September 2016 SPDS Book

S P D S

The AOAC Stakeholder Panel on Dietary Supplements (SPDS)

STAKEHOLDER PANEL MEETING

FRIDAY, SEPTEMBER 16, 2016 (Working Groups: Saturday, September 17) at the

Sh t D ll H t l era on a as o e 400 North Olive Street, Dallas, Texas, 75201 contact: SPDS@AOAC.ORG

S P D S

The AOAC Stakeholder Panel on Dietary Supplements (SPDS)

STAKEHOLDER PANEL MEETING

FRIDAY, SEPTEMBER 16, 2016 (Working Groups: Saturday, September 17) at the

Sh t D ll H t l era on a as o e 400 North Olive Street, Dallas, Texas, 75201 contact: SPDS@AOAC.ORG

SPDS Meeting, September 16-17, 2016 – 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 COFFEE CO. Vice Chair, AOAC Stakeholder Panel on Dietary Supplements

Brian Schaneberg, Ph.D., is the Global Scientific & Regulatory Affairs Director for Starbucks Coffee Company. 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 16-17, 2016 – Chair and Presenter Bios

PRESENTER BIOS

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

WORKING GROUP

SPDS VITAMIN B 12

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. Anton Bzhelyansky holds a Master’s degree in analytical chemistry from the University of Maryland Baltimore County. His thesis, under the direction of George M. Murray, was on uranyl-templated polymers. Upon graduation, he spent 13 years working for generic pharmaceutical and dietary supplement companies, primarily as a method developer. Anton’s analytical portfolio includes methodologies for a broad spectrum of analytes, from conventional pharmaceutical APIs to complex dietary supplement formulations, from marine oils to vitamins, chondroitins and botanicals. During his tenure in the dietary supplement industry, he implemented total inspection of incoming raw materials by NIR, established ICP-OES routine analysis, studied sampling of incoming ingredients and in-process blends, worked on formulation of enteric-coated dosage forms, and served as a Waters Empower® administrator. An AOCS Approved Chemist in 2011-2012, Anton developed a 20-minute marine oil GC method (poster at AOAC 125 th Annual Meeting) and optimized Peroxide and Anisidine Value analyses. ANTON BZHELYANSKY, USP CHAIR, GINGER WORKING GROUP

SPDS Meeting, September 16-17, 2016 – Chair and Presenter Bios His most memorable analytical work, however, remains the suite of methods for monitoring glucosinolates and isothiocyanates in formulations involving Cruciferae , including assessment of their enzymatic conversion rate. Anton has been with USP for three-and-a-half years. He is responsible for the majority of botanical monographs in the USP-NF Dietary Supplements section. Anton dedicated a significant effort to development of the USP General Chapter <2251> Screening For Undeclared Drugs and Drug Analogues, and is currently compiling the USP Adulterants database. In line with the USP's “Up-To-Date" policy, he is continuously working to improve compendial analyses. Anton is interested in implementing advanced techniques for challenging analytes such as oligomeric proanthocyanidins and complex polysaccharides, as well as devising a practical route for adoption of chemometric procedures in pharmacopeial monographs. He is a member of AOAC (2004) and AOCS (2008).

SPENCER C ARTER, GENYSIS LABS

SPDS PROTEIN WORKING GROUP

Spencer Carter is Senior Vice President of Genysis Labs. Genysis Labs is a cGMP compliant, full service testing laboratory that has provided comprehensive analytical testing for the Dietary Supplement and Food & Beverage industries since 2008. Additionally, Genysis Labs specializes in developing patentable formulations in the field of sports nutrition. Backed by

an ISO 17025:2005 accreditation and a management team of highly qualified scientists, Genysis Labs is committed to providing accurate and timely testing services while maintaining a laboratory environment consistent with ISO/IEC 17025:2005 requirements. Spencer earned his Ph.D. in Analytical Chemistry from the University of Alberta, in Edmonton, Alberta, Canada. His thesis focused on the analysis of tamoxifen metabolites by non-aqueous capillary electrophoresis and mass spectrometry detection. Prior to Genysis Labs, he was Lab Director at Tandem Labs (now Covance). Tandem Labs is a contract research organization (CRO) in the pharmaceutical industry performing bioanalytical services. His work included method development, validation, and sample analysis of biological samples. He focused on high- throughput analysis and improving efficiencies in the lab, as well as developing and maintaining non-proprietary assays. Previous to that, he was also the Bioanalytical Director at WIL Research and the Director of Bioanalytical Services at Pyxant Labs.”

