Susumu Kanda

Determination of the fluorescent drugs dipyridamole and benzydamine in rat plasma by liquid chromatography with peroxyoxalate chemiluminescence detection

Abstract A facile and sensitive method for the determination of the fluorescent drugs dipyridamole (coronary vasodilator) and benzydamine hydrochloride (anti-inflammatory) in rat plasma is presented. The method consists of the addition of an internal standard (DNS- l -phenylalanine for dipyridamole and dipyridamole for benzydamine hydrochloride), deproteinization of plasma (1 μl for dipyridamole and 10 μl for benzydamine hydrochloride) with 13 volumes of acetonitrile, direct injection of the supernatant into an ODS column, separation by liquid chromatography and peroxyoxalate chemiluminescence detection using bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate and hydrogen peroxide. Calibration graphs were linear over the ranges 2.5–200 nM and 2.5–100 μM for dipyridamole and benzydamine hydrochloride in plasma, respectively. The detection limits (signal-to-noise ratio = 2) were 345 pM for dipyridamole and 147 nM for benzydamine hydrochloride in plasma. The relative standard deviation for four determinations of dipyridamole at 2.5 nM and benzydamine hydrochloride at 2.5 μM in plasma were 3.7% and 2.6%, respectively. The method was applied to real samples and the time courses of the plasma concentration after oral administration of the two drugs [0.50 mg kg −1 (0.99 μ mol kg −1 ) and 10 mg kg −1 (29 μ mol kg −1 ), respectively] were obtained.

Fluorogenic reagents: 4-aminosulphonyl-7-hydrazino-2,1,3-benzoxadiazole, 4-(N,N-dimethylaminosulphonyl)-7-hydrazino-2,1,3-benzoxadiazole and 4-hydrazino-7-nitro-2,1,3-benzoxadiazole hydrazine for aldehydes and ketones

Fluorogenic reagents for aldehydes and ketones, viz., 4-aminosulphonyl-7-hydrazino-2,1,3-benzoxadiazole (ABD-H) and 4-(N,N-dimethylaminosulphonyl)-7-hydrazino-2,1,3-benzoxadiazole (DBD-H) and also purified 4-hydrazino-7-nitro-2,1,3-benzoxadiazole hydrazine (NBD-H.NH2NH2) were synthesised. These reagents are not fluorescent; however, their reaction products with aldehydes and ketones fluoresce at wavelengths from 548 to 580 nm with excitation from 450 to 470 nm. Both ABD-H and DBD-H exhibited similar reactivity and were more reactive than NBD-H.NH2NH2. The respective pseudo-first-order reaction rate constants for the production of the hydrazone of propionaldehyde with ABD-H, DBD-H and NBD-H.NH2NH2 were 8.9 × 10–2, 7.2 × 10–2 and 4.2 × 10–2 min–1(the reaction was carried out in 0.0025% trifluoroacetic acid in acetonitrile at room temperature, 22 °C). The detection limits using the manual method (i.e., measurement of fluorescence intensity) for the hydrazones of aldehydes and ketones with ABD-H, DBD-H and NBD-H.NH2NH2 were in the µM range. The substrate blank fluorescence with ABD-H was half of that with DBD-H and NBD-H.NH2NH2. The reaction products were separated and analysed by reversed-phase high-performance liquid chromatography (HPLC) with spectrofluorimetric detection. The detection limits for propionaldehyde were 1040, 120 and 35.0 fmol with ABD-H, DBD-H and NBD-H.NH2NH2, respectively, and those for heptan-4-one were 2690, 560 and 673 fmol, respectively. Of the three reagents, DBD-H is recommended for the sensitive detection of ketones and NBD-H.NH2NH2 for the detection of aldehydes. The detection limits for aldehydes and ketones by HPLC were in the sub-pmol to pmol range.

Availability of fluorogenic reagents having a benzofurazan structure in the biosciences

Abstract Fluorogenic reagents having a 2,1,3-benzoxadiazole (benzofurazan) moiety are reviewed in terms of reactivity, fluorescence characteristics, sensitivity and applicability in the biosciences. Those included are 4-fluro-7-nitro-benzofurazan (NBD-F), 4-( N,N -dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F), 4-aminosulphonyl-7-fluoro-2,1,3-benzoxadiazole (ABD-F), ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulphonate (SBD-F), 4-hydrazino-7-nitro-2,1,3-benzoxadiazole (NBD-H), 4-hydrazino-7-nitro-2,1,3-benzoxadiazole hydrazine (NBD-H NH 2 NH 2 ), 4-( N,N -dimethylaminosulphonyl)-7-hydrazino-2,1,3-benzoxadiazole (DBH-H), 4-aminosulphonyl-7-hydrazino-2,1,3-benzoxadiazole (DBD-PZ), (+)-4-nitro-7-(2-aminophyrrolidin-1-yl)-2,1,3-benzoxadiazole ( d -NBD-APy), (–)-4-nitro-7-(3-aminopyrrolidin-1-yl)-2-1,3-benzoxadizole ( l -NBD-APy), 4- N,N -dimethylamino-7 N -piperazino-2,1,3-benzoadiazole (DBD-PZ), 4- N,N -dimethylamino-7- N -cadaverino-2,1,3-benzoxadiazole (DBD-CD), 4-(aminosulphonyl)-7-(1-piperazynyl)-2,1,3-benzoxadiazole (ABD-PZ), 4-(aminosulphonyl)-7-(5-aminopentylamino)-2,1,3-benzoxadiazole (ABD-AP), 4-(aminosulphonyl)-7-(2-aminoethyl)-2,1,3-benzoxadiazole (ABD-AE) and ammonium 7- N -piperazino-2,1,3-benzoxadiazole-4-sulphonate (SBD-P2). Their application data in liquid chromatography are listed in tables. Future trends for the benzofurazan reagents are discussed.

