Reference Protocol to Assess Analytical Performance of Higher Order Structural Analysis Measurements: Results from an Interlaboratory Comparison.

Measurements of protein higher order structure (HOS) provide important information on stability, potency, efficacy, immunogenicity, and biosimilarity of biopharmaceuticals, with a significant number of techniques and methods available to perform these measurements. The comparison of the analytical performance of HOS methods and the standardization of the results is, however, not a trivial task, due to the lack of reference protocols and reference measurement procedures. Here, we developed a protocol to structurally alter and compare samples of somatropin, a recombinant biotherapeutic, and describe the results obtained by using a number of techniques, methods and in different laboratories. This, with the final aim to provide tools and generate a pool of data to compare and benchmark analytical platforms and define method sensitivity to structural changes. Changes in somatropin HOS, induced by the presence of zinc at increasing concentrations, were observed, both globally and at more localized resolution, across many of the methods utilized in this study and with different sensitivities, suggesting the suitability of the protocol to improve understanding of inter- and cross-platform measurement comparability and assess analytical performance as appropriate.

Selectivity in capillary electrophoresis: the use of proteins.

Proteins, by their very diverse nature, provide a wide variety of options for generating selectivity in capillary electrophoresis (CE). Their use in different modes of CE will be considered in this review. Proteins added in solution to the background electrolyte allow separations to be made in a similar fashion to other electrokinetic chromatography methods, e.g., micellar separations. Alternatively, different immobilization schemes can be used to secure proteins within the capillary; these have included capillary electrochromatography with the protein grafted onto a silica support, or immobilization of the protein within a gel structure. Compounds varying in size from small inorganic ions to biopolymers may be bound by proteins. There is the potential for any sort of intermolecular interaction to play a role in the binding process (e.g., hydrophobic interactions, electrostatic interactions, etc.). Very specific high-affinity binding often occurs, but also there is often weaker, non-selective binding. Frequently the interactions of chiral compounds with proteins are stereoselective. Obtaining chiral selectivity has been one of the main applications of protein selectors in CE, and this use will be emphasized here in a discussion structured by type of protein. As well as utilizing the selectivity of proteins to develop separations, the role of CE in investigating ligand-protein interactions will be emphasized.

Immobilized penicillin G acylase as reactor and chiral selector in liquid chromatography.

In this paper, the use of penicillin G acylase (PGA) as a biocatalyst and as a chiral selector is described. Penicillin G-acylase is an interesting enzyme used in the manufacture of semisynthetic antibiotics and, in particular, in the production of 6-APA by hydrolysis of penicillin G. Five PGA-based HPLC columns have been prepared by using two different silica supports by employing two immobilization methods, namely "in situ" and "in batch". The effects of the immobilization techniques and of different silica pore size on the catalytic properties of the enzyme as well as the applicability of the PGA-bonded stationary phases as chiral selectors for a number of chiral drugs have been investigated. The HPLC columns based on immobilized PGA combine the hydrolytic activity and the chiral recognition properties of PGA, therefore they have been used for the development of a combined reaction-separation system for chiral and achiral substrates.

Validation of a RP-LC method for the simultaneous determination of isoniazid, pyrazinamide and rifampicin in a pharmaceutical formulation.

A simple and accurate liquid chromatographic method was developed and validated for estimation of isoniazid (ISN), pyrazinamide (PYR) and rifampicin (RIF) in combined dosage forms. Drugs were chromatographed on a reverse phase C18 column using a mobile phase gradient and monitored at the corresponding maximum of each compounds. Peaks were identified with retention time as compared with standards and confirmed with characteristic spectra using diode-array detector. Solution concentrations were measured on a weight basis to avoid the use of an internal standard. The method does not require any specific sample preparation except the use of a guard column. The method is linear (r(2)>0.999), precise (RSD%: 0.50% for ISN, 0.12% for PYR and 0.98% for RIF), accurate (overall average recovery yields: 98.55% for ISN, 98.51 for PYR and 98.56% for RIF) and selective. Due to its simplicity and accuracy the method is suitable for routine quality control analysis of antitubercolosis combination dosage form.

Design of chiral LC separations for calcium antagonists on alpha 1-acid glycoprotein and ovomucoid columns.

