Spectrofluorimetric Detection Research Articles

A flow-injection renewable surface sensor for the fluorimetric determination of vanadium(V) with Alizarin Red S

Vanadium(V) is determined by a simple bead injection spectroscopy–flow-injection analysis (BIS–FIA) system with spectrofluorimetric detection using a commercially available flow cell (Hellma 176-QS). The 500 μl of a homogeneous bead suspension of an anionic resin (Sephadex QAE A-25) previously loaded with the fluorogenic reagent 1,2-dihydroxyanthraquinone-3-sulfonic acid (Alizarin Red S) was injected to fill the flow cell. Next, V(V) is injected into the carrier and reacts with the immobilized reagent on the active solid support placed in the flow cell to form a fluorescent chelate in the absence of surfactant agents. The complex is so strongly retained on the beads that the regeneration of the solid support becomes extraordinarily difficult, so needing the renovation of the sensing surface which is achieved by means of bead injection. At the end of the analysis, beads are automatically discarded from the flow cell and transported out of the system by reversing the flow. The measurement of fluorescence is continuously performed at an excitation wavelength of 521 nm and an emission wavelength of 617 nm. Using a low sample volume of 800 μl, the analytical signal showed a very good linearity in the range 2–60 ng ml −1, with a detection limit of 0.45 ng ml −1 and a R.S.D. (%) of 4.22 for 50 ng ml −1 of V(V) concentration ( n = 10). The sensor showed both a good selectivity, which could also be increased by the addition of EDTA and F − as masking agents, and applicability to the determination of V(V) in waters, physiological samples (serum and urine) and mussel tissues.

Esterases as a marker for growth of BY-2 tobacco cells and early somatic embryos of the Norway spruce

Growth is one of the basic properties of biological systems. The methods which are commonly used for the determination of growth are usually difficult and not very accurate. In the present work we decided to use esterase activity as a growth marker in tobacco suspension culture (BY-2 line) and in early somatic embryos of Norway spruce (clone 2/32) grown on a semi-solid medium. Esterase activity correlates well with the classical growth characteristics of BY-2 and spruce early somatic embryos. Determination of esterase activity is based on spectrophotometric and spectrofluorimetric detection of reaction products, which arise from the enzymatic hydrolysis of two substrates (p -nitrophenyl acetate and fluorescein diacetate) by esterase. The spectrophotometric method enabled us to detect approximately 104 BY-2 cells and 25 spruce embryos whereas the more sensitive spectrofluorimetric method allowed us to detect approximately 800 BY-2 cells and 5 early somatic embryos of Norway spruce.

Nanoliter detectors for flow systems

The new fiber-based optical microfluidic detectors, designed for nano-volume sample measurement, are presented. The silicon-glass microdetectors were fabricated by use of micromechanical techniques. Spectrofluorimetric and spectrophotometric methods of detection were applied. Optical fibers, integrated into microchannels, ensured very low dead-volume (17 nl for spectrofluorimetric and 900 nl for spectrophotometric detector). The microdevices were made as separate modules and may be connected to any microanalytical system, in which the fluorescence or absorbance is measured. The fabrication methods of microdetectors are compatible with micro-total analysis system (μTAS) technology. The preliminary tests showed the linear detection range 0–0.2 mg/ml of fluorescein and erythrosine (spectrofluorimetric detector), and 0.2–1.6 μg P/ml of phosphate in water solutions (spectrophotometric detector).

Determination of milnacipran, a serotonin and noradrenaline reuptake inhibitor, in human plasma using liquid chromatography with spectrofluorimetric detection

Milnacipran is an antidepressant drug belonging to the class of serotonin and noradrenaline reuptake inhibitors. A sensitive high performance liquid chromatographic during the development method coupled with a fluorimetric detection was set up, validated and then used routinely of the drug. After liquid–liquid extraction, milnacipran and its internal standard were analyzed by reversed-phase liquid chromatography (LC). The drug was derivatized with fluorescamine for fluorescence detection. The identity of the liquid chromatography peaks was controlled using mass spectrometry. The assay linearity was validated up to 1000 ng/ml. The limit of quantification was set at 5 ng/ml. Precision values (relative standard deviations) were lower than 5.4%, whereas the mean accuracy was higher than 95%. The extraction recoveries were higher than 70% for both milnacipran and the internal standard. In clinics, the LC-fluorescence method was routinely used to investigate the pharmacokinetics of milnacipran in patients and proved to be robust and capable of quantifying milnacipran in plasma for at least 36 h (four- to five-fold the elimination half-life).

