Meeting of the AOAC Stakeholder Panel on Dietary ... · Draft, Do Not Distribute Meeting of the AOAC Stakeholder Panel on Dietary Supplements Atlanta Marriott Marquis 8:30 a.m. –

Draft, Do Not Distribute

Meeting of the AOAC Stakeholder Panel on Dietary Supplements

Atlanta Marriott Marquis

8:30 a.m. – 5:00 p.m. ET, October 6, 2017

Stakeholder Attendees:

Darryl Sullivan, Covance Laboratories (Chair) Cristina Amarillas, Traditional Medicinals Melissa Phillips, US NIST Karen Andrews, USDA Prasad Rallabhandi, US FDA Gisele Atkinson, CRN Klaus Reif, PhytoLab GmbH & Co. KG John Austad, Covance Laboratories Lars Reimann, Eurofins Joseph Betz, US NIH John Reuther, Eurofins Simon Bevis, R-Biopharm Rhone Kate Rimmer, US NIST Sneh Bhandari, Silliker Laboratories Joe Romano, Waters Corporation Paula Brown, BCIT Leila Saldanha, ODD/CRN Paul Burns, Eurofins Scientific Richard Sanders, Covance Laboratories Anton Bzhelyansky, USP Andre Santos, Agilent Technologies Kelsey Chandler, Covance Laboratories Brian Schaneberg, Starbucks (Vice-Chair) Chung Cho, US FDA Jeff Shippar, Covance Laboratories Bob Clifford, Shimadzu Aniko Solyom, GAAS Analytical Jean-Luc Deborde, SCL Joan Stevens, Agilent Technologies Steven Dentali, Dentali Botanical Sciences Sidney Sudberg, Alkemist Labs Eric Gordon, Covance Laboratories John Szpylka, Mérieux NutriSciences Philip Haselberger, Abbott Nutrition Melissa Thompson, Covance Laboratories Adam Horkey, Schwabe North America Son Tran, National Institute for Food Control Martha Jennens, Covance Laboratories John Travis, NSF International David Ji, Covance Laboratories Richard van Breemen, Linus Pauling Institute, OSU Holly Johnson, Alkemists Labs Wayne Wargo, Abbott Nutrition Ron Johnson, bioMérieux Stephen Wise, US NIH/ODS Scott Krepich, Phenomenex Laura Wood, US NIST Mary Krogull, Eurofins Scientific, Inc. Jinchuan Yang, Waters Corporation Adam Kuszak, NIH Office of Dietary Supplements Zhang Yi, Shenzhen CIQ Hao Le, National Institute for Food Control Kurt Young, Nutra Manufacturing/GNC John Lee, Agilent Technologies Zhenfeng Yue, Shenzhen CIQ Katerina Mastovska, Covance Laboratories Hui Zhao, Covance Laboratories Linda Messick, Covance Laboratories Joseph Zhou, Sunshineville Maria Monagas, USP Garrett Zielinski, Covance Laboratories Maria Ofitserova, Pickering Laboratories Jerry Zweigenbaum, Agilent Technologies

AOAC Staff Attendees:

Delia Boyd, Scott Coates, Christopher Dent, Dawn Frazier, Nora Marshall, Tien Milor, La’Kia Phillips

Meeting Minutes

I. Welcome and IntroductionsAOAC President Ron Johnson opened the meeting highlighting the AOAC standards developmentprocess and he invited attendees to review the AOAC policies and procedures, in the meeting book.

II. Ingredient UpdatesDarryl Sullivan, SPDS Chair, then began a presentation1 describing SPDS and the ingredients forwhich the stakeholders have discussed to date. He reviewed all completed and in-progressStandard Method Performance Requirements® (SMPR®) developed for this project, as well as allcurrent and past Calls for Experts and Calls for Methods.

III. SMPR Approval PresentationsA. Ginseng-

Paula Brown, Ginseng Working Group Chair, took the floor to present the draft SMPR forGinseng. The stakeholders made some editorial changes within the document. After furtherdiscussion, the group agreed to modify the applicability statement to make P. notoginsengoptional.

MOTION to approve the SMPR for Determination of Select Ginsenosides in Dietary Supplementsand Dietary Ingredients. (Brown/Bhandari)17 in favor, 1 opposed (Rimmer) and 1 abstention (Betz). The motion passed.2

B. Echinacea-Brian Schaneberg, SPDS Vice-Chair, presented the draft SMPR on behalf of the EchinaceaWorking Group and its Chair, Stefan Gafner. His presentation3 included background informationon the working group, key points in the draft SMPR, and a motion to approve. Discussion on thedraft SMPR followed. The panel agreed to re-title Table 3 to “Examples of Dietary Ingredientand Dietary Supplements Matrices” and also to remove “oils” from the list because essential oilsare out of scope. A few other minor changes were made and Sullivan called for a vote.

MOTION to approve the SMPR for Determination of Phenolic Compounds in DietarySupplements and Dietary Ingredients Containing Echinacea (Schaneberg / Brown).19 in favor, 0 opposed, 0 abstentions. The motion passed.4

C. SAMe-Joseph Zhou, SAMe Working Group Chair, then took the floor with a presentation5 on the draftSMPR. Zhou reviewed background information of the working group, including all work to date,as well as the key points in the proposed SMPR. The panel spent some time reviewing the key

1 SPDS Overview Presentation (Sullivan) 2 Ginseng SMPR as Approved 3 Echinacea Approval Presentation (Gafner) 4 Echinacea SMPR as Approved 5 SAMe Approval Presentation (Zhou)

points of the method performance table. With all points agreed, Zhou made a motion to approve the SMPR. MOTION to approve the SMPR for Determination of SAM-e in Dietary Ingredients and Dietary Supplements (Zhou / Solyom).

IV. Launch of Set 8 Working Groups

A. Kavalactones- Steven Dentali took the floor with a presentation6 introducing stakeholders to Kavalactones. He reviewed the history and uses, preparations, biochemistry, pharmacology, analytical significance, and existing methods. He concluded the presentation with a proposed fitness for purpose statement, to which there were no objections. The Fitness for Purpose for Kavalactones was agreed as follows:

“The method identifies and quantifies the six primary kavalactones derived from the underground portions of kava (Piper methysticum), namely desmethoxyangonin, dihydrokavain, yangonin, kavain, dihydromethysticin, and methysticin in plant material, dietary ingredients and dietary supplements. The method identifies and quantifies flavokavains A, B, and C in kava plant material, dietary supplements and dietary ingredients.

