Fluorescamine Derivatives Research Articles

Correction

A copy-editing error occurred in our September issue in the article “Fluorescence of Aromatic Amines and Their Fluorescamine Derivatives for Detection of Explosive Vapors”, by DeLyle Eastwood, Carlos Fernandez, Byunghoon Yoon, Chrystal N. Sheaff, and Chien M. Wai [Appl. Spectrosc. 60, 958–963 (2006)]. The third author, Byunghoon Yoon, had his name misspelled as B. Yunghoon Yoon. This has been corrected in our end-of-year Cumulative Author Index, appearing in this issue. A number of author errors also occurred in our February issue in the article “Two-Dimensional Correlation Study of Uniaxially Drawn Poly(Ethylene Terephthalate) Films by Using Attenuated Total Reflection Based Dynamic Compression Modulation Step-Scan Fourier Transform Infrared in Combination with Spectral Simulation Analysis by Density Functional Theory”, by Yuji Nishikawa, Tatsuhiko Nakano, and Isao Noda [Appl. Spectrosc. 60, 145–154 (2006)]. The order of signals was incorrectly assigned in a number of places. Please note that the overall conclusions of the paper are not affected. The corrections are as follows. On page 147, right-hand column, the last sentence in the first paragraph reads: “The signs of the asynchronous 2D peaks also indicated that the spectral intensity change at 1234 cm−1 occurs before the changes at 1290, 1265, and 1248 cm−1.” That sentence should read: “The signs of the asynchronous 2D peaks also indicated that the spectral intensity change at 1234 cm−1 occurs after the changes at 1290, 1265, and 1248 cm−1.” The next line reads: “1234 cm−1 → 1290, 1265, and 1248 cm−1.” It should read: “1290, 1265, and 1248 cm−1 → 1234 cm−1.” On page 148, the last sentence on the page reads: “The sequence analysis carried out with the signs of the cross peaks indicates that the intensity change around 1234 cm−1 occurs before the signals around 1718, 1709, 1290, 1265, and 1248 cm−1.” That sentence should read: “The sequence analysis carried out with the signs of the cross peaks indicates that the intensity change around 1234 cm−1 occurs after the signals around 1718, 1709, 1290, 1265, and 1248 cm−1.” On page 149, the first line reads: “1234 cm−1 → 1718, 1709, 1290, 1265, and 1248 cm−1.” It should read: “1718, 1709, 1290, 1265, and 1248 cm−1 → 1234 cm−1.” Also on page 149, the fourth line reads: “1234 cm−1 → 1709 cm−1 → 1718, 1290, 1265, and 1248 cm−1.” It should read: “1709 cm−1 → 1718, 1290, 1265, and 1248 cm−1 → 1234 cm−1.” On page 149, right-hand column, the last sentence of the first full paragraph reads: “The signs of the asynchronous 2D peaks may indicate that the spectral intensity changes at 1220 and 1290 cm−1 occur before those at 1261 and 1244 cm−1.” It should read: “The signs of the asynchronous 2D peaks may indicate that the spectral intensity changes at 1220 and 1290 cm−1 occur after those at 1261 and 1244 cm−1.” The next line reads: “1220, 1290 cm−1 → 1261, and 1244 cm−1.” It should read: “1261 and 1244 cm−1 → 1220 and 1290 cm−1.” On page 150, the last full sentence on the page reads: “The signs of the asynchronous 2D peaks indicate that the C=O stretching bands consist of at least three components, and the spectral intensity change at 1717 cm−1 occurs before those at 1743 and 1709 cm−1.” It should read: “The signs of the asynchronous 2D peaks indicate that the C=O stretching bands consist of at least three components, and the spectral intensity change at 1717 cm−1 occurs after those at 1743 and 1709 cm−1.” The next line reads: “1717 cm−1 → 1743 and 1709 cm−1.” It should read: “1743 and 1709 cm−1 → 1717 cm−1.” On page 151, the second to the last sentence of the first paragraph (beginning at line 8) currently reads: “The sequence analysis carried out with the signs of the cross peaks indicates that the intensity changes around 1717, 1220, and 1743 cm−1 occur before the signals around 1709, 1290, 1261, and 1244 cm−1, and the intensity change around 1717 cm−1 occurs before the signals around 1220 and 1743 cm−1.” It should read: “The sequence analysis carried out with the signs of the cross peaks indicates that the intensity changes around 1743 and 1709 cm−1 occur before the signals around 1220, 1290, 1261, and 1244 cm−1, and the intensity change around 1717 cm−1 occurs after the signals around 1220 and 1743 cm−1.” The overall sequence that follows currently reads: “1717 cm−1 → 1743, 1220 cm−1 → 1709 cm−1 → 1290, 1261, and 1244 cm−1.” It should read: 1743, 1709 cm−1 → 1261, 1244 cm−1 → 1220, 1290 cm−1 → 1717 cm−1.” On page 153, line 9 should read “The order of crystalline and amorphous responses are reversed, such that amorphous chains orient first. However, the overall conclusions about the side-group and backbone responses are not changed.”

