SPDS Set 1 ERP Book

AUGUST 3-4, 2015 AOAC STAKEHOLDER PANEL on DIETARY SUPPLEMENTS (SPDS)

EXPERT REVIEW PANEL (SET 1) MEETING BOOK

dfrazier@aoac.org

cdent@aoac.org

Appendix W

POLICY AND PROCEDURES ON VOLUNTEER CONFLICT OF INTEREST

Statement of Policy

While it is not the intention of AOAC INTERNATIONAL (AOAC) to restrict the personal, professional, or proprietary activities of AOAC members nor to preclude or restrict participation in Association affairs solely by reason of such activities, it is the sense of AOAC that conflicts of interest or even the appearance of conflicts of interest on the part of AOAC volunteers should be avoided. Where this is not possible or practical under the circumstances, there shall be written disclosure by the volunteers of actual or potential conflicts of interest in order to ensure the credibility and integrity of AOAC. Such written disclosure shall be made to any individual or group within the Association which is reviewing a recommendation which the volunteer had a part in formulating and in which the volunteer has a material interest causing an actual or potential conflict of interest. AOAC requires disclosure of actual or potential conflicts of interest as a condition of active participation in the business of the Association. The burden of disclosure of conflicts of interest or the appearance of conflicts of interest falls upon the volunteer. A disclosed conflict of interest will not in itself bar an AOAC member from participation in Association activities, but a three-fourths majority of the AOAC group reviewing the issue presenting the conflict must concur by secret ballot that the volunteer's continued participation is necessary and will not unreasonably jeopardize the integrity of the decision-making process. Employees of AOAC are governed by the provision of the AOAC policy on conflict of interest by staff. If that policy is in disagreement with or mute on matters covered by this policy, the provisions of this policy shall prevail and apply to staff as well. 1. A volunteer who is serving as a committee member or referee engaged in the evaluation of a method or device; who is also an employee of or receiving a fee from the firm which is manufacturing or distributing the method or device or is an employee of or receiving a fee from a competing firm. 2. A volunteer who is requested to evaluate a proposed method or a related collaborative study in which data are presented that appear detrimental (or favorable) to a product distributed or a position supported by the volunteer's employer. 3. A referee who is conducting a study and evaluating the results of an instrument, a kit, or a piece of equipment which will be provided gratis by the manufacturer or distributor to one or more of the participating laboratories, including his or her own laboratory, at the conclusion of the study. 4. Sponsorship of a collaborative study by an interest (which may include the referee) which stands to profit from the results; such sponsorship usually involving the privilege granted by the investigator to permit the sponsor to review and comment upon the results prior to AOAC evaluation. Illustrations of Conflicts of Interest

5. A volunteer asked to review a manuscript submitted for publication when the manuscript contains information which is critical of a proprietary or other interest of the reviewer.

The foregoing are intended as illustrative and should not be interpreted to be all-inclusive examples of conflicts of interest AOAC volunteers may find themselves involved in.

Do's and Don't's

Do avoid the appearance as well as the fact of a conflict of interest.

Do make written disclosure of any material interest which may constitute a conflict of interest or the appearance of a conflict of interest.

Do not accept payment or gifts for services rendered as a volunteer of the Association without disclosing such payment or gifts.

Do not vote on any issue before an AOAC decision-making body where you have the appearance of or an actual conflict of interest regarding the recommendation or decision before that body.

Do not participate in an AOAC decision-making body without written disclosure of actual or potential conflicts of interest in the issues before that body.

Do not accept a position of responsibility as an AOAC volunteer, without disclosure, where the discharge of the accepted responsibility will be or may appear to be influenced by proprietary or other conflicting interests.

Procedures

Each volunteer elected or appointed to an AOAC position of responsibility shall be sent, at the time of election or appointment, a copy of this policy and shall be advised of the requirement to adhere to the provisions herein as a condition for active participation in the business of the Association. Each volunteer, at the time of his or her election or appointment, shall indicate, in writing, on a form provided for this purpose by AOAC, that he or she has read and accepts this policy. Each year, at the spring meeting of the AOAC Board of Directors, the Executive Director shall submit a report certifying the requirements of this policy have been met; including the names and positions of any elected or appointed volunteers who have not at that time indicated in writing that they have accepted the policy. Anyone with knowledge of specific instances in which the provisions of this policy have not been complied with shall report these instances to the Board of Directors, via the Office of the Executive Director, as soon as discovered.

