Stepwise Elution Research Articles

Enhanced Separation Capability of Sequential Injection Chromatography for Fluorimetric Determination of Intracellular Dissolved Free Amino Acids in Marine Microalgae.

This chapter describes improvements in a sequential injection method to automate the fluorimetric determination of amino acids by pre-column derivatization with o-phthaldialdehyde in presence of 2-mercaptoethanol. Separation is achieved by reversed-phase liquid chromatography in a 50×4.6mm C18 silica-based monolithic column. The method is low-priced, and the separation is performed by stepwise gradient elution using six mobile phases. The mobile phase used for the first elution step is composed of methanol/tetrahydrofuran/10mM phosphate buffer (pH7.2) at volumetric ratio 8:1:91. Additional elution steps use mobile phases containing methanol and 10mM phosphate buffer at volumetric ratios of 17.5:82.5, 25:75, 35:65, 50:50, and 65:35. Nineteen chromatographic peaks are observed in a mixture of twenty amino acids. The only complete co-elution is between tryptophan and methionine. The entire cycle of amino acid derivatization, chromatographic separation, and column conditioning at the end of separation lasts for 30min. The method is successfully applied to quantify the major intracellular dissolved free amino acids in the marine microalgae Tetraselmis gracilis, Phaeodactium tricornutum, and Synechococcus elongatus.

Isolation of high-purity peptide Val-Val-Tyr-Pro from Globin Peptide using MCI gel column combined with high-speed counter-current chromatography.

Peptides have gained increased interest over the past several decades because of their therapeutics. In this research, a strategy combining MCI gel column chromatography and high-speed countercurrent chromatography was developed for the separation of high-purity peptide Val-Val-Tyr-Pro from Globin Peptide. First, the fraction of Val-Val-Tyr-Pro mixtures with a purity of 15.8% was obtained by using MCI gel column with a mixture of ethanol/water (20:80, v/v/v). Then, the high-purity Val-Val-Tyr-Pro was separated by high-speed countercurrent chromatography with a aqueous two phase systems of ethanol/acetonitrile/iso-propyl alcohol/(NH4 )2 SO4 Saturated solution /H2 O (0.5:0.5:0.25:1.5:0.7,v/v). The ammonium sulfate from high-speed countercurrent chromatography fractions was removed from target compound by MCI gel column chromatography using ethanol/water in stepwise elution mode. A 78mg of Val-Val-Tyr-Pro was successfully purified with the purities of 98.80% from 30g crude Globin Peptide. The amino acid sequence of the Val-Val-Tyr-Pro was determined by electrospray ionization high resolution tandem mass spectrometry. The method presents a practical strategy for the large-scale separation of pure peptide Val-Val-Tyr-Pro from Globin Peptide, and provides a reference method for obtaining high-purity peptide from other polypeptide mixtures.

Simple and efficient approach for enrichment of major isoflavonoids from Astragalus membranaceus with macroporous resins and their nephroprotective activities

Isoflavonoids are abundant in Astragalus membranaceus and exhibit multiple pharmacological activities, including nephroprotective activity. In the present study, a simple, green and effective method for the simultaneous enrichment of four major bioactive isoflavonoids from A. membranaceus with macroporous resin was established. Among ten widely used macroporous resins, AB-8 resin exhibited the highest adsorption capacity and desorption ratio in static tests. The static equilibrium adsorption data fit to the Langmuir isotherm, and the adsorption kinetics followed the pseudo-second order model. Dynamic adsorption and desorption experiments were performed to optimize the separation parameters. After only one cycle of treatment with a stepwise elution on AB-8 resin, the contents of calycosin-7-O-β-d-glucoside (CAG), ononin (ONO), calycosin (CAL) and formononetin (FOR) in the final product were 7.71-, 10.64-, 10.50- and 9.49-fold increased with recovery yields of 70.89, 83.14, 85.01 and 74.62%, respectively. Furthermore, the target isoflavonoid-enriched product exhibited better nephroprotective activity than the crude extract, and both the purity of the isoflavonoids and their bioactivities were significantly enhanced after enrichment with macroporous resin. The developed method is a promising foundation for the scaled-up preparation of bioactive isoflavonoids for their wide use in the pharmaceutical industry.

