Investigation of Chlorpyrifos and Its Transformation Products in Fruits and Spices by Combining Electrochemistry and Liquid Chromatography Coupled to Tandem Mass Spectrometry

The identification of transformation products (TPs) of pesticides in food is a crucial task difficult to tackle, due to the lack of standards. In this work, we present a novel methodology to synthesize five main TP standards of the insecticide chlorpyrifos (CPF) and to investigate their occurrence in selected fruits and spices. TPs were electrochemically (EC) synthesized using a boron-doped diamond electrode (BDD) and identified by EC coupled online to mass spectrometry, LC-MS/MS, and high-resolution mass spectrometry. CPF and its TPs were analyzed in the food samples by LC-MS/MS on multiple reaction monitoring (MRM) after dispersive solid-phase extraction. A good recovery of 83–103% for CPF and 65–85% for TPs was obtained. Matrix effects, which cause signal suppression, ranged between 81 to 95% for all targeted analytes. The limit of detection and quantification for CPF were 1.6–1.9 and 4.9–5.7 μg/kg, respectively. Among investigated samples, CPF was determined in fresh lemon (104 μg/kg), fenugreek seed (40 μg/kg), and black pepper (31 μg/kg). CPF content in all samples was lower than the EU maximum residue level (MRL). The most frequently detected TPs were diethylthiophosphate and diethylphosphate. Other TPs, CPF oxon and trichloropyridinol, were also detected. Hence, EC is a versatile tool to synthesize TP standards which enables the determination of contaminants and residues in foodstuffs even if no commercial standards are available.     

Uncertainties in the determination of pyrrolizidine alkaloid levels in naturally contaminated honeys and comparison of results obtained by different analytical approaches

The contamination of honey with hepatotoxic pyrrolizidine alkaloids (PAs) is a well-known hazard for food safety. While management strategies and controls of the honey industry aim to reduce the PA levels, uncertainties remain with regard to the safety of regionally produced and marketed honey. In addition, a previous study showed large differences of results obtained after various periods of storage and apparent differences between the analytical results of different laboratories. Therefore, this study aimed at examining these uncertainties by monitoring the impact of storage on the PA and PA N-oxide (PANO) content of two freshly harvested honeys and on possible demixing effects caused by pollen settling. Additionally, three analytical approaches – target analysis with matrix-matched calibration or standard addition and a sum parameter method – were applied for a comparative analysis of 20 honeys harvested in summer 2016. All samples originated from Schleswig-Holstein in Northern Germany where the PA plant Jacobaea vulgaris is currently observed on a massive scale. The results of the time series analyses showed that PANO levels markedly decreased within a few weeks and practically reached the LOD 16 weeks after harvest. Tertiary PAs, by contrast, remained stable and did not increase as a consequence of PANO decrease. The experiments on a putative demixing, which may result in a heterogeneous distribution of PAs/PANOs, revealed that there was no such effect during storage of up to 12 weeks. A comparison of the PA/PANO levels obtained by different analytical approaches showed that in some cases the sum parameter method yielded much higher levels than the target approaches, whereas in other cases, the target analysis with standard addition found higher levels than the other two methods. In summary, the results of this study highlight uncertainties regarding the validity and comparability of analytical results and consequently regarding health risk assessment.     

Optimal conditions for mycelial growth of Schizosaccharomyces japonicus cells in liquid medium: it enables the molecular investigation of dimorphism

The non‐pathogenic dimorphic fission yeast, Schizosaccharomyces japonicus, could be a suitable model organism for investigation of the genetic background of mycelial growth, as it has a haploid chromosome set and its genome is sequenced. Since earlier results have suggested that its morphological transition required solid substrates, but molecular biological experiments would require hyphae production in a liquid medium, we wanted to find circumstances which would enable hyphae production in liquid media. Several external conditions were investigated, but the strongest inducer was fetal bovine serum (FBS). Its positive effect could be hampered by heat and was dependent on pH, temperature and concentration of the serum. Other protein‐containing compounds, such as peptone and bovine serum albumin or amino acids, proved to be ineffective or weak. Generally, the uninduced and induced mycelial growth of Sz. japonicus could be improved by lower external pH and higher temperature. Copyright © 2014 John Wiley & Sons, Ltd.     

