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    • HPLC Method Development and Transfer Utilizing Fused-Core® Particle Technologies
      HPLC Method Development and Transfer Utilizing Fused-Core® Particle Technologies David S. Bell, Ph.D. Recorded: Dec 5 2012 4:00 pm UTC 31 mins
    • Superficially porous (Fused-Core, Core-shell) particle technologies have gained acceptance in general high-performance liquid chromatography (HPLC) and ultra-high pressure liquid chromatography (UHPLC) practice over the past several years due to improved efficiency relative to comparably sized fully porous particles. The Fused-Core option has also been shown to be a superior approach toward improving column efficiency as compared to smaller porous particle (sub-2 µm, UHPLC) technologies owing to the lessened backpressure penalties that are paid for the efficiency gains. Ascentis® Express HPLC columns were initially introduced that employed Fused-Core particles with an overall 2.7 µm diameter. These columns provide efficiencies equal to sub-2 µm particles at much lower backpressures as well as superior efficiencies when compared to fully porous 3 µm phases. There are still instances, however, where the backpressure generated by a 2.7 µm particle may preclude their use and there are also situations based on available equipment or regulatory guidance where larger particles are preferred. For these reasons, a new generation of 5 µm Fused-Core columns has been developed.
      In this seminar we will provide an introduction to the new line of Ascentis Express 5 µm HPLC columns and explore several scenarios practicing analytical chemists might encounter:

      • Desire to develop method on the more efficient 2.7 µm column, but need to be able to transfer to different location or lab
      1. Can I easily transfer methods from 2.7 µm to 5 µm?
      2. Can I easily transfer methods from 5 µm to 2.7 µm?

      • Wish to transfer methods based on fully porous column technologies to Fused-Core
      1. Can this be done easily?
      2. What gains should I expect? What can be done to optimize the gains?
      3. Can I transfer both 3 µm and 5 µm fully porous particle method to Fused-Core?

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    • New! Introducing Ascentis Express 5 um HPLC Columns with Fused-Core Technology
      New! Introducing Ascentis Express 5 um HPLC Columns with Fused-Core Technology R. A Henry Recorded: Sep 21 2012 3:00 pm UTC 53 mins
    • Core-type particles are competing strongly with small porous particles to improve the speed and resolution of HPLC and UHPLC experiments. The pioneering Ascentis Express column with Fused-Core® 2.7µm particles has exploded in popularity because it operates more ruggedly at much lower pressure than current sub-2µm porous particles, yet delivers the same ultra-high performance. This unique performance has been largely attributed to very narrow particle size distribution. Fused-Core® design advantages have also become popular for LC-MS because Ascentis Express columns surpass performance of columns with 3µm porous particles and operate ruggedly at higher velocities and similar pressures.

      With new, narrow-distribution 5µm Fused-Core® particles, the same design advantages can now be realized over traditional 5µm and 3µm porous particles that remain very popular for HPLC columns. An Ascentis Express 5µm column brings 3µm performance and extreme ruggedness at 5µm pressures to your laboratory. Like Ascentis Express 2.7µm particles, the 5µm particles show flatter van Deemter plots than same-size porous particles and allow separation speed to be maximized with minimal loss of resolution. Extremely high plates per pressure are observed. The core-type 5µm design should replace porous 5µm columns in routine HPLC applications with traditional instruments, and should also compete with porous 3µm columns in many LC-MS applications. Performance will be compared to 5µm and 3µm particle columns, and examples of method transfer will be shown. Ascentis Express 5µm will be available in the same phase modifications as the original Ascentis Express 2.7µm ultra-high performance column. Highly stable columns are available in various IDs and lengths up to 25cm.

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    • HPLC of Peptides:  Speed and Resolution with Fused-Core Columns
      HPLC of Peptides: Speed and Resolution with Fused-Core Columns Hillel Brandes, Ph.D. Recorded: Jan 25 2011 7:45 pm UTC 24 mins
    • HPLC columns featuring 2.7 µm Fused-Core (superficially porous) particles with 90 Å pores demonstrate very fast separations of small molecules because of high efficiency and a flat van Deemter plot. These particles rival the efficiency of sub-2 µm totally porous UHPLC particles, but show only about one-half the backpressure. Fused-Core 2.7 µm particles with wider (160 Å) pores have been optimized for the rapid separation of peptides and small proteins. The higher efficiency and lower pressure drop of Fused-Core particles allows preparation of longer columns with very large numbers of theoretical plates. This dramatically increases the peak capacity of the column system, which facilitates qualitative and quantitative HPLC and LC-MS analysis.

