Fluorescence Derivatization Reagent Research Articles

Enantiomeric determination of amines by high-performance liquid chromatography using chiral fluorescent derivatization reagents.

4-(2-carboxypyrrolidin-1-yl)-7-nitro-2,1,3-benzoxadiazole (NBD-Pro), 4-(2-carboxypyrrolidin-1-yl)-7-(N,N-dimethylamino-sulphonyl)-2,1,3 - benzoxadiazole DBD-Pro), 4-(N-1-carboxyethyl-N-methyl)amino-7-nitro-2,1,3-benzoxadiazole NBD-N-Me-Ala), 4-(N-1-carboxyethyl-N-methyl) amino-7-(N,N-dimethylamino-2,1,3-benzoxadiazole. (DBD-N-Me-Ala) have been synthesized for the resolution of enantiomers of amines by high performance liquid chromatography (HPLC). The reagents react with amino group at room temperature in the presence of activation agents, 2,2'-dipyridyl disulphide (DPDS) and triphenylphosphine (TPP) to produce the corresponding diastereomers. The derivatives were detected at lambda ex = 469, lambda em = 569 for DBD-moeity and lambda ex = 469, lambda em = 535 for NBD moeity. The resulting diastereomers were efficiently resolved using reversed-phase column with aqueous acetonitrile and aqueous methanol as the mobile phase. The elution order of the derivatives were D and L when proline was used as the chiral selector but the order was reversed when the diastereomers were prepared with the reagent containing N-methyl alanine as the chiral selector. DBD-Pro and NBD-Pro seem to give better separation as compared to DBD-N-Me-Ala and NBD-N-Me-Ala.

N-(4-nitro-2,1,3-benzoxadiazoyl-7-yl)- N-methyl-2-aminoacetohydrazide (NBD-CO-Hz) as a precolumn fluorescent derivatization reagent for carboxylic acids in high-performance liquid chromatography

A new fluorescent reagent for carboxylic acids, N-(4-nitro-2,1,3-benzoxadiazoyl-7-yl)- N-methyl-2-aminoacetohydrazide (NBD-CO-Hz) was synthesized and its applicability as a precolumn derivatization reagent in high-performance liquid chromatography was examined. NBD-CO-Hz reacted with 2-arylpropionic acids (2-APAs), a group of non-steroidal antiinflammatory drugs (NSAIDs) in the presence of a condensing agent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and pyridine at room temperature for 2 h to give fluorescent adducts. The reaction solution was subjected to a reversed phase or a chiral stationary phase HPLC and the derivatives were detected fluorometrically at a wavelength of 530 nm with an exitation of 475 nm. The detection limits were in the fmol range on column.

Enantiomeric determination of amines by high‐performance liquid chromatography using chiral fluorescent derivatization reagents

4-(2-carboxypyrrolidin-1-yl)-7-nitro-2,1,3-benzoxadiazole (NBD-Pro), 4-(2-carboxypyrrolidin-1-yl)-7-(N,N-dimethylamino-sulphonyl)-2,1,3-benzoxadiazole DBD-Pro), 4-(N-1-carboxyethyl-N-methyl)amino-7-nitro-2,1,3-benzoxadiazole NBD-N-Me-Ala), 4-(N-1-carboxyethyl-N-methyl) amino-7-(N,N-dimethylamino-2,1,3-benzoxadiazole. (DBD-N-Me-Ala) have been synthesized for the resolution of enantiomers of amines by high performance liquid chromatography (HPLC). The reagents react with amino group at room temperature in the presence of activation agents, 2,2′-dipyridyl disulphide (DPDS) and triphenylphosphine (TPP) to produce the corresponding diastereomers. The derivatives were detected at λ ex = 469, λ em = 569 for DBD-moeity and λ ex = 469, λ em = 535 for NBD moeity. The resulting diastereomers were efficiently resolved using reversed-phase column with aqueous acetonitrile and aqueous methanol as the mobile phase. The elution order of the derivatives were DL when proline was used as the chiral selector but the order was reversed when the diastereomers were prepared with the reagent containing N-methyl alanine as the chiral selector. DBD-Pro and NBD-Pro seem to give better separation as compared to DBD-N-Me-Ala and NBD-N-Me-Ala. © 1998 John Wiley & Sons, Ltd.