Fluorescence of Aromatic Amines and Their Fluorescamine Derivatives for Detection of Explosive Vapors

Nitroaromatics (such as dinitrotoluene, trinitrotoluene, and nitrobenzene) found in explosive vapors from buried landmines can be reduced to aminoaromatics by a novel process involving Pd metal nanocatalysts prepared in supercritical fluid carbon dioxide and supported on multi-walled carbon nanotubes. These aminoaromatics are fluorescent and, if desired, the fluorescence yield can be increased and the fluorescence maxima shifted further toward the red by reaction with appropriate derivatizing agents such as fluorescamine. Corrected spectra for these chemicals and their derivatives are included. Subpicomolar detection limits have already been achieved using a laboratory spectrofluorometer with a 150 W Xe arc lamp. Using lasers as excitation sources, this approach has the potential for developing a field sensor competitive with other methods currently used for detecting explosive vapors from land mines.

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 ( λ ex, 360 nm; λ em, 530 nm for AG-DNS derivatives; λ ex, 395 nm, λ 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.

Homogeneous liquid–liquid extraction method for the selective spectrofluorimetric determination of trace amounts of tryptophan

Twenty-one amino acids were derivatized with fluorescamine (FLA) under basic conditions (pH 9) and the extraction of the amino acid-FLA derivatives was investigated using a homogeneous liquid-liquid extraction with perfluorooctanoic acid (HPFOA) based on phase separation under strongly acidic conditions. Under the optimum concentration conditions for the reagents ([PFOA]T = 3 x 10(-3) mol dm-3, [acetone]T = 3 vol.%, [HCl]T = 1.8 mol dm-3), the concentration factor was approximately 1000-fold (i.e., 30 microliters of the sedimented liquid phase was produced from 33 ml of the homogeneous aqueous solution). The percentage extraction (E) was determined for the 21 amino acid-FLA derivatives; the value for the tryptophan (Trp)-FLA derivative was 80.9%, whereas the other derivatives were not almost extracted (E < 0.4%). The Trp-FLA derivative was selective for the extraction using the homogeneous liquid-liquid extraction method with HPFOA. After the sedimented liquid phase containing Trp-FLA has been placed on a polytetrafluoroethylene filter-paper, the fluorescence intensity was determined using a spectrofluorimeter with filter-paper as the solid-sample holder. The calibration graph of Trp was linear over the range 1.0 x 10(-8)-1.5 x 10(-6) mol dm-3. The relative standard deviation for the central value of the calibration graph was 4.5% (five determinations) and the detection limit (S/N = 3) was 8.9 x 10(-9) mol dm-3. When the proposed method was applied to the highly sensitive spectrofluorimetric determination of Trp in animalin-L syrup, the results were satisfactory.

Investigation of amine and polyol functionality in extracts of polyurethane wound management dressings using MALDI-MS.

Polyurethane (PU) foams used in wound management are produced by a reaction between aromatic diisocyanates and polyether polyols. There is concern that residues of these starting materials, which may contain aromatic amine functionality, may leach from the finished polymer during in vivo applications. Furthermore, oligomers and additives may be leached from the PU system after the polymerization process is complete. Finished polymers have, therefore, been extracted with a range of solvents, such as water, diethyl ether and dilute HCl. The extracts were subjected to MALDI-MS (matrix-assisted laser desorption ionization mass spectrometry) analysis in an attempt to determine the amine and polyol functionality. Direct MALDI-MS analysis of the wound dressing extracts indicated the presence of components based on the polyols [corrected] used in the formulation of the foam. The spacing between the peaks identified the base monomer used in the polyol. MALDI-MS analysis of the fluorescamine derivatives of model amine compounds has demonstrated the anticipated increase in mass (278 for monoamines and 278 and 556 for diamines). Similar results were obtained from the derivatization of model polyols with phenyl isocyanate, where the mass shift (n x 119) was a direct measure of the number of active hydroxyl groups. Fluorescamine labelling of PU foams shows the colour change which could be [corrected] indicative of the presence of an amine, but the subsequent MALDI-MS analysis was unable to demonstrate the anticipated increase in mass.

