Zsuzsanna Eke

Development and validation of a liquid chromatographic-tandem mass spectrometric method for determination of eleven coccidiostats in milk

A reversed phase liquid chromatographic-tandem mass spectrometric method with simple solvent extraction and purification by solid phase extraction (SPE) has been developed for the determination of coccidiostats in milk. For sample preparation matrix solid phase dispersion, extraction by organic solvent and SPE with different cartridges were also tested. The compounds determined include lasalocid, narasin, salinomycin, monensin, semduramicin, maduramicin, robenidine, decoquinate, halofuginone, nicarbazin and diclazuril. Main steps of the method are addition of acetonitrile to the milk samples, centrifugation, removal of matrix by SPE, concentration by evaporation and LC-MS-MS determination. During a 15 min time segmented chromatographic run compounds are ionised either positively or negatively. Calculated recoveries range between 77.1% and 118.2%. Maximum levels are in the range of 1-20 μg/kg. The developed method was validated in line with the requirements of Commission Decision 2002/657/EC (2002). It is applicable for control of coccidiostat residues in milk as indicated in Regulation 124/2009/EC (2009).

Determination of antihypertensive and anti-ulcer agents from surface water with solid-phase extraction-liquid chromatography-electrospray ionization tandem mass spectrometry.

Pharmaceuticals are emerging contaminants in surface water and they must be measured to follow their effects on the aquatic environment. We developed a solid-phase extraction and liquid chromatography-electrospray ionization tandem mass spectrometry (SPE-LC-ESI-MS/MS) method for the determination of twenty-six pharmaceutical compounds - which belong to antihypertensive and anti-ulcer agents - from surface water samples. The selection of pharmaceuticals was based on usage frequency in Hungary. During method development Oasis HLB, SampliQ Polymer SCX and Si-SCX SPE cartridges were tested. As LC eluent ammonium formate, ammonium acetate buffers at pH 3 and 5 were investigated and for quantitation both matrix-matched and internal standard calibration was used. For matrix effect assessment post-extraction spike method was applied which can separate the extraction efficiency from ion suppression for better determination of recovery. Method detection limits (MDLs) varied between 0.2 and 10 ng/L. Precision of the method, calculated as relative standard deviation (RSD), ranged from 0.2 to 14.6% and from 1.2 to 22.4% for intra- and inter-day analysis, respectively. The method was applied to analyze Danube water samples. Measured average concentrations varied between 2 and 39 ng/L for eleven compounds and another one could be detected under LOQ.

Comparison of spray drying, electroblowing and electrospinning for preparation of Eudragit E and itraconazole solid dispersions

Three solvent based methods: spray drying (SD), electrospinning (ES) and air-assisted electrospinning (electroblowing; EB) were used to prepare solid dispersions of itraconazole and Eudragit E. Samples with the same API/polymer ratios were prepared in order to make the three technologies comparable. The structure and morphology of solid dispersions were identified by scanning electron microscopy and solid phase analytical methods such as, X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC) and Raman chemical mapping. Moreover, the residual organic solvents of the solid products were determined by static headspace-gas chromatography/mass spectroscopy measurements and the wettability of samples was characterized by contact angle measurement. The pharmaceutical performance of the three dispersion type, evaluated by dissolution tests, proved to be very similar. According to XRPD and DSC analyses, made after the production, all the solid dispersions were free of any API crystal clusters but about 10 wt% drug crystallinity was observed after three months of storage in the case of the SD samples in contrast to the samples produced by ES and EB in which the polymer matrix preserved the API in amorphous state.

Determination of Coccidiostats in Milk Products by LC–MS and Its Application to a Fermentation Experiment

A liquid chromatographic tandem mass spectrometric (LC–MS–MS) method for the determination of five chemical coccidiostats (decoquinate, diclazuril, halofuginone, nicarbazin, and robenidine) and five ionophore coccidiostats (maduramicin, monensin, narasin, salinomycin, and semduramicin) in yoghurt, kefir, and sour cream is presented. Lasalocid, the sixth ionophore listed in 124/2009/EC was not included because of its extremely dissimilar behavior during sample preparation. Main steps of the method include extraction with acetonitrile, centrifugation, clean-up on Oasis HLB solid phase extraction cartridge, evaporation under nitrogen stream, and LC–MS–MS determination. Selectivity, linearity, sensitivity, accuracy, repeatability, within-laboratory reproducibility, limit of determination, and limit of quantitation were determined during the validation procedure. The method proved to be applicable for both qualitative and quantitative determination of the ten above-mentioned target compounds. In our in-house fermentation experiments, milk fortified with coccidiostats was fermented to get yoghurt, kefir, and sour cream. Our results show that the coccidiostat content did not change significantly during fermentation for any of the target compounds.

Analysis of hydrocarbon contamination with membrane-assisted solvent extraction: comparison of agitation and sonication methods.

