A high-performance liquid chromatography assay of brain adenylate cyclase using [3H]ATP as substrate

A sensitive, reproducible assay for adenylate cyclase is described which separates labeled cyclic AMP from ATP and other nucleotides by high-performance liquid chromatography (HPLC) on reverse-phase columns. The technique utilizes [³H]ATP as substrate, and the principal compound contaminating the [³H]cyclic AMP peak, adenosine, is removed by incubation of assay tubes with small amounts of adenosine deaminase. The HPLC elution utilizes high resolution (3 m) short (10 cm) C-18 columns for increased resolution and decreased flow rates. Since cyclic AMP elutes at 4 min following injection, this procedure can easily process large numbers of samples per day when combined with automated techniques of sample injection and collection.     

Chromatographic quantitation of territrems A,B, and C,tremorgenic mycotoxins from Aspergillus terreus

This paper describes assay procedures for territrems A, B, and C by thin layer chromatography (TLC)-fluorodensitometry and reverse phase high performance liquid chromatography (HPLC).     

Effect of tortilla-preparation process on aflatoxins B1 and B2 in corn

Naturally contaminated corn containing 450 and 54 ppb aflatoxins Bi and B2, respectively was treated with Ca(OH)₂ for making tortillas. The cleaned corn and tortillas were analyzed for aflatoxins B1 and B2 by high performance liquid chromatography (HPLC) and confirmed by thin layer chromatography (TLC). The average concentrations of aflatoxins B1 and B2 in the final products (tortillas) were only 40% and 28% lower than that in starting materials (corn kernels), respectively. Aflatoxins G1 and G2 were not detected in either corn or tortilla samples.     

A heterogeneous kinetic model for the cutinase-catalyzed hydrolysis of cyclo-tris-ethylene terephthalate

The kinetics of enzyme-catalyzed hydrolysis of the polyester oligomer cyclo-tris-ethylene terephthalate, commonly known as cyclic trimer, using a developmental cutinase is reported. The effect of substrate surface area and enzyme concentration, in a largely aqueous medium, on the rate of hydrolysis was measured via spectrophotometric measurement using high performance liquid chromatography (lambda 254 nm) at 60 degrees C in a glycine buffer (pH 8). The rate was strongly dependent on the substrate's surface characteristics. When the substrate surface area was relatively small and the substrate was relatively low in crystallinity, the reaction followed zero order kinetics, whereas a first order rate constant was obtained when the substrate surface area was increased considerably and the crystallinity was relatively high.     

Fumonisin B1: Isolation from corn culture,and purification by high performance liquid chromatography

A method is described to isolate fumonisin B₁ (FB₁) from corn cultured for 18 days at 25°C withFusarium moniliforme. Cultured corn was extracted with aqueous methanol and purified with XAD-2 column chromatography and high performance liquid chromatography (HPLC). About 450 mg of FB₁ were obtained from 800g cultured corn. Its identity was established by fast-atom bombardment (FAB) mass spectrometry, and infrared spectrum and nuclear magnetic spectrum. Its purity was estimated to be 95% by gas chromatography/mass spectrometry (GC/MS).References     

Auxin levels in single half nodes ofAvena fatua estimated using high performance liquid chromatography with coulometric detection

A high performance liquid chromatography (HPLC) based micro-method for estimation of indole-3-acetic acid (IAA) levels in single half nodes from the flowering stalks ofAvena fatua has been developed; this features a dual electrode coulometric electrochemical detector operating at a detection limit of c. 2 pg. Samples were prepared by solvent partitioning and preliminary fractionation with C¹⁸ Sep-Pak cartridges. Two stages of reversed phase ion pair HPLC were employed; the first was gradient elution with fluorescent detection, the second, isocratic elution with coulometric detection. The lower limit for estimation of IAA levels in purified extracts was c. 5 pg.     

Immobilized horse liver alcohol dehydrogenase as an on‐line high‐performance liquid chromatographic enzyme reactor for stereoselective synthesis

Horse liver alcohol dehydrogenase (HLADH) has been non‐covalently immobilized on an immobilized artificial membrane (IAM) high‐performance liquid chromatography (HPLC) stationary phase. The resulting IAM‐HLADH retained the reductive activity of native HLADH as well as the enzyme's enantioselectivity and enantiospecificity. HLADH was also immobilized in an IAM HPLC stationary phase prepacked in a 13 × 4.1 mm ID column to create an immobilized enzyme reactor (HLADH‐IMER). The reactor was connected through a switching valve to a column containing a chiral stationary phase (CSP) based upon p‐methylphenylcarbamate derivatized cellulose (Chiralcel OJR‐CSP). The results from the combined HLADH‐IMER/CSP and chromatographic system demonstrate that the enzyme retained its activity and stereoselectivity after immobilization in the column and that the substrate and products from the enzymatic reduction could be transferred to a second column for analytical or preparative separation. The combined HLADH‐IMER/CSP system is a prototype for the preparative on‐line use of cofactor‐dependent enzymes in large‐scale chiral syntheses. Chirality 11:39–45, 1999. © 1999 Wiley‐Liss, Inc.     

