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PubMed1993

Functions of interleukin-8 are mediated through thiol group(s) of IL-8 receptor in human polymorphonuclear neutrophils. Effects of 5,5'-dithio-bis(2-nitrobenzoic acid) on IL-8 receptor.

Interleukin-8, a neutrophil chemotactic agent causes excessive accumulation of the cells in a number of inflammatory diseases. The activity has been shown to be mediated through a specific functional receptor present on the surface of neutrophils. No information is available about the amino acids constituting the IL-8 binding domain of the receptor. Treatment of neutrophils with 5,5'-dithio-bis(2-nitrobenzoic acid), a thiol-specific modifier, at the concentrations of 0.4 mM and 1 mM reduced IL-8 binding ability and IL-8-induced migration of the cells by 45% and 65%, respectively. Dithiothreitol could regenerate the binding capacity and the ligand could protect the receptor from the effect of the reagent. All the evidence suggests that one or more critical thiol residues are located in the IL-8 binding site of the receptor which are indispensible for normal functions of IL-8.

Chemotaxis↗
PubMed1991

Kinetics of the reaction between 5,5'-dithiobis[2-nitrobenzoic acid] and the sulphydryl group in Zn(2+)-dependent beta-lactamase II.

The kinetics of the reaction of the thiol residue in Zn(2+)-dependent beta-lactamase II with 5,5'-dithiobis[2-nitrobenzoic acid], and the concomitant inactivation revealed that both events take place at the same rate. The inactivation could not be reverted by incubation with Zn2+ or by using a substrate concentration about eight times the Km of the enzyme. EDTA incubation also produced inactivation of the enzyme, although it was reverted by increasing the substrate concentration in the assay. A dual role is proposed for Zn2+ in beta-lactamase. The kinetic analysis of the thiol modification and the concomitant inactivation is in agreement with previous reports on the implication of the metal ion in catalysis. A role in stabilizing the native structure of the enzyme is also suggested.

Bacillus↗
PubMed1993

The oxidation of rabbit liver metallothionein-II by 5,5'-dithiobis(2-nitrobenzoic acid) and glutathione disulfide.

Because metallothionein (MT) may undergo thiol-disulfide or other redox reactions under certain cellular conditions, the partially and completely oxidized products of the reactions of Cd7-MT-II with the electrophile 5,5'-dithiobis(2-nitrobenzoic acid), ESSE, and oxidized glutathione, GSSG, were characterized. Reaction with the stoichiometric quantity of ESSE (1 ESSE per MT thiolate) generates monomeric and polymeric MTs with three types of disulfide bonds: intra- and intermolecular CyS-SCy linkages and a small number (2-3/MT) of mixed disulfides, CyS-SE, involving thionitrobenzoate (ES-). Reaction with substoichiometric quantities of ESSE (0.02 or 0.1 per MT thiolate) causes the formation of intra- and intermolecular CyS-SCy disulfides, but no mixed disulfides. In the latter reactions, two equivalents of ES- are released per mole of ESSE, but the release is described by a single first-order rate constant (k = 3.0 +/- 0.5 sec-1). Substantial amounts of cadmium remained bound to the MT monomers and polymers after reaction with the substoichiometric quantities. Despite the Cd bound to the MT after reaction with 0.1 ESSE per MT thiolate, no 111Cd NMR signals were detected, indicating rapid equilibration of the remaining metal ions among the disrupted binding sites. Large excesses of the endogenous aliphatic disulfide, GSSG, displace Zn+2 from Zn7-MT slowly. The reaction is complete after 24 hours with 5000 microM GSSG, but only 25% complete after 72 hours with 250 microM GSSG. Approximately one Cd+2 is displaced rapidly from Cd7MT by 5000 microM GSSG and half as much by 250 microM GSSG, but no further reaction occurs. It is unlikely that GSSG oxidation of MTs would be physiologically significant.

Animals↗
PubMedDate not supplied

Relationships among direct-acting mutagenicity, nitro group orientation and polarographic reduction potential of 6-nitrobenzo[a]pyrene, 7-nitrobenz[a]anthracene and their derivatives.