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    • Using Silica HPLC Columns in HILIC Mode:  A Practical Guide
      Using Silica HPLC Columns in HILIC Mode: A Practical Guide Merlin K. L. Bicking, Ph.D. Recorded: Apr 27 2011 3:00 pm UTC 44 mins
    • Silica is a popular option for use in “HILIC” mode, but developing new methods can be difficult because the retention patterns are so different from C18. This presentation will provide some practical guidelines for using silica in a HILIC mode – with buffer/acetonitrile mobile phases. Some compound classes are retained under these conditions, and some are not. We will discuss which compounds are good candidates for this system, and summarize how to get started with your method development experiments.

      You will learn:
      •What compounds can be separated on silica in HILIC mode, and what compounds cannot be separated.
      •Starting points for separations, and suggestions for how to adjust conditions for your separation problem.
      •Advantages and limitations of silica in this mode.

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    • A Practical 2D-LC System Developed for Complex Pharmaceuticals
      A Practical 2D-LC System Developed for Complex Pharmaceuticals Lianjia Ma, Ph.D.* and Anthony Alexander, Ph.D. Recorded: Mar 23 2011 3:00 pm UTC 31 mins
    • A comprehensive two-dimensional liquid chromatograph (LC × LC) was constructed from commercially available conventional HPLC equipments. This system utilizes two independently configurable 2nd dimension binary pumping systems to deliver independent flow rates, gradient profiles and mobile phase compositions to dual Fused-Core secondary columns. Very fast gradient separations (30 seconds total cycle time) were achieved at ambient temperature without excessive backpressure and without compromising optimal 1st dimension sampling rates by using superficially porous stationary phases. A practical approach to optimize the various inter-related instrumental parameters will also be presented.

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    • Ultra-Fast LC/MS/MS in Bioanalysis Using Fused-Core Columns
      Ultra-Fast LC/MS/MS in Bioanalysis Using Fused-Core Columns Ethan R. Badman, Ph.D. Recorded: Feb 16 2011 4:00 pm UTC 39 mins
    • With recent advances in HPLC columns and LC/MS hardware, it is possible to increase the throughput of bioanalytical assays without sacrificing quality. By using fused-core columns, it is possible to decrease the run time from ~4 min to 1 min or less, without the use of UHPLC hardware. High quality methods at high flow rates (1-3 mL/min) using non-ballistic gradients as short as 20 seconds were developed that provide comparable or better performance for accuracy, precision, sensitivity, and specificity than traditional slower LC methods. Data will be presented that show that these assays meet regulatory requirements for bioanalytical work. Limitations in the ultimate speed possible for these assays will also be discussed.

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    • Rapid LC/MS/MS-Based Methods For Opiates & Benzodiazepines
      Rapid LC/MS/MS-Based Methods For Opiates & Benzodiazepines Dwight R. Stoll, Ph.D., Assistant Professor - Gustavus Adolphus College Recorded: May 18 2011 3:00 pm UTC 26 mins
    • Increasing case loads and budget and staffing cuts in forensic laboratories continue to motivate the development of higher throughput methods, particularly for confirmatory analysis of regulated intoxicants. In this work, we have focused on the development of rapid LC/MS/MS methods for the determination of nine opiates including two glucuronide metabolites, and 16 benzodiazepines, including two amino- metabolites. Here we aim to analyze both the parent compounds and important polar metabolites in a single analysis. To this end we have compared the retention of the target compounds on two different reversed-phase HPLC stationary phases: a conventional C18 type phase, and a perfluorinated phenyl (PFP or F5) phase built upon the increasingly popular Fused-Core particle morphology. We see that the F5 phase not only generally exhibits higher retention than the C18 type phase, but also exhibits very different selectivity such that the nine opiates can be nearly completely resolved in under four minutes. We find that the mixture of 16 benzodiazepines cannot be completely resolved in a reasonable (i.e., less than 20 min.) time, however we have developed a separation with no more than three overlapping peaks in an analysis time of five minutes.

