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

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

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

Analytical methodologies optimisation for antibiotics determination in aqueous systems

Les antibiotiques sont des polluants présents dans les écosystèmes aquatiques, réceptacles ultimes des substances anthropiques. L’étude de ces composés porte sur leur rémanence dans le milieu ou leurs effets sur des organismes naturels. De nombreux efforts ont été faits à l’échelle mondiale pour l’évaluation de la qualité environnementale des différentes ressources en eau pour la survie des espèces aquatiques mais aussi pour la consommation humaine et le risque sanitaire lié. Dans ce but, l’optimisation des techniques analytiques pour ces composés dans les systèmes aquatiques demeure une nécessité. Notre objectif est de développer des méthodes d’extraction et de détection pour 12 molécules appartenant à la famille des aminoglycosides et de la colistine dans les eaux des stations d’épuration et les eaux hospitalières. L’absence des méthodes d’analyse pour ces composés ainsi que le manque des études permettant leur détection dans l’eau sont les raisons de leur étude. L’Extraction sur Phase Solide (SPE) en mode classique (hors ligne) ou en ligne, suivie d’une analyse par la Chromatographie Liquide couplée à la Spectrométrie de Masse (LC/MS/MS) est la méthode la plus couramment employée pour ce type d’analyse. Les paramètres sont optimisés et validés afin d’assurer les meilleures conditions utilisées dans les analyses environnementales. Cette technique a été appliquée sur des échantillons réels des eaux des stations d’épuration à Bordeaux et au Liban.Antibiotics are pollutants present in aquatic ecosystems ultimate receptacles of anthropogenic substances. These compounds are studied as their persistence in the environment or their effects on natural organisms. Numerous efforts have been made worldwide to assess the environmental quality of different water resources for the survival of aquatic species, but also for human consumption and health risk related. Towards goal, the optimization of analytical techniques for these compounds in aquatic systems remains a necessity. Our objective is to develop extraction and detection methods for 12 molecules of aminoglycosides and colistin in sewage treatment plants and hospitals waters. The lack of analytical methods for analysis of these compounds and the deficiency of studies for their detection in water is the reason for their study. Solid Phase Extraction (SPE) in classic mode (offline) or online followed by Liquid Chromatography analysis coupled with Mass Spectrometry (LC/MS/ MS) is the most method commonly used for this type of analysis. The parameters are optimized and validated to ensure the best conditions for the environmental analysis. This technique was applied to real samples of wastewater treatment plants in Bordeaux and Lebanon

Analytical methodologies optimisation for antibiotics determination in aqueous systems

Les antibiotiques sont des polluants présents dans les écosystèmes aquatiques, réceptacles ultimes des substances anthropiques. L’étude de ces composés porte sur leur rémanence dans le milieu ou leurs effets sur des organismes naturels. De nombreux efforts ont été faits à l’échelle mondiale pour l’évaluation de la qualité environnementale des différentes ressources en eau pour la survie des espèces aquatiques mais aussi pour la consommation humaine et le risque sanitaire lié. Dans ce but, l’optimisation des techniques analytiques pour ces composés dans les systèmes aquatiques demeure une nécessité. Notre objectif est de développer des méthodes d’extraction et de détection pour 12 molécules appartenant à la famille des aminoglycosides et de la colistine dans les eaux des stations d’épuration et les eaux hospitalières. L’absence des méthodes d’analyse pour ces composés ainsi que le manque des études permettant leur détection dans l’eau sont les raisons de leur étude. L’Extraction sur Phase Solide (SPE) en mode classique (hors ligne) ou en ligne, suivie d’une analyse par la Chromatographie Liquide couplée à la Spectrométrie de Masse (LC/MS/MS) est la méthode la plus couramment employée pour ce type d’analyse. Les paramètres sont optimisés et validés afin d’assurer les meilleures conditions utilisées dans les analyses environnementales. Cette technique a été appliquée sur des échantillons réels des eaux des stations d’épuration à Bordeaux et au Liban.Antibiotics are pollutants present in aquatic ecosystems ultimate receptacles of anthropogenic substances. These compounds are studied as their persistence in the environment or their effects on natural organisms. Numerous efforts have been made worldwide to assess the environmental quality of different water resources for the survival of aquatic species, but also for human consumption and health risk related. Towards goal, the optimization of analytical techniques for these compounds in aquatic systems remains a necessity. Our objective is to develop extraction and detection methods for 12 molecules of aminoglycosides and colistin in sewage treatment plants and hospitals waters. The lack of analytical methods for analysis of these compounds and the deficiency of studies for their detection in water is the reason for their study. Solid Phase Extraction (SPE) in classic mode (offline) or online followed by Liquid Chromatography analysis coupled with Mass Spectrometry (LC/MS/ MS) is the most method commonly used for this type of analysis. The parameters are optimized and validated to ensure the best conditions for the environmental analysis. This technique was applied to real samples of wastewater treatment plants in Bordeaux and Lebanon

