Ikue Morita

Capillary electrophoresis with on-line sample pretreatment for the analysis of biological samples with direct injection

Abstract A method of capillary electrophoresis (CE) using on-line sample pretreatment was developed. The system utilized on-line solid-phase extraction and capillary column switching. To explore the advantages of the method, propranolol in serum was determined by direct injection. lie pretreatment part of the system consisted of an injection capillary including a small bed of protein-coated ODS. The protein-coated ODS concentrated the analyte by hydrophobic interactions, and the serum proteins were allowed to flow into a drain capillary. As samples up to ca . 1 μl could be loaded without a decrease in resolution, the detection limit was improved by two orders of magnitude (0.15 μg/ml). Propranolol was detected in a separation capillary by UV absorbance measurement without any interfering peaks. A linear response was obtained between 100 fg and 2 ng per injection. This method provides novel possibilities for extending the application of capillary electrophoresis.

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.

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.

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.

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.

Simultaneous determination of free and bound adriamycin, adriamycinol, adriamycinone and duanorubicin in plasma using column-switching technique and protein-coated pre-columns☆

Adriamycin, adriamycinol, adriamycinone and duanorubicin were simultaneously determined by the development of an on-line plasma clean-up system. A short protein-coated Lichrosorb, RP-8, RP-2, CN and muBondapak phenyl as well as ODS silica have been examined for their performance as pre-columns. The drugs and metabolites were separated from weakly retained plasma components through two steps; phosphate buffer saline, pH 7.4 and 15% acetonitrile in 0.1 M sodium dihydrogen phosphate, pH 3. The chromatographic conditions were: ODS/TM column, flow rate 1 ml/min, 35% acctonitrile in 0.1 M sodium dihydrogen phosphate (pH 3) containing 0.3% heptafluorobutyric acid as mobile phase. The detection was carried out using fluorescence monitor operated at an emission 555 nm and excitation 460 nm. Good resolution was obtained within 13 min. This method is reproducible for analysis of drugs and metabolites (99.3-100.1%, CV < 2%) in plasma.

Effect of cyclodextrins on the stability of adriamycin, adriamycinol, adriamycinone and daunomycin

The stability of adriamycin (ADR), adriamycinol, adriamycinone (ADR-ONE) and daunomycin in the presence of alpha-, beta- and gamma-cyclodextrins (CDs) was studied using high-performance liquid chromatography. It was found that alpha-CD did not affect the degradation of tested compounds, beta-CD caused a little effect and gamma-CD resulted in pronounced stabilizing effect. The formation of complexes between ADR and ADR-ONE with CDs was monitored by fluorescence spectroscopy. The fluorescence spectrum of ADR-gamma-CD complex had an activation maximum at 460 nm, emission maximum at 555 nm and a shoulder at 585 nm. A similar finding was observed in case of alpha-CD. In case of beta-CD, the fluorescence intensity at 580 nm peak enhanced less than in case of gamma-CD. With ADR-ONE, alpha-CD did not cause any significant change compared with the spectrum of free molecule. On the other hand, it was noticed that, the fluorescence spectra of ADR-ONE with both beta- and gamma-CD were the same but showed a significant difference to the spectrum of free molecule, especially the molar fluorescence of the 585 nm emission peak.

Utility of Ion-Pair Chromatography for Analysis of Some Anthracyclines in Plasma and Urine

Abstract A high performance liquid chromatographic assay was developed and validated for a simultaneous determination of adriamycin and its metabolites adriamycinol and adriamycinone as well as daunomycin in plasma. Acetonitrile and trichloroacetic acid were employed in removing proteins and extracting the drugs and metabolites into the supernatant for HPLC. Enhancement of chemical stability of the anthracyclines during sampling procedure was investigated using 0.01 M γ-cyclodextrin. The processed samples were chromatographed using an ODS/TM column (150 × 4.6 mm I.D.) eluted with a mobile phase composed of 35% acetonitrile and 65% (0.1M) monobasic phosphate, containing 0.3% heptafluorobutyric acid, pH 3. The detection was performed fluorimetrically at emission and excitation wavelengths of 555 and 460 nm, respectively. The described method could be directly applied for analysis of tested anthracyclines in human urine without sample pretreatment.