[Study on ratiometric determination of peroxynitrite and its scavenger based on synchronous fluorescence spectroscopy].

A ratiometric fluorimetry is proposed for the determination of peroxynitrite (ONOO-) and the evaluation of its scavenger based on synchronous fluorescence spectroscopy. L-tyrosine, an intrinsic fluorescent aminoacid, reacts with peroxynitrite and carbon dioxide, yielding a highly fluorescent dimer of tyrosine in pH 8.5 PBS buffer solution. By synchronous fluorescence scanning, both monomer and dimer emission bands of L-tyrosine appeared at 364 and 406 nm, respectively. The ratio of F406/ F364 is quantitatively related to the concentration of peroxynitrite, where F406 represents the intensity of synchronous emission band of dimer and F364 represents that of monomer. The method has been showing merit of being insensitive to the changes in experimental parameters and offers a higher sensitivity and a broader responding linear range of the analyte concentration, compared to fluorimetric method, giving a LOD of 1.84 X 10(-8) mol x L(-1) and a linear range of 1.60 X 10(-7) mol x L(-1)-6.00 X 10(-6) mol x L(-1) for ONOO-, respectively, with a deviation of 2.4% for 1.00 X 10(-6) mol x L(-1) ONOO- (n = 8). A IC50 of 0.065 microg x mL(-1) for mitoxantrone, an antioxidant and anticancer drug, was also obtained. This method has proved to be simple and speedy. It would be easily used in the determination of ONOO- and its scavenger.

Screening of tetracycline residues in fish muscles by CCD camera-based solid-surface fluorescence.

A methodology for the screening of tetracyclines (TCs), including tetracycline (TC), oxytetracycline (OTC), and chlorotetracycline (CTC), in different fish muscle matrices has been proposed. This method was based on in situ fluorescent derivation of TCs, transferring weakly fluorescing TCs to highly fluorescent species, on alkaline-activated solid silica gel G plates (SGGPs). By coupling solid-surface fluorescence (SSF) with charge-coupled device (CCD) camera imaging, a CCD camera-based SSF (CCD-SSF) methodology has been developed. Calibration curve, repeatability, selectivity, limit of detection (LOD), and limit of quantification (LOQ) have been explored for evaluating the performance of the method itself. Linear calibration curves were obtained over a range of 0.20-1.0 ng/spot for all three TCs. The LODs, defined as 3sigma, for TC, OTC, and CTC were 0.14, 0.15, and 0.16 ng/spot, respectively. The trueness of method was validated by HPLC, and no significant difference between CCD-SSF and HPLC was found, on a basis of 95% confidence level. By spiked recovery studies, a linear calibration curve ranging from 20 to 300 microg/kg of TC in fish muscle samples with a correlation coefficient (R 2) equal to 0.994 was obtained. The total average recovery for TC in fish muscle samples from six different fish matrices, fortified with TC at 50, 100, and 200 microg/kg levels, was 75.7% with average relative standard deviations (RSDs) ranging from 2.0 to 7.7%. RSDs ranged from 2.5 to 5.8% and from 5.2 to 7.6% for in-day and interday repeatability, respectively. The detection and quantification limits in fish muscle matrices were 16 and 53 microg/kg of TCs, respectively. The newly developed CCD-SSF method has been applied to the screening of the TC residues in fish muscle samples. The method has been demonstrated to bear some advantages, such as its simplicity, high throughput, low cost, use of fewer pollutants, and reasonable sensitivity.

[Determination of tetracyclines by a new spectrum technique].

Tetracyclines are light-fluorescence substances, which cannot be detected directly by fluorometry. Herein a new spectrum method was proposed to detect tetracyclines directly by fluorometry. Under optimal conditions, the calibration graph is linear over the range 0.10-9.00 microg x mL(-1) for tetracyclines, and the detection limits of tetracycline, oxytetracycline, chlortetracycline and doxycycline are 0.065, 0.067, 0.068 and 0.070 microg x mL(-2), respectively.

