<p><strong>In the week from January 25, 2021, the following webinars await you in the field of LC and LCMS. More <a href="https://lcms.labrulez.com/webinars">details and registration can be found in the WEBINARS section.</a>:</strong></p>
<p><strong>Search and filter</strong> in more than <strong>600 GC and LC webinars</strong>.</p>
<p><strong>1. Thermo Fisher Scientific:</strong> The Successful Leap of Faith for an Established Chromatography Lab</p>
<p><strong>2. Thermo Fisher Scientific:</strong> Making Instrument Equivalency Assessments and Actions Successful</p>
<p><strong>3. Waters Corporation:</strong> Introduction to Ion Mobility and the SELECT SERIES Cyclic IMS</p>
<p><strong>4. C&amp;EN/Wyatt Technology:</strong> Characterizing Polymers and Polymer Aggregates Using SEC-MALS and Batch-Mode MALS</p>
<p><strong>5. Agilent Technologies:</strong> Increase Your IQ for Petrochemical Applications with the Agilent LC/MSD iQ</p>
<p><strong>6. Agilent Technologies:</strong> Sensitive Techniques for Targeted PFAS Analysis in Water</p>
<p><strong>7. Waters Corporation:</strong> Determination of Haloacetic Acids and Acrylamide in Drinking Water by Direct Injection Using Liquid Chromatography-Tandem Quadrupole Mass Spectrometry</p>
<p><strong>8. Agilent Technologies:</strong> Transitioning from nanoflow to standard flow LC/MS: High throughput protein biomarker quantification for clinical research</p>
<p><strong>9. VWR: Back to Basics 2:</strong> Mobile Phase Selection In HPLC &amp; UHPLC </p>
<p><strong>10. C&amp;EN/Agilent Technologies:</strong> Automated Sample Preparation to Overcome Key Challenges in the Analysis of Nitrosamines in Drug Substance and Drug Product</p>
<p><strong>11. Evosep Biosystems:</strong> A beginner’s guide to Evosep</p>
<p><strong>12. Thermo Fisher Scientific:</strong> Panel Discussion: Lessons Learned from Transitioning to New Chromatography Instrumentation and Vendors </p>
<p><strong>13. Shimadzu Scientific Instruments:</strong> Dissecting the impact of gut microbiome on human health by mass spectrometry-based metabolomics</p>
<p><strong>14. Phenomenex:</strong> Determining a Rational HPLC/UHPLC Selectivity Starting Point</p>


LC and LCMS webinars - Week 4

<p><strong>In the first half of February 2021, so far the following webinars await you in the field of LC and LCMS. More <a href="https://lcms.labrulez.com/webinars">details and registration can be found in the WEBINARS section.</a>:</strong></p>
<p><strong>Search and filter</strong> in more than <strong>600 LC and GC webinars</strong>.</p>
<h5><strong>1. Agilent Technologies: Recent Developments in Ion Mobility - Mass Spectrometry: Enabling Structural and Isomeric Separation for Large and Small Molecules</strong></h5>
<p>The talk will present on two specific areas of developments in Ion Mobility - Mass Spectrometry. Structural differences in native proteins and also focusing on the achievements in high resolution ion mobility.</p>
<h5><strong>2. Agilent Technologies: MassHunter Study Manager: streamline your LC/MS workflow and increase analytical productivity</strong></h5>
<p>MassHunter Study Manager is a software tool that automates LC/MS analyses for multiple sample sets and studies queued up for analysis on a single instrument.</p>
<h5><strong>3. Shimadzu Scientific Instruments: Analysis of Steroid Hormones by LC-MS/MS: Advancing Research in HIV/AIDS and Reproductive Sciences</strong></h5>
<p>This presentation will focus on ways in which the Endocrine Technologies Core applies LC-MS/MS analysis of steroid hormones to advancing research in HIV/AIDS, reproductive disorders, and contraception. </p>
<h5><strong>4. SelectScience/Waters Corporation: Compliance-ready intact mass analysis of modified oligonucleotides and impurities using the BioAccord LC-MS System</strong></h5>
<p>In this webinar, we will talk about how the BioAccord LC-MS system has helped users analyze a diverse range of modified oligonucleotides and known impurities using the compliance-ready workflow.</p>
<h5><strong>5. SelectScience/Agilent Technologies/Protein Metrics: Comprehensive adeno-associated virus critical quality attributes analysis with LC-MS</strong></h5>
<p>In this expert webinar we will highlight a workflow to characterize AAVs at the intact and peptide level, including PTM identification, host cell protein analysis and sequence variants.</p>
<h5><strong>6. Agilent Technologies: Technological Innovations to Speed Protein Characterization</strong></h5>
<p>In this presentation we present several mass spectrometry-based technologies used in a stage appropriate manner to dramatically shorten timelines and improve throughput in biologics research.</p>
<h5><strong>7. Agilent Technologies: Method Development for Multi-class, Multi-residue Analysis in Matrices</strong></h5>
<p>Five main phases of method development and optimization will be demonstrated including MS/MS conditions for individual compounds; LC conditions; sample preparation; validation or data acceptance.</p>
<h5><strong>8. Bruker Daltonics: High-Throughput Proteomics During a Pandemic</strong></h5>
<p>In this webinar will show the application and limitations of HT timsTOF Pro MS on large scale clinical sample sets in order to stratify patients and contribute to a multi-omics blood atlas of COVID-19 patients.</p>
<h5><strong>9. C&amp;EN/Agilent Technologies: Agilent OpenLab Remote Access Solutions to increase productivity</strong></h5>
<p>This webinar introduces using &quot;Thin Client&quot; technology which enables all Masshunter, ChemStation or OpenLab CDS to be accessed outside the lab, whether this is at from nearby desks, across sites, or from home. </p>
<h5><strong>10. Shimadzu Scientific Instruments: Affinity Selection-Mass Spectrometry for the Discovery of Pharmacologically Active Natural Products</strong></h5>
<p>In this webinar we will talk about Affinity Selection - Mass Spectrometry for the Discovery of Pharmacologically Active Natural Products.</p>


