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PubMed1979

Formaldehyde-fluorescamine-induced fluorescence of mammary carcinoma cells. Lack of concordance with occurrence of oestrogen-binding receptor proteins.

The combined formaldehyde-fluorescamine technique demonstrates fluorescence by mammary carcinoma cells whereas cells of the normal gland or of benign tumours do not show fluorescence. We have studied the correlation between histological staging of the disease, concentrations of oestrogen-binding proteins and the occurrence of formaldehyde-fluorescamine (FF)-inducible fluorescence. Our results demonstrate that the FF-technique detects all types of mammary carcinoma cells irrespective of their concentration of oestrogen receptors. Hence, the FF-technique represents a valuable tool for detecting both hormone-responsive and hormone-unresponsive malignant cells of the mammary gland.

Breast Neoplasms↗
PubMed2005

Fluorimetric determination of histamine in fish using micellar media and fluorescamine as labelling reagent.

An analytical method based on the use of fluorescamine to produce a fluorescent derivative with histamine and combined with micellar-enhanced fluorescence detection of the formed complex is developed for the sensitive and rapid determination of histamine in fishes. The fluorescence properties of the obtained complex in water and micellar solutions of sodium dodecyl sulfate (SDS), cetyltrimethylammonium chloride (CTAC) and brij-700 are reported. Physicochemical variables influencing the sensitivity of the method (pH, micellar, fluorescamine and NaCl relative concentrations) have been optimized. The stability of the formed complex, as shown by kinetic study, depends on the pH of the solution. Linear calibration curves allowing an effective histamine determination were established with large linear dynamic range (LDR), and low limits of detection (LOD) between 0.5 and 33 ng mL-1, according to the solvent. Application to the analysis of fish samples (sardines) yielded satisfactory results. The method seems to be suitable for environmental fish quality control.

Animals↗
PubMed1991

Pre-column derivatization of sulfa drugs with fluorescamine and high-performance liquid chromatographic determination at their residual levels in meat and meat products.

A rapid, sensitive and selective high-performance liquid chromatographic method is described for simultaneous determination of eight sulfa drugs in meat and meat products using pre-column derivatization with fluorescamine. The drugs are sulfisomidine, sulfadiazine, sulfamerazine, sulfadimidine, sulfamonomethoxine, sulfamethoxazole, sulfadimethoxine and sulfaquinoxaline. The method includes blender extraction of 3-g samples with chloroform, partition with 3 M hydrochloric acid, derivatization with fluorescamine at pH 3.0 and subsequent high-performance liquid chromatographic analysis on a C18 column with fluorescence detection at an excitation wavelength of 405 nm and an emission wavelength of 495 nm. The drugs were separated with a mobile phase of acetonitrile-2% acetic acid (3:5) at 55 degrees C. The average recovery from samples fortified at 0.1 ng/g was 92.6% with a coefficient of variation of 6.2%. The detection limit was 0.01 ng/g for sulfaquinoxaline and 0.005 ng/g for the other seven drugs. The method was field-tested in a survey of 37 samples including beef (five), pork (seven), chicken (seven), ham (five), sausage (eight), bacon (two) and roast beef (three). Sulfadimidine was detected in one pork sample at the level of 0.295 ng/g and in ham at 0.178 ng/g.

Animals↗
PubMed1992

Assay of protein drug substances present in solution mixtures by fluorescamine derivatization and capillary electrophoresis.

A method is described to enhance the resolution and detection sensitivity of proteins, peptides, and amino acids in capillary electrophoretic analysis of solution mixtures. The method consists of derivatizing the analytes with fluorescamine, which is normally used as a fluorogenic reagent for compounds containing a reactive primary amine functional group, and then using the derivative as an ultraviolet chromophore to enhance detection sensitivity (measured at 280 nm) in capillary electrophoresis. The results demonstrated a significant improvement in the separation and detection sensitivity of the derivatized analytes as compared to their underivatized counterparts. The use of chromophores, such as fluorescamine, in capillary electrophoresis facilitates the analysis of components of solution mixtures, such as pharmaceutical formulations, that could not be resolved and/or detected by conventional capillary electrophoresis procedures.

Antibodies, Monoclonal↗
PubMed1991

Fluorimetric determination of mexiletine in serum by high-performance liquid chromatography using pre-column derivatization with fluorescamine.

