Fluorescence Derivatization Reagent Research Articles

Analysis and Evaluation of Fatty Acid in Rattan Tea by Highly Selective and Sensitive HPLC- FLD-MS Method Coupled with Pre-Column Fluorescent Labeling

Rattan tea regarded as one of the most promising dietary supplements for the prevention and treatment of many chronic diseases has gained extensive attention. Free fatty acids play an important role in human body as essential nutrients. Contents of 31 free fatty acids in Rattan Tea were firstly investigated using toluene-4-sulfonic acid 2-(naphthalene-1-yl-phenyl-amino)-ethyl ester as pre-column fluorescence derivatization reagent by RE-HPLC coupled with an atmospheric chemical ionization-mass spectrometry source (APCI/ MS). The results indicated that lower values of LOD (0.52-1.53 ng mL(-1) ) provided the guarantee for trace analysis and evaluation of free fatty acids. The experimental recoveries were in the range of 88.9-103.6 %. Results of the present study indicated the highest free fatty acids content of Rattan Tea was in April. Thirteen free fatty acids were found, including C18:2 omega 6, C18:3 omega 3, omega 6 and C20:4 omega 6 essential to life. The developed method exhibited powerful potential for the systematic detection of bioactive components of medicinal plants.

Determination of nitrofuran metabolites in shrimp by high performance liquid chromatography with fluorescence detection and liquid chromatography–tandem mass spectrometry using a new derivatization reagent

A high performance liquid chromatography with fluorescence detection (HPLC–FLD) method for the simultaneous determination of total nitrofuran metabolite residues (furazolidone, furaltadone, nitrofurantoin, and nitrofurazone) in shrimp was developed. The method involves the acid hydrolysis of protein-bound metabolites, followed by the derivatization of the freed metabolites with the new fluorescent derivatization reagent 2-hydroxy-1-naphthaldehyde (HN) and subsequent liquid–liquid extraction (LLE). Separation is achieved on a YMC-Pack Polymer C18 column under alkaline conditions, and the high fluorescence intensity of the derivatives at an emission wavelength Em=463nm (Ex=395nm) enables, for the first time, their simultaneous determination in shrimp at concentrations as low as 1μg/kg by HPLC–FLD. The method was validated using blank shrimp fortified with all four metabolites at 0.5, 1.0 and 2.0μg/kg. Recoveries were >87% with relative standard deviations of <8.1% for all four metabolites. Furthermore, the results obtained by HPLC–FLD were in very good agreement with those obtained by LC–MS/MS analysis.

A new fluorescent derivatization reagent and its application to free fatty acid analysis in pomegranate samples using HPLC with fluorescence detection

A new fluorescent labeling reagent has been developed for the determination of fatty acids (FAs) by HPLC with fluorescence detection. The derivatization conditions including the amount of derivatization reagent, temperature, and type of catalyst were investigated, the results indicated that the reaction proceeded within 30 min at 90°C in the presence of K2CO3 catalyst. The maximal yield was obtained with a four- to fivefold molar reagent excess. The derivatives exhibited strong fluorescence with an excitation maximum at λex = 245 nm and an emission maximum at λem = 410 nm. Twenty-five FA derivatives were well separated by RP-HPLC on a Hypersil BDS C8 column in combination with gradient elution. All FAs were found to give excellent linear responses with correlation coefficients >0.9992. The method gave a low LOQ of 0.85–5.5 ng/mL (S/N of 10). The developed method was employed to analyze free FAs (FFAs) composition in pomegranate samples without any purification. FFAs in samples were doubly identified by HPLC retention time and protonated molecular ion corresponding to m/z [M+H]+. This newly developed method allows a highly sensitive determination of trace FFAs from pomegranate and other foodstuffs.

Concerted derivatization and concentration method with dispersive liquid–liquid microextraction for liquid chromatographic analysis of 5-hydroxyindoles in human serum

