Post-column Modification Research Articles

Amino acid composition of pollen <i>Pinus sylvestris</i> L. and <i>Pinus sibirica</i> Du Tour growing in the Baikal region

The purpose of the study was to determine the qualitative and quantitative amino acid composition of pollen Pinus sylvestris L. and Pinus sibirica Du Tour. Pine pollen was collected in June 2021 at natural sites on the southeastern coast of Lake Baikal. The mass fraction of crude protein was determined by the Kjeldahl method; the protein composition and individual amino acid content were determined by high-performance liquid chromatography with post-column modification of ninhydrin on an LA8080 automatic analyser (Hitachi, Japan). The crude protein content of the pollen was 14.38–15.94%. Pine pollen protein is shown to contain 17 amino acids, including 9 essential amino acids: valine, isoleucine, leucine, threonine, methionine, phenylalanine, lysine, histidine, and arginine. The content of the sum of amino acids was 141.4–156.5 mg/g, including essential amino acids 45.9-48.4%. The following amino acids are dominant in Pinus sylvestris and Pinus sibirica pollens (mg/g): monoaminodicarboxylic acids – glutamic (21.3–24.2) and aspartic (13.0–14.2), diaminocarboxylic acid arginine (17.0–17.4) and heterocyclic amino acid proline (14.7–16.2). The obtained results can be useful in the development of drugs and biologically active additives based on pollen Pinus sylvestris and Pinus sibirica, which, due to the presence of the above amino acids, have a nootropic, immunomodulatory, cardiac stimulating, and detoxifying effect.

New and vintage solutions to enhance the plasma metabolome coverage by LC-ESI-MS untargeted metabolomics: the not-so-simple process of method performance evaluation.

Although LC-MS untargeted metabolomics continues to expand into exiting research domains, methodological issues have not been solved yet by the definition of unbiased, standardized and globally accepted analytical protocols. In the present study, the response of the plasma metabolome coverage to specific methodological choices of the sample preparation (two SPE technologies, three sample-to-solvent dilution ratios) and the LC-ESI-MS data acquisition steps of the metabolomics workflow (four RP columns, four elution solvent combinations, two solvent quality grades, postcolumn modification of the mobile phase) was investigated in a pragmatic and decision tree-like performance evaluation strategy. Quality control samples, reference plasma and human plasma from a real nutrimetabolomic study were used for intermethod comparisons. Uni- and multivariate data analysis approaches were independently applied. The highest method performance was obtained by combining the plasma hybrid extraction with the highest solvent proportion during sample preparation, the use of a RP column compatible with 100% aqueous polar phase (Atlantis T3), and the ESI enhancement by using UHPLC-MS purity grade methanol as both organic phase and postcolumn modifier. Results led to the following considerations: submit plasma samples to hybrid extraction for removal of interfering components to minimize the major sample-dependent matrix effects; avoid solvent evaporation following sample extraction if loss in detection and peak shape distortion of early eluting metabolites are not noticed; opt for a RP column for superior retention of highly polar species when analysis fractionation is not feasible; use ultrahigh quality grade solvents and "vintage" analytical tricks such as postcolumn organic enrichment of the mobile phase to enhance ESI efficiency. The final proposed protocol offers an example of how novel and old-fashioned analytical solutions may fruitfully cohabit in untargeted metabolomics protocols.

Quantitative speciation of arsenic compounds when using organic solvent gradients in HPLC-ICPMS

Quantitative analysis with HPLC-ICPMS under gradient elution conditions with increasing content of organic solvents is severely restricted because the elemental response can vary significantly with the amount of carbon reaching the plasma. The present work offers a solution to this challenge to elemental species quantification by introducing a volatile organic solvent directly into the spray chamber whereby the sample introduction system is saturated with organic vapours and no change in sensitivity is observed during a methanol gradient separation. This gradient compensation method was tested with a newly developed chromatographic separation of 12 arsenic species comprising arsenite, arsenate, methylarsonate, dimethylarsinate, four oxo-arsenosugars and four thio-arsenosugars within a run of 25 min using a Hamilton PRP-X100 column applying a methanol gradient up to 50% (v/v). Under these conditions 10 of the 12 species could be separated, whereas arsenite and oxo-arsenosugar glycerol co-elute near the front. Species independent quantification was performed against dimethylarsinate as calibrant and the results were compared with isocratic quantifications. Without compensation arsenic species eluting with more methanol in the organic phase were overestimated by factors of 8 to 10, whereas with gradient compensation the results for all arsenic species were comparable with results obtained from isocratic separations. The method was applied to the determination of arsenosugars in the BCR 710 oyster tissue candidate reference material. Results from these experiments underline the applicability of the proposed gradient compensation method for species independent quantification under reversed phase conditions. The method enables accurate quantification without post column modification.

