Determination of 1-aminocyclopropane-1-carboxylic Acid in Apple and Peach Extracts by High Performance Liquid Chromatography Coupled to a Fluorescence Detector

A new, sensitive, and simple HPLC method was described for the determination of 1-aminocyclopropane-1-carboxylic acid (ACC) in apple and peach extracts. The method was based on the derivatization of ACC with fluorescamine in borate buffer systems of pH 8.0 to yield a highly fluorescent product. The experimental parameters affecting the derivatization reaction efficiency were optimized by fluorimetric analysis. Under optimum derivatization conditions, the derivative product of ACC in apple and peach extracts without extra purification was successfully chromatographed on a C-18 column by HPLC coupled to fluorescence detection. The derivative product of ACC with fluorescamine could be well separated from other concomitant substances or their derivatives that might interfere with the determination of ACC. The linearity of ACC was measured in the range of 23.82–238.82 µg · L^?1 with a good correlation coefficient of 0.9997. Based on signal-to-noise ratio of 3, a low detection limit of 5.0 µg · L^?1 could be reached. The proposed method was applied successfully to the determination of ACC in the crude apple and peach extracts without extra purification with low RSDs of 0.19–1.9% and good recoveries of 90.89–104.4%. The sensitive HPLC quantitative method is of great significance for the investigations of ACC metabolism in plants.     

Determination of 1-aminocyclopropane-1-carboxylic acid and its structural analogue by liquid chromatography and ion spray tandem mass spectrometry

Liquid chromatography coupled to ion spray tandem mass spectrometry was developed as a method for the simultaneous analysis of the amino acid 1-aminocyclopropane-1-carboxylic acid (ACC) and its structural analogue, cyclopropane-1,1-dicarboxylic acid (CDA). ACC and CDA fragmentation as well as optimization of MS parameters were investigated in positive ion mode. In selective reaction monitoring mode the protonated molecule [M+H]+ was selected as parent ion for both ACC and CDA, while the immonium ion from ACC and the [M+H-H2O]+ ion from CDA were selected, respectively, as product ions. In spite of the high selectivity of MS/MS among the 20 protein amino acids potentially present with ACC and CDA in the plant material analyzed, Glu and Thr can interfere with the signal of ACC. As a result, their chromatographic separation is necessary. This was achieved in less than 4 min by ion-pair reversed-phase chromatography with nonafluoropentanoic acid as ion-pair reagent. A linear response within a concentration range of 1-5 mg l(-1) was observed for this LC method and the detection limit was found to be 20 pmol for ACC and 150 pmol for CDA (using a 20-microl loop). This methodology was successfully applied to the detection of ACC in apple tissue.     

Qualitative analysis of some carboxylic acids by ion-exclusion chromatography with atmospheric pressure chemical ionization mass spectrometric detection

A simple, selective and sensitive method for the determination of carboxylic acids has been developed. A mixture of formic, acetic, propionic, valeric, isovaleric, isobutyric, and isocaproic acids has been separated on a polymethacrylate-based weak acidic cation-exchange resin (TSK gel OA pak-A) based on an ion-exclusion chromatographic mechanism with detection using UV-photodiode array, conductivity and atmospheric pressure chemical ionization mass spectrometry (APCI-MS). A mobile phase consisting of 0.85 mM benzoic acid in 10% aqueous methanol (pH 3.89) was used to separate the above carboxylic acids in about 40 min. For LC-MS, the APCI interface was used in the negative ionization mode. Linear plots of peak area versus concentration were obtained over the range 1-30 mM (r2=0.9982) and 1-30 mM (r2=0.9958) for conductimetric and MS detection, respectively. The detection limits of the target carboxylic acids calculated at S/N=3 ranged from 0.078 to 2.3 microM for conductimetric and photometric detection and from 0.66 to 3.82 microM for ion-exclusion chromatography-APCI-MS. The reproducibility of retention times was 0.12-0.16% relative standard deviation for ion-exclusion chromatography and 1.21-2.5% for ion-exclusion chromatography-APCI-MS. The method was applied to the determination of carboxylic acids in red wine, white wine, apple vinegar, and Japanese rice wine.     