Simple and sensitive high-performance liquid chromatographic determination of methamphetamines in human urine as derivatives of 4-(4,5-diphenyl-1H-imidazol-2-yl) benzoyl chloride, a new fluorescence derivatization reagent

A simple, rapid and highly sensitive high-performance liquid chromatographic method for the determination of methamphetamine and its metabolites in human urine was developed. Without tedious pretreatment procedures, aliquots of methamphetamine spiked or abusers' urine samples were simply acidified, dried under a stream of N 2 gas, and then derivatized with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride in acetonitrile in the presence of traces of triethylamine. Five derivatives were isocratically separated within 33 min by an ODS column, and the effluent was monitored at 440 nm ( λ e x, 330 nm). Using 1-phenylethylamine as an internal standard calibration curves were confirmed to be linear up to at least 2×10 − 5 M in urine with correlation coefficients of 0.998–1.000, while the detection limits at S/ N=3 were 0.6–5.2 fmol per 5-μl injection. The relative standard deviations ( n=5) of inter- and intra-day variations were less than 8.9%. The correlation between the concentrations of methamphetamine and amphetamine in urine determined by the proposed method and another currently accepted one was satisfactory. The method was successfully applied to urine samples collected from methamphetamine addicts.

Determination of methamphetamine and related compounds by capillary electrophoresis with UV and laser-induced fluorescence detection

Methods for the simultaneous determination of methamphetamine (MP) and its related compounds (MPs) using capillary electrophoresis (CE) with UV absorbance and laser-induced fluorescence (LIF) detection are described. In UV detection, MPs were applied to CE without any derivatization procedure and detected at 210 nm for a rapid and simple analysis. Capillary zone electrophoresis (CZE) and electrokinetic capillary electrophoresis (MEKC) were used. MP, amphetamine (AP), 1-phenylethylamine (1-PA as an I.S.), 2-phenylethylamine (2-PA), 4-hydroxymethamphetamine (4-HMP) and 4-hydroxyamphetamine (4-HAP) were separated within 15 min by both CZE and MEKC. Detection limits of MPs were in the range 48–72 fmol/injection for CZE and 85–191 fmol/injection for MEKC. MEKC was successfully applied to the determination of MPs in urine. For a highly sensitive analysis, LIF detection was also examined using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) as a fluorescent derivatization reagent. By the method, in which MPs derivatives were separated within 45 min by MEKC, 22–40 amol/injection of primary amines (AP, 4-HAP and 2-PA) and 690 amol/injection of MP and 300 amol/injection of 4-HMP were detected. The concentration of MP and AP in 50 μl urine from MP addicts were successfully determined. A comparison of the characteristics for both UV and LIF detections was also discussed.

高感度キラル蛍光分析試薬開発の戦略

The strategies for the development of fluorescent derivatization reagents for highly sensitive analysis of biomolecules, for example AE-OTf for carboxylic acids, (S) -2A1P-OTf for chiral carboxylic acids having chiral center at the remote position from carboxyl group and (S) -TBME carboxylic acid for “on-line HPLC-exciton CD analysis” of vicinal diols, diamines, and amino alcohols are discussed.

4-(5',6'-Dimethoxybenzothiazolyl)phenylisothiocyanate as Sensitive Fluorescence Derivatization Reagent for Amines in High-Performance Liquid Chromatography.

4-(5',6'-Dimethoxybenzothiazolyl)phenylisothiocyanate as Sensitive Fluorescence Derivatization Reagent for Amines in High-Performance Liquid Chromatography.

4-(7-Diethylaminocoumarin-3-yl)benzoyl Cyanide (DACB-CN): A Highly Sensitive Fluorescent Derivatization Reagent for Alcohols in High-Performance Liquid Chromatography.

4-(7-Diethylaminocoumarin-3-yl)benzoyl cyanide (DACB-CN) was synthesized as a new fluorescent derivatization reagent for alcohols for use in high-performance liquid chromatography. Saturated alcohols (C8-C22) were derivatized in good yields to give the corresponding fluorescent DACB-esters by treatment with DACB-CN. The DACB-esters of these alcohols were clearly separated on a reversed-phase HPLC column. The detection limit (S/N=3) of cetyl alcohol, a test compound, was 1-2 fmol/10 μl.