* * * * * *

Adopted: March 2, 1989 Revised: March 28, 1990 Revised: October 1996 Reviewed by outside counsel March 2000 (Fran Dwornik) and found to be current and relevant

Appendix U

ANTITRUST POLICY STATEMENT AND GUIDELINES

Introduction

It is the policy of AOAC INTERNATIONAL (AOAC) and its members to comply strictly with all laws applicable to AOAC activities. Because AOAC activities frequently involve cooperative undertakings and meetings where competitors may be present, it is important to emphasize the on-going commitment of our members and the Association to full compliance with national and other antitrust laws. This statement is a reminder of that commitment and should be used as a general guide for AOAC and related individual activities and meetings.

Responsibility for Antitrust Compliance

The Association's structure is fashioned and its programs are carried out in conformance with antitrust standards. However, an equal responsibility for antitrust compliance -- which includes avoidance of even an appearance of improper activity -- belongs to the individual. Even the appearance of improper activity must be avoided because the courts have taken the position that actual proof of misconduct is not required under the law. All that is required is whether misconduct can be inferred from the individual's activities. Employers and AOAC depend on individual good judgment to avoid all discussions and activities which may involve improper subject matter and improper procedures. AOAC staff members work conscientiously to avoid subject matter or discussion which may have unintended implications, and counsel for the Association can provide guidance with regard to these matters. It is important for the individual to realize, however, that the competitive significance of a particular conduct or communication probably is evident only to the individual who is directly involved in such matters. In general, the U.S. antitrust laws seek to preserve a free, competitive economy and trade in the United States and in commerce with foreign countries. Laws in other countries have similar objectives. Competitors (including individuals) may not restrain competition among themselves with reference to the price, quality, or distribution of their products, and they may not act in concert to restrict the competitive capabilities or opportunities of competitors, suppliers, or customers. Although the Justice Department and Federal Trade Commission generally enforce the U.S. antitrust laws, private parties can bring their own lawsuits. Penalties for violating the U.S. and other antitrust laws are severe: corporations are subject to heavy fines and injunctive decrees, and may have to pay substantial damage judgments to injured competitors, suppliers, or customers. Individuals are subject to criminal prosecution, and will be punished by fines and imprisonment. Under current U.S. federal sentencing guidelines, individuals found guilty of bid rigging, price fixing, or market allocation must be sent to jail for at least 4 to 10 months and must pay substantial minimum fines. Antitrust Guidelines

Since the individual has an important responsibility in ensuring antitrust compliance in AOAC activities, everyone should read and heed the following guidelines.

1. Don't make any effort to bring about or prevent the standardization of any method or product for the purpose or intent of preventing the manufacture or sale of any method or product not conforming to a specified standard 2. Don't discuss with competitors your own or the competitors' prices, or anything that might

affect prices such as costs, discounts, terms of sale, distribution, volume of production, profit margins, territories, or customers.

3. Don't make announcements or statements at AOAC functions, outside leased exhibit space, about your own prices or those of competitors.

4. Don't disclose to others at meetings or otherwise any competitively sensitive information.

5. Don't attempt to use the Association to restrict the economic activities of any firm or any individual.

6. Don't stay at a meeting where any such price or anti-competitive talk occurs.

7. Do conduct all AOAC business meetings in accordance with AOAC rules. These rules require that an AOAC staff member be present or available, the meeting be conducted by a knowledgeable chair, the agenda be followed, and minutes be kept.

8. Do confer with counsel before raising any topic or making any statement with competitive ramifications.

9. Do send copies of meeting minutes and all AOAC-related correspondence to the staff member involved in the activity.

10. Do alert the AOAC staff to any inaccuracies in proposed or existing methods and statements issued, or to be issued, by AOAC and to any conduct not in conformance with these guidelines.

Conclusion

Compliance with these guidelines involves not only avoidance of antitrust violations, but avoidance of any behavior which might be so construed. Bear in mind, however, that the above antitrust laws are stated in general terms, and that this statement is not a summary of applicable laws. It is intended only to highlight and emphasize the principal antitrust standards which are relevant to AOAC programs. You must, therefore, seek the guidance of either AOAC counsel or your own counsel if antitrust questions arise.