Test results can be used in chemotype identification but this specific determination is outside the method scope. Individual kavalactones should be quantifiable within the range of 0.1 to 50 percent by weight in forms that include liquid, soft, and dry extracts as well as in softgels, capsules, and tablets in the presence of common excipients. The ability to address kavalactones in beverages is an advantage but not a requirement. No limit on analysis time is imposed.”

The stakeholder panel agreed by straw poll and the Kavalactones Working Group was formally launched.

B. Resveratrol-

Richard van Breemen took the floor with a presentation7 on Resveratrol. He reviewed the background of the analyte, sources, appropriate dietary levels, analytical techniques, and a proposed fitness for purpose statement: The method for resveratrol dietary supplement analysis must quantitate both cis and trans forms in a variety of dosage forms. After a group discussion, the panel refined this to state:

The method must separate the cis and trans forms of resveratrol and quantitate the trans isomer in dietary supplements and dietary ingredients.

6 Kavalactones Launch Presentation (Dentali) 7 Resveratrol Launch Presentation (van Breemen)

By straw poll, SPDS accepted this revised fitness for purpose statement and formally launched the Resveratrol working group.

C. Scullcap-Holly Johnson took the floor with a presentation8 on the final Set 8 ingredient, Scullcap (alsospelled Skullcap). She reviewed the goals of this working group, the background of the plant,issues including adulteration, and current methodologies. She then opened the floor to adiscussion on what would make an appropriate fitness for purpose statement. Followingdiscussion on the different analytical challenges facing this ingredient, the panel agreed that twoSMPRs will be required, and agreed to allow the working group to develop the fitness forpurpose at their first meeting.

V. Adjourn

The group agreed to the plan of action. Actions were assigned and the meeting adjourned atapproximately 4:30 pm, ET.

Attachments

• SPDS Overview Presentation (Sullivan)

• Ginseng SMPR as Approved

• Echinacea Approval Presentation (Gafner)

• Echinacea SMPR as Approved

• SAMe Approval Presentation (Zhou)

• SAMe SMPR as Approved

• Kavalactones Launch Presentation (Dentali)

• Resveratrol Launch Presentation (van Breemen)

• Scullcap Launch Presentation (Johnson)

8 Scullcap Launch Presentation (Johnson)

AOAC Stakeholder Panel on Dietary Supplements (SPDS):

Background and Updates(SPDS)

Darryl Sullivan, ChairStakeholder Panel on Dietary Supplements

Covance Laboratories

September 22, 2017

AOAC SPDS History

• AOAC INTERNATIONAL signed a 5‐year contract with theNational Institutes of Health‐Office of Dietary Supplements(NIH/ODS) to establish voluntary consensus standards forhigh‐priority ingredients.

• Develop standard method performance requirements(SMPRs) for 25 priority dietary supplement ingredients.

• Deliver First Action Official MethodsSM for the prioritizeddietary supplement ingredients

• Encourage participation with the dietary supplementsindustry 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 Inhibitormethod

– Call for Methods for Anthocyanins is currently OPEN


• 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


• 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 TeaDietary Ingredients and Supplements (2015.014)

• Determination of Aloin A and Aloin B in Dietary Supplement Products andIngredients (2015.015)

• Determination of Vitamin D in Dietary Supplement Finished Products andIngredients (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.

• 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 andCollagen.



• Set 5 Ingredients: Aloe Vera, Protein, Vitamin B12

– 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 B12 in Dietary Supplements and

Ingredients (2016.017).

– Call for Methods and Experts are currently OPEN.


• Set 6 Ingredients: Amino Acids, Ginger, Vitamins K1 and K2

– 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 K1 and K2 in Dietary Supplements and Dietary

Ingredients (2017.013)

– Call for Methods and Experts are currently OPEN.


• 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 confirmedthe 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 atwww.aoac.org

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

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

Contact Information

Darryl Sullivan, Chair SPDS

Covance Laboratories

Tel: 608.242.2711Email: [email protected]

Brian Schaneberg, Vice Chair, SPDS

Starbucks Corporation

Email: [email protected]

Contact AOAC Staff:

Tel: 301.924.7077Web: www.aoac.org• Deborah McKenzie, Sr. Director, Standards Development and AOAC Research Institute,

[email protected], ext. 157• Dawn Frazier, Sr. Executive for Scientific Business Development, [email protected], ext.

117

AOAC INTERNATIONALSTAKEHOLDER PANEL ON DIETARY SUPPLEMENTSStefan Gafner, American Botanical Council

Echinacea Working Group ‐ SMPR PresentationSeptember 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 andapproval

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 andtreatment of common cold (oral intake), or topically for thetreatment of small superficial wounds

Background (continued)

• The phytochemicals responsible for theimmunostimulant properties of Echinacea spp.are not known

• The following compound classes have beenlinked to bioactivity:– Alkylamides (alkamides)– Phenolic compounds– Polysaccharides– LPS and lipoproteins produced by bacterialendophytes

Main Echinacea phenolics

Cichoric acid Caftaricacid

Echinacoside Chlorogenicacid

Cynarine1

E. angustifolia <LOD – 0.05 <LOD – 0.02 0.13 – 1.70 <LOD – 0.15 0.07 – 0.34

E. pallida <LOD – 0.22 0.01 – 0.08 0.13 – 1.27 <LOD – 0.30 <LOD

E. purpura root 0.33 – 2.78 0.35 – 0.80 <LOD <LOD – 0.19 <LOD

E. purpurea tops 0.52 – 2.20 0.18 – 0.85 <LOD <LOD – 0.03 <LOD

Concentrations in % of dried plant part

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.

Cynarine

Cichoric acid

Caftaric acid

Echinacoside

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

Existing Methods (General)

• Abundance of published methods, mainly using HPLC‐UVor HPLC‐MS

• UV/Vis spectrophotometry (Folin‐Ciocalteu) used for totalphenolic compounds

• HPTLC, CE‐UV infrequently used• Established methods include:

– Official methods of the United States Pharmacopeia and EuropeanPharmacopoeia

– 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 herbby 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 forphenolic compounds

– Echinacea pallida root (whole or cut): HPLC‐UV for phenoliccompounds

– Echinacea purpurea root (whole or cut): HPLC‐UV forphenolic compounds

– Echinacea purpurea dried herb (whole or cut): HPLC‐UV forphenolic compounds

SMPR Key Points

• Amounts of individual phenolics vary substantiallyamong species and plant parts: methodperformance criteria will depend on concentration

• Chlorogenic acid ‐ if present – is found at lowconcentrations: larger amounts may indicateaddition of extraneous material

• Echinacea products combined with other plantextracts will require additional validation (e.g.,selectivity)

• Analyte standards and vouchered botanicalmaterials are commercially available (no NISTstandards)

Comments Submitted (if any)

• No comments submitted.