We developed a concerted derivatization and concentration method based on dispersive liquid-liquid microextraction (DLLME) for the liquid chromatography (LC) determination of 5-hydroxyindoles (5-HIs; serotonin, 5-hydroxyindole-3-acetic acid, N-acetylserotonin, and 5-hydroxytryptohol). Concerted derivatization and concentration could be affected by adding a mixture of an ionic liquid (1-hexyl-3-methylimidazolium hexafluorophosphate, extraction solvent), methanol (disperser), and water containing fluorescence derivatization reagents [benzylamine and potassium hexacyanoferrate(III)] into the sample. The resulting sedimented phase was injected into a reversed-phase LC column using a mixture of acetonitrile and 250 mM acetate buffer (pH 4.3) as the mobile phase for gradient elution, and the derivatives obtained were fluorometrically detected at excitation and emission wavelengths of 345 nm and 452 nm, respectively. The derivatization (reagent concentrations and pH) and extraction (extraction and disperser solvent type) conditions were optimized simultaneously. The limits of detection of the 5-HIs were in the range of 0.08-0.33 nM. The method was validated for 10 and 50 pmol/mL human serum levels, and the recovery of 5-HIs was between 66% and 98%, within a relative standard deviation of 9.5%. The proposed method is well suited for the highly sensitive analysis of trace amounts of 5-HIs in human serum samples.

Determination of thiol compounds by HPLC and fluorescence detection with 1,3,5,7‐tetramethyl‐8‐bromomethyl‐difluoroboradiaza‐s‐indacene

Altered levels of thiols in biological fluids are considered to be an important indicator for several diseases. In this article, 1,3,5,7-tetramethyl-8-bromomethyl-difluoroboradiaza-s-indacene is proposed as a fluorescent derivatization reagent for the determination of thiols including glutathione, cysteine, N-acetylcysteine, and homocysteine by HPLC. Under the optimized derivatization and separation conditions, a baseline separation of all the four derivatives has been achieved using isocratic elution on an RP C(8) column within 26 min. With fluorescence detection at 505 and 525 nm for the excitation and emission, respectively, the LODs (S/N = 3) are from 0.2 nM (glutathione) to 0.8 nM (cysteine). The feasibility of this method in real samples has been evaluated by the determination of thiols in human plasma from the healthy persons and hypertensive patients with recoveries of 92-105.3%.

Fluorescein-core piperazine as a fluorescent labeling reagent for highly sensitive determination of jasmonic acid by high-performance liquid chromatography

In the present work, a pre-column fluorescent derivatization reagent, fluorescein-core piperazine (FCP) was prepared for labeling jasmonic acid (JA). Based on the enhanced fluorescence after derivatization, a simple and sensitive method was developed for determination of JA by high performance liquid chromatography with fluorescence detection (HPLC/FD). Several key derivatization conditions were examined. The results indicated that FCP labeled JA could be well separated from FCP in the sample within 13 min on a C18 reversed-phase column using a mobile phase of methanol–water (55 : 45, v/v) with fluorescence detection at excitation and emission wavelength of 507 nm and 526 nm, respectively. The detection limit obtained was 0.4 nM (signal to noise ratio of 3), which was lower than most of the existing methods. The method is reproducible and yields single peak for JA regardless of isomer. The specificity of the proposed method was confirmed by high-performance liquid chromatography-ion trap mass spectrometry (HPLC/IT-MS). Finally, it has been demonstrated that the proposed HPLC/FD method can be applied to the assay of JA in plant samples.

Amino acid analysis using core–shell particle column

In this study, the separation efficiency of a core-shell particle column was compared with particle-packed and monolithic silica columns, which showed that the core-shell particle column had a smaller theoretical plate height and that its separation efficiency was not affected significantly by the increase in flow rate. A fast HPLC method using a core-shell particle column was developed for the determination of amino acids. 4-Fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was used as a fluorescence derivatization reagent for amino acids, followed by separation on a core-shell Kinetex C18 column. The analysis time for 21 NBD-amino acids was within 7min, which was faster than that in our previous studies with conventional particle-packed columns or monolithic silica columns. The linearities of the calibration curves for all the amino acids were found to be good over a range of injection amounts from 40fmol to 40pmol. The accuracies for the amino acid determinations were 90.9-107%. The method was proved to have potential for the fast determination of amino acids in biological samples.

The Micro-Flow Reaction System Featured the Liquid–Liquid Interface Created with Ternary Mixed Carrier Solvents in a Capillary Tube

A micro-flow reaction system was developed in which liquid-liquid interface was created based on the tube radial distribution of ternary mixed carrier solvents. The system was constructed from double capillary tubes having different inner diameters (100 and 250 µm i.d.). The smaller tube was inserted into the larger one through a T-type joint. The reaction of a protein with a fluorescence derivatizing reagent was adopted as a model. A water-acetonitrile mixture (3:1 volume ratio) including bovine serum albumin (hydrophilic) was delivered into the large tube from the inside through the small tube and an acetonitrile-ethyl acetate mixture (7:4 volume ratio) containing fluorescamine (hydrophobic) as a derivatizing reagent was delivered from the outside through the joint. Solutions were mixed through the double capillary tubes to promote ternary mixed carrier solvents (water-acetonitrile-ethyl acetate; 1:2:1 volume ratio). The liquid-liquid interface was created based on the tube radial distribution of ternary solvents in the larger tube. The derivatization reaction was performed in the larger, or reaction, tube in the micro-flow system. The fluorescence intensity of the fluorescamine-derivatized bovine serum albumin obtained by the system, which specifically included the kinetic liquid-liquid interface in the tube, was greater than that obtained through a batch reaction using a homogeneous solution of water-acetonitrile (1:2 volume ratio).

