the Stakeholder Panel on Dietary Supplements

presents…

the Stakeholder Panel on Dietary Supplements (SPDS)

STAKEHOLDER MEETING #7: September 22, 2017, 8:30 am – 5:00 pm

ATLANTA MARRIOTT MARQUIS 265 Peachtree Center Ave NE Atlanta, Georgia, 30303 CONFERENCE ROOM: M103-M104

contact: spds@aoac.org

presents…

the Stakeholder Panel on Dietary Supplements (SPDS)

STAKEHOLDER MEETING #7: September 22, 2017, 8:30 am – 5:00 pm

ATLANTA MARRIOTT MARQUIS 265 Peachtree Center Ave NE Atlanta, Georgia, 30303 CONFERENCE ROOM: M103-M104

contact: spds@aoac.org

SPDS Meeting, September 22-23 – Chair and Presenter Bios

STAKEHOLDER PANEL CHAIRS

DARRYL SULLIVAN, COVANCE LABORATORIES Chair, AOAC Stakeholder Panel on Dietary Supplements

Darryl Sullivan is a Fellow of AOAC and has been an active member since 1980. He has served terms as secretary, president-elect, president, past president, and director of the Board of Directors, and previously served a three-year term as chair of the Official Methods Board, and is currently serving as Chair of the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals. In 2012 Darryl lead a very successful AOAC engagement with government and industry thought leaders in India and China on behalf of SPIFAN. He is also active with the Stakeholder Panel for Strategic Food Analytical Methods and the Stakeholder Panel for Agent Detection Assays. Sullivan also served a three-year term as a director on the AOAC Research Institute Board of Directors. He was a founding member and chair of the Presidential Task Force on Dietary Supplements and a member of the Task Force on Bacillus anthracis, as well as the AOAC Task Force on Nutrition Labeling and the AOAC Task Force on Sulfites. Prior to chairing the OMB, he served as a member and chair of the Methods Committee on Commodity Foods and Commodity Products. Sullivan was a founding member of the AOAC Technical Division on Reference Materials and served three terms on the Division's Executive Board. A staunch supporter of the Association, Sullivan was active in the e-CAM and Scholar I projects at AOAC, has exhibited at the annual meetings for many years, has presented hundreds of papers and posters at AOAC meetings and regularly publishes his research in the journal of the AOAC. He has also presented a significant number of papers on behalf of AOAC at other scientific meetings in many different parts of the world.

BRIAN SCHANEBERG, STARBUCKS CORPORATION Vice Chair, AOAC Stakeholder Panel on Dietary Supplements

Brian Schaneberg, Ph.D., is the Global Scientific & Regulatory Affairs Director for Starbucks Corporation. Brian participates in the execution of company strategies while ensuring compliance and regulatory guidelines are met and followed by the company across all products: Starbucks, Teavana, Tazo, Evolution Fresh, La Boulange, and Ethos. Brian has over 15 years of natural products experience in the area of dietary supplements and herbals. Brian was also the Quality & Food Saftey and Scientific & Regulatory Affairs Director for Mars Botanical, a division of Mars, Inc. focusing on cocoa flavanol science and products. Before Mars Botanical, he was the Director of Technical Services at ChromaDex, Inc. in Irvine, California and was an Associate Research Scientist at the National Center for Natural Products Research at the University of Mississippi under the guidance of Dr. Ikhlas Khan, in a position funded by the US FDA for the development of methods to ensure the quality and safety of botanicals and dietary supplements. Over the years, Brian has worked closely with trade groups, industry, academia and government leaders. He has been a member of various review committees including NIH grants, analytical validation ERPs at AOAC and the Registry of Carcinogens. Brian also had the pleasure of holding an adjunct faculty position at the University of Colorado, Denver, advising a student that received his MS in Analytical Chemistry isolating phytochemicals and developing analytical testing procedures for Horse Chestnut. Brian has a Ph.D. in Organic Chemistry from Virginia Commonwealth University and a B.A. in Chemistry with a minor in Biology from Central College in Iowa. He has authored or co-authored more than 50 publications and presentations.

SPDS Meeting, September 22-23 – Chair and Presenter Bios

PRESENTER BIOS

RICHARD B. VAN BREEMEN, PHD, UNIVERSITY OF ILLINOIS COLLEGE OF PHARMACY

Chair, SPDS Resveratrol Working Group

Richard B. van Breemen is the Matthias C Lu Collegiate Professor of Pharmacy

and Professor of Medicinal Chemistry and Pharmacognosy at the University of

Illinois College of Pharmacy. He serves as Director of the UIC/NIH Center for Botanical Dietary

Supplements Research and leads the Mass Spectrometry, Metabolomics and Proteomics Facility for the

University of Illinois Cancer Center. Prof. van Breemen received his B.A. in chemistry from Oberlin

College in 1980 and Ph.D. in Pharmacology and Experimental Therapeutics from the Johns Hopkins

University in 1985. He carried out post-doctoral research in laser desorption mass spectrometry at Johns

Hopkins before joining North Carolina State University in 1994 and then the University of Illinois College

of Pharmacy. He is a Regional Editor of Biomedical Chromatography and on the editorial board of Assay

and Drug Development Technologies. Prof. van Breemen has received an Expert Methods Panel award

from the AOAC International for his work on analytical methods for dietary supplements, the Harvey W.

Wiley Award from the AOAC International, and the 2015 Researcher of the Year Award from the

University of Illinois at Chicago. His research concerns the discovery and development of natural

products as chemoprevention agents and the investigation of botanical dietary supplements as

alternatives to hormone therapy for menopausal women.

STEVEN DENTALI

Chair, SPDS Kavalactones Working Group

Steven Dentali, Ph.D., consulting as Dentali Botanical Sciences and widely

considered an international authority on botanical ingredients, previously served as VP and Research

Fellow, Botanical Sciences at Herbalife after eleven years as VP and Chief Science Officer for the

American Herbal Products Association. Dr. Dentali holds a Ph.D. in Pharmaceutical Sciences, with a

Natural Products Chemistry specialization, from the University of Arizona, where he was the American

SPDS Meeting, September 22-23 – Chair and Presenter Bios Foundation for Pharmaceutical Education Edwin Leigh Newcomb Memorial Fellow.