LC and LCMS webinars - February 2021

<p><strong>In the week from February 8, 2021, the following webinars await you in the field of LC and LCMS.  More <a href="https://lcms.labrulez.com/webinars">details and registration can be found in the WEBINARS section.</a>:</strong></p>
<p><strong>Search and filter</strong> in more than <strong>600 LC and GC webinars</strong>.</p>
<h5><strong>1. SelectScience/Agilent Technologies/Protein Metrics: Comprehensive adeno-associated virus critical quality attributes analysis with LC-MS</strong></h5>
<p>In this expert webinar we will highlight a workflow to characterize AAVs at the intact and peptide level, including PTM identification, host cell protein analysis and sequence variants.</p>
<h5><strong>2. Agilent Technologies: Technological Innovations to Speed Protein Characterization</strong></h5>
<p>In this presentation we present several mass spectrometry-based technologies used in a stage appropriate manner to dramatically shorten timelines and improve throughput in biologics research.</p>
<h5><strong>3. Agilent Technologies: Method Development for Multi-class, Multi-residue Analysis in Matrices</strong></h5>
<p>Five main phases of method development and optimization will be demonstrated including MS/MS conditions for individual compounds; LC conditions; sample preparation; validation or data acceptance.</p>
<h5><strong>4. Agilent Technologies: Analytical Labs: SLIMS Introductory Demonstration</strong></h5>
<p>This demonstration will focus on the implementation of a workflow and includes a complete round trip, from the original request to final reporting. </p>
<h5><strong>5. Bruker Daltonics: High-Throughput Proteomics During a Pandemic</strong></h5>
<p>In this webinar will show the application and limitations of HT timsTOF Pro MS on large scale clinical sample sets in order to stratify patients and contribute to a multi-omics blood atlas of COVID-19 patients.</p>
<h5><strong>6. C&amp;EN/Agilent Technologies: Agilent OpenLab Remote Access Solutions to increase productivity</strong></h5>
<p>This webinar introduces using &quot;Thin Client&quot; technology which enables all Masshunter, ChemStation or OpenLab CDS to be accessed outside the lab, whether this is at from nearby desks, across sites, or from home. </p>
<h5><strong>7. Shimadzu Scientific Instruments: Affinity Selection-Mass Spectrometry for the Discovery of Pharmacologically Active Natural Products</strong></h5>
<p>In this webinar we will talk about Affinity Selection - Mass Spectrometry for the Discovery of Pharmacologically Active Natural Products.</p>


