Sensitive determination of aliphatic amines by high-performance liquid chromatography with a new fluorogenic probe 3-(4-fluorinebenzoyl)-2-quinoline carboxaldehyde

A new fluorogenic reagent 3-(4-fluorinebenzoyl)-2-quinoline carboxaldehyde (FBQCA) has been synthesized and used as a derivatizing reagent for the determination of aliphatic amines with HPLC. The reagent is nonfluorescent, but forms highly fluorescent isoindole upon the reaction with primary amines in alkaline medium. Eleven amine derivatives were baseline separated in 8 min using a gradient elution on a C(8) column and detected with fluorescence detection at lambda(ex)/lambda(em) = 480/546 nm. The detection limits were in the range of 0.5-2 nM (S/N = 3). The proposed method has been successfully applied to the analysis of aliphatic amines in food and environmental samples, including white wine, soybean oil, soil, and tap water with satisfactory recoveries in the range of 94-106%.     

Separation of Amines as their 6-Methyl-2-phenyl-4-quinolinecarboxylic Acid N-Hydroxysuccinimide Ester Derivatives by High Performance Liquid Chromatography

6-Methyl-2-phenyl-4-quinolinecarboxylic acid N-hydroxysuccinimide ester (MPQC-OSu) has been developed for precolumn derivatization of aliphatic amines in HPLC. The great difference between the fluorescence quantum yield of MPQC-OSu derivative and that of MPQC-OSu hydrolysate, 6-methyl-2-phenyl-4-quinolinecarboxylic acid (MPQC) makes this reagent suitable for amine-labeling, followed by chromatographic separation. In pH8.5 borate buffer, MPQC-OSu reacted with amines at 60°C for 12 min to form highly fluorescent carboxamides and excess reagent hydrolysed to MPQC. The chromatographic behavior of amine derivatives with MPQC-OSu has been investigated using HPLC on C₁₈ and C₈ columns, respectively, with fluorescence detection at 340–402nm. A baseline separation of isopropanolamine, methylamine, ethylamine, n-propylamine, n-butylamine, n-amylamine and n-hexylamine was obtained in 25min using isocratic elution on a C₁₈ column using methanol-water (70:30, v/v) as eluent. Detection limits were in the range 0.13–1 nM.Acknowledgements This research was supported by the National Natural Science Foundation of China, the Foundation of Education Ministry of China and Chengguang Project of Wuhan, China.     

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 25 min. The baseline separation of these derivatives was carried out on a C₈ column with methanol-tetrahydrofuran-50 mM 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 510 nm, respectively, the detection limits were obtained in the range of 0.01-0.04 nM (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.     

6-Oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl)fluorescein as a new fluorescent labeling reagent for aliphatic amines in environmental and food samples using high-performance liquid chromatography

6-Oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl)fluorescein (SAMF), a new fluorescein-based amine-reactive fluorescent probe was well designed, synthesized and used as a pre-column derivatizing reagent for the determination of aliphatic amines in HPLC. It exhibited relatively pH-independent fluorescence (pH 4-9) and excellent photostability. The derivatization was performed at room temperature in 6min. On a C18 column, the derivatives of SAMF with eight aliphatic amines were baseline separated in 28 min with a mobile phase of methanol-water (57:43, v/v) containing 10 mmol l(-1) pH 5.0, H3Cit3-NaOH buffer. With fluorescent detection at lambda(ex)/lambda(em) = 484/516 nm, the detection limit could reach 2-320 fmol (signal-to-noise = 3), which was equivalent to or better than the detection limits obtained from other analytical methods of aliphatic amines. The proposed method has been applied to the determination of the aliphatic amines in environmental and food samples such as lake water, red wine, white wine, and cheese with satisfying recoveries varying from 95 to 106%.     

