Sonoko Uzu

Fluorogenic reagent for thiols: 4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole

4-(N,N-Dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) was synthesised for use as a more reactive, thiol-specific fluorogenic reagent than 4-(aminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). The former had negligible fluorescence whereas its thiol derivatives fluoresced intensely at about 510 nm (excitation occurred at about 380 nm). The DBD-F reacted quantitatively with thiols after 10 min at 50 degrees C and pH 8.0 and the reaction rates were several times higher than those with ABD-F; it is suggested that the electron withdrawing effect of the dimethylsulphonamide group (SO2NMe2) is larger than that of the sulphonamide group (SO2NH2). No reaction occurred with alanine, proline, cystine or cysteic acid under the same conditions. The fluorescence intensities of the derivatives were found to be higher in neutral and acidic media than in alkaline solutions. The thiol derivatives of DBD-F were separated by high-performance liquid chromatography and detected fluorimetrically, the detection limits being 0.92, 0.16, 0.13, 0.16 and 0.32 pmol for cysteine, glutathione, homocysteine, N-acetylcysteine and alpha-mercaptopropionylglycine, respectively. The method was applied to the determination of thiols in rat tissues.

Fluorogenic reagents, having benzofurazan structure, in liquid chromatography☆

Newly synthesized fluorescent reagents, NBD-F and PBD-SO2F for amines, DBD-F for thiols and amines, and ABD-F and SBD-F for thiols are reviewed in terms of their reactivity, fluorescence characteristics, stability, selectivity, and their applicability to analysis.

Evaluation of benzofurazan derivatives as fluorogenic reagents for thiols and amines using high-performance liquid chromatography

The reactivities of three reagents, 4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F), 4-(aminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) and 4-(fluorosulphonyl)-7-phenoxy-2,1,3-benzoxadiazole (PBD-SO2F), towards thiols and amines were compared. All three reacted with thiol compounds to give derivatives which fluoresced at ca. 515 nm (excitation at ca. 388 nm). However, the PBD-SO2 derivatives were not very stable. The rate of reaction of DBD-F with proline was faster than that of PBD-SO2F or ABD-F. The three reagents and their hydrolysates exhibited extremely low fluorescence. The fluorescence intensities of the proline derivatives synthesised were higher in neutral and acidic solutions than in alkaline solution. The strength of the fluorescence intensities was in the order PBD-SO2-proline > DBD-proline > ABD-proline at all pH values tested. The mean values of the maximum wavelengths of DBD-proline, ABD-proline and PBD-SO2-proline were 596 nm (λex. = 466 nm), 602 nm (λex. = 466 nm) and 566 nm (λex. 453 nm), respectively. Of the three reagents, DBD-F seemed to be applicable to the determination of amino acids. Nine amino acid derivatives of DBD-F were separated by reversed-phase high-performance liquid chromatography and detected with fluorescence at 590 nm (λex. = 450 nm). The detection limits were in the sub-picomole range.

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.

Use of 4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole as a labelling reagent for peroxyoxalate chemiluminescence detection and its application to the determination of the β-blocker metoprolol in serum by high-performance liquid chromatography

Fluorogenic reagents having a benzofurazan moiety, viz., 4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F), 7-fluoro-4-nitro-2,1,3-benzoxadiazole and 4-(aminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole, were compared for the sensitive analysis of their derivatives by high-performance liquid chromatography with peroxyoxalate chemiluminescence detection. Of the proline derivatives, DBD-proline was the most sensitive with a detection limit of 2 fmol. The optimum concentrations of bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate and H2O2 for the post-column reaction were 0.5 and 75 mmol dm-3 respectively and amino acids and beta-blockers derivatized with DBD-F were detected in the range 0.2-40 fmol (signal-to-noise ratio = 3) using the proposed method. The lower detection limit of metoprolol (a beta-blocker having an isopropylamino group) spiked in serum was 0.8 ng ml-1 using 20 microl of serum (signal-to-noise ratio = 5).

Development and clinical application of high performance liquid chromatography for the simultaneous determination of plasma levels of theophylline and its metabolites without interference from caffeine

A high performance liquid chromatography (HPLC) method has been developed for the simultaneous determination of plasma levels of theophylline and its metabolites without interference from caffeine or caffeine metabolites. The method is simple and of practical use because it is applicable even to plasma samples from patients who take caffeine-containing beverages. The method was also reproducible with a coefficient of variation of less than 5% for each analyte. The levels of theophylline, determined by HPLC, were validated by their high correlation to the levels obtained by fluorescence polarization immunoassay. HPLC was used to determine theophylline levels in patients with bronchial asthma. The data revealed that the ratio of 1,3-dimethyluric acid, the major metabolite of theophylline, to theophylline concentration in the plasma was within a narrow range in most patients (0.055 +/- 0.01, n = 66), regardless of the method of theophylline administration or the time of blood sampling. Conversely, this ratio was as low as 0.027 +/- 0.005 in the patient with a long plasma half-life of theophylline. These results suggest that it may be possible to predict the plasma half-life of theophylline for each patient from a single blood sample. This may be useful when planning theophylline administration, especially in patients with abnormal theophylline metabolism.

Determination of medroxyprogesterone acetate in serum by HPLC with peroxyoxalate chemiluminescence detection using a fluorogenic reagent, 4-(N,N-dimethylaminosulphonyl)-7-hydrazino-2,1,3-benzoxadiazole

The high-performance liquid chromatographic determination of medroxyprogesterone acetate (MPA) with peroxyoxalate chemiluminescence (PO-CL) detection is described. The spiked serum containing MPA was extracted on Bond-Elut C18 columns and derivatized with 4-(N,N-dimethylaminosulphonyl)-7-hydrazino-2,1,3-benzoxadiazole (DBD-H). The hydrazone of MPA with DBD-H was confirmed to be a mono-DBD-derivative. The reaction mixture was separated by direct injection onto a C18 analytical column, and quantified by PO-CL detection. The linear range of the standard curve, in serum, was 15.6-96.6 ng ml-1 with a detection limit of 9 ng ml-1 using only 100 microliters of serum, while the detection limit of standard MPA derivatized with DBD-H was 8.7 fmol per injection. The relative standard deviation of the method was 7.4% at 19.3 ng and 1.7% at 77.3 ng ml-1.

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.

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.