High performance liquid chromatography of glucose using a post-column reactor of immobilized enzyme followed by electrochemical detection (glucose-LCEC)

A sensitive and selective, reasonably fast method for the determination of glucose content has been developed. A glucose oxidase immobilized column was coupled to a small-size anion exchange column/borate buffer chromatograph. The hydrogen peroxide produced in the enzyme reaction was detected directly by an amperometric detector using a platinum working electrode. The detection limit was 0.03 ppm (1.5 x 10(-7) M, 3 pmol/injection). The linear dynamic range was three orders of magnitude at least. The system was stable and reproducible both in short- and long-term operation. The proposed method is suitable for analysis of complicated matrices of biological samples because of its good selectivity and sensitivity.     

High performance liquid chromatography of spermidine and spermine using a postcolumn reactor of immobilized polyamine oxidase (Aspergillus terreus) followed by electrochemical detection.

A highly sensitive and selective method for the determination of spermidine and spermine has been developed. A polyamine oxidase (Aspergillus terreus) immobilized column was used as a postcolumn reactor. The detection limit was 0.2 pmol/injection for both spermidine and spermine with a linear range of three orders of magnitude.     

高效液相色谱-柱后固定化酶反应器-电化学检测法分析大鼠脑微透析液中的乙酰胆碱和胆碱

 建立了用高效液相色谱分离－柱后固定化酶反应器酶解－电化学检测器检测酶解最终产物Ｈ２Ｏ２的方法，分析了麻醉和自由活动大鼠脑微透析液中乙酰胆碱（ＡＣｈ）和胆碱（Ｃｈ）的含量。至少在０．２～１００μｍｏｌ／Ｌ范围内ＡＣｈ和Ｃｈ的浓度与其响应的线性关系良好，它们的检测极限都可达５０ｆｍｏｌ。对高效液相色谱结合固定化酶反应器的分析方法作了简要的讨论。     

Analysis of alkyl organoiodide mixtures by high-performance liquid chromatography using electrochemical detection with a post-column photochemical reactor

A method for the analysis of six alkyl organoiodides (iodomethane, iodoethane, 1-iodopropane, 1-iodobutane, 1-iodopentane, 1-iodohexane) commonly found in acetic acid process was developed. In this method the target analytes were determined by high-performance liquid chromatography (HPLC) using a post-column photochemical reactor with electrochemical detection (ED) in less than 30 min. HPLC was performed in ODS C18 reversed-phase column (5 microm, 250 x 4.6 mm I.D.) under isocratic conditions with methanol-0.067 M acetate buffer (70:30, v/v), pH 6.2 as mobile phase at flow-rate 1.1 ml/min. Alkyl organoiodides, which are electrochemically inactive, were made oxidizable at potential of 120 mV after post-column irradiation with low-pressure mercury lamp in a knitted PTFE tube. The photoreactor was placedin an aluminum housing full of nitrogen in order to prevent from the interference of oxygen. The detection limit for most analytes was of the order of 1-2 microg/l. The HPLC-ED method with a post-column photochemical reactor has good precision and linearity and can be readily applied to the routine determination of alkyl organoiodides in real acetic acid samples.     

High-performance liquid chromatography of hydroxysteroids detected with post-column immobilized enzyme reactors

