Comprehensive study of retention influencing gas chromatographic parameters affecting linear retention indices.

Retention in gas chromatographic systems has a central role in the identification of compounds even if detectors providing spectral information are used. But linear retention indices (LRI) of a single compound originating from multiple sources tend to vary greatly, probably due to differences in the experimental settings of the determinations. The effect of gas chromatographic parameters on LRI has been investigated using 41 compounds - previously identified from food contact plastics - and n-alkanes (n-C7-n-C40) used as reference series. As the reproducibility of LRIs under the same conditions is generally very good, the smallest changes in the settings often caused statistically significant, though irrelevant changes in the LRI values. Therefore, a multicriterial scoring-ranking system has been worked out to highlight the LRI value differences. Our results highlight that column length, heating rate, and film thickness can all be the reasons of the varying published LRI values. We also demonstrated that for the reproduction of LRI data, the chemistry (and not simply the polarity) of the stationary phase is crucial.

Oligonucleotide Formulations Prepared by High-Speed Electrospinning: Maximizing Loading and Exploring Downstream Processability.

The aim of this study was to develop antisense oligonucleotide tablet formulations using high-speed electrospinning. Hydroxypropyl-beta-cyclodextrin (HPßCD) was used as a stabilizer and as an electrospinning matrix. In order to optimize the morphology of the fibers, electrospinning of various formulations was carried out using water, methanol/water (1:1), and methanol as solvents. The results showed that using methanol could be advantageous due to the lower viscosity threshold for fiber formation enabling higher potential drug loadings by using less excipient. To increase the productivity of electrospinning, high-speed electrospinning technology was utilized and HPßCD fibers containing 9.1% antisense oligonucleotide were prepared at a rate of ~330 g/h. Furthermore, to increase the drug content of the fibers, a formulation with a 50% drug loading was developed. The fibers had excellent grindability but poor flowability. The ground fibrous powder was mixed with excipients to improve its flowability, which enabled the automatic tableting of the mixture by direct compression. The fibrous HPßCD-antisense oligonucleotide formulations showed no sign of physical or chemical degradation over the 1-year stability study, which also shows the suitability of the HPßCD matrix for the formulation of biopharmaceuticals. The obtained results demonstrate possible solutions for the challenges of electrospinning such as scale-up and downstream processing of the fibers.

Coupling of large volume injection with flow modulated two-dimensional gas chromatography.

The coupling of large volume injection (LVI) with comprehensive two-dimensional gas chromatography (GC × GC) can be a powerful technique in the analysis of trace-level complex samples. The coupling of LVI and GC × GC using a cost efficiently operable pneumatic modulator based on capillary flow technology has been examined. The aim was to optimize the LVI parameters in the case of samples with compounds covering a wide boiling range. For the optimization of LVI 25 microliters of a solution containing 27 target compounds modelling the composition and the boiling range of diesel oils was used. The injection parameters were evaluated for peak shapes, reproducibility and peak volumes relative to peak volumes obtained using cold splitless injection. For all GC × GC experiments a non-polar first column (Rxi-5ms) and a polar second column (HP-INNOWax) were applied. Through extensive method optimization solvent vent proved to be an unsuitable technique for the injection of compounds covering a wide boiling range: at lower vent times peaks split, while higher vent times caused severe losses of highly volatile compounds. Therefore, a split-splitless LVI method was optimized. Injection speed, split vent time, splitless vent time and vent flow during split vent have been optimized. The developed method is suitable for the coupling of LVI with flow modulated GC × GC. Using the optimized split-splitless LVI parameters no peak distortion of the target compounds was observed. The relative peak volumes were between 60-120% for all compounds (80-120% for 13 compounds).

Eggshell Biliverdin and Protoporphyrin Pigments in a Songbird: Are They Derived from Erythrocytes, Blood Plasma, or the Shell Gland?

Biliverdin and protoporphyrin pigments are deposited into the eggshell when the developing egg is in the shell gland. However, the site of synthesis of eggshell pigments is still uncertain, although it may influence the possible costs and potential functions of eggshell coloration in avian species. Eggshell pigments may be derived from red blood cells or be produced in other organs and then transferred to the shell gland, or they may be synthesized de novo in the shell gland. We studied in the canary (Serinus canaria) whether eggshell blue-green and brown pigmentations are associated with experimentally elevated anemia, female hematocrit level, immature erythrocyte percentage, and feces and plasma pigment levels during egg laying to find out the possible origin of eggshell pigments. We found no significant effects of hematocrit level or experimentally elevated anemia on intensity of eggshell blue-green and brown pigmentations; therefore, we consider it less likely that eggshell pigments are derived from erythrocytes. In addition, we found no significant associations between female feces biliverdin concentration during egg laying and intensity of eggshell blue-green pigmentation, suggesting that eggshell biliverdin may not originate from the spleen or liver. We found a negative association between plasma and feces protoporphyrin concentrations during egg laying and eggshell brown chroma. This result suggests that an increased production of protoporphyrin in the liver, which could have elevated plasma and feces protoporphyrin concentrations, could inhibit eggshell protoporphyrin pigmentation, probably through affecting enzymatic activities. We suggest that both pigments are produced de novo in the shell gland in the canary, but circulating pigment levels may influence shell gland pigment synthesis, thus connecting the physiological status of the female to eggshell coloration.