Quantification of Cefepime, Meropenem, Piperacillin, and Tazobactam in Human Plasma Using a Sensitive and Robust Liquid Chromatography-Tandem Mass Spectrometry Method, Part 1: Assay Development and Validation.

The highly variable pharmacokinetics of β-lactam antibiotics and β-lactamase inhibitors poses a significant challenge to clinicians in ensuring appropriate antibiotic doses in critically ill patients. Therefore, routine monitoring of plasma concentrations is important for individualization of antimicrobial therapy. Accordingly, a simple and robust analytical method for the simultaneous measurement of multiple β-lactam antibiotics and β-lactamase inhibitors is highly desirable to ensure quick decisions on dose adjustments. In this study, a sensitive, simple, and robust method for the simultaneous quantification of cefepime, meropenem, piperacillin, and tazobactam in human plasma was developed and rigorously validated according to FDA guidance. Sample extraction was accomplished by simple protein precipitation. Chromatographic separation of analytes was achieved using stepwise gradient elution. Analytes were monitored using tandem mass spectrometry (MS/MS) with a turbo ion spray source in positive multiple-reaction-monitoring mode. The calibration curve ranged from 0.5 to 150 μg/ml for cefepime, 0.1 to 150 μg/ml for meropenem and piperacillin, and 0.25 to 150 μg/ml for tazobactam. Inter- and intraday precision and accuracy, sensitivity, selectivity, dilution integrity, matrix effect, extraction recovery, and hemolysis effect were investigated for all four analytes, and the results met the acceptance criteria. Compared to other reported methods, our method is more robust because of the combination of the following features: (i) a simple sample extraction procedure, (ii) a short sample run time, (iii) a wide dynamic range, and (iv) the small plasma sample volume needed. Since our method already covers β-lactams and a β-lactamase inhibitor with highly heterogeneous physicochemical properties, further antibiotic candidates may easily be incorporated into this multianalyte method.

Oncepts in development of fast, simple, stability indicating HPLC method for analysis of atorvastatin related compounds in tablets

New, fast, simple and mild conditioned High Performance Liquid Chromatography (HPLC) method for determination of atorvastatin and its 7 main specified impurities, as well as unspecified impurities that might possibly appear, was developed. Chromatographic runs last between 25 and 40 minutes, with simple stepwise gradient elution. The main accent in our method development strategy was focused on mobile phase, composed of simple binary system composed of phosphate buffer and acetonitrile, at pH 4.1, without use of tetrahydrofuran, ion-pair reagents, trifluoroacetic acid and other modifiers with high Ultraviolet (UV) cut-off like absorptive acetate or formiate buffers or amines. With our concept of mobile phase, different columns from myriad were tested, with different efficiency, dimensions and properties, which resulted in different separation efficiency and run time. The best results, concerning essential critical peak resolution, run time length including column preparation and equilibration and column backpressure, were achieved with: YMC C18 Triart 150mm x 4.6mm, 3µm (YMC America, Inc.), afterwards with Nucleodur 100-3-C18ec 250mm x 4.6mm, 3µm (Macherey-Nagel GmbH & Co., Germany), Waters Symmetry C18 250mm x 4.6mm, 5µm (Waters, USA) and Superspher C18e 125mm x 4mm, 4µm (Merck, Darmstadt, Germany). All this columns achieve excellent results regarding obligated critical resolution between atorvastatin impurity B and atorvastatin (according to European Pharmacopoeia),1 or in some cases between atorvastatin impurity B and atorvastatin impurity C, to be minimum about 1.5, in both cases.

