Fully automated flow-through synthesis of secondary sulfonamides in a binary reactor system

A fully automated flow-through process for the production of secondary sulfonamides is presented. Primary sulfonamides were monoalkylated using a two-step "catch and release" protocol to generate library products of high purity. The automated flow synthesis platform incorporates four independent reactor columns and is able to perform automated column regeneration. A 48-member sulfonamide library was prepared as two 24-member sublibraries, affording library compounds in good yields and high purities without the need for further column chromatographic purification.     

The Analysis of Sulphonamide Drug Residues in Pork Muscle using Automated Dialysis

ABSTRACT

The aim of this study was to assess the suitability of automated dialysis, using a commercial system, for the analysis of sulphonamides in porcine tissue. The system involves automated dialysis, followed by trace enrichment of the dialysate prior to HPLC determination. The procedure was applied to the analysis of nine sulphonamide drug residues using reversed phase HPLC as the method of determination. Muscle samples were blended in saline, centrifuged and the supernatant was filtered before dialysis for an optimised time of 11 min. The resulting dialysate was concentrated on a reversed phase trace enrichment cartridge prior to HPLC analysis with UV detection at 280 nm. The developed method was evaluated by carrying out intra- and inter-assays on fortified porcine muscle. Mean recoveries, evaluated from the inter-assay study, were 80% or higher for the nine sulphonamides studied and the limit of determination for the method was 40 ng g^?1.     

Determination of aflatoxin M1 using a dialysis-based immunoaffinity sample pretreatment system coupled on-line to liquid chromatography. Reusable immunoaffinity columns.

A liquid chromatographic column-switching system containing a dialysis unit and an anti-aflatoxin immunoaffinity precolumn (immuno precolumn) is described for the automated determination of aflatoxin M1 in milk samples. Both a flat membrane dialysis unit working according to the flowing donor-flowing acceptor principle and a laboratory made hollow-fibre dialysis unit working according to the stagnant donor-flowing acceptor principle were evaluated. The hollow-fibre unit is superior with respect to repeatability (3% relative standard deviation) and detection limit (10 ng/l for aflatoxin M1 in milk), in spite of the fact that the overall recovery is only 6%. Interfering compounds, which would destroy the activity of the immuno precolumn, are efficiently removed from the system by the dialysis step; a single immuno precolumn can then be used for over 70 milk analyses. No decrease in the performance of either the immuno precolumn or the hollow-fibre dialysis unit is observed.     

Automated flow-injection determination of sulfonamides by the bratton—marshall reaction for clinical analysis,assays and dissolution studies of formulations

The automated flow-injection determination described for sulfonamides (2–20 mg l^?1, 5×10^?4?5×10^?3 M) is based on the Bratton-Marshall reaction. Destruction of the excess of nitrous acid is not required and the measurement rate is 72 per hour. Precision is excellent. The method is applied for the determination of sulfonamides in control serum and urine samples, in feeds and in formulations (by using the pseudo-titration technique), and for automated dissolution studies of tablets. The method was evaluated by analyzing spiked sample solutions; the recoveries obtained were 94–101% for serum, 98–103% for urine, 98–105% for feeds and 96–105% for formulations. Comparison of the pseudo-titrimetric method for sulfonamide assays with the HPLC method gave a mean difference of 1.3%.     

Validation of a Biacore method for screening eight sulfonamides in milk and porcine muscle tissues according to European decision 2002/657/EC

Sulfonamides are commonly used for prophylactic or therapeutic purposes in veterinary medicine. A maximum residue limit (MRL) for sulfonamides has been set at 100 microg/kg in milk and muscle. A multisulfonamide antibody was used for the development of 2 different Biacore protocols, one for the screening of milk samples, the other for muscle samples. Two different Biacore systems were used: Biacore X system (milk protocol), which is considered a research and development apparatus, and Biacore 3000 system (muscle protocol), which is a completely automated system used for high-throughput screening. This report describes the validation of semiquantitative immunological methods according to the European Decision 2002/657/EC "concerning the performance of analytical methods." The different performance characteristics (detection capability CCbeta, specificity/selectivity, precision, stability, and applicability) were determined in relation to the European Union MRL of 100 microg/kg for sulfonamides. The applicability of the method to porcine, bovine, and poultry muscle was studied. The detection capabilities CCbeta were calculated to be 40 microg/L in milk and 60 microg/kg in porcine, bovine, and poultry muscles. Eight different sulfonamides, of which 3 (sulfamethazine, sulfamerazine, and sulfadiazine) are authorized in France, were detected simultaneously, at or below the MRL level, with both Biacore systems.     

