Analysing polypeptide antibiotics residues in animal muscle tissues: The crucial role of HRMS.

A confirmatory method for the determination of polypeptide antibiotics (bacitracin, colistin, and polymyxin B) in muscle samples has been developed. Extraction is performed with acidified methanol, and a clean-up step by solid-phase extraction with polymeric cartridges is applied. Separation by ultra-high performance liquid chromatography (UHPLC) is carried out using a solid core C18 column and gradient elution with water/acetonitrile containing 0.2% formic acid. High-resolution mass spectrometry (HRMS) (Q-Orbitrap) detection using different working modes has proved to be highly advantageous in eliminating interfering signals from endogenous matrix components. The analytical method has been successfully validated according to Commission Regulation 2021/808/EU and is currently used in a public health laboratory involved in veterinary medicines residue surveillance activities.

Dietary exposure to total and inorganic arsenic via rice and rice-based products consumption.

Diet is the major route of exposure to arsenic (As), with rice and rice products as food groups with relatively high As levels. This study was aimed at determining the concentrations of total arsenic (total As) and inorganic arsenic (InAs) in rice and rice products. The dietary exposure and health risks for infant and adult population were also estimated. Brown varieties of rice showed higher As levels than white rice (189 vs 132 µg/kg). Toddlers and infants presented the highest dietary exposure to total As (4.08 and 3.99 µg/day, respectively), but unlike the rest of population groups, the main contributor was organic arsenic. Focusing on the contribution of each food item, rice represents the major contributor to InAs exposure by the adult population, while baby cereals and breakfast cereals are the most important contributors for infant exposure. Anyhow, none of the population groups exceeded the lower limit of the BMDL01 range (from 0.3 to 8.0 µg/kg body weight/day) set by EFSA in any of the three exposure scenarios (high, mean, and low) hereby considered. Finally, consumption of white rice varieties or pre-cooked rice, as well as washing rice before cooking, are recommended in order to minimize the exposure to arsenic.

Dietary intake of arsenic, cadmium, mercury and lead by the population of Catalonia, Spain: Analysis of the temporal trend.

In 2017, a monitoring study was conducted in Catalonia (Spain) to analyse, in widely consumed foodstuffs, the concentrations of arsenic (As), cadmium (Cd), mercury (Hg) and lead (Pb), as well as those of inorganic As (InAs) and methylmercury (MeHg). Health risks were estimated for various population groups, classified according to age. A continued reduction of dietary exposure to these elements was observed when comparing the results from the current and previous studies performed during the last 17 years. This reduction would be associated to a decrease of As, Cd, Hg and Pb concentrations in food, as well as by changes in dietary habits. None of the adult groups exceeded the safety threshold established by the EFSA, but toddlers, infants and children exceeded the PTWI for Cd and MeHg. The greatest intake of Cd and MeHg corresponded to infants, being due to the consumption of cuttlefish and hake. The consumption of these species should be reduced to two or three weekly portions, being combined with the intake of other non-predator species. Anyhow, the current results indicate that is necessary to conduct further periodical surveys, paying special attention to the trend in the intake of Cd and MeHg.

Analysis of corticosteroids in samples of animal origin using QuEChERS and ultrahigh-performance liquid chromatography coupled to high-resolution mass spectrometry.

A rapid and sensitive method for the confirmatory analysis of eight synthetic corticosteroids (betamethasone, dexamethasone, prednisolone, 6-methylprednisolone, triamcinolone, flumethasone, beclomethasone, fluocinolone acetonide) is proposed. The method is useful for detecting illegal treatments in different animal species. It consists of an extraction and cleanup using the quick, easy, cheap, effective, rugged, and safe (QuEChERS) strategy. Quantitative determination is achieved by ultrahigh-performance liquid chromatography coupled to high-resolution mass spectrometry with heated electrospray ionization in negative mode. Quantification is performed using surrogate matrix-matched standard calibration curve with dexamethasone-D4 as the internal standard. The method was validated for analyzing liver samples according to the criteria established by Decision 2002/657/EC. Linearity was assessed in the 1-10 µg kg-1 range and linear correlation coefficients were over 0.99 for all the analytes. CCα ranged from 0.04 to 0.16 µg kg-1 for substances without maximum residue limit. The method allows confident quantification and confirmation of corticosteroids in liver samples, and its simplicity makes it suitable for analyzing large numbers of samples.