SPDS Meeting, September 16-17, 2016 – Chair and Presenter Bios

KAN HE, HERBALIFE

SPDS ALOE VERA WORKING GROUP

Kan He is responsible for development of botanical ingredients for Herbalife product line. He has been involved in botanical product design and development from lab scale to commercial production. Before joined Herbalife, Kan He was in charge of research and development at Pure World Botanicals, Inc. and Naturex, Inc. respectively. He was responsible for developing new products and new processes, including scale up of plant extraction, purification, and chemical characterization of standardized herbal extracts. Kan He graduated from the Shanghai University of Traditional Chinese Medicine with BSc and MSc in Pharmacy and Medicinal Chemistry. He received his Ph.D. in pharmacognosy from the Pharmaceutical Sciences, University of Arizona and completed his postdoctoral research at School of Pharmacy, Purdue University. Over the past twenty- five years, he has been working in the area of natural products chemistry and authored or co-authored over 70 research papers on the peer reviewed scientific journals and book chapters. Kan He holds 11 US patents on the development of new herbal ingredients and new herbal manufacturing processes.

INGER REIDUN AUKRUST, KAPPA BIOSCIENCE SPDS Vitamins K 1 and K 2 Working Group

Inger Reidun Aukrust holds a PhD in Organic chemistry in 1995 at the University of Oslo. Established own firm Synthetica in 2000. Synthetica is an CRO in synthetic chemistry within pharma. Established Kappa Bioscience in 2006. Kappa Bioscience is Vitamin K2 MK7 manufacturer and supplier.

GARRETT ZIELINSKI, COVANCE

SPDS FREE AMINO ACIDS WORKING GROUP

Garrett Zielinski is a Program Development Manager at Covance Laboratories in Madison, WI. Mr. Zielinski acts as the primary liaison for dietary supplement clients as well as providing expertise on designing and managing testing programs to meet scientific and regulatory requirements. He also acts as a technical resource for customers as needed for analytical troubleshooting. He has designed and managed raw material, in-process, finished product, stability, and retail audit testing programs. He participates in a number of organizations involved with the dietary supplement industry related to regulation and analytical testing. Mr. Zielinski has over 13 years of experience in organic and analytical chemistry related to pharmaceuticals, foods and dietary supplements. He has authored a number of scientific posters, journal articles, and scientific presentations related to analytical testing of food and dietary supplements.

September 16, 2016 Stakeholder Panel on Dietary Supplements (SPDS) 8:30 a.m. – 5:00 p.m. - Registration Opens at 7:30 a.m. Chair: Darryl Sullivan, Covance | Vice Chair: Brian Schaneberg, Starbucks Sheraton Dallas Hotel | 400 North Olive Street, Dallas, Texas, 75201 Conference Room: Austin 3 A G E N D A

Welcome and Introductions (8:30-8:40am) Jim Bradford, AOAC Executive Director & Darryl Sullivan, Covance (Chair, SPDS) Ingredient Updates (8:45am – 9:00am) a. Ingredient Updates: ERPs, Working Groups, Methods, and the Advisory Panel Darryl Sullivan b. Proposed Vitamin D SMPR Revision John Austad, Covance (Chair, Vitamin D Working Group)

I.

II.

III. SMPR Approval Presentations and Consensus (9:00 am – 12:15 pm) a. Aloe Vera* - Kan He, Herbalife and John Edwards, Process NMR Associates; Co-Chairs, Aloe Vera Working Group (9:00am – 10:00am) b. Protein* – Spencer Carter, Genesys Labs, Chair, Protein Working Group (10:15am – 11:15am) c. Vitamin B 12 * – Richard van Breemen, University of Illinois; Chair, B12 Working Group (11:15 am - 12:15pm) IV. Launch of Set 6 Working Groups (1:30pm – 4:30pm) a. Free Amino Acids* (1:30 pm – 2:30 pm) Garrett Zielinski, Covance (Chair, Free Amino Acids Working Group) b. Ginger* (2:45 pm – 3:45 pm) Anton Bzhelyansky, USP (Chair, Ginger Working Group) 2:45pm – 3:45pm c. Vitamins K 1 and K 2 * (3:45 pm – 4:45 pm) Inger Reidun, Kappa Bio (Chair, Vitamin K Working Group)

Next Steps and Adjourn (4:50pm – 5:00 pm) Darryl Sullivan a. Saturday Working Group Schedule b. Adjourn

V.

Morning Break: 10:00am – 10:15am | Lunch (on your own): 12:15pm – 1:15pm | Afternoon Break 2:30pm – 2:45pm

*Item requires a vote by SPDS

V 5 09/01/2016

AOAC INTERNATIONAL Stakeholder Panel on Dietary Supplements Working Group Sessions – Saturday, September 17, 2016 (Day 2) 8:30 a.m. – 4:30 p.m., Salon CD

I. Free Amino Acids (8:30 a.m. – 10:30 a.m.) Chair: Garrett Zielinski, Covance Laboratories a. Review Fitness for Purpose b. SMPR Development

II. Ginger (11:00 a.m. – 2:00 p.m.**) Chair: Anton Bzhelyansky, USP a. Review Fitness for Purpose b. SMPR Development

III. Vitamins K 1

K 2

(2:30 p.m. – 4:30 p.m.)