As a key opinion leader, Dr. Dentali helps guide the establishment of shared botanical standards via

engagement in governmental and educational standard-setting organizations including AOAC

INTERNATIONAL, the American Botanical Council, the American Herbal Pharmacopoeia, the National

Institutes of Health, and the U.S. Pharmacopeial Convention. He maintains a particular fondness for

communicating basic botanical concepts and the importance of using a shared nomenclature in order to

help drive industry to higher sustainable herbal standards.

STEFAN GAFNER, AMERICAN BOTANICAL COUNCIL

Chair, SPDS Echinacea Working Group

Dr. Stefan Gafner received his degree in pharmacy at the School of Pharmacy,

University of Berne, Switzerland. After obtaining his PhD with focus on

phytochemistry from the University of Lausanne, Switzerland, he conducted postdoctoral research on

cancer chemopreventive natural products at the University of Illinois – Chicago.

For more than a decade, Dr. Gafner has served as director of analytical chemistry in the R&PD

department of Tom’s of Maine. His current position is Chief Science Officer of the American Botanical

Council, an independent, nonprofit organization dedicated to providing reliable information on the

responsible and safe use of medicinal herbs.

HOLLY JOHNSON, ALKEMIST LABS Chair, SPDS Scullcap Working Group

Holly E. Johnson Ph.D., is the Laboratory Director for Alkemist Labs, an ISO

17025 accredited natural products testing lab specializing in identity and

potency testing of botanicals. Dr. Johnson took her Ph.D. in

Pharmacognosy at the College of Pharmacy, University of Illinois – Chicago

(UIC), under renowned Pharmacognosist and researcher Dr. Norman

Farnsworth. Holly was awarded a National Institutes for Health (NIH) Fellowship and trained at the

UIC/NIH Center for Botanical Dietary Supplements. She was a Postdoctoral Research Fellow at the

Institute for EthnoMedicine studying the etiology of neurodegenerative disease, and worked for Waters

Corporation conducting technical training and regulatory consulting for pharmaceutical and

SPDS Meeting, September 22-23 – Chair and Presenter Bios supplements companies. She is currently a Research Associate with the National Tropical Botanical

Garden and has served on AOAC working groups, Stakeholders Panels and Expert Review Panels for

Foods and Dietary Supplements. She is a member of the United States Pharmacoepia’s (USP) Medical

Cannabis Expert Panel, the Editorial Board of the AOAC International Journal, and she serves on the

Advisory Boards of the American Botanical Council and the American Herbal Pharmacoepia. Holly has

over 20 years’ experience in natural products chemistry with botanicals and spent many happy years

conducting research on medicinal plants and giving courses at the University of Hawaii.

JOSEPH ZHOU, SUNSHINEVILLE HEALTH PRODUCTS Chair, SPDS SAMe Working Group

Dr. Joseph Zhou has been working in the dietary supplement industry since 1996.

He is currently the technical director of Sunshineville Health Products, Inc, in charge

of both products development and analytical methods development. He was also a

technical director in a few of other famous brands companies in the US. He has

been actively participating in the AOAC official methods program since 2002. His team established the

AOAC official method of Glucosamine. He was one of the important players in the AOAC single lab

validation projects for Chondroitin Sulfates and MSM, and was involved in many other AOAC methods

projects. Dr. Zhou is the author of the USP monograph of Arginine. He is an adjunct professor of

pharmacognosy at College of Pharmacy, University of Illinois at Chicago. He was awarded by AOAC as

the Study Director of the Year of 2005.

DRAFT, DO NOT DISTRIBUTE

SEPTEMBER 22, 2017 ATLANTA MARRIOTT MARQUIS 265 Peachtree Center Ave NE, Atlanta, GA 30303 CONFERENCE ROOM: M103-M104

8:30am – 5:00pm Eastern Standard Time Registration Opens at 7:30am

STAKEHOLDER PANEL ON DIETARY SUPPLEMENTS (SPDS) Chair: Darryl Sullivan, Covance Vice Chair: Brian Schaneberg, Starbucks A G E N D A

Welcome and Introductions (8:30-8:40am) a. Introductions Darryl Sullivan, Covance (Chair, SPDS) and Brian Schaneberg, Starbucks Corporation (Vice Chair, SPDS) b. Policies and Procedures Sullivan will also review AOAC’s policies and request that participants comply with the AOAC International Antitrust Policy Statement and Guidelines as well as all AOAC Policies and Procedures, found at www.aoac.org , under “About AOAC”.

I.

Ingredient Updates (8:40am – 9:00am) Darryl Sullivan a. Status of Ingredients to Date b. Open Calls for Methods and Calls for Experts

II.

SMPR Presentations and Consensus* (9:00am – 12:15pm)

III.

a. Echinacea (9:00am – 10:00am)

Chair: Stefan Gafner, American Botanical Council

b. Ginseng (10:15am – 11:15am) Chair: Paula Brown, British Colombia Institute of Technology c. SAMe (11:15am – 12:15pm) Chair: Joseph Zhou, Sunshineville Health Products

Launch of Set 8 Working Groups (12:15pm – 4:30pm)

IV.

a. Working Group Launch Presentation: Kavalactones (1:15am – 2:15pm) Chair: Steven Dentali, Dentali Botanical Sciences b. Working Group Launch Presentation: Resveratrol (2:30pm – 3:30pm) Chair: Richard van Breemen, University of Illinois at Chicago c. Working Group Launch Presentation: Scullcap (3:30pm – 4:30pm) Chair: Holly Johnson, Alkemist Labs

Next Steps & Adjourn (4:30pm – 5:00pm)

V.

a. Upcoming Expert Review Panels (4:30pm – 4:45pm) b. Next Steps for the Stakeholder Panel on Dietary Supplements (4:45pm – 5:00pm) Morning Break: 10:00am – 10:15am | Lunch (on your own): 12:15pm – 1:15pm | Afternoon Break 2:15pm – 2:30pm

Version 3 09/13/2017

*Item(s) requires a vote by SPDS

DRAFT, DO NOT DISTRIBUTE

AOAC INTERNATIONAL Stakeholder Panel on Dietary Supplements Working Group Sessions – September 23, 2017 (Day 2) 9:00 a.m. – 4:00 p.m., Room M106 – M107