Simultaneous Quantification of Puerarin and Daidzein in Rat Plasma by High-Performance Liquid Chromatography with Post-Column Modification and Fluorescence Detection

A sensitive HPLC method based on post-column modification and fluorescence detection has been developed for determination of puerarin and daidzein in rat plasma. Chromatographic separation was performed on a C8 column with a linear gradient prepared from 0.5% aqueous acetic acid and 0.5% acetic acid in acetonitrile, delivered at a flow rate of 0.8 mL min−1. Naringin was used as the internal standard. It was necessary to use acetic acid in the mobile phase to achieve good separation, but this led to fluorescence signal suppression, because puerarin and daidzein have native fluorescence at pH 8.0–9.0. To enhance the sensitivity, post-column modification with alkaline buffer was adopted. After this modification, detection sensitivity for puerarin and daizein increased more than 500-fold and 600-fold, respectively, compared with direct fluorescence detection. Signal-to-noise ratios for detection for puerarin were more than 150 times better than for UV detection after use of the same method of sample preparation. This sensitive analytical method was successfully used to determine pharmacokinetic data for puerarin and daidzein in rat plasma after oral administration of a single dose of Puerariae radix extract containing puerarin (approx. 8.4 mg) and daizein (approx. 5.9 mg) to male SD rats.

Determination of piromidic acid residues in trout muscle tissue and in urine by liquid chromatography with post-column modification of pH and fluorimetric detection

A rapid high-performance liquid chromatographic method has been developed to determine piromidic acid in trout muscle tissue and in urine, in the presence of nalidixic, 7-hydroxymethylnalidixic, oxolinic and pipemidic acids and cinoxacin. A Nova-Pak C 18 column was used with acetonitrile–4·10 −4 M oxalic acid (40:60, v/v) as the mobile phase. A post-column change of pH was made with NaOH. Fluorimetric detection at 456 nm ( λ e x 275 nm) was used. The instrumental detection limit was 5.91 ng/ml, based on height of peak. Pretreatment of the urine samples was not necessary and fish samples were extracted with sodium hydroxide solutions and cleaned by means of an extraction with chloroform. Detection limit was 147 ng/ml for urine and 5.91 ng/g for trout muscle. Good separation without interference from any other components was obtained. Recovery was better than 87% in urine and better than 72% in trout muscle tissue.

Cationization in electrospray microcolumn liquid chromatography–mass spectrometry

Post-column modification of the mobile phase in microcolumn liquid chromatography–mass spectrometry has been studied. The modification involved addition of sodium, potassium, rubidium and cesium ions to form charged adduct ions. The target molecules studied included modified cyclodextrins and oligosaccarides, bafilomycins and 18-crown-6. Relative complex constants were determined and the concentration and type of cation was evaluated in order to obtain a good sensitivity. An optimum in sensitivity was found with respect to the cation concentration. The optimum cation concentration for singly charged cations was found to be close to 5×10−5M. This concentration was independent of the alkali metal ion used. In most cases the relative sensitivity increases slightly with the size of the cation. The technique was applied to complex samples for structure elucidation of unknown compounds.

Flow characteristics of a versatile wall‐jet or radial‐flow thin‐layer large‐volume cell for electrochemical detection in flow‐through analytical systems

AbstractFlow characteristics of a versatile large‐volume flow‐through electrochemical cell, which features exchangeable disk, ring‐disk or microdisk working electrodes, are presented. The cell operates in a radial‐flow thin‐layer, wall‐jet or wall‐tube mode, under amperometric or voltammetric conditions, being therefore suitable for flow analytical techniques, such as high performance liquid chromatography (HPLC) and flow injection analysis (FIA). The operation mode is selected by using the inlet capillary either of a flat or conical nozzle and by suitably adjusting the nozzle‐to‐electrode distance. The cell is equipped with a micrometer screw which allows for adjusting of this distance to be as small as 10 ± 5 μm. The inlet solution stream is centered axially against the working electrode surface. Under radial‐flow thin‐layer conditions, flow‐through detection at a ring‐disk electrode benefits from shielding and collecting properties which are enhanced as compared to those for the rotating ring‐disk electrode conditions. The HPLC extracolumn effects due to detection were determined by the peak variance analysis of a mixture of regio isomers of nitrobenzene derivatives. Despite the large (35mL) geometric cell volume, the effective detection volume of the cell is of the microliter order. Therefore, the cell is suitable for detection both in the analytical and microbore‐column HPLC. Due to the large geometric cell volume, solution composition can be different, under wall‐jet conditions, for detection than that for HPLC separation. Hence, in situ postcolumn modification of the mobile phase or analyte is feasible.