Motion

• Move to accept the StandardMethod PerformanceRequirements for Determination ofPhenolic Compounds in DietarySupplements and DietaryIngredients Containing Echinaceaas presented.

Discussion?

DRAFT AOAC SMPR 2017.XXX; Version 9; September 22, 2017 1 2

Method Name: Determination of Phenolic Compounds in Dietary Supplements and 3 Dietary Ingredients Containing Echinacea 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 characteristics10

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. 18

19 2. Applicability:20

Quantitative determination of the individual phenolic compounds including caftaric acid, 21 chlorogenic acid, cichoric acid, cynarin, and echinacoside (table 1) in Echinacea angustifolia 22 DC., E. pallida (Nutt.) Nutt., and E. purpurea (L.) Moench (table 2) in raw materials, and 23 dietary ingredients and dietary supplement products listed in table 3. 24

25 3. Analytical Technique:26

Any analytical technique that meets the method performance requirements in this SMPR is 27 acceptable. 28

29 4. Definitions:30

31 Dietary ingredients.— A vitamin; a mineral; an herb or other botanical; an amino acid; a 32 dietary substance for use by man to supplement the diet by increasing total dietary intake; 33 or a concentrate, metabolite, constituent, extract, or combination of any of the above 34 dietary ingredients. {United States Federal Food Drug and Cosmetic Act §201(ff) [U.S.C. 321 35 (ff)]} 36

37 Dietary supplements.— A product intended for ingestion that contains a “dietary ingredient” 38 intended to add further nutritional value to (supplement) the diet. Dietary supplements may 39 be found in many forms such as tablets, capsules, softgels, gelcaps, liquids, or powders. 40

41 Echinacea angustifolia – see USP 40 Dietary Supplements, Echinacea 6923; or American 42 Herbal Pharmacopoeia® (AHP) Monograph: Echinacea angustifolia root. 43

44 Echinacea pallida - see USP 40 Dietary Supplements, Echinacea 6931; or AHP Monograph: 45 AHP Monograph: Echinacea pallida root 46

47 Echinacea purpurea - USP 40 Dietary Supplements, Echinacea 6937; or AHP Monograph: 48 Echinacea purpurea aerial and AHP Monograph: Echinacea purpurea root. 49

50 51

Limit of Quantitation (LOQ) .— The minimum concentration or mass of analyte in a given 52 matrix that can be reported as a quantitative result 53

54 Raw Materials: Fresh, dried, or cut plant material. 55

56 Repeatability .— Variation arising when all efforts are made to keep conditions constant by 57 using the same instrument and operator and repeating during a short time period. 58 Expressed as the repeatability standard deviation (SDr); or % repeatability relative standard 59 deviation (%RSDr). 60

61 Reproducibility.— The standard deviation or relative standard deviation calculated from 62 among-laboratory data. Expressed as the reproducibility relative standard deviation (SDR); 63 or % reproducibility relative standard deviation (% RSDR). 64

65 Recovery.— The fraction or percentage of spiked analyte that is recovered when the test 66 sample is analyzed using the entire method. 67

68 5. Method Performance Requirements:69

See tables 4 and 5. 70 71

6. System suitability tests and/or analytical quality control:72 Suitable methods will include blank check samples, and check standards at the lowest point 73 and midrange point of the analytical range. A control sample must be included. 74

75 7. Reference Material(s):76

77 See table 2 and 7 for a list and sources of reference and testing materials. 78

79 Refer to Annex F: Development and Use of In-House Reference Materials in Appendix F: 80 Guidelines for Standard Method Performance Requirements, 20th Edition of the AOAC 81 INTERNATIONAL Official Methods of Analysis (2016). Available at: 82 http://www.eoma.aoac.org/app_f.pdf 83

84 8. Validation Guidance:85

86 All target analytes and examples of claimed matrixes listed in Table 1, 2 and 3 shall be 87 considered. Data for at least one analyte per claimed matrix/plant is acceptable provided 88 that all analytes and claimed matrices/plants are represented in the complete evaluation. 89

90 Appendix D: Guidelines for Collaborative Study Procedures tTo Validate Characteristics of a 91 Method of Analysis; 20h Edition of the AOAC INTERNATIONAL Official Methods of Analysis 92 (2016). Available at: http://www.eoma.aoac.org/app_d.pdf 93

94 Appendix K: Guidelines for Dietary Supplements and Botanicals 20th Edition of the AOAC 95 INTERNATIONAL Official Methods of Analysis (2012). Also at: . AOAC Int. 95, 268(2016); DOI: 96 10.5740/jaoacint.11-447 and available at: http://www.eoma.aoac.org/app_k.pdf 97

98 8. Maximum Time-To-Determination: No maximum time.99

100 101

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


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

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


Table 1: Polyphenols 102 103

# Reference Standard CAS # Alternative CAS # Formula FDA UNII # InChI Key PubChem

ID MW,

g/mol

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) 212891-05-9 30964-13-7 C25H24O12 85D81U9JAV YDDUMTOHNYZQPO-RVXRWRFUSA-N 5281769 516.455

6 Powdered Echinacea angustifolia Extract 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

https://pubchem.ncbi.nlm.nih.gov/compound/6440397





Table 2: Echinacea 110 111

Echinacea CAS number Form Source (ID number)

Echinacea angustifolia 84696-11-7

powdered extract USP (1231706)

root Alkemist Labs

herb Alkemist Labs

Echinacea pallida 97281-15-7 powdered extract USP (1231717)

root Alkemist Labs

Echinacea purpurea 90028-20-9

powdered extract USP (1231728)

aerial parts Alkemist Labs

root Alkemist Labs

seed Alkemist Labs

112 113

Table 3: Examples of Dietary Ingredient and Dietary Supplements Matrices 114 115

Ingredients: 116 extracts (including encapsulated) 117 Herb Powders 118 Juice 119

120 121

Supplements : 122 oils 123 powders 124 tablets 125 gummies 126 liquids 127 capsules 128 softgel capsules 129 tinctures 130 gelcaps 131 chewables 132 juice 133

