Intramolecular Excimer-forming Fluorescence Derivatization Research Articles

A highly sensitive and selective method for analysis of biomarkers of diisocyanate exposure in human urine by high-performance liquid chromatography with intramolecular excimer-forming fluorescence derivatization

Urinary aromatic diamines are exposure biomarkers of diisocyanate which are highly irritating to skin, mucosa and the upper respiratory system. In this study, a highly sensitive and selective high-performance liquid chromatography-fluorescence detection (HPLC-FLD) method was developed for determination of three aromatic diamines, 4,4′-methylenedianiline (MDA), 2,4-toluenediamine (4TDA) and 2,6-toluenediamine (6TDA) exposed to corresponding diisocyantes in human urine. The whole idea is based on an intramolecular excimer-forming fluorescence derivatization using 4-(1-pyrene)butanoyl chloride (PBC), followed by performing HPLC-FLD analysis.The resulting polypyrene-labeled derivatives of these aromatic diamines provided intramolecular excimer fluorescence at the emission wavelength of 475 nm wavelengths, which was shifted markedly to the higher emission wavelengths as compared with the wavelengths of the pyrene monomer. Under this condition, interferences such as amino acids and biogenic monoamines gave no peaks. It was found that using PBC derivatization, it was possible to improve the sensitivity and selectivity toward aromatic diamines. The limits of detection (LODs) were in the range of 0.30–0.52 ng/L, while the limits of quantitation (LOQs) between 1.05 and 1.63 ng/L. The intraday and interday precision was in the range of 3.6–4.5% and 4.1–5.7%, respectively. The proposed method was successfully applied to the quantitation of these exposure biomarkers in human urine samples.

Selective and Sensitive Determination of Pheomelanin in Biological Samples Using MEKC with Laser-Induced Fluorescence Detection Based on Intramolecular Excimer-Forming Fluorescence Derivatization

A highly selective micellar electrokinetic capillary chromatography (MEKC) method with laser-induced fluorescence (LIF) detection for the sensitive determination of pheomelanin in diverse biological materials was originally described. The derivatization reagent, 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), allowed for the selective detection of the two aminohydroxyphenylalanines (AHPs) markers for pheomelanin monitored at 500 nm. Multiple labeling of two AHPs with PSE allowed the formation of intramolecular excimers that emit at longer wavelengths (500 nm) than the mono-labeled analytes (360–420 nm) based on intramolecular excimer-forming fluorescence derivatization. Optimal separation of the labeled polyamines was achieved using a separation buffer consisting of 20 mM phosphate pH 7.4, 30 mM cholate, and 30% methanol. Using these conditions, the two AHPs were separated within 12 min, and the relative standard deviations (RSDs) were less than 1.5 and 1.6% (intra-run), 3.8 and 4.6% (inter-run, for a 6-day period) for the migration times and peak areas (n = 10), respectively. This method was successfully applied to the monitoring of pheomelanin in diverse biological samples with the spiked recoveries in the range of 94–101%. At a signal-to-noise ratio of 3, the detection limit for AHPs in the real samples was 31 pM for 3-AHP and 35 pM for 4-AHP, respectively, which are superior to those previously reported in the literature using fluorescence detection.

エキシマー蛍光誘導体化法による河川水中のビスフェノールＡの液体クロマトグラフ分析

Gas chromatography/mass spectrometry (GC/MS) is generally used for analysis of bisphenols in field environments. However, there are the cases that the sensitivity is not enough to determine their concentration. In this work, a liquid-chromatographic determination based on intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butyryl chloride was examined for the analysis of bisphenol A in river waters. This method is highly selective and sensitive fluorometric method for biogenic related compounds, polyamines, polyphenols and dicarboxylic acids though it has never been applied for analysis of bisphenols in field environments. With the method, we determined concentrations of bisphenol A in waters sampled form 6 rivers in Shizuoka Prefecture. As a result, the values determined were ranged from 0.0016-0.17μg/L. They agreed with those determined by conventional method with GC/MS.

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.

Quantification of glutaric and 3-hydroxyglutaric acids in urine of glutaric acidemia type I patients by HPLC with intramolecular excimer-forming fluorescence derivatization

Background Glutaric aciduria type I (GA1) is an autosomal recessive disorder that usually causes neurological damage. Early diagnosis of the disease prior to the appearance of clinical symptoms can lead to better outcomes. Methods We describe a simple and selective HPLC method with intramolecular excimer-forming fluorescence derivatization to diagnose GA1. Glutaric acid (GA) and 3-hydroxyglutaric acid (3HGA) in urine and an internal standard were derivatized with 1-pyrenebutyric hydrazide (PBH). The derivatives were separated on a C 18 column and fluorometrically detected at 475 nm (excitation of 345 nm) with a run time of 18 min. Results Excellent linearity over a wide range, reproducibility (coefficient of variation ≤ 14.5%), and sensitivity (limit of detection 0.4 μmol/l 3HGA and 0.2 μmol/l GA) were obtained. A retrospective study on previously diagnosed GA1 patients' urine from our laboratory archives between 1999 and 2004 was performed by analysts blinded to the study. Conclusions The method enabled us to differentiate GA1 cases ( n = 36) from controls ( n = 99), regardless of the years of urine storage. The method is valuable for both retrospective and prospective diagnoses of GA1.