LC and LCMS webinars - Week 6

<p><strong>In the week from February 15, 2021, the following webinars await you in the field of LC and LCMS.  More <a href="https://lcms.labrulez.com/webinars">details and registration can be found in the WEBINARS section.</a>:</strong></p>
<p><strong>Search and filter</strong> in more than <strong>600 LC and GC webinars</strong>.</p>
<h5><strong>1. Dr. Maisch High Performance LC: LONGLIFE Preparative Scale Column Hardware</strong></h5>
<h5><strong>2. Separation Science/SCIEX: NPS Screening and Identification: Nominal and Accurate Mass Approaches for Ultimate Success</strong></h5>
<h5><strong>3. Thermo Fisher Scientific: Accurate and precise quantification: the next level of proteome profiling with the Orbitrap Exploris 240 mass spectrometer</strong></h5>
<h5><strong>4. Agilent Technologies: Biopharma Symposium 2021 Day 1</strong></h5>
<h5><strong>5. Phenomenex: Method Development Strategies for Purity Analysis of Proteins by Intact and Subunit Analysis</strong></h5>
<h5><strong>6. Agilent Technologies: Developing Multidimensional Skyline Spectral Libraries for Rapid Lipid Analysis</strong></h5>
<h5><strong>7. Agilent Technologies: Biocompatible 1290 Infinity II Bio LC for your most complex Biopharma needs</strong></h5>
<h5><strong>8. Agilent Technologies: Agilent Pharma Virtual Symposium - Day 1</strong></h5>
<h5><strong>9. Waters Corporation: Experience and Perspective on the Cyclic IMS for Characterization of Oligo-/Polysaccharides</strong></h5>
<h5><strong>10. Thermo Fisher Scientific: Confident and sensitive quantitation using data independent acquisition (DIA)</strong></h5>
<h5><strong>11. C&amp;EN/Eurofins CDMO Alphora: A Scientific Journey of a Niche API Achieving Breakthrough Status by the FDA for a Lifesaving Drug for Infants</strong></h5>
<h5><strong>12. Agilent Technologies: WHERE Do I Go From Here: Updating Old HPLC Methods</strong></h5>
<h5><strong>13. Agilent Technologies: Biopharma Symposium 2021 Day 2</strong></h5>
<h5><strong>14. Phenomenex: Improving LC Performance Through Optimization of Complementary Governing Method Components</strong></h5>
<h5><strong>15. Agilent Technologies: Agilent Pharma Virtual Symposium - Day 2</strong></h5>
<h5><strong>16. Thermo Fisher Scientific: An Investigative Metabolomics Study on the Orbitrap Exploris 240 Mass Spectrometer: New Features, Optimized Methods and a Results Discussion</strong></h5>
<h5><strong>17. Thermo Fisher Scientific: Rigorous Measurement and confident identification of the Lipidome</strong></h5>


LC and LCMS webinars - Week 7

<p><strong>MS/MS (Tandem) spectra can be used to identify unknowns employing library searches. This is accomplished in much the same approach as that employed for EI GC-MS with the NIST search software. The much improved NIST Search Version 2.4 is included with the 2020 library release.</strong> </p>
<p>I will attempt to introduce the user to the use of the <strong>NIST Version 2.4 search software</strong> employing NIST, Wiley, “crowd-sourced (MoNA)”, and user libraries for the <strong>identification of unknowns</strong>.</p>
<p>NIST has an ambitious program to extend their already comprehensive, high-quality MS/MS databases. </p>
<p>See the following link: <a href="https://littlemsandsailing.files.wordpress.com/2020/12/xiaoyu_yang_asms2020_presentation.pdf">NIST Pipeline for Extending MS/MS Libraries</a></p>
<p>This involves a very comprehensive process for <strong>selecting</strong> pertinent compounds for purchase and subsequent <strong>analyses</strong>. The problem with variability of the spectra is reduced by analyzing the samples on a <strong>variety of instruments</strong> at a <strong>variety of energy levels</strong> (20 steps). In addition, many different <strong>precursor ions</strong> and <strong>fragment ions</strong> are characterized (MSⁿ). This has already led to a very large high quality library of <strong>accurate mass MS/MS spectra in the 2020 release version</strong> (31K compounds/1.3 M spectra) and the effort is ongoing.</p>
<h3><strong>The Part I will cover:</strong></h3>
<p>Detailed Handouts including tables of content are available in the <a href="https://lcms.labrulez.com/library?search=lc%2Fms%20unknown%20identifications%20using%20msms%20libraries&amp;manufacturerItems=55">LabRulezLCMS library</a>.</p>
<ul>
<li>Introduction of Presenter, James Little</li>
<li>Table of Contents </li>
<li>Series Topics</li>
<li>NIST MS Software and MSMS (Tandem) Libraries</li>
<li>Help Files for NIST Search</li>
<li>NIST Software Windows Compliant Features</li>
<li>Saving and Restoring User Configurations</li>
<li>Main Functions on Toolbar</li>
<li>View of Search Options Employed</li>
<li>Main Window in Lib Search View</li>
<li>Tabs for Other Functions Accessed at Bottom of Main Library Window</li>
<li>Names Search Tab </li>
<li>Setting Up search Parameters for MSMS Searches</li>
<li>InChiKey Field Link to PubChem on Web </li>
<li>Molecular Formulae for Fragments Shown in High Resolution Spectra</li>
<li>Live Demo on YouTube of Software </li>
<li>References (Internet Links)</li>
<li>Acknowledgements</li>
</ul>
<h3><strong>Future parts will focus on:</strong></h3>
<ul>
<li>Part II:   <strong>NIST MSMS Search Software and Libraries</strong></li>
<li>Part III:  <strong>More Detailed Discussion of MSMS Hybrid Search</strong></li>
<li>Part IV:   <strong>Importing MSMS Spectra</strong></li>
<li>Part V:    <strong>NIST Structure Searches</strong></li>
<li>Part VI:   <strong>MS Interpreter Correlation of Substructure to MSMS Ions</strong></li>
<li>Part VII:  <strong>Using and Creating Other MSMS Libraries</strong></li>
<li>Part VIII: <strong>Identification of Unknowns with “Spectraless” Libraries</strong></li>
</ul>


