K. Verheyden

Novel analytical methods for the determination of steroid hormones in edible matrices

This paper reviews recently published multi-residue chromatographic methods for the determination of steroid hormones in edible matrices. After a brief introduction on steroid hormones and their use in animal fattening, the most relevant EU legislation regarding the residue control of these substances is presented. An overview of multi-residue analytical methods, covering sample extraction and purification as well as chromatographic separation and different detection methods, being in use for the determination of steroid hormones (estrogens, gestagens and androgens), is provided to illustrate common trends and method variability. Emphasis was laid on edible matrices and more specifically on meat, liver, kidney, fat and milk. Additionally, the possibilities of novel analytical approaches are discussed. The review also covers specific attention on the determination of natural steroids. Finally, the analytical possibilities for phytosterols, naturally occurring steroid analogues of vegetable origin and a specific group of steroid hormones with a hemi-endogenous status are highlighted.

Residue analysis: future trends from a historical perspective.

A residue is a trace (microg kg(-1), ng kg(-1)) of a substance, present in a matrix. Residue analysis is a relatively young discipline and a very broad area, including banned (A) substances as well as registered veterinary medicinal products (B substances). The objective of this manuscript is to review future trends in the analysis of residues of veterinary drugs in meat producing animals out of historical perspectives. The analysis of residues in meat producing animals has known a tremendous evolution during the past 35-40 years. In the future, it can be foreseen that this evolution will proceed in the direction of the use of more and more sophisticated and expensive machines. These apparatus, and the necessary human resources for their use, will only be affordable for laboratories with sufficient financial resources and having guarantee for a sufficient throughput of samples.

Development and validation of an ultra-high performance liquid chromatography tandem mass spectrometry method for quantifying thyreostats in urine without derivatisation

Thyreostatic drugs, illegally administrated to livestock for fattening purposes, are banned in the European Union since 1981 (Council Directive 81/602/EC). For monitoring their illegal use, sensitive and specific analytical methods are required. In this study an UHPLC-MS/MS method was described for quantitative analysis of eight thyreostatic drugs in urine, this without a derivatisation step. The sample pretreatment involved a reduction step with dithiothreitol under denaturating conditions at 65 degrees C, followed by liquid-liquid extraction with ethyl acetate. This analytical procedure was subsequently validated according to the EU criteria (2002/657/EC Decision), resulting in decision limits and detection capabilities ranging between 1.1 and 5.5 microg L(-1) and 1.7 and 7.5 microg L(-1), respectively. The method obtained for all, xenobiotic thyreostats, a precision (relative standard deviation) lower than 15.5%, and the linearity ranged between 0.982 and 0.999. The performance characteristics fulfill not only the requirements of the EU regarding the provisional minimum required performance limit (100 microg L(-1)), but also the recommended concentration fixed at 10 microg L(-1) in urine set by the Community of Reference Laboratories. Future experiments applying this method should provide the answer to the alleged endogenous status of thiouracil.

Absence of an effect of dietary fibre or clinoptilolite on boar taint in entire male pigs fed practical diets

This study aimed to evaluate the possibility of reducing boar taint in boars (Piétrain×Hybrid) by addition of different feed ingredients (raw potato starch (RPS) 10%, raw potato starch 10%+wheat bran 5% (RPS+WB), lupins 10%, inulin 5%, clinoptilolite 1%) to a standard diet over a period of 4-6 weeks before slaughter. Control boars (CBOAR) as well as barrows were fed the standard diet. Efficacy of the different feed ingredients was evaluated by different boar taint detection methods: hot iron method, consumer panel, expert panel and laboratory analysis. According to all detection methods, clear differences were noticeable between boars and barrows. No differences in boar taint incidence were found between the boars on the different dietary treatments as assessed by consumers, experts, hot iron method or the concentration of skatole in fat. A significant effect on indole level was found, but no further differentiation could be made. The concentration of backfat androstenone was significantly higher for the inulin and control boar group compared to the lupin group. In conclusion, none of the feeding strategies tested in this study reduced boar taint in boars at the given percentages.