A simple liquid chromatographic method based on intramolecular excimer-forming derivatization and fluorescence detection for the determination of tyrosine and tyramine in urine

A liquid chromatographic (LC) method for sensitive and selective fluorometric determination of p-hydroxyphenylethylamino group containing compounds is described. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butanoyl chloride, followed by reversed-phase LC. The analytes, containing an amino moiety and a phenolic hydroxyl moiety in a molecule, were converted to the corresponding dipyrene-labeled derivatives by one-step derivatization. The dipyrene-labeled 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 derivatives of tyrosine and tyramine could be separated by reversed-phase LC on ODS column under conditions of isocratic elution. The detection limits (signal-to-noise ratio = 3) for tyrosine and tyramine were 4.5 and 2.6 fmol per 20 μL injection, which corresponded to analyte concentrations of 0.9 and 0.5 nM, respectively.

Determination of methylmalonic acid in urine by HPLC with intramolecular excimer-forming fluorescence derivatization

We developed and validated an HPLC method with intramolecular excimer-forming fluorescence derivatization to determine methylmalonic acid, a unique biochemical marker for methylmalonic aciduria. Methylmalonic acid in urine and an internal standard were derivatized with pyrenebutyric hydrazide and separated on a C8 column. The derivatives were detected by monitoring the fluorescence at 475 nm (excitation wavelength 345 nm). At a signal-to-noise ratio of 3, the detection limit was 0.33 pmol on the column and the calibration curve was linear up to 1 mmol[sol ]L in urine. In a retrospective study on a relatively large number of known methylmalonic aciduria cases (n = 48), the method enabled us to differentiate methylmalonic aciduria cases from healthy controls (n = 52), regardless of age of patients at sampling or years of specimen storage. No interference was observed from isomeric or other dicarboxylic acids, or other urine constituents. As described, the method can be used retrospectively or prospectively for the diagnosis of methylmalonic aciduria and can be easily adopted by laboratories with no access to gas chromatography-mass spectrometry.

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.

Liquid chromatographic determination of polyamines in human urine based on intramolecular excimer-forming fluorescence derivatization using 4-(1-pyrene)butanoyl chloride.

A liquid chromatographic method for highly sensitive and selective fluorometric determination of polyamines (putrescine, cadaverine, spermidine and spermine) in human urine is described. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butanoyl chloride (PBC), followed by reversed-phase liquid chromatography. The method offers higher sensitivity for determination of spermidine and spermine than previously reported method utilizing 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester as a derivatization reagent. Samples containing free polyamines in diluted human urine were directly derivatized with PBC and separated on an octyl column. The derivatives were detected at excitation 345 and emission 475 nm wavelengths. For determination of total polyamine content, the conjugated polyamines were first hydrolyzed in 4 M HCl. The detection limits (signal-to-noise ratio = 3) for polyamines in urine were 1.1-3.4 pmol/mL. At optimized derivatization and chromatographic conditions, interferences such as biogenic monoamines gave no peaks or the peaks did not interfere with the peaks of polyamine derivatives. In conclusion, the present derivatization method allows direct determination of polyamines in human urine samples without the need for sample clean-up procedures.

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.

Liquid chromatographic determination of ethambutol in serum samples based on intramolecular excimer-forming fluorescence derivatization.

A selective liquid chromatographic method has been developed for the assay of ethambutol in serum samples. The assay involves intramolecular excimer-forming derivatization with 4-(1-pyrene)butanoyl chloride (PBC) and isocratic reversed-phase chromatography with fluorescence detection. After acetonitrile-deproteinization of the serum sample, the derivatization reaction of ethambutol with PBC was completed within 30 min at 50 degrees C. N,N'-Diethylethylenediamine was used as an internal standard. The detection limit of ethambutol was 23 ng/ml serum, corresponding to 180 fmol on column at a signal-to-noise ratio of 3. The present method was selective enough to analyze ethambutol in rabbit serum without any tedious sample clean-up procedure because biogenic monoamines gave no peak in the chromatogram. The method was applicable to drug monitoring in rabbit serum.

