Assessing human urinary clomiphene metabolites after consumption of eggs from clomiphene-treated laying hens using chromatographic-mass spectrometric approaches.

The anti-estrogen clomiphene is prohibited in sports at all times. Yet, adverse analytical findings (AAFs) have increased since 2011. This is possibly due to improved analytical sensitivity, but also contamination of food of animal origin needs to be taken into consideration as a potential source of drug exposure. For instance, studies with laying hens that received orally administered clomiphene have shown a significantly increased egg production rate but, as a consequence, eggs were found to incorporate residues of clomiphene. In order to evaluate if the consumption of clomiphene-contaminated eggs can cause an AAF of a doping control sample, eggs obtained from an animal administration study with clomiphene were consumed by human volunteers. Each volunteer ate two eggs, and urine samples were collected and analyzed using routine doping control procedures. Subsequently, additional volunteers received a microdosed clomiphene capsule to compare the excretion profiles. Maximum urinary concentrations of hydroxy-clomiphene (HC) between 80 and 300 pg mL-1 were detected following the consumption of clomiphene-containing eggs, which would constitute AAFs if observed in athletes' doping control samples. In order to support the differentiation of potential routes of drug exposure, a method was developed which allows for the chromatographic separation of (E)-3-, (Z)-3-, (E)-4-, and (Z)-4-HC using a derivatization step. By comparing the peak areas of these metabolites, characteristic relative distribution patterns were found that assist in identifying AAFs resulting from clomiphene ingested via contaminated eggs and, thus, enable to distinguish clomiphene intake via contaminated eggs from the intake of microdoses or therapeutic dosages, e.g. for doping purposes.

Determination of colistin in luminal and parietal intestinal matrices of chicken by ultra-high-performance liquid chromatography-tandem mass spectrometry.

Justification for continued use of colistin in veterinary medicine, for example medicated water, relies on pharmacokinetic/pharmacodynamic (PK/PD) studies that require accurate measurement of colistin content in the digestive tract. A method for the detection and quantification of colistin in poultry intestinal material was developed and validated. Colistin is not absorbed after oral administration, and the biophase is the gastrointestinal tract. Extraction of colistin from the matrix was achieved using solid-phase extraction with a methanol:water (1:2; v/v) solution. Polymyxin B was used as an internal standard. Colistin A and colistin B, the main components of colistin, were separated, detected and measured using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). The method was validated for linearity/quadraticity between 1.1 (LOQ) and 56.7 mg/kg. Mean accuracy was between 82.7% and 107.7% with inter- and intra-day precision lower than 13.3% and 15% respectively. Freeze-thaw, long-term and bench storage were validated. Incurred samples following colistin treatment in poultry at the approved clinical dose of 75,000 IU/kg in drinking water and oral gavage were quantifiable and in line with expected intestinal transit times. The method is considered appropriately accurate and precise for the purposes of pharmacokinetic analysis in the gastrointestinal tract.

Depletion of clomiphene residues in eggs and muscle after oral administration to laying hens.

The selective oestrogen receptor modulator (SERM) clomiphene is therapeutically used to induce ovulation. While prohibited as a doping agent in sports, it is frequently detected in sports drug testing urine samples. Few reports exist on clomiphene's (illicit) use in the farming industry to increase the egg production rate of laying hens, which creates a risk that eggs as well as edible tissue of these hens contain residues of clomiphene. To investigate the potential transfer of clomiphene into eggs and muscle tissue, laying hens were orally administered with clomiphene citrate at 10 mg/day for 28 days. To determine clomiphene residues in eggs, chicken breast and chicken thigh, the target analyte was extracted from homogenised material with acetonitrile and subjected to ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. The test method reached a limit of quantification (LOQ) of 1 µg/kg and was characterised concerning specificity, precision, trueness and linearity. Analyses were performed on whole egg, egg white and yolk separately, and chicken muscle from breast and thigh. Clomiphene was detectable in eggs two days after the beginning of the drug administration period. The drug concentrations increased to 10-20 µg per egg within one week, and after withdrawal of clomiphene, residues decreased after 4 days, but traces of clomiphene were still detectable until the end of the study (14 days after the last administration). In the chicken's muscle tissue, clomiphene levels up to 150 µg/kg (thigh) and 36 µg/kg (breast) were found. Six days after the last dose, tissue clomiphene concentrations fell below the LOQ. Overall, these results underline the concerns that clomiphene may be transferred into animal-derived food and future research will therefore need to focus on assessing and minimising the risk of unintentional adverse analytical findings in doping controls.

Validation of a two-plate microbiological method for screening antibiotic residues in shrimp tissue.

Microbiological inhibition screening tests could play an important role to detect residues of antibiotics in the different animal food products, but very few are available for the aquaculture products in general, and for shrimps in particular. A two-plate microbiological method to screen shrimp for residues of the most commonly used antibiotics has been developed and validated according to criteria derived from the European Commission Decision 2002/657/CE. Bacillus subtilis was used as a sensitive strain to target antibiotics. Culture conditions on Petri plates (pH of medium) were selected to enhance the capacity of antibiotic detection. Antibiotic residues were extracted from shrimps using acetonitrile/acetone (70/30, v/v) before application on Petri plates seeded with B. subtilis. The method was validated using spiked blank tissues as well as antibiotic treated shrimps with enrofloxacin and tetracycline, two antibiotics often found to be used in shrimp production. For tetracyclines and (fluoro)quinolones, the detection capability was below the maximum residue limit (MRL), while it was around the MRL for sulfonamides. The specificity of the microbiological screening was 100% in all cases while the sensitivity and accuracy was 100% in almost all cases. The capacity of the method to detect contaminated samples was confirmed on antibiotic treated shrimps, analyzed in parallel with a confirmatory method (Liquid Chromatography coupled to mass spectrometry (LC-MS)).