O-Tyrosine hydroxylation by OH [rad] radicals. 2,3-DOPA and 2,5-DOPA formation in γ-irradiated aqueous solution

The reactions of radiolytically generated hydroxyl radicals (OH ) with dl- o-tyrosine ( o-Tyr) in 10 −3 mol dm −3 aqueous solutions, saturated with N 2O/O 2 gas mixture (4:1, v/v), were studied in the dose range between 30 and 270 Gy. Two isomeric o-Tyr hydroxylation products, namely, 2,3-dihydroxyphenylalanine (2,3-DOPA) and 2,5-dihydroxyphenylalanine (2,5-DOPA) have been identified. The chemical structure of both products was confirmed by an electrospray ionization mass spectrometry, UV and NMR- 1H spectroscopy. The quantitative analysis with the use of an electrochemical and a spectrofluorimetric detection showed that the concentration of 2,3-DOPA and 2,5-DOPA increased and the content of substrate decreased linearly with increasing radiation dose. The ratio of the yields of 2,3-DOPA and 2,5-DOPA was equal to 1.3 for the whole range of radiation dose applied. The total yield of 2,3- and 2,5-DOPAs was approximately 50% of the level of o-Tyr decomposition.

Implementation of flow-through multi-sensors with bead injection spectroscopy: fluorimetric renewable surface biparameter sensor for determination of berillium and aluminum

For the first time a new, sensitive, and simple bead injection spectroscopy–flow injection analysis (BIS–FIA) system with spectrofluorimetric detection is described for the sequential determination of two metals. The sensor is based on the alternate use of two carriers and a commercially available flow cell (Hellma 176-QS). The flow cell is filled by injecting in the flow system 500 μl of a homogeneous bead suspension of an appropriate solid support (Sephadex QAE A-25) previously loaded with the fluorogenic reagent morin (2′,3,4′,5,7-pentahydroxyflavone). A sequential reaction of Al(III) and Be(II) with morin (immobilized on beads) to form their fluorescent complexes is performed on the bead sensing support and their respective fluorescence emission monitored, after doing two successive injections from the mixture solution. Firstly, Al(III) could be determined in the sample using 0.5 M NaCl/HCl, pH 6 as carrier. Then, the carrier solution was changed (0.3 M NaCl/NaOH, pH 12) making possible the elution of Al(III) and the restoration of the baseline, then allowing the reaction of Be(II). At the end of the analysis, beads are automatically discarded from the flow cell, by reversing the flow, and transported out of the system. The analytical signals are measured at an excitation wavelength of 440 nm and an emission wavelength of 520 nm. Using a sample volume of 600 μl, the analytical signal showed a very good linearity in the range 0.1–8 ng ml −1 and 0.1–1 μg ml −1 with detection limits of 0.024 ng ml −1 and 0.010 μg ml −1 for Be(II) and Al(III), respectively. R.S.D.s (%) lower than 5% were obtained for both analytes and the selectivity was improved using EDTA as masking reagent. The sensor was satisfactorily applied to the determination of these metals in waters and simulated alloy samples.