Liquid-chromatographic determination of halogenated bisphenols based on intramolecular excimer-forming fluorescence derivatization

A selective determination method for halogenated bisphenols containing tetrabromobisphenol A has been developed. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butanoyl chloride (PBC), followed by reversed-phase chromatography (LC). Bisphenols, containing two phenolic hydroxyl groups in a molecule, were converted to the corresponding dipyrene-labeled derivatives by a reaction with PBC. These derivatives afforded intramolecular excimer fluorescence (440∼540 nm), which could be clearly discriminated from the normal fluorescence (360∼420 nm) emitted from PBC and monopyrene-labeled derivatives. The PBC derivatives could be separated by reversed-phase LC on an ODS column with isocratic elution using aqueous 99% (v/v) acetonitrile as a mobile phase. The detection limits (signal-to-noise ratio = 3) for halogenated bisphenols were femto-mole levels for a 5 μl injection.

A sensitive and selective determination method of histamine by HPLC with intramolecular excimer-forming derivatization and fluorescence detection.

A highly sensitive, selective and simple method is described for the determination of histamine by high-performance liquid chromatography (HPLC) with fluorescence detection. The method is based on an intramolecular excimer-forming fluorescence derivatization of histamine with 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), followed by reversed-phase HPLC. Histamine, having two amino moieties in a molecule, was converted to the dipyrene-labeled derivative by reaction with PSE. The derivative afforded intramolecular excimer fluorescence (450-540 nm), which can clearly be discriminated from the monomer fluorescence (370-420 nm) emitted from PSE. Typically, a 10 micro L sample solution was mixed with 100 micro L of derivatization reagent solution, which was a mixture of 0.5 mm PSE in acetonitrile and 0.5 mm potassium carbonate in water (8:2, v/v). The derivatization was carried out at 100 degrees C for 90 min. The PSE derivative of histamine could be separated by reversed-phase ODS column with isocratic elution using acetonitrile:water (82:18, v/v) containing 0.03% triethylamine. The detection limit (singnal-to-noise ratio = 3) of histamine was 0.5 fmol for a 30 micro L injection. The method was successfully applied to the determination of histamine in human urine, and had enough selectivity and sensitivity for urinary histamine quantification.

Analyses of Biogenic Related Compounds Based on Intramolecular Excimer‐Forming Fluorescence Derivatization

AbstractFor Abstract see ChemInform Abstract in Full Text.

Analyses of biogenic related compounds based on intramolecular excimer-forming fluorescence derivatization

A highly selective and sensitive method based on a novel concept is introduced for the assay of biological substances. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, followed by reverse-phase HPLC. Polyamines, polyphenols, and dicarboxylic acids, which have two or more reactive functional groups in a molecule, were converted to the corresponding polypyrene-labeled derivatives by reaction with the appropriate pyrene reagent. The derivatives exhibited intramolecular excimer fluorescence (440-520 nm), which can clearly be discriminated from the monomer (normal) fluorescence (360-420 nm) emitted by pyrene reagents and monopyrene-labeled derivatives of monofunctional compounds. With excimer fluorescence detection, highly selective and sensitive determination of polyamines, polyphenols, and dicarboxylic acids can be achieved. Furthermore, the methods were successfully applied to the determination of various biological and environmental substances in real samples, which require only a small amount of sample and simple pretreatment.

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

A highly sensitive and selective fluorimetric determination method for dicarboxylic acids (C 5–C 12) 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-μl injection.

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

A highly selective and sensitive fluorometric method for the determination of bisphenols has been developed. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butyryl chloride (PBC), followed by reversed-phase liquid chromatography (LC). The bisphenols, containing two phenolic hydroxyl groups in a molecule, were converted to the corresponding dipyrene-labeled derivatives by reaction with PBC. The derivatives afforded intramolecular excimer fluorescence (440–520 nm) which can clearly be discriminated from normal fluorescence (360–420 nm) emitted from PBC and monopyrene-labeled derivatives of monophenols. The PBC derivatives of bisphenols could be separated by reversed-phase LC on an octyl column with isocratic elution. The detection limits (signal-to-noise ratio = 3) for bisphenols were 3.0–5.0 fmol, for a 20 μl injection. The method was successfully applied to the determination of bisphenol A in hot water in contact with commercially available baby bottle samples after solid-phase extraction.

Liquid chromatographic determination of triethylenetetramine in human and rabbit sera based on intramolecular excimer-forming fluorescence derivatization

A highly selective and simple fluorimetric liquid chromatographic method for the determination of triethylenetetramine (TETA), a therapeutic drug for Wilson’s disease, in human and rabbit sera is described. This method is based on intramolecular excimer-forming fluorescence derivatization, which allows spectrofluorometric discrimination of polyamino compounds from monoamino species, followed by liquid chromatography. TETA and 1,6-hexanediamine (internal standard) were converted to the corresponding excimer-forming derivatives with a pyrene reagent, 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester. The derivatives were separated within 20 min on a reversed-phase column using isocratic elution and detected spectofluorometrically at 480 nm with excitation at 345 nm. This method was successfully applied to the monitoring of TETA in human and rabbit sera with a simple pretreatment. The detection limit for TETA in serum was 18 ng/ml (0.13 nmol/ml) corresponding to 0.2 pmol on column at a signal-to-noise ratio of 3.

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.