Analysis of non-steroidal anti-inflammatory drugs in milk using QuEChERS and liquid chromatography coupled to mass spectrometry: triple quadrupole versus Q-Orbitrap mass analyzers.

We developed a Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) method for the high throughput determination of 10 non-steroidal anti-inflammatory drugs (NSAIDs) in milk samples using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) with a triple quadrupole (QqQ) instrument and an electrospray ionization (ESI) source. The new extraction procedure is highly efficient, and we obtained absolute recoveries in the range 78.1-97.1 % for the extraction and clean-up steps. Chromatographic separation is performed in the gradient mode with a biphenyl column and acidic mobile phases consisting of water and acetonitrile containing formic acid. The chromatographic run time was about 12 min, and NSAID peaks showed a good symmetry factor. For MS/MS detection, we used multiple reaction monitoring (MRM) mode, using ESI in both positive and negative modes. Our method has been validated in compliance with the European Commission Decision 657/2002/EC, and we obtained very satisfactory results in inter-laboratory testing. Furthermore, we explored the use of a hybrid high resolution mass spectrometer, combining a quadrupole and an Orbitrap mass analyzer, for high resolution (HR) MS/MS detection of NSAIDs. We achieved lower NSAID quantification limits with Q-Orbitrap high resolution mass spectrometry (HRMS/MS) detection than those achieved with the QqQ instrument; however, its main feature is its very high selectivity, which makes HRMS/MS particularly suitable for confirmatory analysis.

A false positive case due to matrix interference in the analysis of ronidazole residues in muscle tissue using LC-MS/MS.

In contrast with the information of the inspection body concerning the use of ronidazole, several non compliant muscle samples were detected using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in accordance with confirmation criteria of Decision 2002/657/EC. This led to the suspicion that non compliance could be due to false positive results. In this context, a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method was developed and sample extracts were re-analyzed, resolving the co eluting isobaric interfering peak, which also has an interfering product ion with the transition product (m/z 201>140).

Long-term recovery patterns and limited spillover of large predatory fish in a Mediterranean MPA.

Based on 19 y of visual census data from the Medes Islands MPA (NW Mediterranean), this study analyzes the carrying capacity (K) and population recovery time of six species of fish strongly affected by harvesting pressure along the Mediterranean coast. Three of these species (Epinephelus marginatus, Diplodus cervinus and Dicentrachus labrax) have practically reached carrying capacity in the Medes Islands MPA, while others are still approaching population stabilization (Sciaena umbra) or are still increasing in biomass (Dentex dentex). The one exception to these trends is S. aurata, which tended to decrease inside the MPA, probably due to fishing just outside its borders. These results confirm that fish populations may require decadal time scales to recover from exploitation, both in terms of total abundance (21 to 29 y to exceed 95% K) as well as total biomass (25 to 35 y), and that rates of recovery differ between species (13 to 31 y). The recovery and saturation observed within the no-take zone contrasts with results obtained in the partially protected buffer area and the peripheral area open for fishing, which show much lower biomass values. In general, the spillover from the MPA is very moderate, and its effects extend only to the partially protected area.

Targeted analysis with benchtop quadrupole-orbitrap hybrid mass spectrometer: application to determination of synthetic hormones in animal urine.