Biogeochemistry of Aromatic Surfactants in Microtidal Estuaries

This paper summarizes several studies on input, distribution, and fate of two different types of aromatic surfactants, the anionic linear alkylbenzene sulphonates (LAS) and nonionic alkylphenol polyethoxylates (APnEO), in a karstic estuary characterized by sharp salinity gradients. A combination of reversed-phase and normal-phase HPLC analysis followed by spectrofluorimetric detection allowed detailed characterization of homologue and oligomer compositions of aromatic surfactants in wastewater and estuarine waters. The distribution patterns of homologues and/or oligomers in the wastewater plume reveal a significant alteration of the original composition of both surfactants by biotransformation and physico-chemical partitioning. The vertical distribution of surfactant residues in the estuarine water column is characterized by pronounced maxima at the air–brackish water and brackish water–sea water interface. The brackish water–sea water interface is a very efficient barrier, which prevents spreading of surfactants into the deeper layers. In addition, the interface seems to be a site of high biological activity, including biotransformation of surfactant residues. As a consequence, this layer can contain enhanced levels of lipophilic metabolites of APnEO. Biotransformation is the main mechanism for the removal of both surfactant classes from the water column. However, our studies suggest that a significant percentage of surfactant residues discharged into the stratified estuaries reaches the coastal sea due to the incomplete removal in the estuary. This situation is a consequence of a rather fast flushing of the brackish layer and a very slow biotransformation kinetics in the saline layer, especially in the winter period.

Different glycosyltransferases are differentially processed for secretion, dimerization, and autoglycosylation.

Modification of Golgi glycosyltransferases, such as formation of disulfide-bonded dimers and proteolytical release from cells as a soluble form, are important processes to regulate the activity of glycosyltransferases. To better understand these processes, six glycosyltransferases were selected on the basis of the donor sugars, including two N-acetylglucosaminyltransferases, core 1 beta1,3-N-acetylglucosaminyltransferase (C1-beta3GnT) and core 2 beta1,6-N-acetylglucosaminyltransferase (C2GnT-I); two fucosyltransferases, alpha1,2-fucosyltransferase-I (FucT-I) and alpha1,3-fucosyltransferase-VII (FucT-VII); and two sialyltransferases, alpha2,3-sialyltransferase-I (ST3Gal-I) and alpha2,6-sialyltransferase-I (ST6Gal-I). These enzymes were fused with enhanced green fluorescence protein and stably expressed in Chinese hamster ovary cells. Spectrofluorimetric detection and immunoblotting analyses showed that all of these glycosyltransferases except FucT-VII were secreted in the medium. By examining dimers formed in cells and culture media, we found that all of the enzymes, except ST3Gal-I, form a combination of monomers and dimers in cells, whereas the molecules released in the media are either exclusively monomers (C2GnT-I and ST6Gal-I), dimers (FucT-I) or a mixture of both (C1-beta3GnT). These results indicate that dimerization does not always lead to Golgi retention. Analysis of the N-glycosylation status of the enzymes revealed that the secreted proteins are generally more heavily N-glycosylated and sialylated than their membrane-associated counterparts, suggesting that the proteolytic cleavage occurs before the glycosylation is completed. Using FucT-I and ST6Gal-I as a model, we also show that these glycosyltransferases are able to perform autoglycosylation in the dimeric forms. These results indicate that different glycosyltranferases differ significantly in dimerization, proteolytic digestion and secretion, and autoglycosylation. These results strongly suggest that disulfide-bonded dimerization and secretion differentially plays a role in the processing and function of different glycosyltransferases in the Golgi apparatus.

Feasibility of the Simultaneous Determination of Polycyclic Aromatic Hydrocarbons by Near Infrared Spectroscopy

National Institute of Agricultural Science & Technology, Suwon 441-707, KoreaReceived November 18, 2002Key Words : Polycyclic aromatic hydrocarbons, Partial least squares, Near infrared spectroscopyPolycyclic aromatic hydrocarbons (PAHs) are widelydistributed in the environment and are often implicated aspotential carcinogens. Different methods such as liquidchromatography with spectrophotometric or spectrofluori-metric detection, capillary gas chromatography with massspectrometric detection and supercritical fluid chromatographyhave been used to determine PAHs.

Determination of tenofovir in human plasma by high-performance liquid chromatography with spectrofluorimetric detection

A sensitive high-performance liquid chromatography method was developed with spectrofluorimetric detection for the determination of tenofovir, a new HIV reverse transcriptase inhibitor, in human plasma. After precipitation of 200 μl of plasma samples by methanol and evaporation of the supernatant, fluorescent derivatized compounds were obtained by a 40-min incubation at 80 °C with chloroacetaldehyde 0.34% at pH 4.5. The assay was performed isocratically using 5 m M Na 2HPO 4 (pH 6), containing tetrabutylammonium (TBA) chloride 5 m M, and acetonitrile (85:15, v/v) as mobile phase, and a Cluzeau C 8 plus satisfaction column maintained at 35 °C. Detection was performed at excitation and emission wavelengths set at 236 and 420 nm, respectively. In these conditions, tenofovir can be separated from adefovir, the internal standard, and endogenous substances. The method was found to be linear and has been validated over a concentration range of 5–1000 μg/l. The average coefficient of the limit of quantification (5 μg/l) was 5.38% and at this concentration, a signal-to-noise ratio of 500 was measured

