Determination of phosphorus in small amounts of protein samples by ICP–MS

Inductively coupled plasma mass spectrometry (ICP-MS) is used for phosphorus determination in protein samples. A small amount of solid protein sample (down to 1 micro g) or digest (1-10 micro L) protein solution was denatured in nitric acid and hydrogen peroxide by closed-microvessel microwave digestion. Phosphorus determination was performed with an optimized analytical method using a double-focusing sector field inductively coupled plasma mass spectrometer (ICP-SFMS) and quadrupole-based ICP-MS (ICP-QMS). For quality control of phosphorus determination a certified reference material (CRM), single cell proteins (BCR 273) with a high phosphorus content of 26.8+/-0.4 mg g(-1), was analyzed. For studies on phosphorus determination in proteins while reducing the sample amount as low as possible the homogeneity of CRM BCR 273 was investigated. Relative standard deviation and measurement accuracy in ICP-QMS was within 2%, 3.5%, 11% and 12% when using CRM BCR 273 sample weights of 40 mg, 5 mg, 1 mg and 0.3 mg, respectively. The lowest possible sample weight for an accurate phosphorus analysis in protein samples by ICP-MS is discussed. The analytical method developed was applied for the analysis of homogeneous protein samples in very low amounts [1-100 micro g of solid protein sample, e.g. beta-casein or down to 1 micro L of protein or digest in solution (e.g., tau protein)]. A further reduction of the diluted protein solution volume was achieved by the application of flow injection in ICP-SFMS, which is discussed with reference to real protein digests after protein separation using 2D gel electrophoresis.The detection limits for phosphorus in biological samples were determined by ICP-SFMS down to the ng g(-1) level. The present work discusses the figure of merit for the determination of phosphorus in a small amount of protein sample with ICP-SFMS in comparison to ICP-QMS.     

Synthesis, structures and oxygen atom transfer catalysis of oxo-bridged molybdenum(V) complexes with heterocyclic bidentate ligands (N,X) X = S, Se

The dinuclear μ-oxomolybdenum(V) complexes [Mo₂O₃(PyS)₄] (1), [Mo₂O₃(PySe)₄] (2) and [Mo₂O₃(4-CF₃-PymS)₄] (3) were obtained by similar reactions of the [MoO₂Cl₂(DME)] precursor with the corresponding heterocyclic bidentate (N,X) ligands, X = S, Se, where PyS, PySe and 4-CF₃-PymS are the anions of pyridine-2-thione, pyridine-2-selenolato and 4-trifluoromethyl-2-pyrimidinthiol, respectively. All compounds were characterized by elemental analysis, IR, NMR, EI-MS spectroscopy and X-ray diffraction. The crystal structures of 1–3 all include the common [Mo₂O₃]⁴⁺ core. Compounds 1 and 2 are isostructural. The catalytic oxo-transfer properties of the molybdenum(V) compounds 1 and 2 were studied by the use of PPh₃ in DMSO with a considerably higher catalytic activity for the thionato containing complex 1 than for its selenolato containing analogue 2.     

Effect of cellulase on the pore structure of bead cellulose

An enzymatic treatment with cellulases fromTrichoderma viride was investigated in its effect on the pore structure of different types of bead cellulose. One objective of this study was to establish a suitable procedure for combined enzymatic treatment and solvent exchange that would restore the original pore structure which the beads had before drying without causing major losses in mechanical stability. Another aim was to further increase the accessible pore space and internal surface area for separation of large molecular weight compounds with regard to Chromatographic applications. Finally, an attempt was made to extend the findings for unsubstituted beads to the derivatives carboxymethyl (CM) and diethylaminoethyl (DEAE) cellulose beads. The enzymatically treated samples were characterized by microscopic methods and porosity measurements such as mercury porosimetry, nitrogen sorption and size exclusion chromatography. It was found that under controlled conditions the low-porosity surface layer of dried beads could be removed making the internal pore space accessible without reducing the resistance to deformation of the beads. Additionally, a shift in pore size distribution towards larger pores was observed. Supplementary swelling treatments in solvents of high swelling power could substantially restore the former porosity of the dried beads but did not enhance the accessibility to the cellulases to a considerable extent. Internal pore volume and surface area of the derivatives were dramatically increased in the case of DEAE upon enzymatic hydrolysis, however, at the expense of mechanical stability, whereas CM was found to be less affected.     

