Selective spectrofluorimetric method for determination of Lisinopril in pharmaceutical preparations and in presence of hydrochlorothiazide: Application to content uniformity testing

A novel sensitive and cost‐effective spectrofluorimetric method has been developed and validated for determination of lisinopril (an angiotensin converting enzyme inhibitor) in its pure form and pharmaceutical preparations. The method is based on the reaction of the drug with ninhydrin and phenylacetaldehyde in buffered medium (pH 7.0) to form a highly fluorescent product measured at 460 nm after excitation at 390 nm. Different experimental parameters were optimized and calibration curve was constructed. The fluorescence‐concentration relationship was linear in the range of 0.15–4.0 μg mL^?1. The calculated Limit of detection (LOD) and Limit of quantitation (LOQ) were 0.04 and 0.12 μg mL^?1, respectively. The method was successfully applied for the analysis of pharmaceutical preparations containing the studied drug either alone or co‐formulated with hydrochlorothiazide. The obtained results were in agreement with those of the reported method in respect to accuracy and precession. Moreover, the method was applied content uniformity testing according to United States Pharmacopeia (USP) guidelines.     

Utility of ninhydrin reagent for spectrofluorimetric determination of heptaminol in human plasma

For the determination of heptaminol (HEP) in its authentic and dosage form as well as in human plasma, a new simple, sensitive and cheap fluorimetric method of analysis was developed and validated. The presented method is based on the reaction between aliphatic primary amino moiety present in HEP with ninhydrin and phenylacetaldehyde using Torell and Stenhagen buffer at pH 8.2 that yields a highly fluorescent derivative which after excitation at 390 nm showed a fluorescence emission at 464 nm. The effects of various experimental factors on both the development and stability of the fluorescent product was evaluated and optimized. In the concentration range (0.5–6.0 μg/ml), the constructed calibration curve was linear with a good correlation coefficient (0.9997) and the calculated limit of detection (LOD) and limit of quantitation (LOQ) were 0.14 and 0.43 respectively. The presented method was successfully applied for determination of Corasore® tablets and validated according to ICH guidelines.     

A fluorimetric method for the determination of tryptophan in animal tissues

Tryptophan, tryptamine and peptides containing N-terminal tryptophan give two highly fluorescent products on treatment with dithiothreitol and acid ninhydrin reagent 1 or 2. The first fluorescent product (product A) gives an emission at 500nm on activation at 390–400nm and is stable for 20min. The second product (product B), which gives an emission at 530nm on activation at 470nm, is detectable within 1h after the reaction. It gives almost maximum intensity in 4h and is stable for at least 48h. Except lysine, which in equimolar amounts gives less than 1% of a product similar to product B, no other naturally occurring amino compounds give fluorescent products. A procedure is given for the determination of 0.05–34nmol of tryptophan in tissue extracts. By using this procedure rat brain was found to contain 17.56±0.76 (s.e.m.) nmol/g wet wt.     

Periodate oxidation products of hydroxylysine in the synthesis of 5-substituted prolines

The amino acid hydroxylysine was subjected to oxidation by sodium metaperiodate under various conditions. It was found that in acid and high temperature, the initial oxidation product alpha-aminoglutaric gamma-semialdehyde was converted to glutamic acid with a yield of 60%. The use of alkaline conditions of oxidation favored the cyclization of alpha-aminoglutaric gamma-semialdehyde to form delta 1-pyrroline 5-carboxylic acid. Addition of NaCN to this intermediate generated new proline analogs, likely a mixture of cis- and trans-5-cyanoprolines, with a yield of 30%. Upon hydrolysis, the 5-cyanoprolines were converted to a probable mixture of cis- and trans-5-carboxyprolines. Infrared and high-resolution mass spectral data of the analogs and visual absorption spectra of the ninhydrin products were obtained to confirm the structures.     

Reaction of S-(2-amino-2-carboxyethylsulfonyl)-L-cysteine with thiosulfate: synthesis of L-alanine sulfodisulfane and application to the determination of thiosulfate