Three chiral calcium antagonist drugs, gallopamil and two dihydropyridine derivatives, have been successfully separated within short retention times using both the alpha 1-acid glycoprotein chiral stationary phase (Chiral-AGP) and the ovomucoid column (Ultron ES-OVM). Aqueous buffer at defined pH is modified by the addition of an organic component, in order to modulate the retention properties of each system. Optimization of pH and organic modifier is carried out using the modified simplex method, with Kaiser's peak separation function as a criterion. The influence of pH and percentage of organic modifier on retention, selectivity, resolution and column performance are discussed for the two dihydropyridines analysed on Chiral-AGP and Ultron ES-OVM stationary phases. A new method is proposed as a new chiral system suitability test for these protein-based phases, utilizing a racemic mixture of closely eluting verapamil enantiomers as a probe.

[Evaluation of the changes in glycosylated hemoglobin in a sample of hospitalized diabetic patients. Significance and limitations of this diagnostic method]. / Valutazione delle variazioni dell'emoglobina glicosilata in un campione di soggetti diabetici ospedalizzati. Significato e limiti di questa diagnostica.

Stable glycosylated hemoglobin measurement provides an index for plasma glucose control over an average three month period. It is not clear whether stable HbA1C can undergo rapid changes induced by short-term variations in glucose concentration. The present study was aimed at investigating whether variation of total HbA1C in response to changes in blood glucose, over a short period can be detected by routine methods of determination. Our result show that due to the sensitivity of the methods, stable HbA1C has to be considered only as an index of glucose control over long period of time.

[Clinical experience with a new antibiotic: cefmetazole]. / Esperienza clinica su un nuovo antibiotico: il cefmetazolo.

Cefmetazole is a new semisynthetic derivative with a broad antibacterial spectrum as shown by data from 34 research institutes in Japan. It has antibacterial action against various species of Gram+ and Gram- bacteria and is extremely stable for beta-lactamase because it has a 7-alpha-methoxy group. In addition, it has excellent antibacterial activity against indole-positive Proteus strains against which conventional Cephalosporins are ineffective. Results of clinical trials carried out on urinary tract, respiratory tract and cutaneous infections are reported. A total of 30 patients were examined; the route of administration was intravenous, the most frequent daily-dose was 2 g. Clinical and bacteriological effectiveness of cefmetazole was 80% with excellent activity against E. coli, Proteus, S. aureus and K. pneumoniae. No serious side effect was observed. The above results indicate that CMZ is largely effective and useful for infections due to Gram+ cocci and Gram- bacilli.

[Therapeutic use of glutathione]. / Utilizzo terapeutico del glutatione.

Liver pathology is very frequent in western countries. The numerous therapeutic approaches suggested so far for the prevention or limitation of liver damage in the course of hepatic diseases have often had disappointing results. Considering that at least part of the liver injury is caused by free radicals, the authors have tried an antioxidant, glutathione, as a hepatoprotective agent. Although particularly brilliant results were not achieved, clinical conditions and laboratory findings were improved in hall patients treated with glutathione.

[Evaluation in experimental animals of a mathematical model of foreseeing variations in values of stable glycosylated hemoglobin]. / Valutazione sull'animale da esperimento di un modello matematico per la previsione delle variazioni dei valori di emoglobina glicosilata stabile.

Assay of glycosylated hemoglobin provides reliable information on metabolic control in diabetes mellitus over a period of about 90 days. This is why it is currently used as a parameter of blood glucose control in diabetic patients. However, at present little is known about the kinetics of stable glycosylated hemoglobin variations as a result of circadian changes in blood glucose level. The authors describe a mathematical model which allows to foresee glycosylated hemoglobin variability as a result of alterations of blood glucose equilibrium.

The impact of capillary electrophoresis in drug analysis and bioanalysis.