Discrimination of d/l-mamino acid in peptide sequence based on fluorescent chiral derivatization by reversed-phase liquid chromatography

A fluorescent chiral derivatization reagent, 4-(3-isothiocyanatopyrrolidin-1-yl)-7-( N, N-dimethylaminosulfonyl)-2,1,3benzoxadiazole ( R(-)-DBD-PyNCS), was used for identification of d/l-amino acid in peptide sequence. The reagent reacted with N-terminal amino functional group in the peptide under the conditions of 65 °C for 60 min in 25% pyridine. The resulting thiocarbamoyl derivative of peptide was converted to thiohydantoin via thiazolinone at 55 °C for 1 min in trifluoroacetic acid (TFA). The maximum excitation and emission wavelengths of the derivative were around 460 and 550 nm, respectively. The racemization during the reactions of cleavage and cyclization was low judging from the chromatogram of the reaction solution. The thiohydantoin derivatives of N-terminal amino acid liberated from the peptides labelled were separated by reversed-phase chromatography with acetonitrile/phosphate buffer (pH 6.8) as the eluent. The separation of the thiohydantoins yielded from neutral and aromatic amino acids was good enough for the identification of d/l-amino acid. However, those from aspartic acid, glutamic acid, histidine, serine, proline and cystine were impossible or insufficient for resolution by the solvent composition. The peaks corresponding to d-amino acids were eluted faster than those from l-amino acids using R(-)-DBDPyNCS. The applicability of the proposed procedure to sequential analysis of peptide was demonstrated with [ d-Ala 2]-leucine-enkephalin. l-Tyr in first residue, d-Ala in second residue and Gly in third residue of the peptide were completely identified with the recommended method.

Enantiomeric separation of carboxylic acids having chiral centers remote from the carboxyl group by labelling with a chiral fluorescent derivatization reagent

Enantiomeric separations of 2-, 3-, 4-, 5- and 6-methyl fatty acids and 3-, 4- and 5-hydroxy fatty acids derivatized with ( S)-(+)-2-(anthracene-2,3-dicarboximido)-1-propyl trifluoromethanesulfonate are described. Although there are 4–8 bond distances between the chiral centers of these diastereomeric derivatives, they are separated on HPLC and detected at fmol levels.

Aromatic glycinonitriles and methylamines as pre-column fluorescence derivatization reagents for catecholamines

The reactivity of some catecholamines with aromatic glycinonitriles (AGN) (four species) and aromatic methylamines (AMA) (five species) was investigated in detail, to find pre-column fluorescence derivatization reagents for catecholamines. Of the nine reagents tested, 2-phenylglycinonitrile (PGN) and benzylamine (BA) were shown to be the best reagents in terms of selectivity and sensitivity. The reagents react selectively with catecholamines under mild conditions in the presence of ammonium molybdate and sodium periodate for PGN and potassium hexacyanoferrate(III) for BA to give fluorescent derivatives. The derivatives of four catecholamines (epinephrine (E), norepinephrine (NE), dopamine (DA) and isoproterenol (IP)) could be separated within 13 min by reversed-phase liquid chromatography with isocratic elution and measured fluorimetrically. The detection limits ( signal-to-noise ratio = 3) are in the range 5.2–11.0 fmol for PGN and 1.6–100 fmol for BA in a 50 μl injection volume. The liquid chromatographie (LC) methods with PGN and BA were successfully applied to the determination of some catecholamines in human urine.

4- N, N-Dimethylaminosulfonyl-7- N-(2-aminoethyl)amino-benzofurazan as a new precolumn fluorescence derivatization reagent for carboxylic acids (fatty acids and drugs containing a carboxyl moiety) in liquid chromatography

A new fluorescent reagent for carboxylic acids, 4- N, N-dimethylaminosulfonyl-7- N-(2-aminoethyl) amino-2, l,3-benzoxadiazole (DBD-ED) was synthesized from 4- N, N-dimethylaminosulfonyl-7-chloro-2,1,3-benzoxadiazole (DBD-Cl) and 1,2-diaminoethane. DBD-Cl is also a new compound described in this work for the first time. The applicability of DBD-ED as a precolumn derivatization reagent in liquid chromatography (LC) was examined. DBD-ED reacted with saturated fatty acids and non-steroidal anti-inflammatory drugs (NSAIDs) in the presence of triphenylphosphine and 2,2′-dipyridyl disulfide at room temperature for 30 min for NSAIDs and 1 h for fatty acids to give fluorescent products. The reaction solutions were subjected to LC and the derivatized products were detected spectrofluorimetrically at 560 nm with excitation at 450 nm. The detection limits were in the fmol range