Chair: Inger Reidun, Kappa Bio a. Review Fitness for Purpose b. SMPR Development

**Day 2 Lunch: On your own, 12:00 p.m. – 1:00 p.m.

*Item requires a vote by SPDS

V 5 09/01/2016

Update on the Stakeholder Panel on Dietary Supplements(SPDS)

Darryl Sullivan , Chair Stakeholder Panel on Dietary Supplements Covance Laboratories

September 2016

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 25 standard method performance requirements  (SMPRs) for priority dietary supplement ingredients. • Deliver First Action Official Methods SM for the prioritized  di t l t i di t e ary supp emen ngre en s  • Encourage participation with the dietary supplements  industry to develop voluntary consensus standards.

AOAC SPDS 5 Year Plan

• 5 Advisory Panel Meetings  to identify key stakeholders,  subject matter experts, frames the issues, determine  ingredients, and set priorities for the stakeholder panel.

• 10 Stakeholder Panel Meetings  to deliberate and approve  voluntary consensus standards.

• 25 Total Working Groups  to draft and recommend SMPRs.

• 8 Expert Review Panel Meetings  to review and potentially  adopt fit for purpose First Action Official Methods SM for 25  ingredients.

Stakeholder Panel on Dietary Supplements (SPDS)

• Set 1 Ingredients:  Anthocyanins, Chondroitin, and PDE5 Inhibitors – Launched March, 2014 – SMPRs Approved in September, 2014: • Authentication of Selected Vaccinium species 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  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 ( 2014.009 ) Id tifi ti f Ph h di t en ca on o osp o es erase ype   T 5 (PDE5) I hibit i Di t I di t  n ors n e ary ngre en s  •

Stakeholder Panel on Dietary Supplements (SPDS)

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

Stakeholder Panel on Dietary Supplements (SPDS)

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

Stakeholder Panel on Dietary Supplements (SPDS)

• Set 4 Ingredients:  Collagen, Lutein, Turmeric – 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 ) – Call for Methods & Experts Currently Live: • www.aoac.org

– ERP tentatively scheduled for December, 2016

Stakeholder Panel on Dietary Supplements (SPDS)

• Set 5 Ingredients: Aloe Vera, Protein, Vitamin B 12 – Launched in March, 2016 – SMPRs sent to SPDS for approval in September, 2016 (this meeting):

• DRAFT SMPR for Aloe Vera • DRAFT SMPR for Protein • DRAFT SMPR for Vitamin B 12 – Call for Methods and Experts will follow  approval of SMPRs.

Stakeholder Panel on Dietary Supplements (SPDS)

• Set 6 Ingredients: Amino Acids, Ginger, Vitamins K 1 – Launched in September, 2016 (this meeting) – SMPRs sent to SPDS for approval in September, 2016: • DRAFT SMPR for Aloe Vera • DRAFT SMPR for Protein • DRAFT SMPR for Vitamin B 12 – SMPR Approval Expected March, 2017

and K 2

Stakeholder Panel on Dietary Supplements (SPDS) Advisory Panel

• SPDS Advisory Panel slated for fall 2016 to  prioritize next 6 ingredients for 2017

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

Method Status Chart

– AOAC has prepared a Method Status Chart to keep stakeholders  updated on where ingredients and methods are in process 

– Methods are needed in all ingredient areas – View the status of all submitted methods at  http://tinyurl.com/gv4w35g

Vitamin D SMPR Review

• Chair, John Austad, Covance

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  Di t S l t (SPDS) f l t e ary upp emen s   or comp e e  information about the program

Contact Information

Darryl Sullivan, Chair SPDS Covance Laboratories Tel: 608.242.2711 Email: darryl.sullivan@covance.com

Contact AOAC Staff: Tel: 301.924.7077 Web: www.aoac.org • Jim Bradford , Executive Director/CEO,  jbradford@aoac.org , ext. 102 • 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 John Edwards, Process NMR; Kan He, Herbalife Aloe Vera Working Group September 16, 2016

Sheraton Dallas Hotel, 400 N Olive Street, Dallas, Texas

Fitness for Purpose from 3/17

“The methods are able to qualitatively identify aloe vera; are able to accurately quantitate not only the contents of aloe polysaccharides, but also the molecular weight; are able to accurately quantitate the aloe polysaccharides with different molecular weight ” .