Capillary electrophoresis-chemiluminescence determination of norfloxacin and prulifloxacin

A capillary electrophoresis (CE)–chemiluminescence (CL) method for determining norfloxacin (NFLX) and prulifloxacin (PFLX) was developed based on the enhanced CL intensity of the cerium(IV)–sulfite–fluoroquinolone (FQ) reaction sensitized by terbium(III). The separation was conducted in buffer composed of 20 mM sodium citrate, 4 mM citric acid and 10 mM sodium sulfite at pH 6.1. The CL reagent solution consisted of 2 mM cerium(IV), 4 mM terbium(III) and 1.1 mM hydrochloric acid. NFLX and PFLX were baseline separated within 11 min with detection limits (S/N = 3) of 0.057 and 0.084 μg mL⁻¹, respectively. The maximum intra- and inter-day relative standard deviations (R.S.D.s) of migration time of the analytes were less than 4.0% and 4.2%, respectively. The proposed method was applied to detect NFLX and PFLX in fortified urine sample and the results were comparable to high-performance liquid chromatography (HPLC)–UV method. Moreover, the high selectivity of the CL detection and the high-separation efficiency of CE render the method the potential of quick analyzing fluoroquinolones in real complex matrix.     

Quantitative trace-level speciation of arsenite and arsenate in drinking water by ion chromatography

We describe an improved method for the determination of inorganic arsenic in drinking water. The method is based on comprehensive optimization of the anion-exchange ion chromatographic (IC) separation of arsenite and arsenate with post-column generation and detection of the arsenate-molybdate heteropoly acid (AMHPA) complex ion. The arsenite capacity factor was improved from 0.081 to 0.13 by using a mobile phase (2.0 mL min(-1)) composed of 2.5 mM Na2CO3 and 0.91 mM NaHCO3 (pH 10.5). A post-column photo-oxidation reactor (2.5 m x 0.7 mm) was optimized (0.37 microM potassium persulfate at 0.50 mL min(-1)) such that arsenite was converted to arsenate with 99.8 +/- 4.2% efficiency. Multi-variate optimization of the complexation reaction conditions yielded the following levels: 1.3 mM ammonium molybdate, 7.7 mM ascorbic acid, 0.48 M nitric acid, 0.17 mM potassium antimony tartrate, and 1.0% (v/v) glycerol. A long-path length flow cell (Teflon AF, 100-cm) was used to measure the absorption of the AMHPA complex (818 +/- 2 nm). Figures of merit for arsenite/arsenate include: limit of detection (1.6/0.40 microg L(-1)): standard error in absorbance (5.1 x 10(-3)/3.5 x 10(-3)); and sensitivity (2.9 x 10(-3)/2.2 x 10(-3) absorbance units per ppb). Successful application of the method to fortified surface and ground waters (100 microL samples) is also described.     

Determination of folic acid by capillary electrophoresis with chemiluminescence detection

A rapid and simple method is presented for the determination of folic acid (FA) by capillary electrophoresis (CE) with chemiluminescence (CL) detection. This method was based on enhance effect of FA on the CL reaction between luminol and BrO(-) in alkaline aqueous solution. Optimal separation and determination was obtained with an electrophoretic buffer of 35 mM sodium borate (pH 9.4) containing 0.8 mM luminol, and an oxidizer solution of 1.6 mM NaBrO in 100 mM NaCO(3) buffer solution (pH 12.0). Under the optimal conditions, the determination of FA was achieved in less than 20 min, and the detection limit was 2.0 x 10(-8) M (S/N=3). The relative standard deviations (RSDs) on peak area and migration time were in the 1.5 and 1.1%, respectively. The present CE-CL method was applied to the determination of FA in commercial pharmaceutical tablets, apple juices and human urine.     

