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.     

Suppression of thiol exchange reaction in the determination of reduced-form thiols by high-performance liquid chromatography with fluorescence detection after derivatization with fluorogenic benzofurazan reagent, 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate and 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole

The derivatization of the reduced-form thiols with SBD-F (7-fluoro-2,1,3-benzoxadiazole-4-sulfonate) and ABD-F (4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole) was studied. The yields of the derivatives of the reduced-form thiols (cysteine, homocysteine, reduced-form glutathione) with SBD-F at 60 degrees C for 45 min in the borate buffer (pH 9.3) were significantly decreased in the presence of the oxidized-form thiols (cystine, homocystine, oxidized-form glutathione) because of the thiol exchange reaction between the reduced-form and the oxidized-form thiols. The use of ABD-F at low temperature enabled the suppression of these thiol exchange reactions, and the recommended conditions were below 5 degrees C for 90 min in borate buffer (pH 9.3). These results suggest that ABD-F is a preferred derivatization reagent for the accurate determination of the reduced-form thiols in samples containing the oxidized-form thiols. In addition, it was also suggested that the derivatization of the reduced-form thiols should also be performed at low temperature when derivatization reagents such as o-phthalaldehyde (OPA) and monobromobimane (BrB) are used.     

Amino acid composition analysis of minute amounts of cysteine-containing proteins using 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole and 4-fluoro-7-nitro-2,1,3-benzoxadiazole in combination with HPLC

The simultaneous determination of amino acid composition including cysteine of egg albumin, a model protein containing a/s cysteine residue, is reported. All the thiol groups of the cysteine residue(s) of egg albumin were labelled with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole, a fluorogenic reagent for thiol groups. The labeled egg albumin was hydrolyzed in 6N HCl at 110 degrees C for 24 h. The hydrolysate was lyophilized, derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole, a fluorogenic reagent for amines, and subjected to HPLC. 18 derivatized amino acids including double labelled cysteine were separated within 90 min on a Nucleosil ODS column (150 mm X 4.6 mm i.d.; 5 microns), and detected at 530 nm (ex. 470 nm) in a range from 90 fmol (aspartic acid) to 1.3 pmol (cysteine) (S/N = 3). Composition ratios of amino acids of egg albumin were similar to theoretical values except for methionine, which would be destroyed under the present acid hydrolysis condition. Analytical methods for cysteine residues are reviewed, and the availability of fluorogenic reagents having the benzofurazan structure is also discussed.     

Determination of thiols and disulfides in normal rat tissues and hamster pancreas treated with N-nitrosobis(2-oxopropyl)amine using 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole and ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate

Biological thiols and disulfides in rat and hamster tissues were simultaneously determined by HPLC-fluorescence detection using 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) and ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F). The coefficients of variation (CV) of the method for reduced glutathione (GSH) and oxidized glutathione (GSSG) in liver and for cysteine (CySH) and cystine (CySSCy) in kidney were less than 3.1%. In 11 tissues of Wistar rats (liver, spleen, heart, lung, stomach, bladder, ovary, uterus, adrenal, kidney and pancreas), only CySH, CySSCy, GSH and/or GSSG were detected. Other thiols and disulfides were at extremely low levels in all samples. Both concentrations of CySH and CySSCy in the livers of old rats (111 weeks old, F344) were significantly higher than those of young rats (8 weeks old) (CySH, 0.246 +/- 0.099 vs 0.130 +/- 0.020 mumol/g; CySSCy, 0.051 +/- 0.027 vs 0.013 +/- 0.002 mumol/g). Administration of N-nitrosobis(2-oxopropyl)amine (BOP), a selective carcinogen of hamster pancreatic cancer, to Syrian golden hamsters (38 weeks old) resulted in the increase in the pancreas of GSH to a level 19 times as high and of GSSG to a level 14 times as high as those in untreated hamsters (GSH, 1.173 +/- 0.272 vs 0.062 +/- 0.017 mumol/g; GSSG, 0.155 +/- 0.063 vs 0.011 +/- 0.001 mumol/g).     

