroscopy and HPLC-TOF MS using electrospray ionisation (ESI). The endogenous metabolites were profiled in samples collected from male rats every two weeks from just after weaning at 4 weeks up to 20 weeks of age. Multivariate data analysis enabled clusters to be visualised within the data according to age, with urine collected at 4 and 6 weeks showing the greatest differences by both analytical techniques. Markers detected by (1)H NMR spectroscopy included creatinine, taurine, hippurate and resonances associated with amino acids/fatty acids, which increased with age, whilst citrate and resonances resulting from glucose/myoinositol declined. A number of ions were detected by HPLC-MS that were only present in urine samples at 4 weeks of age in both positive and negative ESI, with a range of ions, including e.g. carnitine, increasing with age. Age predictions by PLS-regression modelling demonstrated an age-related trend within these data, between 4 and 12 weeks for HPLC-MS and 4-16 weeks for NMR. The possible utility of these techniques for metabonomic investigations of age-related changes in the rat is discussed and the importance of employing suitable control animals in pharmacological and toxicological studies is highlighted.     

Ex vivo spontaneous generation of 19-norandrostenedione and nandrolone detected in equine plasma and urine

19-Norandrostenedione (NAED) and nandrolone are anabolic-androgenic steroids (AASs). Nandrolone was regarded solely as a synthetic AAS until the 1980s when trace concentrations of apparently endogenous nandrolone were detected in urine samples obtained from intact male horses (stallions). Since then, its endogenous origin has been reported in boars and bulls; endogenous NAED and nandrolone have been identified in plasma and urine samples collected from stallions. More recently, however, it was suggested that NAED and nandrolone detected in urine samples from stallions are primarily artifacts due to the analytical procedure. The present study was undertaken to determine whether NAED and nandrolone detected in plasma and urine samples collected from stallions are truly endogenous or artifacts from sample processing. To answer this question, fresh plasma and urine samples from ≥8 stallions were analyzed for the two AASs, soon after collection, by liquid chromatography hyphenated to tandem mass spectrometry (LC-MS/MS). NAED and nandrolone were not detected in fresh plasma samples but detected in the same samples post storage. Concentrations of both AASs increased with storage time, and the increases were greater at a higher storage temperature (37°C versus 4°C, and ambient temperature versus 4°C). Although NAED was detected in some fresh stallion urine samples, its concentration (<407 pg/mL) was far lower (<0.4%) than that in the same samples post storage (at ambient temperature for 15 days). Nandrolone was not detected in most of fresh urine samples but detected in the same samples post storage. Based on these results, it is concluded that all NAED and nandrolone detected in stored plasma samples of stallions and most of them in the stored urine samples are not from endogenous origins but spontaneously generated during sample storage, most likely from spontaneous decarboxylation of androstenedione-19-oic acid and testosterone-19-oic acid. To our knowledge, it is the first time that all NAED and nandrolone detected in plasma of stallions and most of them detected in the urine have been shown to be spontaneously generated in vitro during sample storage. This finding would have significant implications with regard to the regulation of the two steroids in horse racing.     

Host plant-dependent metabolism of 4-hydroxybenzylglucosinolate in Pieris rapae: substrate specificity and effects of genetic modification and plant nitrile hydratase

After ingestion of transgenic Arabidopsis thaliana CYP79A1 containing sinalbin (4-hydroxybenzylglucosinolate) due to genetic modification, only one major sinalbin-derived sulphate ester (the sulphate ester of 4-hydroxyphenylacetonitrile) was excreted by Pieris rapae caterpillars (corresponding to 69mol% of ingested sinalbin). An additional sulphate ester (the sulphate ester of 4-hydroxyphenylacetamide) was excreted when the caterpillars were reared on two plant species (Sinapis alba and Sinapis arvensis) that contained sinalbin naturally. Artificial addition of sinalbin to S. arvensis leaves resulted in increased levels of the sulphated amide, and an enzymatic activity (nitrile hydratase) explaining the formation of the sulphated amide from sinalbin was detected in both Sinapis species, but not in A. thaliana. In agreement with the suggested minor metabolic pathway, the caterpillars were able to sulphate 4-hydroxyphenylacetamide offered as part of an artificial diet. In fact, phenol and seven para-substituted phenol derivatives with substituents of moderate size were sulphated and excreted, but all tested phenols devoid of a nitrile functional group were less efficiently sulphated than the primary sinalbin detoxification product, 4-hydroxyphenylacetonitrile. This suggests that the specificity of the sulphation step involved in sinalbin metabolism may be adapted to nitriles formed as metabolites of phenolic glucosinolates. On the contrary, there was no specificity for products (4-hydroxybenzylascorbigen and 4-hydroxybenzylalcohol) derived from the semistable isothiocyanate produced from sinalbin in the absence of nitrile specifier protein.