Assaying the low concentrations of steroid hormones in extracts of body fluids requires detectors that are both highly sensitive to the steroid and relatively insensitive to interfering compounds usually present in much higher concentrations. To explore the use of moderately specific enzymes in post-column reactors, we immobilized 3 alpha- and beta-hydroxysteroid dehydrogenase on controlled pore glass beads, 37 microns in diameter, and constructed 4.6-mm diameter reactor columns, 3-cm long, packed with one of the two kinds of these beads. Hydroxysteroids eluted from the analytical column were mixed with the coenzyme, nicotinamide adenine dinucleotide (NAD), before passing through the reactor. The effluent from the reactor was passed through the 70-microliters flow cell of a fluorometer in which the fluorescence of the NADH produced in the enzyme-catalyzed oxidation of the hydroxysteroid was monitored. At the conventional high-performance liquid chromatography flow-rates used, oxidation of the steroids was almost complete. The yield depended on both the residence time of steroids in the reactor column and the concentration of organic modifier in the reaction mixture. Maximal yield was obtained with buffer having a low organic solvent concentration and passing through the reactor slowly. In assays of mixtures of epimeric hydroxysteroids, the 3 alpha-hydroxysteroids were detected with the 3 alpha-hydroxysteroid dehydrogenase reactor; the beta-hydroxysteroids were not, confirming the specificity of the enzymatic detection. With the fluorometer used, picomole quantities of steroids could easily be distinguished from noise.     

Measurement of bromate in bread by liquid chromatography with post-column flow reactor detection

This method is suitable for the determination of bromate residues in a variety of baked goods. The peer-verified method trial was performed on white bread, multigrain bread, and coffee cake spiked with known levels of potassium bromate. The analytical portion is extracted with deionized water to remove bromate from the bulk of the baked product. The aqueous extract is carried through a series of steps to remove co-extractives that would interfere with the liquid chromatography (LC) in the determinative step or hasten the deterioration of the LC column. The extract is filtered before passing it through a reversed-phase solid-phase extraction (SPE) column and a cation-exchange column in the silver form to remove lipids and chloride, respectively. Ultrafiltration is then used to remove proteins with molecular weights of >30,000 daltons. Finally, a cation-exchange column in the sodium form is used to remove silver ions from the extract. The determinative step uses LC with a reversed-phase column and an ion-pairing agent in the mobile phase. Detection is based on the post-column reaction of bromate with o-dianisidine to form an oxidation product that is quantitated spectrophotometrically at 450 nm. Overall agreement between the submitting and peer laboratories was quite good. For bromate levels of 10-52 ppb, overall mean recoveries were 76.9 and 78.8% for the submitting and peer laboratories, respectively. The standard deviations were higher for the results of the peer laboratory, probably because of the generally higher level of baseline noise present in the chromatograms. The results demonstrate that the method provides adequate accuracy with low-fat as well as high-fat foods. Bromate at levels as low as 5 ppb (ng/g) can be detected with the method.     

Determination of Thiamethoxam residues in honeybees by high performance liquid chromatography with an electrochemical detector and post-column photochemical reactor

A method for the determination of Thiamethoxam in bee samples was set up by means of high performance liquid chromatography with an electrochemical detector and post-column photochemical reactor (HPLC-h nu-ED). Analytical method was based on a rapid sample extraction procedure with acetone, followed by chromatographic separation into a C18-RP column isocratically operated by 60 mM phosphate buffer/acetonitrile (75/25) mobile phase at pH 2.7. A photochemical reactor was used as a tool to verify and eventually quantify the presence of Thiamethoxam in the samples by distinguishing it from interference contribution. Detection was performed with a potential of 880 mV after a photoactivation with a 254 nm light. The least detectable dose was 0.002 mg kg(-1). Recovery rates ranged between 59.88 and 71.62%.     

Specific detection of acetyl-coenzyme A by reversed-phase ion-pair high-performance liquid chromatography with an immobilized enzyme reactor

A selective chromatographic detection system for the determination of acetyl-coenzyme A (CoA) is reported. The short-chain acyl-CoA thioesters were separated by reversed-phase ion-pair high-performance liquid chromatography (HPLC), and then acetyl-CoA was selectively detected on-line with an immobilized enzyme reactor (IMER) as a post-column reactor. Thio-CoA liberated enzymatically from acetyl-CoA was determined spectrophotometrically after reaction with Ellman's reagent in the reagent stream. The IMER with phosphotransacetylase had a substrate specificity sufficient to determine acetyl-CoA and was active and stable in the mobile phase containing methanol and the ion-pair reagent. The calibration graph was linear between 0.2 and 10 nmol, with a detection limit of 0.05 nmol. This HPLC system with detection by IMER allows the selective identification and determination of acetyl-CoA in a mixture of acetoacetyl-CoA and 3-hydroxy-3-methylglutaryl-CoA, which are difficult to separate with ion-pair HPLC.     