Optimisation de techniques analytiques pour caractériser les antibiotiques dans les systèmes aquatiques

Antibiotics are pollutants present in aquatic ecosystems ultimate receptacles of anthropogenic substances. These compounds are studied as their persistence in the environment or their effects on natural organisms. Numerous efforts have been made worldwide to assess the environmental quality of different water resources for the survival of aquatic species, but also for human consumption and health risk related. Towards goal, the optimization of analytical techniques for these compounds in aquatic systems remains a necessity. Our objective is to develop extraction and detection methods for 12 molecules of aminoglycosides and colistin in sewage treatment plants and hospitals waters. The lack of analytical methods for analysis of these compounds and the deficiency of studies for their detection in water is the reason for their study. Solid Phase Extraction (SPE) in classic mode (offline) or online followed by Liquid Chromatography analysis coupled with Mass Spectrometry (LC/MS/ MS) is the most method commonly used for this type of analysis. The parameters are optimized and validated to ensure the best conditions for the environmental analysis. This technique was applied to real samples of wastewater treatment plants in Bordeaux and Lebanon.Les antibiotiques sont des polluants présents dans les écosystèmes aquatiques, réceptacles ultimes des substances anthropiques. L’étude de ces composés porte sur leur rémanence dans le milieu ou leurs effets sur des organismes naturels. De nombreux efforts ont été faits à l’échelle mondiale pour l’évaluation de la qualité environnementale des différentes ressources en eau pour la survie des espèces aquatiques mais aussi pour la consommation humaine et le risque sanitaire lié. Dans ce but, l’optimisation des techniques analytiques pour ces composés dans les systèmes aquatiques demeure une nécessité. Notre objectif est de développer des méthodes d’extraction et de détection pour 12 molécules appartenant à la famille des aminoglycosides et de la colistine dans les eaux des stations d’épuration et les eaux hospitalières. L’absence des méthodes d’analyse pour ces composés ainsi que le manque des études permettant leur détection dans l’eau sont les raisons de leur étude. L’Extraction sur Phase Solide (SPE) en mode classique (hors ligne) ou en ligne, suivie d’une analyse par la Chromatographie Liquide couplée à la Spectrométrie de Masse (LC/MS/MS) est la méthode la plus couramment employée pour ce type d’analyse. Les paramètres sont optimisés et validés afin d’assurer les meilleures conditions utilisées dans les analyses environnementales. Cette technique a été appliquée sur des échantillons réels des eaux des stations d’épuration à Bordeaux et au Liban

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

Innovative SPE-LC-MS/MS technique for the assessment of 63 pharmaceuticals and the detection of antibiotic-resistant-bacteria: A case study natural water sources in Lebanon