Separation and identification of aliphatic amine derivatives with a new fluorescent reagent using high-performance liquid chromatography and thin-layer chromatography

Summary The use of 1,2-naphthoylenebenzimidazole-6-sulphochloride has been proposed as the reagent for derivatization of aliphatic amines prior to their separation, identification and quantitation both in HPLC and in TLC. The reaction of amines with this compound is quantitative and highly fluorescent derivatives are formed that provide favourable detection limits and sensitivity as compared to Dansyl derivatives of aliphatic amines. Actual detection limits achieved correspond to ca. 10^–10 mol of the amine in a spot after elution from the thin-layer plate and to ca. 5·10^–14 mol of the amine in a sample volume of 10 l injected into the liquid chromatograph. The use of this derivatization reagent offers good potential for the analysis of trace amounts of amines in environmental samples and in biological material.Presented at the 14th International Symposium on Chromatography London, September, 1982     

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.     

Spectrofluorimetric determination of aliphatic amines using a new fluorigenic reagent: 2,6-dimethylquinoline-4-(N-succinimidyl)-formate

A new amino fluorescence probe, 2,6-dimethylquinoline-4-(N-succinimidyl) formate (DMQF-OSu) has been synthesized. Based on the selective reaction of DMQF-OSu with primary and secondary aliphatic amines to yield strong fluorescence, a new spectrofluorimetric method for the determination of total aliphatic amines has been developed. At λ_ex/λ_em=324.4/416 nm, the linear calibration range was 6×10⁻⁸-6×10⁻⁶ mol l⁻¹ with the detection limit (3σ) of 1.94×10⁻¹⁰ mol l⁻¹ for the determination of aliphatic amines in weak basic media. The proposed method has been applied to the determination of aliphatic amines in tap water and lake water with the recoveries of 99-104%. Compared with the reported methods, the method presented here is rapid, simple, sensitive and feasible.     

Fluorescence Probe of 10-Phenyl-acridone-2-sulfonyl Chloride and Its Application for Determination of Free Aliphatic Amines in Environmental Samples by HPLC with Fluorescence Detection and APCI-MS

A simple and sensitive method for the determination of free aliphatic amines using 10-phenyl-acridone-2-sulfonyl chloride (PASC) as a labeling reagent by high-performance liquid chromatography with fluorescence detection and online mass spectrometry identification (HPLC-FLD-MS) has been developed. Derivatization conditions including reagent concentration, buffer pH, reaction time and temperature were optimized. PASC reacted with aliphatic amines at 50 °C for 4 min in aqueous acetonitrile (ACN) in the presence of sodiumtetraborate–NaOH buffer (0.10 mol L⁻¹, pH 9.0) to give high yields of PASC-amine derivatives. Derivatives exhibited intense fluorescence with an excitation maximum at λ_ex 265 nm and an emission maximum at λ_em 418 nm. The separation of derivatives was performed by a reversed-phase Hypersil BDS C8 column in combination with a gradient elution. The identification of derivatives was carried out by online post-column mass spectrometry with atmospheric pressure chemical ionization (APCI) source in positive-ion detection mode. Excellent linear responses were observed with the correlation coefficients of larger than 0.9997, and detection limits (at a signal-to-noise of 3:1) were from 3.0 to 24.3 fmol. Comparing with 10-ethyl-acridine-2-sulfonyl chloride (EASC), PASC exhibited more intense fluorescence and ultraviolet absorbance. The proposed method is sensitive and reproducible for the determination of aliphatic amines from water and soil samples.

     

胶束电动毛细管色谱检测鱼肉中的七种生物胺

建立了一种利用胶束电动毛细管色谱同时检测鱼肉中组胺、腐胺、2-苯乙基胺、尸胺、色胺、亚精胺及精胺7种生物胺的方法。样品经6%过氯酸萃取后,由苯甲酰氯衍生化,以含0.06 mol/L脱氧胆酸钠的0.02 mol/L硼酸(pH 9.2)-甲醇(体积比为95∶5)混合液为电泳介质,电泳电压25 kV,温度25 ℃,检测波长214 nm,在12 min内实现了7种生物胺的完全分离。7种生物胺的浓度与其峰面积在一定的范围呈良好的线性关系,检出限除组胺为15 μg/g外,其余均为5 μg/g。迁移时间和峰面积的日内、日间相对标准偏差均小于5%。该法用于海鱼中7种胺类物质含量的测定,结果令人满意。     

Determination of thiol drugs in pharmaceutical formulations as their S-pyridinium derivatives by high-performance liquid chromatography with ultraviolet detection