Adopted by the AOAC Board of Directors: September 24, 1989 Revised: March 11, 1991 Revised October 1996

Appendix V

POLICY ON THE USE OF THE ASSOCIATION NAME, INITIALS, IDENTIFYING INSIGNIA, LETTERHEAD, AND BUSINESS CARDS

Introduction

The following policy and guidelines for the use of the name, initials, and other identifying insignia of AOAC INTERNATIONAL have been developed in order to protect the reputation, image, legal integrity and property of the Association. The name of the Association, as stated in its bylaws, is "AOAC INTERNATIONAL". The Association is also known by its initials, AOAC, and by its logo, illustrated below, which incorporates the Association name and a representation of a microscope, book, and flask. The AOAC logo is owned by the Association and is registered with the U.S. Patent and Trademark Office.

6JG HWNN #UUQEKCVKQP KPUKIPKC KNNWUVTCVGF DGNQY KU EQORTKUGF QH VJG NQIQ CPF VJG VCINKPG 6JG 5EKGPVKHKE #UUQEKCVKQP &GFKECVGF VQ #PCN[VKECN 'ZEGNNGPEG UJQYP DGNQY 6JG V[RGHCEG WUGF KU .CTIQ 6JG #1#% VCINKPG KU QYPGF D[ VJG #UUQEKCVKQP CPF KU TGIKUVGTGF YKVJ VJG 7 5 2CVGPV CPF 6TCFGOCTM QHHKEG

Policy

Policy on the use of the Association's name and logo is established by the AOAC Board of Directors as follows:

“The Board approves and encourages reference to the Association by name, either as AOAC INTERNATIONAL or as AOAC; or reference to our registered trademark, AOAC®, in appropriate settings to describe our programs, products, etc., in scientific literature and other instances so long as the reference is fair, accurate, complete and truthful and does not indicate or imply unauthorized endorsement of any kind. The insignia (logo) of AOAC INTERNATIONAL is a registered trade and service mark and shall not be reproduced or used by any person or organization other than the Association, its elected and appointed officers, sections, or committees, without the prior written permission of the Association. Those authorized to use the AOAC INTERNATIONAL insignia shall use it only for

the purposes for which permission has been specifically granted.

The name and insignia of the Association shall not be used by any person or organization in any way which indicates, tends to indicate, or implies AOAC official endorsement of any product, service, program, company, organization, event or person, endorsement of which, has not been authorized by the Association, or which suggests that membership in the Association is available to any organization.”

The Executive Director, in accordance with the above stated policy, is authorized to process, approve, fix rules, and make available materials containing the Association name and insignia.

It should be noted that neither the Association's name nor its insignia nor part of its insignia may be incorporated into any personal, company, organization, or any other stationery other than that of the Association; nor may any statement be included in the printed portion of such stationery which states or implies that an individual, company, or other organization is a member of the Association.

Instructions

1. Reproduction or use of the Association name or insignia requires prior approval by the Executive Director or his designate.

2. Association insignia should not be altered in any manner without approval of the Executive Director or his designate, except to be enlarged or reduced in their entirety.

3. Artwork for reproducing the Association name or insignia, including those incorporating approved alterations, will be provided on request to those authorized to use them (make such requests to the AOAC Marketing Department). Examples of the types of alterations that would be approved are inclusion of a section name in or the addition of an officer's name and address to the letterhead insignia.

4. When the Association name is used without other text as a heading, it should, when possible, be set in the Largo typeface.

5. Although other colors may be used, AOAC blue, PMS 287, is the preferred color when printing the AOAC insignia, especially in formal and official documents. It is, of course, often necessary and acceptable to reproduce the insignia in black.

6. Do not print one part of the logo or insignia in one color and other parts in another color.

7. The letterhead of AOAC INTERNATIONAL shall not be used by any person or organization other than the Association, elected and appointed officers, staff, sections, or committees; except by special permission.

Correspondence of AOAC official business should be conducted using AOAC letterhead. However, those authorized to use AOAC letterhead shall use it for official AOAC business only.

Copies of all correspondence using AOAC letterhead or conducting AOAC official business,

whether on AOAC letterhead or not, must be sent to the appropriate office at AOAC headquarters.

8. AOAC INTERNATIONAL business cards shall not be used by any person or organization other than the Association, its staff, and elected officials, except by special permission.

Those authorized to use AOAC business cards shall use them for official AOAC business only and shall not represent themselves as having authority to bind the Association beyond that authorized.

Sanctions

1. Upon learning of any violation of the above policy, the Executive Director or a designate will notify the individual or organization that they are in violation of AOAC policy and will ask them to refrain from further misuse of the AOAC name or insignia.