134 135 136

Table 4: LOQ and Analytical Range 137 138

Parameter

Analytical range 0.01% to 5%

LOQ ≤ 0.01%

139 140 141

Table 5: Method Performance Requirements as a Function of Range 142 143

Parameter Range

0.01% - 1% > 1%

Recovery (%) 90 – 107% 95- 105 %

% RSDr ≤ 6% ≤ 3%

% RSDR ≤ 8% ≤ 4%

144 145 146

Table 6: Molecular Structures 147 148

POLYPHENOLS MOLECULAR STRUCTURE

Caftaric Acid

Chicoric (Cichoric) Acid

Chlorogenic Acid

Echinacoside

1,3-Dicaffeoylquinic Acid (Cynarin)

149 150

Table 7: Sources of Phenolic Compound Reference Materials 151 152

POLYPHENOLS CAS NUMBER (alternative) Source PRODUCT NUMBER

Caftaric Acid 67879-58-7

Chromadex ASB-00003028 PhytoLab 89170 Sigma 15029 USP 1086039

Chicoric (Cichoric) Acid

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

Chromadex ASB-00003640 Extrasynthese 4987S PhytoLab 89177 Sigma C7243 USP 1105315

Chlorogenic Acid 327-97-9

Acro 109240000 Alfa Asean J60457 Chromadex ASB-00003450 Extrasynthese 4991S LKT Labs C2943 MP Biomedicals 0215061801 PhytoLabs 89175 Sigma C3878 USP 1115545

Echinacoside 82854-37-3

Chromadex ASB-00005020

Extrasynthese 4988S LKT Labs E0929 PhytoLab 89188 Sigma 01710580 USP 1231750

1,3-Dicaffeoylquinic Acid (Cynarin)

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

Chromadex Extrasynthese PhytoLab Sigma USP

ASB-00003990 4995S 89179 91801

153

DRAFT AOAC SMPR 2017.XXX; Version 6; September 22, 2017. 1 2

Method Name: Determination of Select Ginsenosides in Dietary Supplements and 3 Dietary Ingredients 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). 10

11 1. Purpose12

13 AOAC SMPRs describe the minimum recommended performance characteristics to be used 14 during the evaluation of a method. The evaluation may be an on-site verification, a single-15 laboratory validation, or a multi-site collaborative study. SMPRs are written and adopted by 16 AOAC Stakeholder Panels composed of representatives from the industry, regulatory 17 organizations, contract laboratories, test kit manufacturers, and academic institutions. 18 AOAC SMPRs are used by AOAC Expert Review Panels in their evaluation of validation study 19 data for method being considered for Performance Tested Methods or AOAC Official 20 Methods of Analysis, and can be used as acceptance criteria for verification at user 21 laboratories. [Refer to Appendix F: Guidelines for Standard Method Performance 22 Requirements, Official Methods of Analysis of AOAC INTERNATIONAL (2012) 20th Ed., AOAC 23 INTERNATIONAL, Gaithersburg, MD, USA.] 24


Determination of the individual ginsenosides Rb1, Rb2, Rc, Rd, Rf, Re, Rg1 and optionally 27 pseudoginsenoside (F11) and notoginsenoside R1 in Panax ginseng and P. quinquefolius, and 28 optionally P. notoginseng raw materials, dietary ingredients, and dietary supplements 29 materials as listed in Table 1. 30

Optionally the method should provide guidance for differentiation of species and plant parts 31 based on quantitative ratios of ginsenosides and/or species specific ginsenosides. 32

3. Analytical Technique:33 Any analytical technique that meets the following method performance requirements is 34 acceptable. 35


38 Dietary ingredient.— A vitamin; a mineral; an herb or other botanical; an amino acid; a 39 dietary substance for use by man to supplement the diet by increasing total dietary intake; 40 or a concentrate, metabolite, constituent, extract, or combination of any of the above 41 dietary ingredients. {United States Federal Food Drug and Cosmetic Act §201(ff) [U.S.C. 321 42 (ff)]} 43

44 Dietary supplement.— A product intended for ingestion that contains a “dietary ingredient” 45 intended to add further nutritional value to (supplement) the diet. Dietary supplements may 46 be found in many forms such as tablets, capsules, softgels, gelcaps, liquids, or powders. 47

48 49

50 Ginsenosides 51 See figure 1. 52

53 54

Limit of Quantitation (LOQ) .— The minimum concentration or mass of analyte in a given 55 matrix that can be reported as a quantitative result 56

57 Pseudoginsenoside (F11). 58 See figure 2. 59

60 Raw Materials – Fresh, dried or cut plant materials 61




74 75

5. Method Performance Requirements:76 77

Table 2: Analytical Range & LOQ Based on Matrix 78 Parameter

Analytical range (mg/g) 0.5 - 200

Limit of Quantitation (mg/g) ≤ 0.5 Reported as individual ginsenosides . 79

80 Table 3: Method Performance Requirements as a Function of Range 81

82

Parameter Acceptance Criteria

% Recovery 90 – 110

% RSDr ≤ 7.5

% RSDR ≤ 10

Reported as individual ginsenosides. 83 It is recognized that the individual ginsenosides will vary. Individual presence and levels are dependent upon species. 84

85

86 87

6. System suitability tests and/or analytical quality control:88 Suitable methods will include blank check samples, and check standards at the lowest point 89 and midrange point of the analytical range. A control sample must be included. 90

91 92 93

7. Reference Material(s):94 95

NIST Standard Reference Material (SRMs) 3384 96 NIST Panax ginseng (Asian Ginseng) Rhizome 97 NIST Standard Reference Material 3385 Panax ginseng (Asian Ginseng) Extract 98

99 See table 4 for a list of sources of plant materials. 100

101 See table 5 for a list of sources of individual ginsenosides, protopanaxdiol, and 102 pseudoginsenosides. 103

104 Refer to Annex F: Development and Use of In-House Reference Materials in Appendix F: 105 Guidelines for Standard Method Performance Requirements, 19th Edition of the AOAC 106 INTERNATIONAL Official Methods of Analysis (2012). Available at: 107 http://www.eoma.aoac.org/app_f.pdf 108


111 All target analytes and all matrixes listed in Table 3 plus all claimed matrices shall be 112 evaluated. One analyte per matrix is acceptable provided all analytes are represented in the 113 complete evaluation. 114

115 Appendix D: Guidelines for Collaborative Study Procedures To Validate Characteristics of a 116 Method of Analysis; 19th Edition of the AOAC INTERNATIONAL Official Methods of Analysis 117 (2012). Available at: http://www.eoma.aoac.org/app_d.pdf 118


