Natural Product Application Solution with UNIFI (NPAS) Application Presentation Warren B Potts III Senior Director Americas Marketing warren_potts@waters.com 2017 Waters Corporation 1
Diverse World of Natural Products Natural products are mainly focused on secondary metabolites derived or isolated from plants, terrestrial and marine organisms, bacteria and fungi. Natural products have been an important source for drug discovery and been used for the maintenance of health and well-being in the practice of traditional medicine throughout the world. 2017 Waters Corporation 2
Natural Products = multiple-component mixture! Phenylpropanoids Alkaloids Terpenes Phenolics Glucosinolates Polyketides Terpenoids Can contain >1000s of unique compounds! http://www.puremedic.com/images/img-products.jpg?bc_t=7ta9buifddkkc3x5mmntww 2017 Waters Corporation 3
Concerns in Natural Products NATURAL SAFE! Adulteration is a major concern with natural products in the market Deliberately substituted (financial incentive) Increase efficacy ~903 adulterated natural health products that have been detected in the industry! (US FDA) 2017 Waters Corporation 4
Liquid chromatography coupled with high resolution mass spectrometry LC-MS screening gained popularity with advent of electrospray ionization High sensitivity to detect low abundance compounds in extracts (nm sensitivity) Ability to detect multiple compounds without isolation Accurate mass screening What is Accurate Mass? Provides information on isotope ratios which can ensure the correct assignment of elemental composition Enables rapid determination of molecular formula for peak of interest Nominal Mass: CO = 28 N 2 = 28 C 2 H 4 = 28 Accurate mass: CO= 27.9949 N 2 = 28.0061 C 2 H 4 = 28.0313 ID of compounds 2017 Waters Corporation 5
Waters Natural Product Application Solution with UNIFI ACQUITY UPLC I-Class, ACQUITY UPLC HSS T3 Column Fast, robust and reproducible for all of your assays UNIFI Natural Product Application solution Interactive workflow-driven data platform Xevo G2-XS QTof MS Ultra sensitive exact mass MS E Waters ASR Green Tea Extract & Catechin in Standard Mix Optimized for Natural Product LC/MS experiments Ingredient Profiling Application Traditional Medicine Library Workflow Template Processing/ Acquisition methods Reports Synthetic Adulterant Application Synthetic Adulterant Library Workflow Template Processing/ Acquisition methods Reports 2017 Waters Corporation 6
Natural Product Application Solution Overview Total application solution Sample prep Data acquisition Data processing Review and Confirm Report ASR Green Tea extract Catechin Mix ACQUITY UPLC I-Class/Xevo G2-XS QTof MS ACQUITY UPLC HSS T3 column MS E acquisition mode Data Acquisition Templates UNIFI Informatics Platform Data Processing Templates TM Library Green Tea Library Synthetic adulterant Library Ingredient Profiling workflow Synthetic Adulterant Screening Workflow MassFragment TM Automatic fragment ion confirmation 3 Report Templates for ingredient profiling 1 Report Template for Synthetic Adulterant Screening Workflow 2017 Waters Corporation 7
Natural Product Application Solution Traditional Medicine Library Available Item Tags: Chinese Name (in Mandarin) Classification based on chemical class Plant of origin Latin Name: o o Latin name for the exact use of the herb In NP research, Latin Name is the standard search format for plant of origin Contains about 600 herbs All herbs listed in 2010 Chinese Pharmacopoeia 2017 Waters Corporation 8
Natural Product Application Solution Traditional Medicine Library Compound links back to Plant Origin and Chemical class! Ex: Salviae, Schizonepeta Stem, Herba Glechomae, Organic Esters Compound properties Chemical formula, Accurate mass, CAS No. Structure Plants of origin Chemical classification 2017 Waters Corporation 9
Natural Product Application Solution Synthetic Adulterant Library Compound properties Compound structure Other names of compound Compound identifiers (CAS number, Chemspider ID and PubChem ID ) Item tags (Pharmacological action) 2017 Waters Corporation 10
Natural Product Application Solution Synthetic Adulterant Library Diagnostic experimental fragment ions 2017 Waters Corporation 11
Natural Product Application Solution Custom library creation Researchers can import or create their own custom natural products library for component screening Easy import format such as excel documents A specific library for the researcher s natural product samples o Saves publications and other detection data Example: Microbial Natural Products library (Collaboration project with Professor Roger Linington) Botanical standard library (Collaboration project with Dr. Ikhlas A. Khan) 2017 Waters Corporation 12
