An oxazine reagent for derivatization of carboxylic acid analytes suitable for liquid chromatographic detection using visible diode laser-induced fluorescence

Sv, Rahavendran; Ht, Karnes

doi:10.1016/0731-7085(96)01819-5

Cited by 18 publications

(10 citation statements)

References 21 publications

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“…50 The oxazine dyes display surprising long-wavelength absorption and emission maxima, given their small size. 51 long absorption and emission wavelengths, as shown for IR-125 and Thiazole Orange. Dyes of this class have been extensively characterized for use as long-wavelength probes and in DNA sequencing.…”

Section: Red and Near-infrared (Nir) Dyesmentioning

confidence: 79%

Fluorophores

Lakowicz¹

1999

Principles of Fluorescence Spectroscopy

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FluorophoresFluorescence probes represent the most important area of fluorescence spectroscopy. One can spend a great deal of time describing the instrumentation for fluorescence spectroscopy, including light sources, monochromators, lasers, and detectors. However, in the final analysis, the wavelength and time resolution required of the instruments are determined by the spectral properties of the fluorophores. Furthermore, the information available from the experiments is determined by the properties of the probes. Only probes with nonzero anisotropies can be used to measure rotational diffusion, and the lifetime of the fluorophore must be comparable to the correlation time of interest. Only probes which are sensitive to pH can be used to measure pH. And only probes with reasonably long excitation and emission wavelengths can be used with tissues which display autofluorescence at short excitation wavelengths.Thousands of fluorescent probes are known, and it is not practical to describe them all. This chapter contains an overview of the various types of fluorophores, their spectral properties, and their applications. Fluorophores can be broadly divided into two main classes, intrinsic and extrinsic. Intrinsic fluorophores are those which occur naturally. These include the aromatic amino acids, NADH, flavins, and derivatives of pyridoxal and chlorophyll. Extrinsic fluorophores are added to the sample to provide fluorescence when none exists or to change the spectral properties of the sample. Extrinsic fluorophores include dansyl chloride, fluorescein, rhodamine, and numerous other substances. 3.1.A. Fluorescent Enzyme CofactorsEnzyme cofactors frequently are fluorescent (Figure 3.1). NADH is highly fluorescent, with absorption and emission maxima at 340 and 460 nm, respectively (Figure 3.3). The oxidized form of NADH, NAD+, is nonfluorescent. The lifetime of NADH in aqueous buffer is near 0.4 ns. The 63 J. R. Lakowicz, Principles of Fluorescence Spectroscopy

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Section: Red and Near-infrared (Nir) Dyesmentioning

confidence: 79%

Fluorophores

Lakowicz¹

1999

Principles of Fluorescence Spectroscopy

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“…Equation [4] is valid only when the donor decay is a single exponential. For the D-A pair shown in Scheme 2 the donor will display a lifetime ( DA ) of 100 ns in the presence of acceptor.…”

Section: Theorymentioning

confidence: 99%

“…Long wavelength excitation and emission are valuable because the background autofluorescence from biological samples and optical components typically decreases at longer incident wavelengths. Red and NIR probes are now routinely used in protein labeling (2,3), chromatography (4,5), measurements in blood (6,7), noninvasive medical testing (8 -10), and DNA analysis (11)(12)(13)). An advantage of the presently available red-NIR fluorophores is their high extinction coefficient, which in turn allows high sensitivity detection.…”

confidence: 99%

On the Possibility of Long-Wavelength Long-Lifetime High-Quantum-Yield Luminophores