LC Determination of Thiols Derivatized with 4-(Aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole after SPE

Solid-phase extraction (SPE) coupled with high-performance liquid chromatography?Cfluorescence detection (LC?CFL) was developed for the determination of three thiol compounds including glutathione, cysteine and acetylcysteine. 4-(Aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole was used for derivatization of thiols. Factors affecting derivatization and extraction efficiency were optimized. Sample solution (2?mL) was extracted on a SPE column for 2?min and then eluted with 400???L methanol. The analytes were injected onto the LC system for separation on a C₁₈ column, and eluted with methanol?Cacetate buffer. The analytes were detected by fluorescence at an emission wavelength of 515?nm with excitation at 385?nm. The linearity of the method was in the range of 0.1?C60???M, with correlation coefficients ranging from 0.9979 to 0.9990. The detection limits of the method were in the range of 5?C20?nM. The proposed method was applied to the analysis of human plasma samples with recoveries of 86?C112.9%.     

Fluorescent detection of peptides and amino acids for capillary electrophoresis via on-line derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole

An in-capillary derivatization of amino acids and peptides with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was developed for their subsequent capillary electrophoretic analysis with laser-induced fluorescence detection (λ _ex=488 nm). The in-capillary derivatization was achieved in zone-passing mode by introducing successive plugs of sample and NBD-F into a fused silica capillary previously equilibrated with an alkaline borate buffer. To prevent NBD-F hydrolysis and to achieve a reliable derivatization, NBD-F was prepared daily in absolute ethanol and a plug of absolute ethanol was introduced between the sample and NBD-F reagent plugs. Various parameters influencing the derivatization efficiency were investigated and the optimum conditions were as follows: background electrolyte (BGE), 20 mM borate buffer (pH 8.8); introduction time, 4 s for sample and 2 s for NBD-F; molar ratio of NBD-F/sample, above 215; temperature, 45 °C for amino acids and 35 °C for peptides; applied voltage, +15 kV. The validation of the in-capillary derivatization method under optimal conditions showed a good linearity between the heights of the derivative peaks and the concentrations of the amino acids. The intra-day relative standard deviations of the migration times and the peak heights were less than 1.3% and 4.6%, respectively. The efficient derivatization and separation of a mixture of valine, alanine, glutamic acid and aspartic acid were achieved using this technique. Peptides such as buccaline and β-protein fragment 1–42 could also be derivatized using the developed in-capillary derivatization procedure. In‑capillary derivatization and separation of amino acids with different concentrations. From the top to bottom the concentrations are 1.11×10⁻⁵ M, 5.55×10⁻⁶ M, 2.78×10⁻⁶ M, 6.95×10⁻⁷ M. for valine; 1.26×10⁻⁵ M, 6.30×10⁻⁶ M, 3.15×10⁻⁶ M, 7.88×10⁻⁷ M for alanine; 3.78×10⁻⁵ M, 1.89×10⁻⁵ M, 9.45×10⁻⁶ M, 2.36×10⁻⁶ M for glutamic acid;, 4.27×10⁻⁵ M, 2.14×10⁻⁵ M, 1.07×10⁻⁵ M, 2.68×10⁻⁶ M for aspartic acid. Experiment conditions: injection order: 4s for sample, 1s for absolute ethanol, and then 2s for 5.24×10⁻² M NBD‑F; BGE: 20 mM borate pH 8.77; Applied voltage: 15 kV.     

Chiral separation of homocysteine by derivatization with 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole followed by capillary electrophoresis using gamma-cyclodextrin

Homocysteine was derivatized with 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (ABD-F) to form an inclusion complex with cyclodextrin and to facilitate UV detection. ABD-homocysteine showed interaction with beta- and gamma-cyclodextrin in capillary electrophoresis at pH 2.25 as indicated by the decreased migration time. However, chiral separation of D,L-ABD-homocysteine was observed using gamma-CD only. Optimal separation was obtained at pH 2.25, 50 mM gamma-CD concentration, and 20 kV applied voltage. L-ABD-Homocysteine migrated faster than the D-isomer as demonstrated by a spiking experiment using dithiothreitol-reduced L-homocystine.     