2. If the misuse is by an Individual Member or Sustaining Member of the Association, and the misuse continues after notification, the Board of Directors will take appropriate action.

3. If continued misuse is by a nonmember of the Association or if a member continues misuse in spite of notification and Board action, ultimately, the Association will take legal action to protect its property, legal integrity, reputation, and image.

* * * * * *

Adopted by the AOAC Board of Directors: September 24, 1989 Revised: June 13, 1991; February 26, 1992; March 21, 1995; October 1996

Expert Review Panels

Online Technical Resources Method Development, Optimization & Validation   OMA ‐ Appendix F ‐ Guidelines for Standard  Method Performance Requirements    Homogeneity   Guide for Writing Methods in AOAC Format   Statistics Protocol Review Form   OMA ‐ Appendix D:  Guidelines for Collaborative  Study Procedures to Validate Characteristics of a  Method of Analysis   OMA ‐ Appendix G:  Procedures and Guidelines for  the Use of AOAC Voluntary Consensus Standards to  Evaluate Characteristics of a Method of Analysis   OMA ‐ Appendix I: AOAC INTERNATIONAL Methods  Committee Guidelines for Validation of Biological  Threat Agent   Methods and/or Procedures    OMA ‐ Appendix J:  AOAC INTERNATIONAL Methods  Committee Guidelines for Validation of  Microbiological Methods for Food and  Environmental Surfaces   OMA ‐ Appendix K:  Guidelines for Dietary  Supplements and Botanicals   OMA ‐ Appendix L:  AOAC Recommended  Guidelines for Stakeholder Panel on Infant Formula  and Adult Nutritionals (SPIFAN) Single‐Laboratory  Validation   OMA ‐ Appendix M ‐ Validation Procedures for  Method Review   Examples of Statistical Analysis   Statistics Manuscript Review Form   OMA ‐ Appendix A:  Standard Solutions and  Reference Materials   OMA ‐ Appendix D:  Guidelines for Collaborative  Study Procedures to Validate Characteristics of a  Method of Analysis   OMA ‐ Appendix H:  Probability of Detection (POD)  as a Statistical Model for the Validation of  Qualitative Methods Miscellaneous   Definition of Terms and Explanatory Notes   OMA ‐ Appendix B:  Laboratory Safety   OMA ‐ Appendix E:  Laboratory Quality Assurance   OMA ‐ Appendix C:  Reference Tables  Quantitative Food Allergen ELISA Methods:  Community Guidance and Best Practices   Safety Checklist 

The ERPs review and approve appropriate methods (as submitted or modified)  for adoption as First Action Official Methods or for further validation.  ERPs  also make recommendations regarding Final Action Official Methods status.  Expert Review Panels  Must be supported by relevant stakeholders.   Constituted for the review of methods, not for Standard Method  Performance Requirements (SMPR) purposes or as an extension of a  Working Group.   Consist of a minimum of seven (7) members representing a balance of  expert stakeholders. Quorum is a minimum of 7 members present or 2/3 of  the total vetted members, whichever is greater.  ERP constituency must be approved by the Official Methods Board (OMB).   Holds transparent public meetings only.   Remains in force as long as method in First Action Status. First Action Official Method Status decision  Must be made by an ERP constituted or reinstated post 2011‐03‐28 for First  Action Official Method Approval (FAOMA).   Must be made by an ERP vetted for FAOMA purposes by OMB post 2011‐03‐ 28.   Method adopted by ERP must perform adequately against the SMPR set  forth by the stakeholders. Or demonstrate performance or characteristics  that meet the scope, applicability and/or claims of the method.   Method must be adopted by unanimous decision of ERP on first ballot, If  not unanimous, negative votes must delineate scientific reasons.   Negative voter(s) can be overridden by 2/3 of non‐negative voting ERP  members after due consideration   Method becomes First Action Official Methods on date when ERP decision is  made.   Methods to be drafted into AOAC format by a knowledgeable AOAC staff  member or designee in collaboration with the ERP and method author.   Report of FAOMS decision complete with ERP report regarding decision  including scientific background (references etc) to be published  concurrently with method in traditional AOAC publication venues.  Method in First Action Status and Transitioning to Final Action Status  Further data indicative of adequate method reproducibility (between  laboratory) performance to be collected. Data may be collected via a  collaborative study or by proficiency or other testing data of similar  magnitude.   Two years maximum transition time (additional year(s) if ERP determines a  relevant collaborative study or proficiency or other data collection is in  progress).   Method removed from First Action Official Methods and OMA if no  evidence of method use available at the end of the transition time.   Method removed from First Action Official Methods and OMA if no data  indicative of adequate method reproducibility is forthcoming as outlined  above at the end of the transition time.   ERP to recommend Method to Official Final Action Status to the OMB.   OMB decision on First to Final Action Status 