I. Kavalactones (9:00 am – 11:00 am) Chair: Steven Dentali, Dentali Botanical Sciences a. Review Fitness for Purpose b. SMPR Development II. Resveratrol (11:00 a.m. – 1:00 p.m.) Chair: Richard van Breemen, University of Illinois at Chicago a. Review Fitness for Purpose b. SMPR Development III. Scullcap (2:00 pm – 4:00 pm) Chair: Holly Johnson, Alkemist Labs a. Review Fitness for Purpose b. SMPR Development

Version 3 09/13/2017

*Item(s) requires a vote by SPDS

AOAC Stakeholder Panel on Dietary Supplements (SPDS): Background and Updates (SPDS)

Darryl Sullivan , Chair Stakeholder Panel on Dietary Supplements Covance Laboratories

September 22, 2017

AOAC SPDS History

• AOAC INTERNATIONAL signed a 5‐year contract with the  National Institutes of Health‐Office of Dietary Supplements  (NIH/ODS) to establish voluntary consensus standards for  high‐priority ingredients.  • Develop standard method performance requirements  (SMPRs) for 25 priority dietary supplement ingredients. • Deliver First Action Official Methods SM for the prioritized  dietary supplement ingredients  • Encourage participation with the dietary supplements  industry to develop voluntary consensus standards.

Stakeholder Panel on Dietary Supplements (SPDS)

• Set 1 Ingredients:  Anthocyanins, Chondroitin, and PDE5 Inhibitors – Working Groups Launched March, 2014 – SMPRs Approved in September, 2014: • Authentication of Selected Vaccinium spp in Dietary Ingredients and Dietary  Supplements ( 2014.007 ) • Screening Method for Selected Adulterants in Dietary Ingredients and  Supplements Containing Chondroitin Sulfate ( 2014.008 ) • Determination of Total Chondroitin Sulfate in Dietary Ingredients and Supplements  ( 2014.009 ) • Determination of Total Chondroitin Sulfate in Dietary Ingredients and Supplements  ( 2014.009 ) • Identification of Phosphodiesterase Type 5 (PDE5) Inhibitors in Dietary Ingredients  and Supplements ( 2014.010 )  • Determination of Phosphodiesterase Type 5 (PDE5) Inhibitors in Dietary  Ingredients and Supplements ( 2014.011 ) – First Action OMAs for one (1) Chondroitin and one (1) PDE5 Inhibitor  method – Call for Methods  for Anthocyanins is currently OPEN

Stakeholder Panel on Dietary Supplements (SPDS)

• Set 2 Ingredients:  Ashwagandha, Cinnamon, Folin C and Kratom

– Working Groups Launched September, 2014 – SMPRs Approved in March, 2015: • Withanolide Glycosides and Aglycones of Ashwagandha ( 2015.007 ) • Alkaloids of Mitragyna speciosa  (Kratom) ( 2015.008 ) • Estimation of Total Phenolic Content Using the Folin‐C Assay ( 2015.009 ) • Identification of Selected Cinnamomum spp. Bark in Dietary Supplement Raw  Materials and/or Finished Products ( 2015.010 )

– First Action OMA for One (1) Ashwagandha Method

– Call for Methods and Experts are currently OPEN for Cinnamon,  Folin C, and Kratom

Stakeholder Panel on Dietary Supplements (SPDS)

• Set 3 Ingredients:  Aloin, Tea, and Vitamin D

– Working Groups Launched in March, 2015 – SMPRs Approved in September, 2015: • Determination of Catechins, Methyl Xanthines, Theaflavins, and Theanine in Tea Dietary Ingredients and Supplements  ( 2015.014 )  • Determination of Aloin A and Aloin B in Dietary Supplement Products and  Ingredients ( 2015.015 ) • Determination of Vitamin D in Dietary Supplement Finished Products and  Ingredients ( 2015.016 ) – First Action OMAs for one (1) Aloin and one (1) Tea method

– Calls for Methods and Experts are currently OPEN for Vitamin D.

Stakeholder Panel on Dietary Supplements (SPDS)

• Set 4 Ingredients:  Collagen, Lutein, Turmeric – Working Groups Launched in September, 2015 – SMPRS Approved in March, 2016: • Quantitation of Curcuminoids ( 2016.003 )

• Quantitative Measurement of β‐Cryptoxanthin, Lutein, and Zeaxanthin in  Ingredients and Dietary Supplements ( 2016.004 ) Quantitation of Collagen ( 2016.005 ) – First Action OMAs for one (1) Curcuminoids in Turmeric Method

– Call for Methods and Experts are currently OPEN for Lutein and  Collagen.

Stakeholder Panel on Dietary Supplements (SPDS)

• Set 5 Ingredients: Aloe Vera, Protein, Vitamin B 12

– Working Groups Launched in March, 2016

– SMPRs Approved in September, 2016:

• Identification of Proteins in Dietary Supplements – Animal Derived ( 2016.015 ) and Non‐Animal Derived ( 2016.016 ) • Identification and Quantitation of Proteins in Dietary Supplements – Animal Derived ( 2016.013 ) and Non‐Animal Derived ( 2016.014 ) • Quantitative Measurement of Vitamin B 12 in Dietary Supplements and  Ingredients ( 2016.017 ). – Call for Methods and Experts are currently OPEN.

Stakeholder Panel on Dietary Supplements (SPDS) • Set 6 Ingredients: Amino Acids, Ginger, Vitamins K 1 and K 2

– Launched in September, 2016 

– SMPRs approved in March, 2017: • Identification and Quantitation of Free Alpha Amino Acids in Dietary  Ingredients and Supplements  (2017.011) • Quantitation of Select Nonvolatile Ginger Constituents  (2017.012) • Determination of Vitamins K 1 and K 2 in Dietary Supplements and Dietary  Ingredients  (2017.013) – Call for Methods and Experts are currently OPEN.

Stakeholder Panel on Dietary Supplements (SPDS)

• Set 7 Ingredients: Echinacea, Ginseng, and SAMe

– Launched in March, 2016

– Met via teleconference from April, 2017 to June, 2017

– SMPRs posted for public comment July 7 2017 to August 11, 2017 

– To be reviewed and voted on during this meeting.