Determination of polycyclic aromatic hydrocarbons in infant formula using solid state urea clathrate formation with gas chromatography - tandem mass spectrometry.

An analytical method has been developed for the quantitative determination of the 16 EU priority polycyclic aromatic hydrocarbons (PAHs) in infant formula. Prior to analysis infant formula is subjected to sample preparation including the extraction of PAHs from the sample, transesterification of the triglyceride content of the extract to fatty acid methyl esters (FAMEs), and solid state urea clathrate formation to remove the FAMEs. Measurements were carried out with gas chromatography - tandem mass spectrometry. The developed method has been evaluated in terms of trueness, precision (repeatability), limit of detection (LOD), limit of quantification (LOQ), selectivity, sensitivity and linearity. Measurement range of 200-1500ngkg-1 was selected in accordance with the limit values set by the European Commission (835/2011/EC). LOD values were 75ngkg-1 for dibenzo(a,e)pyrene, dibenzo(a,i)pyrene and dibenzo(a,h)pyrene while 50ngkg-1 for the rest of the PAHs (benzo(c)fluorene, benzo(a)anthracene, cyclopenta(cd)pyrene, chrysene, 5-methylchrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(j)fluoranthene, benzo(a)pyrene, indeno(1,2,3-cd)pyrene, dibenzo(a,h)anthracene, benzo(g,h,i)perylene, dibenzo(a,l)pyrene), LOQ was 200ngkg-1 for all 16 PAHs. Trueness ranged between -29.7 and 29.8% while precision values were between 0.5 and 24.7% (RSD, n=3).

Combined cluster and discriminant analysis: An efficient chemometric approach in diesel fuel characterization.

Combined cluster and discriminant analysis (CCDA) as a chemometric tool in compound specific isotope analysis of diesel fuels was studied. The stable carbon isotope ratios (δ13C) of n-alkanes in diesel fuel can be used to characterize or differentiate diesels originating from different sources. We investigated 25 diesel fuel samples representing 20 different brands. The samples were collected from 25 different service stations in 11 European countries over a 2 year period. The n-alkane fraction of diesel fuels was separated using solid-state urea clathrate formation combined with silica gel fractionation. The stable carbon isotope ratios of C10-C24 n-alkanes were measured with gas chromatography-isotope ratio mass spectrometry (GC-IRMS) using perdeuterated n-alkanes as internal standards. Beside the 25 samples one additional diesel fuel was prepared and measured three times to get totally homogenous samples in order to test the performance of our analytical and statistical routine. Stable isotope ratio data were evaluated with hierarchical cluster analysis (HCA), principal component analysis (PCA) and CCDA. CCDA combines two multivariate data analysis methods hierarchical cluster analysis with linear discriminant analysis (LDA). The main idea behind CCDA is to compare the goodness of preconceived (based on the sample origins) and random groupings. In CCDA all the samples were compared pairwise. The results for the parallel sample preparations showed that the analytical procedure does not have any significant effect on the δ13C values of n-alkanes. The three parallels proved to be totally homogenous with CCDA. HCA and PCA can be useful tools when the examining of the relationship among several samples is in question. However, these two techniques cannot be always decisive on the origin of similar samples. The initial hypothesis that all diesel fuel samples are considered chemically unique was verified by CCDA. The main advantage of CCDA is that it gives an objective index number about the level of similarity among the investigated samples. Thus the application of CCDA supplemented by the traditionally used multivariate methods greatly improves the efficiency of statistical analysis in the CSIA of diesel fuel samples.

Determination of particulate phase polycyclic aromatic hydrocarbons and their nitrated and oxygenated derivatives using gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry.

