Investigation of urinary steroid metabolites in calf urine after oral and intramuscular administration of DHEA

DHEA (3 beta-hydroxy-androst-5-en-17-one) is a natural steroid prohormone. Despite a lack of information on the effect, DHEA and other prohormones are frequently used as a food supplement by body-builders. DHEA is suspected for growth promoting abuse in cattle as well. Considering the latter, urine samples from a previous exposure study in which calves were exposed to 1 g DHEA per day for 7 days, were used. The calves were divided in three groups: one orally treated, one intramuscularly injected, and a control group. The effect of this treatment on the urinary profile of several precursors and metabolites of DHEA was investigated. Urine samples were collected several days before and during the 7 days of administration and were submitted to a clean-up procedure consisting of a separation of the different conjugates (free, glucuronidated, and sulfated forms) of each compound on a SAX column (Varian). An LC-MS/MS method was developed for the detection and quantification of several metabolites of the pathway of DHEA including 17 alpha- and 17 beta-testosterone, 4-androstenedione, 5-androstenediol, pregnenolone, and hydroxypregnenolone. Elevated levels of DHEA, 5-androstenediol, and 17 alpha-testosterone were observed in the free and sulfated fraction of the urine of the treated calves, thus indicating that the administered DHEA is metabolized mainly by the a dagger(5)-pathway with 5-androstenediol as the intermediate. Sulfoconjugates of DHEA and its metabolites were found to constitute the largest proportion of the urinary metabolites. The free form was also present, but in a lesser extent than the sulfated form, while glucuronides were negligible

The use of library identification and common fragments for the identification of corticosteroids in forensic samples

The detection of corticosteroids and sex steroids in samples with no content indication, which are confiscated for forensic investigation, is a challenge in doping analysis. A screening method based on the identification of androgens, estrogens, gestagens, and their esters by means of a mass spectral library, along with a fast ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) method, was recently developed in our lab for the analysis of dietary supplements. However, for forensic investigations, it is important to extend the scope of the method to corticosteroids in various matrices. Therefore, 36 corticosteroids were added to the mass spectral library, and the sample preparation step was modified so that androgens, gestagens, corticosteroids, and their esters could be analyzed with only one injection with the UPLC-MS method. A complementary tool to the existing library identification was found in the extraction of common fragment ions out of the full scan data obtained for the library search. The fragment ion with m/z 147 was found to be a good marker for the detection of steroids. Extra confirmation was obtained from the fragment ions with m/z 135 (for all steroids) and 237 (specific for corticosteroids) or from the fragment ions with m/z 77, 91, and 105. The effectiveness of this approach was evaluated on some samples previously screened for forensic investigation with thin-layer chromatography and confirmed with a targeted gas chromatography-mass spectrometry method. This study shows that the combination of the library identification and the common fragment ions approach can be a valuable tool in the detection of steroids without defining any target at the start of the analysis

Impact of fertilization with pig or calf slurry on antibiotic residues and resistance genes in the soil

Antibiotic residues and antibiotic resistance genes can enter the environment via fertilization with calf and pig manure. In a longitudinal study, nine antibiotic resistance genes (tet(B), tet(L), tet(M), tet(O), tet(Q), tet(W), erm(B), erm(F) and sul2) and 56 antibiotic residues were investigated in 288 soil samples and 8 corresponding slurry samples from 6 pig farms and 2 veal farms using qPCR and LC-MS/MS, respectively. A significant increase in gene copy number of tet(M), erm(B), erm(F) and sul2 was observed in all the soil layers between sampling times prior to (T1) and 2-3 weeks after fertilization (T3). Tet(B), tet(Q) and tet(L) were least abundant in the soil among the genes tested. From 7 classes of antibiotics, 20 residues were detected in soil and slurry using an optimized and validated extraction method. Flumequine was detected in all soil samples in concentrations below 100 mu g/kg despite being detected in only half of the corresponding slurry samples. Doxycycline, oxytetracycline, lincomycin and sulfadiazine were also frequently detected in concentrations ranging from 0.1 mu g/kg to 500 mu g/kg and from 2 mu g/kg and 9480 mu g/kg in soil and slurry, respectively. Furthermore a positive association between the presence of antibiotic residues (total antibiotic load) and antibiotic resistance genes in soil was found. One possible explanation for this is a simultaneous introduction of antibiotic residues and resistance genes upon application of animal slurry