Stakeholder Panel on Dietary Supplements (SPDS) Advisory Panel

• SPDS Advisory Panel met in March, 2017 and confirmed  the last set (Set 8) of ingredients for the current contract: – Kavalactones – Resveratrol – Scullcap – These working groups will be launched at this meeting, with in  person meetings tomorrow.

• The Advisory Panel includes representatives from AHPA,  CRN, CHPA, NSF, NPA, NIH, USP, and Herbalife

How do you get involved?

• Submit methods on the Call for Methods tab at  www.aoac.org

• Volunteer for Expert Review Panels on the Call for  Experts tab at www.aoac.org

• SPDS site at www.aoac.org , click “Standards”, then  Stakeholder Panel on Dietary Supplements (SPDS)  for complete information about the program

Contact Information

Darryl Sullivan, Chair SPDS Covance Laboratories Tel:  608.242.2711 Email: darryl.sullivan@covance.com Brian Schaneberg, Vice Chair, SPDS Starbucks Corporation Email:   bschaneb@starbucks.com

Contact AOAC Staff: Tel: 301.924.7077 Web: www.aoac.org • Deborah McKenzie , Sr. Director, Standards Development and AOAC Research Institute,  dmckenzie@aoac.org , ext. 157 • Dawn Frazier , Sr. Executive for Scientific Business Development, dfrazier@aoac.org , ext.  117

AOAC INTERNATIONAL STAKEHOLDER PANEL ON  DIETARY SUPPLEMENTS Stefan Gafner, American Botanical Council Echinacea Working Group ‐ SMPR Presentation September 22, 2017

Atlanta Marriott Marquis, Atlanta, Georgia, USA

Fitness for Purpose As Agreed March 17, 2017

Primary objective: Quantitation of phenolic compounds (to include caftaric acid, chlorogenic acid, cichoric acid, cynarine, and echinacoside) in Echinacea angustifolia , Echinacea pallida , and Echinacea purpurea raw materials and finished dietary supplement products. Secondary Objective: Identification of Echinacea angustifolia , Echinacea pallida , and Echinacea purpurea raw materials and finished dietary supplement products by phenolic compound profile.

SPDS Echinacea Working Group Members

• Stefan Gafner, American Botanical Council • Anton Bzhelyansky, USP • Kan He, Herbalife • Adam Horkey, Nature’s Way

• Holly Johnson, Alkemist • Adam Kuszak, NIH/ODS • Jungmin Lee, USDA

• Tom Phillips, State of MD • Klaus Reif, PhytoLab GmbH • Kate Rimmer, NIST • Darryl Sullivan, Covance • Anikó Solyóm, GAAS Analytical • John Szpylka, Mérieux NutriSciences • John Travis, NSF International • Jinchaun Yang, Waters • Garrett Zielinski, Covance

Echinacea Working Group Work to Date

• 6 teleconferences  (April 2017 – June 2017) • 1 SMPR Drafted  • Public comment period (July, 2017) • SMPRs made ready for SPDS review and  approval

Background • The genus Echinacea contains nine species ( E. angustifolia , E. atrorubens ,  E. laevigata , E. pallida , E. paradoxa , E. purpurea , E. sanguinea , E. simulata ,  E. tennessensis ) • The main Echinacea used in commerce are as follows: • Echinacea angustifolia root • Echinacea pallida  root • Echinacea purpurea fresh herb,  • Echinacea purpurea dried herb • Echinacea purpurea rhizome and root • Therapeutic indications include the short‐term prevention and  treatment of common cold (oral intake), or topically for the  treatment of small superficial wounds

Background (continued)

• The phytochemicals responsible for the  immunostimulant properties of Echinacea spp.  are not known • The following compound classes have been  linked to bioactivity: – Alkylamides (alkamides) – Phenolic compounds  – Polysaccharides – LPS and lipoproteins produced by bacterial  endophytes

Main Echinacea phenolics

Concentrations in % of dried plant part

Cichoric acid

Caftaric acid

Echinacoside Chlorogenic acid

Cynarine 1

E. angustifolia

0.13 – 1.70

E. pallida

0.13 – 1.27

E. purpura  root

0.33 – 2.78 0.35 – 0.80

E. purpurea tops 0.52 – 2.20 0.18 – 0.85

1 Syn. 1,3-dicaffeoylquinic acid (1 R ,3 R ,4 S ,5 R )-1,3-bis[[( E )-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy]-4,5-dihydroxycyclohexane-1-carboxylic acid

Cynarine

Caftaric acid

Cichoric acid

Echinacoside

References: Brown et al . 2011, JAOAC Int . 94(5): 1400–1410; Perry et al . 2001, J Agric Food Chem . 49(4): 1702-1706; Laasonen et al . 2002, Planta Med . 68: 572 – 574; Pellati et al . 2005, Phytochem Anal . 16(2): 77 – 85. Blaschek. In: Wichtl – Teedrogen und Phytopharmaka, 2016.

Existing Methods (General)

• Abundance of published methods, mainly using HPLC‐UV  or HPLC‐MS • UV/Vis spectrophotometry (Folin‐Ciocalteu) used for total  phenolic compounds  • HPTLC, CE‐UV infrequently used • Established methods include: – Official methods of the United States Pharmacopeia and European  Pharmacopoeia – American Herbal Pharmacopoeia  • HPLC‐UV for phenolic compounds in Echinacea angustifolia root • HPLC‐UV for phenolic compounds in Echinacea pallida  root  • HPLC‐UV for phenolic compounds in Echinacea purpurea root and herb • SLV for phenolic compounds in Echinacea angustifolia ,  Echinacea pallida , and Echinacea purpurea root and herb  by Brown et al. (2011)

Official Methods

• United States Pharmacopeia – Echinacea angustifolia root, powdered root, and powdered  extract: HPLC‐UV for phenolic compounds – Echinacea pallida  root, powdered root, and powdered  extract: HPLC‐UV for phenolic compounds  – Echinacea purpurea root, powdered root, and powdered  extract: HPLC‐UV for phenolic compounds • European Pharmacopoeia – Echinacea angustifolia root (whole or cut): HPLC‐UV for  phenolic compounds  – Echinacea pallida  root (whole or cut): HPLC‐UV for phenolic  compounds  – Echinacea purpurea root (whole or cut): HPLC‐UV for  phenolic compounds – Echinacea purpurea dried herb (whole or cut): HPLC‐UV for  phenolic compounds • Amounts of individual phenolics vary substantially  among species and plant parts: method  performance criteria will depend on concentration • Chlorogenic acid ‐ if present – is found at low  concentrations: larger amounts may indicate  addition of extraneous material • Echinacea products combined with other plant  extracts will require additional validation (e.g.,  selectivity) • Analyte standards and vouchered botanical  materials are commercially available (no NIST  standards) SMPR Key Points

Comments Submitted (if any)

• No comments submitted.