      Full Title
      DEVELOPMENT OF RAPID LC/MS/MS-BASED METHODS FOR CONFIRMATORY ANALYSIS OF OPIATES AND BENZODIAZEPINES

      Authors
      SPENCER BONNERUP, D. CHRISTOPHER HARMES, TOMAS LISKUTIN, JONNA BERRY, AND DWIGHT R. STOLL

      Department of Chemistry
      Gustavus Adolphus College
      800 West College Avenue
      St. Peter, MN 56082

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    • iPSCs: The Future of Disease Research
      iPSCs: The Future of Disease Research Dr. Sharon Bahia, Product and Distributor Manager, The European Collection of Authenticated Cell Cultures (ECACC) Upcoming: Nov 9 2017 4:00 pm UTC 75 mins
    • The development of human iPSC technology offers researchers the ability to more accurately generate physiologically relevant models of disease and normal tissues in the laboratory. Advances in iPSC generation have allowed many laboratories to make their own cell lines; however, researchers rarely have the resources needed to establish stocks, undertake quality control and share their own de novo iPSC cell lines with other laboratories. A pre-existing and established iPSC collection therefore allows iPSC researchers to obtain “off the shelf” access to a large, robust and reliable supply of iPS cell lines that represent diverse donor to donor variability and which include disease status normal controls and gene edited cell lines. iPSCs from ECACC are standardised and quality controlled and have the benefit of coming from a trusted and internationally recognised collection with worldwide distribution.

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    • No More LC:  Microextraction Coupled with Direct MS Analysis
      No More LC: Microextraction Coupled with Direct MS Analysis Olga Shimelis, Principal R&D Scientist and R&D Supervisor, Merck KGaA, Darmstadt, Germany Upcoming: Oct 26 2017 1:30 pm UTC 90 mins
    • Mass spectrometry (MS) is an accepted research tool for both academic and industrial laboratories. As MS continues to gain ground in clinical and industrial testing, the requirements for high throughput, high sensitivity and high accuracy analyses put more emphasis on sample preparation. Solid Phase Microextraction (SPME) is well suited for this purpose as it requires minimal sample, provides pre-concentration of analytes, and allows for quantitative determinations.
      This webinar will focus on the use of microextraction devices for direct MS analysis for applications that do not require chromatographic separation. Such analysis often results in very high throughput and more immediate results in comparison to traditional methods. Several Direct MS interfaces will be reviewed. The relevant overview of the literature as applied to the direct MS analysis of microextracted samples will be presented. The presentation also will discuss in more detail the coupling of Direct Analysis Real Time (DART) with a new type of solid phase microextraction devices (known as BioSPME).

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    • Characterization of sub-visible particles with Amnis imaging flow cytometry
      Characterization of sub-visible particles with Amnis imaging flow cytometry Christine Probst, Application Scientist - Merck KGaA, Darmstadt, Germany Upcoming: Nov 30 2017 4:00 pm UTC 75 mins
    • Multi-spectral imaging flow cytometry (MIFC) is an established analytical method for cellular analysis, however has only recently been evaluated for characterization of sub-visible particles in therapeutic formulations despite numerous favorable attributes including:

      • Simultaneous collection of bright-field, side-scatter, and fluorescent imagery
      • Sensitive detection of particles 100 nm-100 μm
      • High image quality using 20X-60X magnification objectives
      • 100% sampling efficiency using hydrodynamic focusing
      • Small sample volume requirement (20 μL)
      • Linear concentration range up to 100 million/mL
      • Wide flow cell (250 μm) minimizes clogs

      Assorted case studies using MIFC for analysis of protein and vaccine formulations will be presented, with an emphasis on measurements and samples that pose challenges for current techniques- including detection of small and transparent particles, direct analysis of highly concentrated formulations, and fluorescence characterization of particle type, chemical composition, and heterogeneous interactions.

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    • Stable Isotope-Labeled Protein Internal Standards
      Stable Isotope-Labeled Protein Internal Standards James Walters, Ph.D. Recorded: Sep 21 2017 7:15 pm UTC 62 mins
    • Mass spectrometry-based protein assays impart increased specificity and more rapid development times versus traditional methods, such as ELISA. Coupled with immunoaffinity enrichment, LC-MS/MS is becoming a powerful tool for the quantitation of proteins in plasma. Such methods typically rely on synthetic stable isotope labeled (SIL) peptide internal standards to correct for instrumental variability. For more accurate protein quantitation by LC-MS/MS, experimental variations throughout the entire sample preparation workflow, including protein fractionation, immunoaffinity enrichment, and enzymatic digestion, must be accounted for. An ideal way of improving assay reproducibility is to add a full-length stable isotope labeled recombinant protein, that is equivalent to the native target protein, to the sample at the initial stage of the assay workflow. We have developed a set of stable-isotope-labeled monoclonal antibodies expressed in CHO cells as well as SIL versions of several clinically-relevant human proteins expressed in E. coli, such as IGF1, and in mammalian HEK293 cells, such as Thyroglobulin (manufactured as a Certified Reference Material). We will present data to demonstrate that the use of full-length SIL proteins and antibodies as internal standards allows for more accurate and rapid quantitation of biotherapeutic antibodies and clinically-relevant human protein biomarkers in plasma by LC-MS/MS.