LC/MS Unknown Identifications Using MSMS Libraries Part I: Overview of Software and User-Customized Configurations

<p><strong>In the week from February 22, 2021, the following webinars await you in the field of LC and LCMS.  More <a href="https://lcms.labrulez.com/webinars">details and registration can be found in the WEBINARS section.</a>:</strong></p>
<p><strong>Search and filter</strong> in more than <strong>600 LC and GC webinars</strong>.</p>
<h5><strong>1. Phenomenex: Overview of USP and Ph.Eur. Allowable Adjustments for HPLC Monograph Methods</strong></h5>
<h5><strong>2. Agilent Technologies: Characterization and Quantification of Synthetic Oligonucleotides Using HRAM and QQQ</strong></h5>
<h5><strong>3. PerkinElmer: Food Safety: Advances in Methods for Determination of Allergens in food by LC-MS/MS</strong></h5>
<h5><strong>4. Shimadzu Scientific Instruments: The Role of LC-MS in Glycoscience Research</strong></h5>
<h5><strong>5. Technology Networks/Ascension Sciences: Formulating Cannabinoid Particles for Therapeutics</strong></h5>
<h5><strong>6. Agilent Technologies: Scale Up in Minutes: A Practical Example of Preparative HPLC Scale Up from an Analytical Method</strong></h5>
<h5><strong>7. VWR: Getting the best out of solid core technology for small and large molecule analysis</strong></h5>
<h5><strong>8. Evosep Biosystems: Covid-19 research with Evosep One</strong></h5>


LC and LCMS webinars - Week 8

<p><strong>MS/MS (Tandem) spectra can be used to identify unknowns employing library searches. This is accomplished in much the same approach as that employed for EI GC-MS with the NIST search software. The much improved NIST Search Version 2.4 is included with the 2020 library release.</strong> </p>
<p>I will attempt to introduce the user to the use of the <strong>NIST Version 2.4 search software</strong> employing NIST, Wiley, “crowd-sourced (MoNA)”, and user libraries for the <strong>identification of unknowns</strong>.</p>
<p>NIST has an ambitious program to extend their already comprehensive, high-quality MS/MS databases. </p>
<p>See the following link: <a href="https://littlemsandsailing.files.wordpress.com/2020/12/xiaoyu_yang_asms2020_presentation.pdf">NIST Pipeline for Extending MS/MS Libraries</a></p>
<p>This involves a very comprehensive process for <strong>selecting</strong> pertinent compounds for purchase and subsequent <strong>analyses</strong>. The problem with variability of the spectra is reduced by analyzing the samples on a <strong>variety of instruments</strong> at a <strong>variety of energy levels</strong> (20 steps). In addition, many different <strong>precursor ions</strong> and <strong>fragment ions</strong> are characterized (MSⁿ). This has already led to a very large high quality library of <strong>accurate mass MS/MS spectra in the 2020 release version</strong> (31K compounds/1.3 M spectra) and the effort is ongoing.</p>
<h3><strong>The Part II will cover:</strong></h3>
<p>Detailed Handouts including tables of content are available in the <a href="https://lcms.labrulez.com/library?search=lc%2Fms%20unknown%20identifications%20using%20msms%20libraries&amp;manufacturerItems=55">LabRulezLCMS library</a>.</p>
<ul>
<li>Introduction to Presenter, James Little</li>
<li>Table of Contents </li>
<li>Series of Talks: LCMS Unknown Identifications Using MSMS Libraries</li>
<li>Help Files for NIST Search</li>
<li>NIST Software in General is “Windows Compliant” </li>
<li>NIST Tandem Quick Start Guide</li>
<li>Overview: NIST Mass Spectrometry Software and MSMS Libraries </li>
<li>Limitations of MSMS Searches for Unknown Identifications </li>
<li>Three Basic Types of NIST MSMS (Tandem) Searches </li>
<li>Overall Description for Searching Libraries and Displaying Results</li>
<li>NIST MSMS (Tandem) Libraries 2020 Release </li>
<li>Minimizing Number of Results After Search Performed </li>
<li>Two Types of Ions Filtered with MSMS Hit List Filter Option </li>
<li>Setting Default Parameters for Similarity Search (7 Steps)</li>
<li>Setting Default Parameters for Identify MS/MS Search (8 Steps) </li>
<li>Setting Default Parameters for MS/MS Hybrid Search (9 Steps) </li>
<li>Searching Demonstration (Live YouTube) </li>
<li>Presentation References (Internet Hyperlinks)</li>
<li>Acknowledgements</li>
</ul>
<h3><strong>Future parts will focus on:</strong></h3>
<ul>
<li>Part III:  <strong>More Detailed Discussion of MSMS Hybrid Search</strong></li>
<li>Part IV:   <strong>Importing MSMS Spectra</strong></li>
<li>Part V:    <strong>NIST Structure Searches</strong></li>
<li>Part VI:   <strong>MS Interpreter Correlation of Substructure to MSMS Ions</strong></li>
<li>Part VII:  <strong>Using and Creating Other MSMS Libraries</strong></li>
<li>Part VIII: <strong>Identification of Unknowns with “Spectraless” Libraries</strong></li>
</ul>