A new reaction of S-(2-amino-2-carboxyethylsulfonyl)-L-cysteine (ACESC) with thiosulfate is described. The reaction proceeded quantitatively in formic or acetic acid solutions, yielding equimolar amounts of L-alanine sulfodisulfane (2-amino-2-carboxyethyl sulfodisulfane) and L-alanine 3-sulfinic acid. L-Alanine sulfodisulfane was obtained as pure monosodium salt; the yield was 92% of the theoretical. A new method is described for the determination of thiosulfate. The method is based on the quantitative reaction between ACESC and thiosulfate, and L-alanine sulfodisulfane, one of the reaction products, was determined using acid ninhydrin reagent 2 of M. K. Gaitonde (1967, Biochem. J. 104, 627-633). The recovery was over 95%. When samples contained sulfite in addition to thiosulfate, S-sulfo-L-cysteine (T. Ubuka et al., 1982, Anal. Biochem. 126, 273-277) was produced in addition to L-alanine sulfodisulfane by the treatment with ACESC. Both products were separated by a small Dowex 1 column and determined with the acid ninhydrin reagent 2. The recoveries were over 95%. The new method was applied to the thiosulfate sulfurtransferase reaction, in which thiosulfate, a substrate, and sulfite, a product, were determined separately.     

Reaction of S-(2-amino-2-carboxyethylsulfonyl)- -cysteine with thiosulfate: Synthesis of -alanine sulfodisulfane and application to the determination of thiosulfate

A new reaction of S-(2-amino-2-carboxyethylsulfonyl)- -cysteine (ACESC) with thiosulfate is described. The reaction proceeded quantitatively in formic or acetic acid solutions, yielding equimolar amounts of -alanine sulfodisulfane (2-amino-2-carboxyethyl sulfodisulfane) and -alanine 3-sulfinic acid. -Alanine sulfodisulfane was obtained as pure monosodium salt; the yield was 92% of the theoretical. A new method is described for the determination of thiosulfate. The method is based on the quantitative reaction between ACESC and thiosulfate, and -alanine sulfodisulfane, one of the reaction products, was determined using acid ninhydrin reagent 2 of [7.]. The recovery was over 95%. When samples contained sulfite in addition to thiosulfate, S-sulfo- -cysteine ([6.]) was produced in addition to -alanine sulfodisulfane by the treatment with ACESC. Both products were separated by a small Dowex 1 column and determined with the acid ninhydrin reagent 2. The recoveries were over 95%. The new method was applied to the thiosulfate sulfurtransferase reaction, in which thiosulfate, a substrate, and sulfite, a product, were determined separately.     

Peroxynitrite-mediated oxidation of dichlorodihydrofluorescein and dihydrorhodamine

The oxidations of dichlorodihydrofluorescein and dihydrorhodamine by peroxynitrite are zero-order in the indicator between pH 3 and 10. The yield of the oxidized products, dichlorofluorescein and rhodamine, significantly increased at pH values>7, and the maximal molar yields were 0.47 +/- 0.04 mol rhodamine and 0.54 +/- 0.06 mol, dichlorofluorescein per mol peroxynitrite at pH 8.5. The increase in yield of oxidized products as a function of pH indicates that the peroxynitrite anion may form an adduct with the indicator, followed by protonation and oxidation of the indicator. Carbon dioxide decreased the yield of fluorescent products to about 5%, relative to peroxynitrite, and the rate of product formation is again zero-order in the indicator. Given this yield, it is proposed that nitrogen dioxide and trioxocarbonate (*1-) are the reactive species that oxidize the indicators.     

Role of aromatic aldehyde synthase in wounding/herbivory response and flower scent production in different Arabidopsis ecotypes

Aromatic L-amino acid decarboxylases (AADCs) are key enzymes operating at the interface between primary and secondary metabolism. The Arabidopsis thaliana genome contains two genes, At2g20340 and At4g28680, encoding pyridoxal 5'-phosphate-dependent AADCs with high homology to the recently identified Petunia hybrida phenylacetaldehyde synthase involved in floral scent production. The At4g28680 gene product was recently biochemically characterized as an L-tyrosine decarboxylase (AtTYDC), whereas the function of the other gene product remains unknown. The biochemical and functional characterization of the At2g20340 gene product revealed that it is an aromatic aldehyde synthase (AtAAS), which catalyzes the conversion of phenylalanine and 3,4-dihydroxy-L-phenylalanine to phenylacetaldehyde and dopaldehyde, respectively. AtAAS knock-down and transgenic AtAAS RNA interference (RNAi) lines show significant reduction in phenylacetaldehyde levels and an increase in phenylalanine, indicating that AtAAS is responsible for phenylacetaldehyde formation in planta. In A. thaliana ecotype Columbia (Col-0), AtAAS expression was highest in leaves, and was induced by methyl jasmonate treatment and wounding. Pieris rapae larvae feeding on Col-0 leaves resulted in increased phenylacetaldehyde emission, suggesting that the emitted aldehyde has a defensive activity against attacking herbivores. In the ecotypes Sei-0 and Di-G, which emit phenylacetaldehyde as a predominant flower volatile, the highest expression of AtAAS was found in flowers and RNAi AtAAS silencing led to a reduction of phenylacetaldehyde formation in this organ. In contrast to ecotype Col-0, no phenylacetaldehyde accumulation was observed in Sei-0 upon wounding, suggesting that AtAAS and subsequently phenylacetaldehyde contribute to pollinator attraction in this ecotype.     