Determination of Fluorogen-Labeled Neurotransmitters at the Zeptomole Level Using Two-Photon Excited Fluorescence with Capillary Electrophoresis

Two-photon excited (2PE) fluorescence detection is demonstrated to be a highly sensitive means for analyzing fluorogen-labeled neurotransmitters fractionated in submicrometer capillary electrophoresis channels. In this approach, fluorescamine-labeled neurotransmitters that have been electrophoretically separated in 620-nm-i.d. capillaries intersect the focused output from a near-infrared mode-locked titanium−sapphire laser positioned at the capillary outlet. Extremely high peak laser intensities (∼1011−1012 W cm-2) facilitate the nearly simultaneous absorption of two near-IR photons (λex ≈ 780 nm) to excite fluorescamine derivatives ordinarily excited with a single, near-ultraviolet photon (λex ≈ 390 nm). Rapid cycling of analytes through the fluorescent excited state and low background from scatter and out-of-sample luminescence combine to make 2PE fluorescence a highly sensitive approach for detecting minute quantities of neurotransmitters. In these studies, mixtures of the fluorescamine derivatives of ...

A Fluorescamine Assay for Membrane Protein and Peptide Samples with Non-Amino-Containing Lipids

A method is described for determining the concentration of membrane proteins and peptides in the presence of non-amino-containing lipids. The assay is quantitative when used for purified proteins and peptides of known sequence and qualitative when sequences are unknown or samples contain contaminating proteins. In this method, proteins and peptides are hydrolyzed to amino acids followed by derivatization by fluorescamine and spectroscopic detection in a mixed solvent system. A liquid-phase acid hydrolysis separates lipid from the sample and increases the sensitivity and accuracy of the assay. The aqueous–organic solvent, composed of 40% dimethylformamide, has two advantages. First, it suppresses differences in fluorescence between samples with and without residual hydrolyzed lipid, allowing direct comparison of samples and standards regardless of lipid content. Second, the solvent enhances the fluorescence of amino acid derivatives. While the fluorescence intensities of fluorescamine derivatives reach a maximum at approximately 40% dimethylformamide, the emission maximum wavelengths continue to blue-shift at higher concentrations of organic solvent. The selection of an acid hydrolysis mixture based on the fluorescence quenching by different acid mixtures is also reported.

Supercritical Fluid Extraction of Fumonisin B1 from Grain Dust

Fumonisin B1 (FB1) was extracted from corn, corn dust, and Fusarium moniliforme culture samples using supercritical carbon dioxide (SC-CO 2 ). The supercritical fluid extraction (SFE) conditions were optimized regarding the use of different SC-CO 2 modifiers, modifier volume, pressure, temperature, and static extraction time. The addition of the modifier both in the extraction cell (prior to the static extraction step) and on-line with liquid carbon dioxide (during the dynamic SFE step) was found to significantly increase the recovery of FB1. Under the optimized SFE conditions (15 mL of liquid CO 2 , 750 μL of 5% acetic acid per gram of sample, 1200 psi, 20 min static extraction time), the recovery of FB1 was found to be approximately 40 times greater than the recovery using conventional solvent extraction. SFE was faster and more reproducible (RSD = 3-5%) compared to the solvent extraction (RSD = 6.5%). The calculated detection limit was approximately 150 ppb, on the basis of the HPLC analysis of FB 1 -fluorescamine derivatives with UV detection.

Determination of tetrahydro-β-carbolines in rat brain by gas chromatography-negative-ion chemical ionization mass spectrometry without interference from artifactual formation

This paper describes a quantitative method for neuroactive alkaloids, 1-methyl-1,2,3,4-tetrahydro-β-carboline (MTBC) and 1,2,3,4-tetrahydro-β-carboline (TBC), in rat brain by gas chromatography-negative-ion chemical ionization mass spectrometry (GC-NICIMS). After addition of tetradeuterated MTBC and TBC (internal standards), the samples were subjected to deproteinization, reaction with fluorescamine, solvent extractions, trifluoroacetylation and GC-NICIMS analysis. In contrast to the other previous methods, the artifactual formation during analysis did not interfere with the determination of MTBC and TBC because their precursor tryptamine was removed as a fluorescamine derivative from the analytical system at the first step of pretreatment. MTBC and TBC were specifically and reliably determined in the range of pg-ng/sample. Application of the proposed method has revealed that the MTBC and TBC contents in rat brain significantly increase after intraperitoneal administration of MTBC and TBC, indicating their ability to easily cross the blood-brain barrier.