All resources are accessible at http://www.aoac.org/vmeth/guidelines.htm For questions, please contact: P 301-924-7077 x157 E dmckenzie@aoac.org

Revised October 2013  © 2013 Copyright AOAC INTERNATIONAl.

MECHANICS OF AN AOAC EXPERT REVIEW PANEL

ERP OVERVIEW: An Expert Review Panel (ERP) is assembled to review and adopt methods as Official First Action.  ERPs will track Official Methods for two years or until such time as reproducibility has been demonstrated and cumulative feedback on method use and performance are obtained.  ERPs will make a recommendation regarding Final Action method status for all OMAs to the Official Methods Board (OMB).    All ERP members are expected to serve with the highest integrity and without  direct or indirect conflicts of interest.  A method assignment can last two years.   All members of the ERP are expected to actively participate in ERP meetings and  to perform duties and reviews in timely fashion. All members should maintain  strict adherence to review timelines and deadlines.  AOAC staff documents ERP  deliberations. ESTABLISHING AN EXPERT REVIEW PANEL:    AOAC staff issues a Call for Experts:   o Based on voluntary consensus standards and methods submitted to  AOAC INTERNATIONAL that may meet the standards.    o Proprietary and sole source method developers submit individual  methods to the AOAC Research Institute.    o Candidates are asked to submit a CV or information that demonstrates  expertise to AOAC staff if not already part of a recognized pool of  experts.   AOAC Chief Scientific Officer (CSO) reviews the documentation for the  candidates and make recommends a slate for an expert review panel  including the chair to the Official Methods Board.   The candidate list and supporting documentation are forwarded to the Chair  of the OMB who will assign the review to at least two OMB members.    The OMB reviewers will review the candidates for expertise and perceived  conflicts of interest and the OMB may then approve the members of the  ERP. A Chair for the ERP is also approved.  About Expert Review Panels (ERPs) EXPERT REVIEW PANEL (ERP):  Review, discuss and demonstrate consensus on methods for Official First  Action method status.    Participate in the publications process of First Action methods.   Track and discuss feedback all First Action methods for two years.   Reach and demonstrate consensus on recommendations for Final Action  method status.   Actively participate in the broader stakeholder effort. ERP CHAIR:   Lead ERP discussions in the review and adoption of methods for First Action  Official Methods.   Participate in stakeholder panel activities.   Review and approve ERP report.   Work with AOAC staff, working groups and other stakeholder panels to  ensure a thorough understanding of the standard method performance  requirements and the methods to be assessed.   Implement the OMB First Action to Final Action Guidelines with the ERP  members.   Advise and review First Action methods and post First Action publications.   Represent the ERP in presenting the ERPs recommendation to the Official  Methods Board regarding Final Action method status.

 AOAC CSO assigns methods for review to the  expert review panel members.   For each method, 2 ERP members are assigned as  primary and secondary reviewers and present at  the ERP meeting.   All members are expected to actively participate  and review methods for First Action Official  Method status ‐ conducting thorough and prompt  review of methods and being prepared to speak  on assigned methods at ERP meetings   The ERP chair and the 2 reviewers for each  method are expected to participate in the  publications peer review process for First Action  methods.    ERP reviewers track assigned methods that were  adopted as First Action Official Methods and  update ERP on method use during two year period  between First Action and Final Action    ERP members are expected to participant in the  stakeholder panel activities and/or community at  large .   ERPs can work with topic advisors (aka, subject  matter experts)   OMB can recognize a pool of experts from which  ERP members can be selected Eligibility Criteria for Expert Reviewers  Be a key expert and/or thought leader of the method  or priority under consideration.    Demonstrated knowledge in the appropriate  scientific disciplines.   Demonstrated knowledge regarding data relevant  to adequate method performance.    Demonstrated knowledge of practical application  of analytical methods to bona fide diagnostic  requirements.   Be approved by the Official Methods Board    Qualifications must be clearly described and  submitted to AOAC headquarters. 