Acetylcysteine and captopril can be determined in pharmaceutical formulations after precolumn derivatization of the thiol with 1-benzyl-2-chloropyridinium bromide (BCPB) by reversed-phase ion-pair HPLC separation and UV detection. The thiol group of the drugs reacts with BCPB in 0.1 mol/L phosphate buffer (pH 8) to form S-pyridinium derivatives showing an absorption maximum at 314 nm. The S-pyridinium derivative was separated isocratically on an Asahipak ODP-50 column at 45°C with 0.2 mol/L citrate buffer containing 10 mmol/L of sodium octanesulfonate (pH 2.5) and acetone (4:1, v/v). Calibration curves for both analytes were linear over the range of 2–20 μg/mL with variation coefficients of 3.12–1.28% for acetylcysteine and 9.22–1.51% for captopril.     

Application of 10‐ethyl‐acridine‐3‐sulfonyl chloride for HPLC determination of aliphatic amines in environmental water using fluorescence and APCI‐MS

A simple, sensitive method for the determination of aliphatic amines based on a sulfonylation reaction using 10‐ethyl‐acridine‐3‐sulfonyl chloride (EASC) as pre‐column labeling reagent with fluorescence detection and APCI‐MS identification has been developed. The labeled derivatives exhibited high stability and were enough to be efficiently analyzed by HPLC with an excitation maximum at λ_ex 270 nm and an emission maximum at λ_em 430 nm. Identification of derivatives was carried out by online post‐column MS in positive‐ion mode. Comparing with the widely used 5‐dimethylaminonaphthalene‐1‐sulfonylchloride (Dansyl‐Cl), EASC‐amine derivatives not only exhibited high fluorescence but also exhibited excellent MS ionizable potential. Detection limits obtained from 0.10 pmol injection, at a S/N of 3, were 4.0–12.7 fmol. The mean intra‐ and inter‐assay precision for all aliphatic amine levels were <3.84 and 3.21%, respectively. Excellent linear responses were observed with coefficients of >0.9995.     

Quantification of glutathione in plasma samples by HPLC using 4-fluoro-7-nitrobenzofurazan as a fluorescent labeling reagent

A rapid and highly sensitive high-performance liquid chromatograpy method with fluorescence detection has been developed for determination of glutathione (GSH) in human plasma. A simple pre-column derivatization procedure with 7-flouro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) reagent was employed. The separation of the derivatized glutathione was performed using a mobile phase consisting of phosphate buffer (0.02 mol/L, pH 6.0)-acetonitrile (77:23, v/v) at a flow rate of 1.0 mL/min with the column temperature 2°C. The eluted derivatives were fluorometrically detected at an excitation wavelength 470 nm and an emission wavelength 530 nm. Under the optimum chromatographic conditions, the calibration curve was linear over the range of 0.1 μmol/L to 10.0 μmol/L with the correlation coefficient of 0.9988. The precision of the method was satisfactory with the intra- and inter-day coefficient of variation being 6.3%, 6.9%, respectively. This method has been used to determine glutathione concentrations in plasma samples from healthy individuals.     

Simultaneous analysis of free amino acids and biogenic amines in honey and wine samples using in loop orthophthalaldeyde derivatization procedure

This work presents a RP-HPLC method for the simultaneous quantification of free amino acids and biogenic amines in liquid food matrices and the results of the application to honey and wine samples obtained from different production processes and geographic origins. The developed methodology is based on a pre-column derivatization with o-phthaldialdehyde carried out in the sample injection loop. The compounds were separated in a Nova-Pack RP-C(18) column (150 mm x 3.9 mm, 4 microm) at 35 degrees C. The mobile phase used was a mixture of phase A: 10 mM sodium phosphate buffer (pH 7.3), methanol and tetrahydrofuran (91:8:1); and phase B: methanol and phosphate buffer (80:20), with a flow rate of 1.0 ml/min. Fluorescence detection was used at an excitation wavelength of 335 nm and an emission wavelength of 440 nm. The separation and quantification of 19 amino acids and 6 amines was carried out in a single run as their OPA/MCE derivatives elute within 80 min, ensuring a reproducible quantification. The method showed to be adequate for the purpose, with an average RSD of 2% for the different amino acids; detection limits varying between 0.71 mg/l (Asn) and 8.26 mg/l (Lys) and recovery rates between 63.0% (Cad) and 98.0% (Asp). The amino acids present at the highest concentration in honey and wine samples were phenylalanine and arginine, respectively. Only residual levels of biogenic amines were detected in the analysed samples.     