Strategy for the development of automated methods involving dialysis and trace enrichment as on-line sample preparation for the determination of basic drugs in plasma by liquid chromatography

Among the sample preparation techniques, dialysis followed by clean-up and enrichment of the dialysate on a pre-column has proved to be a useful approach for the LC determination of drugs in plasma. By use of sample processors, like the ASTED system, such bioanalytical methods can be fully automated, the dialysis and trace enrichment steps being directly coupled to LC. In order to facilitate the development of such automated methods, a strategy based on a decision tree has been elaborated. After the selection of appropriate conditions for the LC analysis, the decision tree provides information about suggested starting conditions and guidelines for the optimisation of the most important parameters likely to influence analyte recovery and method selectivity. The plasma samples are dialysed on a cellulose acetate membrane in the static-pulsed mode and the dialysate is enriched on a trace enrichment pre-column packed with octadecyl silica or with a strong cation-exchange material. This decision tree is until now restricted to the analysis of basic drugs in plasma. In order to demonstrate the applicability of this method development strategy, an automated procedure based on the coupling of dialysis with trace enrichment has been developed for the LC determination of antifungal agents (clotrimazole, econazole and miconazole) in plasma.     

Automated dilution in flow injection analysis with double on-line dialysis. A system for the determination of chloride in industrial effluents and plating bath solutions

Summary An automated on-line system for the determination of chloride in industrial effluents and plating bath solutions based on the concept of flow injection analysis is described. Samples with a very high chloride content up to 6 g/100 ml (60 g/l) are analysed by using an automated in-manifold double on-line dialysis technique. The sampling rate is 100 samples per hour. The method is suitable for the analysis of chloride with a relative standard deviation of better than 0.8%. The measurement is based on the red-coloured iron thiocyanate complex.     

Binding of sulfonamides to erythrocyte proteins and possible drug-drug interaction

The mode of binding of sulfonamides to erythrocyte proteins and possible drug-drug interaction between those compounds in erythrocytes resulting in changes in tissue levels were studied in rats using zonisamide (a novel antiepileptic agent possessing a sulfonamide group), several other sulfonamides and some antiepileptics without a sulfonamide group. In Michaelis-Menten plottings, the sulfonamide was found to be concentrated into erythrocytes in vitro and in vivo in a saturable high-affinity mode and in a linear low-affinity mode at ordinary therapeutic plasma levels through a simple diffusion process. Concentration in erythrocytes was affected by the presence of albumin in the extracellular medium. The cellular sulfonamide was readily replaced by extracellular sulfonamides in vitro. Even in vivo, erythrocyte levels of zonisamide were lowered by administration of other sulfonamides, although the plasma and tissue levels were not significantly changed since the plasma and tissue compartments of zonisamide were large relative to the erythrocyte compartment at ordinary therapeutic dose levels of zonisamide in animals and man. Therefore, disposition of zonisamide was not significantly influenced by other sulfonamides, but it is suggested that drug-drug interaction affecting the tissue levels may occur for a combination of sulfonamides with extremely different affinities for erythrocytes and low therapeutic plasma levels.     

Titanium(IV) isopropoxide mediated solution phase reductive amination on an automated platform: application in the generation of urea and amide libraries

Amine libraries and their derivatives are important targets for high throughput synthesis because of their versatility as medicinal agents and agrochemicals. As a part of our efforts towards automated chemical library synthesis, a titanium(IV) isopropoxide mediated solution phase reductive amination protocol was successfully translated to automation on the Trident(TM) library synthesizer of Argonaut Technologies. An array of 24 secondary amines was prepared in high yield and purity from 4 primary amines and 6 carbonyl compounds. These secondary amines were further utilized in a split synthesis to generate libraries of ureas, amides and sulfonamides in solution phase on the Trident(TM). The automated runs included 192 reactions to synthesize 96 ureas in duplicate and 96 reactions to synthesize 48 amides and 48 sulfonamides. A number of polymer-assisted solution phase protocols were employed for parallel work-up and purification of the products in each step.     

Combining Organometallic Reagents,the Sulfur Dioxide Surrogate DABSO,and Amines: A One‐Pot Preparation of Sulfonamides,Amenable to Array Synthesis

We describe a method for the synthesis of sulfonamides through the combination of an organometallic reagent, a sulfur dioxide equivalent, and an aqueous solution of an amine under oxidative conditions (bleach). This simple reaction protocol avoids the need to employ sulfonyl chloride substrates, thus removing the limitation imposed by the commercial availability of these reagents. The resultant method allows access to new chemical space, and is also tolerant of the polar functional groups needed to impart favorable physiochemical properties required for medicinal chemistry and agrochemistry. The developed chemistry is employed in the synthesis of a targeted 70 compound array, prepared using automated methods. The array achieved a 93 % success rate for compounds prepared. Calculated molecular weights, lipophilicities, and polar surface areas are presented, demonstrating the utility of the method for delivering sulfonamides with drug‐like properties.     