Development and applications of a chemical method for sequential analysis of reducing oligosaccharides

A new method based on the chemical reaction has been devised for the sequential analysis of reducing oligosaccharides using 8-amino-2-naphthalenesulfonic acid (ANS), a fluorescent precolumn derivatization reagent for reducing saccharides. The procedure established includes 1) the derivatization of a reducing oligosaccharide to produce a Schiff base, 2) the reduction of the base with sodium cyanoborohydride (NaBH(3)CN), 3) the methoxycarbonylation of the resultant secondary amino group, 4) the cleavage of the glycoside bond next to the reducing end, based on the intramolecular acid hydrolysis by the action of a sulfonic acid group of the ANS derivative, 5) the identification of the liberated reducing end by high-per-formance liquid chromatography (HPLC), and finally 6) the recovery of the resultant oligosaccharide fragment from the cleavage reaction mixture. The extensive examination of the conditions for the sequential analysis of reducing oligosaccharides resulted in the procedure of simplicity, high selectivity and high recovery. This procedure was found to be useful for the sequential analysis of di-, tri- and tetrasaccharides.

3,4-Dimethoxybenzylamine as a sensitive pre-column fluorescence derivatization reagent for the determination of serotonin in human platelet-poor plasma

3,4-Dimethoxybenzylamine is shown to be a highly sensitive pre-column fluorescence derivatization reagent for the determination of serotonin in plasma by high-performance liquid chromatography. The reagent reacts selectively with 5-hydroxyindoles including serotonin in slightly alkaline media in the presence of potassium hexacyanoferrate(III) to give highly fluorescent derivatives. The derivatives of six standard 5-hydroxyindoles (5-hydroxytryptophan, 5-hydroxyindole-3-acetic acid, serotonin, 5-hydroxyindole-3-acetamide, N-acetyl-5-hydroxytryptamine and 5-hydroxytryptophol) are separated within 18 min by isocratic elution using acetonitrile–10 m M phosphate buffer (pH 6.0)–50 m M 1-hexanesulfonic acid on a Wakosil II 5C18RS reversed-phase column. The detection limits (signal-to-noise ratio=3) for the indoles were 1.0–5.7 fmol in a 100-μl injection volume. The method was applied to the measurement of serotonin in human platelet-poor plasma.

A Fluorometric Determination Method ford,lConfigurations of Per-O-Methylated Monosaccharides by Anomeric 2-Methyl-2-β-naphthyl-1,3-benzodioxole 4-Carboxylation and High-Performance Liquid Chromatography

Per-O-methylated pentopyranoses and hexopyranoses were converted to their glycosyl chlorides and coupled with cesium salt of a fluorescent chiral derivatization reagent, (+)-2-methyl-2-β-naphthyl-1,3-benzodioxole-4-carboxylic acid [(+)-MNB carboxylic acid], to afford their 1-O-(+)-MNB carboxylates. Thed,lenantiomers were separated by normal-phase HPLC and determined at the picomolar level, and this methodology could be extended to the highly sensitive simultaneous determination of per-O-methylated monosaccharides.

Highly Sensitive Isomeric Determination of Beraprost Sodium in Plasma Using a Fluorescent Chiral Derivatization Reagent.

(S)-(+)-2-(Anthracene-2,3dicarboximido)-1-propyl trifluoromethanesulfonate (AP-OTf) was synthesized as a chiral fluorescence derivatization reagent for carboxylic acids. Beraprost sodium (BPS), which is composed of four optical isomers, was derivatized with this reagent, and the derivatives were separated into each isomer by an HPLC using a silica gel column. This method was applied to determine BPS in human plasma. By the method, the determination range of the four isomers of BPS were 6 - 3000 fmol (0.5 -250 ng/ml) and their detection limits were 3-4 fmol (1.3-1.7 pg, signal-to-noise ratio =3). This method made it possible to detect 0.25-0.33 ng/ml of each isomer of BPS in plasma.

