Nuova metodica LC-MS/MS per la determinazione di peptidi endogeni nel plasma di ratto

Aim: Kinin-kallikrein and renin-angiotensin systems are strictly connected and involved in the control of blood pressure. In this work we quantify the major mediators of these two systems in a rat strain with low urinary kallikrein levels (LKRs). We hypothesize that LKRs has lower amounts of kinins and higher angiotensin II (ANG II) levels whencompared to a normal rat strain (NKRs). Methods: 46 plasma samples were analyzed (23 from LKRs and 23 from NKRs) using radioimmunoassay (RIA) and liquid chromatography coupled to tandem mass spectrometry (LCMS/MS). RIA assay wasdeveloped following RIA kit’s guidelines. An LC-MS/MS assay has been developed, validated and used for the quantification of bradykinin (BK). An elution gradient in a total run time of 12 minutes was used. The mass spectrometer was operated in positive ion-mode. ANG II plasma levels were lower than the sensitivity limit of the LC-MS/MS method developed. Results: We found lower BK concentrations in the LKRs compared to the NKRs; the average plasma BK concentration was 34.3 ± 1.29 ng/mL in NKRs versus 19.2 ± 0.92 ng/mL in LKRs. Furthermore ANG II levels were significantly higher in the LKRs, compared with the NKRs (90 ± 8 versus 74 ± 9 pg/ml in LKRs and NRKs, respectively). Conclusion: We report a new LC-MS/MS assay for BK in rat plasma.RIA assay’s results were not reproducible for BK but reliable for ANG II. We found that LKRs has lower BK levels and greater ANG II levels

The effect of by-pass linseed oil supplementation on the maternal antioxidant system during the embryo-maternal recognition period in ewes

This study analyzed the effects of dietary supplementation with by-pass linseed oil (LO; rich in α-linolenic acid) on maternal antioxidant systems at Days 14 and 16 of pregnancy in Sarda ewes. This trial used sixteen dry ewes. Eight ewes (CT group) were fed with a control diet without LO, and eight ewes (LO group) were fed with a diet supplemented with LO (10.8 g of α-linolenic acid/ewe/day). Both diets had similar crude protein and energy levels. The experiment included 10 days of an adaptation period and 31 days of a supplementation period. This supplementation period was divided into Period −2 (from Day −15 to −8), Period −1 (from Day −7 to −1; before synchronized mating period/Day 0), Period +1 (from Day +1 to + 7 after mating), and Period +2 (from Day +8 to +15 after mating). Estrous synchronization was induced in all the ewes using an intravaginal sponge (45 mg fluorgestone acetate) for 14 days and equine chorionic gonadotropin (350 UI/ewe) at the end of the treatment. On Days 14 (CT, N = 4; LO, N = 4) and 16 (CT, N = 4; LO, N = 4) after mating, the ewes were slaughtered. Samples of plasma, uterine, and luteal tissues were collected. Thiols, total antioxidant activity (TEAC), superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content were measured. On Day 16, thiol and TEAC in luteal tissues were higher in the LO group when compared with the control one (p < 0.05). Moreover, TEAC was higher for the LO group in uterine tissues on Days 14 and 16 (p < 0.05). SOD activity was higher in the LO group in luteal and uterine tissues on Day 14 and Day 16, respectively (p < 0.001). On Day 16, uterine MDA content was lower for the LO group (p < 0.001). No differences were found between groups at the plasmatic level. However, the by-pass LO supplementation enhanced the analyzed antioxidant parameters in luteal and uterine tissues. In conclusion, these results demonstrate that by-pass LO supplementation exerted a positive effect on antioxidative defenses on maternal structures during the embryo-maternal recognition period in ewes. Thus, this could contribute to improving the maternal environment during the embryo-maternal recognition period in mammals

Multi-residue analysis of eight thioamphetamine designer drugs in human urine by liquid chromatography/tandem mass spectrometry