Membrane-assisted solvent extraction (MASE) coupled to large volume injection was applied to the determination of (gasoline-type) hydrocarbon contamination in water samples. Hexane was used as acceptor phase. 50 microL extract was injected in the programmed temperature vaporizer injector using combined split-splitless evaporation. The extraction conditions were optimized both for MASE with agitation and for MASE with sonication. In the course of optimization the effect of extraction time, extraction temperature, agitation speed, solvent volume, pH, ionic strength and the addition of methanol were tested. Over 75% recovery was accomplished in the range of diesel oil hydrocarbons (n-C(9)-n-C(24)). The developed method was validated. Linearity, accuracy and precision were tested. The method showed excellent linearity between 1 and 1000 microgL(-1) for n-alkanes and between 0.05 and 50 mgL(-1) for gasoline. The method was tested with comprehensive GCxGC as well and found to be non-discriminative to all major compounds of diesel oil.

Effects of breeding habitat (woodland versus urban) and metal pollution on the egg characteristics of great tits (Parus major)

In an urban environment, birds are exposed to metals, which may accumulate in their tissues and cause oxidative stress. Female birds may eliminate these pollutants through depositing them into eggs, thus eggs become suitable bioindicators of pollution. In this study, we aimed to analyse whether eggshell spotting pattern, egg volume, eggshell thickness and egg yolk antioxidant (lutein, tocopherol, retinol and selenium) levels were related to the breeding area (woodland versus urban) and the metal levels in the eggshell of a small passerine species, the great tit (Parus major). In the urban habitat, soil and eggshells contained higher concentrations of metals, and soil calcium level was also higher than that in the woodland. Eggshell spotting intensity and egg volume did not differ between eggs laid in the woodland and the urban park, and these traits were not related to the metal levels of the eggshell, suggesting that these egg characteristics are not sensitive indicators of metal pollution. A more aggregated eggshell spotting distribution indicated a higher Cu concentration of the eggshell. We found that eggshells were thinner in the less polluted woodland habitat, which is likely due to the limited Ca availability of the woodland area. Great tit eggs laid in the urban environment had lower yolk lutein, retinol and selenium concentrations, however, as a possible compensation for these lower antioxidant levels, urban females deposited more tocopherol into the egg yolk. It appears that females from different breeding habitats may provide similar antioxidant protection for their offspring against oxidative damage by depositing different specific dietary antioxidants. Egg yolk lutein and retinol levels showed a negative relationship with lead concentration of the eggshell, which may suggest that lead had a negative impact on the amount of antioxidants available for embryos during development in great tits.

Analysis of Potential Migrants from Plastic Materials in Milk by Liquid Chromatography–Mass Spectrometry with Liquid–Liquid Extraction and Low-Temperature Purification

A simple and fast analytical method was developed for the determination of six UV stabilizers (Cyasorb UV-1164, Tinuvin P, Tinuvin 234, Tinuvin 326, Tinuvin 327, and Tinuvin 1577) and five antioxidants (Irgafos 168, Irganox 1010, Irganox 3114, Irganox 3790, and Irganox 565) in milk. For sample preparation liquid-liquid extraction with low-temperature purification combined with centrifugation was used to remove fats, proteins, and sugars. After the cleanup step, the sample was analyzed with high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). External standard and matrix calibrations were tested. External calibration proved to be acceptable for Tinuvin P, Tinuvin 234, Tinuvin 326, Tinuvin 327, Irganox 3114, and Irganox 3790. The method was successfully validated with matrix calibration for all compounds. Method detection limits were between 0.25 and 10 μg/kg. Accuracies ranged from 93 to 109%, and intraday precisions were <13%.

Darker eggshell spotting indicates lower yolk antioxidant level and poorer female quality in the Eurasian Great Tit (Parus major)

ABSTRACT Protoporphyrin pigment causes the red-brown eggshell colors; however, for many species, the function of this pigment is unknown. It has been proposed that eggshell pigmentation may strengthen the shell or that it may be a sexually selected signal, which advertises the quality of the female and that of her offspring to the male parent. In this study, we aimed to discover whether protoporphyrin-based eggshell pigmentation patterns of Eurasian Great Tits (Parus major) were related to female or egg quality. Additionally, we tested whether different methods of eggshell pigmentation estimation could be reliable predictors of eggshell protoporphyrin levels. We found that spot intensity, spot size, spotting coverage, and brown spot chroma indicated the protoporphyrin pigment concentration of the eggshell. Our results revealed that Eurasian Great Tit females that laid eggs with darker pigmentation had more lymphocytes in their circulation and had paler yellow breast and lower UV plumage reflectance, possibly indicating poorer health and individual quality. However, we did not find evidence that eggshell pigmentation patterns indicated the body condition, body size, or plasma oxidative status of females. Furthermore, we found that eggs with darker spots contained lower concentrations of antioxidants in the yolk. High protoporphyrin levels may be detrimental to females as they may cause oxidative damage, and this may be why birds that laid eggs with darker spots deposited lower amounts of antioxidants into the egg yolk. Shell spot darkness may also indicate territory quality, as females that laid smaller clutches also laid eggs with higher eggshell pigmentation levels. Thus, our results suggest that shell spot darkness may indicate the state of health of the female, egg yolk antioxidant level, and possibly also the quality of the territory.

