Selective determination of cysteines through precolumn double-labeling and liquid chromatography followed by detection of intramolecular FRET

In this paper we introduce a novel approach for highly selective and sensitive analysis of cysteines (glutathione, cysteine, and homocysteine). This method is based on the detection of intramolecular fluorescence resonance energy transfer (FRET) in a liquid chromatography (LC) system after double-labeling of the amino and sulfhydryl groups of the cysteines. In this detection process, we monitored the FRET between the amine-derivatized and thiol-derivatized fluorophores. We screened 16 combinations of fluorescent reagents. As a result, FRET occurred most effectively when the sulfhydryl and amino groups of the cysteines were derivatized with 7-diethylamino-3-[{4'-(iodoacetyl)amino}phenyl]-4-methylcoumarin (DCIA, Ex/Em 390/480 nm) and 4-fluoro-7-nitrobenz-2-oxo-1,3-diazole (NBD-F, Ex/Em 480/540 nm), respectively, in this order. The double-labeled cysteines emitted NBD-F fluorescence (540 nm) through an intramolecular FRET process when they were excited at the wavelength of maximum excitation of DCIA (390 nm). The generation of FRET was confirmed by comparison with analysis of n-amylamine or tryptophan (amines without a sulfhydryl group) and 6-mercaptohexanol (thiol without an amino group) performed using LC and a three-dimensional fluorescence detection system. We were able to separate the double-labeled cysteines (DCIA and NBD-F) when performing LC on an ODS column with isocratic elution. The limits of quantification (signal-to-noise ratio = 10) and detection (signal-to-noise ratio = 3) for the cysteines, for a 20-μL injection volume, were in the range 150-670 fmol and 46-200 fmol, respectively. The sensitivity of the intramolecular FRET-forming derivatization method is higher than that of a system which takes advantage of conventional detection of the derivatives. Furthermore, this method provides sufficient selectivity and sensitivity to determine the total cysteines present in the plasma of healthy humans.     

Fluorogenic derivatization of aryl halides based on the formation of biphenyl by Suzuki coupling reaction with phenylboronic acid

The fluorogenic derivatization method for aryl halide was developed for the first time. This method was based on the formation of fluorescent biphenyl structure by Suzuki coupling reaction between aryl halides and non-fluorescent phenylboronic acid (PBA). We measured the fluorescence spectra of the products obtained by the reaction of p-substituted aryl bromides (i.e., 4-bromobenzonitrile, 4-bromoanisole, 4-bromobenzoic acid ethyl ester and 4-bromotoluene) with PBA in the presence of palladium (II) acetate as a catalyst. The significant fluorescence at excitation maximum wavelength of 275–290 nm and emission maximum wavelength of 315–350 nm was detected in all the tested aryl bromides. This result demonstrated that non-fluorescent aryl bromides could be converted to the fluorescent biphenyl derivatives by the coupling reaction with non-fluorescent PBA. We tried to determine these aryl bromides by HPLC-fluorescence detection with pre-column derivatization. The aryl bromide derivatives were detected on the chromatogram within 30 min without any interfering peak derived from the reagent blank. The detection limits (S/N = 3) for aryl bromides were 13–157 fmol/injection.     

Liquid chromatographic determination of dicarboxylic acids based on intramolecular excimer-forming fluorescence derivatization

A highly sensitive and selective fluorimetric determination method for dicarboxylic acids (C5-C12) has been developed. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butyric acid hydrazide (PBH), followed by reversed-phase liquid chromatography (LC). The carboxylic acids were converted to the corresponding dipyrene-labeled derivatives by reaction with PBH in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The derivatives afforded intramolecular excimer fluorescence (450-550 nm) which can clearly be discriminated from the normal fluorescence (370-420 nm) emitted from PBH and monopyrene-labeled derivatives of monocarboxylic acids. The structures of the derivatives and the emission of excimer fluorescence were studied by LC with mass spectrometry and with spectrofluorimetry, respectively. The PBH derivatives of the carboxylic acids could be separated by reversed-phase LC on an ODS column with isocratic elution. The detection limits (signal-to-noise ratio = 3) were 1.3 fmol to undetectable for a 20-microl injection.     

Liquid chromatographic determination of ornithine and lysine based on intramolecular excimer-forming fluorescence derivatization.