Comparison of Rapid Methods for Detection of Giardia spp. and Cryptosporidium spp. (Oo)cysts Using Transportable Instrumentation in a Field Deployment

Reliable, sensitive, quantitative, and mobile rapid screening methods for pathogenic organisms are not yet readily available, but would provide a great benefit to humanitarian intervention units in disaster situations. We compared three different methods (immunofluorescent microscopy, IFM; flow cytometry, FCM; polymerase chain reaction, PCR) for the rapid and quantitative detection of Giardia lamblia and Cryptosporidium parvum (oo)cysts in a field campaign. For this we deployed our mobile instrumentation and sampled canal water and vegetables during a 2 week field study in Thailand. For purification and concentrations of (oo)cysts, we used filtration and immunomagnetic separation. We were able to detect considerably high oo(cysts) concentrations (ranges: 15-855 and 0-240 oo(cysts)/liter for Giardia and Cryptosporidium, respectively) in 85 to 300 min, with FCM being fastest, followed by PCR, and IFM being slowest due to the long analysis time per sample. FCM and IFM performed consistently well, whereas PCR reactions often failed. The recovery, established by FCM, was around 30% for Giardia and 13% for Cryptosporidium (oo)cysts. It was possible to track (oo)cysts from the wastewater further downstream to irrigation waters and confirm contamination of salads and water vegetables. We believe that rapid detection, in particular FCM-based methods, can substantially help in disaster management and outbreak prevention.     

产品开发过程中的危机管理

产品开发过程的危机通常无法避免,而应对危机并非一件易事．欲使危机的负面影响降至最低,运用规范的方法应对危机尤显重要．针对目前产品开发的行为特征,提出产品开发过程中应对危机的行为模式．     

Zum Einfluß von Dauerbelastung,Temperatur- und Klimawechsellagerung auf die Querzugfestigkeit von Fichtenholz

For timber the tensile stress perpendicular to the grain is a critical type of stress. The influence of long-term loading, temperature and climate changes upon the tensile strength perpendicular to the grain of timber is shown. The results prove that the greatest loss of strength happened after long-term outdoors loading. While storing under alternating climate conditions, great crepp strains appeared, and it is to be presumed that the damages of wooden components which fail across the grain are less a problem of “tensile stress perpendicular to the grain” than a problem of “strain under tension perpendicular to the grain”.     

Effect of pretreatment with prostaglandin F2alpha before resynchronization of ovulation on fertility of lactating dairy cows

Our objective was to assess the effect of pretreatment with PGF_2α 12 d before initiation of a protocol for resynchronization of ovulation (Resynch) using an Ovsynch protocol. Lactating Holstein cows diagnosed not pregnant 31 d after a timed artificial insemination (TAI) were randomly assigned to initiate the Resynch protocol 32 d after TAI (n = 255; RES), or receive 25 mg of PGF_2α 34 d after TAI and initiate the Resynch protocol 12 d later at 46 d after TAI (n = 272; PGF+RES). Within each treatment, a subset of cows were examined using transrectal ultrasonography to determine ovulatory response to the first GnRH injection of the Resynch protocols or a blood sample was collected to determine serum progesterone (P₄) at initiation of the Resynch protocol, or both. Overall, PGF+RES cows had more pregnancies per artificial insemination (P/AI) than RES cows 66 d after TAI (35.2 vs. 25.6%), whereas pregnancy loss from 31 to 66 d after TAI was greater for RES than PGF+RES cows (17.1 vs. 7.6%). Although P/AI was greater for cows with high (≥1.0 ng/mL) vs. low (<1.0 ng/mL) P₄ at the first GnRH injection of the Resynch protocols, treatment did not affect the proportion of cows with low P₄ at the first GnRH injection of the Resynch protocols. Overall, no effect of treatment on ovulatory response to the first GnRH injection of the Resynch protocols was detected. We conclude that pretreatment with PGF_2α 12 d before initiation of the Resynch protocol increased P/AI 66 d after TAI for cows with serum P₄ concentration >1.0 ng/mL at the first GnRH injection of the Resynch protocol and decreased pregnancy loss from 31 to 66 d after TAI. This modified resynchronization protocol may be a useful strategy for reproductive management of lactating dairy cows.     

Regulation of genes encoding proteolytic enzymes during mammary gland development

The mammary gland undergoes extensive tissue remodelling during each lactation cycle. During pregnancy, the epithelial compartment of the gland is vastly expanded (Benaud et al. 1998). At the end of lactation the epithelial cells undergo apoptosis and adipocyte differentiation is induced (Lilla et al. 2002). Ductal and alveolar growth during puberty and pregnancy, and the involution process require the action of proteolytic enzymes (including matrix metalloproteinases, plasminogen and membrane-peptidases) and the corresponding genes are activated during these periods (Benaud et al. 1998; Alexander et al. 2001). Matrix metalloproteinases (MMP) are expressed in several cell types of the mammary gland including stromal fibroblasts (e.g., MMP3, MMP2), epithelial cells (e.g., MMP7 or MMP9), adipocytes (e.g., MMP2) and lymphoid cells (e.g., MMP9) (Crawford et al. 1996; Lund et al. 1996; Wiseman et al. 2003). A number of knock-out mice, which are deficient for individual MMP genes (e.g., MMP2, MMP3) or plasminogen, display alterations to mammary gland structure and impairment of lactation (Lund et al. 1999; Wiseman et al. 2003).