Stoichiometry of formation of Ruhemann's purple in the ninhydrin reaction

The course and mechanism of reaction of ninhydrin with amines has both bioanalytical and bioorganic significance since the reaction is widely used for analysis of amino groups and serves as a model for several biochemical reactions that occur in metabolism of phosphonic acid derivatives, deamination, transamination, and transpeptidation. In many cases, e.g., with lysine, cysteine, proteins, the yield of the ninhydrin product, Ruhemann's purple, does not correspond exactly to the expected 1 equiv. per amino group. Possible reasons for this apparent nonideal stoichiometry include slow formation, side reactions, hydrolytic, oxidative, and photolytic instability, and interfering color. The origin and contributions of each of these factors are examined.     

A new type of carbohydrate-protein linkage in a glycopeptide from normal human urine.

A glycopeptide, 3-O-beta-D-glucopyranosyl-alpha-L-fucopyranosyl-L-threonine, has been isolated from normal human urine. The glycopeptide was isolated by gel chromatography, preparative zone electrophoresis, paper chromatography, and high voltage electrophoresis. The average yield of the glycopeptide was in the range of 0.2 to 0.3 mg/liter of urine. Sugar analysis and amino acid analysis gave equimolar amounts of glucose, fucose, and threonine. Linkages and sequential order were established by methylation analysis of the glycopeptide after degradation of the amino acid residue with ninhydrin. The permethylated product was analyzed on gas liquid chromatography and mass spectrometry. Anomeric configuration was deduced from optical rotation.     

Interfering with nitric oxide measurements. 4,5-diaminofluorescein reacts with dehydroascorbic acid and ascorbic acid

4,5-Diaminofluorescein (DAF-2) is widely used for detection and imaging of NO based on its sensitivity, noncytotoxicity, and specificity. In the presence of oxygen, NO and NO-related reactive nitrogen species nitrosate 4,5-diaminofluorescein to yield the highly fluorescent DAF-2 triazole (DAF-2T). However, as reported here, the DAF-2 reaction to form a fluorescent product is not specific to NO because it reacts with dehydroascorbic acid (DHA) and ascorbic acid (AA) to generate new compounds that have fluorescence emission profiles similar to that of DAF-2T. When DHA is present, the formation of DAF-2T is attenuated because the DHA competes for DAF-2, whereas AA decreases the nitrosation of DAF-2 to a larger extent, possibly because of additional reducing activity that affects the amount of available N(2)O(3) from the NO. The reaction products of DAF-2 with DHA and AA have been characterized using capillary electrophoresis with laser-induced fluorescence detection and electrospray mass spectrometry. The reactions of DAF-2 with DHA and AA are particularly significant because DHA and AA often colocalize with nitric-oxide synthase in the central nervous, cardiovascular, and immune systems, indicating the importance of understanding this chemistry.     

New approach for stability study and determination of fluvoxamine in raw materials and pharmaceuticals through condensation with 2,2‐dihydroxyindane‐1,3‐dione

The present study describes the validation of a selective spectroscopic method for assay of fluvoxamine maleate (FXM). The validated method relies on condensation of FXM with 2,2‐dihydroxyindane‐1,3‐dione and phenylacetaldehyde using Teorell–Stenhagen buffer (pH 6.6) to give coloured fluorescent product measured at 482 nm using 386 nm as the excitation wavelength. The parameters influencing the reaction were studied precisely and adjusted accurately. The constructed calibration graph appeared rectilinear over the following range (0.8–14 μg ml^?1) and the estimated limit of detection was 0.25 μg ml^?1. Two pharmaceutical products from the Egyptian market were assayed using the suggested method and the final results agreed with measurements from other reported methods. Moreover, the drug was subjected to diverse stress conditions including acidic, alkaline, thermal, and photolytic degradation to examine the FXM stability. Directives from the International Conference on Harmonisation guidelines were applied to establish the validity of the work.     

Uptake and metabolism of a fluorescent sulfatide analogue in cultured skin fibroblasts.