Aloe Vera Working Group Members

• John Edwards, Process NMR • Kan HE, Herbalife • Joseph Betz, NIH • Jasen Lavoie, Pharmachem Labs • Barry McCleary, Megazyme • Charles Metcalfe, Custom  Analytics • Elizabeth Mudge, BCIT

• Maria Ofitserova, Pickering Labs • Catherine Rimmer, ATCC • Brian Schaneberg, Starbucks • Aniko Solyom, GAAS Analytical

• Darryl Sullivan, Covance • Jinchaun Yang, Waters • Kurt Young, GNC / Nutra Manufacturing

Aloe Vera Working Group Work to Date

• 1 In Person Meeting • 3 teleconferences (March 2016 – June 2016) • 1 SMPR Drafted  • Public comment period (August, 2016) • SMPRs made ready for SPDS review and  approval

Background

Definition: • The major polysaccharide in aloe is glucomannan  h h d f ( ) d l w ic is consiste o mannose major an g ucose  (minor) with 1,4‐β‐linked backbone.   • The mannose moieties are highly acetylated and are  referred to Acemannan in literature. 

2/3/6-OAc

2/3/6-OAc

6

HO

HO

HOH 2 C O

HOH 2 C

O

HOH 2 C

O

O

1

3 4 5

O

O

O

Structure of the major aloe polysaccharides

HO

2

HO

HO

OH

Glc p

Man p

y

x

Man p

z

Background

Definition ( cont’d ): • The molecular weight (MW) of aloe polysaccharides can be in  th f 10 000 2 000 000 D lt e range o , ‐ , ,  a ons: – Unprocessed fresh aloe polysaccharides usually has the  MW around  or over 2 million Daltons; – Less processed commercial aloe polysaccharide MW can be in the  range of 200,000‐500,000 Daltons; – Highly processed commercial aloe products contain aloe  polysaccharide MW can be lower to 10,000‐100,000 Daltons; • Degrees of acetylation on the aloe polysaccharides are varied  depending on process; • High MW polysaccharides may affect the accuracy of results  due to the effect of solubility and viscosity, etc.

Background

Summary of current methods used in Aloe  composition analysis:

• NMR ‐ quantification of polysaccharides and  providing aloe fingerprint for identification; – Quantification of polysaccharides by analysis of the  content of acetyl groups; – Quantification of organic acids including acetic acid, lactic  acid,  malic acid and isocitric acid;  • GC – quantification of aloe organic acid, such as acids  of lactic, malic, citric, isocitric, etc.; quantification of  monosaccharides including existed monosaccharides  or hydrolyzed from polysaccharides;

Background

Summary of current methods used in Aloe  composition analysis ( cont’d ): • Colorimetric – quantification of aloe polysaccharides  by photometric analysis;   • GPC – providing fingerprint of aloe polysaccharides  and their molecular weight and size; • HPLC – quantification of organic acids, including  oxalic, malic, isocitric, lactic, citric, fumaric acid, and  isocitric acid lactone; providing aloe fingerprint for  identification;

SMPR Key Points

• Quantitation of water soluble Aloe vera  polysaccharides and the following organic acids  (acetic acid, lactic acid, malic acid and isocitric acid)    including the matrix(es) in which the polysaccharides  and the acids are found); • NMR, GC, Colorimetric, GPC, HPLC; or any analytical  technique that meets the following method  performance requirements is acceptable.   • It is expected that more than one technique will be  required; • May require developing aloe polysaccharide  standards;

SMPR Key Points

Table 1: Method performance requirements

Ingredients (Raw  Materials)

Finished Products  – Liquid (Freeze dried  samples) 0.15 ‐ 15 0.15 ‐ 0.5 ≥ 0.5 ‐ 100

Finished  Products ‐ Solid

Parameter

Analytical  Range (%) LOQ  (%)

1 ‐ 100

1 ‐ 100

< 0.5

< 0.5

0.15

Recovery (%) 

90 ‐ 110

90 ‐ 110

≥ 50 90 ‐ 110

% RSD r % RSD R

≤ 10 ≤ 15

≤ 10 ≤ 15

≤ 20 ≤ 30

≤ 10 ≤ 15

Comments Submitted (if any)

• No comments submitted

Motion

• Move to accept the Standard Method  Performance Requirements for Quantitation  of Aloe Vera Polysaccharides in Dietary  Supplements as presented.

Discussion?