Capillary electrophoresis (CE) has penetrated a wide variety of areas of separation science during the last ten years. The relative strengths and weaknesses of this technique are here overviewed, by citing the most recent literature and updated Medline search, keeping an eye on technology news, as technology and the development of CE and its applicability progress in parallel. The issues discussed include micellar electrokinetic chromatography (MEKC), which permits non-ionic solutes to be solubilized and separated. Enantioselective applications in CE exploit addition to the run buffer of cyclodextrins, macrocyclic antibiotics and proteins for analysis of chiral drugs and the state of the art of this field will be emphasized. CE has significant potential for drug metabolism studies, especially coupled with MS for high sensitivity detection and structural characterisation. Strategies for the analysis of drugs and metabolites in body fluids include on-capillary methods of sample concentration and single-step analyses with direct injection of body fluids on-column, where detection techniques other than conventional UV are essential to achieve adequate sensitivity. The potential contribution of the hybrid technique of capillary electrochromatography (CEC), which couples the separating power of reversed-phase HPLC and the high efficiency of CE, has recently attracted growing interest and is therefore discussed. Finally, validation issues which are peculiar to CE are illustrated.

Comparison of drug binding sites on rat and human serum albumins using immobilized-protein stationary phases as a tool for the selection of suitable animal models in pharmacological studies.

Immobilized serum albumins (rat and human) HPLC stationary phases have been used to investigate and to compare the binding sites of a number of analytes of pharmaceutical interest assessing the retention time as the chromatographic parameter which correlates the percentage of drug-binding of the corresponding free protein. Anti-inflammatory drugs with different molecular structure were chromatographed and the k' values were determined with a mobile phase based on 100 mM sodium phosphate buffer (pH 6.9) containing 6% n-propanol (v/v). The effect of the addition to the mobile phase of octanoic, decanoic and dodecanoic acids on the k' values enabled to elucidate the site of interaction between the solutes and the two albumins. The results indicate that there are some structural differences in the binding sites of the two albumins and also some quantitative differences with respect to the extent of drug-binding. Moreover the different stereoselective behaviour of the two albumins was studied by analysing some chiral anti-inflammatory drugs much as aryl-propionic acids.

Synthesis and chromatographic evaluation of molecularly imprinted polymers prepared by the substructure approach for the class-selective recognition of glucuronides.

Two series of molecularly imprinted polymers (MIPs) for the class-selective recognition of glucuronides have been prepared by using lipophilic substructures of the target analyte as template molecule and potent host monomers against oxyanions, that are expected to establish a strong stoichiometric interaction with the single carboxylic group of the template. The polymers were tested as stationary phases in liquid chromatography for specific recognition. A preliminary investigation of the imprinting properties of eleven MIPs was carried out, by comparing the retention time of the template and of structurally related compounds on the MIP column with that on the corresponding non-imprinted polymer (NIP). The two polymers showing the best performance were selected to further test cotinine, mycophenolic acid, testosterone and their respective glucuronides as model compounds. The high specificity obtained against glucuronides and the different chemical structure of the parent drug make the two MIPs class-selective imprinted receptors, also suitable for SPE application.

Chromatographic characterisation, under highly aqueous conditions, of a molecularly imprinted polymer binding the herbicide 2,4-dichlorophenoxyacetic acid.

The affinity of a 2,4-dichlorophenoxyacetic acid (2,4-D) molecularly imprinted polymer (MIP), which was synthesised directly in an aqueous organic solvent, for its template (2,4-D) was studied and compared with the affinity exhibited by two other reference (control) polymers, NIPA and NIPB, for the same analyte. Zonal chromatography was performed to establish the optimal selectivity, expressed as imprinting factor (IF), under chromatographic conditions more aqueous than those described so far in the literature. Frontal analysis (FA) was performed on columns packed with these polymers, using an optimized mobile phase composed of methanol/phosphate buffer (50/50, v/v), to extract adsorption isotherm data and retrieve binding parameters from the best isotherm model. Surprisingly, the template had comparable and strong affinity for both MIP (K = 3.8x10(4) M(-1)) and NIPA (K = 1.9x10(4) M(-1)), although there was a marked difference in the saturation capacities of selective and non-selective sites, as one would expect for an imprinted polymer. NIPB acts as a true control polymer in the sense that it has relatively low affinity for the template (K = 8.0x10(2) M(-1)). This work provides the first frontal chromatographic characterization of such a polymer in a water-rich environment over a wide concentration range. The significance of this work stems from the fact that the chromatographic approach used is generic and can be applied readily to other analytes, but also because there is an increasing demand for well-characterised imprinted materials that function effectively in aqueous media and are thus well-suited for analytical science applications involving, for example, biofluids and environmental water samples.

