Ernst Küsters

HMG-CoA reductase inhibitors and P-glycoprotein modulation

Five 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase inhibitors (statins), (e.g. atorvastatin, fluvastatin, lovastatin, pravastatin and simvastatin), were investigated for their ability to reverse P‐glycoprotein (P‐gp) mediated rhodamine 123 (R123) transport in a murine monocytic leukaemia cell line that over‐expresses the multi‐drug resistance protein 1a/b (mdr1a/1b). P‐gp modulation was studied by a fluorimetric assay and confocal microscopy by means of R123 efflux and uptake experiments, respectively. Atorvastatin acid, methyl ester and lactone, lovastatin lactone and simvastatin lactone inhibited R123 transport in a concentration‐dependent manner. Lovastatin acid, simvastatin acid, fluvastatin and pravastatin did not show a significant inhibition of the R123 transport in our cell system. Atorvastatin methyl ester and lactone showed the highest affinities for P‐gp and results were comparable for both methods. In conclusion, monitoring of R123 transport in living cells by confocal microscopy in addition to fluorimetric assay is a sensitive tool to study P‐gp affinity in drug screening that is especially useful for early phases of drug development.

Design of the simulated moving bed process based on adsorption isotherm measurements using a perturbation method

Abstract The design of a simulated moving bed (SMB) chromatography process for the enantioseparation of 1-phenoxy-2-propanol with Chiralcel OD as stationary phase is described using equilibrium theory and a dispersion model. The most essential prerequisite for reliable process simulation is the proper experimental determination of the corresponding adsorption isotherms. This paper evolved from the need to: (i) elaborate a technique for adsorption isotherm measurement based on a perturbation method; and (ii) to demonstrate the applicability of the equilibrium-dispersion model for quick process design. The accuracy of the obtained adsorption isotherms was evaluated by comparison with results that have been obtained independently using the classical adsorption–desorption procedure. As the main result, it turned out that the suggested SMB design concept based both on adsorption isotherms measured with the perturbation method and on the equilibrium-dispersion model could be verified experimentally.

Influence of temperature on the enantioseparation of rolipram and structurally related racemates on Chiracel-OD

Abstract The temperature dependence of the chiral separations of rolipram and structurally related racemates was investigated in liquid chromatography with Chiralcel-OD as staionary phase. The thermodynamic data reveal that the enantioseparation of rolipram and two other racemates belong to the unusual case of entropy-controlled separations whereas for the remaining racemates the expected enthalpy-controlled separations were observed. In particular, at 20°C not even a partial separation is obtained for rolipram whereas a complete baseline resolution is achieved at 65°C.

Rapid and highly automated determination of morphine and morphine glucuronides in plasma by on-line solid-phase extraction and column liquid chromatography.

A high-performance liquid chromatography (HPLC) method has been developed for the determination of morphine and its main metabolites, morphine-6-glucuronide (M-6-G) and morphine-3-glucuronide (M-3-G), in plasma or cerebrospinal fluid. Samples were extracted using on-line solid-phase extraction followed by reversed-phase HPLC with fluorescence detection. Recoveries of 20 ng morphine and morphine glucuronides in plasma were over 95%. The limit of detection using 400 microliters of a biological matrix was 0.85, 3.4 and 1.0 ng/ml of M-3-G, M-6-G and morphine, respectively. Inter- and intra-day assay precision was better than 10%. The main advantages of the present described method are increased recoveries (> 95%) and a high degree of automation allowing a high speed in routine analysis. The time required for the fully automated analysis of one sample was less than 26 min.

Synthesis of regioselectively substituted cellulose derivatives and applications in chiral chromatography

Various cellulose-2,3-bis-arylcarbamate-6-O-arylesters and cellulose-2,3-bis-arylester-6-O-arylcarbamates, designed to test the possible combined effects of the known tris-arylcarbamate and tris-arylester classes, were synthesized with high regioselectivity at O-C(6), and their use as CSPs in liquid chromatography for enantiomeric separations was investigated. The separations obtained with the synthesized CSPs were compared to the separations achieved on a self-packed reference column, consisting of cellulose-tris-(3,5-dimethylphenyl-carbamate) as CSP standard. Among the synthesized, regioselectively substituted cellulose derivatives, 2,3-bis-O-(3,5-dimethylphenylcarbamate)-6-O-benzoate-cellulose and 2,3-bis-O-(benzoate)-6-O-(3,5-dichlorophenylcarbamate)-cellulose gave the best CSPs for the separation of the test racemates. CSPs from regioselectively substituted cellulose derivatives seem to exhibit higher selectivities than cellulose-tris-(3,5-dimethylphenylcarbamate) for certain classes of racemic compounds. Chirality 10:294–306, 1998.