123 8. Maximum Time-To-Determination: No maximum time.124

125 126 127






Figure 1: Molecular structures of Ginsenosides 128 129 130 131

132 133

Figure 2: Molecular Structure of Pseudoginsenoside (F11) 134

135

Table 1: Required Matrices 136 137 138 Powdered root 139 Powdered extract 140 Tablets 141 Capsules 142 Combination – ginseng and one of: (e.g. Ginkgo biloba, Eleutherococcus senticosus, Rhodiola 143 rosea) 144

145 146 147

Table 4: Sources of of Plant Materials 148 149

Product Name Product Description Manufacturer

Product Code

AHP Monograph: American Ginseng Root (PDF)

AHP Monograph for Panax quinquefolius American Herbal Pharmacopoeia 502

Elutherococcus senticosus, root

Common Name: Siberian ginseng Genus Species: Eleutherococcus senticosus Plant Part: root Physical Form: cut and sifted Latin name: Eleutherococcus senticosus (Rupr. & Maxim.) Maxim. [Araliaceae]

Alkemist 903528

Elutherococcus senticosus, root (AHP)

Common Name: Eleuthero (Siberian ginseng); ci wu jia Genus Species: Eleutherococcus senticosus Plant Part: Root Physical Form: whole

American Herbal Pharmacopoeia 596473

Panax ginseng (red), root

Common Name: Asian ginseng (hong shen; ren shen) Genus Species: Panax ginseng (red) Plant Part: Root Physical Form: whole


Panax ginseng (white), root

Common Name: Asian ginseng (bai ren shen; ren shen) Genus Species: Panax ginseng (white) Plant Part: Root Physical Form: whole


Panax ginseng, root

Common Name: Asian Ginseng Genus Species: Panax ginseng Plant Part: Root Physical Form: Cut and sifted Latin Name: Panax ginseng C.A. Mey [Araliaceae]

Alkemist 945031

Panax pseudoginseng, root

Common Name: Tienchi ginseng (tien qi; san qi) Genus Species: Panax pseudoginsengPlant Part: Root Physical Form: Whole


Panax quinquefolius, root

Common Name: American ginseng Genus Species: Panax quinquefoliusPlant Part: Root Physical Form: whole/dry Latin Name: Panax quinquefolius L. [Araliaceae]

Alkemist 964728

Panax quinquefolius, root (AHP)

Common Name: American ginseng (xi yang shen) Genus Species: Panax quinquefoliusPlant Part: Root Physical Form: Whole


150 151

Table 5: Sources of Individual Ginsenonsides, Protopanaxdiol, and Pseudoginsenosides. 152 153 154

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]

155

AOAC INTERNATIONALSTAKEHOLDER PANEL ON DIETARY SUPPLEMENTSJoseph Zhou, Sunshineville Health Products

SAMe Working Group ‐ SMPR PresentationSeptember 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

N

NN

N

NH2

O

HO OH

SOOC

H2N

+_

H

(S) (S)

CH3

Molecular Structure of

(S, S) S - Adenosyl-L-Methionine

Background

• 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 …

SAMe Finished Products (Dietary Supplements)

Challenge: SAMe’s Extreme Instability

SAMeBio-Active, (S,S)

Degradation

• S-adenosyl-L-homocysteine(SAH)

• Adenosine (ADE)• Deoxy-methylthioadenosine

(DMTA)• Etc… Irreversible,

Significant

(1)

SAMeBio-Inactive, (R,S)

Reversible,Insignificant

(2)

SMPR Key Points

Method NameDetermination of SAMe in Dietary Supplements and Dietary Ingredients

Intended UseFor quality assurance and compliance to current good manufacturing practices

ApplicabilityDetermination of total (S,S and R,S isomers) SAM-e in dietary ingredients and dietary supplements

Analytical TechniqueAny analytical technique that meets the method performance requirements is acceptable

SMPR Key Points

Table 1: Analytical Range & LOQ Based on Matrix

ParameterDietary Supplements and

Ingredients

Analytical range (mg/g) ≥ 5 to ≤ 800

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

ParameterRange

≤ 15 mg/g > 15 mg/g

Recovery (%) ≥ 90 ≥ 98

% RSDr ≤ 8 ≤ 5

% RSDR ≤ 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‐methionineenriched) Product number: A2308, CAS Number17176‐17‐9

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


2. Synthetic Precursors / Stabilizing Agents:

• Methionine,• ATP (adenosine triphosphate),• Toluene• Sulfonic Acid

SMPR Key Points

Validation Guidance


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.

SMPR Key Points

SAMe Stability

Maximum Time‐To‐Determination

Method developers must specify amaximum time‐to‐determination; and

Less than the stability time of SAMe samples

Comments Submitted

• No comments submitted.

Motion

• Move to accept the StandardMethod PerformanceRequirements forDetermination of SAMe inIngredients and Supplements aspresented.

Discussion?

DRAFT AOAC SMPR 2016.XXX; Version 7; September 22, 2017. 1 2

Method Name: Determination of SAM-e in Dietary Ingredients and Dietary 3 Supplements 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 characteristics10

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. 18


Determination of total (S,S and R,S isomers) SAM-e in dietary ingredients and dietary 21 supplements. 22

3. Analytical Technique:23

Any analytical technique that meets the following method performance requirements is 24 acceptable. 25


28 Dietary ingredients.— A vitamin; a mineral; an herb or other botanical; an amino acid; a 29 dietary substance for use by man to supplement the diet by increasing total dietary intake; 30 or a concentrate, metabolite, constituent, extract, or combination of any of the above 31 dietary ingredients. {United States Federal Food Drug and Cosmetic Act §201(ff) [U.S.C. 321 32 (ff)]} 33

34 Dietary supplements.— A product intended for ingestion that contains a “dietary ingredient” 35 intended to add further nutritional value to (supplement) the diet. Dietary supplements may 36 be found in many forms such as tablets, capsules, softgels, gelcaps, liquids, or powders. 37

38 Limit of Quantitation (LOQ) .— The minimum concentration or mass of analyte in a given 39 matrix that can be reported as a quantitative result 40




53 SAM-e – S-Adenosyl methionine, IUPAC name: (2S)-2-Amino-4-[[(2S,3S,4R,5R)-5-(6-54 aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl] methyl-methylsulfonio]butanoate. CAS number: 55 97540-22-2. 56

57 58

5. Method Performance Requirements:59 60

Table 1: Analytical Range & LOQ Based on Matrix 61 62

Parameter

Analytical range (mg/g) 5 to 800

LOQ (mg/g) 1

Note: Results are in Total SAMe 63 64 65

Table 2: Method Performance Requirements as a Function of Range 66 67

Parameter Range

≤ 15 mg/g > 15 mg/g

Recovery (%) 90 - 110 98 - 105

% RSDr ≤ 8 ≤ 5

% RSDR ≤ 10 ≤ 8

Note: Results are in Total SAMe 68 69

6. System suitability tests and/or analytical quality control:70 71

Method must be able to separate SAMe from decomposition products (Table 3) and 72 synthetic precursors (Table 4), and quantify total SAM-e in the presence of stabilizing agents 73 (Table 5). 74