Binding of sulfonamides to erythrocytes and their components

Binding of zonisamide, a new antiepileptic sulfonamide derivative, was examined to human erythrocytes, their lysate and their carbonic anhydrase by centrifugation for cells or by ultrafiltration for the others. Scatchard plots revealed that the binding to intact and lysed cells was composed of high- and low-affinity components and that to carbonic anhydrase, of the high-affinity component alone. Parameters for high-affinity binding were similar in all three preparations and those for low-affinity binding were similar in the former two preparations. Dissociation constants for these bindings to erythrocytes were smaller than the dissociation constant for serum albumin. These results may explain the concentration of sulfonamides in red cells, and suggest the participation of cellular protein component(s) in addition to previously known carbonic anhydrase in the binding. Acetazolamide, sulthiame, zonisamide, hydrochlorothiazide and sulfanilamide inhibited carbonic anhydrase in a non-competitive manner to different extents. The Ki values of these sulfonamides were of the order of 0.1--0.2 of their respective Kd values determined by ultrafiltration, suggesting that under the present conditions, physicochemical interactions between sulfonamides and carbonic anhydrase primarily occur at common sites that affect the activity of the enzyme.     

Combining Organometallic Reagents,the Sulfur Dioxide Surrogate DABSO,and Amines: A One‐Pot Preparation of Sulfonamides,Amenable to Array Synthesis

We describe a method for the synthesis of sulfonamides through the combination of an organometallic reagent, a sulfur dioxide equivalent, and an aqueous solution of an amine under oxidative conditions (bleach). This simple reaction protocol avoids the need to employ sulfonyl chloride substrates, thus removing the limitation imposed by the commercial availability of these reagents. The resultant method allows access to new chemical space, and is also tolerant of the polar functional groups needed to impart favorable physiochemical properties required for medicinal chemistry and agrochemistry. The developed chemistry is employed in the synthesis of a targeted 70 compound array, prepared using automated methods. The array achieved a 93 % success rate for compounds prepared. Calculated molecular weights, lipophilicities, and polar surface areas are presented, demonstrating the utility of the method for delivering sulfonamides with drug‐like properties.     

自动化样品预处理的一项新技术——吉尔森ASTED系统及其应用（续完）

自动化样品预处理的一项新技术——吉尔森ＡＳＴＥＤ系统及其应用（续完）陈小华（华运有限公司香港上环永乐街１７２—１７６号永富商业大厦三楼）３应用吉尔森ＡＳＴＥＤ系统已经被广泛地应用在许多领域，如药理学、药物动力学、药物－蛋白质结合研究、毒理学、临床医学...     

Automated on-Line Dialysis and Column-Switching HPLC Determination of Flumequine and Oxolinic Acid in Fish Liver

Abstract

An automated method for residue analysis of oxolinic acid and flumequine in liver of Atlantic salmon is described. Oxolinic acid and flumequine are extracted from liver with 0.4 M phosphate buffer pH 10 and the extracts are automatically analysed by on-line dialysis and column-switching in an HPLC system. The limit of detection was 4 μg/kg for oxolinic acid and 7 μg/kg for flumequine with fluorescence detection. The on-line combination of dialysis and column-switching HPLC was shown to be a reliable technique for residue control of these drugs in fish liver.     

Fully automated column-switching HPLC determination of aflatoxin M1 in milk using dialysis as sample preparation

A procedure has been developed for the automated determination of aflatoxin M1 in decreamed milk, by using on-line dialysis and subsequent trace enrichment on a reverse phase column. After foreflush to the analytical column the determination is performed with fluorescence detection. Fully automated analysis within 10 min is thus possible with reproducible dialysis recoveries above 50% (CV is 3.3%, n = 20) and detection levels of 50 ng/kg.     