Duties of Expert Reviewers  Members of the Pool of Experts will be called upon to serve  on ERPs as needed and to review documents .These  documents may include:  

Procedural documents on how methods will be  selected and how single laboratory validation  studies will be done;   Methods submitted for consideration as First  Action Official Methods;   Methods submitted for selection for further  validation studies;   Protocols to be used for single laboratory  validation studies;   Selection of methods to be considered for full 

collaborative studies; and   Validation study reports  reports to bona fide diagnostic requirements

Revised October 2013 © 2013 Copyright AOAC INTERNATIONAl.

AOAC Stakeholder Panel on Dietary Supplements SET 1 INGREDIENTS EXPERT REVIEW PANEL (Anthocyanins, Chondroitin, PDE5 Inhibitors)

Monday, August 3, 2015 – Tuesday, August 4, 2015

A G E N D A

EXPERT REVIEW PANEL CHAIR: Brian Schaneberg, Starbucks Coffee Company

1. Welcome and Introductions Brian Schaneberg, Starbucks Coffee Company

2. Review a. AOAC Volunteer Policies & ERP Proccess Overview and Guidelines (8 :10 a.m. – 8 :30 a.m.) Deborah McKenzie

3. Review of Methods

For each method the assigned ERP members will present a review of the revised method manuscripts, after which the ERP will discuss the method and render a decision on the status for each method.

A. Chondroitin (August 3, 08 :30 a.m. – 12 :30 p.m.) a. CHON-001 b. CHON-002 c. CHON-003 d. CHON-004

e. Final Action Requirements for Approved Method(s)

--------------------------------- Lunch 12 :30 p.m. – 1 :00 p.m. ---------------------------------

B. Anthocyanins (August 3, 1 :00 p.m. – 5 :00 p.m.) a. ANTH-001 b. ANTH-002 c. ANTH-003

d. Final Action Requiremets for Approved Method(s)

SPDS Set 1 ERP 07/10/2015 – v1.0

SPDS ET 1 ERP Agenda

3. Review of Methods (continued)

C. PDE5 Inhibitors (August 4, 9 :00 a.m. – 3 :00 a.m.) a. PDE5-001 b. PDE5-002 c. PDE5-003 d. PDE5-004 e. PDE5-005

f. Final Action Requirements for Approved Method(s)

4. Adjourn

MEMORANDUM

D ATE :

A UGUST 3, 2015

T O :

SPDS SET 1 E XPERT R EVIEW P ANEL M EMBERS (C HONDROITIN )

F ROM :

AOAC INTERNATIONAL

S UBJECT : C HONDROITIN M ETHOD S UBMISSIONS AND R EVIEWS ____________________________________________________________________________ BACKGROUND: Four methods were submitted in response to the Chondroitin Call for Methods. The reviews submitted are provided in this meeting book, and links to the Standard Method Performance Requirements SM (SMPRs) and the candidate methods themselves are provided below.

AOAC Candidate Method Number

Submitter

Primary Reviewer

Secondary Reviewer(s)

CHON-001 CHON-002

USP

Jana Hildreth

Curtis Phinney Curtis Phinney

Institute of Chemical Technolgy, Czech Republic

Kelly Reins

CHON-003

Amway

Aniko Solyom

Nour Eddine Es Safi Phillip Koerner

CHON-004

Analytical Laboratories in Anaheim, Inc.

Darryl Sullivan

SMPRs:

• AOAC SMPR 2014.008 - Screening Method for Selected Adulterants in Dietary Ingredients and Supplements Containing Chondroitin Sulfate • AOAC SMPR 2014.009 - Determination of Total Chondroitin Sulfate in Dietary Ingredients and Supplements

CHON-001

Question

Response

General

AOAC Method Number:

CHON-001

Selected Adulterants in Dietary Ingredients and Dietary Supplements Containing Chodroitin Sulfate

Method Title:

Method Author(s):

United States Pharmaopeia

Reviewer Name:

Jana Hildreth

Cellulose acetate membranes e;ectophoresis with Toluidine Blue staining based on the characteristic migration pattern of chondroitin sulfate on cellulose acetate membranes when subject to a voltaic current, producing visible very well defined bands after staining the developed membranes with a cationic dye (Toluidine Blue) . Screening method for selected adulterants in dietary ingredients claiming to contain chondroitin sulfate: Sodium hexametaphosphate Sodium alginate Propylene glycol alginate sulfate sodium Over-sulfated polysaccharides This method has a proven track record of finding adulterants in chondroitin sulfate raw material and has been in pharmacopieas worldwide. This is an extremely straight forward method that does not require much training. The cost of running this method is very cheap. It has proven to find new adulterants that more sophisticated methods missed. The cost of the equipment is under $2000 and training is minimal. Currently only a couple of labs have the set up to run this method in the US. Supporting Data