75 Suitable methods will include blank check samples, and check standards at the lowest point 76 and midrange point of the analytical range. A control sample must be included. 77

78 7. Reference Material(s):79

80 USP Ademetionine Disulfate Tosylate (S-Adenosyl-L-Methionine Disulfate Tosylate; 81 Catalog no. 1012134. 82

83 Sigma (detail to be added) 84

85

Refer to Annex F: Development and Use of In-House Reference Materials in Appendix F: 86 Guidelines for Standard Method Performance Requirements, 19th Edition of the AOAC 87 INTERNATIONAL Official Methods of Analysis (2012). Available at: 88 http://www.eoma.aoac.org/app_f.pdf 89

90




93 Method must be able to separate SAMe from decomposition products (Table 3) and 94 synthetic precursors (Table 4), and quantify total SAM-e in the presence of stabilizing agents 95 (Table 5). 96

97 Appendix D: Guidelines for Collaborative Study Procedures to Validate Characteristics of a 98 Method of Analysis; 19th Edition of the AOAC INTERNATIONAL Official Methods of Analysis 99 (2012). Available at: http://www.eoma.aoac.org/app_d.pdf 100


105 9. Maximum Time-To-Determination:106

Method developers must specify a maximum time-to-determination; and less than the 107 stability time of SAMe samples 108

109 110 111 112




Figure 1: Molecular structures of SAM-e. 113 114 115

116 117 118

119 120 121

Table 3: Decomposition products 122 123

p-toluenesulfonic acid (PTSA),124 S-adenosylhomocysteine (SAH),125 Adenosine,126 5'-deoxy-5'-methylthioadenosine (DMTA)127

128 Table 4: Synthetic precursors and Stabilizing Agents 129

130 Methionine, 131 ATP (adenosine triphosphate) 132 Toluene 133 Sulfonic Acid 134

135 Table 5: Dosage Forms 136 Tablets 137 Capsules 138

139 140 141 142 143 144 145

Stakeholder Panel on Dietary Supplements

Background and Fitness for Purpose for Quantifying Selected Kavalactones

Steven Dentali, Ph.D.AOAC Annual Meeting, Atlanta, GA22 September 2017

Kavalactones Begin with Kava

Kava is…a lot of things.

1. A Polynesian name for the plant known as Piper methysticumG.Forst (Piperaceae), intoxicating pepper.

2. A domesticated plant cultivated in the South Pacific Islands withscores of cultivars.

3. The beverage prepared from the plant.

4. The associated ceremony.

5. Root and rhizome raw material for ingredient manufacture.

6. An ingredient for dietary supplement finished products.

7. A variety of finished dietary supplement product forms.

Kava History

▪ Distribution of the plant is limited to the Pacific Islands

▪ Its use predates local written languages (2,000-3,000 yrs est.)

▪ Its ritual use, central to many local cultures, was recorded by Dutch explorers in 1616 and Cook’s first voyage in 1769.

▪ Its cultivation, use, and traditional ceremony are interwoven.

▪ All cultivars are sterile and related to the wild P. wichmannii.

▪ Wide variety of cultivar phenotype (color and shape appearance).

▪ ~ 120-150 known and named cultivars that are propagated based on subjective evaluation of pharmacological effects.

Kava Uses

▪ A natural alternative to anti-anxiety drugs

▪ Does not impair mental function (unlike anti-anxiety drugs)

▪ Sleep aid – mild sedative effects

▪ Relief of muscle tension or spasm due to stress

▪ Production of mild euphoria

▪ Affords mild pain relief

▪ May increase sociability

Kava and Kavalactones

1992 1997

Kava Chemistry

Bioactive constituents are known- Contains six major “kavalactones” (pyrones)

- arylethylene-α-pyrenes (also chalcones and other flavones, and conjugated diene ketones)

- At least a dozen other kavalactones identified

- Constituent composition varies among cultivars

- Relationship exists between “chemotype” and traditional status

- Kava lipid soluble resin forms an emulsion (milk) in water

Kavalactones

From: Chua et al. Kavain, the Major Constituent of the Anxiolytic Kava Extract, Potentiates GABAA Receptors: Functional Characteristics and Molecular Mechanism. PLoS One. 2016 Jun 22;11(6):e0157700. doi: 10.1371/journal.pone.0157700.

Kava Preparations

▪ Whole or powdered rootstock (root, rhizome, stump, laterals)

▪ 3%–20% dry weight kavalactones in underground plant material

▪ Traditional non-fermented drink (more than 1,000 yrs of use)

▪ ~ 250-300 mg kavalactones per serving

▪ Liquid, soft, and dry extracts: 30%–90% kavalactones

▪ Capsules or tablets: 50-250 mg kavalactones per serving

▪ Tea bags, Instant powdered drink mix

▪ In combination with other ingredients

▪ DSLD: 40 named products, 27 ingredients, 79 w/kava on label

▪ ABC Market Report, Natural Channel, #29, $3.2 mil, 10.3% incr.

Finished Kava Products

Kava Pharmacology

- Pharmacology depends on kavalactonemake up, not plant’s visual appearance

- Only known anxiolytic substance w/o causing impairment of mental function

- Possibly also direct muscle relaxant action

- High percentage kavain and low percentage dihydrokavain & dihydromethysticin preferred

Kava Rituals

Kava Adverse Effects

▪ Kava dermopathy – reversible scaly skin condition

▪ Formerly considered a sign of royalty (able to drink kava all day)

▪ Cases of liver damage and liver failure associated with use of commercial extract preparations

▪ Analysis of 78 case reports from 1990-2002 concluded that four established a certain probability from kava ingestion

▪ During the kava boom several non-traditional forms ingested

▪ Use of undesirable cultivars (two-day), plant parts (stem peelings), solvents (acetone), and production issues (mold) were known to occur around this time.

German Kava Regulations▪ German Federal Institute of Drugs and Medical Devices

(BfArM) made the decision to withdraw all drug registrations for all products containing kava on 14 June 2002 (previously approved for treatment of situational anxiety).

