Gen Tamai

On-line deproteinization of serum sample for HPLC analysis of hydrophilic compounds and its application to gentamicin

SummaryThe binding of serum proteins with Butyl Toyopearl (BT) 650-M has been investigated and applied to on-line deproteinization for the HPLC determination of gentamicin components, c1, c1a, c2, in serum. It was found that in 0.4% perchloric acid medium about 36mg of BSA was adsorbed on 1ml of wet gel. Under this condition hydrophilic components such as gentamicin passed through the pre-column packed with BT 650-M, while serum proteins and hydrophobic components were trapped in the pre-column. The ion pair between gentamicin components and pantanesulfonate anion was effectively trapped in a reversed-phase analytical column. It was then eluted and fluorometrically determined by post-column derivatization with o-phthalaldehyde. The recovery was quantitative with good reproducibility at therapeutic concentrations in sera. Several clinical samples were analyzed by the method.

High-performance liquid chromatographic drug analysis by direct injection of whole blood samples. II. Determination of hydrophilic drugs.

The determination of hydrophilic drugs in whole blood by direct injection high-performance liquid chromatography was investigated. A pre-column equipped with an inlet filter of pore size 40 microns and an outlet filter of pore size 2 microns was packed with Butyl Toyopearl 650-M. A whole blood sample was injected directly into the pre-column to trap proteins, hydrophobic compounds and blood cytomembranes, and hydrophilic compounds emerged into an analytical column (Nucleosil 5SA, particle size 5 microns) and were determined after column switching. Proteins in 40 microliters of rabbit whole blood were adsorbed in the pre-column (0.63 ml of wet gel) in 0.4% perchloric acid solution. The recovery of procainamide and N-acetylprocainamide from whole blood was quantitative with good reproducibility (coefficient of variation less than 4%). It was shown that procainamide added to rabbit whole blood was subjected to N-acetylation by N-acetyltransferase in blood cells.

High-performance liquid chromatographic drug analysis by direct injection of whole blood samples: I. Determination of moderately hydrophobic drugs incorporated into blood corpuscles

Amounts of a moderately hydrophobic drug incorporated into blood corpuscles were determined by column switching high-performance liquid chromatography with direct injection of whole blood samples. Some modifications were required in order to apply the conventional direct injection method to whole blood samples, as follows. Clogging of the pre-column by blood corpuscles or cytomembranes was avoided by using an end-fitting filter with pore size larger than 40 micron, and accordingly a packing with particle size larger than 40 micron was used. Haemoglobin was decomposed in contact with ODS silica gel and reduced the column efficiency. The protein-coated ODS pre-column (pore size less than 7 nm) was restored by washing with 0.1 M phosphate buffer (pH 3.0) containing 50% acetonitrile. Sodium dodecyl sulphate solution (0.5%, w/v) was preferable for rinsing and removes material remaining in the pre-column. A whole blood sample containing carbamazepine was directly injected into the pre-column, then blood cells were haemolysed promptly in contact with a mobile phase of low salt concentration. The total amount of carbamazepine could be determined with a coefficient of variation of 1.9% (within-run). Carbamazepine incorporated into rabbit blood cells (haematocrit value 33.6%) was determined to be 1.1-1.3 times higher than the concentration in plasma. Adsorption on the cytomembranes was not observed.

High-performance liquid chromatographic drug analysis by direct injection of whole blood samples: III. Determination of hydrophobic drugs adsorbed on blood cell membranes☆

The determination of strongly hydrophobic drugs in whole blood by high-performance liquid chromatography was investigated and the amount adsorbed on cytomembranes was measured. A polyvinyl resin, TSK Gel HW-65, was used as the pre-column packing. Proteins, cytomembranes and endogenous hydrophilic components flowed through the pre-column in aqueous medium, but strongly hydrophobic substances such as chlorpromazine were adsorbed and then eluted by backflushing into an ODS analytical column. The recovery of chlorpromazine from whole blood was 103.3% with a coefficient of variation of 3.4% (n = 10). This method gave the total amounts in blood, representing not only the amount bound to proteins but also that bound to cytomembranes. The difference in the concentrations determined in whole blood and the supernatant of haemolysed whole blood gave the value adsorbed on cytomembranes.

Mild interaction of proteins with butyl and hydroxyl groups on the surface of polymer gels TSK HW-65 and butyl toyopearl 650-M

Interaction of bovine serum albumin (BSA) with butyl and hydroxyl groups on adsorbent gel surfaces was investigated by using TSK HW-65 and Butyl Toyopearl 650-M gels. It was found that BSA was adsorbed on the gel not only from highly concentrated ammonium sulphate but also from dilute perchloric acid, trichloroacetic acid, etc. Some eluent modifiers, such as organic solvents (30% aqueous methanol or acetonitrile), salt solutions (0.18-0.2 M phosphate) and hydrogen-bond breaking reagents (3-7 M urea, 10 M ethylene glycol, 0.1% triethylamine) were found to be effective in facilitating the elution of trapped BSA from the gel. The conformational change of BSA in these solutions was slight except for urea, and it was reversibly recovered after removal of the modifier from the aqueous solutions, except for the hydrogen-bond breaking reagents.