Applicability of a yeast bioassay in the detection of steroid esters in hair

The aim of the present study was to demonstrate the applicability of a yeast androgen and estrogen bioassay in the detection of steroid esters in hair samples of animals treated with a hormone ester cocktail. The outcome of the activity screenings was critically compared with the results previously obtained with LC-MS/MS analysis. Hair samples of one pour-on treated animal, 10 ml DMSO containing 25 mg estradiol benzoate (EB), 60 mg testosterone decanoate (TD) and 60 mg testosterone cypionate (TC), were selected and analyzed with the androgen and estrogen yeast bioassay. Results showed that by the introduction of a hydrolysis step, bioassays can be used to screen for the presence of hormone esters in hair samples. Based on the difference in fluorescence responses between the non-hydrolyzed and the hydrolyzed hair samples, it was possible to detect the presence of EB up to at least 56 days after a single pour-on treatment and to detect the presence of TC and TD up to at least 14 days after the treatment. Although the LC-MS/MS analysis could detect TC and TD up to 49 days after treatment, bioassays have the advantage that they can also detect any (un) known steroid ester

The impact of antibiotic residues on resistance patterns in leek at harvest

When crops are cultivated on fields fertilized with animal manure, the risk exists that plants may take up antibiotic residues and may be exposed to antibiotic resistance genes and antibiotic resistant bacteria. During cultivation in a greenhouse pot experiment, leek (Allium porrum) was fertilized with either pig slurry or mineral fertilizer and exposed to either no antibiotics, doxycycline (10,000 μg/kg manure), sulfadiazine (1000 μg/kg manure), or lincomycin (1000 μg/kg manure). At harvest, 4.5 months later, lincomycin, sulfadiazine or doxycycline were not detected in any of the leek samples nor in their corresponding soil samples. Further, antimicrobial susceptibility testing was performed on 181 Bacillus cereus group isolates and 52 Pseudomonas aeruginosa isolates from the grown leek. For the B. cereus group isolates, only a small shift in MIC50 for lincomycin was observed among isolates from the lincomycin and control treatment. For P. aeruginosa, only in the setup with doxycycline treatment a higher MIC50 for doxycycline was observed compared to the control, specifically the isolates selected from growth media supplemented with 8 mg/L doxycycline. Nine antibiotic resistance genes (tet(B), tet(L), tet(M), tet(O), tet(Q), tet(W), erm(B), erm(F) and sul2) were investigated at harvest in the leek and soil samples. In the leek samples, none of the antibiotic resistance genes were detected. In the soil samples fertilized with pig slurry, the genes erm(B), erm(F), tet(M), sul2, tet(W) and tet(O) were detected in significantly higher copy numbers in the lincomycin treatment as compared to the other antibiotic treatments. This could be due to a shift in soil microbiota induced by the addition of lincomycin. The results of this study indicate that consumption of leek carries a low risk of exposure to antibiotic residues or antibiotic resistance to doxycycline, sulfadiazine or lincomycin

Comparison of the wetting properties of three commonly used simulated intestinal fluids used as dissolution media in the characterization of drugs

Simulated intestinal fluids (SIFs) are described in the United States Pharmacopoeia (USP) and the International Pharmacopoeia (IntPh) as recommended dissolution media for the characterization of solid drugs. This study was carried out to compare their wettability, properties. Six different model substrates were used as surrogates for solid drugs. These surrogate solid substrates were characterized by the Lifshitz-van der Waals, electron donor and electron acceptor terms, which are the three components of the total surface energy obtained by the van Oss model, which is based upon Young's equation. Contact angles of the SIFs on the surrogate solid-substrates were determined dynamically by image analysis up to five minutes after applying a drop of the fluid on the solid surface. Observed time-dependent spreading behavior was accounted for by a linear extrapolation of the experimental data to zero contact time to obtain the apparent equilibrium contact angle at zero time. Additionally, the pH, osmolality and buffer capacity of the SIFs were experimentally determined. Although some differences in osmolality and buffer capacity were observed, no statistically significant differences in the wetting properties of the three SIFs were detected. This confirms recent findings that there are no observed differences in dissolution behavior among these simulated intestinal fluids. Harmonization of these SIFs is thus recommended