Summary of Method

Method Scope / Applicability:

General Comments About the Method:

Method Clarity

Pros/Strengths

Cons/Weaknesses

General Comments

I feel this is a great screening tool for incoming chodroitin

CHON-001

raw material that any lab can run and can afford the equipment.

Method Optimization

None needed. Performance Characteristics

Sodium Alginate: 0.3 % Hexametaphosphate: ~1 %

Analytical Range:

Propylene glycol Alginate Sulfate Sodium: 0.6 % Oversulfated Chondroitin: Between 1 and 2 %

LOQ:

See above-this is a meant to be a qualitative method

Accuracy/Recovery

N/A

Precision (RSDr)

N/A

Reproducibility (RSDR)

N/A

System Suitability

N/A

Safety Review

Is the method safe to use as written? Yes Are there any safety concerns with any component, instrument, reagent or instruction of the method? If yes, please clearly state the concerns. Does the method include all necessary safety precautions and/or warning statements? Yes or No. If No, please suggest potential precautions or warnings that can be added to the method for users or recommend safer option(s) if warranted. Yes

Electric shock if someone does not understand the basics of electricity.

I feel this is a great screening tool that labs worldwide can afford and apply to screen out adulterated materials.

Final Recommendation:

CHON-002

Question

Response

General

AOAC Method Number:

CHON-002

Isotachophoretic Determination of Glucosamine and Chondroitin Sulphate in Dietary Supplements, Czech Journal of Food Science 31:55-65

Method Title:

Method Author(s):

Eliska Vaclavikova and Frantisek Kvasnicka

Reviewer Name:

Kelly Reins

Capillary isotachophoretic method for the determination of chondroitin suphate in dietary supplements. Anionic analysis of chondroitin sulphate, CS, was performed using a leading electrolyte solution consisting of a mixture of 5mM HCl + 10mM glycylglycine+ 0.05% 2-hydroxyethylcellulose. The terminating solution was 10mM citric acid. The separation compartment contained a pre-separation Fluorinated ethylene propylene (FEP) capillary coupled with an analytical FEP capillary. The separation was carried out with the electro- osmotic flow suppressed. The analytes were detected by conductivity and UV (254nm) detectors. Method is for Glucosamine and CS determination. All reviewer comments are for CS only. The authors performed the validation on tablets with stated daily doses of 600 and 1200 mg CS. Capsules, syrups and powders obtained from manufacturers and domestic markets were analyzed, however, were not part of the validation. • Raw data was not submitted for the reviewer. • The validation was performed using analytical grade chemicals and reagents. The standards were purchased from Sigma and do not appear to be properly characterized. Authors reported impurities being found in the reference materials; CS-A being a mix of CS-A and CS-C. • Accuracy was performed using standard addition of CS-A at 50% and 100%. • Data on the effectiveness on sonication was not provided. Stability of sample solution in a sonicator for 30 minutes was not evaluated.

Summary of Method

Method Scope / Applicability:

General Comments About the Method:

CHON-002

• Extraction was assumed to be finished upon complete disintegration of the tablet in water. Data was not presented for determination of complete disintegration. • Samples were filtered using unspecified filter paper. Filter recovery studies were not reported. • pH is critical for the success of this type of analysis. Data was not given for leading or terminating solutions at various pH levels. • Authors used enzymes for the digestion of CS. The chondroitinase is pH, concentration and temperature dependent. The authors did not describe the sample preparation in the original paper. They did however, discuss following the procedure published by Volpi in 2007 with some modifications. It’s not clear if these modifications were consistent. Miscellaneous typos, cation vs anion among others. The method lacks details for step by step sample preparation used for analysis. This could be due to the 10-page limitation for articles to be included in this particular journal. Sample preparation dilution on p. 59 appears to be out of the calibration curve but in Table 3 the validation samples were stated to be diluted into the calibration curve range. It’s unclear if this was done with routine samples as well. Sample filtration was performed with an unspecified filter type. Filter studies not mentioned. Method was used to test multiple dosage forms but the validation was only for tablets. There is no suggested system suitability requirements. The method refers to analysis of CS but sample concentration estimation appears to be based on CS-A during the validation. It’s unclear which reference material are used in the preparation of stock and working standards, page 59 P2.