A highly sensitive and selective fluorometric determination method for ornithine and lysine has been developed. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), followed by reversed-phase liquid chromatography (LC). The analytes, containing two amino moieties in a molecule, were converted to the corresponding dipyrene-labeled derivatives by reaction with PSE. The derivatives afforded intramolecular excimer fluorescence (450-550 nm) which can clearly be discriminated from the normal fluorescence (370-420 nm) emitted from PSE and monopyrene-labeled derivatives of monoamines. The structures of the derivatives and the emission of excimer fluorescence were confirmed by LC with mass spectrometry and with three-dimensional fluorescence detection system, respectively. The PSE derivatives of ornithine and lysine could be separated by reversed-phase LC on ODS column with isocratic elution. The detection limits (signal-to-noise ratio = 3) for ornithine and lysine were 3.5 and 3.7 fmol, respectively, for a 20-microl injection. Furthermore, this method had enough selectivity and sensitivity for the determination of ornithine and lysine in normal human urine.     

On-line post-column fluorescence detection for N-terminal tyrosine-containing peptides in high-performance liquid chromatography

A detection system based on on-line post-column fluorescence derivatization is described for the determination of N-terminal tyrosine-containing peptides by reversed-phase high-performance liquid chromatography. The peptides are automatically converted into fluorescent derivatives by reaction with hydroxylamine, cobalt (II) and borate after peptide separation on a reversed-phase column (TSKgel ODS-120T) followed by passage through an ultraviolet absorbance detector. The reaction system permits the fluorescence detection at 435 nm (emission) with excitation at 335 nm for N-terminal tyrosine-containing synthetic peptides in as little as picomole amounts. The facile fluorescence detection of N-terminal tyrosine-containing fragments produced from methionine-enkephalin by enzymatic degradation using a rat brain homogenate was achieved by comparison with the ultraviolet absorption detection at 215 nm.     

Liquid chromatographic determination of polythiols based on pre-column excimer fluorescence derivatization and its application to α-lipoic acid analysis

We developed an LC method for the sensitive and selective fluorometric determination of polythiols. This method employs pre-column intramolecular excimer-forming fluorescence derivatization with N-(1-pyrene)iodoacetamide followed by LC separation. Polythiols were converted to the corresponding dipyrene-labeled derivatives, and the derivatives afforded intramolecular excimer fluorescence (440–540 nm). After the optimization using dithiothreitol and dimercaprol as model polythiols, α-lipoic acid (LA) and α-lipoamide were determined with high sensitivity and selectivity. The detection limits for polythiols were 0.6–3.5 fmol on column. Furthermore, this method could be successfully applied to the determination of LA in commercial dietary supplements and in human urine.     

Determination of catecholamines and indoleamines in human urine based on intramolecular excimer-forming derivatization and fluorescence detection.

A liquid chromatographic (LC) determination of catecholamines and indoleamines is described. This is based on intramolecular excimer-forming fluorescence derivatization with 4-(1-pyrene)butanoyl chloride, followed by reversed-phase LC. The analytes, containing an amino moiety and phenolic hydroxyl moieties in a molecule, were converted to the corresponding polypyrene-labeled derivatives by one-step derivatization. They afforded intramolecular excimer fluorescence, which can clearly be discriminated from the normal fluorescence emitted from reagent blanks. The detection limits (S/N = 3) for catecholamines and indoleamines were femto-mole levels per 20-microL injection. Furthermore, this method was applied to a urine assay.     

Simultaneous determination of histamine and histidine by liquid chromatography following intramolecular excimer-forming fluorescence derivatization with pyrene-labeling reagent.

A highly sensitive and selective fluorometric method for the determination of histamine and histidine has been developed. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent followed by reversed-phase liquid chromatography. The analytes, containing two amino moieties in a molecule, were converted to the corresponding dipyrene-labeled derivatives by derivatization. The derivatives afforded intramolecular excimer fluorescence (440 - 540 nm), which can clearly be discriminated from the normal fluorescence (360 - 420 nm) emitted from reagent blanks. The detection limits (signal-to-noise ratio = 3) were femto mole levels.     

Screening method for organic aciduria by spectrofluorometric measurement of total dicarboxylic acids in human urine based on intramolecular excimer-forming fluorescence derivatization

A simple screening method of organic aciduria by spectrofluorometric measurement of total dicarboxylic acids in human urine is described. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butanoic acid hydrazide (PBH). Dicarboxylic acids in urine were converted to the corresponding dipyrene-labeled derivatives by reaction with PBH in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and pyridine, and the derivatives afforded intramolecular excimer fluorescence (420-540 nm) which can clearly be discriminated from the normal fluorescence (360-420 nm) emitted from reagent blanks. The technique is so selective that it permits spectrofluorometric measurement of total amount of dicarboxylic acids by the direct derivatization of diluted urine samples. The same reaction mixture has also served as a liquid chromatographic (LC) sample for the separative determination of individual dicarboxylic acids. The spectrofluorometric data did not contradict with the LC data. These methods were usefully applied to preliminary screening test of glutaric aciduria. In conclusion, the present derivatization method allows rapid and direct determination of total amount of dicarboxylic acids in human urine samples.     