Time-based multisyringe flow injection system for the spectrofluorimetric determination of aluminium

A time-based multisyringe flow injection procedure with spectrofluorimetric detection is proposed in this paper for the determination of aluminium in drinking water. The flow methodology is based on the simultaneous or sequential injection of sample and chelating reagent (viz. 8-hydroxyquinoline-5-sulphonic acid) plugs using a multicommutation approach so that three successive injections may be performed with a sole displacement of the piston driver bar of the burette. Thus, an injection throughput as high as 154 h −1 is achieved by sampling a 182 μl sample zone. In order to enhance the luminescence, the reaction is carried out in micellar medium using hexadecyltrimethylammonium chloride as surfactant. The influence of geometric and hydrodynamic variables as well as several parameters such as multicommutation timing, ligand and surfactant concentration and reagent pH was assessed. Under the selected working conditions, a linear dynamic range from 10 to 500 μg l −1 Al(III), a 3 σ detection limit of 0.5 μg l −1 and a coefficient of variation of 0.6% at the 30 μg l −1 level were obtained. The analytical features were compared with those reported in previous flow injection and sequential injection methods. The multisyringe technique was successfully applied to the determination of aluminium in drinking water at low mineralisation levels, validating the results by inductively coupled plasma atomic emission spectrometry.

POST-CHROMATOGRAPHIC DETECTION OF OLIGOPEPTIDES USING TERBIUM-SENSITIZED LUMINESCENCE

This report investigated the potential use of oligopeptide complexation with terbium III as a high-performance liquid chromatography method with time-resolved spectrofluorimetric detection for analysis of milk-derived products. The study focused on glycyl-leucyl-phenylalanine, a compound with immunostimulatory properties isolated from milk casein, and also considered tyrosine and tryptophan residues in peptides. As the water content of the mobile phase was highly restrictive for detection, acetate ions were introduced as a potentiation agent. Only post-chromatographic complexation proved feasible, because passage through a column destroyed oligopeptide-terbium chelation. Validation of glycyl-leucylphenylalanine, glycyl-phenylalanine, and leucyl-phenylalanine assays showed linearity in the 8 × 10−5 to 5 × 10−4mol l−1 range (correlation coefficients of 0.96, 0.99 and 0.99 respectively). Results for the assay at 10−5 mol l−1 showed a 7% intra-day (repeatability) coefficient of variation (CV) and a 15% inter-day (reproducibility) CV. No interferences were observed in the presence of various water-soluble vitamins present in whey. This method, when tested in the bromatology field on yoghurts with different milk origins, showed that goat wheys had phenylalanine, tyrosine, and tryptophan residues twice those of cow wheys and three times those of sheep or soja bean wheys.

Selenium in municipal wells in the Irapuato City — spectrofluorimetric determination with 2,3-diaminonaphthalene

Abstract A chromatographic procedure with spectrofluorimetric detection (λ = 257 nm, λ = 548 nm) is proposed in this work for Se determination in well water after formation of a piazselenol compound. The low detection limit obtained (15 ng·l-1 ) enabled minimisation of the sample size and, consequently, smaller amounts of reagents were needed. Selenium content was confirmed using standard reference material. The proposed procedure was applied for the determination of Se in municipal wells in Irapuato city (Lerma-Chapala basin) and the obtained results showed that 52.5% of the analysed wells contained amounts higher than the limit 10 μg·l-1 of selenium as recommended by the World Health Organisation for drinking water. The high selenium levels were found only in the wells of the city of Irapuato (concentration range 12 – 223 μg l-1 ), not in the wells in other cities of the State of Guanajuato. No contamination by other trace elements (arsenic, cadmium, chromium, iron, manganese, lead) was found in Irapuato wells.