New approach for the detection of peptide- and protein-based radicals using a pre-fluorescent probe.

A novel application for pre-fluorescent probes in the detection of peptide- and protein-based radicals is proposed. Pre-fluorescent probes combine a fluorescent moiety labeled with a paramagnetic nitroxide that acts as a fluorescence quencher. Trapping of a radical by the nitroxide group restores the fluorescence properties. The increase in fluorescence intensity with time reflects the formation and quenching of free radicals and can be employed for the quantitative evaluation of yields and kinetics. In this test system, the pre-fluorescent probe 4-(9-acridinecarbonate)-2,2,6,6-tetramethylpiperidinyl-1-oxyl radical (Ac-Tempo), in which an acridine moiety was labeled with 2,2,6,6-tetramethylpiperidinyl-1-oxy (Tempo), was employed to probe peptide- and protein-based radicals. Peptide-based radicals were generated through the reaction between horseradish peroxidase (HRP)/H(2)O(2) and a derivative of tyrosine. Protein-based radicals were generated through the reaction between myoglobin (Mb) and H(2)O(2). In both cases the Ac-Tempo was found, using a combination of high-performance liquid chromatography (HPLC) and mass spectrometry, to be converted into fluorescent acridine (Ac)-piperidine (4-(9-acridinecarbonate)-2,2,6,6-tetramethylpiperidine).

Probing the hydroxyl radical-mediated reactivity of peroxynitrite by a spin-labeling fluorophore.

The decomposition of peroxynitrite at physiological pH yielded a hydroxyl radical, which reacted rapidly with dimethyl sulfoxide (DMSO) to produce a methyl radical (*CH3), which was then trapped by a spin-label fluorophore nitroxide-linked naphthalene (NTEMPO), a carbon-centered radical probe with a low fluorescence intensity, and transformed to a stable diamagnetic O-alkoxyamine, a high-fluorescence compound. The fluorescence increment was proportional to the concentration of the hydroxyl radical, and then to the concentration of peroxynitrite. NTEMPO therefore was demonstrated to be capable of detecting hydroxyl radicals generated from peroxynitrite, and the method was proved to be simple and sensitive. The hydroxyl radical-mediated reactivities of peroxynitrite to several amino acids, such as tyrosine, phenylalanine and histidine, were then evaluated by the spin-labeling fluorophore NTEMPO at pH 7.4 and, 37 degrees C. The obtained data were in good agreement with the reference values, respectively.

Fluorimetric determination of hemoglobin using spiro form rhodamine B hydrazide in a micellar medium.

A novel fluorimetric method for the determination of hemoglobin (Hb) using spiro form rhodamine B hydrazide (RBH) as fluorogenic reagent in the presence of sodium dodecylbenzene sulfonate (SDBS) surfactant micelles is described. The method employs the reaction of Hb with RBH, a colorless, non-fluorescent spirolactam hydrazide in SDBS micelles to generate highly fluorescent product rhodamine B and hence leads to a large increase in fluorescence intensity. The fluorescence increase is linearly related to the concentration of Hb in the range 0.2-12.0 nmol l(-1) with a detection limit of 0.086 nmol l(-1) (3sigma). The optimal conditions for the detection of Hb are evaluated and the possible detection mechanism is also discussed. The proposed method has been applied to the determination of Hb in human blood.

Development of a novel rhodamine-type fluorescent probe to determine peroxynitrite.

A novel method for the determination of peroxynitrite using rhodamine B hydrazide as a fluorogenic probe is described. The method is based on the oxidation of rhodamine B hydrazide, a colorless, non-fluorescent substance, by peroxynitrite to give rhodamine B-like fluorescence emission. The fluorescence increase is linearly related to the concentration of peroxynitrite in the range of 7.5x10(-8)-3.0x10(-6) mol l(-1) with a detection limit of 2.4x10(-8) mol l(-1) (3sigma). The optimal conditions for the detection of peroxynitrite were evaluated and the possible detection mechanism was also discussed in this paper.