An automated analyzer for methylated arginines in rat plasma by high-performance liquid chromatography with post-column fluorescence reaction

A fully automated analyzer for methylated L-arginine metabolites [N,N-dimethyl-L-arginine (ADMA), N-methylarginine (NMMA) and N,N'-dimethyl-L-arginine (SDMA)] by high-performance liquid chromatography with post-column fluorescence derivatization was developed. This system consists of an on-line extraction, a separation on a reversed phase ion-pair chromatograph, a post-column derivatization by o-phthaladehyde (OPA) and thiol reaction, and fluorescence detection. NMMA, ADMA and SDMA were separated in 40 min with isocratic elution by a combination of octanoate and cyclohexane carboxylate as ion-pair reagents. The eluate was monitored at 450 nm with excitation at 337 nm. The calibration curves for NMMA, ADMA and SDMA showed linearity over the range from 0.05 micromol l(-1) (0.5 pmol on column) to 5.0 micromol l(-1) (50 pmol on column). This method does not require any time-consuming pre-treatment and requires only 10 microl of plasma sample for assay.     

新型荧光衍生试剂用于脂肪胺的反相高效液相色谱-荧光检测分析

利用新型荧光试剂4-(1H-菲并[9,10-d]咪唑-2-)苯甲酸(PIBA)进行柱前衍生并经荧光检测对脂肪胺进行了高效液相色谱(HPLC)分离和在线质谱定性。激发和发射波长分别为ex=261nm,em=443nm。80℃下在吡啶溶剂中用N-乙基-N’-[(3-二甲氨基)丙基]碳二亚胺盐酸盐(EDC)做催化剂,衍生反应10min后获得稳定的荧光产物。在EclipseXDB-C8色谱柱(4.6150mm,5mm)上,梯度洗脱对12种游离脂肪胺衍生物进行了优化分离。采用大气压化学电离源(APCI)正离子模式,实现了各种脂肪胺衍生物的测定。多数脂肪胺的线性回归系数大于0.9999,检测限为10.5～53.4fmol。     

2,6-Dimethyl-4-quinolinecarboxylic acid N-hydroxysuccinimide ester: a fluorogenic hydrophilic derivatizing reagent for liquid chromatographic analysis of aliphatic amines

The use of a fluorogenic, hydrophilic, and amine-reactive reagent, 2,6-dimethyl-4-quinolinecarboxylic acid N-hydroxysuccinimide ester (DMQC-OSu) has been investigated in the procolumn derivatization for the LC separation of aliphatic amines. In pH 8.0 aqueous medium, DMQC-OSu reacted with amines at 50 degrees C within 20 min to form highly fluorescent carboxamides and the excess reagent hydrolyzed to the corresponding carboxylic acid. The separation of representative amine derivatives with DMQC-OSu has been performed using a C18 column with the fluorescence detection at 326/409 nm. The detection limits reached nanomolar level. The proposed method has been applied to the analysis of real samples with recoveries of 94-108%. Compared with other succinimidyl esters used in the derivatization of amino compounds, DMQC-OSu and its hydrolysate had negligible fluorescence (phi(fl) = 0.09 and 0.02, respectively), which implied that small peaks appeared in chromatograms and slight interference was introduced to the determination.     

Sensitization of lanthanides by nonnatural amino acids

The sensitization of Eu(III) and Tb(III) by ethylenediaminetetraaceticacid (EDTA)-derivatized tryptophan (Trp), 7-azatryptophan (7AW) and 5-hydroxytryptophan (5HW) has been examined. These Trp analogs were utilized in the present study because they can be incorporated into proteins in place of native Trp residues and because they absorb strongly beyond 305 nm (where Trp absorbance goes to zero), allowing selective excitation of such species in the presence of other Trp-containing proteins. All three indole derivatives were able to sensitize Tb(III) luminescence, with the relative sensitization being in the order Trp > 5HW > 7AW. On the other hand, only the 7AW-EDTA complex was able to sensitize Eu(III) luminescence, likely owing to a better spectral overlap between 7AW emission and Eu(III) absorbance. The sensitized emission of Tb(III) and Eu(II) displayed the expected long emission lifetimes at 545 nm [for Tb(III)] and 617 nm [for Eu(III)], indicating that long-lifetime lanthanide emission could be produced using nonnatural amino-acid donors. Thus, 7AW- and 5HW-sensitized lanthanide emissions should prove to be useful in biophysical studies, such as the use of fluorescence energy transfer to probe biomolecular interactions in vivo.     

