Daniel Maume

Exposure assessment of French women and their newborn to brominated flame retardants : Determination of tri-to deca-polybromodiphenylethers (PBDE) in maternal adipose tissue, serum, breast milk and cord serum

In the frame of a French monitoring program, tri- to deca- polybromodiphenylethers (PBDE) have been measured in maternal and cord serum, adipose tissue, and breast milk samples, collected from 93 volunteer women during caesarean deliveries. The seven major tri- to heptaBDE (BDE-28, 47, 99, 100, 153, 154, and 183) were detected in adipose tissue and breast milk with cumulated median values of 2.59 and 2.51 ng g(-1) l w. Nine highly brominated octa- to decaBDE (BDE-196, 197, 201, 202, 203, 206, 207, 208 and 209) was performed in the same samples, with cumulated median values of 2.73 and 3.39 ng g(-1) l w in adipose tissue and breast milk, respectively. At this opposite, median levels of octa- to decaBDE in maternal and cord serum appeared significantly higher than the levels of tri- to heptaBDE in the same matrices, i.e. 8.85 and 12.34 versus 0.98 and 0.69 ng g(-1) l w, respectively.

Exposure assessment of French women and their newborns to tetrabromobisphenol-A: Occurrence measurements in maternal adipose tissue, serum, breast milk and cord serum

A French monitoring study was initiated to evaluate the exposure of fetus and newborn to brominated flame retardants (BFR). A previously developed multi-residue analytical method was used for measuring the main classes of BFR (tetrabromobisphenol-A, and tri- to decabomodiphenyl ethers) in various human biological matrices. Analyzed samples (maternal and cord serum, adipose tissue and breast milk) were collected from 93 volunteer women during caesarean deliveries. TBBPA was detected in 44% of the analyzed breast milk samples, at levels varying from 0.06 to 37.34 ng g(-1) lipid weight, but was not detected in adipose tissue. This compound was also detected in 30% of the analyzed serum samples, with similar average values in maternal and cord serum (154 pg g(-1) fresh weight versus 199 pg g(-1) fresh weight, respectively). The interpretation of the collected data permitted the demonstration of (1) a significant exposure to TBBPA both for mothers and fetuses and (2) a possible risk of overexposure of newborns through breastfeeding.

Exposure assessment of fetus and newborn to brominated flame retardants in France: preliminary data

Brominated flame retardants (BFR) are chemicals extensively used in many manufactured products to reduce the risk of fire, but also environmental pollutants. In order to assess the potential risk linked to these compounds in human, a French monitoring study was initiated to evaluate the exposure of fetus and newborn. A previously described multi-residue analytical method was used, for measuring the main classes of BFR (hexabromocyclododecane, tetrabromobisphenol-A, and tri- to deca-polybromodiphenylethers) in various biological matrices. These analyzed samples (maternal and umbilical serum, adipose tissue and breast milk) were collected on volunteer women during caesarean deliveries. Preliminary results obtained on 26 individuals (mother/newborn pairs) mainly demonstrated the presence of polybromodiphenylethers (PBDE) and tetrabromobisphenol A both in maternal and fetal matrices, and a possible risk of overexposure of newborns through breastfeeding. Contaminations levels were found globally in the ng/g lipid weight range, consistent with other published European data. Exposure results regarding highly brominated PBDE congeners (octa- to deca-BDE) appeared particularly informative and non-commonly reported, these compounds accounting for around 50% of the total PBDE load. Additional data collection and metabolism investigations are now on-going. A more complete statistical analysis related to this BFR exposition study will be provided in a next future.

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

The occurrence of the main steroid hormones (oestrone, 17α-oestradiol, 17β-oestradiol, 17α-testosterone, 17β-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 17β-oestradiol levels were estimated to be near 20 ng l−1. 17α-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 17β-oestradiol were found at 1.5 and 0.9 µg kg−1, respectively, while 17α-oestradiol was found to be in lower concentrations (i.e. around 0.55 µg kg−1). Regarding androgens, 17α- and 17β-testosterone were estimated at 1.9 and 1.3 µg 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.

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.

A novel glycosphingolipid expressed in pig kidney: Galα1-3Lewisx hexaglycosylceramide

Immunodetection of thin layer chromatograms of neutral glycosphingolipids of pig kidney cortex with a polyclonal antibody directed against the Galα1-3Gal 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 Galα1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glcβ1-Cer by fast atom bombardment- and desorption-chemical ionization-mass spectrometry, methylation analysis and hydrolysis with α-galactosidase followed by immunostaining with an anti-Lewisx 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 Galα1-3Lewisx determinant appeared to be developmentally regulated as it increased with age. The characterization of Galα1-3Lex 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 Lex-based glycolipids. Abbreviations: HPTLC, high performance thin layer chromatography; FAB-MS, fast atom bombardment mass spectrometry; DCI, desorption-chemical ionization; Me2SO-d6, hexadeuterated dimethyl sulfoxide

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]‐17β‐estradiol in cattle (1–2). Various classes of free estradiol and conjugates were separated: estradiol −17β and −17α, 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 17β −estradiol −d3 standard and was validated at the ng · 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.

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 µg 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 µg 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.

Detection and identification of thyreostats in the thyroid gland by gas chromatography-mass spectrometry

Abstract Because thyreostatic compounds, also named thyreostats, are banned in Europe (directive 86/469/EEC), methods have to be developed to prevent the illegal use of these substances. The analytical procedure described herein involves the detection and identification at the low ng g−1 level of the main thyreostats known to be used for growth promotion by gas chromatography coupled to mass spectrometry (GC-MS). The assay is based on a liquid/liquid extraction of the thyroid gland, derivatization with pentafluorobenzyl bromide (PFBBr), purification on a silica solid phase extraction column and finally a trimethylsilylation prior to GC-MS. Good thyreostat recoveries were obtained (from 40% to 70%) as well as at acceptable repeatability. The target analytes were detectable below the 1 ng g−1 level on a quadrupole mass spectrometer with negative chemical ionization (NCI) using ammonia as reagent gas and the selected ion monitoring (SIM) acquisition mode. This limit of detection was also reached in the SIM high resolution mode. An improved specificity (more diagnostic ions) was obtained under electronic impact (EI) conditions and positive chemical ionization (PCI) with methane as reagent gas. Identification of thyreostats according to the EU (European Union) criteria (93/256/EEC decision) was made on the basis of two independent GC-MS techniques; the limit of identification was close to 5 ng g−1 for most thyreostats, which represents a real improvement for their control.

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.