Total selenium and selenomethionine in pharmaceutical yeast tablets: assessment of the state of the art of measurement capabilities through international intercomparison CCQM-P86.

Results of an international intercomparison study (CCQM-P86) to assess the analytical capabilities of national metrology institutes (NMIs) and selected expert laboratories worldwide to accurately quantitate the mass fraction of selenomethionine (SeMet) and total Se in pharmaceutical tablets of selenised-yeast supplements (produced by Pharma Nord, Denmark) are presented. The study, jointly coordinated by LGC Ltd., UK, and the Institute for National Measurement Standards, National Research Council of Canada (NRCC), was conducted under the auspices of the Comité Consultatif pour la Quantité de Matière (CCQM) Inorganic Analysis Working Group and involved 15 laboratories (from 12 countries), of which ten were NMIs. Apart from a protocol for determination of moisture content and the provision of the certified reference material (CRM) SELM-1 to be used as the quality control sample, no sample preparation/extraction method was prescribed. A variety of approaches was thus used, including single-step and multiple-step enzymatic hydrolysis, enzymatic probe sonication and hydrolysis with methanesulfonic acid for SeMet, as well as microwave-assisted acid digestion and enzymatic probe sonication for total Se. For total Se, detection techniques included inductively coupled plasma (ICP) mass spectrometry (MS) with external calibration, standard additions or isotope dilution MS (IDMS), inductively coupled plasma optical emission spectrometry , flame atomic absorption spectrometry and instrumental neutron activation analysis. For determination of SeMet in the tablets, five NMIs and three academic/institute laboratories (of a total of five) relied upon measurements using IDMS. For species-specific IDMS measurements, an isotopically enriched standard of SeMet (76Se-enriched SeMet) was made available. A novel aspect of this study relies on the approach used to distinguish any errors which arise during analysis of a SeMet calibration solution from those which occur during analysis of the matrix. To help those participants undertaking SeMet analysis to do this, a blind sample in the form of a standard solution of natural abundance SeMet in 0.1 M HCl (with an expected value of 956 mg kg(-1) SeMet) was provided. Both high-performance liquid chromatography (HPLC)-ICP-MS or gas chromatography (GC)-ICP-MS and GC-MS techniques were used for quantitation of SeMet. Several advances in analytical methods for determination of SeMet were identified, including the combined use of double IDMS with HPLC-ICP-MS following extraction with methanesulfonic acid and simplified two-step enzymatic hydrolysis with protease/lipase/driselase followed by HPLC-ICP-IDMS, both using a species-specific IDMS approach. Overall, satisfactory agreement amongst participants was achieved; results averaged 337.6 mg kg(-1) (n = 13, with a standard deviation of 9.7 mg kg(-1)) and 561.5 mg kg(-1) (n = 11, with a standard deviation of 44.3 mg kg(-1)) with median values of 337.6 and 575.0 mg kg(-1) for total Se and SeMet, respectively. Recovery of SeMet from SELM-1 averaged 95.0% (n = 9). The ability of NMIs and expert laboratories worldwide to deliver accurate results for total Se and SeMet in such materials (selensied-yeast tablets containing approximately 300 mg kg(-1) Se) with 10% expanded uncertainty was demonstrated. The problems addressed in achieving accurate quantitation of SeMet in this product are representative of those encountered with a wide range of organometallic species in a number of common matrices.

The hydrophobin EAS is largely unstructured in solution and functions by forming amyloid-like structures.

BACKGROUND: Fungal hydrophobin proteins have the remarkable ability to self-assemble into polymeric, amphipathic monolayers on the surface of aerial structures such as spores and fruiting bodies. These monolayers are extremely resistant to degradation and as such offer the possibility of a range of biotechnological applications involving the reversal of surface polarity. The molecular details underlying the formation of these monolayers, however, have been elusive. We have studied EAS, the hydrophobin from the ascomycete Neurospora crassa, in an effort to understand the structural aspects of hydrophobin polymerization. RESULTS: We have purified both wild-type and uniformly 15N-labeled EAS from N. crassa conidia, and used a range of physical methods including multidimensional NMR spectroscopy to provide the first high resolution structural information on a member of the hydrophobin family. We have found that EAS is monomeric but mostly unstructured in solution, except for a small region of antiparallel beta sheet that is probably stabilized by four intramolecular disulfide bonds. Polymerised EAS appears to contain substantially higher amounts of beta sheet structure, and shares many properties with amyloid fibers, including a characteristic gold-green birefringence under polarized light in the presence of the dye Congo Red. CONCLUSIONS: EAS joins an increasing number of proteins that undergo a disorder-->order transition in carrying out their normal function. This report is one of the few examples where an amyloid-like state represents the wild-type functional form. Thus the mechanism of amyloid formation, now thought to be a general property of polypeptide chains, has actually been applied in nature to form these remarkable structures.

Determination of defensin HNP-1, HNP-2, and HNP-3 in human saliva by using LC/MS.

The mass spectral profiling of saliva by liquid chromatography mass spectrometry in relation to particular types of pain is being examined. The aim is to develop a profile that could be useful for the assessment of patients and their treatment programs, as well as identifying unknown compounds observed in saliva. Defensin human neutrophil peptide-1 (HNP-1) and defensin HNP-2 were identified and confirmed, whereas defensin HNP-3 was tentatively identified. Linear calibration range of defensin HNP-1 and HNP-2 was 0.25 to 3 microg/ml with R(2) values of > 0.99 for both. The detection limit for defensin HNP-1 and HNP-2 was estimated at 0.1 microg/ml. The healthy subjects surveyed in this study had readily measurable salivary concentrations of defensin HNP-1 (8.6 +/- SD 8.0 microg/ml) and defensin HNP-2 (5.6 +/- SD 5.2 microg/ml).