Determination of biogenic amines in food samples using derivatization followed by liquid chromatography/mass spectrometry

A liquid chromatography (LC)/mass spectrometry method was developed for the determination of selected biogenic amines in various fish and other food samples. It is based on a precolumn derivatization of the amines with succinimidylferrocenyl propionate under formation of the respective amides and their reversed-phase liquid-chromatographic separation with subsequent electrospray ionization mass-spectrometric detection. Deuterated putescine, cadaverine, and histamine are added prior to the derivatization as internal standards that are coeluted, thus allowing excellent reproducibility of the analysis to be achieved. Depending on the analyte, the limits of detection were between 1.2 and 19.0 mg/kg, covering between 2 and 3 decades of linearity. The limit of detection and the linear range for histamine are suitable for the surveillance of the only defined European threshold for biogenic amines in fish samples. Compared with the established ortho-phthalaldehyde (OPA)/LC/fluorescence method, the newly developed method allows an unambiguous identification of the biogenic amines by their mass spectra in addition to only retention times, a fivefold acceleration of the separation, and independency from the sample matrix owing to the isotope-labeled internal standards. Various fish, calamari, and salami samples were successfully analyzed with the new method and validated with an independent OPA/LC/fluorescence method.     

Fluorescence Detection in High Performance Liquid Chromatography

Abstract

The principles and applications of fluorescence detection and fluorescence introducing reagents and methods in HPLC are reviewed. The design and requirements for fluorescence detectors, flow cells and excitation sources and the conversion of non-fluorescent compounds into fluorescent products by pre-column and post-column derivatization reactions are discussed. For the applications the emphasis is on drug analysis, where possible in biological fluids (serum, urine, etc.). The last paragraphs are divided in a number of sections in which newly developed and some scarcely used reagents are mentioned shortly; a more complete treatment is given of the reagents and labels most frequently used in the derivatization of certain functional groups. In this discussion the methods of derivatization as well as the selectivity, stability, fluorescence behaviour of the reagents/labels and derivatives and the reaction conditions are included. An up-to-date survey of the applications of fluorescence detection in liquid chromatography (TABLE III, TABLE IV and TABLE V), ends this review paper.     

Capillary electrophoresis with a UV light-emitting diode source for chemical monitoring of native and derivatized fluorescent compounds

We report the utilization of a high power UV light-emitting diode for fluorescence detection (UV-LED-IF) in CE separations. CE-UV-LED-IF allows analysis of a range of environmentally and biologically important compounds, including PAHs and biogenic amines, including neurotransmitters, amino acids, proteins, and peptides, that have been derivatized with UV-excited fluorogenic labels, e.g., o-phthalic dicarboxaldehyde/beta-mercaptoethanol (OPA/beta-ME). The 365 nm UV-LED was used as a stable, low cost source for detection of UV-excited fluorescent compounds. UV-LED-IF was used with both zonal CE separations and MEKC. Native fluorescence detection of PAHs was accomplished with detection limits ranging from 10 nM to 1.3 microM. Detection limits for OPA/beta-ME-labeled glutamic acid and aspartic acid were 11 and 10 nM, respectively, for off-line labeling, and 47 and 47 nM, respectively, for on-line labeling, comparable to UV-laser-based systems. Analysis of OPA/beta-ME-labeled proteins and peptides was performed with 28 and 47 nM detection limits for BSA and myoglobin, respectively.     