Determination of tetrahydro-β-carbolines in urine by high-performance liquid chromatography with suppression of artefact formation

A high-performance liquid chromatographic method has been developed for the determination of urinary tetrahydro-beta-carbolines. When standing tryptamine with formaldehyde and acetaldehyde under extraction conditions, the significant amounts of artefact 1,2,3,4-tetrahydro-beta-carboline (TBC) and 1-methyl-1,2,3,4-tetrahydro-beta-carboline (MTBC) were formed in a short time. Urine samples added with 2-ethyl-1,2,3,4-tetrahydro-beta-carboline (an internal standard) were treated with fluorescamine, and then with glycine, followed by serial solvent extractions. Such a pretreatment using two-step reactions removed a precursor (trypamine) by extracting its fluorescamine derivative, and enhanced the detection response by consuming excess fluorescamine. It solved the analytical problem that artefact TBC and MTBC are formed during analysis. Reversed-phase ion-pair chromatography using a C8-column and trifluoroacetic acid as a counter ion completed a base-line separation of three analytes within 10 min. The calibration graphs showed a good linearity in the range 0.1-50.0 ng ml-1 of urine samples spiked with standard TBC and MTBC. In the spike experiment, the recovery and relative standard deviation were almost 100% and less than 3.0%, respectively, for both TBC and MTBC. The proposed method enables the determination of the genuine urinary concentrations of TBC and MTBC without involving their artefacts.

Analysis of Diethylenetriamine in Water and Soil at PPB Levels by High Performance Liquid Chromatography with Fluorescence Detection

Abstract A fast and sensitive method for the determination of diethylenetriamine in water and soil by High Performance Liquid Chromatography (HPLC) with Fluorescence Detection is described. Diethylenetriamine is converted to its fluorescamine derivatives through pre-column derivatization and is separated by an isocratic reverse phase chromatographic system (Nucleosil C18) with acetonitrile and borate buffer at pH 8 as the mobile phase. Water samples at concentrations of diethylenetriamine from 20–100 μg/L were derivatized with fluorescamine and chromatographed directly through direct aqueous injections. Recoveries averaged 94 ± 7% for a population of nine samples. Soil samples at concentrations of diethylenetriamine from 0.24–1.0 μg/g were extracted with CaCl2 solution (2 M) followed by derivatization with fluorescamine. Derivatized soil extracts were then analyzed by HPLC. Recoveries averaged 21 ± 2% for a population of nine samples.

Concentrations of fumonisin B1 in feeds associated with animal health problems

Ninety-eight samples of feeds associated with 44 cases of equine leukoencephalomalacia (ELEM) and 83 samples of feed associated with 42 cases of a porcine pulmonary edema syndrome (PPE) were analyzed for fumonisin B1 (FB1). For comparison purposes, 51 feed samples not associated with PPE or ELEM were also analyzed. Feed associated with ELEM contained FB1 ranging from less than 1 microgram/g to 126 micrograms/g with 75% of the cases having at least 1 sample above 10 micrograms/g. Feeds associated with PPE ranged from less than 1 microgram/g to 330 micrograms/g with 71% of the cases having at least 1 sample greater than 10 micrograms/g. Quantitation was by high performance liquid chromatography (HPLC)/fluorescence using the fluorescamine derivative with confirmation by thin layer chromatography (TLC) and/or gas chromatography/mass spectroscopy (GC/MS).

Tussock Moth Dermatitis: Evidence for Histamine Involvement

Moth hairs shed by the few tussock moths caught during the first documented outbreak of pruritic dermatitis in Singapore were investigated to provide evidence for the presence of histamine. Aqueous extract of the moth hairs was used for the test and analyses. In the isolated guinea pig ileum, the extract, like histamine, produced responses that were blocked by mepyramine. The high performance liquid chromatographic (HPLC) analysis of the fluorescamine derivative of the moth extract resulted in a fluorescent peak with exactly the same retention. time as the histamine fluorescamine derivative. The mass spectrum of the pentafluoropropionic acid (PFP) derivative of the extract was also identical to that obtained from the reference histamine-PFP derivative. We concluded that histamine was present in the tussock moth hairs and was involved as one of the inflammatory mediators responsible for the pruritic skin rashes.

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.

Analysis and Heat Stability of Taurine in Milk

A method based on formation of the fluorescamine derivative of taurine and HPLC was developed for analysis of taurine in milk. Taurine in milk ranged from 2.4 to 12.0 mg/L. The degradation of taurine in taurine-fortified milk and in a buffered taurine and lactose solution (pH 6.7) was determined by heating at 80, 100, and 120°C. First-order reaction kinetics were observed for taurine losses in milk and buffered solution. Activation energies were 20.5 and 21.0 kcal/mol for milk and buffered solution, respectively. The taurine loss in milk seems to proceed through browning with the same degradation rate as lysine.