Sensitive assay system for bile acids and steroids having hydroxyl groups utilizing high-performance liquid chromatography with peroxyoxalate chemiluminescence detection.

3 alpha- or 3 beta-hydroxysteroids, such as bile acids (free and glycine and taurine conjugates), 3 beta-hydoxy-5-cholenic acid, pregnanediol, 5-pregnene-3 beta, 20 beta-diol and 5-pregnene-3-beta,20 alpha-diol, were converted to 3-oxosteroids by enzymatic reaction using immobilized hydroxysteroid dehydrogenase, derivatized with dansylhydrazine to the corresponding dansyl hydrazones and purified by gel permeation chromatography. The dansyl hydrazones were chromatographed on a C18 column with a tetrahydrofuran-containing eluent and detected at the level of a few femtomoles by a peroxyoxalate chemiluminescence post-column reaction using bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate as a chemilumigenic reagent. The dansyl hydrazones of chenodeoxycholic acid and deoxycholic acid (free and glycine and taurine conjugates) in particular, which coeluted under the chromatographic conditions above, were separated using an eluent including acetonitrile and 2,6-di-O-methyl-beta-cyclodextrin and detected in the same way.

Effect of tetrahydrofuran on reversed-phase liquid chromatographic separation of dansylhydrazones of oxo-steroids

The oxo-steroids corticosterone, testosterone and progresterone were converted with 5-N,N-dimethylaminonaphthalene- 1 -sulphonohydrazide (DNS-hydrazine) into DNS-hydrazones, which were separated by reversed-phase liquid chromatography and detected fluorimetrically. The anti and syn conformers of the hydrazones were recognized on a C18 column with eluents consisting of imidazole buffer (nitrate, pH 6.0) containing acetonitrile, methanol, acetone or dioxane, and thus two peaks were observed for each oxo-steroid derivative. On the other hand, a single peak was obtained when an eluent containing tetrahydrofuran was used, affording highly sensitive detection of oxo-steroids. A further improvement in the detection of oxo-steroids down to subpicomole level was achieved by adding a gel permeation chromatographic clean-up procedure to remove the excess reagent before the analysis by reversed-phase liquid chromatography.

Effect of additives to the reaction solvents in a peroxyoxalate chemiluminescence detection system for liquid chromatography

Abstract A facile means of optimizing solvent compositions and reagent concentrations for the peroxyoxalate chemiluminescence (PO-CL) detection of fluorescent compounds with bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate (TDPO) and hydrogen peroxide in liquid chromatography (LC) was developed. The reaction time course of CL was easily obtained by changing the PTFE tube lengths in the mixing device in the LC flow system in which the column effluent and CL reagent solution flow. The addition of a phthalate ester to the basic solvent mixture for the chemiluminogenic reagents, ethyl acetate-acetonitrile (50 + 50, v/v) containing TDPO and hydrogen peroxide, resulted in a considerable reduction in the baseline noise level without decreasing the signal of a fluorescent compound, dipyridamole. Phthalate esters having branched-chain moieties were more effective, di-2-ethylhexyl phthalate (DOP) being the best in reducing the baseline noise. A mixture of 0.25 mM TDPO and 25 mM hydrogen peroxide in ethyl acetate-acetonitrile-DOP (50 + 49.75 + 0.25, v/v/v) (1.3 ml min −1 as a chemiluminogenic reagent solution for the eluent [acetonitrile-50 mM imidazole nitrate buffer (pH 6.0) (45 + 55, v/v) (1.0 ml min −1 )] was optimum for the detection of amol levels of fluorescent compounds. The detection limit for dipyridamole was 10 amol (signal-to-noise ratio = 2). Under the same conditions, the detection limit for dansylated phenylalanine was 800 amol.

Dynamic analytical chemistry: a trial study of the interaction of fluorogenic reagents with living chinese hamster ovary cells

Abstract The concept of dynamic analytical chemistry, which deals with the qualitative localization or the quantitative transportation of a known or unknown component or its conversion into a product within living cells, is introduced. The concept was tested using cultured Chinese hamster ovary cells in a phosphate-buffered saline medium in the presence of fluorogenic reagents for amines and thiols. The phenomenon was observed and recorded with a fluorescence microscope equipped with a SIT camera. Among the reagents, 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) added to the culture medium was first trapped at the surface of the cells and then passed inside the cells to react with the mitochondria to give yellow fluorescence, o -Phthalaldehyde and 4-aminosulphonyl-7-fluoro-2, 1,3-benzoxadiazole reacted with the cells to give a blue florescence at certain sites inside the cells. One of the two major components labelled with NBD-F appeared to be phosphatidylethanolamine, a component of the plasma membrane. Its identification and quantitation were effected by conventional analytical techniques, such as thin-layer and liquid chromatography, following enzymatic hydrolysis. The fate of the N -NBD-phosphatidylethanolamine is discussed.