SPIN-TRAPPING and ESR STUDIES OF THE DIRECT PHOTOLYSIS OF AROMATIC AMINO ACIDS, DIPEPTIDES, TRIPEPTIDES and POLYPEPTIDES IN AQUEOUS SOLUTIONS—III. TRYPTOPHAN and RELATED COMPOUNDS

Abstract— The UV photolysis of tryptophan (Trp) and Trp-containing peptides in aerated aqueous solutions has been studied by ESR and spin-trapping techniques using f-nitrosobutane as the spin-trap. The photolysis of Trp alone at 290 nm gave rise to the addition of the spin-trap to carbon 3 of the indole ring. A large ESR signal from the hydronitroxide spin-adduct was also observed revealing the formation of hydrated electrons. Generally, the photolysis of Trp-containing dipeptides generated the deamination radical of the N-terminal amino acid followed by addition to the spin-trap. In the case of lysyl-Trp, a deamination radical from the side chain of lysine was proposed. A sensitization experiment with Trp as sensitizer and glycine (Gly) as substrate led to the generation of the deamination radical of Gly. Most of the observed free radicals resulting from the photolysis of Trp-containing peptides can be explained in terms of hydrated electrons reacting with the carbonyl group followed by deamination of the N-terminus.     

Determination of multiple drugs of abuse in human urine using capillary electrophoresis with fluorescence detection

Methods for separation and determination of multiple drugs of abuse in biological fluids using capillary electrophoresis (CE) with native fluorescence and laser-induced fluorescence (LIF) detection are described herein. Using native fluorescence, normorphine, morphine, 6-acetyl morphine (6-AM), and codeine were analyzed by CE without any derivatization procedure and detected at an excitation wavelength of 245 nm with a cut-off emission filter of 320 nm, providing a rapid and simple analysis. The detection limits were in the range of 200 ng/mL. For a highly sensitive analysis, LIF detection was also examined using a two-step precolumn derivatization procedure. In this case, drugs extracted from human urine were first subjected to an N-demethylation reaction involving the use of 1-chloroethyl chloroformate (ACE-Cl) and then derivatized using fluorescein isothiocyanate isomer I (FITC) and analyzed by CE coupled to a LIF detector. Variables affecting this derivatization: yield of demethylation reaction, FITC concentration, reaction time and temperature, were studied. The estimated instrumental detection limits of the FITC derivatives were in the range of 50-100 pg/mL, using LIF detection with excitation and emission wavelengths of 488 nm and 520 nm, respectively. The linearity, reproducibility and reliability of the methods were evaluated. In addition, a comparison of the characteristics for both native fluorescence and LIF detections was also discussed.     

Nonaqueous capillary electrophoresis coupled with laser-induced native fluorescence detection for the analysis of berberine, palmatine, and jatrorrhizine in Chinese herbal medicines

LIF detection is one of the most sensitive detection methods for CE. However, its application is limited because the analyte is usually required to be derivatized with a fluorescent label. As a result, LIF is seldom used to analyze active ingredients in plants. In this work, we introduce a rapid, simple, and sensitive method of nonaqueous CE (NACE) coupled with laser-induced native fluorescence detection for the simultaneous analysis of berberine, palmatine, and jatrorrhizine. This method skillfully utilizes the native fluorescence of these alkaloids and requires no troublesome fluorescent derivatization. As these alkaloids can fluoresce to some degree, they were simply detected by a commercially available 488 nm Ar+ laser. The native fluorescence of the analytes was greatly enhanced by nonaqueous media. Compared with the reported UV detection method, much lower LOD was achieved (6.0 ng/mL for berberine, 7.5 ng/mL for palmatine, and 380 ng/mL for jatrorrhizine). This method was successfully applied to analyze berberine, palmatine, and jatrorrhizine in two Chinese herbal medicines, Rhizoma coptidis and Caulis mahoniae.     

Derivatization, stabilization and detection of biogenic amines by cyclodextrin-modified capillary electrophoresis-laser-induced fluorescence detection

o-Phthalaldehyde (OPA) derivatives of eight biogenic amines were stabilized at 5 degrees C by forming inclusion complexes with methyl-beta-cyclodextrin (MBCD). The derivatives were separated and detected by cyclodextrin-modified capillary electrophoresis (CE) with UV or laser-induced fluorescence (LIF) detection. Using a borate buffer, pH 9.0 consisting of ethanol and a mixture of negatively charged sulfobutylether-beta-cyclodextrin and neutral MBCD, baseline separation of the eight OPA derivatives was achieved within 25 min with high separation efficiencies. The detection limits (S/N=3) obtained by UV and LIF detection were determined to be 10 microM and 0.250 microM, respectively. Glutamic acid was added after the initial derivatization step to neutralize residual OPA which otherwise caused a significant interference, particularly when analysis was performed around the detection limit of the OPA derivatives. Important biogenic amines in fish, wine and urine were then derivatized and determined by CE-LIF. In the case of sole and rainbow trout, the results obtained were validated by an enzymatic assay using putrescine oxidase.