LC/MS Unknown Identifications Using MSMS Libraries Part II: NIST Search Software and Libraries

<p><strong>In the week from March 1, 2021, the following webinars await you in the field of LC and LCMS.  More <a href="https://lcms.labrulez.com/webinars">details and registration can be found in the WEBINARS section.</a>:</strong></p>
<p><strong>Search and filter</strong> in more than <strong>600 LC and GC webinars</strong>.</p>
<h5><strong>1. Thermo Fisher Scientific: FreeStyle, the qualitative Explorer</strong></h5>
<p>Making the transition from Qualbrowser to Freestyle? Thermo Fisher Scientific FreeStyle application is used to visualize and qualitatively analyze RAW data.</p>
<h5><strong>2. Agilent Technologies: Electron Fragmentation-Based Workflows for Characterizing Proteoforms with Agilent Q-ToFs</strong></h5>
<p>The ExD cell preserves labile post-translational modifications, differentiate isobaric leucine from isoleucine as well as isoaspartate from aspartate, and can cleave disulfide bonds in cysteine knot proteins.</p>
<h5><strong>3. LCTech GmbH: Cannabis analysis - From Plant to Product Solution</strong></h5>
<p>In this webinar we will cover Aflatoxins and other Mycotoxins in Cannabis and Sample Clean-up for the Determinaton of Pesticide using non-dispersive QuEChERS Method.</p>
<h5><strong>4. Waters Corporation: Characterization of Viral Glycopeptides and Support of Global SARS-CoV-2 Efforts</strong></h5>
<p>Join us for a user’s perspective on the characterization of the SARS-CoV-2 glycopeptides by using Ion Mobility Separation (IMS) and mass spectrometry (MS). </p>
<h5><strong>5. C&amp;EN/Agilent Technologies: Enabling Automated Metabolite and Lipid Extraction with the Agilent Bravo Liquid Handler</strong></h5>
<p>The Bravo Metabolomics Sample Prep Platform is built on the Bravo liquid handling system and specifically designed to prepare samples as part of the Agilent Metabolomics workflow solution.</p>
<h5><strong>6. Agilent Technologies: Electron Fragmentation for the Agilent Q-TOF Family</strong></h5>
<p>Here we are going to discuss how ECD facilitates the characterization of post-translational modifications (PTMs) without loss of labile groups.</p>


LC and LCMS webinars - Week 9

<p><strong>In the week from January 18, 2021, the following webinars await you in the field of LC and LCMS. More <a href="https://lcms.labrulez.com/webinars">details and registration can be found in the WEBINARS section.</a>:</strong></p>
<p><strong>Search and filter</strong> in more than <strong>600 GC and LC webinars</strong>.</p>
<h5><strong>1. Agilent Technologies: Simple, Accurate and Reliable Quantification of THC and CBD in Cannabis-Infused Chocolate Edibles, baked goods and Candies</strong></h5>
<p>In this seminar, you will learn a simple and optimized procedures to grind, extract cannabinoids and to quantify them by LC-UV.</p>
<h5><strong>2. Agilent Technologies: HOW Do I Determine the Best Chromatography for My Sample</strong></h5>
<p>In this webinar, we will highlight not only the different column chemistries, particle types, and the sample types they are suitable for, but will also give you practical steps to follow.</p>
<h5><strong>3. GERSTEL: Automation: The next critical tool for scientists’ survival during the pandemic and during recovery.</strong></h5>
<p>This seminar, we will discuss how GERSTEL helps its customers automate their sample preparation and sample introduction methods in order increase throughput and free up resources when they are needed.</p>
<h5><strong>4. Phenomenex: LC Accessories: A Look at the Unsung Heroes of Chromatography</strong></h5>
<p>In this webinar we will reviewing case studies and understanding how a few accessories work, you’ll learn about the added security they can provide in your laboratory.</p>
<h5><strong>5. Shimadzu Scientific Instruments: A systematic analysis of commercial e-liquids: The evolution of GRAS to Inhaled Toxins</strong></h5>
<p>The chemical composition of e-liquids for electronic cigarettes has been evaluated since 2015, using TOF MS, HS-GC-FID, GC-MS, and LC-MS/MS.</p>
<blockquote>
<p>In the week from January 25, 2021, another <a href="https://lcms.labrulez.com/webinars"><strong>7 webinars</strong></a> are waiting for you in the field of LC and LCMS.</p>
</blockquote>