DRAFT AOAC SPDS Aloe Vera SMPR, v4, July 21, 2016. 1 2 Quantitation of Aloe Vera Polysaccharides in Dietary Supplements 3 4 Intended Use : Reference method for cGMP compliance. 5 6 1. Purpose: AOAC SMPRs describe the minimum recommended performance characteristics 7 to be used during the evaluation of a method. The evaluation may be an on-site 8 verification, a single-laboratory validation, or a multi-site collaborative study. SMPRs are 9 written and adopted by AOAC Stakeholder Panels composed of representatives from the 10 industry, regulatory organizations, contract laboratories, test kit manufacturers, and 11 academic institutions. AOAC SMPRs are used by AOAC Expert Review Panels in their 12 evaluation of validation study data for method being considered for Performance Tested 13 Methods or AOAC Official Methods of Analysis , and can be used as acceptance criteria for 14 verification at user laboratories.

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 50

2. Applicability :

Quantitation of water soluble Aloe Vera main constituents and degradation products in the

matrices listed in Table 3.

3. Analytical Technique :

NMR, GC, Colorimetric, GPC; or any analytical technique that meets the following method performance requirements is acceptable. It is expected that more than one technique will

be required.

4. Definitions :

Aloe Vera Main Constituents and Degradation Products

Aloe Vera Polysaccharides ( Acetylated 1, 4 beta Glucomannan) is the signature component of Aloe Vera. Acetic acid is a degradation product of Aloe Vera, quantified as a measure of the level of de-acetylation of Aloe Vera polysaccharide (degradation product). Malic acid is a necessary component of Aloe Vera. Lactic acid is a product of malolactic fermentation (degradation product). Isocitrate is the component utilized to identify and

quantify whole leaf markers.

Limit of Quantitation (LOQ)

The minimum concentration or mass of analyte in a given matrix that can be reported as a

quantitative result.

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 standard deviation (SD R

); or % reproducibility relative

standard deviation (% RSD R ).*

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 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99

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 table 1.

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.

7. Potential Reference Material(s):

Custom Analytics (add Charlie’s info) Low Molecular Weight Pure Polysaccharides (80,000

daltons)

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 :

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 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

Appendix K

Appendix K: Guidelines for Dietary Supplements and Botanicals, Official Methods of Analysis (current edition), AOAC INTERNATIONAL, Rockville, MD, USA (http://www.eoma. aoac.org/app_k.pdf). Also at: J. AOAC Int . 95 , 268(2012); DOI: 10.5740/jaoacint.11-447 Data demonstrating that the candidate method meets the performance criteria should be submitted for the adulterants listed in Table 2 and the matrices listed in Table 3.

Pharmachem Labs may provide materials for evaluation.

9. Maximum Time-To-Result: None

100 101 102

103 104

Table 1: Method performance requirements.

Finished Products - Solid

Finished Products – Liquid (Freeze dried samples)

Ingredients (Raw Materials)

Parameter

LOQ (%)

≤ 0.5

≤ 0.5

≤ 0.15

Analytical Range (%)

1 – 100

1 – 100

0.15 – 0.5

≥ 0.5 - 100

Recovery (%)

90 - 110

90 - 110

≤ 50

90 - 110

% RSD

≤ 10

≤ 10

≤ 20

≤ 10

r

% RSD

≤ 15

≤ 15

≤ 30

≤ 15

R

105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124

Table 2: Potential Adulterants

Maltodextrin Carragennan Gum acacia Locust gum

Table 3 : List of Matrices

Tablets Capsules Liquids Powders Extracts

Plant products

f:\spds\working groups\set 5\aloe vera\smpr\aloe smpr v4.docx

AOAC INTERNATIONAL STAKEHOLDER PANEL ON  DIETARY SUPPLEMENTS Spencer Carter, Genysis Labs Protein Working Group September 16, 2016

Sheraton Dallas Hotel, 400 N Olive Street, Dallas, Texas

Fitness for Purpose from 3/17

“Method must identify and quantify specific proteins in presence of other proteins and potential adulterants. Quantitative method must provide accurate and precise concentrations of specific intact proteins in ingredients and finished goods.”

Protein Working Group Members

• Spencer Carter, Genysis Labs • Joseph Betz, NIH St D t li H b lif • even en a , er a e • Jason Hendrickson, Bodybuilding.com • Martha Jennens, Covance • Suvash Kafley, Milk Specialties  • Adam Kuszak, NIH • John Lawry, Covance • Katerina Maastovska, Covance • Elizabeth Mudge, BCIT

• Brian Schaneberg, Starbucks • Aniko Solyom, GAAS Analytical D l S lli C • arry u van, ovance • James Sullivan, Natures Products • John Spzylka, Mérieux NutriSciences • Barry Tulk, DuPont • Robert Wildman, Dymatize

• Jason Wubben, ADM • Jinchuan Yang, Waters • Kurt Young, GNC • Joseph Zhou, Sunshineville • Garrett Zielinski, Covance