A simple, specific and sensitive micro-scale method for the assay of the antiarrhythmic agent mexiletine in human serum is described. The method uses high-performance liquid chromatography, with pre-column fluorimetric derivatization by fluorescamine. Following extraction with diethyl ether, mexiletine and 4-methylmexiletine (an internal standard) were derivatized with fluorescamine under weakly alkaline condition (pH 9.0) and chromatographed on a reversed-phase column with aqueous methanol-2-propanol as the mobile phase. The two fluorescent derivatives of mexiletine and the internal standard were separated as clear single peaks, and no interfering peaks were observed on the chromatograms. The detection limit for mexiletine was 0.005 micrograms/ml from only 100 microliters of serum, and the calibration curves in the range 0.01-5 micrograms/ml were linear, with an overall coefficient of variation of less than 5%. The analytical recovery of a known amount of mexiletine added to serum was almost 100%. This method proved to be effective in the rapid monitoring of the serum concentrations in patients who received this potent antiarrhythmic drug.

Anti-Arrhythmia Agents↗
PubMed1994

Quantification of 3'-amino-3'-deoxythymidine, a toxic catabolite of 3'-azido-3'-deoxythymidine (zidovudine) in human plasma by high-performance liquid chromatography using precolumn derivatization with fluorescamine and fluorescence detection.

A specific and sensitive high-performance liquid chromatographic (HPLC) assay with precolumn derivatization and fluorescence detection was developed for the determination of 3'-amino-3'-deoxythymidine (AMT), a recently characterized toxic catabolite of 3'-azido-3'-deoxythymidine (zidovudine, AZT). After extraction with tert.-butyl methyl ether-1-butanol (6:4, v/v) and back-extraction into basified aqueous phase, the solution was neutralized with phosphate buffer and the compound derivatized with fluorescamine. Fluorescamine-labeled AMT was chromatographed on a reversed-phase C18 column using a mixture of phosphate buffer-methanol-acetonitrile (47:48:5, v/v/v) as mobile phase and fluorescence detection (excitation wavelength 265 nm, emission wavelength 475 nm). The limit of quantification was 3 ng/ml using a 200-microliters plasma sample. The recovery of the extraction procedure averaged at 94.4% in the range of 3-200 ng/ml. The HPLC analysis time required per sample was 16 min. Replicate analyses of quality control samples (5-80 ng/ml) gave satisfactory intra- and inter-assay precision (coefficient of variation varied from 1.9 to 6.7%) and accuracy.

Chromatography, High Pressure Liquid↗
PubMed1999

Characterisation of the binding interaction between poly(L-lysine) and DNA using the fluorescamine assay in the preparation of non-viral gene delivery vectors.

A major factor limiting the development of non-viral gene delivery systems is the poor characterisation of polyelectrolyte complexes formed between cationic polymers and DNA. The present study uses the fluorescamine reagent to improve characterisation of poly(L-lysine) (pLL)/DNA complexes post-modified with a multivalent hydrophilic polymer by determining the availability of free amino groups. The results show that the fluorescamine reagent can be used to monitor the self-assembly reaction between pLL and DNA and the degree of surface modification of the resultant complexes with a hydrophilic polymer. This experimental approach should enable the preparation of fully defined complexes whose properties can be better related to their biological activity.

Animals↗
PubMed1979

High-performance liquid chromatographic determination of free and total polyamines in human serum as fluorescamine derivatives.

A highly sensitive and simple fluorimetric method for the determination of free and total polyamines, spermidine, spermine, putrescine and cadaverine, in human serum by high-performance liquid chromatography is described. The polyamines, obtained after clean-up of deproteinized serum by Cellex P column chromatography, are converted to their fluorescamine derivatives in the presence of nickel ion which inhibits the reaction of interfering amines with fluorescamine, and the derivatives are separated simultaneously by reversed-phase chromatography (LiChrosorb RP-18) with a linear gradient elution. The lower limits of detection are 10 and 15 pmole for spermine and the others in 0.5 ml of serum, respectively.

Adult↗
PubMed2002

LC determination of aminoglutethimide enantiomers as dansyl and fluorescamine derivatives in tablet formulations.

Determination of dansyl (AG-DNS) and fluorescamine (AG-F) derivatives of rac-aminoglutethimide in tablet formulation by HPLC has been achieved on a cellulose tris-(3,5-dimethylphenyl carbamate), known as Chiralcel OD and OD-R under normal and reversed phase columns, respectively, using a fluorescence detector (lambda(ex), 360 nm; lambda(em), 530 nm for AG-DNS derivatives; lambda(ex), 395 nm, lambda(em), 495 nm for fluorescamine derivatives (AG-F)). The best results were obtained with mobile phase ethanol:cyclohexane:methanol (95:5:2 v/v/v) for AG-DNS derivatives and acetonitrile:0.5% ortho-phosphoric acid (85:15 v/v) containing 0.26 mM 1-hexanesulfonic acid sodium salt (HSA) for AG-F, respectively. The lower limit of detection (signal to noise ratio of 3:1) were found to be 20 ng ml(-1) for each enantiomer for AG-DNS and 20.5 ng ml(-1) for each diastreoisomer for AG-F.