The direct-acting mutagenicity in Salmonella typhimurium strains TA98 and TA100 and the half-wave reduction potentials of 6-nitrobenzo[a]pyrene (6-nitro-BaP), 7-nitrobenz[a]anthracene (7-nitro-BA), and a series of their derivatives were compared. The common structural feature of these compounds is that their nitro substituents, in order to minimize steric hindrance, preferentially adopt an orientation perpendicular or nearly perpendicular to the aromatic rings. All of the compounds, except 7-hydroxy-6-nitro-BaP and 7-acetoxy-6-nitro-BaP, were found to exhibit very weak or no direct-acting mutagenicity. 7-Acetoxy-6-nitro-BaP and 7-hydroxy-6-nitro-BaP were also found to have the lowest reduction potentials among the tested compounds. The results suggest that a combination of the orientation of the nitro substituent and the first half-wave reduction potential of the compound may correlate with the direct-acting bacterial mutagenicity of nitro-polycyclic aromatic hydrocarbons.

Benz(a)Anthracenes↗
PubMed1989

Modification of the (Ca2+ + Mg2+)-ATPase protein of sarcoplasmic reticulum with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole.

The (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase (Ca2+-transporting), EC 3.6.1.38) protein of rabbit skeletal sarcoplasmic reticulum (SR) rapidly incorporated 2 mol of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) per 10(5) g of protein with little change in the Ca2+-dependent ATPase activity. When 2 additional mol of the reagent were bound the Ca2+-ATPase, activity was inhibited. The same pattern was found for modified intact SR and the Ca2+ uptake ability was inhibited. MgATP, CaATP and MgADP protected the Ca2+-ATPase activity concurrent with a decrease of about 1 mol of the NBD group per 10(5) g protein, but the Ca2+ uptake ability was not protected. Calcium alone had no effect on the modification. The modified ATPase protein or SR formed non-serial oligomers or aggregates, but the ATPase protein remained the predominant species present. In the presence of MgATP, oligomer formation was reduced partially but the major changes in the Ca2+-ATPase activity were due to the modification of the ATPase monomer. Thiolysis of the NBD-ATPase protein with dithiothreitol did not restore the Ca2+-ATPase activity, although more than 1 mol of the NBD group was removed from cysteine residues. Cysteine residues were modified in the NBD-ATPase protein or SR when the enzyme activity was inhibited. Trypsin digestion of NBD-SR or its ATPase protein released the A, B, A1, and A2 fragments. The A fragment and its subfragment A2 contained most of the label. Substrate MgATP protection studies showed that the A1 and A2 fragments were involved in maintaining the Ca2+-ATPase activity. Reagent-induced conformational changes of these fragments rather than direct active site group labeling accounted for the loss of ATPase activity.

4-Chloro-7-nitrobenzofurazan↗
PubMed1994

Reaction of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole with the (Ca2+ + Mg2+)- ATPase protein of sarcoplasmic reticulum at low temperature.

Modification of the (Ca2+ + Mg2+)-ATPase protein of rabbit skeletal sarcoplasmic reticulum (SR) with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, NBD-Cl, at 4 degrees C for 5 min caused a 63% loss of the Ca(2+)-dependent ATPase activity when 1 mol of the adenine analog was incorporated per 10(5) g of protein. At 25 degrees C, above the lipid phase transition, the extent of labeling was 3-fold higher although the Ca(2+)-ATPase activity was inhibited to the same extent. MgATP protected the ATPase activity at 4 degrees C and 25 degrees C but there was little change in the extent of labeling at 4 degrees C suggesting that changes in the fluidity of the lipid moiety made different sites on the ATPase protein accessible to the reagent. At 4 degrees C, addition of sodium deoxycholate enhanced the inactivation (6% ATPase activity remained) but the labeling of the SR-ATPase protein did not increase significantly. Incubation with MgATP prior to solubilization with deoxycholate resulted in the protection of the Ca(2+)-ATPase activity and only a small decrease in the labeling occurred. At 25 degrees C, a similar pattern was found with deoxycholate but the loss of ATPase activity was less dramatic and the extent of labeling by NBD-Cl was greater than that at 4 degrees C. MgATP induced changes in the conformation of the ATPase protein protecting essential cysteine residues while shifting the reaction of NBD-Cl with the ATPase protein to non-essential sites in the absence or presence of deoxycholate. An analysis of tryptic digests of the NBD-ATPase protein showed that MgATP shifted the labeling from the A2 subfragment to the A1 subfragment in the absence of deoxycholate and from the A1 subfragment to the A2 subfragment in the presence of deoxycholate. The reagent, NBD-Cl, can distinguish between different temperature dependent conformational states of the ATPase protein.