An analytical method has been developed for the quantitative determination of polycyclic aromatic hydrocarbons (PAHs) and their nitrated and oxygenated derivatives (nitro- and oxy-PAHs respectively) in particulate matter (PM) samples. The sample preparation procedure included only a simple and quick sonication-assisted extraction step, clean-up based on addition of water and centrifugation as well as pre-concentration under N2 stream. The determination of 16 PAHs and 4 oxy-PAHs was carried out by gas chromatography-mass spectrometry, while liquid chromatography-tandem mass spectrometry was used in the case of the 11 investigated nitro-PAHs. The optimized method was fully evaluated in terms of trueness, precision (repeatability), limit of detection (LOD), limit of quantification (LOQ), sensitivity and linearity. The LOQ values ranged at pgm-3 level for the investigated PAHs (42pgm-3), oxy-PAHs (either 42 or 83pgm-3) and nitro-PAHs (either 83 or 167pgm-3) as well. The developed method was applied for the quantitative determination of PAHs, nitro- and oxy-PAHs in urban PM2.5 (particles with aerodynamic diameter smaller than 2.5µm) samples (n=36) collected in Budapest, Hungary. Almost 100% of the PM2.5 samples contained the investigated PAHs and oxy-PAHs in detectable and quantifiable amounts; however, the concentration of the nitro-PAHs was generally lower than the corresponding LOD/LOQ values. According to our results, during the 3-year long sampling campaign the concentration of benzo(a)pyrene never exceeded the limit value (1ngm-3) set by the European Commission.

Effects of Temperature and Duration of Storage on the Stability of Antioxidant Compounds in Egg Yolk and Plasma.

Antioxidants help protect tissues from oxidative damage caused by reactive oxygen species. In view of the widespread interest in egg yolk and plasma antioxidants in relation to environmental and life-history variables, there is a need for knowledge on the necessary conditions for sample storage, which is currently lacking. In this study, our purpose was to examine the kinetics of the degradation of lutein, retinol, and tocopherol in egg yolk samples and the total antioxidant capacity in plasma samples stored at three different temperatures (-20°, -50°, and -80°C) for 24 mo. We found that yolk lutein was stable during the study period. Yolk retinol and tocopherol showed a steep early decline and then remained relatively stable, but retinol showed significant losses at the end of the study period too. In contrast to our expectations, there was no difference in the stability of antioxidant compounds of egg yolk samples stored at different temperatures. Plasma OXY level was stable during the first 6 mo, showed a slight decline between 6 and 12 mo, and declined more intensely after 12 mo of storage. We suggest that studies focusing on the analysis of egg yolk retinol or tocopherol concentrations and total plasma antioxidant capacity should analyze the samples in the first 6-7 mo after collection. For the analysis of yolk lutein, samples can be stored for 24 mo without significant degradation. The storage temperature of -20°C seemed to be sufficient, as a lower temperature did not significantly affect the slope of degradation of the samples.

Solubility determination as an alternative to migration measurements.

Solubility values for six UV stabilisers (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) were determined in all the liquid food simulants (3% (m/V) acetic acid-water mixture, 10% (V/V), 20% (V/V), 50% (V/V) ethanol-water mixture and vegetable oil) proposed in European Union Regulation No. 10/2011/EC, as well as in fruit juice and cola drink. The applied method was obtained by modification of the method for the determination of water solubility as described in OECD guideline Test No. 105. By using ultrasonication and shorter equilibration time, the time demand of the solubility determinations were decreased notably. Solubility values proved to be lower than the specific migration limits (as specified in 10/2011/EC) at 25 °C for almost all target compounds in food simulants A, B, C and D1 as well as in fruit juice and cola drink. The exceptions were Tinuvin P and Irganox 3790 in simulant D1. The solubility in food simulant D2 was higher than 1000 µg ml(-1) for all target compounds. These results show that the solubility of some additives in food simulants can be so low that it makes migration studies for certain additive-food simulant pairs dispensable.

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.

Migration of Tinuvin P and Irganox 3114 into milk and the corresponding authorised food simulant.

Migration of Tinuvin P (UV stabiliser) and Irganox 3114 (antioxidant) from high-density polyethylene (HDPE) was studied. HDPE pieces were soaked in either milk (1.5% or 3.5% fat content) or 50% (v/v) ethanol-water mixture - the food simulant for milk as specified in Regulation No. 10/2011/EC. The obtained extracts were analysed by LC-MS/MS. For statistical assessment variography was used. It proved to be a useful tool for making a distinction between the early migration range and the equilibrium, despite the variance of the data. Regulation No. 10/2011/EC specifies 10 days of contact time for milk at 5°C. Our experiments with the food simulant with 24 dm(2) kg(-1) surface/mass ratio showed that both Tinuvin P and Irganox 3114 need less than 1 h to reach equilibrium. Furthermore, 10-day experiments with daily sampling showed that these additives are stable in milk, as well as in the food simulant. The effect of the concentration of the additives in HDPE was studied in the 0.01-5% (m/m) range. For both Tinuvin P and Irganox 3114 and all three extractants the migrated amount became independent of the concentration of the additive in the HDPE approximately at 1% (m/m). For Tinuvin P the food simulant gave a close estimate for the milk samples. However, using the food simulant for modelling the migration of Irganox 3114 into milk gave an overestimation with a factor of minimum 3.5. In the case of Tinuvin P special care must be taken, since the recommended amount in the HDPE can result in additive concentrations near or even over the specific migration limit (SML). However, Irganox 3114 cannot reach the SML either in milk or in the food simulant.

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%.