• Melissa Phillips, NIST • Curtis Phinney, Consultant • Catherine Rimmer, NIST

Protein Working Group Work to Date

• 1 In Person Meeting • 3 teleconferences (March 2016 – June 2016) • 4 SMPRs Drafted  • Public comment period (August, 2016) • SMPRs made ready for SPDS review and  approval

Background

• Proteins are polypetides made of individual amino acids in  a linear chain • Form the basis of life and perform functions in every  system of the human body

• Enzymes catalyze biochemical reactions • Hormones are used for cell signaling and  communication • Synthesize and repair DNA • Tranport materials across the cell • Respond to stimuli • Provide structural support

Significance

• Estimated that 4 billion metric tons of food  protein is produced globally • Estimated that $94M was lost by changing the  nitrogen‐to‐protein factor for dairy products from  6.38 to 6.25 in Europe in 2006 • Proteins make up $4.7B dollars in the Sports  Nutrition industry which represents 70% of the ,  total revenue in that category

Adulteration

• Non‐selective protein methods have fueled  the potential to adulterate samples with  non‐proteins and give inaccurate results • Melamine, urea, free amino acids cannot  be differentiated using Kjeldahl, Dumas  methods and have been the source of  d l scan a s • Public health is still at risk; Economics still  push adulteration

Existing Methods (Qualitative)

• Some proteins have FCC monographs.  For example,  Whey Protein is identified by testing for: – Ash – Fat – Lactose – Loss on drying – Nitrogen (and apply conversion factor) • DNA Analysis • LC/MS/MS

Existing Methods (Quantitative)

• Kjeldahl 1. Wet digestion converts nitrogen to ammonium sulfate 2. Neutralize to convert to free ammonia 3. Distill ammonia into boric acid 4. Back titrate with alkali 5. Convert nitrogen concentration to protein using  conversion ratio – “True Protein” can be determined by precipitating out  protein, analyzing remaining nitrogen, subtracting from  total nitrogen content

Existing Methods (con’t)

• Dumas 1 Combust samples at high temp with oxygen to form . water, carbon dioxide, nitrogen 2. Remove water and carbon dioxide using column 3. Nitrogen is measured using a thermal conductivity  detector 4. Convert nitrogen concentration to protein using  conversion ratio

Existing Methods (con’t)

• Amino Acids

1 Hydrolyze protein into amino acids . 2. Derivatize amino acids 3. Determine protein by summing individual amino acids • Dye‐binding 1. Form complex with dye and protein using ionic or  electrostatic forces. 2. Determine dye concentration using spectrophotometer

Existing Methods (con’t)

• Copper‐Binding 1 Copper ions react with proteins to form complex . 2. Measure absorbance at 540 nm • Others – UV absorption – Infrared

Challenges with Existing Methods

• Kjeldahl, Dumas: not selective to protein; • True Protein Kjeldahl: non‐protein, nitrogen‐containing  compounds may precipitate or form complex with  precipitated protein • Amino Acid: Inaccurate quantitation due to variable  recovery of amino acids • Copper, Dye‐Binding: other constituents besides proteins  form complexes • Lack of Standards – Protein biosynthesis is expensive, time‐consuming, not robust – Proteins samples vary widely and usually include multiple proteins

SMPR Key Points

• Definition of Protein is same as IUPAC:  “Naturally occurring and synthetic  l d h l l h po ypepti es aving mo ecu ar weig ts greater  than about 10000 daltons (the limit is not  precise)” • Four SMPRs are being proposed: – Quantitative (i.e. Determination) and Qualitative  (i e Identification) . .  – Plant and Meat‐Derived Proteins

Method Performance Requirements for Determination in Plant & Meat-Derived Proteins

Parameter

Criteria

Analytical Range (%)

0 1 – 100 . 

LOQ  (%)

0.050

LOD  (%)

0.025

90‐110 (0.1 – 1% range) 97‐103 (>1 – 100% range) ≤ 10 (0.1 – 1% range) ≤ 6 (>1 – 100% range) ≤ 12 (0.1 – 1% range) ≤ 8 (>1 – 100% range)

Recovery (%)

% RSD r

% RSD R

Single-Lab Method Performance Requirements for Identification in Plant & Meat-Derived Proteins

Parameter  Requirements

Target Test  Conc

Minimum Acceptable Results 90% POI (95%  confidence interval) of the pooled data for  all target compounds  and matrices.