LC and LCMS webinars - Week 3

<p><strong>The plant Stevia rebaudiana (Stevia sweet) is becoming a favourite sweetener in the food industry. Sweet taste is caused by diterpenic glycosides, so-called steviol glycosides, which lack calories while being 300 times sweeter than sucrose. While all the other glycosides are slightly or even disagreeably bitter, rebaudioside A possesses desirable sensorial properties. Because this sweetener is used to sweeten beverages including mixed beer, a fast routine analytical method was developed using HPLC with UV detection for determination of rebaudioside A in mixed beer and lemonade. The HILIC principle, which is suitable for separation of polar and hydrophilic analytes, was used for separation of steviol glycosides. The method is selective, reproducible and has a high recovery of 90-100 %.</strong></p>
<h3><strong>1 INTRODUCTION</strong></h3>
<p>Sweet stevia (Stevia rebaudiana) is native to South America, plentifully spread in Asia, especially in Japan and is used as a non-calorifi c sugar substitute.</p>
<p>French chemists isolated basic glycosides stevioside and rebaudioside A from plant leafs in 1931. They are diterpenic compounds with high sweetness, approximately 300 times sweeter than sucrose, and are contained in all parts of this plant with the exception of roots. Dry leaves contain about 10% of stevioside and 2–4% rebaudioside A. The stevia leaves contain altogether eight diterpenic steviol glycosides: stevioside, steviolbioside, rebaudioside (A, B, C, D, E) and dulcoside A; their concentration is minor. Diterpenic steviol glycosides have a slightly bitter taste except for rebaudioside A, which has minimal bitterness. The major produced compound, stevioside, exhibits appreciable unpleasant bitterness (Prakash et al., 2008) and rebaudioside A extract of is therefore the only one used in food and beverage industry. Rebaudioside A extracts can differ in the content and purity of the final product depending on plant origin and extraction technique. Apart from steviol glycosides, extracts of stevia can contain also other compounds without any sweetness.</p>
<p>The worldwide “stevia rediscovery” as a food supplement required the overcoming of numerous obstacles presented by lobbyist groups since it represented a significant competition to other artificial sweeteners then in use. The US Food and Drug Administration FDA recognized stevia as a food supplement only in 1995; before that sloustevia could be used only in cosmetic industry although its safe use was known more than 1500 years. The position of the FDA was influenced by the misleading studies by the Brazilian Prof. Mauro Alvarez in 1988, which described stevia as having contraceptive properties and even causing infertility of men and women. A number of studies had to be performed to disprove this myth. Nearly 40 years thus elapsed before FDA approved stevia as a common sweetener without any contraceptive effect. None of the current studies has confirmed its effect on human fertility (Kumar et al., 2008).</p>
<p>Also in European Union extensive discussions and often mutually contradictory studies preceded the recognition of stevia as food supplement with designation E960.</p>
<p>Steviol glycosides were registered by EU as permitted sweetener only on December 2, 2011. This approval concerns rebaudioside A with the content of at least 90% of pure substance and mixtures of sterol glycosides with a total content higher than 95%. </p>
<p>According to the Joint Committee of Experts for Food Additives (JEFCA) of World Health Organization (WHO) and of Food and Agriculture Organization of the United Nations (FAO) the acceptable daily intake of steviol glycosides is 4 mg of steviol per kg body mass a day (US Food and Drug Administration, 2008, JEFCA 2008). The sweetener rebaudioside A has found application in all branches of food industry as a non-calorific sweetener; it is used especially for sweetening of lemonades and syrups in beverage industry. Fruit beers sweetened with rebaudioside A have recently appeared increasingly often on the market. The slightly bitter taste of rebaudioside A harmonizes with the bitter beer character and it replaces more and more artifi cial sweeteners such as aspartam.</p>
<p>The widespread method for determination of steviol glycosides is HPLC, though there exist also some methods using thin layer chromatography, capillary electrophoresis or spectroscopic methods (NIR, VIS). The choice of HPLC separation conditions is dependent on the polar character of the glycosides, the choice of UV detection restricts the range of applicable absorbance to short wavelengths. An overview of earlier methods used for determination of steviol glycosides was written by Bovanová (1998). The HPLC methods describe a separation of steviol glycosides especially on columns with NH2 or C18 functional groups; the mobile phases include mostly acetonitril or methanol with water in various rations. Afandi et al (Afandi, 2013) described the optimization of conditions to obtain rebaudioside A from stevia leaves using Soxhlet extractor with subsequent HPLC determination on C18 column with mobile phase acetonitrile/water 80/20 (v/v). Bovanová et al. (Bovanová, 1998) published the preparation of liquid samples by SPE prior to HPLC analysis.</p>