In‐depth insight into the methods of plasma protein‐drug interaction studies: Comparison of capillary electrophoresis‐frontal analysis,isothermal titration calorimetry,circular dichroism and equilibrium dialysis

Plasma protein‐drug binding assays are routinely performed during the early stages of drug discovery and development, which creates demand for an automated high‐throughput screening assay to increase laboratory efficiency. A comprehensive comparison of the four methods typically used for determining the binding parameters is presented in this study with respect to the above demand. Capillary electrophoresis‐frontal analysis, isothermal titration calorimetry, circular dichroism and equilibrium dialysis were used to study the affinity of human serum albumin for diclofenac and lidocaine. These model drugs were chosen due to their different physico‐chemical properties and different binding sites on the albumin molecule, also resulting in different binding strength. The binding parameters estimated under the conditions as similar as possible were comparable among all these approaches as well as to the literature values. Besides this, the comparison of the results and especially other considerations demonstrated the benefits and drawbacks of the selected methods, with capillary electrophoresis‐frontal analysis being the best candidate for such studies.     

Dialysis cells for controlled DNA: drug binding studies.

An efficient small volume accurate dialysis system has been designed, built and tested for proflavine binding to DNA based upon the side by side design used in the Franz diffusion cell. In a typical experiment 3 cm3 of DNA solution is added to one side and 3 cm3 of ligand to the other with a dialysis membrane between the two sides of 1 cm in diameter, thereby minimizing the area of dialysis membrane that the solutions are in contact with.     

Preliminary Evaluation of the Asted XL Dialysis System Towards its Applicability In Ligand Binding Assays

Abstract

In ligand binding assays, the separation of bound and free fraction of the labeled ligand is very important. Dialysis is generally overlooked as separation technique since it requires large volumes and long analysis times. The availability of the ASTED-system (Automated Sequential Trace Enrichment of Dialysates) might open ways for a complete automation of immuno assays including the separation step.

A well-documented radio-immuno assay for 3-keto-desogestrel (Org3236) was used to test the potentials of this system. The tritiated analog of Org3236 not only served as label in the immuno assay but was also used to trace this compound in the entire procedure. The dialysis efficiency increased with the dialysis time and with the flush rate of the recipient solvent (tris-HCI or phosphate buffer). Addition of methanol to recipient solvent had spectacular effects on the recovery. With tris-HCI buffer, 0.18 mL/min and 1.0 mL recipient solvent 2.5% of the label was collected. Addition of 50% methanol resulted in a 5-fold increase to 12%. Replacement of buffer by 100% methanol resulted in another 5% increase in dialysis efficiency which was accompanied by a reduction in the antibody binding in the donor compartment due to denaturation of the antibody.

The commercial availability of other types of membranes is essential to find optimal conditions for each analyte. A serious problem is the carryover effect between subsequent samples. Roughly 0.25% of label was collected in the next run which may have a substantial impact on the accuracy and precision of the assay. Renewal of the dialysis membrane might exclude this carry-over effect but is not a serious option with the available instrumentation.

Automated dialysis systems can be very valuable for ligand binding assays as soon as membranes become available for the analytes of interest which provide high recoveries (>40%) in 1 mL recipient solvent. Moreover their carry-over effect should be negligible or eliminated by more efficient rinsing procedures of the entire dialysis system. Temperature control is favourable for the immuno assays as well as for the dialysis process in that the kinetics are temperature dependent.     

Monitoring of veterinary drug residues by a combination of continuous flow techniques and column-switching high-performance liquid chromatography. I. Sulphonamides in egg, meat and milk using post-column derivatization with dimethylaminobenzaldehyde

By using a combination of a low-pressure continuous flow module and a column-switching high-performance liquid chromatographic system, procedures have been developed for the automated residue analysis of sulphonamides and selected other drugs in meat, egg and milk. Aqueous extracts are purified by on-line dialysis and subsequent trace enrichment on a short column containing silica-based or polymer material. After backflush to the analytical column, detection is performed either at 280 nm or, after post-column derivatization with p-dimethylaminobenzaldehyde, at 450 nm. With these procedures the fully automated determination of both polar and apolar sulphonamides and dapsone above 5-20 micrograms/kg is possible. Coefficients of variation are 4-10%, recoveries compared to standards are 85-90%. The methods developed were tested in routine veterinary drug control.     

Membrane separation in flow injection systems

A critical review of membranes for on-line separations in flow-through dialysers is given. Historical perspectives are briefly outlined. The fundamental aspects of dialysis as the selective separation of species through a semi-permeable membrane are discussed. Work done on theoretical principles by a number of authors is described and a simplified model is given. The basic components, locations thereof and the influence of various parameters on dialysis efficiency are highlighted. The value of dialysis as sampling preparation system for chromatography and reactor systems is outlined and the suitability of dialyser/flow injection systems in dilution, preconcentration and multicomponent analysis is evaluated. The review is concluded with a brief look at the use of dialysis in proteinligand binding studies and microdialysis.