Study

Parameter 

POI @ low conc

≥ 33 reps representing  all listed target analytes

0.1 %

≥ 5 reps per matrix type  spiked at 10x the  designated low level  target test conc

Matrix

POI @ high conc

10%

100% correct analyses  are expected

POI @  zero conc

≥ 5 reps per matrix type 

0 %

Evaluate samples  containing  non‐protein ingredients  and listed adulterants

False positive  rate

10 %

≤ 5%

Selectivity

Multiple-Lab Method Performance Requirements for Identification in Plant & Meat-Derived Proteins

Parameter  Requirements

Target Test 

Minimum

Study

Parameter

Conc

Acceptable Results

0.1 %

≥ 0.85

Use ISPAM Guidelines  for Validation of  Qualitative Binary  Chemistry Methods

LPOI

Matrix

10 %

≥ 0.95

0 % 

≤ 0.05

LPOI (0)

SMPR Sources of Proteins

Plant‐Derived

Meat‐Derived 

• Algae

• Casein • Egg • Whey • Milk

• Canola (Rapeseed) • Flax • Hemp • Pea • Potato P ki

• ump n • Quinoa • Rice • Soy • Wheat

SMPR Non-Protein Ingredients Including Adulterants

• Melamine • Urea • Free amino acids

• Creatine • Caffeine • Taurine S f t t • ur ac an s  • Peptides (less than 10,000 daltons)

Comments Submitted

• Comment: “Though the given protein  definition is recommended by IUPAC, it will  h l l l f h f d d ave itt e practica use or t e oo in ustry.  For instance, in the newly revised FDA food  and supplement nutrition fact labeling, FDA  recognizes all peptides as proteins (a merely  amino acid source). And 10000 sounds over  h d ” emp asize . • Proposed Change: “Polymeric chains of amino  acid residues connected with peptide bonds.”

Motion

Move to accept the Standard Method  Performance Requirements for: – Determination of Meat Derived Proteins – Determination of Plant Derived Proteins – Identification of Meat Derived Proteins – Identification of Plant Derived Proteins

….as presented.

Discussion?

DRAFT AOAC SPDS Animal-Derived Protein SMPR, v4, 6/30/2016 1 2 Identification and Quantitation of Animal-Derived Proteins in Dietary Supplements 3 4 Intended Use : Reference method for cGMP compliance. 5 6 1. Purpose: AOAC SMPRs describe the minimum recommended performance characteristics 7 to be used during the evaluation of a method. The evaluation may be an on-site 8 verification, a single-laboratory validation, or a multi-site collaborative study. SMPRs are 9 written and adopted by AOAC Stakeholder Panels composed of representatives from the 10 industry, regulatory organizations, contract laboratories, test kit manufacturers, and 11 academic institutions. AOAC SMPRs are used by AOAC Expert Review Panels in their 12 evaluation of validation study data for method being considered for Performance Tested 13 Methods or AOAC Official Methods of Analysis , and can be used as acceptance criteria for 14 verification at user laboratories.

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 50 51

2. Applicability :

Method must identify and quantify animal-derived proteins and their corresponding sources in the presence of potential adulterants in ingredients and finished dietary supplements.

3. Analytical Technique :

Any analytical technique is acceptable.

4. Definitions :

Protein

Naturally occurring and synthetic polypeptides having molecular weights greater than about

10000 daltons (the limit is not precise) (IUPAC Definition)

Limit of Quantitation (LOQ)

The minimum concentration or mass of analyte in a given matrix that can be reported as a

quantitative result.

Limit of Detection (LOD)

The minimum concentration or mass of analyte that can be detected in a given matrix with no greater than 5% false-positive risk and 5% false-negative risk. 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

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 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.

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

5. Method Performance Requirements :

See table 1 and 2.

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.

7. Potential Reference Material(s):

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 :

Data demonstrating method performance for the animal-derived proteins listed in table 3 in the presence of the potential non-protein ingredients

including adulterants listed in table 4 is recommended.

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 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

Appendix K: Guidelines for Dietary Supplements and Botanicals, Official Methods of

Analysis (2016) 20th Ed., AOAC INTERNATIONAL.

9. Maximum Time-To-Result: None

Table 1: Method performance requirements (part 1)

Parameters

Acceptable Criteria

Analytical Range (%)

0.1 - 100

LOQ (%)

0.05

LOD (%)

0.025

88 89 90 91

92 93 94 95

Table 2: Method performance requirements (part 2)

Ranges (%)

0.1-1

>1

Recovery (%)

90-110

97-103

% RSD r

≤ 10

≤ 6

% RSD

≤ 12

≤8

R

96 97 Table 3 : Recommended Animal-Derived Proteins from these sources

98 99

100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115

Casein

Egg

Whey

Milk

Table 4: Non-Protein Ingredients Including Adulterants

Melamine

Urea

Free amino acids

Creatine Caffeine Taurine

Surfactants

Peptides (less than 10,000 daltons)

