Determination of naturally occurring oestrogens and androgens in retail samples of milk and eggs.

The occurrence of the main steroid hormones (oestrone, 17alpha-oestradiol, 17beta-oestradiol, 17alpha-testosterone, 17beta-testosterone, dehydroepiandrosterone, 4-androstenedione), especially in milk and eggs, was investigated. An analytical method based on GC-MS/MS was developed for steroid measurement at an ultra-trace level in food products. The limits of detection for oestrogens were about 5 and 30 ng kg(-1) in milk and eggs, respectively. For androgens, the limits of detection were around 10 and 50 ng kg(-1) in milk and eggs, respectively. The method was applied to milk and egg samples collected in a French supermarket. In milk, oestrone was found at levels between 100 and 300 ng l(-1), while 17beta-oestradiol levels were estimated to be near 20 ng l(-1). 17alpha-testosterone was found to be from 50 ng l(-1) in skimmed milk to 85 ng l(-1) in whole milk. In egg samples, oestrone and 17beta-oestradiol were found at 1.5 and 0.9 microg kg(-1), respectively, while 17alpha-oestradiol was found to be in lower concentrations (i.e. around 0.55 microg kg(-1)). Regarding androgens, 17alpha- and 17beta-testosterone were estimated at 1.9 and 1.3 microg kg(-1), respectively. These results represent a first attempt to estimate the food exposure to steroid hormones. In the future, the collection of additional data should permit the comparison between this exogenous dietary intake and the daily endogenous production in pre-pubertal children as a basis of risk assessment regarding endocrine disruption linked to these molecules for this critical population.

Evidence that urinary excretion of thiouracil in adult bovine submitted to a cruciferous diet can give erroneous indications of the possible illegal use of thyrostats in meat production.

Thyrostats have been banned for use as veterinary drugs in Europe since 1981 because of their carcinogenic and teratogenic properties. Until now, the identification of thiouracil in animal biological matrices has been interpreted as the consequence of an illegal administration. The present paper studies the influence of a cruciferous-based feed on the occurrence of thiouracil as a residue in urine. Urine samples collected from two heifers fed on cabbage or rapeseed cakes were analysed for the presence of thiouracil by 3-iodobenzylbromide derivatization and liquid chromatography-electrospray ionization tandem mass spectroscopy (LC-ESI(-)-MS/MS) analysis. Urine collected after cabbage or rapeseed feeding showed thiouracil concentrations in the range 3-7 and 2-9 microg l-1, respectively, demonstrating a relationship between a diet based on cruciferous vegetables and the occurrence of thiouracil in urine. Thiouracil was excreted in urine in the hours following cruciferous intake. Complete elimination (<0.8 microg l-1) of the compound occurred within 5 days. The precursors in cruciferous vegetables responsible for the thiouracil excretion in urine were proved not to be thiouracil itself.

Assessment of estradiol and its metabolites in meat.

Most studies related to research on steroids in main edible tissues (muscle, liver or kidney) have focused on measurement of parent or major metabolite residues. In order to evaluate the estradiol content in bovine edible tissues, a multi-step extraction procedure was developed in conjunction with parallel metabolism studies of [14C]-17beta-estradiol in cattle (1-2). Various classes of free estradiol and conjugates were separated: estradiol -17beta and -17alpha, estradiol-17-fatty acid esters, estradiol 17-glycoside, estradiol 3-glucuronide, estradiol-17-glycoside and 3- glucuronide (diconjugates) were separated. No sulphates conjugated forms have been found at the detection level of the method. The quantification was realized by calibration with deuterated 17beta -estradiol -d3 standard and was validated at the ng x kg(-1) (ppt) level. Muscle, liver, kidney and fat samples from control or Revalor S single (licensed implantation) or multi-implanted steers have been assayed. The results show a wide variation between animals, but both the highest value and the mean of total estradiol content in each group proportionally increase from untreated to multi-implanted animals. In accordance with international rules, a calculation of the daily food supply of estradiol by such edible tissues in comparison with the acceptable daily intake was performed.

Biosynthesis of 6beta-hydroxymethyltestosterone using bovine hepatocyte cultures.

Usually performed to investigate biotransformations of xenobiotics, in vitro liver models could become useful tools for the synthesis of not commercially available compounds. In this study, bovine hepatocyte cultures were used to biosynthesise, on the laboratory scale, one major metabolite of methyltestosterone: 6beta-hydroxymethyltestosterone. After incubation of bovine hepatocytes with methyltestosterone for 24 h, culture medium was removed and stored at -20 degrees C until analysis. The sample was extracted and purified on a reversed-phase HPLC system. The metabolite of interest was then analysed in LC-MS and GC-MS for structural identification. The purity and the isomery of the 6 and 17 positions were confirmed by NMR analyses. This first success in producing purified 6beta-hydroxymethyltestosterone from bovine hepatocyte cultures allowed us to consider that in vitro liver models could be reliable tools for standard biosynthesis.