L-asparaginase tubes: kinetic behavior and application in physiological studies.

ArticleL-asparaginase tubes: kinetic behavior and application in physiological studies.C Horvath, A Sardi, and J S WoodsC Horvath, A Sardi, and J S WoodsPublished Online:01 Feb 1973https://doi.org/10.1152/jappl.1973.34.2.181MoreSectionsPDF (2 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited ByPoly(2-Vinyl-4,4-dimethylazlactone)-Functionalized Magnetic Nanoparticles as Carriers for Enzyme Immobilization and Its Application12 November 2014 | ACS Applied Materials & Interfaces, Vol. 6, No. 23References[43] Enzyme reactors for the removal of amino acids from plasmaFLOW INJECTION ANALYSIS IN CLINICAL CHEMISTRYReviews in Analytical Chemistry, Vol. 8, No. 3Enzymatic preparations in analytical continuous-flow systemsAnalytica Chimica Acta, Vol. 145Effect of diffusional resistances on the performance of substrate inhibited immobilized enzymatic CFSTR systems15 December 2008 | Journal of Environmental Science and Health . Part A: Environmental Science and Engineering, Vol. 18, No. 2Immobilized Enzymes as Precolumn and Postcolumn Modification Reagents in Liquid ChromatographyOpen Tubular Heterogeneous Enzyme Reactors in Continuous-Flow AnalysisThe modern state and prospects for the use of immobilized physiologically active protein substances in medicine (survey)Pharmaceutical Chemistry Journal, Vol. 14, No. 4Whole-cell hollow-fiber reactor: Effectiveness factorsBiotechnology and Bioengineering, Vol. 21, No. 10A Model Enzymic Extracorporeal Detoxification System12 November 2008 | Artificial Organs, Vol. 3, No. 2Hepatic microsomal enzymes: Potential applicationsEnzyme and Microbial Technology, Vol. 1, No. 1Non-Conventional Treatment of Hepatic Failures16 April 2018 | The International Journal of Artificial Organs, Vol. 1, No. 6Immobilized Enzymes in Tubes and Hollow Fibers for Clinical ApplicationsThe kinetic behavior of α-chymotrypsin immobilized inside hydrogel-grafted tubesJournal of Molecular Catalysis, Vol. 2, No. 4Perfusion trails with a collagen-immobilized enzyme in an extracorporeal reactor: Activity, stability, and biocompatibilityJournal of Biomedical Materials Research, Vol. 11, No. 1Immobilized Enzymes for Therapeutic Applications and for Large-Scale Production of Biologically Active CompoundsSome in Vivo and in Vitro Studies of Biologically Active Molecules on Organic Matrixes for Potential Therapeutic ApplicationsTherapeutic Perspectives of Enzyme ReactorsRationale and Strategies for the Therapeutic Applications of Immobilized EnzymesPreparation, characteristics, and application of an asparaginase tubeJournal of Solid-Phase Biochemistry, Vol. 1, No. 1Design and Analysis of Immobilized-Enzyme Flow Reactors[46] Methods for the therapeutic applications of immobilized enzymesDetermination of l-asparagine and l-arginine with open tube enzyme reactorsInternational Journal of Biochemistry, Vol. 6, No. 9Immobilized electric eel acetylcholinesteraseBiochimica et Biophysica Acta (BBA) - Enzymology, Vol. 377, No. 2Biochemical and pharmacologic properties of l-asparaginase bonded to dacron vascular prosthesesBiochemical Pharmacology, Vol. 24, No. 4APPLICATIONS OF IMMOBILIZED ENZYMESEnzymes as Therapeutic AgentsFlow kinetics ofL-asparaginase attached to nylon tubingBiotechnology and Bioengineering, Vol. 16, No. 1Open Tubular Heterogeneous Enzyme ReactorsUse of Collagen Immobilized Enzymes in Blood TreatmentSome Systems Coupling Enzymic Reactions and Other Phenomena; Energy Conversions More from this issue > Volume 34Issue 2February 1973Pages 181-7 https://doi.org/10.1152/jappl.1973.34.2.181PubMed4686352History Published online 1 February 1973 Published in print 1 February 1973 Metrics