4-Chloro-7-nitrobenzofurazan↗
PubMed1987

Metabolism of 6-nitrobenzo[a]pyrene in rat lung preparations.

6-Nitrobenzo[alpha]pyrene (6-NBaP) occurs in our environment. Since human exposure to environmental contaminants may occur via the inhalation route, we examined the metabolites of 6-NBaP formed in lung preparations, and compared the metabolite profile to that which was found with liver. The metabolites formed in both liver and lung preparations consisted of ring-hydroxylated 6-NBaP, 6-hydroxybenzo[a]pyrene (6-OHBaP) and small amounts of benzo[a]pyrene. In the lung experiments, 6-OHBaP most frequently oxidized to quinones. The mechanism for the formation of 6-OHBaP from 6-NBaP remains to be elucidated.

Animals↗
PubMed1995

Sensitive detection of selected cholinergic channel activators derivatized with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole.

A high-performance liquid chromatographic method with fluorescence detection was developed for a series of cholinergic channel activators using 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) as a fluorescent labeling reagent. This method includes three separate steps: extraction of parent compound and internal standard with organic solvent from plasma, reaction of parent and internal standard in NBD-F precolumn to yield a fluorescent product and extraction of the resultant fluorophores with organic solvents. The extraction and reaction procedures were optimized for this series of structurally new compounds. The method showed high sensitivity, selectivity and reproducibility and proved useful for the determination of ng ml-1 plasma levels of selected cholinergic channel activators.

4-Chloro-7-nitrobenzofurazan↗
PubMed1995

Enzymatic reduction of 5,5'-dithiobis-(2-nitrobenzoic acid) by lysate of rat liver mitochondria.

The oxidation of mitochondrial sulphydryl groups is known to increase the permeability of mitochondrial membranes and to be a key event in oxidative stress. Resistance to this damage is thought to involve thioredoxin reductase. In this study, the reduction of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) by a lysate of rat liver mitochondria was used to assay the mitochondrial disulphide reducing capacity. NADPH-dependent reduction of DTNB was used to distinguish enzymatic reduction from the non-enzymatic reduction. Enzymatic reduction by the mitochondrial lysate was suppressed by DTNB at concentrations exceeding 0.25 mM and by pH above 7.0. It was strongly inhibited by Zn2+ and Mn2+ (IC50 about 2.5 and 20 microM, respectively) and was more weakly inhibited by Mg2+ and Ca2+ (IC50 about 1.8 and 2.1 mM, respectively). As a consequence of inhibition by divalent cations, the reaction was stimulated by both physiological (ATP, ADP, pyrophosphate and citrate) and non-physiological (EDTA and EGTA) chelators. Reduction of insulin disulphides by the mitochondrial lysate was dependent on the presence of a divalent cation chelator during the isolation of mitochondria and during the enzyme assay. Our results suggest that stimulation of mitochondrial disulphide reducing activity by lowered pH, as well as by increased levels of ATP, ADP and citrate, has the potential to contribute to the maintenance of mitochondrial sulphydryl groups under oxidative conditions.

Adenosine Diphosphate↗
PubMed2005

Spectrophotometric and fluorometric assay of superoxide ion using 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole.

