The statistical basis of standardization designs for diagnostic assays

Accuracy and long-term quality of laboratory diagnostic assays depend critically on the standardization process. In this note we review statistically typical procedures and designs used in standardization. Issues of practical relevance such as the number of systems, runs, and replicates to be involved in the standardization design, and quality control aspects, are addressed.     

Novel Heterospirocyclic 3-Amino-2H-azirines as Synthons for Heterocyclic α-Amino Acids

The heterospirocyclic N-methyl-N-phenyl-2H-azirin-3-amines (3-(N-methyl-N-phenylamino)-2H-azirines) 1a - d with a tetrahydro-2H-thiopyran, tetrahydro-2H-thiopyran, and a N-protected piperidine ring, respectively, were synthesized from the corresponding heterocyclic 4-carboxamides 2 by consecutive treatment with lithium diisopropylamide (LDA), diphenyl phosphorochloridate (DPPCI), and sodium azide (Scheme 4). The reaction of these aminoazirines with thiobenzoic acid in CH₂Cl₂ at room temperature gave the thiocarbamoyl-substituted benzamides 13a - d in high yield. The azirines 1a-d were used as synthons for heterocyclic α-amino acids in the preparation of tripeptides of the type Z-Aib-Xaa-Aib-N(Ph)Me ( 18 ) by following the protocol of the ‘azirine/oxazolone method’: treatment of Z-Aib with 1 to give the dipeptide amide 15 , followed by selective hydrolysis to the corresponding acid 16 and coupling with the 2,2-dimethyl-2H-azirin-3-amine 17 gave 18 , again in high yield (Scheme 5). With some selected examples of 18 , the selective deprotection of the amino and the carboxy group, respectively, was demonstrated (Scheme 6). The solid-state conformations of the protected tripeptides 18a - d , as well as that of the corresponding carbocyclic analogue 18e , were determined by X-ray crystallography (Figs. 1-3 and Tables 1-3). All five tripeptides adopt a β-turn conformation of type III or III′. The solvent dependence of the chemical shifts of the NH resonances (Fig. 6) suggests that there is an intramolecular H-bond between H-N(4) and O(11) in all cases, which is an indication that a relatively rigid β-turn structure also persists in solution. Surprisingly, the tripeptide acid 20a shows no intramolecular H-bond in the crystalline state (Fig. 7); O(11) is involved in an intermolecular H-bond with the OH group of the carboxy function.     

Synthesis of chiral self-assembling rhombs and their characterization in solution, in the gas phase, and at the liquid-solid interface

Chiral, enantiopure metallo-supramolecular rhombs self-assemble in solution through coordination of bis-pyridyl-substituted ligands with (en)M(NO3)2 (en = ethylenediamine, M = Pd(II), Pt(II)). Characterization by NMR and CD spectroscopy in solution and by ESI-FT-ICR mass spectrometry in the gas phase suggests that an equilibrium exists in water/methanol of a major 2:2 complex and a minor 3:3 complex of ligands and metal corners. In the gas phase, doubly charged 2:2 complexes fragment into two identical singly charged halves followed by metal-mediated C-H and C-C bond activation reactions within the ethylenediamine ligands. Electrochemical scanning tunneling microscopy (EC-STM) provides in situ imaging of the complexes even with submolecular resolution. Flat-lying rhombs are deposited under potential control from an aqueous electrolyte on a Cu(100) electrode surface precovered by a tetragonal pattern of chloride anions from the supporting electrolyte. Chirality induces the formation of only one domain orientation. Density functional calculations help to interpret the STM images.     

Absolute Configuration of Gilmaniellin

The absolute configuration of dechlorogilmaniellin ( 2 ) could be determined from its chiroptical data by applying coupled-oscillator theory to the CD couplet around 272 nm and from the sign of its n →π^* Cotton effect. Gilmaniellin ( 1 ) has the same absolute configuration.     

Ag5GeO4, das erste subvalente ternäre Silberoxid

Ag₅GeO₄, the First Subvalent Ternary Silver Oxide Applying high oxygen pressure the first subvalent ternary silver oxide Ag₅GeO₄ was obtained (for crystallographic data c.f. “Inhaltsübersicht”). Ag₅GeO₄ contains tetrahedral GeO₄^4?-ions besides [Ag₆]⁴⁺ clusters, which have not been observed in ternary silver oxides, so far. The electrical and magnetical properties prove the localisation of two paired electrons in each silver octahedron. The thermal decomposition occurs in two steps at 432.7 °C and 524.5°C.     

Spectrophotometric study of the acid-base equilibria of an imidazobenzodiazepine,midazolam, and its determination in pharmaceutical formulations

The acid-base behaviour of midazolam, a psychotropic drug derived from imidazobenzodiazepine family, has been studied spectro-photometrically. This compound hydrolyzes at pH values lower than 4. Reversible cleavage of the azomethine bond takes place and the open-ring compound is in equilibrium with the closed-ring compound (protonated form of the parent drug). Absorbance-time data (measured at 225 nm and for different pH values) have been evaluated by a pseudo-first order logarithmic approach, leading to different apparent kinetic constants, depending on pH and temperature. A simple mechanism of hydrolysis, corresponding to fast protonation and slow hydrolysis with opening of the ring is in good agreement with the kinetic results. From data obtained at pH values greater than 4, the deprotonation constant of the nitrogen atom at position 2 of the imidazole ring has been calculated and a pK_a value of 5.50 ± 0.05 obtained. In addition, a Spectrophotometric method has been developed which allows the determination of midazolam at concentrations from 1.23 × 10^–6 M to 3.38 × 10^–5 M. This method has been applied to a pharmaceutical formulation midazolam; the Dormicum error, in terms of relative standard deviation, was lower than 1.5%.     

Thermal analysis of thione compounds

The thermal behavior of thiourea (Tu), N-methylthiourea (MeTu), N, N-dimethylthiourea (Me₂Tu) and N, N, N, N-tetramethylthiourea (Me₄Tu) was investigated by means of conventional thermal analysis in O₂ and N₂ and IR spectroscopy of the residues on heating in air at various temperatures.At ordinary pressure, the compounds are thermally stable up to the melting points. After melting, they show high vapor pressure, combustion in O₂ atmosphere and isomerization to ammonium thiocyanate, with the exception of Me₄Tu.Financially supported by the Italian MURST.     

Uncertainty calculation for calibrators of the IFCC HbA1c standardization network

Within the last decade, the IFCC HbA1c standardization network has established the metrologically highest reference measurement procedure for HbA1c testing. Based on this procedure, reference calibrators are produced which in turn provide the starting point for the standardization of the manufactures routine HbA1c assays. According to the IVD directive, the uncertainty of the reference calibrators must be calculated and reported together with their assigned values. Within this article, we elaborate the uncertainty calculation according to GUM (guide to the expression of uncertainty in measurement) in detail. Finally, the results are validated by a simulation study.     

Solid phase extraction conditions for the selective isolation of drugs from biological fluids predicted using liquid chromatography

Summary A new method to predict the most suitable conditions for the solid phase extraction of 1,4-benzodiazepines and related compounds using C18 Sep-Pak carridges is proposed. The composition of the washing and elution solvents for the solid phase extraction of a test compound can be obtained from its capacity factor on a C18 HPLC column and an equation which relates capacity factors and solid phase extraction data of other similar compounds. The solid phase extraction data given in this paper can be used by others, there by saving considerable time and effort in the development of sample preparation methods. The suitability of the method was checked with two test compounds, showing good results.