Motion

• Move to accept the Standard  Method Performance  Requirements for Determination of  Phenolic Compounds in Dietary  Supplements and Dietary  Ingredients Containing Echinacea  as presented.

Discussion?

DRAFT AOAC SMPR 2017.XXX; Version 9; June 29, 2017 1 2 Method Name: 3 4 5 Approved by: Stakeholder Panel on Dietary Supplements (SPDS) 6 7 Intended Use : For quality assurance and compliance to current good manufacturing practices 8 9 1. Purpose: AOAC SMPR’s describe the minimum recommended performance characteristics 10 to be used during the evaluation of a method. The evaluation may be an on-site 11 verification, a single-laboratory validation, or a multi-site collaborative study. SMPRs are 12 written and adopted by AOAC Stakeholder Panels composed of representatives from the 13 industry, regulatory organizations, contract laboratories, test kit manufacturers, and 14 academic institutions. AOAC SMPRs are used by AOAC Expert Review Panels in their 15 evaluation of validation study data for method being considered for Performance Tested 16 Methods or AOAC Official Methods of Analysis , and can be used as acceptance criteria for 17 verification at user laboratories. Determination of Phenolic Compounds in Dietary Supplements and Dietary Ingredients Containing Echinacea

18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51

2. Applicability :

Quantitative determination of the individual phenolic compounds including caftaric acid, chlorogenic acid, cichoric acid, cynarin, and echinacoside (table 1) in Echinacea angustifolia DC., E. pallida (Nutt.) Nutt., and E. purpurea (L.) Moench (table 2) in raw materials, and dietary ingredients and dietary supplement products listed in table 3.

3. Analytical Technique :

Any analytical technique that meets the method performance requirements in this SMPR is

acceptable.

4. Definitions :

Dietary ingredients .— A vitamin; a mineral; an herb or other botanical; an amino acid; a dietary substance for use by man to supplement the diet by increasing total dietary intake; or a concentrate, metabolite, constituent, extract, or combination of any of the above dietary ingredients. {United States Federal Food Drug and Cosmetic Act §201(ff) [U.S.C. 321 Dietary supplements .— A product intended for ingestion that contains a “dietary ingredient” intended to add further nutritional value to (supplement) the diet. Dietary supplements may be found in many forms such as tablets, capsules, softgels, gelcaps, liquids, or powders. Echinacea angustifolia – see USP 40 Dietary Supplements, Echinacea 6923; or American Herbal Pharmacopoeia ® ( AHP) Monograph: Echinacea angustifolia root. (ff)]}

Echinacea pallida - see USP 40 Dietary Supplements, Echinacea 6931; or AHP Monograph:

AHP Monograph: Echinacea pallida root

Echinacea purpurea - USP 40 Dietary Supplements, Echinacea 6937; or AHP Monograph: Echinacea purpurea aerial and AHP Monograph: Echinacea purpurea root.

52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98

Limit of Quantitation (LOQ) .— The minimum concentration or mass of analyte in a given

matrix that can be reported as a quantitative result

Raw Materials: Fresh, dried, or cut plant material.

Repeatability .— Variation arising when all efforts are made to keep conditions constant by using the same instrument and operator and repeating during a short time period.

Expressed as the repeatability standard deviation (SD r

); or % repeatability relative standard

deviation (%RSD r ).

Reproducibility .— The standard deviation or relative standard deviation calculated from among-laboratory data. Expressed as the reproducibility relative standard deviation (SD R );

or % reproducibility relative standard deviation (% RSD R ).

Recovery .— The fraction or percentage of spiked analyte that is recovered when the test

sample is analyzed using the entire method.

5. Method Performance Requirements :

See tables 4 and 5.

6. System suitability tests and/or analytical quality control:

Suitable methods will include blank check samples, and check standards at the lowest point and midrange point of the analytical range. A control sample must be included.

7. Reference Material(s):

See table 2 and 7 for a list and sources of reference and testing materials.

Refer to Annex F: Development and Use of In-House Reference Materials in Appendix F: Guidelines for Standard Method Performance Requirements , 20 th Edition of the AOAC

INTERNATIONAL Official Methods of Analysis (2016). Available at:

http://www.eoma.aoac.org/app_f.pdf

8. Validation Guidance :

All target analytes and all claimed matrixes listed in Table 1, 2 and 3 shall be evaluated. Data for at least one analyte per claimed matrix/plant is acceptable provided that all analytes and

claimed matrices/plants are represented in the complete evaluation.