Application of a Fluorescent Derivatization Reagent 9-Chloromethyl Anthracene on Determination of Carboxylic Acids by HPLC

A simple and sensitive high-performance liquid chromatography (HPLC) method is proposed for the analysis of some carboxylic acids in food samples and the environment. The use of 9-chloromethyl anthracene as a fluorescence-labeling reagent has been investigated. The derivatization reagent reacts with unitary carboxylic acids and tetrabutylammonium bromide as a catalyst within 50 min in acetonitrile to give esters, which can be separated by HPLC employing fluorescence detection at λ(ex) = 365 and λ(em) = 410 nm. The optimum conditions for derivatization, fluorescence detection and chromatographic separation are established. The method shows good sensitivity, with a detection limit from 0.18 to 2.53 pmol, and good linearity between 1-250 nmol/mL of each analyte. The practical applicability of the method was demonstrated by analyzing samples that were spiked with the acid standards, environment and food samples.

Rapid enantiomeric separation and simultaneous determination of phenethylamines by ultra high performance liquid chromatography with fluorescence and mass spectrometric detection: application to the analysis of illicit drugs distributed in the Japanese market and biological samples

A rapid enantiomeric separation and simultaneous determination method based on ultra high performance liquid chromatography (UHPLC) was developed for phenethylamine-type abused drugs using (R)-(-)-4-(N,N-dimethylaminosulfonyl)-7-(3-isothiocyanatopyrrolidin-1-yl)-2,1,3-benzoxadiazole ((R)-(-)-DBD-Py-NCS) as the chiral fluorescent derivatization reagent. The derivatives were rapidly enantiomerically separated by reversed-phase UHPLC using a column of 2.3-µm octadecylsilica (ODS) particles by isocratic elution with water-methanol or water-acetonitrile systems as the mobile phase. The proposed method was applied to the analysis of products containing illicit drugs distributed in the Japanese market. Among the products, 1-(3,4-methylenedioxyphenyl)butan-2-amine (BDB) and 1-(2-methoxy4,5-methylenedioxyphenyl)propan-2-amine (MMDA-2) were detected in racemic form. Furthermore, the method was successfully applied to the analysis of hair specimens from rats that were continuously dosed with diphenyl(pyrrolidin-2-yl)methanol (D2PM). Using UHPLC-fluorescence (FL) detection, (R)- and (S)-D2PM from hair specimens were enantiomerically separated and detected with high sensitivity. The detection limits of (R)- and (S)-D2PM were 0.12 and 0.21 ng/mg hair, respectively (signal-to-noise ratio (S/N) = 3).

Highly sensitive capillary electrophoresis analysis of N‐linked oligosaccharides in glycoproteins following fluorescence derivatization with rhodamine 110 and laser‐induced fluorescence detection

We describe a highly sensitive CE with laser-induced fluorescence (LIF) detection for the analysis of N-linked oligosaccharides in glycoproteins using rhodamine 110 as a fluorescence derivatization reagent. One CE separation is performed using a fused-silica capillary and neutral pH buffer conditions and allows for the separation of sialo-oligosaccharides according to the number of sialic acids. An alternate separation is performed using the same capillary and acidic pH buffer conditions, enabling the separation of asialo-oligosaccharides according to their sizes. The derivatization and separation conditions for the analysis of sialo- and asialo-oligosaccharides were optimized. Furthermore, we applied the proposed method for the analyses of N-linked sialo- and asialo-oligosaccharides in glycoproteins (ribonuclease B, fetuin, and recombinant human erythropoietin).