Chemiluminescence high performance liquid chromatography of corticosteroids using lucigenin as post-column reagent

A chemiluminescence high performance liquid chromatographic method for the determination of corticosteroids and tetrahydrocorticosteroids has been developed. Corticosteroids and their metabolites extracted from urine samples were separated using an ODS column and a mixture of methanol + water + 0.01 M sodium acetate solution (70:30:5) as eluent. The eluent from the column was mixed with the chemiluminescent solution containing lucigenin and Triton X-100 and a 0.28 M KOH solution by pumps and monitored by a chemiluminescence detector. No interference was encountered and the method is both precise and reproducible.     

Post-column derivatization in liquid chromatography using immobilized enzyme reactors and amperometric detection

The advantages to be gained by conducting enzyme assays of carbohydrates in liquid chromatography are discussed. The enzymes are contained in immobilized enzyme reactors and used in the post-column mode. The product formed in the reactor is selectively detected amperometrically at a chemically modified electrode mounted in a flow-through detector. Selected examples are given to illustrate the advantages obtained.     

Phenols removal by immobilized tyrosinase reactor in on-line high performance liquid chromatography

The development of an immobilized enzyme reactor (IMER) based on tyrosinase immobilized on aminopropyl-controlled pore glass (AP-CPG) for the removal of phenols from model aqueous solutions was reported. To elucidate the influence of the substrate nature, the apparent (V'max, K'm) and the inherent (Vmax, Km) Michaelis-Menten constants were determined by Lineweaver-Burk method and the external diffusional contributions on measured enzyme activities were removed by a graphical method. The dephenolization process was realized by recycling the phenol solutions through the bioreactor connected to a chitosan trap in order to remove the colored quinone-type products of the tyrosinase reactions. The results indicated that a complete removal of phenol derivatives in the range of 150-300 min, with the exception of 60% removal for phenol reached in 400 min, was obtained. The observed sequence: cresol > 4-methylcathecol > catechol > 4-Cl-phenol phenol was in accordance to the V'max/K'm values.     

Simultaneous determination of allopurinol and oxypurinol by liquid chromatography using immobilized xanthine oxidase with electrochemical detection

A novel liquid chromatographic method using an immobilized xanthine oxidase reactor and an electrochemical detector was developed for the simultaneous determination of allopurinol and oxypurinol in rat plasma, intestinal wash and bile. Xanthine oxidase was immobilized on 5-microns aldehyde silica (prepacked into a 2 mm x 10 mm cartridge) in a simple procedure. Allopurinol eluted from an analytical column was converted to oxypurinol in the enzyme reactor with the eluent as the reaction medium and detected with high selectivity using an amperometric detector with a glassy carbon electrode at the applied potential of +0.85 V. High specificity of the enzymatic reaction combined with selectivity of the electrochemical detection eliminated the need for an extensive sample preparation. The assay was linear in the range 15-500 ng/ml of rat plasma, intestinal wash and bile with a low limit of detection of 10 pg on-column (signal-to-noise ratio = 4) for both allopurinol and oxypurinol.     

Novel quartz flow-cell as a post-column photochemical reactor for high-performance liquid chromatography

The construction of a new post-column photochemical reactor with quartz flow cells in series for high-performance liquid chromatography (HPLC) is described. The performance of the new reactor was compared with a conventional open tubular PTFE coil reactor. The sensitivity, accuracy and precision obtained with both reactors are comparable. The new reactor has the obvious advantages of smaller cell volume as well as inertness and resistance to not only light and heat produced by the UV lamp, but also to organic solvents in the mobile phases, which results in greatly improved durability, reduced peak broadening and shorter chromatographic run times. Application of the new reactor to the fluorescence detection of DU-6859a, a new fluoroquinolone antimicrobial agent, in human serum is reported.     