Generic HPLC platform for automated enzyme reaction monitoring: Advancing the assay toolbox for transaminases and other PLP‐dependent enzymes

Methods for rapid and direct quantification of enzyme kinetics independent of the substrate stand in high demand for both fundamental research and bioprocess development. This study addresses the need for a generic method by developing an automated, standardizable HPLC platform monitoring reaction progress in near real‐time. The method was applied to amine transaminase (ATA) catalyzed reactions intensifying process development for chiral amine synthesis. Autosampler‐assisted pipetting facilitates integrated mixing and sampling under controlled temperature. Crude enzyme formulations in high and low substrate concentrations can be employed. Sequential, small (1 µL) sample injections and immediate detection after separation permits fast reaction monitoring with excellent sensitivity, accuracy and reproducibility. Due to its modular design, different chromatographic techniques, e.g. reverse phase and size exclusion chromatography (SEC) can be employed. A novel assay for pyridoxal 5'‐phosphate‐dependent enzymes is presented using SEC for direct monitoring of enzyme‐bound and free reaction intermediates. Time‐resolved changes of the different cofactor states, e.g. pyridoxal 5'‐phosphate, pyridoxamine 5'‐phosphate and the internal aldimine were traced in both half reactions. The combination of the automated HPLC platform with SEC offers a method for substrate‐independent screening, which renders a missing piece in the assay and screening toolbox for ATAs and other PLP‐dependent enzymes.     

Purification of a 51 kDa endo-β-N-acetylglucosaminidase from Staphylococcus aureus

A bacteriolytic enzyme obtained from the culture fluid of Staphylococcus aureus FDA 209P was purified to homogeneity utilizing dye-ligand affinity column chromatography, hydrophobic interaction high pressure liquid chromatography (HPLC) and hydroxyapatite HPLC. Subsequent characterizations indicated that the purified enzyme acted as endo-beta-N-acetylglucosaminidase. The molecular weight determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was 51,000 and the isoelectric point was higher than 10. The optimum pH for the enzyme activity on whole cells of Micrococcus luteus as a substrate was 8.0. Some heavy metal cations (Cu2+ and Zn2+) inhibited the enzyme activity at a concentration of 0.1 mM and others (Ba2+, Mg2+ and Co2+) showed a stimulating effect at a concentration of 1 mM.     

An electrochemiluminescent method for glutamate measurement in small microdialysate samples in asphyxiated young rats

Glutamate (Glu) quantification has been performed by a combination of intracerebral microdialysis through which the samples are obtained and analyzed by high performance liquid chromatography (HPLC); its measurement requires a large expenditure of time (15–30 min per sample) and special training. Therefore, an alternative method is presented here, based on the electrochemiluminescence produced by the use of an enzymatic reactor, containing glutamate‐oxidase, mixed and incubated with microdialysate from dorsal striatum (DS) and prefrontal cortex (PFC) of young rats asphyxiated during the neonatal period, under a global asphyxia model in order to test this method. Using this approach, we found high extracellular Glu concentration in the DS of asphyxiated animals, but only during K⁺ stimulation, while in the PFC, only a delay in the rise of Glu after K⁺ stimulation was observed, without any difference in extracellular Glu content when compared with controls. This new method permitted a fast measurement of Glu in brain dialysate samples, it significantly reduces the cost of the analysis per sample, since only a single device and pump are needed without using columns and high pressure inside the system or complex hardware and software to control pumps, detector, fraction collector or any other peripheral used in HPLC.     

Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon. III. Comparison of shikonin derivatives of cultured cells and ko-shikon

A method for quantitative analysis of shikonin derivatives using high pressure liquid chromatography (HPLC) was established. With this method the composition of shikonin derivatives in cultured cells and roots of Lithospermum erythrorhizon (ko-shikon) was compared. The composition of shikonin derivatives produced by cell suspension cultures was similar to that of the ko-shikon, and the composition in cultured cells was found to fluctuate less than that of the ko-shikon.     

