Takashi Nishikawa

On-column enantiomerization of 3-hydroxybenzodiazepines during chiral liquid chromatography with optical rotation detection

Abstract Oxazepam and lorazepam, 3-hydroxybenzodiazepines that undergo reversible enantiomerization at ambient temperature, were eluted on a chiral HPLC column at various temperatures and various flow-rates. The profiles obtained by a UV detector were various, such as two peaks, a plateau sandwiched between two peaks, and one fused peak. The profiles obtained by an optical rotation detector were also various, such as two sharp peaks with opposite chirality (−, +), two peaks (−, +) with a great tailing or leading, two small peaks (−, +) next to each other. These peculiar profiles could be explained by on-column enantiomerization. Unexpectedly, the elution order of the (−)- and (+)-enantiomers was reversed for oxazepam and lorazepam. The simultaneous detection by UV and chiroptical measurements allowed us to plot HPLC profiles of the individual enantiomers.

On-column isomerization of sugars during high-performance liquid chromatography: analysis of the elution profile.

Four monosaccharides (glucose, galactose, mannose and fructose) and one disaccharide (maltose) were subjected to high-performance liquid chromatography with UV or refractive index detection. Various profiles such as broad, tailed and splitted peaks were produced, depending on column temperature and eluent flow-rate because these saccharides underwent isomerization. In contrast, alpha-methylglucoside, a non-converting derivative, always produced a sharp peak. By analyzing these profiles kinetic constants of the isomerization were obtained and compared with the literature data.

Elevated S100B level in cerebrospinal fluid could predict poor outcome of carbon monoxide poisoning

OBJECTIVE S100B is a calcium-binding protein produced by astroglia in the brain and has been used as a marker of neuronal damage after brain trauma. We investigated the utility of S100B in cerebrospinal fluid (CSF) measured during the early phase of carbon monoxide (CO) poisoning in predicting the subsequent clinical course. METHODS The study included 31 patients who were admitted to the hospital with loss of consciousness following CO poisoning. S100B levels were measured by enzyme-linked immunosorbent assay in CSF, and serum samples collected simultaneously within 24 hours and on the fourth day after CO exposure. All patients were followed for at least 3 months and divided into 3 groups based on the clinical course: persistent vegetative state (PVS), delayed encephalopathy (DE), and complete recovery with no complications (NC). RESULTS During the 3-month period, 3 patients developed PVS, 5 developed DE, and 23 were classified as NC. The mean S100B levels in the CSF within 24 hours after CO exposure were higher in the PVS group (9.25 ng/mL) than in the DE (2.03 ng/mL) and NC groups (1.86 ng/mL). However, the mean serum S100B levels were not elevated in the 3 groups (0.21, 0.59, and 0.16 ng/mL, respectively). CONCLUSION Early elevation of S100B in CSF after CO poisoning could be a suitable predictor of subsequent development of PVS.

Kinetic Analysis of Molecular Interconversion of Immunosuppressant FK506 by High-Performance Liquid Chromatography

High-performance liquid chromatography of FK506, a macrolide immunosuppressant, was performed on a reversed-phase column. The peak was broad with the column kept at room temperature, which was accounted for by slow interconversion between the two forms of FK506. With the use of a heated column, a sharp peak was observed because of the rapid interconversion at high temperature. When the column was cooled to 0°C, two sharp peaks were observed because essentially no interconversion occurred at 0°C during elution. Analysis of the chromatograms obtained at various eluant flow rates indicated that the conversion of the two forms follows first-order kinetics, and the apparent activation energies for the conversions were calculated. The interconvertibility between two molecular forms may be related to the immunosuppressive activity.

Interconversion of cyclosporin molecular form inducing peak broadening, tailing and splitting during reversed-phase liquid chromatography

SummaryReversed-phase liquid chromatography of cyclosporin A, a peptide immunosuppressant, at various temperatures produced remarkably different chromatograms. At 60°C one sharp peak was obtained, at 23°C this became a single broad peak and between 15° and 0°C this became one high sharp peak followed by a tailing or low plateau. Remarkably different chromatograms were produced also by varying the mobile phase flow-rate. The effects of both temperature and flow-rate on the chromatogram could be accounted for by interconversion between two forms of the cyclosporin molecule. Kinetic analysis showed that one form was converted by first-order kinetics with a half-life of 2.0 min at 20°C and that the apparent activation energy for the conversion was about 18 kcal/mol. The other two immunosuppressants, cyclosporin C and D, were also shown to undergo interconversion. This kinetic analysis of the interconversion should be helpful in clarifying the relationship between molecular structure and activity of the immunosuppressants.

Production of a reactive metabolite of troglitazone by electrochemical oxidation performed in nonaqueous medium.

In order to confirm the existence of reactive metabolites by LC-MS/MS analysis, they should be modified into stable compounds, because some reactive metabolites generated by biotransformation induce drug toxicity; however, they are unstable, with very short lives, and cannot be detected in their intact forms. To overcome these problems, electrochemical oxidation of troglitazone was performed in nonaqueous medium, since such reactive compounds are stable in the absence of water. Troglitazone, an antidiabetic agent, was withdrawn from the market because of serious hepatotoxicity in some patients. It has been considered that one or more reactive metabolites are involved in hepatotoxicity, although the mechanism of the adverse reaction is unclear. Using our method of electrochemical oxidation in nonaqueous medium, we obtained a product of troglitazone derivative that may be a clue to clarify the mechanism of toxicity. The product in the reaction mixture was separated by HPLC without chemical modification and detected using UV and ESI-MS. The mass spectrum of its molecular ion showed that it was an o-quinone methide derivative of troglitazone and identified as a reactive metabolite generated by liver microsome oxidation of the drug. The product was stable over 24 h at room temperature in anhydrous acetonitrile, but it reacted with N-(tert-butoxycarbonyl)-L-cystein methylester to produce an adduct that could be identified by its m/z value. Thus, the method of electrochemical oxidation in nonaqueous medium is considered to be useful to prepare and predict reactive metabolites of drugs that are unstable in aqueous medium or in vivo.