A novel and effective mode-switching triple quadrupole mass spectrometric approach for simultaneous quantification of fifteen ginsenosides in Panax ginseng

BackgroundPanax ginseng (PG) is one of the most valuable and frequently used phytomedicine in Asia. In the current Chinese Pharmacopeia, only three ginsenosides, Rg1, Re and Rb1, were set as standard compounds for the quality evaluation of PG. However, only these three compounds could hardly reflect the quality and therapeutic efficacy of this traditional Chinese medicine (TCM). PurposeQuantification analysis of quality markers (Q-markers) in PG is meaningful for determining the quality of this herbal medicine. MethodBy combining the modes of multiple reaction monitoring (MRM) and single ion monitoring (SIM) of tripe quadrupole mass spectrometry (QqQ-MS) through a mode-switching function, a novel, sensitive and effective LC-MS/MS method has been established to simultaneous quantify fifteen Q-markers in PG in one run time cycle. In order to comprehensively evaluate the quality of ten batches of PG, hierarchical clustering analysis (HCA) and the complete linkage method were conducted on the data of the contents of fifteen ginsenosides. ResultsThirteen Q-markers, including four pairs of isomers with the same product ions and approximately the same retention times, have been well-separated by MRM. Meanwhile, the other two Q-markers with no fragments have also been quantified by SIM. Chromatographic separation was carried out on a reversed-phase C18 column by stepwise gradient elution with a mobile phase of water (containing 0.1% formic acid, v/v) and acetonitrile. Good linearity was observed with the correlation coefficients (r2) greater than 0.99. The intra- and inter-day precisions as well as repeatability of all of the investigated Q-markers were all no more than 5.91%. The average recoveries varied from 83.06% to 116.42%, with relative standard deviation values (RSDs) less than 6.73%. The total contents of the fifteen ginsenosides in ten batches of PG were in the range of 15.54–24.03 mg/g. The results indicated that the growing region has a significant impact on the contents of ginsenosides in PG. ConclusionThe proposed approach could be readily utilized as a comprehensive approach for determining the consistency of the quality and therapeutic efficacy of PG, and it might be an example for the selection of Q-marker standards for the Chinese Pharmacopoeia.

Preparative Separation and Purification of Trichothecene Mycotoxins from the Marine Fungus Fusarium sp. LS68 by High-Speed Countercurrent Chromatography in Stepwise Elution Mode.

The contamination of foods and animal feeds with trichothecene mycotoxins is a growing concern for human and animal health. As such, large quantities of pure trichothecene mycotoxins are necessary for food safety monitoring and toxicological research. A new and effective method for the purification of trichothecene mycotoxins from a marine fungus, Fusarium sp. LS68, is described herein. Preparative high-speed countercurrent chromatography (HSCCC) was utilized for the scalable isolation and purification of four trichothecene mycotoxins for the first time in stepwise elution mode, with a biphasic solvent system composed of hexanes–EtOAc–CH3OH–H2O (6:4:5:5, v/v/v/v) and (8.5:1.5:5:5,v/v/v/v). This preparative HSCCC separation was performed on 200 mg of crude sample to yield four trichothecene mycotoxins, roridin E (1), roridin E acetate (2), verrucarin L acetate (3), and verrucarin J (4) in a single run, with each of >98% purity. These compounds were identified by MS, 1H NMR, 13C NMR, and polarimetry. The results demonstrate an efficient HSCCC method for the separation of trichothecene mycotoxins, which can be utilized to produce pure commercial and research standards.

High-yield and high-throughput single-chirality enantiomer separation of single-wall carbon nanotubes

Single crystals of single-wall carbon nanotubes are desired for precise analysis of the physical properties of carbon nanotubes, necessitating preparation of single-chirality enantiomers in large quantities. In this work, by investigating the effects of surfactant concentration and temperature on the enantiomer separation, we achieved large-scale single-chirality enantiomer separation for the first time using triple surfactant stepwise elution chromatography with a naturally produced dextran-based gel as the column medium. Through one-round programmed separation, milligram-scale of (11,–5) (equivalent to (5,6)) and (6,5) enantiomers were separated from CoMoCAT carbon nanotube soot. The separation yields estimated from the optical absorbance at 280 nm were 5.6% (11,−5) and 2.6% (6,5) per load of all SWCNTs, which correspond 28% and 13% per load of total (11,–5) and (6,5) enantiomers, respectively. This high yield is mainly attributed to the narrow chirality distribution and high concentrations of (11,–5) and (6,5) in CoMoCAT and the high-resolution selectivity of the triple surfactant system for two enantiomers. The high throughput is sufficient to prepare high-purity (11,–5) and (6,5) bucky papers.