▪ Kava was officially judged as potentially dangerous and its efficacy was denied. The Society for Medicinal Plant Research neither accepted the official conclusions drawn from the presentation of fragmentary safety data, nor a negative benefit-risk-ratio (DOI: 10.1055/s-2003-45140).

▪ June of 2014, a German court ruled the “ban” illegal (data did not support alleged hepatotoxicity, and ban unjustified merely based on the perception of an inconclusive efficacy). Ruling confirmed (and finalized) 25 February 2015.

▪ The impact of ban on island economies was substantial.

Kava Regulations▪ 25 March 2002 US FDA advised consumers of the potential

risk of severe liver injury associated with the use of kava-containing dietary supplements. Urged consumers and their health care professionals to report any cases of liver and other injuries that may be related to the use of kava-containing dietary supplements.

▪ “FDA will continue to investigate the relationship, if any, between the use of DSs containing kava and liver injury.”

▪ NIH: Kava Kava in Generalized Anxiety: A Double-Blind Trial

▪ This study will compare KAV, the drug venlafaxine-XR (VEN), and placebo for the treatment of Generalized Anxiety Disorder

▪ This study was to last 10 weeks with 16 participants randomly assigned to receive kava, venlafaxine (Effexor), or placebo for 8 weeks, then undergo a 1-week tapering of medication followed by an additional week of observation.

▪ This study has been terminated. European reports of liver toxicity from kava meant that the study had to stop.

Kava Regulations

▪ Legal in most countries

▪ Australia: restrictions introduced in 2007

▪ Canada: 2002 ban lifted in 2012, products virtually absent

▪ EU: Regulated in Switzerland, France, and Netherlands

▪ Poland: Outright ban (probably the only one)

▪ UK: Ban on selling, supplying, importation

▪ New Zealand: Regulated as a Dietary Supplement

▪ Only traditional plant parts prepared in a traditional way

▪ Vanuatu: Legislation passed to regulate quality

▪ Export of non-noble cultivars and aerial parts prohibited

Safety Review Conclusion

When used in normal … doses, kava appears to offer safe and effective anti-anxiety and muscle relaxant actions without depressing centers of higher thought(emphasis added).

The safe use of kava … in cultures that do not have historical experience with its use depends on responsible manufacturing, marketing, individual consumption patterns, and education.

Significance of Analysis

▪ Need to know strength of raw materials, ingredients, and finished products

▪ Quantitate total kavalactones – list in order or perdominance

▪ Perceived need to determine cultivar type of starting material (noble cultivar vs. two-day)

▪ Quantitate amounts of individual six major kavalactones

▪ Different cultivars are regulated differently in Vanuvatu

▪ Concern for controlling/limiting amount of suspected hazard

▪ Limits for flavokavain B (< 2 mg/g) have been proposed.*

* Planta Med. 2015 Dec;81(18):1647-53. doi: 10.1055/s-0035-1558295.

General Analytical Needs

▪ Individual quantitation of six major kava kavalactones in raw materials, ingredients, and finished products.

▪ Quantitation of flavokavain B?

▪ Ability to differentiate noble from two-day cultivars?

Existing Kavalactone Methods

▪ Spectrophotometric

▪ 1g root/rhizome 3 ml acetone

▪ Dominate wavelength said to differentiate noble, two day, and mixtures of the two.

▪ ≤ 577.5 nm for noble kava

▪ ≥ 581.0 nm for non-noble kava

▪ Premised on unknown constituent differences

▪ Possibly chalconesFrom left to right, acetonic extracts of noble (test tubes 1–4), two-days (5–9) and wichmannii varieties (10–12).


▪ NIR Spectroscopy

▪ For routine analysis of dry export material

▪ Reportedly helpful in determining chemotype

▪ NIR predictions correlate well to HPLC values


▪ FTIR (and GC)

▪ Determines the content of six kavalactones

▪ Enables assignment of chemotype

▪ Partial least square regression used to model

▪ Comparable to GC


▪ HPTLC

▪ USP, HPTLC Association

▪ Generally used for ID and qualitative purposes

▪ Detection of flavokavins A, B, C

▪ Validation of High-Performance Thin-Layer Chromatographic Methods for the Identification of Botanicals in a cGMP Environment

▪ J AOAC Int. 2008;91(1):13–20.


▪ RP HPLC - MS

▪ Baseline separations of six major kavalactones

▪ Reports by the major US extract producers

▪ East Earth Herb 1997

▪ Madis Botanicals 1998

▪ Then Pure World

▪ Then Naturex

▪ USP (UV) NLT 4.5%


▪ Supercritical Fluid Chromatography

▪ CO2 & methanol w/diethylamine

▪ Commercial column

▪ LOD below 1.5 ng on-column

▪ Baseline separation

▪ Under 4 minute run time

▪ Method of choice?

Challenges

▪ Cost effectively addressing stakeholder needs

▪ Focus methods solely on kavalactone quantitation or include other considerations for kava quality control?

▪ Deciding to accept methods that may help differentiate cultivars (noble vs. two day) based on chemotype or presence of flavokavains A, B, and C

▪ Include qualitative methods?

▪ Obtain sufficient stakeholder engagement to best understand needs of the whole kava industry supply chain

▪ Collaborate with WHO Proposal to develop a Regional Codex Standard for Kava Products for Use as a Beverage?

Kava Industry Stakeholders

▪ American Kava Association http://americankavaassociation.org/

▪ Kava growers, manufacturers, distributors, retailers and consumers who advocate for the safe and responsible distribution of kava in the United States.

▪ Sets minimum quality control standards for the distribution of Kava in North America through lab testing and responsible marketing bylaws. HPLC and HPTLC mentioned.

▪ True Kava http://www.truekava.com/

▪ A non-profit corporation dedicated to the traditional use of kava

▪ HPLC: Identification, purity, strength, chemotype. Method: USP Piper methysticum Root and Rhizome Powder

▪ Qualitative: Indicator of noble/two day, adulteration of noble kava with two day. Method: Colorimetric assessment of kava quality, Lebot 2017

First Annual International Botanical Symposium on Kava, May 1997, Kona

Proposed Fitness for Purpose

▪ The method identifies and quantifies the six primary kavalactones derived from the underground portions of kava (Piper methysticum), namely desmethoxyangonin, dihydrokavain, yangonin, kavain, dihydromethysticin, and methysticin in plant material, dietary ingredients and dietary supplements. The ability of the method to also identify and quantify flavokavains A, B, and C is optional. Test results can be used in chemotype identification but this specific determination is outside the method scope. Individual kavalactones should be quantifiable within the range of 0.1 to 50 percent by weight in forms that include liquid, soft, and dry extracts as well as in softgels, capsules, and tablets in the presence of common excipients. The ability to address kavalactones in beverages is an advantage but not a requirement. No limit on analysis time is imposed.