Sensitive and unequivocal determination of analytes/contaminants in complex matrices is a challenge in the field of food safety control. In this study, various acquisition modes (Full MS/AIF, Full MS+tMS/MS, Full MS/dd MS/MS and tSIM/ddMS/MS) and parameters of a quadrupole-orbitrap hybrid mass spectrometer (Q Exactive) were studied in detail. One of the main conclusions has been that, reducing the scan range for Full MS (using the quadrupole) and targeted modes give higher signal-to-noise (S/N) ratios and thereby better detection limits for analytes in matrix. The use of Q Exactive in a complex case, for the confirmatory analysis of hormones in animal urine is presented. A targeted SIM data dependent MS/MS (tSIM/ddMS/MS) acquisition method for determination of eight synthetic hormones (trenbolone, 17α ethinylestradiol, zeranol, stanozolol, dienestrol, diethylstilbestrol, hexestrol, taleranol) and a naturally occurring hormone (zearalenone) in animal urine were optimized to have sensitive precursors from targeted SIM mode and trigger MS/MS scans over the entire chromatograph peak. The method was validated according to EC/657/2002. CCα (decision limit) for the analytes ranged between 0.11 µg L(-1) and 0.69 µg L(-1) and CCß (detection capability) ranged between 0.29 µg L(-1) and 0.90 µg L(-1).

The Mediterranean benthic herbivores show diverse responses to extreme storm disturbances.

Catastrophic storms have been observed to be one of the major elements in shaping the standing structure of marine benthic ecosystems. Yet, little is known about the effect of catastrophic storms on ecosystem processes. Specifically, herbivory is the main control mechanism of macrophyte communities in the Mediterranean, with two main key herbivores: the sea urchin Paracentrotus lividus and the fish Sarpa salpa. Consequently, the effects of extreme storm events on these two herbivores (at the population level and on their behaviour) may be critical for the functioning of the ecosystem. With the aim of filling this gap, we took advantage of two parallel studies that were conducted before, during and after an unexpected catastrophic storm event. Specifically, fish and sea urchin abundance were assessed before and after the storm in monitored fixed areas (one site for sea urchin assessment and 3 sites for fish visual transects). Additionally, we investigated the behavioural response to the disturbance of S. salpa fishes that had been tagged with acoustic transmitters. Given their low mobility, sea urchins were severely affected by the storm (ca. 50% losses) with higher losses in those patches with a higher density of sea urchins. This may be due to a limited availability of refuges within each patch. In contrast, fish abundance was not affected, as fish were able to move to protected areas (i.e. deeper) as a result of the high mobility of this species. Our results highlight that catastrophic storms differentially affect the two dominant macroherbivores of rocky macroalgal and seagrass systems due to differences in mobility and escaping strategies. This study emphasises that under catastrophic disturbances, the presence of different responses among the key herbivores of the system may be critical for the maintenance of the herbivory function.

Determination of aminoglycoside residues in kidney and honey samples by hydrophilic interaction chromatography-tandem mass spectrometry.

Two methods based on liquid chromatography-tandem mass spectrometry were developed for the determination of ten aminoglycosides (streptomycin, dihydrostreptomycin, spectinomycin, apramycin, paromomycin, kanamycin A, gentamycin C1, gentamycin C2/C2a, gentamycin C1a, and neomycin B) in kidney samples from food-producing animals and in honey samples. The methods involved extraction with an aqueous solution (for the kidney samples) or sample dissolution in water (for the honey samples), solid-phase extraction with a weak cation exchange cartridge and injection of the eluate into a liquid chromatography-tandem mass spectrometry system. A zwitterionic hydrophilic interaction chromatography column was used for separation of aminoglycosides and a triple quadrupole mass analyzer was used for detection. The methods were validated according to Decision 2002/657/EC. The limits of quantitation ranged from 2 to 125 µg/kg in honey and 25 to 264 µg/kg in the kidney samples. Interday precision (RSD%) ranged from 6 to 26% in honey and 2 to 21% in kidney. Trueness, expressed as the percentage of error, ranged from 7 to 20% in honey and 1 to 11% in kidney.

Multiple processes regulate long-term population dynamics of sea urchins on Mediterranean rocky reefs.