Aminoglutethimide↗
PubMed1975

Application of fluorescamine to the study of protein-DNA interactions.

The reactivity of alpha-amino groups of basic proteins towards fluorescamine is essentially abolished if salt linkages with DNA phosphate groups are formed. This observation prompted the elaboration of a very general assay which allows the determination of binding parameters for the interaction of proteins with DNA and chromatin. Protamines, labeled with fluorescamine prior to their binding by DNA appear to be useful probes to monitor the formation and nature of DNA-protein complexes.

Animals↗
PubMed1985

Study of human chromosomes. IV. Labeling of chromosomal proteins with the amino group specific fluorescent reagent fluorescamine.

We describe a method for labeling chromosomal proteins with an amino-group-specific fluorescent reagent, fluorescamine. Chromosomes thus labeled appear either as uniformly fluorescent or as haloes in structure depending on the proteins remaining after treatment with acid-alcohol fixation. Using fluorescamine as a probe, we demonstrate that there is a substantial loss of labeled proteins during the chromosomal preparation and also during the trypsin treatment used in the banding of chromosomes.

Amnion↗
PubMedDate not supplied

The inhibition of the calcium transport ATPase of the sarcomplasmic reticulum by fluorescamine: evidence for an oligomeric functional unit of the calcium transport system.

The labeling of the protein moiety of the sarcoplasmic calcium transport ATPase by fluorescamine suppresses calcium transport, calcium dependent ATPase activity, protein phosphorylation by [gamma-32P]ATP and [32P]phosphate at different extent of amino group substitution. For the hydrolysis of para nitrophenylphosphate by the calcium transport ATPase, it is shown that the relationship between the extent of amino group labelling can considerably be altered by the temperature and the presence of ethyleneglycol. It is shown that the amino residues of the phosphatidylethanolamine moiety do not contribute to the inhibiting effect of fluorescamine labelling. The observations suggest that the different functions of the calcium transport system are based on the cooperation of a varying number of calcium transport ATPase molecules.

4-Nitrophenylphosphatase↗
PubMed2000

Detection, quantitation, and identification of residual aminopenicillins by high-performance liquid chromatography after fluorescamine derivation.

A high-performance liquid chromatographic (HPLC) method with fluorescence detection after precolumn fluorescamine derivation was developed to detect residues of two aminopenicillins, amoxicillin (AMPC) and ampicillin (ABPC), in bovine serum. Proteins in serum samples spiked with each of these penicillins were precipitated with sodium tungstate and sulfuric acid, centrifuged, and removed by passage through a C18 solid-phase extraction cartridge. After precolumn treatment of the extraction products of AMPC and ABPC with fluorescamine solution, HPLC analysis with fluorescence spectrophotometric detection at an excitation wavelength of 390 nm and an emission wavelength of 485 nm was performed to identify these products. Two mobile phases were used for residual analysis by the isocratic HPLC system. An ODP column (polyvinyl alcohol bonded with an octadecyl functional group) that can be used with strongly alkaline mobile phases (pH 2.0 to 13) was selected, and the column temperature was set at 40 degrees C. A mobile phase comprising 100-mM K2HPO4 solution and acetonitrile (72:28, vol/vol), which yielded AMPC and ABPC retention times of 4.1 and 7.9 min, respectively, was suitable for detection of residual ABPC but not for residual AMPC because interference was caused by peaks of other extracted substances. When a mobile phase comprising a different ratio of 100-mM K2HPO4 solution and acetonitrile (78:22, vol/vol) was used, the retention times of AMPC and ABPC were 7.3 and 26.3 min, respectively, and both penicillins could be analyzed using this system. The calculated standard curves of the reaction products with both mobile phases were linear, and the correlation coefficients were greater than 0.999. The lower limit of detection was 10 ng/ml for both penicillins. Analysis of extracts from bovine serum spiked with AMPC and ABPC at a concentration of 1 microg/ml yielded recovery rates of 102.2 +/- 5.5% and 79.0 +/- 5.2%, respectively. This detection method may be useful for routine laboratory testing of AMPC and ABPC.

Amoxicillin↗
PubMedDate not supplied

[Structural changes in erythrocyte ghosts after irradiation and the initiation of lipid oxidation detectable using 2,6-TNS and fluorescamine].

It was shown on erythrocyte ghosts that the parameters of fluorescence of 2,6-toluidine-naphthalene-sulfonate (2,6-TNS) and fluorescamine undergo similar changes after irradiation. After a dose of 100 Gy the equally effective concentrations of Fe2+ were 1-5 microM and 50-100 microM with regard to changes in the rate of fluorescence of fluorescamine and 2,6-TNS, respectively, and greater than 100 microM with regard to fluorescence anisotropy.

