H. Noppe

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.

Biological removal of 17α-ethinylestradiol by a nitrifier enrichment culture in a membrane bioreactor.

Increasing concern about the fate of 17alpha-ethinylestradiol (EE2) in the environment stimulates the search for alternative methods for wastewater treatment plant (WWTP) effluent polishing. The aim of this study was to establish an innovative and effective biological removal technique for EE2 by means of a nitrifier enrichment culture (NEC) applied in a membrane bioreactor (MBR). In batch incubation tests, the microbial consortium was able to remove EE2 from both a synthetic minimal medium and WWTP effluent. A maximum EE2 removal rate of 9.0 microg EE2 g(-1)biomass-VSS h(-1) was achieved (>94% removal efficiency). Incubation of the heterotrophic bacteria isolated from the NEC did not result in a significant EE2 removal, indicating the importance of nitrification as driving force in the mechanism. Application of the NEC in a MBR to treat a synthetic influent with an EE2 concentration of 83 ng EE2 L(-1) resulted in a removal efficiency of 99% (loading rates up to 208 ng EE2 L(-1)d(-1); membrane flux rate: 6.9 L m(-2) h(-1)). Simultaneously, complete nitrification was achieved at an optimal ammonium influent concentration of 1.0 mg NH(4)(+)-N L(-1). This minimal NH(4)(+)-N input is very advantageous for effluent polishing since the concomitant effluent nitrate concentrations will be low as well and it offers opportunities for the nitrifying MBR as a promising add-on technology for WWTP effluent polishing.

Influence of manganese and ammonium oxidation on the removal of 17α-ethinylestradiol (EE2)

Flow-through reactors with manganese oxides were examined for their capacity to remove 17 alpha-ethinylestradiol (EE2) at microg L(-1) and ng L(-1) range from synthetic wastewater treatment plant (WWTP) effluent. The mineral MnO(2) reactors removed 93% at a volumetric loading rate (B(V)) of 5 microg EE2 L(-1) d(-1) and from a B(V) of 40 microg EE2 L(-1) d(-1) on, these reactors showed 75% EE2 removal. With the biologically produced manganese oxides, only 57% EE2 was removed at 40 microg EE2 L(-1) d(-1). EE2 removal in the ng L(-1) range was 84%. The ammonium present in the influent (10 mg N L(-1)) was nitrified and ammonia-oxidizing bacteria (AOB) were found to be of prime importance for the degradation of EE2. Remarkably, EE2 removal by AOB continued for a period of 4 months after depleting NH(4)(+) in the influent. EE2 removal by manganese-oxidizing bacteria was inhibited by NH(4)(+). These results indicate that the metabolic properties of nitrifiers can be employed to polish water containing EE2 based estrogenic activity.

Microbiological detection of 10 quinolone antibiotic residues and its application to artificially contaminated poultry samples

To assess if microbiological inhibition tests for detection of antibiotic residues are suitable for routine screening for quinolone residues, the limit of detection (LOD) of 10 different quinolones and fluoroquinolones was determined. Two media were tested, one at pH 6 and the other at pH 8, each seeded with one of the following test strains: Bacillus subtilis, Escherichia coli or Bacillus cereus. LODs of the 10 substances were highest on plates seeded with B. cereus, intended for selective detection of tetracycline residues. The pattern of zones on the other four plates differed for the targeted quinolones: flumequine and oxolinic acid were detected at lower concentrations at pH 6, while the LODs of ciprofloxacin, enrofloxacin, danofloxacin, marbofloxacin, sarafloxacin and norfloxacin were lower at pH 8. Nine of the 10 quinolones were detected more easily with E. coli, but the LOD of difloxacin was lower with B. subtilis. Finally, the three most sensitive media were selected and fluid from chicken meat, spiked with eight quinolones near maximum residue limits (MRL), analysed on each plate. The plate seeded with E. coli at pH 8 detected five of eight quinolones at levels of interest, but an additional E. coli plate at pH 6 was necessary for detection of flumequine in species other than poultry and fish. None of the plates detected oxolinic acid and difloxacin at MRLs in muscle tissue.

Endogenous occurrence of some anabolic steroids in swine matrices.

Following findings of 17β-19-nortestosterone (150–200 µg kg−1) in pigs of unspecified gender imported into the European Union, a study to determine steroid and hormone levels in swine from six age/gender categories (uncastrated ‘old’ boars, cryptorchids, one intersex, barrows, gilts and sows) was initiated. Indeed, for some hormones there has been a discussion about their being endo- or exogenous. Tissue and urine samples from swine from each of the six categories were obtained in Belgium, France, the Netherlands and the USA. Samples were analysed in three laboratories. Quantitation was obtained for norandrostenedione, 19-nortestosterone and boldenone. The results give a well-documented overview of the status of the presence of these hormones in swine. The data illustrate that uncastrated ‘old’ boars produce the highest percentage of ‘positive’ matrices, followed by the cryptorchids. Concentrations in the matrices of the barrows and the gilts are lower. Also, sow matrices contain low amounts of nor-steroids. Furthermore, urine samples from an intersex pig contains a higher concentration of nortestosterone than sows and can therefore be suspected for illegal use of these hormones. Veterinarians taking samples in pig farms for the analysis of hormones need to be aware of the presence and concentrations of these substances in the different categories.

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.

Androstadienetrione, a boldenone-like component, detected in cattle faeces with GC-MSn and LC-MSn

Boldenone (1,4-androstadiene-17-ol-3-one, Bol) has been the subject of a heated debate because of ongoing confusion about its endogenous or exogenous origin when detected in one of its forms in faecal or urine samples from cattle. An expert report was recently written on the presence and metabolism of Bol in various animal species. Androstadienedione (ADD) is a direct precursor of 17β-boldenone (βBol). It is a 3,17-dione; ßBol is a 17-ol-3-one. Not much is published on 1,4-androstadiene-3,17-diol, which is a 3,17-diol (ADL). If animals were exposed for a longer period to one of these analytes, a metabolic pathway would be initiated to eliminate these compounds. Similar to recent testosterone metabolism studies in the aquatic invertebrate Neomysis integer, ADD, ßBol and ADL could also be eliminated as hydroxymetabolites after exposure. The presence of 11-keto-steroids or 11-hydroxy-metabolites in faecal samples can interfere with a confirmation method by gas chromatography-negative chemical ionization mass spectrometry (GC-NCI-MS), after oxidation of corticosteroids with a double bond in the A-ring (e.g. prednisolone or its metabolite prednisone). The presence of androstadienetrione (ADT) in faecal samples of cattle has never been reported. The origin of its presence can be explained through different pathways, which are presented in this paper.

Biological and Chemical Approaches for the Detection and Identification of Illegal Estrogens in Water-based Solutions

The continuous introduction of new products used as growth promoters in animal husbandry, for sports doping and as products for body-building requires residue laboratories to initiate research on developing a strategy for the identification of ’unknown’ components. In this study, a strategy is presented for elucidating the identity, the structure and the possible effects of illegal estrogenic compounds in an unidentified water-based solution. To obtain complete information on the composition and activity of the unidentified product, a multidisciplinary approach was needed. A case-study is described with a ’solution X’ found during a raid. First, in vivo techniques (animal trials with mice, anatomical and histological research) were combined with in vitro techniques (the yeast estrogenic screen (YES)). In a later stage of the investigation, HPLC-fractionation, liquid chromatography–multiple mass spectrometry (LC-MS n and gas chromatography-multiple mass spectrometry (GC-MS n were used. Finally, the identity of ’solution X’ was confirmed in a very low concentration range (10 ng/L estrone and 400 ng/l ethinyloestradiol).