Rapid quantitative analysis of a gibberellin-sterol inhibitor using high-performance liquid chromatographic cartridge columns

A rapid, sensitive, and economical chemical analysis of the triazole, gibberellin-inhibitor, paclobutrazol (PP333, [(2RS,3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4 triazol-1-yl) pentan-3-ol]) was sought, featuring high-performance liquid chromatography (HPLC) as the final quantitation step. Three C₁₈-reverse phase columns (conventional, 250×4.6 mm; cartridge type, 125×4.6 mm; and minicolumn, 33×4.6 mm) were evaluated for their performance in HPLC separation and quantitation of PP333 applied to soil and plant foliage. The 125-mm Whatman Partisil 5 ODS-3 cartridge column was superior to the standard 250-mm DuPont Zorbax ODS unit, and provided enhanced resolution and reduced solvent consumption, analysis time, and cost. A Perkin-Elmer Pecosphere 3×3C-C₁₈ cartridge system was also superior to the 125-mm column with respect to these parameters. Although this minicolumn necessitated an additional purification step prior to HPLC analysis, its exceptionally fast analysis time and recovery period coupled with a high degree of sensitivity rendered it the most superior unit. This HPLC technology provided an efficient means of assaying for PP333 in large-scale experiments dealing with the chemical's absorption, translocation, and physiological response.     

An immobilized cell reactor with simultaneous product separation. I. Reactor design and analysis

A four-phase reactor-separator (gas, liquid, solid, and immobilized catalyst) is proposed for fermentations characterized by a volatile product and nonvolatile substrate.In this reactor, the biological catalyst is immobilized onto a solid column packing and contacted by the liquid containing the substrate.A gas phase is also moved through the column to strip the volatile product into the gas phase. The Immobilized Cell Reactor-Separator (ICRS) consists of two basic gas-liquid flow sections: a cocurrent "enricher" followed by a countercurrent-"stripper".In this article, an equilibrium stage model of the reactor is developed to determine the feasibility and important operational variables of such a reactor-separator. The ICRS concept is applied to the ethanol from whey lactose fermentation using some preliminary immobilized cell reactor performance data. A mathematical model for a steady-state population based on an adsorbed monolayer of cells is also developed for the reactor. The ICRS model demonstrated that the ICRS should give a significant increase in reactor productivity as compared to an identically sized Immobilized Cell Reactor (ICR) with no separation. The gas-phase separation of the product also allows fermentation of high inlet substrate concentrations. The model is used to determine the effects of reactor parameters on ICRS performance including temperature, pressure, gas flow rates, inlet substrate concentration, and degree of microbial product inhibition.     

Determining specific biomass activity in anaerobic wastewater treatment processes

An experimental method for the measurement of specific gas production rate was developed and tested with biomass samples taken from anaerobic fluidized bed reactors, operating with a variety of carriers with molasses, condensate from cellulose production and brewery wastewater as feeds. The method is based on reactor sampling and offline gas volume measurement during a known time interval. Important factors are biomass and liquid sampling under oxygen-free conditions, using the liquid from the reactor as substrate, providing sufficient mixing and maintaining the physical integrity of the biomass. The method was developed in such a way that small samples (20 ml) were taken under anaerobic conditions (poising agent) for short-term (2–3 min.) gas rate measurements in a small fluidized bed (25 ml) batch reactor with U-tube. Biomass content was measured by an instrumental nitrogen method (Dumas), followed by weight determination of the carrier. The gas rates measured with the test system, and their dependence on substrate concentration, were in good agreement with those directly measured from the continuous fluidized bed reactor. Additions of molasses and acetate to the sample proved that the influence of concentration on the biomass activity can be obtained only by operating the continuous reactor at the concentration levels of interest. Comparison between the reactors showed large differences in the specific activity and the total reactor activity. It was found when comparing two reactors, that the values of the specific and the total activities permitted the calculation of the relative biomass quantities. In this way the influence of the carrier-type could be evaluated.     

A continuous fluorescence assay for cyclic nucleotide phosphodiesterase hydrolysis of cyclic GMP

The fluorescent 2'-methylanthraniloyl derivative of cyclic GMP undergoes a 45% decrease in fluorescence when it is cleaved by brain phosphodiesterase in the presence of calmodulin. This fluorescence decrease is dependent upon calcium, calmodulin, and phosphodiesterase, and correlates well (r = 0.996) with the disappearance of substrate as monitored by high-performance liquid chromatography. The Kd values determined by this fluorescence method and HPLC suggest that cyclic GMP and its fluorescent derivative exhibit similar kinetic parameters in their hydrolysis.     