Appendix D: Guidelines for Collaborative Study Procedures tT o Validate Characteristics of a Method of Analysis; 20 h Edition of the AOAC INTERNATIONAL Official Methods of Analysis

(2016). Available at: http://www.eoma.aoac.org/app_d.pdf

Appendix K: Guidelines for Dietary Supplements and Botanicals 20 th Edition of the AOAC INTERNATIONAL Official Methods of Analysis (2012). Also at: . AOAC Int. 95, 268(2016); DOI: 10.5740/jaoacint.11-447 and available at: http://www.eoma.aoac.org/app_k.pdf

99 100 101

8. Maximum Time-To-Determination: No maximum time.

102 103

Table 1: Polyphenols

Alternative CAS #

PubChem ID

MW, g/mol

#

Reference Standard

CAS #

Formula

FDA UNII #

InChI Key

1

Caftaric Acid

67879-58-7

C13H12O9

WCV7W3174L

SWGKAHCIOQPKFW-JTNORFRNSA-N

6440397

312.230

2

Chicoric (Cichoric) Acid

70831-56-0

6537-80-0

C22H18O12

S4YY3V8YHD

YDDGKXBLOXEEMN-IABMMNSOSA-N

5281764

474.374

3

Chlorogenic Acid

327-97-9

C16H18O9

318ADP12RI

CWVRJTMFETXNAD-JUHZACGLSA-N

1794427

354.311

4

Echinacoside

82854-37-3

C35H46O20

I04O1DT48T

FSBUXLDOLNLABB-ISAKITKMSA-N

5281771

786.733

5

1,3-Dicaffeoylquinic Acid (Cynarin) Powdered Echinacea angustifolia Extract

212891-05-9

30964-13-7

C25H24O12

85D81U9JAV

YDDUMTOHNYZQPO-RVXRWRFUSA-N

5281769

516.455

6

84696-11-7

7

Powdered Echinacea pallida Extract

97281-15-7

8

Powdered Echinacea purpurea Extract

90028-20-9

104 105 106 107 108 109

110 111

Table 2: Echinacea

Source (ID number)

Echinacea

CAS number

Form

powdered extract

USP (1231706)

Echinacea angustifolia

root

Alkemist Labs

84696-11-7

herb

Alkemist Labs

powdered extract

USP (1231717)

Echinacea pallida

97281-15-7

root

Alkemist Labs

powdered extract

USP (1231728)

aerial parts

Alkemist Labs

Echinacea purpurea

90028-20-9

root

Alkemist Labs

seed

Alkemist Labs

112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135

Table 3: Dietary Ingredient and Dietary Supplements Matrices

Ingredients:

powders

oils

extracts

encapsulated

Supplements :

powders tablets gummies liquids capsules tinctures gelcaps chewables oils

softgel capsules

136 137

Table 4: LOQ and Analytical Range

Parameter Analytical range

≥ 0.01% to ≤ 5%

LOQ

≤ 10 µg/ml

138 139 140 141 142

Table 5: Method Performance Requirements as a Function of Range

Range*

Parameter

0.01% - 1%

≥ 1%

Recovery (%)

90 – 107%

95- 105 %

% RSD r

≥ 6%

≥ 3%

% RSD R

≥ 8%

≥ 4%

143 144 145

146 147

Table 6: Molecular Structures

POLYPHENOLS

MOLECULAR STRUCTURE

Caftaric Acid

Chicoric (Cichoric) Acid

Chlorogenic Acid

Echinacoside

1,3- Dicaffeoylquinic Acid (Cynarin)

148 149

150 151

Table 7: Sources of Phenolic Compound Reference Materials

CAS NUMBER (alternative)

POLYPHENOLS

Source

PRODUCT NUMBER

Chromadex

ASB-00003028

PhytoLab

89170 15029

Caftaric Acid

67879-58-7

Sigma

USP

1086039

Chromadex

ASB-00003640

Extrasynthese

4987S 89177 C7243

Chicoric (Cichoric) Acid

70831-56-0 (6537-80-0)

PhytoLab

Sigma

USP Acro

1105315

109240000

Alfa Asean Chromadex

J60457

ASB-00003450

Extrasynthese

4991S C2943

Chlorogenic Acid

LKT Labs

327-97-9

MP Biomedicals

0215061801

PhytoLabs

89175 C3878

Sigma

USP

1115545

Chromadex

ASB-00005020

Extrasynthese

4988S E0929 89188

LKT Labs PhytoLab

Echinacoside

82854-37-3

Sigma

01710580 1231750

USP

Chromadex Extrasynthese PhytoLab Sigma USP

ASB-00003990 4995S 89179 91801

1,3- Dicaffeoylquinic Acid (Cynarin)

212891-05-9 (30964-13-7)

152

DRAFT AOAC SMPR 2016.XXX; Version 5; June 29, 2017. 1 2 Method Name: 3 4 5 Approved by: Stakeholder Panel on Dietary Supplements (SPDS) 6 7 Intended Use : For quality assurance and compliance to current good manufacturing practices 8 and possibly detection of adulterants and enforcement of Convention of International Trade in 9 Endangered Species of Wild Fauna and Flora (CITES). Determination of Ginseng in Dietary Supplements and Dietary Ingredients AOAC SMPRs describe the minimum recommended performance characteristics to be used during the evaluation of a method. The evaluation may be an on-site verification, a single- laboratory validation, or a multi-site collaborative study. SMPRs are written and adopted by AOAC Stakeholder Panels composed of representatives from the industry, regulatory organizations, contract laboratories, test kit manufacturers, and academic institutions. AOAC SMPRs are used by AOAC Expert Review Panels in their evaluation of validation study data for method being considered for Performance Tested Methods or AOAC Official Methods of Analysis , and can be used as acceptance criteria for verification at user laboratories. [Refer to Appendix F: Guidelines for Standard Method Performance Requirements , Official Methods of Analysis of AOAC INTERNATIONAL (2012) 20th Ed., AOAC 1. Purpose

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

INTERNATIONAL, Gaithersburg, MD, USA.]

2. Applicability :

Determination of the individual ginsenosides Rb1, Rb2, Rc, Rd, Rf Re, Rg1 and optionally pseudoginsenoside (F11) in Panax ginseng and P. quinquifolius, and P. notoginseng raw materials, dietary ingredients, and dietary supplements materials as listed in Table 1. Optionally the method should provide guidance for differentiation of species and plant

parts.

3. Analytical Technique :

Any analytical technique that meets the following method performance requirements is

acceptable.

4. Definitions :

Dietary ingredient .— A vitamin; a mineral; an herb or other botanical; an amino acid; a dietary substance for use by man to supplement the diet by increasing total dietary intake; or a concentrate, metabolite, constituent, extract, or combination of any of the above dietary ingredients. {United States Federal Food Drug and Cosmetic Act §201(ff) [U.S.C. 321

(ff)]}

Dietary supplement .— A product intended for ingestion that contains a “dietary ingredient” intended to add further nutritional value to (supplement) the diet. Dietary supplements may be found in many forms such as tablets, capsules, softgels, gelcaps, liquids, or powders.