HPLC coupled with fluorimetric detection for the identification of natural resins in archaeological materials

For better identification of chemical compounds in natural resins HPLC with UV-visible detection has been complemented with spectrofluorimetric detection. The fluorimetric response of many standards has been studied and typical patterns were obtained for several resins which are also found in ancient samples.

Synthesis of ascorbate and urate in the ovary of water buffalo

Blood flow interruption is associated with oxygen depletion and loss of factors for function and survival in downstream tissues or cells. Hypoxia and absence of gonadotropins trigger apoptosis and atresia in the ovary. We studied the antioxidant response of follicular cells to plasma deprivation in ovaries dissected from water buffalo. Aliquots of follicular fluid were aspirated from each antral follicle, before and during incubation of the ovaries at 39°C. Urate, ascorbate, retinol and α-tocopherol in the fluid were, titrated by High Performance Liquid Chromatography (HPLC) with spectrophotometric or spectrofluorimetric detection. The total antioxidant capacity of follicular fluid was determined as absorbance decrease, following addition of a source of radical chromophores. The more the incubation progressed, the higher levels of urate, ascorbate and total antioxidant capacity were found. Conversely, changes in concentration of the liposoluble antioxidants were not observed. Ascorbate synthesizing activity in the follicle was demonstrated by detecting the enzyme L-gulono-γ-lactone oxidase in microsomes prepared from granulosa cells. These cells were also analyzed for the expression of the enzyme CPP32. The enzyme level, measured as DEVD-p-nitroanilide cleaving activity, was found related with the immunoreactivity to anti-CPP32 antibodies. Negative correlation between the enzyme activity (which is known to be induced by peroxynitrite) and the follicular level of urate (which scavenges peroxynitrite) was also observed. The amount of nitrotyrosine, a product of peroxynitrite attack on proteins, was measured in follicular fluids by Enzyme Linked ImmunoSorbent Assay (ELISA). This amount was found positively correlated with the CPP32 activity, and negatively correlated with the urate level in follicular fluid. Alterations in concentrations of ascorbate or urate may be associated with oxidative stress during follicular atresia.

DETERMINATION OF TOTAL GLUTATHIONE IN CELL LYSATES BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH O-PHTHALALDEHYDE PRECOLUMN DERIVATIZATION IN THE PRESENCE OF TRIS(2-CARBOXYETHYL)-PHOSPHINE

A very sensitive HPLC method for the determination of total glutathione (GSH) and γ-glutamyl-cysteine (γ-Glu-Cys) content in cell lysates is presented. It is based on a precolumn derivatization with o-phthalaldehyde (OPA) using tris(2-carboxyethyl)-phosphine (TCEP) as a reducing agent. Separation of the peptide derivatives is carried out by reversed-phase chromatography on a C18 column, followed by spectrofluorimetric detection. The fluorescence response of GSH and γ-Glu-Cys derivatives is linear over a range of 0.05 to 100 μM and 1 to 25 μM, respectively, with good precision. The detection limits approach 26 fmol and 60 fmol on column, respectively. In contrast the detection limit for GSH in the presence of DTT, which is often used as the reducing agent, is 250 fmol GSH on column. The results for the GSH concentration of cell lysates are in agreement with a radiotracer method based on metabolic labeling of GSH with [35S]-Cys.

Evaluation of a sensitive HPLC method for the determination of Malondialdehyde, and application of the method to different biological materials

Reactive oxygen species (ROS), important mediators of cell and tissue injury during inflammation, are produced by several types of inflammatory cells. The formation of ROS can be monitored by detection of lipid peroxidation products. The extremely broad spectrum of biological effects of aldehydic lipid peroxidation products has necessitated the development of a technique that enables the sensitive routine quantitation of aldehydes formed in biological materials. Malondialdehyde (MDA) is a by-product of enzymatic eicosanoid formation and an end-product of nonenzymatic peroxidation of polyunsaturated fatty acids with three or more bisallylic double bonds. The determination of the thiobarbituric acid derivative of MDA (TBA-MDA) is a widely used method for estimating overall lipid peroxidation. We describe a rapid, isocratic, simple, and sensitive high-performance liquid chromatographic (HPLC) method with spectrofluorimetric detection for measurement of MDA-TBA in human biological samples such as plasma, urine, wound secretions, amniotic fluid, sputum and tissue samples. By use of this method, picomole quantities of MDA can be readily and specifically detected in different biological materials. Coefficients of variation of repeated MDA-TBA assays were 4.4% within run and 6.9% from run to run. Reference values are given for a variety of human body fluids and for rat tissues.