Determination of triethylenetetramine in plasma of patients by high-performance liquid chromatography.

A sensitive and simple fluorometric method for the determination of N,N'-bis(2-aminoethyl)-1,2-ethanediamine dihydrochloride (triethylenetetramine) in human plasma by high-performance liquid chromatography is described. Free triethylenetetramine (TETA) obtained by passing the TETA-copper chelate compound through a solid-phase cation exchange resin was converted to its fluorescamine derivative in the presence of ethylenediaminetetraacetic acid to mask the interfering metal ions in the reaction solution, and the derivatives were separated on a nitrile high-performance liquid chromatograph column (Nucleosil 5-CN) using isocratic elution. The plasma levels of TETA were measured in eight patients receiving treatment for excess copper. Absorption rates of TETA were relatively slow and the peak levels were significantly different among patients. The bioavailability of TETA in the rat was also examined and the ratio of intestinal absorption was extremely low.

Enhanced fluorescence from o-phthalaldehyde and fluorescamine fluorophores using Triton and β-cyclodextrin

Mexiletine— and lysine hydrochloride— o-phthalaldehyde and mexiletine hydrochloride—, cysteine—, cysteamine—, homocysteine— and lysine hydrochloride—fluorescamine derivatives were subjected to Triton and β-cyclodextrin enhancement treatments. Of several classical fluorescence-enhancing reagents tested (Triton, β-cyclodextrin, sodium dodecyl sulphate, Brij), Triton provided the best results, followed by β-cyclodextrin. Increases in fluorescence emission by a factor of up to about 10 (mexiletine—fluorescamine—Triton X-100) were observed, with a generally negligible influence of the enhancing reagents on the excitation and emission maxima. Fluorescence enhancement by the addition of a suitable reagent solution to the final analyte solution may, in specific instances, enhance the detectability of native or chemically induced fluorophores.

The transbilayer distribution of phosphatidylethanolamine in erythroid plasma membranes during erythropoiesis

Fluorescamine was used to assess the transbilayer distribution of phosphatidylethanolamine in the plasma membrane of murine erythroid progenitor cells, CFU-E (colony-forming unit erythroid), at different stages of their differentiation pathway. Intact cells were exposed to increasing concentrations of fluorescamine and the amount of labeled phosphatidylethanolamine was determined by measuring the fluorescence intensity of its fluorescamine derivative. A semilogarithmic plot of the dose-response curve revealed three different pools of phosphatidylethanolamine, representing its fractions in, respectively, the inner- and outer monolayers of the plasma membrane and subcellular membrane systems. These results show that 9–11% of the total cellular phosphatidylethanolamine is present in the outer leaflet and 9–10% of it is located in the inner leaflet of the plasma membrane in early as well as late erythroblasts. This symmetric distribution of phosphatidylethanolamine over the two halves of the bilayer in the plasma membrane of CFU-E is very similar to that observed earlier in the plasma membrane of friend erythroleukaemic cells (Rawyler, Van der Schaft, Roelofsen and Op den Kamp (1985) Biochemistry 24, 1777–1783). These observations imply that the characteristic asymmetric distribution of phosphatidylethanolamine, as is found in mature erythrocytes, is accomplished at a very late stage of erythropoiesis and possibly during enucleation of the cells or shortly thereafter.

An improved HPTLC method for the rapid identification and quantification of sulfonamides

Rf values of twenty therapeutic sulfonamides have been estimated in seven solvent systems. Based on these data a simple identification scheme was developed demanding only three solvent systems for complete separation. Quantification of corresponding fluorescamine derivatives at nanogram level was performed by the use of an TLC-scanner configured online with a microcomputer. Mathematical treatment of standard curves using the computer program Curve Fitter are discussed. Validity and reproducibility of results are compared with data obtained by HPLC and show good correlation.

Estimation of biogenic amines in foods

AbstractBiogenic amines in foods have been proposed as initiators of dietary‐induced migraine. Studies of levels of 2‐phenylethylamine, p‐tyramine and the diamines histamine, putrescine and cadaverine in a number of Australian and other foods were made by ion exchange and high pressure liquid chromatographic methods. In the latter method the amines were chromatographed as their fluorescamine derivatives. The use of such derivatives also enabled confirmation of the identity of the amine by field desorption‐mass spectrometry. Extraction procedures were devised to give satisfactory recoveries of amines in foods and to overcome problems associated with the binding of amines to other food constituents.