Analysis of thyreostats: A history of 35 years

Thyreostatic drugs (TS), illegally administrated to livestock for fattening purposes, are banned in the European Union since 1981 (Council Directive 81/602/EC). This paper reviews the trends in the analytical approaches for the determination of TS drugs in biological matrices. After a brief introduction on the different groups of compounds with a thyreostatic action, the most relevant legislation regarding the residue control of these compounds is presented. An overview of the analytical possibilities for the determination of TS in animal matrices, covering sample extraction, purification, separation techniques and detection methods is provided. Additionally, a brief outline of animal experiments is described that illustrates the excretion and distribution profiles of TS residues. Finally, the novel developments in TS analysis are highlighted. Also the possible semi-endogenous status of thiouracil is discussed.

Endogenous boldenone-formation in cattle: Alternative invertebrate organisms to elucidate the enzymatic pathway and the potential role of edible fungi on cattle's feed

Although beta-boldenone (bBol) used to be a marker of illegal steroid administration in calves, its endogenous formation has recently been demonstrated in these vertebrates. However, research on the pathway leading to bBol remains scarce. This study shows the usefulness of in vivo invertebrate models as alternatives to vertebrate animal experiments, using Neomysis integer and Lucilia sericata. In accordance with vertebrates, androstenedione (AED) was the main metabolite of beta-testosterone (bT) produced by these invertebrates, and bBol was also frequently detected. Moreover, in vitro experiments using feed-borne fungi and microsomes were useful to perform the pathway from bT to bBol. Even the conversion of phytosterols into steroids was shown in vitro. Both in vivo and in vitro, the conversion of bT into bBol could be demonstrated in this study. Metabolism of phytosterols by feed-borne fungi may be of particular importance to explain the endogenous bBol-formation by cattle. To the best of our knowledge, it is the first time the latter pathway is described in literature.

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 alpha-boldenone, beta-boldenone, boldione, androstenedione, beta-testosterone and alpha-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.

Detection of macrocyclic lactones in porcine liver, meat and fish tissue using LC–APCI–MS–MS

A selective and sensitive method for the simultaneous determination of five avermectins (abamectin, ivermectin, doramectin, emamectin and eprinomectin) and one milbemycin (moxidectin) in porcine liver, bovine meat and fish tissue was developed. The method involved extraction with acetonitrile and purification by C18 solid-phase extraction. Detection was carried out using liquid chromatography coupled to multiple mass spectrometry (LC–MS2) equipped with APCI in the negative mode. This method was validated according to the requirements of Commission Decision EC/2002/657 (Implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. Off J Eur Commun. L221: 8–36.). In addition to the linear response (R 2 between 59 and 97%), good repeatability (CV between 20 and 35%), reproducibility (CV between 20 and 35%) and detection (CCα) and quantification (CCβ) limits were obtained for all compounds in all matrices considered.

Determination of bixin and norbixin in meat using liquid chromatography and photodiode array detection

The development of an analytical method that enables routine analysis of annatto dye, specifically bixin and norbixin, in meat tissue is described. Liquid-solid extraction was carried out using acetonitrile. Analysis was by HPLC with photodiode array detection using two fixed wavelengths (458 and 486 nm). The possibilities of ion trap mass spectrometry (MS) were also assessed. Method performance characteristics, according to Commission Decision 2002/657/EC, were determined, with recoveries between 99 and 102% and calibration curves being linear in the 0.5–10 mg kg−1 range. The limit of quantification was 0.5 mg kg−1.

Predicting the likelihood of developing boar taint: Early physical indicators in entire male pigs

Three potential early-age predictors of which boars are likely to develop boar taint (testes volume, skin lesions and dirtiness) were measured on 102 boars every fortnight from 10 weeks of age until slaughter. These predictors were correlated with the level of boar taint according to the hot iron method and the concentrations of skatole and androstenone as determined by chemical analysis. The chance of no/low boar taint according to the hot iron method decreased with higher testes volume (weeks 22 and 24) and increased with skin lesion score (weeks 12, 16 and 18). For the concentrations of androstenone and skatole, the strongest correlation was found with testes volume in week 12. Skin lesions in week 16 were negatively correlated with skatole levels. Dirtiness was negatively correlated with skatole concentrations (week 18) but positively correlated with androstenone concentrations (weeks 20 and 22). Testes volume has the greatest potential for predicting the likelihood of developing boar taint.