An analytical procedure for the simultaneous determination in human urine of several thioamphetamine designer drugs (2C-T and ALEPH series) is reported. The quantitative analysis was performed by liquid chromatography/tandem mass spectrometry and has been fully validated. The mass spectrometer was operated in positive-ion, selected reaction monitoring (SRM) mode. In order to minimize interferences with matrix components and to preconcentrate target analytes, solid-phase extraction was introduced in the method as a clean-up step. The entire method was validated for selectivity, linearity, precision and accuracy. The method turned out to be specific, sensitive, and reliable for the analysis of amphetamine derivatives in urine samples. The calibration curves were linear over the concentration range of 1 to 100 ng mL-1 for all drugs with correlation coefficients that exceeded 0.996. The lower limits of detection (LODs) and quantification (LOQs) ranged from 1.2 to 4.9 ng mL-1 and from 3.2 to 9.6 ng mL-1, respectively

An Overview of Antibiotics as Emerging Contaminants: Occurrence in Bivalves as Biomonitoring Organisms

Antibiotics are used for therapeutic and prophylactic purposes in both human and veterinary medicine and as growth promoting agents in farms and aquaculture. They can accumulate in environmental matrices and in the food chain, causing adverse effects in humans and animals including the development of antibiotic resistance. This review aims to update and discuss the available data on antibiotic residues, using bivalves as biomonitoring organisms. The current research indicates that antibiotics’ presence in bivalves has been investigated along European, American and Asian coasts, with the majority of studies reported for the last. Several classes of antibiotics have been detected, with a higher frequency of detection reported for macrolides, sulfonamides and quinolones. The highest concentration was instead reported for tetracyclines in bivalves collected in the North Adriatic Sea. Only oxytetracycline levels detected in this latter site exceeded the maximum residual limit established by the competent authorities. Moreover, the risk that can be derived from bivalve consumption, calculated considering the highest concentrations of antibiotics residues reported in the analyzed studies, is actually negligible. Nevertheless, further supervisions are needed in order to preserve the environment from antibiotic pollution, prevent the development of antimicrobial resistance and reduce the health risk derived from seafood consumption

Novel LC-MS/MS method for CJ-023423 (Grapiprant) determination in rabbit plasma

Grapiprant is a new anti-inflammatory drug that preferentially targets the EP4 receptor of prostaglandin E2 limiting the wide range of adverse effects caused by the classical non steroideal anti-inflammatory drugs. The aim of this study was to develop and validate a new, simple, sensitive and rapid Liquid Chromatography tandem Mass Spectrometry method (LC-MS/MS) in order to quantify this novel drug in plasma. The method involved a simple liquid extraction followed by a gradient elution with formic acid 0.2% in water and acetonitrile in reverse phase chromatography. The method was validated according to international guidelines determining selectivity, linearity, sensitivity, recovery, matrix effect and precision. Linearity was obtained over a range of 5-1000 ng mL−1. The obtained Limit of Quantitation (LOQ) and of Determination (LOD) were of 5 and 1.5 ng mL−1respectively. Extraction recovery was >73% for all the tested concentrations. Matrix effect, expressed as ion suppression, was ≤9%. The intraday and inter-day precision results showed good RSD values. All the validation parameters were satisfactory making this new method an interesting tool for scientists to further investigations on pharmacokinetics parameters. This validated method was applied to assess the pharmacokinetic of grapiprant in one rabbit administered with 0.5 mg kg-1

Microcystins Presence in Mussels (M. galloprovincialis) and Water of Two Productive Mediterranean’s Lagoons (Sardinia, Italy)

Microcystins (MCs) are hepatotoxins harmful for animal and human health. The most toxic type between them is MC-LR whose presence has been investigated in different reservoirs all around the world. In this work microcystins were monitored in spring and summer in water and mussels (Mytilus galloprovincialis) of two Sardinia lagoons: Cabras and Calich lagoons. A Solid Phase Extraction method was developed to clean and concentrate samples before the Enzyme Linked Immunosorbent Assay (ELISA) and the following Mass Spectrometry detection. MCs presence was detected using the screening ELISA test in both lagoons. MCs peak was revealed in July for water and mussels belonging to Cabras lagoon (0.75±0.07 ng/L in water and 0.12±0.04 ng/g ww in mussels). In water of Calich lagoon there was a constant trend in the concentration of MCs during the considered months, while there was a MCs peak in July (0.6±0.5 ng/g ww) in mussels. The following LC-MS/MS analysis did not reveal MC-LR presence in all analyzed samples. These results can be useful to enrich knowledge on public health and consumer’s safeguard