50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77

Ginsenosides See figure 1.

Limit of Quantitation (LOQ) .— The minimum concentration or mass of analyte in a given

matrix that can be reported as a quantitative result

Pseudoginsenoside (F11).

See figure 2.

Raw Materials – Fresh, dried or cut plant materials

Repeatability .— Variation arising when all efforts are made to keep conditions constant by using the same instrument and operator and repeating during a short time period.

Expressed as the repeatability standard deviation (SD r

); or % repeatability relative standard

deviation (%RSD r ).

Reproducibility .— The standard deviation or relative standard deviation calculated from among-laboratory data. Expressed as the reproducibility relative standard deviation (SD R );

or % reproducibility relative standard deviation (% RSD R ).

Recovery .— The fraction or percentage of spiked analyte that is recovered when the test

sample is analyzed using the entire method.

5. Method Performance Requirements :

Table 2: Analytical Range & LOQ Based on Matrix

Parameter Analytical range (mg/g)

≤ 0.5 - ≥ 200

Limit of Quantitation (mg/g) Reported as individual ginsenosides .

≤ 0.5

78 79 80 81

Table 3: Method Performance Requirements as a Function of Range

Parameter

Acceptance Criteria

% Recovery

90 – 110

% RSD r

≤ 7.5

% RSD R

≤ 10

82 83 84 85

Reported as individual ginsenosides.

86 87 88 89 90 91 92 93 94 95 96 97 98 99

6. System suitability tests and/or analytical quality control:

Suitable methods will include blank check samples, and check standards at the lowest point and midrange point of the analytical range. A control sample must be included.

7. Reference Material(s):

NIST Standard Reference Material (SRMs) 3384 NIST Panax ginseng (Asian Ginseng) Rhizome

NIST Standard Reference Material 3385 Panax ginseng (Asian Ginseng) Extract

See table 4 for a list of sources of plant materials.

100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125

See table 5 for a list of sources of individual ginsenonsides, protopanaxdiol, and

pseudoginsenosides.

Refer to Annex F: Development and Use of In-House Reference Materials in Appendix F: Guidelines for Standard Method Performance Requirements , 19 th Edition of the AOAC

INTERNATIONAL Official Methods of Analysis (2012). Available at:

http://www.eoma.aoac.org/app_f.pdf

8. Validation Guidance :

All target analytes and all matrixes listed in Table 3 plus all claimed matrices shall be evaluated. One analyte per matrix is acceptable provided all analytes are represented in the

complete evaluation.

Appendix D: Guidelines for Collaborative Study Procedures To Validate Characteristics of a Method of Analysis; 19 th Edition of the AOAC INTERNATIONAL Official Methods of Analysis

(2012). Available at: http://www.eoma.aoac.org/app_d.pdf

Appendix K: Guidelines for Dietary Supplements and Botanicals 19 th Edition of the AOAC INTERNATIONAL Official Methods of Analysis (2012). Also at: . AOAC Int. 95, 268(2012); DOI: 10.5740/jaoacint.11-447 and available at: http://www.eoma.aoac.org/app_k.pdf

8. Maximum Time-To-Determination: No maximum time.

126 127 128 129

Figure 1: Molecular structures of Ginsenosides

130 131 132

Figure 2: Molecular Structure of Pseudoginsenoside (F11)

133

134 135 136 137 138 139 140 141 142 143 144

Table 1: Required Matrices

Powdered root Powdered extract

Tablets Capsules

Combination – ginseng and one of: (e.g. ginkgo, eleuthra, rhodiola)

145 146

Table 4: Sources of of Plant Materials

Product Name

Product Code

Product Description

Manufacturer

AHP Monograph: American Ginseng Root (PDF)

American Herbal Pharmacopoeia

AHP Monograph for Panax quinquefolius

502

Common Name: Siberian ginseng Genus Species: Eleutherococcus senticosus Plant Part: root Physical Form: cut and sifted Latin name: Eleutherococcus senticosus (Rupr. & Maxim.) Maxim. [Araliaceae] Common Name: Eleuthero (Siberian ginseng); ci wu jia Genus Species: Eleutherococcus senticosus Plant Part: Root Physical Form: whole Common Name: Asian ginseng (hong shen; ren shen) Genus Species: Panax ginseng (red) Plant Part: Root Physical Form: whole Common Name: Asian ginseng (bai ren shen; ren shen) Genus Species: Panax ginseng (white) Plant Part: Root Physical Form: whole Common Name: Asian Ginseng Genus Species: Panax ginseng Plant Part: Root Physical Form: Cut and sifted Latin Name: Panax ginseng C.A. Mey [Araliaceae] Common Name: Tienchi ginseng (tien qi; san qi) Genus Species: Panax pseudoginseng Plant Part: Root Physical Form: Whole Common Name: American ginseng Genus Species: Panax quinquefolius Plant Part: Root Physical Form: whole/dry Latin Name: Panax quinquefolius L. [Araliaceae] Common Name: American ginseng (xi yang shen) Genus Species: Panax quinquefolius Plant Part: Root Physical Form: Whole

Elutherococcus senticosus, root

Alkemist

903528

Elutherococcus senticosus, root (AHP)

American Herbal Pharmacopoeia

596473

American Herbal Pharmacopoeia

Panax ginseng (red), root

530358

American Herbal Pharmacopoeia

Panax ginseng (white), root

536137

Panax ginseng, root

Alkemist

945031

Panax pseudoginseng, root

American Herbal Pharmacopoeia

545272

Panax quinquefolius, root

Alkemist

964728

Panax quinquefolius, root (AHP)