A powerful analytical method for simultaneous determination of 63 pharmaceuticals and some metabolites in aqueous samples has been developed. The list of compounds amenable to the methods includes different therapeutic classes belonging to antibiotics, stimulants, antidepressants, mucolytics, and antiparasites. The method involves concentration and clean up by an offline solid phase extraction SPE followed by liquid chromatography coupled to tandem mass spectrometry (LC-ESI-MS/MS). The recovery of the target compounds from water samples was most efficient on Waters Oasis HLB SPE cartridge, while acetonitrile/water (60/40) was shown to be the most suitable solvent for desorbing the compounds from SPE. In addition, acidification of samples prior to SPE was optimized to enhance the recovery of the compounds. In terms of method validation, the recoveries of analytes ranged from 68% to 134%. Repeatability and intermediate precision were  10,000 ng L− 1 and 2000 ng L− 1, respectively. Moreover, bacterial analysis showed that the samples were contaminated by Escherichia coli (23%), intestinal Enterococcus (48%) and Pseudomonas aeruginosa (27%). Therefore, in order to evaluate if a correlation exists between finding antibiotics in water samples and the development of resistant-bacteria, an antimicrobial susceptibility test was conducted to the identified isolates using disk diffusion method. Multiple-antibiotic-resistant strains in both intestinal Enterococcus and E. coli were evident in many water samples, while P. aeruginosa was resistant to only one studied antibiotic

OCCURRENCE OF 63 SELECTED PHARMACEUTICALS AND ISOLATION OF RESISTANT- BACTERIA STRAINS IN NATURAL WATER SOURCES IN LEBANON

A powerful analytical method was developed for the simultaneous determination of traces of 63 pharmaceuticals and their metabolites representing different therapeutic classes: antibiotics, stimulant, antidepressant, mucolytic, antiparasite and dyes using SPE-LC-ESI-MS/MS in water samples. 100 samples from rivers, lakes, fountain and artesian wells were collected for this study overall Lebanon. Monitoring programs show the presence of many antibiotics in several analyzed samples. Therefore, to evaluate if a relation may exist between finding antibiotics in water samples and resistant-bacterial strains; three bacteria (Escherichia coli, Enterococcus faecalis and Pseudomonas aeruginosa) were selected for isolation, then antimicrobial susceptibility was determined using disk diffusion method according to the recommendations of the European Committee on Antimicrobial Susceptibility Testing

Photodegradation of sulfamethazine, sulfamethoxypiridazine, amitriptyline, and clomipramine drugs in aqueous media

International audienceThe photochemical transformation of two antibacterial sulfonamides, namely sulfamethazine (SMT) and sulfamethoxypyridazine (SMP), and two tricyclic antidepressants, namely amitriptyline (AMT) and clomipramine (CMP) were investigated. Experiments conducted in river water under artificial sunlight irradiation show an acceleration of the degradation for SMT, SMP, and CMP of a factor 1.6–7.7 by comparison to purified water. This acceleration is, at least partially, due to photosensitized reactions which can occur in river water. The photodegradation of CMP was particularly fast. In addition, no degradation was observed for AMT in purified water while photosensitized reaction occurs. Under ultra-violet (254 nm) irradiation in purified water, the four drugs were degraded. Calculated quantum yields of photodegradation were of 4.3 × 10−3, 5.1 × 10−3, 7.6 × 10−3, and 65.0 × 10−3 respectively for SMT, SMP, AMT, and CMP. UV coupled with hydrogen peroxide (UV/H2O2) was used as an advanced oxidation process for water depollution. The calculated second order rate constants of reaction with hydroxyl radicals were of 5.0 × 109, 5.0 × 109, 8.0 × 109 and 9.5 × 109 L mol−1 s−1 for SMT, SMP, AMT and CMP, respectively. Finally, the structures of photoproducts were proposed according to LC–MS/MS analyses. The elimination of SO2 was the main photochemical process for SMT and SMP. In the case of AMT and CMP, hydration and hydroxylation, respectively, were observed