Lakowicz

Piszczek

Kang

2001

Analytical Biochemistry

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We describe an approach to creating a new class of luminophores which display both long wavelength emissions exceeding 600 nm and long lifetimes. These luminophores are based on resonance energy transfer (RET) from a long lifetime donor to a short lifetime but long wavelength acceptor. We demonstrated the possibility of obtaining these desirable spectral properties using donors and acceptors noncovalently bound to DNA. The donor was a ruthenium (Ru) metal-ligand complex in which one of the diimine ligands intercalated into double-helix DNA. The acceptors were either nile blue, TOTO-3, or TO-PRO-3. Upon binding of the acceptor to donor-labeled DNA, we found that the acceptor quantum yield was remarkably enhanced so that the wavelength-integrated intensities of the donor and acceptor bound to DNA were many-fold greater than the intensity of the donor and acceptor alone when separately bound to DNA. The origin of this effect is efficient energy transfer from the donor. Under these conditions the effective overall quantum yield approaches that of the acceptor. Importantly, the increased quantum yield can be obtained while maintaining usefully long apparent acceptor lifetimes of 30 to 80 ns. The effect of an increased quantum yield from a low quantum yield donor may find use in assays to detect macromolecular binding interactions. These results suggest the synthesis of covalently linked donor-acceptor pairs with the desirable spectral properties of long wavelength emission, high quantum yield, and moderately long lifetimes for gated detection.

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“…Method 2 . The NB−PAA derivative was also synthesized by reacting NB with phenylacetyl chloride in the presence of triethylamine for 20 min at 40 °C as described elsewhere …”

Section: Exprimental Sectionmentioning

confidence: 99%

“…Nile blue (NB), a far-red oxazine dye (λ ex = 633 nm, λ em = 660 nm, ε = 75 000, Φ f = 27%) has been shown, in previous work, to be a useful precolumn derivatization reagent suitable for VDLIF detection of carboxylic acid- containing analytes in neat solution . A limit of detection (S/N = 3) of 5 × 10 -15 M was obtained for underivatized NB using a reversed phase column.…”

mentioning

confidence: 99%

Visible Diode Laser-Induced Fluorescence Detection of Phenylacetic Acid in Plasma Derivatized with Nile Blue and Using Precolumn Phase Transfer Catalysis

Rahavendran

Karnes

1997

Anal. Chem.

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This study reports the application of Nile blue (NB), a farred oxazine label, as a precolumn derivatization reagent for the measurement of free levels of phenylacetic acid (PAA) in plasma. The measurement of PAA in psychiatric populations is important because it provides a marker for 2-phenylethylamine (PEA), which has been implicated in the pathogenesis of schizophrenia and major depression. PAA was derivatized with NB through an amide linkage in the presence of 2-chloro-1-methylpyridinium iodide (carboxylic acid activator, CMP) and triethylamine (base catalyst, TEA), respectively. The formation of the NB-PAA derivative was confirmed using normal phase and reversed phase thin-layer chromatography, reversed phase liquid chromatography, and electrospray mass spectrometry. The formation of the NB-PAA derivative was optimized using a sequential single factor approach. The optimal conditions for the formation and chromatographic separation of the derivative were determined to be 8.0 nmol/mL NB, 390 nmol/mL CMP, 2 mumol/mL TEA, a reaction time of 45 min, and a reaction temperature of 25 degrees C. This derivatization scheme was performed in a phase transfer catalysis mode that enabled the simultaneous extraction, preconcentration, and derivatization of the analyte in a single step. The limit of derivatization of PAA was determined to be 1.0 x 10(-9) M in phosphate-buffered saline, a PAA-free matrix. This derivatization was limited not by the kinetics of the reaction but by the chromatographic separation of the derivative from a side reaction product. The method was used to estimate endogenous free levels of PAA in human plasma samples. The levels of PAA in four sources of plasma were determined to be within 30-70 ng/mL using the method of standard addition and reflected levels that have been reported in the literature. The limit of detection of the derivative was determined to be 7.33 x 10(-11) M using a laboratory-constructed HPLC-VDLIF detector.

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An oxazine reagent for derivatization of carboxylic acid analytes suitable for liquid chromatographic detection using visible diode laser-induced fluorescence

Cited by 18 publications

References 21 publications

Fluorophores

Fluorophores

On the Possibility of Long-Wavelength Long-Lifetime High-Quantum-Yield Luminophores

Visible Diode Laser-Induced Fluorescence Detection of Phenylacetic Acid in Plasma Derivatized with Nile Blue and Using Precolumn Phase Transfer Catalysis

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