Extraction and quantitative analysis of 1-aminocyclopropane-1-carboxylic acid in plant tissue by gas chromatography coupled to mass spectrometry

We developed a new method for the determination of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) using quantitative GC-negative chemical ionisation MS as a detection and quantification system, in combination with isotope dilution using [2H4]ACC and an off-line solid-phase extraction. By derivatisation with pentafluorobenzyl bromide, ACC could easily be detected with m/z 280 being the most abundant ion. Determination of this component resulted in a detection limit of 10 fmol and a linear fit in the 100 fmol-100 pmol range. The combination of a rapid, high yield purification method with a stable derivatisation procedure and a sensitive detection method allowed the detection of ACC in samples as low as 100 mg fresh mass.     

High-performance liquid chromatographic determination of 1,2-diethyl-3-hydroxypyridin-4-one and its 2-(1-hydroxyethyl) metabolite in rat blood.

A sensitive and selective high-performance liquid chromatographic (HPLC) method for the analysis of 1,2-diethyl-3-hydroxypyridin-4-one (CP94, I) and its 2-(1-hydroxyethyl) metabolite (II) in rat blood is described. I, II and the internal standard, 1-propyl-2-ethyl-3-hydroxypyridin-4-one (CP95, III) were extracted into dichloromethane (3 x 5 ml, with the addition of 1 g of sodium chloride) from blood (0.25 ml plus 0.75 ml of pH 7.0 morpholinopropanesulphonic acid buffer). Extractability approached 100% for I and III, and approximately 65% for II under these conditions. Chromatographic analysis was carried out using a Hypercarb porous graphitised carbon HPLC column (10 cm x 0.46 cm). The mobile phase was 14:86 (v/v) acetonitrile-NaH2PO4 buffer (10 mM, containing 2 mM EDTA, pH adjusted to 3 with phosphoric acid) and detection was by ultraviolet at 280 nm. Calibration curves were linear (correlation coefficient greater than 0.99) and reproducible over the concentration range 0-80 micrograms/ml and the coefficient of variation was less than 16% even at low (1 microgram/ml) concentrations. The minimum quantifiable level was 0.5 microgram/ml for both I and II.     

Determination of fumaric acid in apple juice by on-line coupled capillary isotachophoresis-capillary zone electrophoresis with UV detection

An on-line coupled capillary isotachophoresis-capillary zone electrophoresis (cITP-CZE) method for the determination of the fumaric acid content in apple juice is presented. A clear separation of fumaric acid in real samples is achieved within 20 min. The leading, terminating and background electrolyte of the employed system consist of 10 mM HCl+beta-alanine+5 mM beta-cyclodextrin+0.05% hydroxypropylmethylcelullose (HPMC), pH 3, 10 mM citric acid and 20 mM citric acid+beta-alanine+5 mM beta-cyclodextrin+0.1% HPMC, pH 3.3, respectively. The linearity, recovery, repeatability and detection limit of the developed method are 25-1000 ng/ml, 1.07%, 95.4+/-3.5 (+/-s)% and 10 ng/ml, respectively. Low laboriousness (no sample pretreatment), sufficient sensitivity and low running cost are the important attributes of the cITP-CZE method which was successfully applied to analyses of real samples of apple juices.     