American Herbal Pharmacopoeia

514442

147 148

Table 5: Sources of Individual Ginsenonsides, Protopanaxdiol, and Pseudoginsenosides. 149 150 151 Product Name Product Code CAS# Ginsenoside CK 0150S [39262-14-1] Ginsenoside CK 25mg 0150S 25mg [39262-14-1] Ginsenoside Rb1 0105S [41753-43-9] Ginsenoside Rb1 25mg 0105S 25mg [41753-43-9] Ginsenoside Rb2 0104S [11021-13-9] Ginsenoside Rb2 25mg 0104S 25mg [11021-13-9] Ginsenoside Rb3 0151S [68406-26-8] Ginsenoside Rb3 25mg 0151S 25mg [68406-26-8] Ginsenoside Rc 0106S [11021-14-0] Ginsenoside Rc 25mg 0106S 25mg [11021-14-0] Ginsenoside Rd 0102S [52705-93-8] Ginsenoside Rd 25mg 0102S 25mg [52705-93-8] Ginsenoside Re 0103S [52286-59-6] Ginsenoside Re 25mg 0103S 25mg [52286-59-6] Ginsenoside Rf 0107S [52286-58-5] Ginsenoside Rf 25mg 0107S 25mg [52286-58-5] Ginsenoside Rg1 0101S [22427-39-0] Ginsenoside Rg1 25mg 0101S 25mg [22427-39-0] Ginsenoside Rg2 0108S [52286-74-5] Ginsenoside Rg2 25mg 0108S [52286-74-5] Ginsenoside Rg3 0152S [14197-60-5] Ginsenoside Rg3 25mg 0152S 25mg [14197-60-5] Ginsenoside Rh1 0153S [63223-86-9] Ginsenoside Rh1 25mg 0153S 25mg [63223-86-9] Ginsenoside Rh2 0154S [78214-33-2] Ginsenoside Rh2 25mg 0154S 25mg [78214-33-2] Protopanaxadiol 2308 [7755-01-3] Protopanaxatriol 2307 [1453-93-6] Pseudoginsenoside F11 0155S [69884-00-0] 152

AOAC INTERNATIONAL STAKEHOLDER PANEL ON  DIETARY SUPPLEMENTS Joseph Zhou, Sunshineville Health Products SAMe Working Group ‐ SMPR Presentation September 22, 2017

Atlanta Marriott Marquis, Atlanta, Georgia, USA

Fitness for Purpose As Agreed March 17, 2017

Methods for the determination of SAMe in dietary  ingredients and finished products.  Primary  objective:  Method should have capability to  separate SAMe from decomposition products and  synthetic precursors, as well as other joint support  materials.   Secondary objective:  Consider separation of  racemic isomers.

SPDS SAMe Working Group Members

• Joseph Zhou, Sunshineville Health Products • LaVerne Brown, NIH/ODS • Adam Kuszak, NIH/ODS • Punam Patel, Phamravite • Catherine Rimmer, NIST • Aniko Solyom, GAAS Analytical • John Szpylka, Mérieux NutriSciences • John Travis, NSF International • Jinchaun Yang, Waters • Hong You, Eurofins • Garrett Zielinski, Covance • Sunny Zhou, Northeastern University

SAMe Working Group Work to Date

• 5 teleconferences (April 2017 – June 2017) • 1 SMPR Drafted  • Public comment period (July, 2017) • SMPRs made ready for SPDS review and approval

Background

Molecular Structure of (S, S) S - Adenosyl-L-Methionine

NH 2

N

N

CH 3

(S)

_

+

(S)

S

OOC H 2 N

N N

O

H

HO OH

Background

SAMe Finished Products (Dietary Supplements)

• One of the most popular dietary supplements; • Popular Product Format: Tablets in Blister Pack; • Dosage: 200mg-400mg/Tablet, 2-4 Tablets daily; • Principal Structure Function: Methyl Donor • Medical Uses: Depression, Parkinson’s, Osteoarthritis, Liver disease …

Challenge: SAMe’s Extreme Instability

• S-adenosyl-L-homocysteine (SAH) • Adenosine (ADE) • Deoxy-methylthioadenosine (DMTA) • Etc… Irreversible, Significant

Degradation

SAMe Bio-Active, (S,S)

(1)

(2)

SAMe Bio-Inactive, (R,S)

Reversible , Insignificant

SMPR Key Points

Method Name Determination of SAMe in Dietary Supplements and Dietary Ingredients Intended Use For quality assurance and compliance to current good manufacturing practices Applicability Determination of total (S,S and R,S isomers) SAM-e in dietary ingredients and dietary supplements

Analytical Technique Any analytical technique that meets the method performance requirements is acceptable

SMPR Key Points

Table 1: Analytical Range & LOQ Based on Matrix

Dietary Supplements and  Ingredients

Parameter

≥ 5 to ≤ 800

Analytical range (mg/g)

Limit of Quantitation (mg/g)

1

Note: Results are in Total SAMe

SMPR Key Points

Table 2: Method Performance Requirements  as a Function of Range

Range

Parameter

≤ 15 mg/g

> 15 mg/g

Recovery (%)

≥ 90

≥ 98

% RSD r

≤ 8

≤ 5

% RSD R

≤ 10

≤ 8

Note: Results are in Total SAMe

SMPR Key Points

System Suitability Tests and/or  Analytical Quality Control

Suitable methods will include blank check  samples, and check standards at the lowest point  and midrange point of the analytical range.  A  control sample must be included.

SMPR Key Points

Reference Material

• USP Ademetionine Disulfate Tosylate (S‐Adenosyl‐L‐ Methionine Disulfate Tosylate; Catalog no. 1012134

• Sigma S‐(5’‐Adenosyl)‐L‐methionine p‐ toluenesulfonate salt – from yeast (L‐methionine enriched) Product number: A2308, CAS Number  17176‐17‐9

SMPR Key Points

Validation Guidance Method must be able to separate SAMe from:  1. Decomposition Products: • p‐toluenesulfonic acid (PTSA),  • S‐adenosylhomocysteine (SAH),  • Adenosine,  • 5'‐deoxy‐5'‐methylthioadenosine (DMTA).

SMPR Key Points

Validation Guidance Method must be able to separate SAMe from:  2. Synthetic Precursors / Stabilizing Agents: • Methionine,  • ATP (adenosine triphosphate),  • Toluene  • Sulfonic Acid

SMPR Key Points

Validation Guidance Method must be able to separate SAMe from: 3. Other Ingredients Used in the Dietary Supplements  (Finished Products):  Vitamin C  Glutathione  Folic acid  B12

SMPR Key Points

Validation Guidance

STABILITY:  Method  developers must provide data 

demonstrating that SAMe remains stable in test  samples for the maximum time‐to‐determination.