4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole as chemilumigenic reagent for high performance liquid chromatographic peroxyoxalate chemiluminescence detection

4-(N,N-Dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F), presented as a fluorogenic labelling reagent for amines and amino acids, is preferred for peroxyoxalate chemiluminescence (PO-CL) detection in high performance liquid chromatography. Amino acids and epinephrine derivatized with DBD-F were separated on a reversed phase column and detected at the femtomole level by the PO-CL detection system.     

Determination of fluvoxamine in rat plasma by HPLC with pre-column derivatization and fluorescence detection using 4-fluoro-7-nitro-2,1,3-benzoxadiazole

A sensitive, simple and reliable method using high-performance liquid chromatographic (HPLC) assay of fluvoxamine (FLU), a selective serotonin reuptake inhibitor (SSRI), in rat plasma after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was developed in this study. Extracted plasma samples were mixed with NBD-F at 60 degrees C for 5 min and injected into HPLC. Retention times of FLU and an internal standard (propafenone) derivative were 15.5 and 13.5 min, respectively. The calibration curve was linear over the range 0.015-1.5 microg/mL (r2 = 0.9985) and the lower limits of detection and quantification of FLU were 0.008 and 0.015 microg/mL, respectively, in 100 microL of plasma. The derivative sample was stable at 4 degrees C for 1 day. The coefficients of variation for intra-day and inter-day assay of FLU were less than 8.3 and 9.6%, respectively. Other SSRIs and centrally acting drugs did not interfere with the peak of the FLU derivative. The method was applied for analysis of the plasma samples from rats treated with FLU. These results indicate that the method presented is useful to determine the FLU levels in rat plasma of volumes as small as 100 microL and can be applied to pharmacokinetic studies.     

Catecholamines derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole: characterization of chemical structure and fluorescence properties

4-Fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was evaluated as a fluorogenic derivatization reagent for the analysis of the catecholamines, dopamine, epinephrine, norepinephrine, and their naturally occurring metabolites, metanephrine and normetanephrine, homovanillic acid, 3,4-dihydroxyphenyl acetic acid. These compounds reacted rapidly with NBD-F under mild conditions to form stable derivatives. The optimal reaction conditions were found to be 12.5 mM borate buffer pH 8.0 in water:acetonitrile (1:1) at 50 °C for 5 min. New NBD derivatives of all the catecholamines and metabolites were prepared and purified and were shown by electrospray mass spectrometry to be fully reacted at all available catechol and amine sites, resulting in di- or tri-substituted derivatives. Homovanillic acid and 3,4-dihydroxyphenyl acetic acid reacted with NBD-F but gave non-fluorescent derivatives. The fluorescence excitation wavelength maximum demonstrated a red shift for the derivatives with increasing polarity of the solvent and the fluorescence intensity increased linearly with increasing organic ratio in the solvent-aqueous buffer complex. The presence of electrolyte in the solvent and the electrolyte concentration in the solvent-electrolyte complex had little effect on the fluorescent intensity. The fluorescence quantum yields in acetonitrile were also obtained. The separation behavior of the NBD-catecholamines was determined by high-performance liquid chromatography (HPLC). The studies demonstrated good potential for the application of NBD-F derivatization to the quantitative analysis of catecholamines and related compounds in biological matrices.     

Reversible labeling of tyrosine residue in peptide using 4-fluoro-7-nitro-2,1,3-benzoxadiazole and N-acetyl-L-cysteine.