Cardiovascular response and stress reaction to flumazenil injection in patients under infusion with midazolam.

Objectives: To evaluate the cardiovascular response and acute stress reaction after arousal induced by a benzodiazepine antagonist, flumazenil, in patients sedated with midazolam. Design: Prospective study. Setting: Emergency center in a university hospital. Patients: A total of 12 patients were ventilated mechanically under sedation with midazolam. Interventions: We monitored the consciousness level, heart rate, systemic blood pressure, pulmonary artery pressure, and pulmonary artery occlusion pressure before and after a bolus injection of 0.5 mg of flumazenil. The score for the consciousness level represents the sum of the scores for eye opening and best motor response, as determined by the Glasgow Coma Scale. We measured the cardiac output, concentrations of norepinephrine, epinephrine, and 3‐methoxy‐4‐hydroxyphenylethyleneglycol in plasma, and concentration of cortisol in serum. We calculated the left ventricular ejection fraction, cardiac index, systemic vascular resistance index, pressure‐rate product, systemic oxygen delivery, and systemic oxygen consumption at 0, 10, 30, and 60 mins after injection of flumazenil. Measurements and Main Results: The serum benzodiazepines receptor binding activity in serum was in the range from 50 to 1000 ng/mL before injection of flumazenil. Flumazenil improved the consciousness level from 6.7 ± 2.0 to 8.9 ± 1.6 and induced transient elevations in heart rate, blood pressure, systolic pulmonary artery pressure, and pulmonary artery occlusion pressure. Left ventricular ejection fraction, oxygen delivery index, and pressure‐rate product increased significantly, from 61% ± 8%, 640 ± 170 mL/min/m2, and 13,300 ± 2600 mm Hg/min at 0 mins to 67% ± 5%, 710 ± 220 mL/min/m2, and 16,500 ± 4400 mm Hg/min at 10 mins, respectively. Concentrations of norepinephrine and epinephrine in plasma increased significantly, from 890 ± 840 pg/mL and 220 ± 360 pg/mL, respectively, at 0 mins to 990 ± 850 pg/mL and 270 ± 300 pg/mL, respectively, at 10 mins. There were no significant changes in the plasma concentration of 3‐methoxy‐4‐hydroxyphenylethyleneglycol, the serum concentration of cortisol after the administration of flumazenil. Conclusions: Flumazenil did not result in a significant acute stress reaction in midazolam‐sedated patients, but it increased myocardial oxygen consumption by enhancing sympathetic nervous activity or antagonizing cardiovascular depression induced by midazolam.

Fluorometric determination of isoniazid and its metabolites in urine by high-performance liquid chromatography using in-line derivatization

A rapid, simple, and accurate method has been developed for the determination of isoniazid and its metabolites (isonicotinic acid, isonicotinyl-glycine, and acetylisoniazid) in human urine by high-performance liquid chromatography. Isoniazid and its metabolites are separated by reversed-phase ion-exchange chromatography with a mobile phase containing hydrogen peroxide as a fluorogenic reagent and butanesulfonate as a hydrophobic ion exchanger, and are detected by fluorometry (excitation at 317 nm and emission at 415 nm) using in-line derivatization at high temperature (160°C). The detection limits are isonicotinic acid, 0.5 μmol/L; isonicotinylglycine, 1 μmol/L; acetylisoniazid, 1 μmol/L; and isoniazid, 1.5 μmol/L. This method can be applied for acetylator phenotyping.

Competitive nephelometric immunoassay of theophylline in plasma

We have developed an accurate, simple, and rapid method for the determination of theophylline in plasma. The principle of the method is based on inhibition of immunoprecipitation by hapten. A nephelometer is used to measure the scattered light from the immunoprecipitate. The macromolecule, which possesses numerous theophylline moieties and forms immunoprecipitate with anti-theophylline antibodies, can be easily prepared and used as the reagent for the assay. Theophylline in the assay mixture competitively inhibits the immunoprecipitation of the macromolecule. Therefore, theophylline can be determined by the measurement of the decrease of the scattered light. The assay is rapid (incubation time within 15 min), requires as little as 10 microliter of plasma, and requires neither troublesome pretreatment nor separation of antibody-bound from free antigen. Estimations of theophylline levels in patient plasma specimens correlate well to those obtained by the high performance liquid chromatography (correlation coefficient 0.971).

Fluorometric determination of isoniazid and its metabolites in urine by high performance liquid chromatography

SummaryA rapid, simple, and accurate method was developed for the determination of isoniazid and its metabolites (isonicotinic acid, acetylisoniazid and isonicotinylglycine) in urine by high-performance liquid chromatography. Urine is diluted with the mobile phase. After centrifugation, an aliquot of the supernatant is injected into the chromatograph. Isoniazid and its metabolites are separated by reversed-phase ionpairing chromatography with a mobile phase containing propanesulfonate and detected by fluorometry using postcolumn derivatization at high temperature (150°C) with hydrogen peroxide. The method was applied to the analysis of urine from patients receiving isoniazid therapy.