Two highly sensitive spectrophotometric methods are developed and described for the measurement of superoxide ion radical derived from KO2 as well as O2*- generated either from the xanthine-xanthine oxidase reaction or by the addition of nicotinamide adenine dinucleotide (NADH) to skeletal muscle sarcoplasmic reticulum (SR) vesicles. These methods allow quantification of superoxide ion concentration by monitoring its reaction with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), either by recording absorbance of the final reaction product at a wavelength of 470 nm or by measuring its fluorescence emission intensity at 550 nm using an excitation wavelength of 470 nm. The extinction coefficient of the active product was determined to be 4000 M(-1) cm(-1). A lower limit second-order bimolecular rate constant of 1.5+/-0.3x10(5) M(-1) s(-1) was estimated from kinetic stopped-flow analysis for the reaction between NBD-Cl and KO2. A plot of absorbance versus concentration of superoxide was linear over the range 2 to 200 microM KO2, whereas higher sensitivities were obtained from fluorometric measurements down into sub-micromolar concentrations with a limit of detection of 100 nM KO2. This new spectrophotometric assay showed higher specificity when compared with some other commonly used methods for detection of superoxide (e.g., nitroblue tetrazolium). Results presented showed good experimental agreement with rates obtained for the measurement of superoxide ion when compared with other well-known probes such as acetylated ferri cytochrome c and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT). A detailed discussion of the advantages and limitations of this new superoxide ion probe is presented.

4-Chloro-7-nitrobenzofurazan↗
PubMed2004

Implications of the role of reactive cystein in arginine kinase: reactivation kinetics of 5,5'-dithiobis-(2-nitrobenzoic acid)-modified arginine kinase reactivated by dithiothreitol.

The reduction of 5,5'-dithiobis-(2-nitrobenzoic acid)-modified arginine kinase by dithiothreitol has been investigated using the kinetic theory of the substrate reaction during modification of enzyme activity. The results show that the modified arginine kinase can be fully reactivated by an excess concentration of dithiothreitol in a monophasic kinetic course. The presence of ATP or the transition-state analog markedly slows the apparent reactivation rate constant, while arginine shows no effect. The results of ultraviolet (UV) difference and intrinsic fluorescence spectra indicate that the substrate arginine-ADP-Mg2+ can induce conformational changes of the modified enzyme but adding NO3- cannot induce further changes that occur with the native enzyme. The reactive cysteines' location and role in the catalysis of arginine kinase are discussed. It is suggested that the cysteine may be located in the hinge region of the two domains of arginine kinase. The reactive cysteine of arginine kinase may play an important role not in the binding to the transition-state analog but in the conformational changes caused by the transition-state analog.

Arginine Kinase↗
PubMed2005

Reversible reaction of 5,5'-dithiobis(2-nitrobenzoate) with the hemoglobins of the domestic cat: acetylation of NH3+ terminal group of the beta chain transforms the complex pH dependence of the forward apparent second order rate constant to a simple form.

We demonstrate kinetically that the reaction of 5,5'-dithiobis(2-nitrobenzoate) with the CysF9[93]beta sulfhydryl group of domestic cat hemoglobins is a reversible process. In the major hemoglobin, in which the NH3+ terminal group of GlyNA1[1]beta is free, kf, the apparent forward second order rate constant, has a complex pH dependence profile. In the minor hemoglobin, the NH3+ terminal group of SerNA1[1]beta is acetylated, and the pH dependence profile of kf is simple. These results support the proposal that the positively charged groups at the organic phosphate binding site are electrostatically linked to CysF9[93]beta. Quantitative analyses of the complex profiles enabled us to estimate pKas of 7.47 +/- 0.3; 6.53 +/- 0.03 and 8.49 +/- 0.3 for GlyNA1[1]beta, HisH21[143]beta and other histidines within 2 nm of the sulfhydryl, and CysF9[93]beta, respectively, of the major hemoglobin. Analyses of the simple profiles gave pKas of 6.33 +/- 0.17 and 8.54 +/- 0.5 for HisH21[143]beta and other histidines within a distance of 2 nm of the sulfhydryl, and CysF9[93]beta of the minor hemoglobin, respectively.

Acetylation↗
PubMed2006

Reversible reaction of 5,5'-dithiobis(2-nitrobenzoate) with the CysF9[93]beta sulfhydryl groups of the hemoglobins of the domestic cat: variation of the equilibrium and reverse rate constants with pH.