<p>A new technique based on hydrophilic interactions in liquid chromatography (HILIC) is currently used to separate very polar and hydrophilic compounds. Alpert (Alpert, 1990) suggested term “chromatography of hydrophilic interactions” (HILIC) for chromatography on columns typical for systems with normal phases, which however are using water-organic mobile phases like the RP systems. The word “hydrophilic” characterizes the affinity to water added to mobile phases in HILIC separations on polar columns. The HILIC technique was first used for separation of saccharides, amino acids and peptides (Strege,1998). However, the number of newly developed columns for HILIC and applications for separations in the area of environment, foods, natural compounds, synthetic drugs and nonionic surfactants has grown very quickly in the last years. An important benefit of HILIC separations is higher sensitivity of LC-MS analyses because of improved electrospray ionization (ESI) in mobile phases with high content of acetonitrile (Brown at al., 2002; Jandera, 2011).</p>
<p>This principle can be used for HILIC separation of steviol glycosides with UV detection (Thermo, 2012). Compared to UV detection, the use of ELS detector (evaporative light scattering) provides improvement of the limits of quantification and detection. The disadvantage of this detector is the necessity to use volatile mobile phase. </p>
<p>Though the relevant literature contains many methods for determination of rebaudioside A and other steviol glycosides, only a few food laboratories use this method routinely. This is reflected in the cost of this analysis. This study therefore aimed at setting up an analytical method for the determination of rebaudioside A (Fig. 1) in mixed beer and lemonade using an available HPLC technique with UV detection and HILIC separation principle.</p>
<p>The structure of rebaudioside A with prevailing –OH groups determines its high polarity. The probable value of logarithm of distribution coefficient between water and octanol (log P) is – 3.12. Values lower than 1 characterize the polar compounds (author’s comment, see the Dictionary of Food Compounds on CD-ROM, 2004).</p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Obr_1_Struktura_rebaudiosidu_A_38e60bf9ca/Obr.-1-Struktura-rebaudiosidu-A.png" alt="Fig. 1 Structure of rebaudioside A"/></p>
<h3><strong>2 MATERIAL AND METHODS</strong></h3>
<h5><strong>2.1. Chemicals and solvents</strong></h5>
<ul>
<li>Rebaudioside A, HPLC standard &gt; 96% (Sigma – Aldrich)</li>
<li>Deionized water, content of organic compounds &lt; 4 ppb (Millipore, USA)</li>
<li>Acetonitrile, Chromasolv, gradient grade, for HPLC &gt; 99.9%, Sigma Aldrich</li>
<li>Methanol, Chromasolv, gradient grade, for HPLC &gt; 99.9%, Sigma Aldrich</li>
<li>Syringe cellulosic filters 0.2 μm porosity (SISw, Prague).</li>
</ul>
<h5><strong>2.2. Sample preparation</strong></h5>
<p>Samples of lemonades are only pre-purified before analysis in view of the higher concentration of rebaudioside A. Samples of mixed beer, with much lower content of the sweetener, must be pre-concentrated so that the measured values will fi t into the range of the calibration curve of rebaudioside A. The extraction columns C18, E 1000 mg/6 ml (Strata, Phenomenex, USA) were used for purification and pre-concentration. The extraction column was first conditioned step by step with10 ml of methanol, 10 ml of deionized water and after that a sample of lemonade (5 ml) or mixed beer (25 ml) was applied on the column. The sample captured on the adsorbent was washed (10 ml of 20% acetonitrile in deionized water) and eluted into 5 ml of the mixture of acetonitrile – methanol in a 1:1 (v/v) ratio. The purified sample was loaded on the column after filtration on cellulose filter.</p>
<h5><strong>2.3. Preparation of calibration solutions</strong></h5>
<p>The range of the calibration curve was chosen to comply with determination of rebaudioside A in both lemonades and in mixed beers with concentration lower than 1 mg/l (these samples were preconcentrated). The calibration solutions were prepared by dissolving a standard in mobile phase. The stock solution of rebaudioside A was prepared by weighing 100 mg of rebaudioside A standard with accuracy ± 1 mg and its dissolution in 100 ml in graduated flask. </p>
<p>The other calibration levels of analyte, i.e. 1, 5, 10, 20 and 50 mg/100 ml were prepared by dissolving stock solution in appropriate volumes of mobile phase.</p>
<h5><strong>2.4. HPLC determination</strong></h5>
<p>HPLC with UV detection at 210 nm on Dionex Ultimate 3000 liquid chromatograph (Thermo Scientific) with Chromeleon 7 control and processing software was used for determination of rebaudioside A. The column Acclaim Mixed – Mode WAX 1 (2.1 x 150 mm; 5 μm; Thermo Scientific) in HILIC mode was used for separation of polar rebaudioside A from other compounds present in beer and lemonade matrices. The mobile phase consisted of acetonitrile and 10 mM ammonium formate at pH 3.00 ± 0.05 in a ratio of 80/20 (v/v) in isocratic mode. The flow rate of mobile phase was 0.5 ml/min, the column was temperature-controlled at 40 °C, and injection volume was 5 μl.</p>
<h3><strong>3 RESULTS AND DISCUSSION</strong></h3>
<p>The method was optimized for mixed beer and lemonade matrices, and its validation involved verification of linearity, repeatability and recovery of the method.</p>
<h5><strong>3.1 Selectivity</strong></h5>
