Hisanobu Yoshida

Some characteristics of a protein-coated ODS column and its use for the determination of drugs by the direct injection analysis of plasma samples

SummaryIt was found that an ODS column of small pore (120Å) which was coated with denatured plasma proteins (protein-coated ODS) no longer adsorbed plasma proteins from aqueous solution but retained the characteristics of native ODS for small hydrophobic molecules. Elemental analysis and nitrogen desorption (BET) analysis showed that the protein-coated ODS contained ca 25 mg proteing−1 dry resin and that the pore diameter or pore volume was similar to that of native ODS. The coated denatured proteins, which seemed to be adsorbed on the external surface of the porous resin, were not eluted under usual reverse-phase elution conditions. Operating as either an analytical column or a pre-column, this protein-coated ODS column was used to analyse spiked-drugs in plasma. The recovery of all the spiked-drugs (such as doxorubicin, methotrexate) was almost quantitative (98–102%) with good reproducibilities (c.v., less than 2%). The present method was useful for the determination of total, that is, free + bound-to-plasma-proteins, hydrophobic drugs in plasma in view of its accuracy and simplicity.

Direct plasma injection method for the analysis of tryptophan metabolites by high-performance liquid chromatography coupled with precolumn deproteinization

Reversed-phase HPLC method by direct plasma injection has been developed for the analysis of major tryptophan metabolites (both metabolites in kynurenine pathways and in indole pathways). Two columns were used: one was a short precolumn of protein-coated octadecylsilane (ODS) for deproteinization and also for trapping of tryptophan metabolites, and the other was an analytical column of the usual ODS. By a column-switching method, the metabolites trapped in the precolumn were allowed to be eluted through the analytical column. The recovery of the spiked metabolites in plasma by the present method was almost quantitative (98-102%) with good reproducibility (CV less than 3%, within-run), and the method is determined to be simple and reproducible for the analysis of total (free + protein-bound) tryptophan metabolites in plasma. The analysis of rabbit plasma showed several peaks corresponding to kynurenine, kynurenic acid, 5-hydroxyindole-3-acetic acid, indole-3-lactic acid, indole-3-acetic acid, indole-3-propionic acid, and 5-hydroxy-tryptamine in addition to tryptophan.

HPLC analysis of human epidermal growth factor using immunoaffinity precolumn. I. Optimization of immunoaffinity column

SummaryThe preparation of support-coupled antibodies for high-performance immunoaffinity chromatography has been demonstrated. Diol silica had the least non-specific adsorption of all supports investigated. The optimum activation conditions for diol silica with 1,1′-carbonyl-diimidazole and the optimum coupling conditions for anti-human epidermal growth factor antibody as a function of salt concentration, pH and ligand concentration were selected. The pore size of the diol silica was an important factor in achieving good results. Diol silica with 500Å pore size had high loading capacity against the antigen. The use of F(ab′)2 or Fab as a ligand showed almost the same chromatographic characteristics as IgG.

Determination of tryptophan and its metabolites in human plasma and serum by high-performance liquid chromatography with automated sample clean-up system

An automated high-performance liquid chromatographic method that incorporates direct injection of biological samples followed by chromatographic sample clean-up in a precolumn is described for the determination of tryptophan and its metabolites in human plasma and serum. The system gave reproducible data with a coefficient of variation of less than 3% with a sample size of 100 microliters of human plasma. The major tryptophan metabolites found in 100 microliters of human plasma were kynurenine, indolelactic acid, indoleacetic acid, indolepropionic acid, serotonin and 5-hydroxyindoleacetic acid. The level of tryptophan and kynurenine in individuals was constant in comparison with other metabolites. Analysis of samples from normal controls, diabetics, gravida and their foetuses showed a tendency for tryptophan metabolites to be low in maternal plasma.

HPLC analysis of human epidermal growth factor using immunoaffinity precolumn. II. Determination of hEGFs in biological fluids

SummaryA high-performance liquid-chromatographic, column-switching system for automated sample pre-treatment and determination of human epidermal growth factor and its degradation products (hEGFs) is described. The system consists of an immunoaffinity precolumn (4.0×10mm) packed with diol silica immobilized with antibody against hEGF and an analytical ODS column (4.6×250mm). Samples such as cultured media ofE. coli, human urine, milk, seminal fluid and saliva can be directly injected on the immunoaffinity precolumn and the analytes of interest are trapped by the immobilized anti-hEGF antibody. After washing this precolumn with aqueous solvents, the analytes are desorbed with an aqueous solution of low pH and transferred to the analytical column to allow their separation in reversed phase mode. The recoveries of hEGFs spiked in these biological fluids were over 98%. The detection limit was 1 ng for a 1ml sample injection. This method was applied for the determination of hEGF levels in cultured media ofE. coli and biological fluids. Degradation of hEGF in human serum and urine was also examined.

Difference in the concentration of tryptophan metabolites between maternal and umbilical foetal blood.

Maternal and umbilical foetal blood at delivery were analysed for tryptophan metabolites by using fully automated high-performance liquid chromatography. The metabolites detected in 100 microliters of maternal plasma were kynurenine, serotonin, 5-hydroxyindoleacetic acid, indolelactic acid, indoleacetic acid and indolepropionic acid. These metabolites were present in various amounts in the protein-bound form. Except for indolepropionic acid, the concentrations of tryptophan metabolites were significantly higher in umbilical foetal plasma than in maternal plasma. In addition, 3-hydroxyanthranilic acid was present in umbilical foetal blood, but not in maternal blood. Furthermore, kynurenic acid was also detected in amniotic fluids.

Enrichment and high-performance liquid chromatography analysis of tryptophan metabolites in plasma

Abstract Tryptophan metabolites with an indole ring are enriched by adsorption either as an ion pair with a trichloroacetic acid anion or as its undissociated form on porous polystyrene polymer (TSK 2000 S) from strongly acidic plasma deproteinized by trichloroacetic acid, and after washing with water, they are eluted with a 90% methanol solution. Following the removal of the solvent, the residue is dissolved in a small amount of water and then subjected to high-performance liquid chromatography (hplc) analysis. Using 0.2 ml of adsorbent, the recovery of the 500 pmol added for each of the tryptophan metabolites into 1.5 ml of deproteinized plasma is above 70%. This method is used for the analysis of normal rabbit and rat plasma. The hplc analysis, with native fluorescence detection, shows several peaks corresponding to tryptophan, 5-hydroxytryptophan, serotonin, 5-hydroxyindole-3-acetic acid, indole-3-acetic acid, and indole-3-propionic acid. Peak identification and cross reactivity were checked by the retention time with two hplc systems, fluorometric characterization, and electrochemical characterization. This method is easy and is simple enough for routine analysis.

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.

Single Column HPLC Method for Drug Level Monitoring by Direct Plasma Sample Injection. Application to 6-Mercaptopurine, Theophylline and Chlorpromazine

Abstract An HPLC method with direct plasma sample injection onto a reverse phase column and stepwise elution was applied to the drug level monitoring of 6-mercaptopurine, theophylline and chlorpromazine by using spectrophotometric or electrochemical detection. The analysis of the drug spiked in human plasma was quantitative, and 100% of the drug was recovered regardless of the entity free or bound to plasma protein. Owing to a preliminary procedure of protein coating on the ODS silica gel the column characteristics were somewhat deteriorated, but accurate analyses could be achieved by a single column method including the simultaneous determination of some metabolites of the drug.