A simple and sensitive HPLC-fluorescence method for quantification of MDMA and MDA in blood with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a label

A sensitive high-performance liquid chromatographic method with fluorescence detection to determine 3,4-methylenedioxymethamphethamine (MDMA) and 3,4-methylenedioxyamphethamine (MDA) in human and rat whole blood or plasma samples was developed by using 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a label. MDMA and MDA in a small amount of blood sample (ca 100 microL) were extracted by liquid-liquid extraction with ethyl acetate, and were derivatized with DIB-Cl under mild conditions (10 min at room temperature). A good separation of DIB-derivatives could be achieved within 45 min using a commercially available ODS column with an isocratic eluent of 10 mM citric acid-20 mM Na(2)HPO(4) aqueous buffer (pH 4.0)-CH(3)CN-CH(3)OH (50:45:5, v/v/v %). The calibration curves prepared with 1-methyl-3-phenylpropylamine (MPPA) as an internal standard showed good linearity (r = 0.999) with 0.36-0.83 ng/mL detection limit at a signal-to-noise ratio of 3. MDMA and MDA in rat whole blood could be monitored for 6 h after a single administration of MDMA (2.2 mg/kg, i.p.). The pharmacokinetic parameters for MDMA and MDA obtained by triplicate measurements were 426 +/- 23 and 39 +/- 6 ng/mL (C(max)), 20 +/- 5 and 100 +/- 10 min (T(max)), respectively.     

Determination of phosphoamino acids by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection

A micellar electrokinetic capillary chromatography (MEKC) method with laser-induced fluorescence detection (LIF) was developed for analyzing three phosphoamino acids including phosphotyrosine (P-Tyr), phosphoserine (P-Ser), and phosphothreonine (P-Thr). 3-(2-Furoyl)quinoline-2-carboxaldehyde (FQ), a fluorogenic dye, was employed for derivatization of these phosphoamino acids. Results indicated that the complete baseline resolution of each phosphoamino acid was obtained within 10 min, using 20 mmol l⁻¹ sodium borate buffer (pH 9.35) containing 20 mmol l⁻¹sodium deoxycholate (SDC) and 10 mmol l⁻¹ Brij35. Other common amino acids, especially Glu and Asp, did not disturb the assay of these phosphoamino acids. There was a linear relationship between the peak area for analyte and its concentration, with correlation coefficients in the range of 0.9966-0.9996. The concentration detection limits (signal-to-noise = 3) for P-Tyr, P-Ser, and P-Thr were 10, 40, and 75 nmol l⁻¹, respectively. The developed method was successfully applied for determining phosphoamino acids in the hydrolysis sample of a phosphorylated protein kinase.     

Determination of atmospheric nitrobenzanthrones by high-performance liquid chromatography with chemiluminescence detection.

A method using high-performance liquid chromatography with chemiluminescence detection was developed for analyzing mutagenic nitrobenzanthrone (NBA) isomers in airborne particulates. The method was a modification of our previously described method for analyzing nitropolycyclic aromatic hydrocarbons (NPAHs). The pretreatment and reducing conditions for 1-, 2-, 3- and 10-NBAs were the same as those for NPAHs. In order to separate these NBA isomers, we used a polymeric-type ODS column (Cosmosil 5C-18MS); a mixture of 40% acetonitrile and 60% 10 mM imidazole-HClO4 buffer was employed as the mobile phase at a flow rate of 1 mL/min. The isomers of 1-, 2-, 3- and 10-NBA were determined in chemiluminescence with linear calibration graphs from 0.1 to 4 pmol, from 200 to 4000 pmol, from 1 to 50 pmol and from 10 to 400 pmol, respectively. The detection limits (S/N = 3) of 1-, 2-, 3- and 10-NBA isomers were 0.02 pmol, 35 pmol, 0.3 pmol and 3 pmol, respectively. The method was used to analyze airborne particulates at a heavy traffic site in Kanazawa. 2- and 3-NBAs were detected in the extracts of the particulates, while 1-NBA and 10-NBA were not detected. The atmospheric concentrations of 2- and 3-NBAs were 1.83 pmol/m3 and 24.7 fmol/m3, respectively.     

Labeling effects on the isoelectric point of green fluorescent protein.

