Reliable on-line sample preparation of basic compounds from plasma using a reversed phase restricted access media in column-switching LC.

We investigated on-line sample preparation of basic compounds from plasma using a methylcellulose-immobilized reversed-phase restricted-access media in column-switching liquid chromatography (LC). Dilution of the plasma sample with phosphate buffered saline prevented or delayed the formation of fibrin clots at 4 degrees C and resulted in reproducible on-line sample preparation over a 30-h period. The use of an ion-pair reagent in the extraction LC enhanced recoveries of hydrophilic basic compounds. The ability of the methods to quantify compounds in plasma were validated and the method was successfully applied to the pharmacokinetic study of a hydrophilic basic compound injected into the bloodstream of rats.

Effective on-line purification for cationic compounds in rat bile using a column-switching LC technique.

An on-line purification method for cationic compounds and their metabolites in rat bile was investigated using a column-switching technique. 8-Hydroxyquinoline and its glucuronide were used as test compounds. Bile samples were injected directly into the system and successful on-line extraction with high purification efficiency for analytes was achieved using two-dimensional extraction LC; that is, reversed-phase chromatography followed by cation-exchange chromatography. After removal of the endogenous component by extraction LC, chromatographic separation of the target analyte was performed on an analytical ODS column, followed by identification using UV detection. The quantitative ability of the method was evaluated on the basis of injection repeatability, linearity and accuracy. This novel method was also applied to LC/MS analysis in order to characterise the pharmacokinetics of propranolol in rats, and the metabolites were successfully identified.

Novel methylcellulose-immobilized cation-exchange precolumn for on-line enrichment of cationic drugs in plasma.

We developed a novel methylcellulose-immobilized strong cation-exchange (MC-SCX) precolumn for direct analysis of drugs in plasma. MC-SCX consists of silica gel with a methylcellulose outer-surface and a 2-(4-sulfophenyl) ethyl phase inner-surface. The MC-SCX precolumn was evaluated by direct analysis using pyridoxine, atenolol and sulpiride spiked in plasma, using a column-switching HPLC system. Each drug was retained and enriched on MC-SCX using an acidic mobile phase, which resulted in good linearity, sufficient reproducibility, intra- and inter day precision, and accuracy in analytical ion-pair LC with trifluoroacetic acid. The analytical methods for model drugs were applied to pharmacokinetics of atenolol and sulpiride in rats.

A rapid method for pKa determination of drugs using pressure-assisted capillary electrophoresis with photodiode array detection in drug discovery.

We developed a rapid screening method for determination of pK(a) of candidate drugs by pressure-assisted capillary electrophoresis (CE) coupled with a photodiode array detector. Application of pressure during CE analysis allowed completion of one CE run in less than 1 min, and the obtained pH-metric mobility shifts as well as pH-metric UV spectrum were analyzed by a nonlinear regression fitting software to determine pK(a) values. The difference between pK(a) values by this method and by other conventional methods is within 0.25 units for 82 ionic functional groups of 77 drugs. The pK(a) values of 96 compounds in dimethylsulfoxide solution on a 96-well microplate could be measured in 1 day. Our method provides rapid and accurate determination of pK(a) value.

[Development of solubility screening methods in drug discovery].

We developed two methods for solubility screening of drug candidates in drug discovery. The first is a solution-precipitation (SP) method, in which the sample solutions are prepared by adding the drug solution in dimethylsulfoxide (DMSO) to buffers followed by filtering off the precipitate using 96-well filterplate. The second is a powder-dissolution (PD) method, in which the solid samples are dissolved to the buffer in the HPLC vial equipped with the filter membrane in the HPLC autosampler. An HPLC equipped with a photodiode array detector is used to measure the concentration of the sample solutions in both methods. The SP method was used for high throughput screening the solvating process of the candidates in aqueous solutions with lower sample consumption, and the PD method was used for screening both inter-molecular interaction in solid state and solvation in aqueous solution with more sample amount than that of SP method. Therefore, the solubility screening from early to final stage of lead optimization process would be successfully accomplished by using both methods complementarily.