Rapid, selective and direct spectrophotometeric determination of aliphatic amines with m-dinitrobenzene

Color reaction has been studied for identification and spectrophotometric determination of aliphatic amines at room temperature by m-dinitrobenzene (m-DNB) as reagent. The λ_max value ranges from 458 to 570 nm. This is a simple and rapid method for determination of aliphatic amines in the acidic, water and acetone medium. Beer's law is verified for methylamine, dimethylamine, trimethylamine and n-butylamine in the range of 0.5–8 mg l⁻¹. The effect of pH on the molar absorptivity is investigated for a representative primary amine i.e. methylamine and it was observed that molar absorptivity increases from acidic to basic pH, with a sharp increase at pH 12. The kinetic of reaction was also studied and found that reaction time has marked effect on the molar absorptivity of electron donor–acceptor (EDA) complex. The detection of methyl amine has been reported in three real samples of water.     

Determination of amino acid neurotransmitters in rat hippocampi by HPLC‐UV using NBD‐F as a derivative

A simple, rapid and accurate high‐performance liquid chromatography method with ultraviolet–visible detection was developed for the determination of five amino acid neurotransmitters – aspartate, glutamic acid, glycine, taurine and γ‐aminobutyric acid – in rat hippocampi with pre‐column derivatization with 4‐fluoro‐7‐nitrobenzofurazan. Several conditions which influenced derivatization and separation, such as pH, temperature, acetonitrile percentage mobile phase and flow rate, were optimized to obtain a suitable protocol for amino acids quantification in samples. The separation of the five neurotransmitter derivatives was performed on a C₁₈ column using a mobile phase consisting of phosphate buffer (0.02 mol/L, pH 6.0)–acetonitrile (84:16, v/v) at a flow rate of 1.0 mL/min with the column temperature at 30°C. The detection wavelength was 472 nm. Without gradient elution, the five neurotransmitter derivatives were completely separated within 15 min. The linear relation was good in the range from 0.50 to 500 µmol/L, and the correlation coefficients were ≥0.999. Intra‐day precision was between 1.8 and 3.2%, and inter‐day precision was between 2.4 and 4.7%. The limits of detection (signal‐to‐noise ratio 3) were from 0.02 to 0.15 µmol/L. The established method was used to determine amino acid neurotransmitters in rat hippocampi with satisfactory recoveries varying from 94.9 to 105.2%. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of trace biogenic amines with 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene derivatization using high-performance liquid chromatography and fluorescence detection

A reversed-phase high-performance liquid chromatographic method based on chemical derivatization with fluorescence detection has been developed for analyzing biogenic amines in food and environmental samples. A BODIPY-based fluorescent reagent, 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene (TMBB-Su), was employed for the derivatization of these biogenic amines at 20 °C for 20 min in pH 7.20 borate buffer after careful investigation of the derivatization conditions including reagent concentration, buffer solution, reaction temperature and reaction time. Separation of biogenic amines with gradient elution was conducted on a C8 column with methanol-tetrahydrofuran-water as mobile phase. The detection limits were obtained in the range from 0.1 to 0.2 nM (signal-to-noise=3). This procedure has been validated using practical samples. The study results demonstrated a potential of employing high-performance liquid chromatography (HPLC) with 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene labeling as a tool for quantitative analysis of biogenic amines involved in various matrices.     