We studied the effects of fluorescent labeling on the isoelectric points (pI values) of proteins using capillary isoelectric focusing with laser-induced fluorescence detection (cIEF-LIF). Specifically, we labeled green fluorescent protein (GFP) from the jellyfish Aequorea victoria with the fluorogenic dye 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ). cIEF-LIF was used to monitor the native fluorescence of GFP and showed pI changes in GFP's FQ-labeled products. Multiple labeling of GFP with FQ produced a series of products with pI values shifted towards a low pH. We verified cIEF-LIF results with traditional slab gel IEF. Our cIEF-LIF technique can routinely detect 10(-11) M of FQ-labeled protein, whereas traditional slab gel IEF with silver stain detection gives detection limits of 10(-7) M in the same samples.     

Measurement of nitrate and chlorate in swimming pool water by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) of nitrate and chlorate in swimming pool water are described. Nitrate and chlorate were determined simultaneously with an indirect detection method in an electrolyte containing 10 mM chromate and 0.1 mM cetyltrimethylammonium bromide (CTAB). Where chloride concentration was so high that nitrate could not be determined satisfactorily because of interference, a direct detection technology was developed in which 10 mM sulfate and 0.1 mM CTAB were used as the buffer. The wavelength for indirect detection was 254 nm and 214 nm for direct detection. Relative standard deviations of the quantification of nitrate and chlorate in real samples were below 6%. The detection limits were 7 mug ml(-1) for chlorate, and 4 mug ml(-1) (indirect detection) and 0.4 mug ml(-1) (direct detection) for nitrate.     

Application of matrix solid-phase dispersion to the determination of a new generation of fungicides in fruits and vegetables

A method based on matrix solid-phase dispersion (MSPD) and gas chromatography to determine eight fungicides in fruits and vegetables is described. Fungicide residues were identified and quantified using nitrogen-phosphorus detection and electron-capture detection connected in parallel and confirmed by mass spectrometric detection. The method required 0.5 g of sample, C18 bonded silica as dispersant sorbent, silica as clean-up sorbent and ethyl acetate as eluting solvent. Recoveries from spiked orange, apple, tomato, artichoke, carrot and courgette samples ranged from 62 to 102% and relative standard deviations were less than 15% in the concentration range 0.05-10 mg kg(-1). Detection and quantitation limits ranged 3-30 microg kg(-1) and 10-100 microg kg(-1), respectively, with linear calibration curves up to 10 mg kg(-1). The analytical characteristics of MSPD compared very favourably with the results of a classical multiresidue method, which uses ethyl acetate and anhydrous sodium sulphate for the extraction.     

Development of a method for quantitative analysis of the major whey proteins by capillary electrophoresis with on-capillary derivatization and laser-induced fluorescence detection

The main whey proteins have been derivatized on-capillary with 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) and analyzed using a laboratory-made capillary electrophoresis apparatus provided with a laser-induced fluorescence detector. Several parameters controlling on-capillary derivatization of proteins, including pH, mixing time, reaction time, concentration of the reagents (potassium cyanide and FQ), and reaction temperature, were optimized. Coefficient variations were lower than 1% for migration time and 7% for peak height. Assay detection limits for the different proteins were in the range 5 nM to 10 nM. The method developed was applied to the separation of the major whey proteins in a laboratory-made cheese whey and in an infant food formulated with milk. In addition, the beta-LG content of these samples was quantitated. The results showed good agreement with those given by an RP-HPLC method and with those reported in the literature.     

High-performance liquid chromatographic determination of 3-hydroxykynurenine with fluorimetric detection; comparison of preovulatory phase and postovulatory phase urinary excretion.

A high-performance liquid chromatographic assay for 3-hydroxykynurenine in human urine is described. A fluorescent derivative of 3-hydroxykynurenine was prepared, based on the reaction of the compound with p-toluenesulphonyl chloride in a basic medium. The analytical method for the measurement of the fluorescent compound employed a Tosoh ODS 80 column eluted with 10 mM potassium dihydrogenphosphate (pH 4.5) and acetonitrile (3:2, v/v) and detection at an excitation wavelength of 375 nm (10 nm bandpass) and an emission wavelength of 455 nm (10 nm bandpass). The column temperature was maintained at 25 degrees C. The detection limit was 3 pmol (673 pg) at a signal-to-noise ratio of 5:1. The fluorescent derivative of 3-hydroxykynurenine was eluted at ca. 12.5 min. The technique was applied to the analysis of human urine. The total analysis time was ca. 15 min.     