Determination of the cytokinin complement in healthy and witchesbroom malformed proteas

Cytokinins from normal and witchesbroom malformed stems of proteas were determined by radioimmunoassay following sample resolution by high-performance liquid chromatography (HPLC). Material from the early stages of shoot malformation had increased cytokinin concentrations which, over time, declined to the concentrations found in normal-growing stems. The cytokinin complement of the malformed structures was different from that of normal stems. The high concentrations of isopentenyladenosine detected appear to be related to the loss of the correlative inhibition of lateral buds and the development of the witchesbroom structures and may result from localized changes in cytokinin biosynthesis and/or metabolism.     

Comparative chemical analysis and antifungal activity of Ochtodes secundiramea (Rhodophyta) extracts obtained using different biomass processing methods

The activities of bioactive compounds can be influenced by countless factors, including post-collection sample treatment. In this study, the activities of Ochtodes secundiramea extracts obtained using different processes are compared; the biomass was macerated immediately, lyophilized in liquid nitrogen, or dried in an oven at 50 °C after collection. The chemical profiles of the extracts were evaluated using gas chromatography-mass spectrometry (GC-MS), and their biological activities were assessed using qualitative and quantitative bioautography thin-layer chromatography (TLC) assays against phytopathogenic fungi. Preparative high-performance liquid chromatography (HPLC) was employed to isolate the major compound observed in semi-quantitative (CG-MS). The biomass processing methods were shown to influence the chemical profiles of the extracts that promote the oxidation of halogenated monoterpenes; however, C₁₀H₁₅Br₂Cl was the major compound identified in all of the extracts regardless of the preparation method used. Similarly, changes in qualitative antifungal activity were observed depending on the preparation method, even though the minimum amount of material required for the inhibition of fungal growth (the activity detection limit) was 5 μg in all cases. The activity detection limits for the HPLC-purified majority compound were 5 and 10 μg against Cladosporium sphaerospermum and Colletotrichum lagenarium, respectively; these values are similar to those obtained for the crude extracts. These results point towards the possibility of using these crude extracts for the control of anthracnose in cucumbers post-harvest.     

A high-performance liquid chromatography-based radiometric assay for acyl-CoA:alcohol transacylase from jojoba.

Acyl-CoA:alcohol transacylase catalyzes the final step in the biosynthesis of storage liquid wax esters from acyl-CoA fatty acids and fatty alcohols in a limited number of microbes, algae, and Simmondsia chinensis Link (jojoba). An improved and automated method of enzyme assay for this catalyst from cotyledons of jojoba is described. The assay method uses reversed-phase C18 high performance liquid chromatography (HPLC) to separate the labeled C30:1 liquid wax product, [14C]-dodecanyl-octadecenoate, from the unreacted substrate, [14C]octadecenoyl-CoA (oleyl-CoA), and other components produced from enzymes present in the crude homogenate of jojoba cotyledons, including [14C]-octadecenoic acid (oleic acid) and [14C]octadecenol (oleyol). Methods are also described for microscale chemical synthesis in one vessel of 14C-radiolabeled substrates and products for the transacylase. These labeled reagents are required to confirm the HPLC separations of reaction products. The radioactive components are quantitated using an on-line flow-through scintillation detector enabling sensitive and precise analysis of the reaction products.     

New trends in sample preparation: on-line microextraction in packed syringe (MEPS) for LC and GC applications Part III: Determination and validation of local anaesthetics in human plasma samples using a cation-exchange sorbent, and MEPS-LC-MS-MS

The need for on-line sample preparation for high-throughput applications in bioanalysis has increased during the past decade. In this paper a robust and on-line sample preparation technique, micro extraction in packed syringe (MEPS) has been developed and validated. The method is a miniaturized, fully automated, solid-phase extraction (SPE) technique that can be connected on-line to GC or LC without any modification of the chromatographs. The performance of MEPS as sample preparation method is illustrated by the determination of local anaesthetics in human plasma samples on-line with high performance liquid chromatography (HPLC) and tandem mass spectrometry. The sampling sorbent was 1mg silica based benzenesulphonic acid cation exchanger that was inserted in a 250 microl syringe. Ropicavine and two of its metabolites (PPX and 3-OH-ropivacine), lidocaine and bupivacine were used as model substances. The accuracy values of quality control samples (QC) were between 95% and 109%, and precision (relative standard deviation, R.S.D.) had a maximum deviation of 9% for the analytes.