An LC—MS—MS method for quantitation of four new phenethylamines (BOX series) in plasma: in vivo application

The appearance of new “designer drugs” in the illicit market poses a serious health risk because they have unknown safety profiles, have a high potential for abuse, high potency, and can lead to devastating health consequences. For this reason, it is desirable to develop validated and reliable analytical screening tests that allow detection of amphetamines and related designer drugs in biological samples. We report a method for separation and quantitation of four new phenethylamines, 4-bromo-2,5-beta-trimethoxyphenethylamine (BOB), 4-methyl-2,5-beta-trimethoxyphenethylamine (BOD), 3,4-methylenedioxy-beta-methoxyphenethylamine (BOH), and 4-methyl-2,5-dimethoxy-beta-hydroxyphenethylamine (BOHD), in plasma. Quantitation was achieved via liquid chromatography—tandem mass spectrometry (LC—MS—MS) in the multiple reaction monitoring mode, using 2,3-dimethoxyphenethylamine-d 3 as internal standard. The method was validated according to international guidelines. The parameters determined were selectivity, sensitivity, matrix effect, linearity, precision, recovery, and stability. All parameters were satisfactory. To remove matrix interference, solid-phase extraction was introduced in the method as clean-up step. The same method was applied in a pharmacokinetic study to monitor the target compounds in rat plasma after a single oral administration. The developed and validated LC—MS—MS method is the first available for quantitation of BOB, BOH, BOD, and BOHD in a biological matrix. This method is recommended for use in forensic and clinical toxicology, because of its sensitivity, selectivity, and simplicity. An important extension of this method could involve its application to other complex matrices

Determination of Praziquantel in Sparus aurata L. after Administration of Medicated Animal Feed

Praziquantel (PZQ) is an anthelmintic drug used in humans and animals against Platyhelminthes and in aquaculture in the Far East. Medicated feed is one of the most convenient forms of oral administration of drugs in aquaculture because it allows to treat a large population of fish in an easy way. However, this treatment may lead to residues in fish intended for human consumption. In this study, a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed in order to verify the presence of PZQ in samples of Sparus aurata after oral administration of feed treated with PZQ. The method was validated according to international guidelines. It showed good recoveries, selectivity and sensitivity (LOD and LOQ were 3.0 and 9.3 ng/g, respectively), with precision and matrix effect values ≤ 15%. This method could also be applied to determine PZQ residue in other fish species and thus to evaluate the appropriate withdrawal time in treated fish intended for human consumption

Quantitative assay for bradykinin in rat plasma by liquid chromatography coupled to tandem mass spectrometry

An assay to quantify bradykinin in rat plasma has been developed and validated, using liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). Sar-d-Phe8-des-Arg9-bradykinin was used as internal standard. Aprotinin was added to rat plasma to inhibit the activity of proteinases. Recoveries for solid-phase extraction (SPE) on Strata X reversed phase were greater than 80%. Multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer equipped with an electrospray source (ESI), operating in the positive ion-mode, was used for detection. The assay was validated and stability was explored. Bradykinin (10–500 ng/mL) was quantified with accuracy values (% RE) below 10% and intra- and inter-day precisions (% RSD) below 12 and 16%, respectively, for all concentrations. The method was successfully applied to several plasma samples from low levels kallikrein rats (LKRs) compared with normal kallikrein rats (NKRs)

Poisoning by <i>Nerium oleander</i> L. in Franconia Geese

This study describes the acute poisoning of four 3-month-old Franconia geese (Anser anser) by oleander plants (Nerium oleander). After the accidental ingestion of oleander clippings, the geese exhibited a rapid onset of severe symptoms, leading to mortality within 15–90 min. Necropsy revealed cardiac and renal lesions. Specifically, interstitial edema, red blood cell infiltration, and myofibril loss were observed in the cardiac muscle, and tubular epithelial degeneration, interstitial edema, and hemorrhages were evident in the kidneys. Oleandrin, a glycoside with cardiac effects, was detected in the liver, kidneys, heart, brain, and muscles. The clinical implications underscore the urgency of veterinary intervention upon oleander ingestion, and the specific findings contribute valuable insights into the pathological effects of acute oleander poisoning in geese, aiding veterinarians in prompt diagnosis and treatment