VALIDATION OF CONFIRMATORY MULTI-RESIDUE METHOD FOR THE DETERMINATION OF THIRTEEN HORMONES RESIDUES IN SEVERAL BOVINES MATRICES USING HPLC-MS/MS

Synthetic and natural hormones are used in the production and intensive breeding of animals as growth factors and reproductive regulators. However, the illegal and inappropriate use of these substances increases the risk of introducing residues into the food chain. Also because of their carcinogenic and teratogenic effects, the European Union has banned the use of these substances since 1985. For this reason, the development of sensitive and reliable analytical methods for the monitoring of their residues in food has become a necessity. A liquid chromatography analysis method coupled to mass spectrometry and based on Quechers extraction has been developed to analyse thirteen synthetic and natural hormones in muscles. In order to study the reliability of this method, its validation has been carried out in different bovine matrices (liver, kidney, bile, and hair) according to European Decision 2002/657 / EC. The method demonstrates good linearity (R2> 0.99) as well as accuracy with coefficients of variation for repeatability and reproducibility less than 23%. The values of CCα and CCβ were determined for each analyte indicating values ranging from 0.13 to 0.86 μg.kg-1 and 0.25 to 1.72 μg.kg-1 respectively for most of the analytes. Higher values were obtained for 17β-estradiol, estriol, 17α-ethinyl-estradiol ranging from 1.73 to 13.95 μg.kg-1 for CCα and from 3.47 to 23.87 μg.kg-1 for CCβ. The recovery rate in the different matrices (liver, kidney, bile, and hair) varies from 51.5 to 107 %. The matrix effect of the method was evaluated, indicating significant suppression values for the liver and the kidney, which varied respectively from -45 to -15.5 % and -35 to - 2.5%. In the same way, the other two matrices hair and bile show smaller matrix effects than the others. Finally, this method has been successfully applied to detect anabolic hormones in nighty one samples (muscle, liver, kidney, bile) available from different local butchers. Progesterone was found in 41 samples at 0.11 to 11.7 µg.kg−1 otherwise testosterone was observed in 43 samples ranging from 0.5 to 9.72 µg.kg−1

LC-MS/MS Method for the Detection of Hormones: Development, Validation, and Application in Various Bovine Matrices

A liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of fourteen hormones belonging to different families in various bovine matrices (muscle, liver, kidney, bile, and hair). The modified QuEChERS method was applied to ensure the extraction of the analytes. The additives of the mobile phase, the parameters of the mass spectrometer, as well as those of the ionization source were optimized to enhance detection sensitivity. On the other hand, the parameters influencing sample extraction including the choice of the extraction solvent and its acidification, the solvent water ratio, the use of buffer salts, and the purification of the extract were also optimized. The method was validated according to the Commission Decision 2002/657/EC. Good recoveries were obtained (from 60 to 116.8 %) with coefficients of variation in terms of repeatability and reproducibility lower than 23%. The values of the decision limit CCα and the detection capability CCβ were lower than 1 and 2 μg/kg respectively in muscle and liver for most of the analyte. The matrix effect was also evaluated. Finally, this method was applied to detect hormones in two hundred and thirty-four (234) real samples. A preliminary long-term exposure assessment was evaluated based on the obtained data

Pre-Treatment of Swine Oviductal Epithelial Cells with Progesterone Increases the Sperm Fertilizing Ability in an IVF Model

Mammalian spermatozoa are infertile immediately after ejaculation and need to undergo a functional modification, called capacitation, in order to acquire their fertilizing ability. Since oviductal epithelial cells (SOECs) and progesterone (P4) are two major modulators of capacitation, here we investigated their impact on sperm functionality by using an IVF swine model. To that, we treated SOECs with P4 at 10, 100, and 1000 ng/mL before the coincubation with spermatozoa, thus finding that P4 at 100 ng/mL does not interfere with the cytoskeleton dynamics nor the cells' doubling time, but it promotes the sperm capacitation by increasing the number of spermatozoa per polyspermic oocyte (p < 0.05). Moreover, we found that SOECs pre-treatment with P4 100 ng/mL is able to promote an increase in the sperm fertilizing ability, without needing the hormone addition at the time of fertilization. Our results are probably due to the downregulation in the expression of OVGP1, SPP1 and DMBT1 genes, confirming an increase in the dynamism of our system compared to the classic IVF protocols. The results obtained are intended to contribute to the development of more physiological and efficient IVF systems

Hormones residues in bovine animals: sampling, analysis and health risk assessment

: The use of hormones for breeding of animal livestock has been banned since 1981 under the Council Directive 81/602/EC. So far, each country should monitor the use of anabolic hormones in animal production in order to protect the consumer's health against these unwanted residues. This paper presents the research results on steroid and non-steroid hormones residues carried out in Lebanon from 2018 to 2020. Using a new developed and validated LC-MS/MS method, the detection and the quantification of hormones in bovine matrices were done. The targeted matrices were muscle, liver, kidney, and bile. A total of two-hundred and forty-seven samples were collected from different slaughterhouses located in six different cities in Lebanon. Interestingly, only four hormones were found: testosterone, progesterone, epitestosterone, and 6 propyl 2thiouracil . based on the obtained data, the estimated daily intake, hazard quotient, and hazard index were calculated to evaluate an exposure assessment

CD200 as a Potential New Player in Inflammation during Rotator Cuff Tendon Injury/Repair: An In Vitro Model

Rotator cuff tendon (RCT) disease results from multifactorial mechanisms, in which inflammation plays a key role. Pro-inflammatory cytokines and tendon stem cell/progenitor cells (TSPCs) have been shown to participate in the inflammatory response. However, the underlying molecular mechanism is still not clear. In this study, flow cytometry analyses of different subpopulations of RCT-derived TSPCs demonstrate that after three days of administration, TNFα alone or in combination with IFNγ significantly decreases the percentage of CD146+CD49d+ and CD146+CD49f+ but not CD146+CD109+ TSPCs populations. In parallel, the same pro-inflammatory cytokines upregulate the expression of CD200 in the CD146+ TSPCs population. Additionally, the TNFα/IFNγ combination modulates the protein expression of STAT1, STAT3, and MMP9, but not fibromodulin. At the gene level, IRF1, CAAT (CAAT/EBPbeta), and DOK2 but not NF-κb, TGRF2 (TGFBR2), and RAS-GAP are modulated. In conclusion, although our study has several important limitations, the results highlight a new potential role of CD200 in regulating inflammation during tendon injuries. In addition, the genes analyzed here might be new potential players in the inflammatory response of TSPCs