Development of Novel Fluorescence-Derivatization-HPLC Methods Enabling Highly Sensitive and Selective Analysis of Biological Compounds

Fluorescence-derivatization-HPLC methods are powerful tools for performing the analysis of bioactive compounds with high sensitivity and selectivity. In this paper, the author reviews the development of the following four types of novel fluorescence-derivatization-HPLC analytical systems: (1) simultaneous HPLC analysis of melatonin and its related compounds through post-column electrochemical demethylation and fluorescence derivatization, (2) HPLC analysis of 5-hydroxyindoles based on fluorescence derivatization by online pre-column photocatalytic oxidation with benzylamine, (3) reagent peak-free HPLC analysis for aliphatic amines and amino acids using F-trap pyrene as a fluorous tag-bound fluorescence derivatization reagent, and (4) reagent peak-free HPLC analysis for carboxylic acids using a fluorous scavenging-derivatization method. The authors have also successfully applied these systems to biological and pharmaceutical analyses.

4-Carbomethoxybenzaldehyde as a highly sensitive pre-column fluorescence derivatization reagent for 9,10-phenanthrenequinone

9,10-Phenanthrenequinone (PQ) is harmful environmental pollutant that is detected in airborne particulates. The measurement of PQ in the air should be necessary to evaluate the potential adverse effects of PQ on human health. We have recently developed a determination method for PQ based on the fluorescence derivatization of PQ using benzaldehyde and ammonium acetate as a reagent. In this study, in order to obtain more sensitive and selective fluorescence derivatization reaction, we measured the fluorescence of the reaction mixture of PQ with 21 kinds of aromatic aldehydes in the presence of ammonium acetate. Among the tested aldehydes, 4-carbomethoxybenzaldehyde was found to be the best reagent in regard to fluorescence intensity and emission wavelength maximum. Based on the fluorescence derivatization with 4-carbomethoxybenzaldehyde, a highly sensitive chromatographic method was developed for the determination of PQ with the detection limit (S/N=3) of 1.2 fmol/injection.

Determination of primary and secondary aliphatic amines with high performance liquid chromatography based on the derivatization using 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene

In this article, the simultaneous determination of primary and secondary aliphatic amines including dimethylamine (DMA), diethylamine and eleven primary aliphatic amines by high performance liquid chromatography (HPLC) with fluorescence detection has been achieved using a BODIPY-based fluorescent derivatization reagent, 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene (TMBB-Su). The derivatization reaction of TMBB-Su with aliphatic amines was optimized with orthogonal design experiment and the derivatization reaction proceeded at 15°C for 25min. The baseline separation of these derivatives was carried out on a C8 column with methanol–tetrahydrofuran-50mM pH 6.50 HAc–NaAc buffer (55/5/40, v/v/v) as a mobile phase. Detected at the excitation and emission of 490 and 510nm, respectively, the detection limits were obtained in the range of 0.01–0.04nM (signal-to-noise ratio=3). The proposed method has been applied to the determination of trace aliphatic amines in viscera samples from mice without complex pretreatment or enrichment method. The recoveries ranged from 95.1% to 106.8%, depending on the samples investigated.

ChemInform Abstract: Synthesis of Readily Accessible Bodipy‐Based Fluorescence Derivatization Reagents for Amines.

AbstractTwo highly sensitive borondipyrromethene‐based fluorescence derivatization reagents (FDRs) for amines are synthesized showing prompt reactivity, even with sterically hindered amines, to provide the FDR‐labeled products (III) in very good yields.

HPLC enantioseparation of α,α‐diphenyl‐2‐pyrrolidinemethanol and methylphenidate using a chiral fluorescent derivatization reagent and its application to the analysis of rat plasma

Enantioseparation of α,α-diphenyl-2-pyrrolidinemethanol (D2PM) and methylphenidate (MPH; Ritalin(®)) using (R)-(-)-4-(N,N-dimethylaminosulfonyl)-7-(3-isothiocyanatopyrrolidin-1-yl)-2,1,3-benzoxadiazole as the chiral derivatization reagent has been achieved for the first time, and a simple, reliable detection method using HPLC with fluorescence detection has been developed. D2PM and MPH have been derivatized with (R)-(-)-4-(N,N-dimethylaminosulfonyl)-7-(3-isothiocyanatopyrrolidin-1-yl)-2,1,3-benzoxadiazole at 55°C for 15 min. The derivatives of D2PM and MPH have been separated, completely and rapidly, using a reversed-phase system within 16 min (resolution factor (R(s))=1.60 and 2.53, respectively). The detection limits of (R)- and (S)-D2PM were found to be 6.8 and 13 ng/mL, respectively, and those of D- and L-threo-MPH were 61 and 66 ng/mL, respectively (S/N=3). The proposed method was successfully applied to the analysis of rat plasma, where the rats were separately dosed with D2PM and MPH (Ritalin).

