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.

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.

An LC-MS screening method with library identification for the detection of steroids in dietary supplements.

For many years anabolic-androgenic steroids (AAS) are by far the most frequently detected pharmacological substances in doping control. In order to improve their performances, professional sportsmen are often tempted to take dietary supplements. However, due to the frequent and widespread occurrence of contaminated supplements, the use of such products is not without risk for the athletes involved. In order to minimize the chances of an unattended positive doping test or serious health problems, fast and reliable screening methods for the detection of anabolic steroids in dietary supplements are needed. A general screening procedure requires the fast and unambiguous detection of a large range of steroids. Gas chromatography-mass spectrometry (GC-MS) has been used intensively in the detection of doping substances for the past 40 years. Over time, many laboratories have delivered spectra to be included in standard reference databases, one of which is maintained by the National Institute of Standards and Technology (NIST) (Gaithersburg, MD, USA). In recent years, however, liquid chromatography coupled to mass spectrometry (LC-MS) has gained popularity. Unfortunately, existing GC-MS libraries are not applicable to LC-MS analysis. In the present study, a new mass spectral library of 88 steroids was developed, along with a fast UPLC-MS method. For the construction of this mass spectral library, three different mass spectra were measured for each steroid, with a sample cone voltage of 30, 60 and 100 V, respectively. This method was then successfully tested on contaminated dietary supplements which had previously been tested by means of a targeted LC-MS/MS method. Overall, the library search was shown to identify the same compounds as the MRM method.

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

DHEA (3beta-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 17alpha- and 17beta-testosterone, 4-androstenedione, 5-androstenediol, pregnenolone, and hydroxypregnenolone. Elevated levels of DHEA, 5-androstenediol, and 17alpha-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 Delta(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.