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    • Stable Isotope-Labeled Protein Internal Standards
      Stable Isotope-Labeled Protein Internal Standards James Walters, Ph.D. Recorded: Sep 20 2017 3:00 pm UTC 56 mins
    • Mass spectrometry-based protein assays impart increased specificity and more rapid development times versus traditional methods, such as ELISA. Coupled with immunoaffinity enrichment, LC-MS/MS is becoming a powerful tool for the quantitation of proteins in plasma. Such methods typically rely on synthetic stable isotope labeled (SIL) peptide internal standards to correct for instrumental variability. For more accurate protein quantitation by LC-MS/MS, experimental variations throughout the entire sample preparation workflow, including protein fractionation, immunoaffinity enrichment, and enzymatic digestion, must be accounted for. An ideal way of improving assay reproducibility is to add a full-length stable isotope labeled recombinant protein, that is equivalent to the native target protein, to the sample at the initial stage of the assay workflow. We have developed a set of stable-isotope-labeled monoclonal antibodies expressed in CHO cells as well as SIL versions of several clinically-relevant human proteins expressed in E. coli, such as IGF1, and in mammalian HEK293 cells, such as Thyroglobulin (manufactured as a Certified Reference Material). We will present data to demonstrate that the use of full-length SIL proteins and antibodies as internal standards allows for more accurate and rapid quantitation of biotherapeutic antibodies and clinically-relevant human protein biomarkers in plasma by LC-MS/MS.

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    • Developing Simplified Methods for Quantifying Analytes using  SPME
      Developing Simplified Methods for Quantifying Analytes using SPME Robert E. Shirey, M.S.; Principal R&D Scientist Recorded: Jul 13 2017 1:30 pm UTC 75 mins
    • Solid phase microexatraction or SPME is a green method for extraction of analytes out of a sample. Since SPME is a non-exhaustive extraction technique, some analysts believe that SPME is not quantifiable. This presentation will provide basic information for developing a method to extract and quantify analytes using SPME. Examples will be given on the extraction and quantification of analytes out of various matrices, and SPME will be compared to other extraction techniques such as QuEChERS and SPE. In this webinar, we will discuss some new SPME technologies such as SPME-OC (over-coated) fibers and BioSPME that help to isolate and quantify analytes from interfering compounds in the matrix. Guidelines will be provided for enhancement of precision using SPME.

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    • Automation of the in vitro micronucleus assay using imaging flow cytometry
      Automation of the in vitro micronucleus assay using imaging flow cytometry Matthew. A Rodrigues, PhD., Research Scientist with Amnis-Merck KGaA, Darmstadt, Germany Upcoming: Oct 24 2017 3:00 pm UTC 75 mins
    • The in vitro micronucleus assay is one of the most widely used tests to quantify genotoxicity and cytotoxicity, especially as a screening tool in the development of chemicals and pharmaceuticals. Micronuclei (MN) are formed from whole chromosomes or chromosome fragments that lag behind during the metaphase-anaphase transition and are excluded from the main nucleus following division. MN form into small, rounded bodies surrounded by their own nuclear envelope and represent chromosomal mutations that can be used as an endpoint in genotoxicity testing. Typically performed by manual microscopy, the MN assay is laborious and can be subject to scorer bias. To overcome this, automated microscopy and conventional flow cytometry methods have been developed. However, these methods suffer from several limitations such as the requirement to create high quality slides in the case of microscopy and the lack of visual confirmation of MN in the case of flow cytometry. The ImageStream®X (ISX) imaging flow cytometer has the potential to overcome these limitations as it combines the speed, statistical robustness and rare event capture capability of conventional flow cytometry with high resolution fluorescent imagery.
      In this webinar, adaptation of the in vitro MN assay to an imaging flow cytometry-based method will be described. Using the ISX Mark II imaging flow cytometer, images of micronucleated mono- and binucleated cells as well as polynucleated cells can be captured at a high flow rate and automatically identified and scored in the Image Data Exploration and Analysis Software (IDEAS®) that accompanies the ISX. A data analysis template created specifically for this application allows for the determination of both genotoxicity and cytotoxicity following treatment with known clastogens and aneugens. This work is the first demonstration of fully automated method for performing the in vitro MN assay on an imaging flow cytometry platform.

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