DRAFT AOAC SPDS Plant-Derived Protein SMPR, v4, 6/30/2016 1 2 Identification and Quantitation of Non-animal-Derived Proteins in Dietary Supplements 3 4 Intended Use : Reference method for cGMP compliance. 5 6 1. Purpose: AOAC SMPRs describe the minimum recommended performance characteristics 7 to be used during the evaluation of a method. The evaluation may be an on-site 8 verification, a single-laboratory validation, or a multi-site collaborative study. SMPRs are 9 written and adopted by AOAC Stakeholder Panels composed of representatives from the 10 industry, regulatory organizations, contract laboratories, test kit manufacturers, and 11 academic institutions. AOAC SMPRs are used by AOAC Expert Review Panels in their 12 evaluation of validation study data for method being considered for Performance Tested 13 Methods or AOAC Official Methods of Analysis , and can be used as acceptance criteria for 14 verification at user laboratories.

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

2. Applicability :

Method must identify and quantify non-animal -derived proteins and their corresponding sources in the presence of potential adulterants in ingredients and finished dietary

supplements.

3. Analytical Technique :

Any analytical technique is acceptable.

4. Definitions :

Protein

Naturally occurring and synthetic polypeptides having molecular weights greater than about

10000 daltons (the limit is not precise) (IUPAC Definition)

Limit of Quantitation (LOQ)

The minimum concentration or mass of analyte in a given matrix that can be reported as a

quantitative result.

Limit of Detection (LOD)

The minimum concentration or mass of analyte that can be detected in a given matrix with no greater than 5% false-positive risk and 5% false-negative risk. 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

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 standard deviation (SD R

); or % reproducibility relative

standard deviation (% RSD R ).

Recovery

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 78 79 80 81 82 83 84 85 86 87 88

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 table 1 and 2.

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.

7. Potential Reference Material(s):

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 :

Data demonstrating method performance for the non-animal-derived proteins listed in table 3 in the presence of the potential non-protein ingredients

including adulterants listed in table 4 is recommended.

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 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

Appendix K: Guidelines for Dietary Supplements and Botanicals, Official Methods of Analysis

(2016) 20th Ed., AOAC INTERNATIONAL.

9. Maximum Time-To-Result: None

Table 1: Method performance requirements (part 1)

Parameters

Acceptable Criteria

Analytical Range (%)

0.1 - 100

LOQ (%)

0.05

LOD (%)

0.025

89

90 91 92 93 94 95 96

Table 2: Method performance requirements (part 2)

Ranges (%)

0.1-1

>1

Recovery (%)

90-110

97-103

% RSD r

≤ 10

≤ 6

% RSD

≤ 12

≤8

R

97 98 Table 3 : Recommended Non-Animal-Derived Proteins from these sources 99 100 Algae 101 Canola (Rapeseed) 102 Flax 103 Hemp 104 Pea 105 Potato 106 Pumpkin 107 Quinoa 108 Rice 109 Soy 110 Wheat

111 112 113 114 115 116 117 118 119 120 121 122 123 124

Table 4: Non-Protein Ingredients Including Adulterants

Melamine

Urea

Free amino acids

Creatine Caffeine Taurine

Surfactants

Peptides (less than 10,000 daltons)

DRAFT AOAC SPDS Identification of Animal-derived Proteins V 4, 6.30.2016 1 2 Identification of Animal-Derived Proteins in Dietary Supplements 3 4 Intended Use : Reference method for cGMP compliance. 5 6 1. Purpose: AOAC SMPRs describe the minimum recommended performance characteristics 7 to be used during the evaluation of a method. The evaluation may be an on-site 8 verification, a single-laboratory validation, or a multi-site collaborative study. SMPRs are 9 written and adopted by AOAC Stakeholder Panels composed of representatives from the 10 industry, regulatory organizations, contract laboratories, test kit manufacturers, and 11 academic institutions. AOAC SMPRs are used by AOAC Expert Review Panels in their 12 evaluation of validation study data for method being considered for Performance Tested 13 Methods or AOAC Official Methods of Analysis , and can be used as acceptance criteria for 14 verification at user laboratories.

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

2. Applicability :

Method must identify animal-derived proteins and their corresponding sources in the presence of potential adulterants in ingredients and finished dietary supplements.

3. Analytical Technique :

Any analytical technique is acceptable.

4. Definitions :

Protein

Naturally occurring and synthetic polypeptides having molecular weights greater than about

10000 daltons (the limit is not precise) (IUPAC Definition)

Probability of Identification (POI)

The proportion of positive analytical outcomes for an identification method for a given matrix at a given analyte level or concentration. LPOI is the Laboratory Probability of

Identification.

5. Method Performance Requirements :

See table 1.

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.

7. Potential Reference Material(s):

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