Optimization strategy for simulated moving bed systems

Abstract Simulated moving bed (SMB) systems are of rising interest in the purification of pharmaceuticals or specialty chemicals (racemic mixtures, proteins, organic acids, etc.). This is particularly due to their advantage in solvent reduction, obtained productivity and purities as well as investment costs in comparison to eluent chromatography. This paper evolved from the need for a readily available algorithm in order to find optimal operating conditions for SMB chromatography systems with nonlinear or coupled adsorption isotherms. The herein developed algorithm is based on a semi-deterministic two-step approach. First, optimal operating conditions with regard to an objective function are found by knowing adsorption measurements only. In a second step actual SMB results are used to adapt the initial isotherm measurements and match the simulation with the experiment. The algorithm is verified on a bench-scale SMB unit applied for the separation of a racemic epoxide with Chiralcel-OD as stationary phase. The developed algorithm improved the productivity of the investigated experimental design by 24%.

Application of centrifugal counter-current chromatography to the separation of macrolide antibiotic analogues. II. Determination of partition coefficients in comparison with the shake-flask method

The separation of compounds by centrifugal counter-current chromatography (CCC) is based on the difference in partition behaviour of solutes between the two immiscible liquid phases. Therefore, Δlog K, defined as the difference of partition coefficients of the solutes, is a key criterion in the solvent system selection for the CCC. Partition coefficients (log K) of macrolide antibiotic analogues can be measured by different ways, such as direct shake-flask method or indirect HPLC analysis. However, there are also some clear limitations and shortcomings with these methods. As an alternative, a Quattro Counter-Current Chromatograph was examined to measure log K for a series of macrolide antibiotic analogues and compared with the classical shake-flask method. The experimental results shows that partition coefficients measured by CCC are highly correlated with those determined by shake-flask method. The major advantages of the CCC method are rapidity, precision, and the possibility to determine log K of analytes in mixtures. Therefore, CCC is a promising alternative to directly measure the partition coefficients of macrolide antibiotics analogues in purification process development.

Chromatographic resolution of synthetically useful chiral glycine derivatives by high-performance liquid chromatography

Abstract The preparation of one oxo-oxazolidine and two dihydro-imidazole derivatives, useful chiral building blocks for amino acid syntheses, in racemic form is described. The racemic mixtures were separated on an analytical and preparative scale using high-performance liquid chromatography on chiral stationary phases. Detailed procedures for the separations are given, the results obtained are discussed, and the enantiopure compounds are fully characterized.

Application of coil centrifugal counter-current chromatography to the separation of macrolide antibiotic analogues. III. Effects of flow-rate, mass load and rotation speed on the peak resolution

As the third part of our methodology studies on the application of centrifugal counter-current chromatography to the preparative separation of macrolide antibiotic analogues, we have investigated the effects of various parameters on the retention of stationary phase and peak resolution. Our results show that the retention percentage of the stationary phase has linear relationships with both flow-rate at 1 to 3 ml/min and rotation speed at 100 to 700 rpm, but their correlation coefficients are negative (-1.000) and positive (0.9821), respectively. The peak resolution (Rs) is inversely proportional to the flow-rate (Fr) and mass load (Ml), but directly proportional to the rotation speed (Rrev). Their correlation coefficients in linear regression for the preparative separation in laboratory scale are -0.981 to -1.000 for Rs=a+bFr at flow-rates of 1 to 3 ml/min, -0.929 to -0.993 for Rs=a+bMl at mass loads of 12.5 to 100 mg, and 0.975 to 0.998 for Rs=a+bRrev at rotation speeds of 300 to 700 rpm, respectively. Preparative separation of six very closely related macrolide antibiotics, which belong to ascomycin and rapamycin analogues, has also been successfully achieved under optimized conditions.

Application of centrifugal counter-current chromatography to the separation of macrolide antibiotic analogues: I. Selection of solvent systems based on solubility and partition coefficient investigations

As the first part of our studies on counter-current chromatography, the methodology for selecting suitable solvent systems was established based on detailed investigations of solubility and partition coefficients (log K) of macrolide antibiotic analogues. The solubility of two important macrolides, ascomycin and FK-506, was measured in a series of common solvents, where their polarities were ranked with dielectric constants. The partition coefficients of the two macrolides were compared in various binary, ternary, quaternary solvent systems. Hexane-tert.-butyl methyl ether-methanol-water system was selected based on suitable log K of solutes and hydrogen-bonding properties of solvents. In the further optimisation of composition proportions in the multicomponent solvent system, hexane-tert.-butyl methyl ether-methanol-water (1:3:6:5) showed the best solvent selectivity by giving the most prominent difference of partition coefficient (delta log K) between ascomycin and FK-506. With this solvent system, a baseline preparative separation of these two very closely related 23-membered macrolide antibiotics was successfully achieved by employing the newly introduced Quattro counter-current chromatograph.