Validation of a LC method for the determination of 5-aminosalicylic acid and its metabolite in plasma and urine

The choice of a proper analytical method for the quantification of drugs and/or their metabolites in biological samples plays a significant role in the evaluation and interpretation of bioavailability, bioequivalence and pharmacokinetic data. The aim of this study was validation of a method for the identification and quantitative determination of 5-aminosalicylic acid (5-ASA) and its metabolite N-acetyl-5-aminosalicylic acid in human plasma and urine. According to previous studies on the disposition of 5-ASA (mesalazine) in a patient with inflammatory bowel diseases, we have developed a rapid, sensitive method for the determination of 5-ASA and its acetylated metabolite, N-acetyl-5-aminosalicylic acid (Ac-5-ASA). The advantage of this method is that it measures both compounds, and is more rapid, reproducible and credible than the previous studies [C. Fischer, K. Maier, U. Klotz, J. Chromatogr. Biomed. Appl. 225 (1981) 498–503; P.N. Shaw, A.L. Sivner, L. Aarons, J.B. Houston, J. Chromatogr. Biomed. Appl. 274 (1983) 393–397; B. Norlander, R. Gotthard, M. Strom, Aliment. Pharmacol. Ther. 3 (1989) 333–342; U. Klotz, G.L. Stracciari, Arzneim.-Forsch. Drug Res. II 43 (12) (1993) 1357–1359]. 5-ASA was quantitatively determined in human plasma and urine samples by liquid chromatography following prior derivatization to its acetylated metabolite (Ac-5-ASA). N-Acetyl-anthranilic acid was used as the internal standard. The detection was performed with a spectrofluorimetric detector, excitation at 311 nm, cut-off at 449 nm. The method was validated for the following parameters: linearity, recovery, sensitivity, precision, accuracy, selectivity and stability, limits of quantification and of detection. It showed good linearity ( r 2≥0,996) in the range 0.1 ng/ml to 8 μg/ml using a Lichrospher 60 RP-select B column. The lower limit of detection was 20 ng/ml in plasma and urine. The within-run relative standard deviations (R.S.D.) were below 6.7% at all concentration levels and the between-run R.S.D. were below 25.4% at all concentration levels.

Study of biologically active amines in grapes and wines by HPLC

The biologically active amines agmatine, cadaverine, histamine, phenethylamine, putrescine, spermidine, and tyramine have been determined in different varieties of grape, aszu grape, wine and aszu wine from the Tokaj region of Hungary. Ion pairs formed between the amines and octanesulphonic acid were separated by liquid chromatography on a μBondapak C18 reversed-phase column, and spectrofluorimetric detection was performed after post-column derivatization witho-phthalaldehyde and 2-mercaptoethanol. The method was linear for the amines between 0.1 and 10 mg L−1, and for spermidine between 1 and 30 mg L−1.

Photodissociation/gas-diffusion separation and fluorimetric detection for the analysis of total and labile cyanide in a flow system

Total cyanide species are determined in a flow injection system which includes UV-photodissociation, gas-diffusion separation and spectrofluorimetric detection. Without the irradiation step, only cyanide easily released in acid medium, i.e. labile cyanide, is determined. Cyanide diffuses through a microporous PTFE membrane from an acid donor stream to a sodium hydroxide acceptor stream. Then, the transferred cyanide reacts with ¶o-phthalaldehyde and glycine to form a highly fluorescent isoindole derivative. Complete cyanide recoveries were obtained for the most important metal cyanide complexes found in environmental samples, excepting cobaltocyanide. The sampling frequency for total cyanide was 4 samples h–1 and the detection limit was 0.4 μg L–1. Recoveries of total cyanide from river water obtained with this method are about 90% of those obtained with APHA Method 4500-CN C for total cyanide.