The reversible labeling of tyrosine (Tyr)-containing peptide, which involves detection and recovery, is described in this paper. The phenolic-OH in Tyr structure reacted with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) under a mild condition at room temperature in alkaline medium. The resulting derivative absorbed at around 280 nm and 380 nm. However, the fluorescence was very weak. The NBD moiety in the derivative was efficiently removed by the treatment of N-acetyl-L-cysteine (N-AcCys), and the original peptide before the labeling was completely recovered. The proposed procedure was successfully applied to the reversible labeling of N-terminal amine-blocked peptides, i.e., N-AcTyr-Val-Gly, Z-Glu-Tyr, Z-Phe-Tyr, N-Formyl-Met-Leu-Tyr, and N-AcArg-Pro-Pro-Gly-Phe-Ser-Pro-Tyr-Arg. Although the proposed method could not recover the N-terminal amine-free peptides without blocking, the selective detection and the recovery of Tyr-containing peptide fragments were possible by the combination with enzyme digestion. The reversible labeling of Tyr-containing peptide was demonstrated with [Tyr8]-bradykinin as a model for high-molecular-mass peptides and proteins. The peptide fragments containing NBD-O-Tyr moiety, obtained after the digestion, were easily discriminated from various peptides with the monitoring of UV and FL, because the target peptide did not fluoresce, but absorbed at both 280 nm and 380 nm. The peptide fragment containing Tyr was finally recovered from the de-labeling reaction with N-AcCys. The proposed method hence provides a novel technique for the reversible labeling of Tyr-containing peptides, which will enable the selective detection and the recovery of the original peptide.     

Development of 7-(N,N-dimethylaminosulfonyl)-5-N-(4-N-aminoethyl)piperazino-2,1,3-benzoxadiazole as a water-soluble fluorogenic reagent for the sensitive liquid chromatographic determination of saturated carboxylic acids

A reversed-phase high-performance liquid chromatographic (HPLC) method for the femtomole determination of nine saturated carboxylic acids, n-butyric (C4), n-hexanoic (C6), n-caprylic (C8), n-decanoic (C10), lauric (C12), n-tetradecanoic (C14), palmitic (C16), stearic (C18) and arachidic (C20), based on the condensation reaction of these acids with a newly synthesized water-soluble benzofurazan fluorescent reagent, 7-(N,N-dimethylaminosulfonyl)-4-N-(4-N-aminoethyl)piperazino-2,1,3-benzoxadiazole (DBD-PZ-NH2), was developed. The derivatization reaction proceeds with 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC) in the presence of the catalyst 4-(dimethylamino)pyridine (DMAP). A model derivative of the reagent with n-caprylic acid (C8) was synthesized for fluorescence excitation/emission characterization. Depending on the solvents, including water, methanol, acetonitrile, 1,4-dioxane or N.N-dimethyformamide (DMF), the C8 derivative has a fluorescence emission with a fluorescence quantum yield (phi) ranging from 0.01 to 0.20 in the region from 545 to 580 nm. An exponential increase in phi was observed with increasing acetonitrile content. The calculated detection limits (signal-to-noise ratio = 3:1) of the proposed method for the above nine carboxylic acids were 9.1, 4.0, 2.5, 2.2, 2.0, 1.8, 1.2, 1.0 and 1.3 fmol, respectively. Biological samples including Intralipos 20% and rat plasma were analysed satisfactorily.     

Sensitive determination of 4-(4-chlorophenyl)-4-hydroxypiperidine, a metabolite of haloperidol, in a rat biological sample by HPLC with fluorescence detection after pre-column derivatization using 4-fluoro-7-nitro-2,1,3-benzoxadiazole

4-(4-Chlorophenyl)-4-hydroxypiperidine (CPHP), one of the metabolites of haloperidol, is considered to exhibit brain toxicity. CPHP concentrations in plasma and tissue homogenates (each 200 microL) from rats were analyzed by HPLC fluorescence detection after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). After basic extraction of the samples with benzene, the derivatization with NBD-F was conducted in borate buffer (pH 8.0) at 60 degrees C for 3 min. Mexiletine was carried through the procedure as an internal standard. The regression equation for CPHP showed a good linearity in the range of 0.03-1 microg/mL with a detection limit of 0.008 microg/mL. The coefficient of variation was less than 11.6%. Plasma concentration-time courses of CPHP after intraperitoneal or per oral administration of CPHP, haloperidol or reduced haloperidol were examined, and the pharmacokinetic parameters were estimated. Additionally, CPHP levels in various tissues at 8 h after intraperitoneal administration of these compounds were compared. The method was simple and sensitive, useful for determination of CPHP in rat biological samples using as little as 200 microL of sample volume and could be applied for pharmacokinetic study.     