The sulfatide fluorescent analogue N-lissamine rhodaminyl-(12-aminododecanoyl) cerebroside 3-sulfate was administered in the form of albumin complex to normal human skin fibroblasts and its metabolic fate was investigated. Ceramide, galactosylceramide, glucosylceramide, sphingomyelin and free acid, all containing the fluorophore lissamine rhodamine, have been synthesized as reference standards for the identification of the metabolic products. Ceramide appeared to be the main metabolic product present both in cell extract and medium, followed by galactosylceramide and sphingomyelin. Fluorescence microscopy of cells showed a marked perinuclear fluorescence.     

Mechanism-based inhibition of dopamine beta-monooxygenase by aldehydes and amides

A mechanism for beta-chlorophenethylamine inhibition of dopamine beta-monooxygenase has been postulated in which enzyme-bound alpha-aminoacetophenone is generated, followed by an intramolecular redox reaction to yield a ketone-derived radical cation as the enzyme inhibitory species (Mangold, J. B., and Klinman, J. P. (1984) J. Biol. Chem. 259, 7772-7779). If correct, additional compounds capable of producing enzyme-bound (formula; see text) reductant should inhibit dopamine beta-monooxygenase. Phenylacetaldehyde was chosen to test this model, since beta-hydroxyphenylacetaldehyde is expected to function as a reductant in a manner analogous to alpha-aminoacetophenone. Phenylacetaldehyde exhibits the properties of a mechanism-based inhibitor. Kinetic parameters are comparable to beta-chlorophenethylamine under both initial velocity and inactivation conditions. Since phenylacetaldehyde bears little resemblance to beta-chlorophenethylamine, its analogous inhibitory action provides support for an intramolecular redox reaction (via beta-hydroxyphenylacetaldehyde oxidation to a radical cation) in dopamine beta-monooxygenase inactivation. beta-Hydroxyphenylacetaldehyde was identified as the enzymatic product of phenylacetaldehyde turnover. As predicted, this product behaves both as a time-dependent inhibitor of dopamine beta-monooxygenase and as an electron donor in enzyme-catalyzed hydroxylation of tyramine to octopamine. Phenylacetamide and p-hydroxyphenylacetamide are also found to be mechanism-based inhibitors of dopamine beta-monooxygenase. In this case the product of hydroxylation (beta-hydroxyphenylacetamide) is redox inactive and, therefore, is unable to function as either a reductant or an inhibitor. Thus, mechanism-based inhibitors are divided into two types: type I, which undergoes hydroxylation prior to inactivation, and type II, which only requires hydrogen atom abstraction. A general mechanism for dopamine beta-monooxygenase inactivation is described, in which a common mechanistic radical intermediate is formed from both pathways.     

Microsatellite analysis using a two-step procedure for fluorescence labeling of PCR products.

A method for fluorescent labeling of PCR products has been developed. This method consists in a two-step procedure in which a first exponential classical PCR is followed by a "linear amplification". This second step relies on incorporation of fluorescent dNTP (dUTP or dCTP) in order to label the product on only one strand. The products can be applied without prior purification directly to a gel on a fluorescence-based automated DNA sequencer, for length and allele determination. The reliability of the results equals those of the classical 32P or fluorescent primer labeling methods, and the method is definitely less costly. Since the interpretation of the results is easier than with the method consisting in a fluorescent dNTP uptake in both strands in a single PCR, the present strategy should prove useful in mapping projects requiring analysis of a large number of microsatellites.     

Estimating cell and product yields

The balance equations for carbon, reduction potential, and energy during cell growth and product formation are rederived in a general form. Cells are treated simply as a very complex product, and the Y(ATP) concept is extended to products. Limitations on the theoretical yield are discussed for different product types. Simple aerobic products cannot be energy limited unless the maintenance requirement is large, while complex products cannot be reduction limited. A maximum yield is defined for products much more oxidized than their substrate (carbon limited) because the theoretical yield conditions may violate the energy balance. For reduced complex products the yield on available electrons is related to Y(ATP), the P/O ratio, and the product composition. Narrow bounds are established on the actual yields in simple anaerobic fermentations, and the significance of the yields in the linear growth equation is discussed.     

A spectrophotometric method for the direct determination of cysteine in the presence of other naturally occurring amino acids

1. An acid ninhydrin reagent was found to react specifically in forming a pink product (E(max.) 560mmu) with cysteine. 2. The method was highly sensitive for the determination of cysteine (in 28.0x10(3)). Homocysteine, glutathione, proline, ornithine and other naturally occurring amino acids tested did not give a similar reaction. 3. The reaction product was stable for at least 3-4hr. at room temperature and the extinction was proportional to the concentration in the range 0.05-0.5mumole of cysteine. 4. The acid ninhydrin reagent also gave yellow products (E(max.) 370-404mmu) with tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine and indol-3-ylacetic acid. 5. The method was applied for the determination of cysteine in perchloric acid extracts of rat brain, liver and blood.     