Expression and purification of biologically active bovine Interferon λ3 (IL28B) in Pichia pastoris

Interferon lambda-3 (IFNλ3) which is also known as IL28B is a member of type III Interferons which are structurally and genetically different from type I Interferons. These Interferons induce signal transduction pathways similar to type I Interferons which results in the activation of Interferon Stimulated Genes (ISGs). This group of Interferons are tissue specific and reported to have antiviral activity. In the present communication, we report the expression of bovine IFNλ3 gene (coding for the mature protein) in Pichia pastoris, purification of the expressed protein and evaluation of its biological activity. About 19 kDa protein expressed by the transformed Pichia cells, secreted into the media and the protein was purified by SP-Sepharose ion exchange chromatography with NaCl stepwise gradient elution. Specificity of the protein was confirmed by Western blotting. Pichia expressed IFNλ3 was found to be biologically active, as it induced ISGs (Mx protein, OAS and PKR genes) in bovine PBMCs. Further it was also found to modulate Th1/Th2 cytokines expression in the stimulated bovine PBMCs.

Implementing Stepwise Solvent Elution in Multisyringe Liquid Chromatography (MSC) to Separate Norepinephrine, Epinephrine and Dopamine

A multisyringe liquid chromatographic (MSC) method by using stepwise solvent elution and reverse-phase liquid chromatography C18 monolithic column to separate Epinephrine (E), Norepinephrine (NE) and Dopamine (DA) was developed. The first step employs the mixture sodium1-hexanosulfonate monohydrate (HS) 7.5mM: methanol (98:2) to separate NE and E and the second step uses HS (5.0 mM): methanol (92:8) to elute DA. The three analytes were separated on 6 min spending only 10 mL of mobile phases. The detection limits were between 1.3 μg mL-1 (DA) and 2.04 μg mL-1 (NE).

High‐Efficiency Separation of (6,5) Carbon Nanotubes by Stepwise Elution Gel Chromatography

We adopted stepwise elution gel chromatography method for CoMoCAT single‐walled carbon nanotubes that is well known as narrow chirality distribution. High‐efficiency separation of (6,5) single‐chirality is realized. Because of high throughput of this method, even a lab scale apparatus can separate 0.43 milligram of (6,5) per one‐round separation procedure (4.5 h) with a separation yield of 24% with respect to the loaded (6,5) nanotubes. This method can also separate high‐purity metallic and rest of semiconducting carbon nanotubes simultaneously. Because of the high‐scalability of column chromatography, this high‐efficiency method is suitable for the large scale production of single chirality single‐walled carbon nanotubes in an industrial scale.

Effect of Yacon (Smallanthus sonchifolius) fructooligosaccharide purification technique using activated charcoal or ion exchange fixed bed column on recovery, purity and sugar content

SummaryA rich in FOS and simple sugar yacon extract was clarified and decoloured and subsequently purified in an activated charcoal column at 25 and 40 °C using two or three stepwise elution with yields of 72.0–86.7% and 90–92.4% purity. Best results were obtained at 25 °C and two stepwise elution, with yield and purity of 81.5% and 92% but 9% sucrose and no glucose and fructose. Different cationic exchange resins on a clarified and demineralised extract at 25 and 60 °C had yields and purity of 73.1–99.4% and 75.0–88.2%, respectively. Best results were obtained with Diaion UBK530Na at 60 °C (99.4% yield and 88.2% purity) but 1.6, 2.7 and 7.5% of fructose, glucose and sucrose. The by‐product was rich in sugars (59–60.1% fructose, 28.3–32.8% glucose, 7.2–8.4% sucrose) and could be used by the feed and food industry.