<p>As follows from Fig. 2, the elution zone of rebaudioside A is distinctly separated from other polar analytes even for its very low concentration in the sample. The last minority component elutes at 7.04 min while the elution zone of rebaudioside A is at 7.95 min. The total separation of rebaudioside A from other components complies with the requirements for a good selectivity of the method. This is given by the use of HILIC mode which has a markedly better selectivity to polar compounds and thus to rebaudioside A than the usual non-polar columns. Another advantage of the HILIC mode is a higher sensitivity; it is 10 – 1000-fold higher for hydrophilic analytes than reverse mode techniques.</p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Obr_2_Chromatogram_separace_vzorku_piva_obsahujici_rebaudiosid_A_14e5fe876d/Obr.-2-Chromatogram-separace-vzorku-piva-obsahujici-rebaudiosid-A.png" alt="Fig. 2 Chromatogram of separation of beer sample containing rebaudioside A. Column Acclaim Mixed – Mode WAX 1 (2.1 x 150 mm; 5 μm), mobile phase acetonitrile and 10 mM ammonium formate 80/20 (v/v) in isocratic mode. Flow rate of mobile phase 0.5 ml/min, temperature of column 40 °C, injection volume 5 μl. The retention time of rebaudioside A is 9.75 min"/></p>
<h5><strong>3.2 Linearity</strong></h5>
<p>The linearity was verified in a calibration range of 1-50 mg/l where the detector response of rebaudioside A has clearly a linear dependence. It is evident from table 1 that the concentrations of rebaudioside A in the original mixed beer appear in the close proximity of the lower point of the calibration curve.</p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Tab_1_Opakovatelnost_metody_ca557696e5/Tab.-1-Opakovatelnost-metody.png" alt="Table 1 Repeatability of the method"/></p>
<h5><strong>3.3 Repeatability and recovery of the method</strong></h5>
<p>A Cola-type lemonade and mixed beer flavored with fruit extract, both with the stated content of rebaudioside A, were used for determination of repeatability and recovery of the method. The concentration of the monitored analyte was determined for both samples. Each sample was extracted on a SPE column and subsequently analyzed on a chromatographic column. The determined content of rebaudioside A in each sample and repeatability (expressed as a limit of repeatability) are given in Table 1. The permissible difference between two parallel determinations (limit of repeatability) in both the beer and the lemonade is 0.42 mg/100 ml, which attests to the good precision of the method.</p>
<p>The repeatability of the method was determined using standard addition of rebaudioside A to samples of mixed beer and lemonade. Two distinct concentrations of the standard were prepared to eliminate mistakes in the dosage of different volumes of standard added into the beer and the lemonade. A solution having the concentration of 1000 mg/100 ml water was prepared for spiking of lemonade while a solution of 100 mg/100 ml water was prepared for spiking beer. The standard was added into 100 ml volumetric flasks to achieve two different concentrations. With both matrices, an adequate volume of the standard was filled up to the graduation mark.</p>
<p>The dosage of 1 ml or 2 ml of standard solution into lemonade caused an increase of the analyte concentration to about 10 resp. 20 mg/100 ml. The dosage of 2 ml resp. 4 ml of standard solution into beer signified the increasing the concentration of analyte by about 2 or 4 mg/100 ml. Every concentration level was determined twice. The results are given in Table 2. The recovery of the method for lemonades and mixed beer ranges in the interval from 101.9% to 102.8% and from 90.2% to 93.1%, respectively. This fact is apparently caused by larger matrix effect in beer and by pre-concentration of beer before analysis. Due to the solubility of the analyte in alcohol its partial leak occurs during of sorption and washing of SPE column. </p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Tab_2_Vyteznost_metody_0c8925b9c2/Tab.-2-Vyteznost-metody.png" alt="Table 2 Recovery of the method"/></p>
<p>The HILIC technique permits the separation of rebaudioside A from other polar compounds also in the very complicated beer matrix, which would not be possible using conventional polar column due to the high polarity of rebaudioside A. The elimination of co-elution of rebaudioside A with other polar compounds assures the accuracy of determination without overrating of the results. All other analytical parameters are in the agreement with the results of other published methods with UV detection in the range of short wavelengths.</p>
<h3><strong>4 CONCLUSION</strong></h3>
<p>The newly developed method to determination of rebaudioside A shows satisfactory precision (repeatability 0.42 mg/100 ml) and accuracy (recovery 90–100%). The solubility of rebaudioside A in alcohol explains the equilibrium shift on extraction column, and thus also a lower recovery from beer in comparison to non-alcoholic lemonade. The concentration of rebaudioside A in mixed beer (or a beer-based beverage) and lemonade after its treatment on the SPE column complies with a linear calibration relationship in the range from 1 to 50 mg/100 ml of the analyte.</p>


Determination of Rebaudioside A in Mixed beer, Beer-based Beverages and Lemonades

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Vědecký recenzovaný online časopis Kvasný průmysl (eISSN 2570-8619), vychází jako samostatné periodikum v angličtině. Od ročníku 2015 je obsah indexovaný databází Web of Science (edice ESCI), dále je indexován v databázích CAS, CAB Abstracts, DOAJ.
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