Natural Product Application Solution Overview Review and Confirm Sample prep Data acquire Data process Review and confirm Report Workflow templates (Ingredient Profiling) Ensures data quality Component identification What is it? Unique data processing and review workflow Uses data filters to automatically screen componentised data Filters can be saved as a routine workflow. Comparison of two samples 2017 Waters Corporation 13
Natural Product Application Solution Overview Review and Confirm Sample prep Data acquire Data process Review and confirm Report Workflow templates (Adulterants Screening) Ensures data quality Component identification What is it? Unique data processing and review workflow Uses data filters to automatically screen componentised data Filters can be saved as a routine workflow. 2017 Waters Corporation 14
Natural Product Application solution Screening Applications Sample Place of Origin Ingredients on Label 1) Oprah (Weight loss) Ganoderma lucidum Panax Ginseng Fructus Mume Dioscorea opposita Euryale Ferox Poria Cocos Semen Pruni Cassia tora L. 2) DaiDaiHua (Weight loss) Aurantii fructus immaturus 3) D.N. Capsule (Pain Relief) Picrorhiza kurroa (Kutki) Strychnos nuxvomica (poison nut) Commiphora mukul (guggul) 2017 Waters Corporation 15
Natural Product Application solution Screening Applications LC conditions LC System: Waters ACQUITY UPLC I-Class Column: HSS T3 C18 2.1x100mm, 1.8µm Column temp: 40 ºC Flow rate: 600 µl/min Injection volume: 1 µl Mobile phase A:H 2 O in 0.1% formic acid Mobile phase B:ACN Gradient: 2-100% B in 14.5 min and 2.5 min 98% A equilibration MS E conditions MS System: Waters Xevo G2-XS QTof Ionization: ESI positive and negative Capillary voltage: 1.5 kv(+ve) & 1.5 kv(-ve) Trap CE Ramp 10-40 ev Acquisition range: 50-1500 m/z ACQUITY UPLC I-Class G2-XS QTof MS 2017 Waters Corporation 16
Natural Product Application solution Oprah- Screening Application All the chemical constituents in each of the traditional medicines from the product label are imported into the method for screening Items tags help with understanding more about the compounds 2017 Waters Corporation 17
Natural Product Application solution Oprah- Screening Application Components list Item tags Low CE Workflow templates Chromatograph for each component High CE A comprehensive view using workflows to identify compounds Three fragments identified 2017 Waters Corporation 18
Natural Product Application solution Oprah- Screening Application Overview plot of components Five major compounds were detected from Fructus mume and cassiae tora Verified through fragment ions, error limits and isotopic match intensity Ingredients Present Ingredients Absent Ganoderma lucidum Panax Ginseng Dioscorea opposita Cassia tora L. Fructus Mume Euryale Ferox Poria Cocos Semen Pruni 2017 Waters Corporation 19
Natural Product Application solution Oprah- Screening Application Components list Item tags Low CE Chromatograph for each component High CE Workflow templates One fragments identified 2017 Waters Corporation 20
Natural Product Application solution Oprah- Screening Application Overview plot of components Three adulterants were detected with the highest response. Nine major compounds were detected from Aurantii fructus immaturus and Nelumbinis folium Verified through fragment ions, error limits and isotopic match intensity Adulterants Present Ingredients Present New Ingredients Present Sibutramine Nor-acetildenafil Cyproterone acetate Aurantii fructus immaturus Nelumbinis folium 2017 Waters Corporation 21
Summary of UNIFI NP workflow Application workflows Sample Prep Import Components from Waters TM and/or Adulterant Library UPLC/QTof MS E Data Acquisition UNIFI Data Processing and Component ID Single software (UNIFI) to execute all the identification steps Identified Components Unidentified Components MVA of chemical profile MS/MS Fragments Verification using MassFragment TM Search against Waters internal/ custom libraries Structure Elucidation Tool + Search Internet Libraries Structure elucidation and online database search Marker identification + Search Internal/ Internet Libraries MVA analysis of chemical profiles of samples Confirm Component Identity 2017 Waters Corporation 22
In Summary Natural Products are complex mixtures and require comprehensive analytical technologies identify and confirm chemical components Natural Product application solution with UNIFI is a powerful approach in screening for: Ingredients in natural products Illegal synthetic adulterants in commercial natural products 2017 Waters Corporation 23
Acknowledgements Dr. Roger Linington Dr. Ihklas Khan Simon Fraser University University of Mississippi National Center for Natural Product Research Waters Natural Products team Dr. Dhaval Patel Waters Singapore Dr. Lirui (Kevin) Qiao Waters, China Dr. Jimmy Yuk Waters Corporaiton Dr. Giorgis Isaac Waters Corporation Dr. Kate Yu Waters Corporation Dr. Kerri Smith Waters Corporation Mr. James Traub Waters Corporation 2017 Waters Corporation 24
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