Effects of environmental conditions on the egg mass, yolk antioxidant level, eggshell thickness and eggshell spotting patterns of Great Tits (Parus major)

AbstractAvian mothers can influence the fitness of their offspring by resource investment into the egg. Allocation of macro- and micronutrients into the eggs may be costly for the female, therefore, we expect that resource investment may be affected by the environmental and social conditions the mother experiences during egg formation. Here, we investigated whether environmental circumstances experienced by the reproducing female exert an influence on egg mass, yolk antioxidant (lutein and tocopherol) concentration, eggshell thickness, and the eggshell spotting patterns of Great Tits (Parus major). Our study showed that when caterpillars were less abundant, in colder weather and when breeding in an area of higher local breeding density, female Great Tits laid eggs of lower mass, suggesting that adverse environmental circumstances constrain the macronutrient investment into the eggs. However, we found no evidence that yolk antioxidant concentration and eggshell thickness were affected by environmental factors. Female Great Tits may use their endogenous stores or spend more time and energy in finding sufficient amounts of dietary antioxidants and calcium under unfavourable environmental conditions, which may have a cost effect on their own conditions. We found that birds that bred in the beginning of the season laid eggs with darker eggshell spots. Moreover, females laid more spotted eggs in colder weather and when breeding in higher density areas. Our results suggest that Great Tits deposit more of the potentially harmful pro-oxidant protoporphyrin pigment into the eggshell under unfavourable environmental conditions.ZusammenfassungAuswirkungen von Umweltbedingungen auf Eimasse, Antioxidantiengehalt des Dotters, Schalendicke und Fleckung der Eischale bei Kohlmeisen (Parus major) Vogelmütter können die Fitness ihrer Nachkommenschaft durch die Ressourcen beeinflussen, die sie in ein Ei investieren. Die Abgabe von Makro- und Mikronährstoffen an die Eier kann für das Weibchen kostspielig sein, daher ist zu erwarten, dass die Investition von Ressourcen durch Umweltbedingungen und soziale Faktoren beeinflusst wird, denen die Mutter während der Eibildung ausgesetzt ist. Hier untersuchten wir, inwieweit die Umweltbedingungen, denen das Weibchen während der Fortpflanzung ausgesetzt war, einen Einfluss auf die Eimasse, die Konzentration von Antioxidantien (Lutein und Tocopherol) im Dotter, die Schalendicke und die Fleckung der Eischalen von Kohlmeisen (Parusmajor) ausübten. Unsere Studie zeigte, dass Kohlmeisen-Weibchen in Zeiten geringeren Raupenangebots, bei kälterem Wetter und in Brutgebieten mit höherer lokaler Brutdichte Eier von geringerer Masse legten, was darauf hindeutet, dass ungünstige Umweltbedingungen die Investition von Makronährstoffen in die Eier beschränken können. Allerdings fanden wir keine Hinweise darauf, dass die Konzentration von Antioxidantien im Dotter und die Schalendicke durch Umweltfaktoren beeinflusst wurden. Unter ungünstigen Umweltbedingungen können Kohlmeisen-Weibchen körpereigene Reserven angreifen oder mehr Zeit und Energie auf die Suche nach ausreichenden Mengen nahrungsgebundener Antioxidantien und Kalzium aufwenden, was zu Lasten ihrer eigenen Kondition gehen kann. Wir stellten fest, dass früh in der Saison brütende Vögel Eier mit dunklerer Schalenfleckung legten. Außerdem legten die Weibchen bei kälterem Wetter und in dichter besetzten Brutgebieten mehr gefleckte Eier. Unsere Ergebnisse deuten an, dass Kohlmeisen unter ungünstigen Umweltbedingungen mehr des potenziell schädlichen prooxidativen Pigments Protoporphyrin in der Eischale ablagern.

Identification of uranium signatures relevant for nuclear safeguards and forensics

The paper describes the applicability of different characteristics (signatures) in nuclear safeguards and forensics for assessment of uranium material provenance in terms of production process. The study follows a uranium ore concentrate production from an ore to a U3O8 product. It turned out that rare-earth elemental pattern, radiochronometry (age of ore body and material production date), sulphur and organic impurities are useful to find out the origin or history of the material, while certain trace-elements and isotopics of Pb or Sr were found to be inconclusive. The results will be important to understand the signatures in nuclear safeguards and forensics.