A Simple Isocratic High-Performance Liquid Chromatographic (HPLC) Determination of Naproxen in Human Plasma using a Microbore Column Technique

Abstract

A simple and sensitive HPLC method was developed for the determination of naproxen in human plasma. The assay employs a microbore column packed with a C18 reversed-phase material (5 μm ODS Hypersil) with an isocratic mixture of acetonitrile and 10 mM phosphate buffer, pH 2.5 (40:60, v/v) as the mobile phase. The mobile phase was pumped at a flow rate of 0.5 ml/min. For sample analysis 200 μl of acetonitrile containing internal standard (flurbiprofen) was added to 100 μl of plasma. After centrifugation 10 mM phosphate buffer, pH 7.4 (200 μl) was added to the tube, then vortexed and centrifuged. The supernatant (20 μl) was injected onto the HPLC column. The chromatographic separation was monitored by a fluorescence detector at an emission wavelength of 350 nm with an excitation wavelength of 225 nm. The direct precipitation of plasma protein using acetonitrile gave a good recovery for both naproxen and the internal standard. The detection limit was 0.1 μg/ml for naproxen. The intra- and inter-assay coefficients of variation at different concentrations evaluated were less than 10%.     

Spectrofluorimetric Evaluation of Total Aliphatic and Aromatic Amines in Well Waters and Wastewaters

Abstract

A simple and rapid spectrofluorimetric method is described for the determination of aliphatic and aromatic amines on the basis of ammonia and aniline, respectively. Aromatic amines in samples were reacted at pH 5.5 with fluram immoblised on an Octadecylsilane Solid Phase Extraction (ODS-SPE) cartridge. The produced pyrrolinones were adsorbed on SPE and separated from the aliphatic amines. Analysis of these compounds was carried out by elution of SPE with 1 ml Tetrahydrofuran (THF) and determination of fluorescence intensity at excitation wavelength 400 nm and emission wavelength 475 nm. Aliphatic amines after passing from SPE were collected and reacted with fluram at pH 9.2, and extracted into dichloromethane at pH 3 and quantitated fluorimetrically.

Linear dynamic ranges and detection limits (LOD) were 1–20, 0.43 mg 1^?1 and 1-200, 0.39 μg 1^?1 for ammonia and aniline, respectively. The proposed method was successfuly applied for the evaluation of these compounds in local well waters and municipality wastewaters.     

Sensitive determination of biogenic amines by capillary electrophoresis with a new fluorogenic reagent 3-(4-fluorobenzoyl)-2-quinolinecarboxaldehyde

An effective approach was proposed to the derivatization of seven biogenic amines using 3-(4-fluorobenzoyl)-2-quinolinecarboxaldehyde (FBQCA) as a fluorogenic reagent. The sensitive determinations of these derivatives were achieved by micellar electrokinetic capillary chromatography (MEKC) with laser-induced fluorescence (LIF) detection. The derivatization and electrophoretic conditions have been optimized. A running buffer was composed of mixtures of 25 mM pH 9.5 boric acid, 25 mM SDS, and 27% ACN. At 25 °C and 22.5 kV, the baseline separation of the derivatives was accomplished in 13 min. The detection limit (S/N = 3) was found as low as 0.4 nM. The proposed method was validated by the linearity of two orders magnitude and correlation coefficient in the range 0.9969–0.9998. Also, the procedure was successfully applied to the determination of biogenic amines in soy sauce, fish and wine samples.     

Determination of aspartate and glutamate in rabbit retina using polymer monolith microextraction coupled to high-performance liquid chromatography with fluorescence detection

A simple, sensitive and low-cost method was developed for the determination of aspartate (Asp) and glutamate (Glu) in rabbit retina. Polymer monolith microextraction (PMME) using a poly(acrylamide–vinylpyridine–N,N′-methylene bisacrylamide) (AA–VP–Bis) monolithic column was combined with derivatization of Asp and Glu using 8-phenyl-(4-oxy-acetic acid N-hydroxysuccinimide ester)-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (TMPAB-OSu), and this was used to analyze the derivatives of Asp and Glu by high-performance liquid chromatography (HPLC) with fluorescence detection. The conditions for the derivatization and the subsequent extraction of Asp and Glu derivatives were optimized. The enrichment factors for the derivatives of Asp and Glu were found to be 14.1 and 14.7, respectively, by PMME. The limits of detection of Asp and Glu were 0.14 and 0.53 nmol/L, respectively. The precision and recovery were evaluated with spiked retina. The inter- and intraday relative standard deviations were less than 10%. The proposed method was successfully applied to the determination of Asp and Glu levels in rabbit retina samples with different stages of intraocular hypertension.