High performance liquid chromatography with chemiluminescence detection of methamphetamine and its related compounds using 4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole.

A high performance liquid chromatographic assay of methamphetamine (MP) and its related compounds, i.e. ephedrine (EP), norephedrine (NE), p-hydroxymethamphetamine (p-HMP), p-hydroxyamphetamine (p-HAP) and amphetamine (AP), with peroxyoxalate chemiluminescence detection has been developed. 4-(N,N-Dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) was used as a fluorescent labeling reagent. A mixture of hydrogen peroxide and bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate in acetonitrile was used as a postcolumn chemiluminogenic reagent. DBD derivatives of MP and its related compounds were separated by a gradient elution with acetonitrile and 0.01 M imidazole buffer (pH 7.0) within 65 min. The detection limits (S/N = 3) for the proposed method for MP, AP, EP, NE, p-HMP and p-HAP were 27, 100, 40, 133, 25 and 133 fmol on column, respectively. The recoveries of these compounds with normal urine samples were 87.4-106.4%. The method was successfully applied to the assay of MP and its metabolites in urine samples from MP addicts. A good linear correlation for the resulted amounts of MP or AP between the proposed method and gas chromatography was obtained (r = 0.993 for MP or 0.991 for AP).     

Capillary electrophoresis with laser induced-fluorescence detection of profens derivatized with the water-soluble fluorogenic reagent 4-N-(4-N'-aminoethyl)piperazino-7-nitro-2,1,3-benzoxadiazole

Profens, including pranoprofen, fenoprofen, flurbiprofen, ketoprofen and ibuprofen (Ib), were derivatized by a water-soluble benzofurazan fluorescent reagent, 4-N-(4-N'-aminoethyl)piperazino-7-nitro-2,1,3-benzoxadiazole and then were run on capillary electrophoresis in a NH4Ac-HAc buffer of pH 3.1 containing 2.4 mM beta-cyclodextrin. At room temperature, the derivatization reaction was catalyzed by triphenyl phosphine and diphenyl disulfide in acetonitrile medium, and the derivatives fluoresce around 530 nm when excited at 488 nm. With the CE running on a 50 cm x 50 microm I.D. length fused-silica capillary of by using Ar+ laser induced-fluorescence detection, the detection limits attained were in the range of 0.16 to 0.3 fmol.     

Simultaneous liquid chromatographic assay of amantadine and its four related compounds in phosphate-buffered saline using 4-fluoro-7-nitro-2,1,3-benzoxadiazole as a fluorescent derivatization reagent

Simultaneous HPLC assay of 1-adamantanamine hydrochloride (amantadine) and its four related compounds [2-adamantanamine hydrochloride (2-ADA), 1-adamantanmethylamine (ADAMA), 1-(1-adamantyl)ethylamine hydrochloride (rimantadine) and 3,5-dimethyl-1-adamantanamine hydrochloride (memantine)] in phosphate-buffered saline (pH 7.4) after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was developed. Phosphate-buffered saline samples were mixed with borate buffer and NBD-F solution in acetonitrile at 60 degrees C for 5 min and injected into HPLC. Five derivatives were well separated from each other. The lower limits of detection of amantadine, 2-ADA, ADAMA, rimantadine and memantine were 0.008, 0.001, 0.0008, 0.0015 and 0.01 microg/mL, respectively. The coefficients of variation for intra- and inter-day assay were less than 6.4 and 8.2%, respectively. The method presented was applied to a binding study of these compounds to human alpha(1)-acid glycoprotein. While affinity constants and capacities for ADAMA, rimantadine and memantine were calculated by means of Scatchard plots, those for the others were not determined. ADAMA, rimantadine and memantine were bound with different affinities and capacities. These results indicate that NBD-F is a good candidate as a fluorescent reagent to simultaneously determine amantadine and its four related compounds by HPLC after pre-column derivatization. Our method can be applied to binding studies for protein.     