We annually monitored the abundance and size structure of herbivorous sea urchin populations (Paracentrotus lividus and Arbacia lixula) inside and outside a marine reserve in the Northwestern Mediterranean on two distinct habitats (boulders and vertical walls) over a period of 20 years, with the aim of analyzing changes at different temporal scales in relation to biotic and abiotic drivers. P. lividus exhibited significant variability in density over time on boulder bottoms but not on vertical walls, and temporal trends were not significantly different between the protection levels. Differences in densities were caused primarily by variance in recruitment, which was less pronounced inside the MPA and was correlated with adult density, indicating density-dependent recruitment under high predation pressure, as well as some positive feedback mechanisms that may facilitate higher urchin abundances despite higher predator abundance. Populations within the reserve were less variable in abundance and did not exhibit the hyper-abundances observed outside the reserve, suggesting that predation effects maybe more subtle than simply lowering the numbers of urchins in reserves. A. lixula densities were an order of magnitude lower than P. lividus densities and varied within sites and over time on boulder bottoms but did not differ between protection levels. In December 2008, an exceptionally violent storm reduced sea urchin densities drastically (by 50% to 80%) on boulder substrates, resulting in the lowest values observed over the entire study period, which remained at that level for at least two years (up to the present). Our results also showed great variability in the biological and physical processes acting at different temporal scales. This study highlights the need for appropriate temporal scales for studies to fully understand ecosystem functioning, the concepts of which are fundamental to successful conservation and management.

The power of hyphenated chromatography/time-of-flight mass spectrometry in public health laboratories.

Laboratories devoted to the public health field have to face the analysis of a large number of organic contaminants/residues in many different types of samples. Analytical techniques applied in this field are normally focused on quantification of a limited number of analytes. At present, most of these techniques are based on gas chromatography (GC) or liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS). Using these techniques only analyte-specific information is acquired, and many other compounds that might be present in the samples would be ignored. In this paper, we explore the potential of time-of-flight (TOF) MS hyphenated to GC or LC to provide additional information, highly useful in this field. Thus, all positives reported by standard reference targeted LC-MS/MS methods were unequivocally confirmed by LC-QTOF MS. Only 61% of positives reported by targeted GC-MS/MS could be confirmed by GC-TOF MS, which was due to its lower sensitivity as nonconfirmations corresponded to analytes that were present at very low concentrations. In addition, the use of TOF MS allowed searching for additional compounds in large-scope screening methodologies. In this way, different contaminants/residues not included in either LC or GC tandem MS analyses were detected. This was the case of the insecticide thiacloprid, the plant growth regulator paclobutrazol, the fungicide prochloraz, or the UV filter benzophenone, among others. Finally, elucidation of unknowns was another of the possibilities offered by TOF MS thanks to the accurate-mass full-acquisition data available when using this technique.

Hydrophilic interaction chromatography for the analysis of aminoglycosides.

The effect of mobile-phase constituents (pH and ionic strength) and chromatographic behaviour of ten aminoglycosides (streptomycin, dihydrostreptomycin, spectinomycin, apramycin, paramomycin, kanamycin A, gentamycin C1, gentamycin C2/C2a, gentamycin C1a and neomycin) in the bare silica, amino, amide and zwitterionic phases of hydrophilic interaction chromatography (HILIC) were studied systematically. Among the stationary phases studied, the zwitterionic phase provided the best separation of aminoglycosides. The effect of pH, ionic concentration and column temperature on retention time, peak shape and sensitivity was studied using a central composite design. pH affected sensitivity of the detection of analytes but not the retention time. High ionic strength in the mobile phase was necessary to control the ionic interactions between ionised aminoglycosides and the hydrophilic phase, thereby influencing peak shape and retention time. Column temperature affected sensitivity of the detection but not the retention time. During method development, crosstalk between the MS/MS channels of the analytes was observed and resolved.

Determination of synthetic hormones in animal urine by high-performance liquid chromatography/mass spectrometry.

A method was developed for the extraction of stanozolol, taleranol, zeranol, hexestrol, dienestrol, ethynylestradiol, diethylstilbestrol, and trenbolone from animal urine. The analytes were extracted from the matrix by enzymatic hydrolysis, solid-phase extraction, and liquid-liquid extraction. Detection and quantitation were performed on a high-performance liquid chromatography system coupled to a triple quadrupole mass spectrometer. The identification criteria met the European Union regulations. Validation of this method established a decision limit between 0.2 and 0.9 microg/L and a detection capability between 0.3 and 1.0 microg/L, depending on the analyte.