Determination of cyclodextrins in biological fluids by high-performance liquid chromatography with negative colorimetric detection using post-column complexation with phenolphthalein

A rapid and sensitive high-performance liquid chromatographic method for the analysis of beta- and gamma-cyclodextrin in aqueous biological fluids such as plasma, urine, or tissue homogenate is described. The chromatographic system consists of a microBondapak Phenyl column as stationary phase and a mobile phase of water with 10% methanol. After post-column addition of an alkaline solution of phenolphthalein, negative colorimetric detection is used. The elution solvent and post-column reagent were mixed in a capillary tubing of 1.5 m (1.0 mm I.D.). Two methods of sample treatment are given, one for large (1.0 ml) and one for small (0.1 ml) sample volumes. Both methods were shown to be linear and reproducible. The detection limit for beta-cyclodextrin was 1.0 microgram/ml (0.77 nmol/ml). The method was used in the determination of some pharmacokinetic parameters of beta-cyclodextrin in rats after intravenous injection.     

Metallic copper-containing post-column reactor for the detection of thiram and disulfiram in liquid chromatography

A reaction detector has been developed for the selective detection of thiram and disulfiram. The detection is based on the post-column complexation of these analytes on a solid-state reactor packed with finely divided metallic copper to form a coloured copper complex, copper(II) N,N-dimethyldithiocarbamate, with an absorption maximum at 435 nm. The method is combined with a pre-concentration and clean-up step on a pre-column to permit the sub-ppb determination of, e.g., thiram in surface water samples or disulfiram in urine. Separation is achieved by reversed-phase liquid chromatography.     

Determination of tetraethyllead and tetramethyllead using high performance liquid chromatography and electrochemical detection

Summary A new method for the separation of tetramethyllead (TML) and tetraethyllead (TEL) was developed using high-performance liquid chromatography. The electrochemical detection was examined with different electrodes. Amperometric and pulse-amperometric techniques were investigated and the optimal working potential for each electrode was determined. Linearity for the glassy carbon electrode was observed between 350 ng and 30 g; the detection limit is 310 ng (TML) resp. 340 ng (TEL). In case of the mercury gold electrode the linearity range was 300 g–3 g and the detection limit 1.5 m (TML) resp. 1.7 g (TEL).     

Determination of catecholamines in urine using aminophenylboronic acid functionalized magnetic nanoparticles extraction followed by high‐performance liquid chromatography and electrochemical detection

A new method was developed for the simultaneous determination of three catecholamines in urine using aminophenylboronic acid functionalized magnetic nanoparticles extraction followed by high‐performance liquid chromatography with electrochemical detection. Novel aminophenylboronic acid functionalized magnetic nanoparticles were prepared by multi‐step covalent modification, and characterized by transmission electron microscopy, Fourier‐transformed infrared spectroscopy, X‐ray diffraction, and vibrating sample magnetometry. With the help of the high affinity between the boronate and cis‐diol group, the particles were used for the highly selective separation and enrichment of three major catecholamines, norepinephrine, epinephrine, and dopamine. Effects of the pH of the feed solution, the extraction time, the composition of the buffer solution, the amount of the magnetic particles, the elution conditions, and the recycling of aminophenylboronic acid functionalized magnetic nanoparticles were explored. Under the optimized conditions, 13–17‐fold enrichment factors were obtained. The linear ranges were 0.01–2.0 μg/mL for the studied analytes. The limits of detection and quantification were in the range of 2.0–7.9 and 6.7–26.3 ng/mL, respectively. The relative recoveries were in the range of 92–108%, with intraday and interday relative standard deviations lower than 6.8%. This method was successfully applied to analysis of catecholamines in real urine.