A correlation between the secondary structure of DNA and the reactivity of adenine residues with chloroacetaldehyde.

The rate of reaction of chloroacetaldehyde (0.039 M) with the "free" adenine residue in deoxyadenosine-5'-phosphate (dAMP) at pH 6.5 has been found to be nearly equal to that at pH 4.5. Practically 100% of the adenine is converted to a fluorescent product (epsilon-adenine residue) on incubation for 60 h at 37 degrees C and pH 6.5. Of the adenine residues in "single-stranded" DNA, however, only 14% react with chloroacetaldehyde (0.039 M) under the same incubation conditions. The reaction rate of this 14% is nearly equal to that of dAMP, but the fluorescence of the product is appreciably quenched; the quantum yield is only 0.45 times that of the "free" adenine residue. In "double-helical" DNA, on the other hand, no adenine residue has been found to react with chloracetaldehyde. Possible application of these findings to structural studies of DNA is suggested.     

Specificity of a Vibrio vulnificus aminopeptidase toward kinins and other peptidyl substrates

Recently, phosphoglucose isomerase with a lysyl aminopeptidase (PGI-LysAP) activity was identified in Vibrio vulnificus. In this paper, we demonstrate the proteolytic cleavage of human-derived peptides by PGI-LysAP of V. vulnificus using three approaches: (i) a quantitative fluorescent ninhydrin assay for free lysine, (ii) matrix-assisted laser desorption ionization-two-stage time of flight mass spectrometry (MALDI-TOF-TOF), and (iii) Tricine gel electrophoresis. PGI-LysAP hydrolyzed bradykinin, Lys-bradykinin, Lys-(des-Arg9)-bradykinin, neurokinin A, Met-Lys-bradykinin, histatin 8, and a myosin light chain fragment. We detected the proteolytic release of free L-lysine from peptide digests using a rapid, simple, sensitive, and quantitative fluorescent ninhydrin assay, and results were confirmed by MALDI-TOF-TOF. The use of the fluorescent ninhydrin assay to quantitatively detect free lysine hydrolyzed from peptides is the first application of its kind and serves as a paradigm for future studies. The visualization of peptide hydrolysis was accomplished by Tricine gel electrophoresis. Proteolytic processing of kinins alters their affinities toward specific cellular receptors and initiates signal transduction mechanisms responsible for inflammation, vasodilation, and enhanced vascular permeability. By applying novel approaches to determine the proteolytic potential of bacterial enzymes, we demonstrate that PGI-LysAP has broad exopeptidase activity which may enhance V. vulnificus invasiveness by altering peptides involved in signal transduction pathways.     

4-alkyl-o-quinone/2-hydroxy-p-quinone methide isomerase from the larval hemolymph of Sarcophaga bullata. I. Purification and characterization of enzyme-catalyzed reaction

An enzyme which catalyzes the conversion of certain 4-alkyl-o-benzoquinones to 2-hydroxy-p-quinone methides has been purified to apparent homogeneity from the hemolymph of Sarcophaga bullata by employing conventional protein purification techniques. The purified enzyme migrated with an approximate molecular weight of 98,000 on gel filtration chromatography. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it migrated as a single band with a molecular weight of 46,000, indicating that it is made up of two identical subunits. It exhibited a pH optimum of 6.0 and readily converted chemically synthesized as well as enzymatically generated quinones derived from N-acetyldopamine, N-beta-alanyldopamine, and 3,4-dihydroxyphenethyl alcohol to highly unstable 2-hydroxy-p-quinone methides. The quinone methides thus formed were rapidly and nonenzymatically hydrated to form side chain hydroxylated o-diphenols as the stable product. In support of this proposition, when the enzyme reaction with N-acetyldopamine quinone was conducted in the presence of 10% methanol, racemic beta-methoxy-N-acetyldopamine was recovered as an additional product. The quinones of N-acetylnorepinephrine, N-beta-alanylnorepinephrine, and 3,4-dihydroxyphenylglycol were also attacked by the isomerase, resulting in the formation of N-acetylarterenone, N-beta-alanylarterenone and 2-hydroxy-3',4'-dihydroxyacetophenone, respectively as the stable products. The isomerase converted the dihydrocaffeiyl methyl amide quinone to its quinone methide analog which rapidly tautomerized to yield caffeiyl methyl amide. The importance of quinone isomerase in insect immunity and sclerotization of insect cuticle is discussed.