Riboflavin binding proteins as chiral selectors in HPLC and CE.

The term riboflavin binding proteins (RfBPs) is applied to several molecular species that play the important role of vitamin delivery to the developing embryo, thus becoming essential for the survival of the fetus. In addition to this physiological significance, these proteins have recently been found to be successful chiral selectors. In this review, the authors address the use of such proteins, both as columns for high performance liquid chromatography (HPLC) and as additives in capillary electrophoresis (CE), for the enantioseparation of several racemic drugs.

[Evolution of ventricular depolarization in the acute phase of inferior and posteroinferior myocardial infarction (author's transl)]. / Evoluzione della depolarizzazione ventricolare nella fase acuta dell'infarto inferiore e postero-inferiore

Twenty-five patients, hospitalized within 24 hours from the beginning of the precordiaglia due to acute diaphragmatic infarction, with or without a posterior extension, underwent the ECG and VCG recordings for the first consecutive twenty days and on the fortieth day, in order to study the development of ventricular depolarization. To diagnose an infarction of the diaphragmatic and posterior walls the usual ECG and VCG paramaters were used; the ECG was found to be less useful above all for the difficulty of measuring the variations, in the loss of the electrical forces. The electrodes were always placed in the same points suitably signed. Several evolutive modalities of the diaphragmatic infarction were observed; in 52% of the cases the loss of the inferior electrical forces reaches its maximum expression quickly, then it remains almost constant. In 12% of the cases the necrosis increases very clearly, tardily, between the ninth and fourteenth day. In 24% a precocious reduction of the electrical extension of the infarction on the eighth-twelfth day is observed. In 12% of the cases there are irregular oscillations during the whole acute phase. In all cases more or less stressed daily or cyclic oscillations were present. In nine cases an extension of the infarction to the posterior wall was evident; among these in five, two peaks are present; in two an initial increase, in the other two an initial decrease of the electrical extension of the necrosis; then, more or less stressed daily or cyclic oscillations. In another nine cases a single reading of the tracings do not let us diagnose a posterior extension; this is possible only through an "in series" reading. The clinical course and the humoral data do not give any contribution to the interpretation of the founded evolutive variabilities. The necrosis, therefore, must not be considered a zone of homogeneous and global stuffing, but composed of cellular groups in several and variable anatomo-metabolic situations conditioning the observed electrical instabilities.

Evaluation of new adsorbed coatings in chiral capillary electrophoresis and the partial filling technique.

When using chiral selectors and the partial filling technique in capillary electrophoresis, a suitable and reproducible suppression of the electroosmotic flow is still a challenging issue, and there are a number of reasons to find alternatives to the use of covalently coated capillaries for such a particular application. In this paper, new achiral, neutral, and water-soluble polymers are evaluated as adsorbed polymers for the suppression of electroosmotic flow (EOF) when employing chiral capillary electrophoresis and the partial filling technique. Four chiral selectors, namely a cationic cyclopeptide, vancomycin, human serum albumin and riboflavin binding protein have been chosen for this study and some analytes such as derivatized amino acids, promethazine and prilocaine have been used as test compounds. Reproducibility of migration times, resolution, and selectivity as well as efficiency are reported to critically evaluate the performance of the adsorbed coatings. Results are compared to parallel data obtained with fused-silica and polyvinyl alcohol-coated capillaries.

High-performance liquid chromatography post-column derivatization with fluorescence detection to study the influence of ambroxol on dipalmitoylphosphatidylcholine levels in rabbit eustachian tube washings.

In this work an appropriate high-performance liquid chromatography method was set up to guarantee specificity, sensitivity, precision and accuracy in analyzing dipalmitoylphosphatidylcholine (DPPC) in rabbit eustachian tube washings, as well as to determine its varying levels after administration of ambroxol chloride. The procedure is based on a post-column derivatization with fluorescence detection using 1,6-diphenyl-1,3,5-hexatriene which exhibits increased fluorescence in a lipid environment. DPPC was chromatographed on a Hypersil C18. The mobile phase for the isocratic elution consisted of 40 mmol/l choline chloride in methanol-tetrahydrofuran (97:3). Ambroxol was given to a group of New Zealand white rabbits at a dose of 30 mg/kg. A second group receiving vehicle only acted as controls.