Simultaneous determination of bifonazole and tinctures of calendula flower in pharmaceutical creams by reversed-phase liquid chromatography

The aim of this research was to develop and validate a sensitive, rapid, easy, and precise reversed-phase liquid chromatography (LC) method for stability studies of bifonazole (I) formulated with tinctures of calendula flower (II). The method was especially developed for the analysis and quantitative determination of I and II in pure and combined forms in cream pharmaceutical formulations without using gradient elution and at room temperature. The influence on the stability of compound I of temperature, artificial radiation, and drug II used for the new pharmaceutical design was evaluated. The LC separation was carried out using a Supelcosil LC-18 column (25 cm x 4.6 mm id, 5 microm particle size); the mobile phase was composed of methanol-0.1 M ammonium acetate buffer (85 + 15, v/v) pumped isocratically at a flow rate of 1 mL/min; and ultraviolet detection was at 254 nm. The analysis time was less than 10 min. Calibration graphs were found to be linear in the 0.125-0.375 mg/mL (rI = 0.9991) and 0.639-1.916 mg/mL (rII = 0.9995) ranges for I and II, respectively. The linearity, precision, recovery, and limits of detection and quantification were satisfactory for I and II. The results obtained suggested that the developed LC method is selective and specific for the analysis of I and II in pharmaceutical products, and that it can be applied to stability studies.     

Synthesis and ligand properties of thianthrenophane

Thianthrenophane 1 has a cavity which offers enough room to potentially enable endohedral coordination to small ions or molecules. For the complexation of silver(I) perchlorate the complex stability constants of 1 logK1=5.45 +/- 0.13 and of thianthrene logK2=9.16 +/- 0.10 were determined by UV/Vis titration. Single competition transport experiments with ten metal salts demonstrate a very high selectivity of 1 as a carrier for silver(I) and a distinctly higher transport rate compared to carriers such as thianthrene and 1,4,8,11-tetrathiacyclotetradecane (14-ane-S4). Although the X-ray crystal structure analysis of the polymeric [Ag(1)]ClO4.(dioxane)7 complex shows an exohedral coordination to silver(I) we suggest that the formation of an endohedral [Ag(1)]+ complex is the explanation for the unusual carrier selectivity of silver(I) by 1 in bulk liquid membrane.     

Application of dried blood spots combined with high-performance liquid chromatography coupled with electrospray ionisation tandem mass spectrometry for simultaneous quantification of vincristine and actinomycin-D

A sensitive, specific and efficient high-performance liquid chromatography-tandem mass spectrometry assay for the simultaneous determination of vincristine and actinomycin-D in human dried blood spots is presented. Dried blood spots were punched out of a collection paper with a 0.25-in.-diameter punch. The analytes were extracted from the punched-out disc using sonication during 15 min in a mixture of acetonitrile–methanol–water (1:1:1, v/v/v) containing the internal standard vinorelbine. Twenty-microlitre volumes were injected onto the HPLC system. Separation was achieved on a 50 × 2.1 mm ID Xbridge C₁₈ column using elution with 1 mM ammonium acetate–acetonitrile (70:30, v/v) adjusted to pH 10.5 with ammonia and run in a gradient with methanol at a flow rate of 0.4 mL/min. HPLC run time was 6 min. The assay quantifies vincristine from 1 to 100 ng/mL and actinomycine-D from 2 to 250 ng/mL using a blood sample obtained by a simple finger prick. Validation results demonstrate that vincristine and actinomycin-D can be accurately and precisely quantified in human dried blood spots with the presented method. The assay can now be used to support clinical pharmacologic studies with vincristine and actinomycin-D.     

Novel Xylan Gels Prepared from Oat Spelts

Gels and pastes are used in many areas including application in the cosmetic, pharmaceutical and medical industries. A xylan derivative with pasty consistency was prepared in addition to the synthesis of xylan ethers and esters. The water content of the gel can be widely varied. The gel is formed although the xylan contains no cross-linking group. The hydrophobic and hydrophilic nature of the xylan gel can be adjusted by modifying the xylan. All of the gels can be dried using different methods (air drying, solvent exchange as well as freeze drying) and the dried gels can again disperse in water. The gels were characterised by porosity measurements (mercury intrusion), dynamic vapour sorption (DVS), scanning electron microscopy (SEM) and dispersion stability investigation respectively.     

Synthesis of alpha-D-Galp-(1-->3)-beta-D-Galf-(1-->3)-D-man,a terminal trisaccharide of Leishmania type-2 glycoinositolphospholipids

The synthesis of alpha-D-Galp-(1-->3)-beta-D-Galf-(1-->3)-D-Man, present in the type-2 glycoinositolphospholipids and in the core of the lipophosphoglycan of Leishmania, is described. The glycosyl aldonolactone approach, followed by reduction of the lactone with diisoamylborane, was utilized for the introduction of the internal galactofuranosyl unit and the trichloroacetimidate method for the O-glycosidation reaction. A high-yield synthesis of the beta-D-Galf-(1-3)-D-Man unit, also present in the lipopeptidophosphoglycan of Trypanosoma cruzi, is reported.