Development and optimisation of a generic micro LC-ESI-MS method for the qualitative and quantitative determination of 30-mer toxic gliadin peptides in wheat flour for food analysis.

We sometimes see manufactured bakery products on the market which are labelled as being gluten free. Why is the content of such gluten proteins of importance for the fabrication of bakery industry and for the products? The gluten proteins represent up to 80 % of wheat proteins, and they are conventionally subdivided into gliadins and glutenins. Gliadins belong to the proline and glutamine-rich prolamin family. Its role in human gluten intolerance, as a consequence of its harmful effects, is well documented in the scientific literature. The only known therapy so far is a gluten-free diet, and hence, it is important to develop robust and reliable analytical methods to quantitatively assess the presence of the identified peptides causing the so-called coeliac disease. This work describes the development of a new, fast and robust micro ion pair-LC-MS analytical method for the qualitative and quantitative determination of 30-mer toxic gliadin peptides in wheat flour. The use of RapiGest™ SF as a denaturation reagent prior to the enzymatic digestion showed to shorten the measuring time. During the optimisation of the enzymatic digestion step, the best 30-mer toxic peptide was identified from the maximum recovery after 3 h of digestion time. The lower limit of quantification was determined to be 0.25 ng/µL. The method has shown to be linear for the selected concentration range of 0.25-3.0 ng/µL. The uncertainty related to reproducibility of measurement procedure, excluding the extraction step, has shown to be 5.0 % (N = 12). Finally, this method was successfully applied to the quantification of 30-mer toxic peptides from commercial wheat flour with an overall uncertainty under reproducibility conditions of 6.4 % including the extraction of the gliadin fraction. The results were always expressed as the average of the values from all standard concentrations. Subsequently, the final concentration of the 30-mer toxic peptide in the flour was calculated and expressed in milligrams per gram unit. The determined, calculated concentration of the 30-mer toxic peptide in the flour was found to be 1.29 ± 0.37 µg/g in flour (N = 25, s y = 545,075, f = 25 - 2 (t = 2.069), P = 95 %, two-sided).

Hazard identification of inhaled nanomaterials: making use of short-term inhalation studies.

A major health concern for nanomaterials is their potential toxic effect after inhalation of dusts. Correspondingly, the core element of tier 1 in the currently proposed integrated testing strategy (ITS) is a short-term rat inhalation study (STIS) for this route of exposure. STIS comprises a comprehensive scheme of biological effects and marker determination in order to generate appropriate information on early key elements of pathogenesis, such as inflammatory reactions in the lung and indications of effects in other organs. Within the STIS information on the persistence, progression and/or regression of effects is obtained. The STIS also addresses organ burden in the lung and potential translocation to other tissues. Up to now, STIS was performed in research projects and routine testing of nanomaterials. Meanwhile, rat STIS results for more than 20 nanomaterials are available including the representative nanomaterials listed by the Organization for Economic Cooperation and Development (OECD) working party on manufactured nanomaterials (WPMN), which has endorsed a list of representative manufactured nanomaterials (MN) as well as a set of relevant endpoints to be addressed. Here, results of STIS carried out with different nanomaterials are discussed as case studies. The ranking of different nanomaterials potential to induce adverse effects and the ranking of the respective NOAEC are the same among the STIS and the corresponding subchronic and chronic studies. In another case study, a translocation of a coated silica nanomaterial was judged critical for its safety assessment. Thus, STIS enables application of the proposed ITS, as long as reliable and relevant in vitro methods for the tier 1 testing are still missing. Compared to traditional subacute and subchronic inhalation testing (according to OECD test guidelines 412 and 413), STIS uses less animals and resources and offers additional information on organ burden and progression or regression of potential effects.

European external quality control study on the competence of laboratories to recognize rare sequence variants resulting in unusual genotyping results.

BACKGROUND: Depending on the method used, rare sequence variants adjacent to the single nucleotide polymorphism (SNP) of interest may cause unusual or erroneous genotyping results. Because such rare variants are known for many genes commonly tested in diagnostic laboratories, we organized a proficiency study to assess their influence on the accuracy of reported laboratory results. METHODS: Four external quality control materials were processed and sent to 283 laboratories through 3 EQA organizers for analysis of the prothrombin 20210G>A mutation. Two of these quality control materials contained sequence variants introduced by site-directed mutagenesis. RESULTS: One hundred eighty-nine laboratories participated in the study. When samples gave a usual result with the method applied, the error rate was 5.1%. Detailed analysis showed that more than 70% of the failures were reported from only 9 laboratories. Allele-specific amplification-based PCR had a much higher error rate than other methods (18.3% vs 2.9%). The variants 20209C>T and [20175T>G; 20179_20180delAC] resulted in unusual genotyping results in 67 and 85 laboratories, respectively. Eighty-three (54.6%) of these unusual results were not recognized, 32 (21.1%) were attributed to technical issues, and only 37 (24.3%) were recognized as another sequence variant. CONCLUSIONS: Our findings revealed that some of the participating laboratories were not able to recognize and correctly interpret unusual genotyping results caused by rare SNPs. Our study indicates that the majority of the failures could be avoided by improved training and careful selection and validation of the methods applied.

Reference materials (RMs) for analysis of the human factor II (prothrombin) gene G20210A mutation.

The Scientific Committee of Molecular Biology Techniques (C-MBT) in Clinical Chemistry of the IFCC has initiated a joint project in co-operation with the European Commission, Joint Research Centre, Institute of Reference Materials and Measurements to develop and produce plasmid-type reference materials (RMs) for the analysis of the human prothrombin gene G20210A mutation. Although DNA tests have a high impact on clinical decision-making and the number of tests performed in diagnostic laboratories is high, issues of quality and quality assurance exist, and currently only a few RMs for clinical genetic testing are available. A gene fragment chosen was produced that spans all primer annealing sites published to date. Both the wild-type and mutant alleles of this gene fragment were cloned into a pUC18 plasmid and two plasmid RMs were produced. In addition, a mixture of both plasmids was produced to mimic the heterozygous genotype. The present study describes the performance of these reference materials in a commutability study, in which they were tested by nine different methods in 13 expert laboratories. This series of plasmid RMs are, to the best of our knowledge, the first plasmid-type clinical genetic RMs introduced worldwide.