We have determined for the first time the equilibrium constant, Keq, for the reaction of Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoate), with the CysF9[93]beta sulfhydryl groups of the hemoglobins of the domestic cat. In the pH range 5.6 to 9.0 Kequ varies over four orders of magnitude--between ca 10 and 10(-3)--for all hemoglobin derivatives. Using these Kequ values and published data on the dependence of the apparent second order forward rate constant, kf, on pH we have calculated the apparent second order reverse rate constant, kr, as a function of pH. This parameter increases strongly with pH, particularly above pH 7.5. Quantitative analyses of the pH dependence profiles of log10kr indicate that the reverse reaction is coupled to the ionization of two groups on the protein with pKas of 7.2+/-0.2 and 9.4+/-0.1 in the major hemoglobin and 6.7+/-0.3 and 8.4+/-0.1 in the minor hemoglobin.

Animals↗
PubMed2005

The kinetic study of arginine kinase from the sea cucumber Stichopus japonicus with 5,5'-dithiobis-(2-nitrobenzoic acid).

The Stichopus japonicus arginine kinase (AK) is a significant dimeric enzyme. Its modification and inactivation course with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and the reactivation course of DTNB-modified AK by dithiothreitol were investigated on the basis of the kinetic theory of the substrate reaction during the modification of enzyme activity. The results show that the modification is a biphasic course while the inactivation is monophasic, with one essential reactive cysteine per subunit. The Cys274 (numbering from the Stichopus sequence) is exposed to DTNB and is near the ATP binding site. The modified AK can be reactivated by an excess concentration of dithiothreitol in a monophasic kinetic course. The presence of ATP or the transition-state analog markedly slows the apparent reactivation rate constant. The analog components, arginine-ADP-Mg2+ can induce conformational changes of the modified enzyme, but adding NO3- cannot induce further changes that occur with the native enzyme. The reactive cysteines' location and its role in the catalysis of AK are discussed. The results suggest that the cysteine may be located in the hinge area of the two domains of AK. The reactive cysteine of AK, which was proposed to be Cys274, may play an important role not in the binding of the transition-state analog but in the conformational changes caused by the transition-state analog.

Adenosine Diphosphate↗
PubMed2004

Determination of lisinopril in dosage forms and spiked human plasma through derivatization with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) followed by spectrophotometry or HPLC with fluorimetric detection.

Two sensitive, simple and specific methods based on spectrophotometry and reversed-phase HPLC with fluorimetric detection are described for the determination of lisinopril in dosage forms as well as in spiked human plasma using solid phase extraction (SPE) procedures. Both methods are based on the derivatization of lisinopril with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) in borate buffer of pH 9 to yield a yellow, fluorescent product. The spectrophotometric method depends on measuring the formed yellow color at 470 nm after optimization of the reaction conditions. The HPLC method is based on measurement of the derivatized product using fluorescence detection at 540 nm (excitation at 470 nm). The separation of the derivatized drug, the excess reagent and the internal standard (bumetanide) was performed on a reversed-phase ODS column using isocratic elution with methanol-0.02 M sodium dihydrogen phosphate, pH 3.0 (55:45, v/v) at a flow rate of 1.0 ml/min. The calibration graphs were linear over the concentration ranges 2-20 or 0.02-3.2 microg/ml of lisinopril with minimum detectability of 0.3 and 0.008 microg/ml (6.1 x 10(-7) and 1.7 x 10(-8)M) for the spectrophotometric and the HPLC methods, respectively. The proposed methods were applied without any interference from the tablet excipients for the determination of lisinopril in dosage forms, either alone or co-formulated with hydrochlorothiazide. Furthermore, the use of the HPLC method was extended to the in vitro determination of the drug in spiked human plasma. Interference from endogenous amino acids has been overcomed by using the solid phase extraction technique, the percentage recovery (n=6) was 101.6+/-3.35.

4-Chloro-7-nitrobenzofurazan↗
PubMed1999

Effect of NBD chloride (4-chloro-7-nitrobenzo-2-oxa-1,3-diazole) on the pyridine nucleotide transhydrogenase of Escherichia coli.