Micellar electrokinetic capillary chromatographic separation and fluorescent detection of amino acids derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole

Another method has been developed for the separation of amino acids (1 min derivatization plus 22 min separation) by micellar electrokinetic capillary chromatography (MECC) with laser-induced fluorescence detection. Interestingly enough, such work has never been performed on essential amino acids derivatized by 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). Fifteen L-amino acid standards were labelled with NBD-F at 60 degrees C for 1 min, and separated in a buffer system containing 20 mM borate, 25 mM sodium cholate, 10 mM Brij 35 and 2.5% methanol. Methanol was employed to expand the MECC migration time window; whereas Brij 35 was used to improve the fluorescence intensity of amino acid derivatives. This method also indicates that bile salt is effective for MECC separation of ionic analytes. Surprising though, improvements in resolution, sensitivity and speed for amino acids analysis are obtained in this work, which are not initially apparent in just employing another derivatizing reagent. Under optimal conditions, 15 amino acids were separated in a short 22 min analysis time, the shortest ever reported, and detection limits of nanomolar concentration and attomole mass were obtained. Furthermore, RSDs of migration time and peak height are better than 1% and 1.8%, respectively, again the smallest ever reported in the literature.     

Synthesis and application of 4-(N,N-dimethylaminosulfonyl)-7-N-methylhydrazino-2,1,3-benzoxadiazole (MDBDH) as a new derivatizing agent for aldehydes

In a five step synthesis, 4-(N,N-dimethylaminosulfonyl)-7-N-methylhydrazino-2,1,3-benzoxadiazole (MDBDH) was prepared in high yields as a stable new derivatizing agent for carbonyl compounds. Reagent and derivatives have not been described in literature before. Major advantage of this substance compared with similar reagents is its improved solubility in polar solvents, e.g. methanol and ethanol. MDBDH reacts with aldehydes in the presence of an acidic catalyst under formation of the corresponding hydrazones. These are separated by means of reversed-phase liquid chromatography and detected UV/vis spectroscopically at wavelengths around 450 nm, depending on the individual hydrazone. MDBDH reacts with oxidizers as nitrogen dioxide and nitrite to only one product, 4-(N,N-dimethylaminosulfonyl)-7-methylamino-2,1,3-benzoxadiazole (MDBDA), which can easily be separated from the hydrazones of lower aldehydes. Due to large molar absorptivities and absorption maxima at wavelengths > 430 nm, limits of detection range from 4 × 10^–8 to 6 × 10^–8 mol · L^–1, and limits of quantification range from 1 × 10^–7 to 2 × 10^–7 mol · L^–1 for the individual hydrazones. The method was applied to the determination of aldehydes in automobile exhaust. Received: 31 July 2000 / Revised: 9 October 2000 / Accepted: 18 October 2000     

Synthesis and application of 4-(N,N-dimethylaminosulfonyl)-7-N-methylhydrazino-2,1,3-benzoxadiazole (MDBDH) as a new derivatizing agent for aldehydes

In a five step synthesis, 4-(N,N-dimethylaminosulfonyl)-7-N-methylhydrazino-2,1,3-benzoxadiazole (MDBDH) was prepared in high yields as a stable new derivatizing agent for carbonyl compounds. Reagent and derivatives have not been described in literature before. Major advantage of this substance compared with similar reagents is its improved solubility in polar solvents, e.g. methanol and ethanol. MDBDH reacts with aldehydes in the presence of an acidic catalyst under formation of the corresponding hydrazones. These are separated by means of reversed-phase liquid chromatography and detected UV/vis spectroscopically at wavelengths around 450 nm, depending on the individual hydrazone. MDBDH reacts with oxidizers as nitrogen dioxide and nitrite to only one product, 4-(N,N-dimethylaminosulfonyl)-7-methylamino-2,1,3-benzoxadiazole (MDBDA), which can easily be separated from the hydrazones of lower aldehydes. Due to large molar absorptivities and absorption maxima at wavelengths > 430 nm, limits of detection range from 4 x 10(-8) to 6 x 10(-8) mol.L-1, and limits of quantification range from 1 x 10(-7) to 2 x 10(-7) mol.L-1 for the individual hydrazones. The method was applied to the determination of aldehydes in automobile exhaust.