Animals↗
PubMed1990

Differential Inhibition of Tonoplast H-ATPase Activities by Fluorescamine and Its Derivatives.

Corn (Zea mays L.) root tonoplast vesicles were treated with the primary-amine specific reagent, fluorescamine (FL). Modification by FL caused a differential inhibition to the coupled activities of tonoplast H(+)-ATPase. Within the range of 0 to 5 micromoles of FL per milligram of protein, the proton pumping rate was significantly reduced but ATP hydrolysis was only slightly affected. Yet, the membrane H(+) leakage during the pumping stage increased only slightly. FL treatment resulted in (a) a decrease in amine containing phospholipids and (b) an insertion of multiple H-bonding moieties into the membrane. To test which of these two possible effects were responsible for inhibition, FL derivatives of benzylamine, butylamine, and phenylalanine were synthesized. It was found that the acyclic derivatives with high H-bonding potential at concentrations of 10 micromolar inhibited proton pumping by 50% without a significant effect on ATP hydrolysis. Cyclic derivatives were largely ineffectual. Proton leakage during pumping was not affected by these acyclic modifiers. Membrane fluidity, as measured by the polarization of diphenyl hexatriene, decreased upon treatment with either FL or its derivatives. The results suggest that the proton pumping is indirectly linked to ATP hydrolysis in the tonoplast vesicles, and the link between these processes is apparently weakened by the presence of acyclic fluorescamine derivatives in the membrane.

Journal Article↗
PubMed1997

[Medium effects on derivative reaction between fluorescamine and primary arlyamine].

The effects of pH, various organic solvents, surfactants, beta-CD and PVA on derivative reaction and fluorescence properties of its product between fluorescamine and p-amino phenyl sulfonic acid have been studied. The results show that the derivative reaction needs the organic solvent, many kind of organic solvents are available, but its amount should not be too large. The rate of derivative reaction was slowed and fluorescence intensity of product was decresed with increase in amount of organic solvent and precision is not good because of the fluorescence intensity is changing with delay time. Only tritonX-100 enhanced the fluorescence of product in all of the surfactants tested. Span20, Tween80 and Brij35 which same as tritonX-100 are non-ionic surfactant, and PVA, beta-CD do not exhibit sensitization. Ionic Surfactant CTAB, SDS quench the fluorescence. The sensitization of tritonX-100 is dependent on the amount of organic solvent. A competitive effect between tritonX-100 micellae and organic solvent is exhibition. In this paper, the derivative reaction between fluorescamine and p-chloroaniline is compared and studied too.

English Abstract↗
PubMed2000

Determination of taurine in plasma by capillary zone electrophoresis following derivatisation with fluorescamine.

A novel capillary zone electrophoresis method is described for the determination of taurine in plasma. The method is rapidly executed and is highly selective for taurine as separation is based on the difference in ionisation of this amino acid from that of other amino acids. Following addition of homotaurine as internal standard, plasma proteins were precipitated with acetonitrile and the supernatant was derivatised with fluorescamine in the presence of a borate buffer. Capillary electrophoresis (CE) separations were carried out in reverse polarity mode at 27.5 kV on a Beckman P/ACE MDQ CE instrument, equipped with a diode array detector (DAD) set at 266 nm. The sample tray was cooled to 5 degrees C and separations were carried out at 20 degrees C. The fused-silica capillary was 50.2 cm in length (40.2 cm to detector) with an internal diameter of 75 microm. A capillary conditioning solution was applied daily in order to suppress the residual electroosmotic flow (EOF). The method, which was validated using feline plasma as the blank matrix, was shown to be linear and reproducible over the concentration range 2.5-100 microg/mL. The coefficients of variation (CVs) of replicate analyses were less than 4.5% at 1 microg/mL taurine in feline plasma and less than 3% for 2.5 microg/mL in human plasma. Recovery was estimated at 99.2% with a CV of 4.85%. It has been demonstrated that quantitation in aqueous solution yields similar results to those obtained by interpolation on a plasma calibration curve provided that subtraction for the taurine peak in unspiked plasma is carried out and that a suitable internal standard is employed.

Animals↗
PubMed1981

Detection of urinary primary amines through negative chemical ionization mass spectrometry of fluorescamine derivatives.

We report the successful detection of urinary biogenic amines by a procedure which involves isolation of the amines on CG-50 cation exchange resin, derivatization of the amines with fluorescamine while on the resin, followed by direct probe negative chemical ionization mass spectrometry screening of the resin extract. This isolation procedure allowed the detection of tyramine, octopamine, normetanephrine, 3-methoxytyramine and a variety of aliphatic amines. Norepinephrine, dopamine and serotonin were chemically reactive under these conditions and were not detectable at physiological concentrations.

Biogenic Amines↗