Pyridine nucleotide transhydrogenases of bacterial cytosolic membranes and mitochondrial inner membranes are proton pumps in which hydride transfer between NADP(+) and NAD(+) is coupled to proton translocation across cytosolic or mitochondrial membranes. The pyridine nucleotide transhydrogenase of Escherichia coli is composed of two subunits (alpha and beta). Three domains are recognized. The extrinsic cytosolic domain 1 of the amino-terminal region of the alpha subunit bears the NAD(H)-binding site. The NADP(H)-binding site is present in domain 3, the extrinsic cytosolic carboxyl-terminal region of the beta subunit. Domain 2 is composed of the membrane-intrinsic carboxyl-terminal region of the alpha subunit and the membrane-intrinsic amino-terminal region of the beta subunit. Treatment of the transhydrogenase of E. coli with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD chloride) inhibited enzyme activity. Analysis of inhibition revealed that several sites on the enzyme were involved. NBD chloride modified two (betaCys-147 and betaCys-260) of the seven cysteine residues present in the transhydrogenase. Modification of betaCys-260 in domain 2 resulted in inhibition of enzyme activity. Modification of residues other than cysteine residues also resulted in inhibition of transhydrogenation as shown by use of a cysteine-free mutant enzyme. The beta subunit was modified by NBD chloride to a greater extent than the alpha subunit. Reaction of domain 2 and domain 3 was prevented by NADPH. Modification of domain 3 is probably not associated with inhibition of enzyme activity. Modification of domain 2 of the beta subunit resulted in a decreased binding affinity for NADPH at its binding site in domain 3. The product resulting from the reaction of NBD chloride with NADPH was a very effective inhibitor of transhydrogenation. In experiments with NBD chloride in the presence of NADPH it is likely that all of the sites of reaction described above will contribute to the inhibition observed. The NBD-NADPH adduct will likely be more useful than NBD chloride in investigations of the pyridine nucleotide transhydrogenase.

4-Chloro-7-nitrobenzofurazan↗
PubMed1997

Quenching of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole-modified Na+/K+-ATPase reveals a higher accessibility of the low-affinity ATP-binding site.

7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) labeled Na+/K+-ATPase covalently with two different inactivation constants (Ki = 2.5 microM; Ki' = 10 microM). It apparently modified the two different ATP-binding sites of the enzyme since it decreased the activity of the E2ATP site, i.e. the K+-activated para-nitrophenylphosphatase activity, in an enzyme whose high-affinity E1ATP site had been blocked by fluorescein 5'isothiocyanate (FITC). It also reduced the activity of the E1ATP site, i.e. the Na+-activated protein phosphorylation, in an enzyme whose low-affinity E2ATP site had been blocked by Co(NH3)4PO4. Fluorescence quenching experiments with KI, CsCl and MnCl2 of the NBD-Cl-labeled Na+/K+-ATPase revealed two differently accessible types of fluorophores depending on the ATP site: The E2ATP site apparently differs from the E1ATP site in that it is more open because the fluorophore labeling in the E2ATP site was sterically better accessible for quenchers.

4-Chloro-7-nitrobenzofurazan↗
PubMed2001

Use of 7-fluoro-4-nitrobenzo-2-oxo-1,3-diazole (NBD-F) for the determination of ramipril in tablets and spiked human plasma.

Ramipril, as a secondary amine compound, reacts with 7-fluoro-4-nitrobenzo-2-oxo-1,3-diazole (NBD-F) producing the corresponding fluorescent NBD-ramipril. According to this fact, spectrophotometric and fluorimetric methods for the determination of ramipril were developed. The effect of these parameters on the reaction product were carefully studied to optimize reaction conditions. The relationship between the absorbance at 465 nm and the concentration was found to be linear over the range 1-10 microg/ml. Moreover, the fluorescence intensity was also found to be directly proportional at the concentration over the range of 20-100 ng/ml at 530 nm after excitation at 465 nm. The proposed procedure was successfully applied to the determination of ramipril in both tablet dosage form and in plasma. Spectrophotometric determination of ramipril tablets yielded a percentage recovery of 98.66+/-0.38, while the percentage recovery of spectrofluorimetric determination of ramipril in spiked human plasma was 99.08+/-1.11%. The results obtained are in good agreement with those obtained by the reference method. No interference could be observed from the co-administered drug (hydrochlorothiazines).

4-Chloro-7-nitrobenzofurazan↗