Structure Sorting of Large‐Diameter Carbon Nanotubes by NaOH Tuning the Interactions between Nanotubes and Gel

AbstractThe structure separation of synthetic single‐wall carbon nanotube (SWCNT) mixture species with diameters larger than 1.2 nm still remains a challenge. Here, an NaOH‐assisted gel chromatography method is used for the structure separation of the SWCNT mixture with a diameter range of 1.2–1.7 nm, in which NaOH is used to tune the interaction between distinct (n, m) SWCNTs and gel. Incrementally increasing NaOH concentration in SWCNT dispersion selectively enhances the adsorbability of different‐structure SWCNTs and enlarges their interaction difference with gel, leading to their structure separation after applying into a gel column system. On this basis, a two‐step method is developed for further improving the structure purity of the separated SWCNTs by combining overloading and stepwise elution. These results are well demonstrated by the optical spectra of the separated SWCNTs. This work paves a way for single‐chirality separation of large‐diameter SWCNTs using gel chromatography technique and is an advanced progress in the structure control of SWCNTs.

A process for supercoiled plasmid DNA purification based on multimodal chromatography

Abstract Non-viral gene therapy and DNA vaccination currently hold great potential for treatment and prevention of genetic and acquired diseases. The large-scale manufacturing of plasmid DNA (pDNA) vectors, and the downstream processing in particular, is one of the key aspects of process development required to move non-viral gene therapy to the clinic. To address the problematic isolation and purification of pDNA molecules, an alternative and cost effective process based on multimodal chromatography was developed. In particular, the possibility of using a cationic multimodal ligand (Capto™ adhere) to remove RNA impurities from E. coli pre-purified lysates and to isolate supercoiled (sc) pDNA isoforms from open circular (oc) pDNA was explored. The process involves E. coli culture, cell harvesting and alkaline lysis followed by isopropanol, ammonium acetate and PEG-8000 precipitation. Finally, sc pDNA is isolated by multimodal chromatography using a stepwise NaCl elution method that includes washing of unbound oc pDNA at 830 mM NaCl, sc pDNA elution at 920 mM and removal of RNA at 2 M. The method provided baseline separation of isoforms and yielded sc-rich fractions (>90%) that are virtually free from RNA and have levels of gDNA (1.3 ± 0.3% µg gDNA/µg sc pDNA) and protein (4.97 ± 1.34 µg/mL) impurities within specifications. Approximately 376 μg of sc pDNA were obtained from 200 mL of bacterial cell culture (OD600nm ≈ 19). The process was reproducible and performed similarly with differently sized plasmids (2686 bp, 3696 bp and 10,410 bp).

Development and validation of liquid chromatography tandem mass spectrometry method quantitative determination of polymyxin B1, polymyxin B2, polymyxin B3 and isoleucine-polymyxin B1 in human plasma and its application in clinical studies

Polymyxin B (PB) is an antibiotic consisting of a cyclic heptapeptide and a tripeptide side chain used in treatment of infections caused by Gram-negative bacteria. Commercial formulations of PB contain multiple structurally related components with major constituents of PB1, PB2, PB3 and ile-PB1. To understand the pharmacokinetics of these major components, we have developed and validated a LC–MS/MS method to quantify PB1, PB2, PB3 and ile-PB1 in human plasma. PB was extracted from plasma by protein precipitation using trichloroacetic acid followed by chromatographic separation on Zorbax Bonus-RP column (100mm×2.1mm, 1.8μm) using stepwise gradient elution of water containing 0.1% of formic acid and 0.1% of trichloroacetic acid (mobile phase A) and 90% acetonitrile with 0.1% formic acid (mobile phase B). Despite of structural similarities, these PBs were completely resolved in the analytical run time of 6.5min. Detection and quantification of PBs were performed by selected reaction monitoring (SRM) under positive ionization mode in the mass spectrometer. Separation of PB1 and ile-PB1, as well as PB2 and PB3, before quantification is crucial because they are structural isomers detected based the same SRM. Excellent linearity was achieved (r2>0.99) in the calibration curves of PB. The developed method was accurate (95.3–111.7%) and precise (CV<5.1%). Recovery of PB from the plasma extraction was between 53 and 76% and reproducible (CV<4.5%). Matrix effect was not observed by post-column infusion of PB in the mass spectrometer. This methodology has been successfully applied to clinical study of patients dosed with intravenous infusions of PB.