Analysis of dideoxyadenosine triphosphate by CE with fluorescence detection. I. Derivatization through the phosphate group

A CZE method was developed, which separates 2',3'-dideoxyadenosine-5'-triphosphate (ddATP) from other metabolites and endogenous nucleotides at high concentrations (20-200 microg/mL) to allow UV detection. To enhance sensitivity, fluorescence detection which requires prior derivatization of compounds was examined. Precapillary derivatization of ddATP in the presence of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC) with dansyl ethylenediamine (dansyl EDA) was faster and stable compared to that of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylenediamine (BODIPY FL EDA). Reaction conditions, reagent concentrations and detection parameters were optimized and highest derivatization efficiency was achieved in 0.1 M 1-methylimidazole buffer (pH 8.0) with 140 mM EDAC in 1-methylimidazole buffer and 30 mM dansyl EDA in DMF for 90 min at 60 degrees C. Dansyl EDA derivatives of ddATP, 2'-deoxyadenosine-5'-triphosphate (dATP) and ATP were comigrating with the CZE method; therefore, a MEKC method was developed and optimized for repeatable separations. Upon dansylation, sensitivity of ddATP with fluorescence detection (LOQ = 12 ng/mL) was 160 times higher than UV detection (LOQ = 1.9 microg/mL).     

Simultaneous micro-determination of nicotinamide and its major metabolites, N1-methyl-2-pyridone-5-carboxamide and N1-methyl-4-pyridone-3-carboxamide, by high-performance liquid chromatography

A simultaneous micro-determination of nicotinamide and its major metabolites, N1-methyl-2-pyridone-5-carboxamide (2-py) and N1-methyl-4-pyridone-3-carboxamide (4-py) by high-performance liquid chromatography is described. The method employs a 7-ODS-L (250 mm X 4.6 mm I.D., particle size 7 microns) column eluted with 10 mM potassium dihydrogenphosphate-acetonitrile (96:4, v/v; pH adjusted to 3.0 by the addition of concentrated phosphoric acid) at a flow-rate of 1.0 ml/min. The UV detector was set at 260 nm. The detection limits for nicotinamide, 2-py and 4-py were 10 pmol (1.22 ng), 2 pmol (304 pg) and 2 pmol (304 pg), respectively, at a signal-to-noise ratio 5:1. Isonicotinamide was used as an internal standard. The technique was applied to the analysis of rat and human urines. The total analysis time was ca. 15 min.     

Pre-concentration of trace amounts of manganese in water samples based on (1-(2-pyridylazo)-2-naphthol) modified magnetic nanoparticles

A simple procedure based on magnetic nanoparticles has been developed for analytical purposes. In this method, 1-(2-pyridylazo)-2-naphthol (PAN)-modified magnetic nanoparticles (MNPs) were used for separation and pre-concentration of manganese(II) ions from aqueous samples. This method combines the use of a solution solvent with separation of magnetic nanoparticles from sample solution using a magnet. The influence of different parameters, such as amount of extractant (PAN) loaded on the nanoparticles, pH of solution, adsorption time, amount of modified nanoparticle, type and amount of eluents for desorption of manganese from magnetic nanoparticles were evaluated. The effect of various cationic and anionic interferences on the percentage recovery of manganese was also studied. Manganese ions were adsorped from a solution at pH 9.5 and desorbed from nanoparticles with 10?mL of DMSO?:?HNO₃ (1?:?1, v/v). The detection limit of the proposed method was found to be 0.11?µg?L^?1. The method was employed to recover and determine the level of manganese in different water samples.