Microwave-accelerated derivatization for capillary electrophoresis with laser-induced fluorescence detection: A case study for determination of histidine, 1- and 3-methylhistidine in human urine

The feasibility of microwave-accelerated derivatization for capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection was evaluated. The derivatization reaction was performed in a domestic microwave oven. Histidine (His), 1-methylhistidine (1-MH) and 3-methylhistidine (3-MH) were selected as test analytes and fluorescein isothiocyanate (FITC) was chosen as a fluorescent derivatizing reagent. Parameters that may affect the derivatization reaction and/or subsequent CE separation were systematically investigated. Under optimized conditions, the microwave-accelerated derivatization reaction was successfully completed within 150 s, compared to 4–24 h in a conventional water-bath derivatization process. This will remarkably reduce the overall analysis time and increase sample throughput of CE-LIF. The detection limits of this method were found to be 0.023 ng/mL for His, 0.023 ng/mL for 1-MH, and 0.034 ng/mL for 3-MH, respectively, comparable to those obtained using traditional derivatization protocols. The proposed method was characterized in terms of precision, linearity, accuracy and successfully applied for rapid and sensitive determination of these analytes in human urine.

HPLC purification and re-evaluation of chemical identity of two circular bacteriocins, gassericin A and reutericin 6

The study aimed for the complete purification and recharacterization of the highly hydrophobic circular bacteriocins, gassericin A and reutericin 6. Gassericin A and reutericin 6 were purified to homogeneity using previously described method and reverse-phase HPLC with an octyl column and eluents of aqueous acetonitrile and 2-propanol. Mass analysis, N-terminal sequencing and bacteriocin assay of the HPLC-purified bacteriocins showed the two bacteriocins had identical seamless circular structures with the same m/z value (5651) of [M + H](+) and both had the same specific activity. D/L-amino acid composition analysis using two distinct methods with the chiral fluorescent derivatization reagents (+)-1-(9-fluorenyl)ethyl chloroformate and O-phthalaldehyde/N-acetyl-L-cystein revealed neither gassericin A nor reutericin 6 contained D-alanine residues contrary to our previous results. Purified gassericin A and reutericin 6 are chemically identical circular molecules containing no D-alanine residues. The HPLC conditions developed in this study will facilitate advanced purification and correct characterization of other highly hydrophobic bacteriocins.

Synthesis of Readily Accessible BODIPY-Based Fluorescence Derivatization Reagents for Amines

Two BODIPY-based fluorescence derivatization reagents (FDRs) for amines were synthesized in a simple manner with practically acceptable yields via five steps. Both FDRs showed prompt reactivity, even against sterically hindered amines, to afford the corresponding FDR-labeled products in good yields. The solvent effect upon the fluorescence intensity of the derivatized amine was also investigated in a methanol-water binary solvent system to demonstrate that the strong fluorescence intensity was maintained in the solvent with a 0-60% water ratio. The detection limits of the new FDRs were estimated to be 20 fmol, which makes it much more sensitive than commercially available FDRs.

1,3,5,7-Tetramethyl-8-aminozide-difluoroboradiaza-s-indacene as a new fluorescent labeling reagent for the determination of aliphatic aldehydes in serum with high performance liquid chromatography

A BODIPY-based fluorescent derivatization reagent with a hydrazine moiety, 1,3,5,7-tetramethyl-8-aminozide-difluoroboradiaza-s-indacene (BODIPY-aminozide), has been designed for aldehyde labeling. An increased fluorescence quantum yield was observed from 0.38 to 0.94 in acetonitrile when it reacted with aldehydes. Twelve aliphatic aldehydes from formaldehyde to lauraldehyde were used to evaluate the analytical potential of this reagent by high performance liquid chromatography (HPLC) on C(18) column with fluorescence detection. The derivatization reaction of BODIPY-aminozide with aldehydes proceeded at 60 degrees C for 30min to form stable corresponding BODIPY hydrazone derivatives in the presence of phosphoric acid as a catalyst. The maximum excitation (495nm) and emission (505nm) wavelengths were almost the same for all the aldehyde derivatives. A baseline separation of all the 12 aliphatic aldehydes (except formaldehyde and acetaldehyde) is achieved in 20min with acetonitrile-tetrahydrofuran (THF)-water as mobile phase. The detection limits were obtained in the range from 0.43 to 0.69nM (signal-to-noise=3), which are better than or comparable with those obtained by the existing methods based on aldehyde labeling. This reagent has been applied to the precolumn derivatization followed with HPLC determination of trace aliphatic aldehydes in human serum samples without complex pretreatment or enrichment method.