Simultaneous determination of eleven alkaloids in Corydalis decumbens by HPLC with diode-array detection

A high-performance liquid chromatography—diode-array detection method was developed and validated to determine simultaneously eleven major alkaloids in Corydalis decumbens (Thunb.) Pers. The alkaloids detected were corlumidine, protopine, coptisine, tetrahydrojatrorrhizine, palmatine, berberine, sanguinarine, papaverine hydrochloride, tetrahydropalmatine, bicuculline, and corydaline. Chromatographic separation was achieved using a C-18 column with a mobile phase composed of A (0.2% acetic acid solution, adjusted with triethylamine to pH 5.0) and B (acetonitrile), with stepwise gradient elution. Ultraviolet diode-array detection was used; chromatograms were examined at the wavelength of 280 nm. The regression equations showed a good linear relationship between the peak area of each marker and concentration (r = 0.9994–0.9999). The recovery values ranged between 93.66% and 100.54%. The method was fully validated with respect to detection and quantification limits, precision, reproducibility, and accuracy. The described high-performance liquid chromatography (HPLC) method was successfully used for the differentiation and quantification of the eleven major alkaloids in C. decumbens (Thunb.) Pers. and can be considered an effective procedure for the analyses of this important class of natural compounds.

An integrated process for the recovery of high added-value compounds from olive oil using solid support free liquid-liquid extraction and chromatography techniques

An integrated extraction and purification process for the direct recovery of high added value compounds from extra virgin olive oil (EVOO) is proposed by using solid support free liquid-liquid extraction and chromatography techniques. Two different extraction methods were developed on a laboratory-scale Centrifugal Partition Extractor (CPE): a sequential strategy consisting of several “extraction-recovery” cycles and a continuous strategy based on stationary phase co-current elution. In both cases, EVOO was used as mobile phase diluted in food grade n-hexane (feed mobile phase) and the required biphasic system was obtained by adding ethanol and water as polar solvents. For the sequential process, 17.5L of feed EVOO containing organic phase (i.e. 7L of EVOO treated) were extracted yielding 9.5g of total phenolic fraction corresponding to a productivity of 5.8g/h/L of CPE column. Regarding the second approach, the co-current process, 2L of the feed oil phase (containing to 0.8L of EVOO) were treated at 100mL/min yielding 1.03g of total phenolic fraction corresponding to a productivity of 8.9g/h/L of CPE column. The total phenolic fraction was then fractionated by using stepwise gradient elution Centrifugal Partition Chromatography (CPC). The biphasic solvent systems were composed of n-hexane, ethyl acetate, ethanol and water in different proportions (X/Y/2/3, v/v). In a single run of 4h on a column with a capacity of 1L, 910mg of oleocanthal, 882mg of oleacein, 104mg of hydroxytyrosol were successfully recovered from 5g of phenolic extract with purities of 85%, 92% and 90%, respectively. CPC fractions were then submitted to orthogonal chromatographic steps (adsorption on silica gel or size exclusion chromatography) leading to the isolation of additional eleven compounds belonging to triterpens, phenolic compounds and secoiridoids. Among them, elenolic acid ethylester was found to be new compound. Thin Layer Chromatography (TLC), Nuclear magnetic Resonance (NMR) and High Performance Liquid Chromatography – Diode Array Detector (HPLC-DAD) were used for monitoring and evaluation purposes throughout the entire procedure.

Content Determination of Phenylpropanoids and Enhancing Exercise Ability of Effective Fractions in Pedicularis densispica.