Thank you for your attention!

STAKEHOLDER PANEL ON DIETARY SUPPLEMENTS

Background and Fitness for Purpose

Resveratrol

Richard B. van Breemen, Ph.D.

Atlanta, GA

September 22, 2017

Background on Resveratrol

Resveratrol (3,5,4′-trihydroxy-trans-stilbene)

First isolated in 1939 from Veratrum album by Michio Takaoka (J. Chem. Soc. Japan 1939; 60: 1090–1100)

Resveratrol cis/trans Chemistry

Although trans-resveratrol is the biosynthetic product, UV exposure can isomerize it to the cis isomer.The 3-O-glucoside (piceid) is also produced by plants.

3

Resveratrol Bioactivities

• In 1997, Pezzuto (Science, 1987; 275: 218) discovered that resveratrol had cancer chemoprevention activity through multiple mechanisms of action• anti-inflammation (inhibits COX, iNOS, and NF-κB)• anti-oxidation (upregulates quinone reductase,

glutathione, superoxide dismutase, and catalase)• induction of apoptosis

• Subsequently, >20,000 papers have reported multiple other activities including • prevention of cardiovascular disease• anti-aging• neuroprotection

Sources of Resveratrol

▪ Resveratrol is a phytoalexin produced by many plants in response to injury or, when under attack by pathogens.

▪ Dietary sources of resveratrol include peanuts, cocoa, grapes, and Vaccinium species such as blueberries, raspberries, cranberries, bilberries, and mulberries.

▪ Other sources include knotweed (Polygonumcuspidatum) and pine trees.

▪ Resveratrol has also been synthesized.

Dietary Levels of Resveratrol

• Levels of resveratrol in food are low but highly variable.

• Dark berries & red wines are highest in resveratrol.• Red wines contain from <LOD to 14.3 mg/L

(62.7 μM) trans-resveratrol. • Levels of cis-resveratrol are lower in food products

but follow the same trend as trans-resveratrol.• Resveratrol 3-glucoside (piceid) occurs in plants

and can be hydrolyzed to resveratrol enzymatically or non-enzymatically.

Resveratrol in Dietary Supplements

• 187 Dietary supplements containing resveratrol are listed in the NIH Office of Dietary Supplements labels database

• Most of these products contain synthetic resveratrol, others cite resveratrol extracted from Polygonum cuspidatum or, less frequently, red wine.

• Some supplement labels list trans-resveratrol, some list resveratrol-3-glucoside, but most list only resveratrol.

• Range of resveratrol in supplements, 37.5–500 mg/dose.

• Dosage forms include capsules, tablets and liquids.

Analytical Objectives

▪ Quantitatively extract resveratrol from finished products including capsules, tablets and pills and liquids.

▪ Measure both trans-resveratrol and cis-resveratrol

Current Analytical Methods for Resveratrol

▪ HPLC-UV

▪ microcapillary-LC with fluorescence detection

▪ HPLC-MS

▪ HPLC-MS/MS

▪ All LC methods utilized reversed phase

Fitness for Purpose (proposal)

The method for resveratrol dietary supplement analysis must quantitate both cisand trans forms in a variety of dosage forms.

QUESTIONS?

Development of SMPR for Skullcap by AOAC SPDS(Scutellaria lateriflora L.)

Holly E. Johnson, Ph.D.Alkemist Labs

AOAC SPDS Goals for Skullcap working group:

• Develop an SMPR (or two?)• Identity and/or Strength…• Method types• Fitness for Purpose• Scope/Applicability• Analytes

Robert H. Mohlenbrock, hosted by the USDA‐NRCS PLANTS Database / USDA SCS. 1989. Midwest wetland flora: Field office illustrated guide to plant species. Midwest National Technical Center, Lincoln.

Scutellaria lateriflora L.

Patrick J. Alexander, hosted by the USDA‐NRCS PLANTS Database


Patrick J. Alexander, hosted by the USDA‐NRCS PLANTS Database


USDA‐NRCS PLANTS Database / Britton, N.L., and A. Brown. 1913. An illustrated flora of the northern United States, Canada and the British Possessions. 3 vols. Charles Scribner's Sons, New York. Vol. 3: 106.

Skullcap (Scutellaria lateriflora L.)• Aerial parts used in modern westernherbalism as a nervine, sedative, anxiolytic

• First described in Tournefort’s The CompleatHerbal (1719)

• Given current name by Linneaus in 1753

Skullcap (Scutellaria lateriflora L.)• Aerial parts used in modern western herbalism as a nervine, sedative, anxiolytic

• First described in Tournefort’s The CompleatHerbal (1719)

• Given current name by Linneaus in 1753• Native American medicinal use: eyewash, fever, emmenagogue, digestif, laxative, + Cherokee, Miwao, Mendocino, Iriquois, Delaware, Ojibwa (Moerman 1998)

Table with history of use from AHP monograph 2009 (Upton et al. )

Toxicity & Adulteration

• 1980’s reports of hepatotoxicity associated with herbal products containing skullcap

• Suspected adulteration of skullcap supply with Teucrium spp. (germander)

• Germander known toxic – attempts to prohibit sale in 90’s

• Adulteration persists…

Known Adulterant Species • Scutellaria lateriflora• S. alpinia L.• S. baicalensis Georg• S. galericulata L. • S. incana Biehler• S. ovata Hill• Teucrium canadense L.• T. chamaedrys L.

Figures from AHP monograph 2009 (Upton et al. )

Figures from AHP monograph 2009 (Upton et al. )

Figures AHP monograph 2009 (Upton et al.); based on the work of Gafner et al. 2003)

Table from ABC AHP NCNPR Skullcap Adulteration Lab Guidance Document (Gafner2015)

Table from ABC AHP NCNPR Skullcap Adulteration Lab Guidance Document (Gafner2015)

AOAC SPDS: Scope & Fitness for Purpose

• Develop SMPR(s) – one or two?• Identity & Strength methods • ID authentic S. lateriflora• Quantify analytes• Label claims…• Stakeholders?

AOAC SPDS: Fitness for Purpose

?....

Sign up for the working group at AOAC.org and/or show up

tomorrow.

Thank you!

Documents

Meeting of the AOAC Stakeholder Panel on Dietary ... · Draft, Do Not Distribute Meeting of the AOAC Stakeholder Panel on Dietary Supplements Atlanta Marriott Marquis 8:30 a.m. –