4-(5',6'-Dimethoxybenzothiazolyl)benzoyl fluoride and 2-(5',6'-dimethoxybenzothiazolyl)benzenesulfonyl chloride as sensitive fluorescence derivatization reagents for amines in high-performance liquid chromatography.

4-(5',6'-Dimethoxybenzothiazolyl)benzoyl fluoride (BHBT-COF) and 2-(5',6'-dimethoxybenzothiazolyl)-benzenesulfonyl chloride (BHBT-SOCI) have been developed as highly sensitive and selective fluorescence derivatization reagents for primary and secondary aliphatic amines in HPLC. These reactivities were investigated using n-propylamine, n-heptylamine and N-methylhexylamine as model compounds to optimize the derivatization conditions. Both reagents readily reacted with the amines in basic media to give the corresponding fluorescent derivatives, which were separated isocratically by reversed-phase (C18) liquid chromatography with aqueous methanol. The detection limits (signal-to-noise ratio = 3) of BHBT-COF for primary and secondary amines are approximately 3 and 30 fmol, respectively, and those of BHBT-SOCI are approximately 3 and 300 fmol, respectively, for an injection volume of 20 microliters. Both reagents gave no fluorescent derivatives for aromatic amines.

Determination of Amantadine in Human Plasma by High-Performance Liquid Chromatography with Fluorescence Detection.

A simple and highly sensitive fluorimetric high-performance liquid chromatographic (HPLC) method was developed for the determination of amantadine in plasma. After a single-step extraction from plasma (50μl) with toluene, the drug and 1-(1-adamantyl)ethylamine (internal standard) were converted into the corresponding fluorescent derivatives by reaction with 3, 4-dihydro-6, 7-dimethoxy-4-methyl-3- oxo-quinoxaline-2-carbonyl chloride, a fluorescence derivatization reagent for amines, in the presence of triethylamine. The derivatives were separated within 20min on a reversed-phase column (YMC- Pack C8) using isocratic elution with methanol-acetonitrile-water (35:35:30, v/v) and detected spectrofluorimetrically at 500nm with excitation at 400nm. The detection limit for amantadine added to plasma was 360fmol (54pg) ml-1 plasma (18fmol on column) at a signal-to-noise ratio of three.

Determination of amikacin in human plasma by high-performance capillary electrophoresis with fluorescence detection.

A selective and reproducible high-performance capillary electrophoretic (HPCE) method for the quantification of amikacin (AMK), an aminocyclitol antibiotic, in human plasma, has been developed for use in clinical laboratory tests. The method involves ultrafiltration (UF) of plasma before derivatization with the fluorescence derivatization reagent 1-methoxy-carbonylindolizine-3, 5-dicarbaldehyde at room temperature for 15 min in the dark. An aliquot of the derivatives is directly introduced into the fused-silica capillary [75 cm (effective length) x 50 microns I.D.] at the anode side by dynamic compression injection (50 hPa for 6 s). After electrophoresis with 40 mM SDS-20 mM phosphate-borate buffer (pH 7) in the micellar electrokinetic chromatography (MEKC) mode at 30 kV, the derivative had a retention time of 16.7 min and was detected by fluorescence intensity at 482 nm (with irradiation at 414 nm). The precision (n = 5) of the method is 4.08 and 1.59% (C.V.) at the 50 and 100 micrograms AMK/ml plasma levels, respectively. Linearity (r = 0.998) was established over the concentration range 5-100 mg of AMK/ml plasma and the detection limit (at a signal-to-noise ratio of 3) is 0.5 microgram AMK/ml plasma. This assay method could potentially have wider application in the determination of other aminocyclitol antibiotics, such as arbekacin, dibekacin, kanamycin, in human plasma as well as of AMK.

Recent progress in liquid chromatographic enantioseparation based upon diastereomer formation with fluorescent chiral derivatization reagents.

Techniques for the resolution by liquid chromatography of racemic compounds based upon diastereomer formation with a fluorescent chiral reagent are outlined in this review. The tagging reagents for various functional groups, i.e. amine, carboxyl, hydroxyl and thiol, are evaluated in terms of optical purity, handling, flexibility, stability, sensitivity and selectivity. The applicabilities of the reagents to drugs and biologically important substances are included in the text. This review is limited to reagents with fluorophores and reagents that exhibit fluorescence.