Background:Most researches were focused on chemical constituents and bioactivities of Pedicularis. However, there were a few reports on simultaneous determination of the series phenylpropanoids compounds in Pedicularis by High Performance Liquid Chromatography (HPLC).Objective:To establish an HPLC method for simultaneous determination of salidroside, verbascoside, iso-verbascoside, leucoseptoside A, jionoside D and martynoside in Pedicularis densispica (PD), and to assess the enhancing exercise ability of effective fractions of phenylpropanoids (EFP).Materials and Methods:The separation was performed on C18 column with step-wise gradient elution with water (A)-methanol (B) as the mobile phase at a flow rate of 1.0 mL/min, with detection wavelength at 275 nm (0–4 min) and 330 nm (4–40 min). The EFP were obtained from extracts of PD by resin gradient dilution. The enhancing exercise ability of EFP was exerted in exhaustive swimming and anoxia endurance tests in vivo.Results:The contents of six marker compounds had good linear relationship in the ranges of 2.10–8.40, 13.60–54.40, 0.93–3.72, 0.53–2.12, 1.50–6.00, 0.37–1.28, respectively, and the average recoveries of the six phenylpropanoids were all in the range of 98–103%. Total contents of phenylpropanoids in EFP were more than 60%. Three medicine groups of exhaustive swimming and anoxia endurance time were higher than those of the water group.Conclusion:The analytical method is reliable, simple and accurate, and can be used for the comprehensive quality control of PD. This experiment suggests that PD has the effect of promoting the recovery and elimination of fatigue and improving the exercise capacity.SUMMARY A simple, practical and low-cost RP-HPLC method has been developed for the simultaneous determination of six marker phenylpropanoids in Pedicularis densispica.Three effective fractions of phenylpropanoids groups of exhaustive swimming and anoxia endurance time were higher than those of the water group.The separation was performed on C18 column with stepwise gradient elution with water-methanol. The enhancing exercise ability was exerted in exhaustive swimming and anoxia endurance tests in vivo.This plant has the effect of promoting the recovery and elimination of fatigue and improving the exercise capacity. Abbreviation used: PD: Pedicularis densispica, EFP: Effective fractions of phenylpropanoids, DAD: Diode array detector, HPLC: High performance liquid chromatography, LOD: Limits of detection, LOQ: Limits of quantification, RSD: Relative standard deviation, BV: Bed volumes

Purification of foot-and-mouth disease virus by heparin as ligand for certain strains.

The goal of this project was to develop an easily operable and scalable process for the recovery and purification of foot-and-mouth disease virus (FMDV) from cell culture. Heparin resins HipTrap Heparin HP and AF-Heparin HC-650 were utilized to purify FMDV O/HN/CHA/93. Results showed that the purity of AF-Heparin HC-650 was ideal. Then, the O/HN/CHA/93, O/Tibet/CHA/99, Asia I/HN/06, and A/CHA/HB/2009 strains were purified by AF-Heparin HC-650. Their affinity/virus recoveries were approximately 51.2%/45.8%, 71.5%/70.9%, 96.4%/73.5, and 59.5%/42.1%, respectively. During a stepwise elution strategy, the viral particles were mainly eluted at 300mM ionic strength peaks. The heparin affinity chromatography process removed more than 94% of cellular and medium proteins. Anion exchange resin Capto Q captured four FMD virus particles; 40% of binding proteins and 80%-90% of viral particles were eluted at 450mM NaCl. Moreover, ionic strength varied from 30 to 450mM had no effect on the immunity to FMDV. The results revealed that heparin sulfate may be the main receptor for CHA/99 strain attachment-susceptible cells. Heparin affinity chromatography can reach perfect results, especially when used as a ligand of the virus. Anion exchange is useful only as previous step for further purification.

Solid-phase extraction-stepwise elution (SPE-SE) procedure for isolation of dissolved organic matter prior to ESI-FT-ICR-MS analysis

Characterization of dissolved organic matter (DOM) at the molecular level will greatly improve our understanding of its bio-geochemical role in controlling the fate of contaminants in the environment, and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) is the most powerful analytical technique for this purpose. Before FT-ICR-MS analysis, isolation, desalination and concentration of DOM are necessary, and solid-phase extraction (SPE) is the most widely applied pretreatment procedure. However, some molecular information is lost using conventional SPE methods. Here, we propose a novel strategy of SPE enrichment using stepwise elution (SPE-SE). Compounds in DOM were divided into three fractions by this SPE-SE procedure according to their polarity and ionization efficiency. The diversity of DOM molecules identified by ESI-FT-ICR-MS using SPE-SE exceeded those using conventional SPE methods by more than 50%. This method is feasible and has the potential to be used as a pretreatment strategy for complex DOM matrixes prior to ESI-FT-ICR-MS analysis, especially for those rich in nitrogenous molecules, carbohydrates, lipids and/or aromatic compounds.