Preliminary assays to elucidate the structure of oxytetracycline's degradation products in sediments. Determination of natural tetracyclines by high-performance liquid chromatography-fast atom bombardment mass spectrometry.

A very specific high-performance liquid chromatography-mass spectrometric method for the determination of natural tetracyclines was developed in order to characterise the degradation products of oxytetracycline in sediments. First, extraction used a clean up step with a Bond Elut Certify LRC cartridge. A 3 microm Spherisorb ODS1 column was then used with a methanol, acetonitrile and oxalic acid mobile phase gradient. Chromatographic resolution in these conditions was 3.31 between oxytetracycline and tetracycline. Two liquid chromatography-mass spectrometry methodologies based on a particle beam and a frit fast atom bombardment interface were developed. In the first approach, ionisation was performed in the negative chemical mode using methane as reacting gas. In the other case, glycerol-thioglycerol mixture was used as matrix to ensure good sensitivity. MS-MS experiment was performed to determinate oxytetracycline fragmentation pattern in the perspective of degradation product study.

N-methyl-N-alkylsilyltrifluoroacetamide-I2 as a new derivatization reagent for anabolic steroid control.

Analytical methods for the control of growth promoters have to be specific and sensitive. At low concentration levels, it is difficult to identify some molecules unambiguously even with the improved performance of analytical methods. GC-MS analysis of 17 beta-trenbolone and its major metabolite, 17 alpha-trenbolone, is a good example. A new derivatization agent has been developed which is based on silylation of the 3- and 17-oxygenated functions and nucleophilic substitution in the 4-position. The structure of the derivatized products was demonstrated using a simple model, cyclohex-2-en-1-one, by NMR and MS spectrometry. In contrast to data found in the literature, this derivative permitted specific mass spectra for trenbolone, sensitive signals for high mass ions and reproducible gas chromatograms to be obtained. The addition of an N(CH3)COCF3 radical to the steroid nucles allowed highly specific detection in GC-high resolution MS even following extraction from complex matrices; sensitive responses were also observed in the negative chemical ionization mode. Moreover, there are significant differences in the electron ionization mass spectra of the two stereoisomers, 17 alpha- and 17 beta-trenbolone. These preliminary results and those obtained for androsta-1,4-dien-3-one and pregna-4,6-dien-3-one indicate useful advances for the determination of steroids and potential applications for metabolism studies on such compounds.

Gas chromatographic-mass spectrometric identification of main metabolites of stanozolol in cattle after oral and subcutaneous administration.

An analytical method has been developed in order to control the illegal use of stanozolol as growth promoter in livestock. The procedure was based on enzymatic hydrolysis, purification on a Clean Screen DAU column and derivatization with heptafluorobutyric anhydride prior to GC-MS analysis. This method allowed us to study the metabolism of stanozolol in cattle after oral and subcutaneous administrations. Urinary metabolites were identified by mass spectrometry. Stanozolol and 16-hydroxystanozolol were detected after oral administration, while 16-hydroxystanozolol and 4,16-dihydroxystanozolol were found after subcutaneous administration.

A novel glycosphingolipid expressed in pig kidney: Gal alpha 1-3Lewis(x) hexaglycosylceramide.

Immunodetection of thin layer chromatograms of neutral glycosphingolipids of pig kidney cortex with a polyclonal antibody directed against the Gal alpha 1-3Gal determinant revealed several glycosphingolipids reacting with different intensities. A minor glycosphingolipid was isolated by preparative high performance thin layer chromatography. It was characterized as a type 2 hexaglycosylceramide with the following structure Gal alpha 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc beta 1-Cer by fast atom bombardment- and desorption-chemical ionization-mass spectrometry, methylation analysis and hydrolysis with alpha-galactosidase followed by immunostaining with an anti-Lewis(x) monoclonal antibody. The proton NMR spectrum was found compatible with the proposed structure. Two other glycosphingolipids carrying the new determinant were partially characterized as an octa- and a branched-dodecaglycosylceramide. The expression of the Gal alpha 1-3 Lewis(x) determinant appeared to be developmentally regulated as it increased with age. The characterization of Gal alpha 1-3Le(x) in pig kidney indicates a new epitope capable of recognition by human natural antibodies in the context of xenotransplantation of pig organs to man. It also adds new members to the family of Le(x)-based glycolipids.

Developments in residue assay and metabolism study of growth-promoters by mass spectrometric analysis.

The French national reference laboratory, for the control of growth-promoter residues, has developed routine methods of multiresidue analysis. These are mainly for the determination of beta-agonistic and steroid compounds. Apart from this regulatory activity, some research into growth-premotor metabolism and assay has also been carried out. This research concerns the method development of assay and metabolism studies of new compounds, including in vitro metabolism studies on cell (mainly hepatocytes) cultures, as well as studying the organic synthesis of new compounds and metabolites. Three examples of the different areas of research are presented here: the metabolism study of 4-chlorotestosterone in cattle; a methodological study on beta-agonists assay by mass spectrometry; and a residue study of beta-agonists in edible tissues.