Method Clarity

No dangerous chemicals required Simple sample preparation in water Short analysis time

Pros/Strengths

In general, CE has some drawbacks: • Low sensitivity • Sample concentrations must be dramatically increased to obtain the same S/N compared to a typical LC run • pH in the capillary can be affected resulting in countering of the stacking effect. • Lack of data regarding standard retention times and peak area

Cons/Weaknesses

o Inability to quantify analyte o Irreproducible flow rates o Inconsistent injection volumes

CHON-002

• Difficult to determine appropriate test conditions for unknown samples.

HPLCs/UPLCs are common instruments in many labs. CE is not as widely used. Method specific drawbacks: • The method is non-specific. CS-A, B, and C had almost identical mobilities, Figure 3, p 61; therefore could not be separated using cITP. • The method is not able to differentiate CS-B (dermatan sulfate). • Acesulfam K and a high content of hydrolyzed collagen were reported interferences. The author state that Acesulfam K was easily detected in the UV trace at 254 nm. • Carrageenan and Hyaluronic could cause interference with this type of methodology. • Author stated the presence of hyaluronate and heparin did not disturb the analysis of CS but they did not provide any data. The assumption was that hyaluronate has a lower mobility than the terminator (citrate) and the heparin has a higher mobility. • In order to maximized separation multiple pH requirements are required for interfering compounds. Supporting Data o Sample preparation for the validation was one tablet dissolved in 250mL MilliQ water. One tablet, or capsule, or daily dose was used to prepare the test samples. However, the validation used a composite of 20 tablets for homogeneity reasons. There is no validation data for other dosage forms. o For the validation, external calibration solutions of CS-A were prepared at 20 – 200 mg/L to create a calibration curve using step length vs. concentration. It is unclear there was other work performed for total CS. The authors provided supplemental information: Quality Control of Chondroitin Sulphate used in Dietary Supplements. Czech Journal of Food Science 33:165-173. Modifications to the method were pH and the leading electrolyte composition. The scope of method was for raw material. Reference standards were EP CRS grade. cITP was used to determine the free sulphate content in CS. This method is unable to differentiate CS-B (dermatan sulphate)

General Comments

Method Optimization

CHON-002

Capillary zone electrophoresis (CZE) was used for the analysis of unsaturated CS disaccharides. Response factors were used for the disaccharides (Di-0S, Di-4S, and Di-6S), for quantitation of disaccharides in the samples. CS was calculated as the amount of unsaturated disaccharides released by chondroitinase ABC divided by the ratio of 1.036 (ratio of MW of disaccharides and MW of disaccharide unit in CS). The method was run ay 60C to avoid peak splitting of the disaccharides. Size exclusion chromatography (SEC) was used to analyze CS using an RI detector. Dextrans (MW: 10K, 20K, 40K, 70K, and 150K g/mol) were used for calibration standards. The relationship of the respective dextran and log MW was evaluated. The separation was carried out using 3 columns in series. Chondroitinase ABC was used to determine the composition and content of the disaccharides. The authors state they followed a published method with modifications. The authors state the digestion time and inactivation method used was sufficient but did not provide any supporting data. The mixtures were then analyzed using CZE. Safety Review The range of the method was not defined by the authors. The authors used statistical regression for linearity. Calibration curve was evaluated for CS-A (20-200 mg/L). Routine samples were evaluated from 20-100 mg/L. There is no mention of why the curve range was varied. Calculated as 3 times the LOD. LOD was calculated from the calibration equation. LOD corresponds to the concentration of CS which gives the minimum detectable zone length equal to one second. The authors report LOQ at 9mg/mL, I calculated the LOQ at 8 mg/mL based on the information given by the authors. 8 mg/mL is at the 1% level. Its unclear if the LOD concentration was properly evaluated since the calibration curve used to extrapolate LOD concentration was much higher (20-200 mg/L) than the lowest standard concentration (LOD estimate implied at 3 mg/L). P. 59 Assessed by the average of three replicates of sample spiked with CS-A at the 50% and 100% level. 20 tablets composited, weight equivalent to one tablet was used. Accuracy was reported as 99.7 +/- 2.6% (97.1 – 102.3); therefore, not meeting the SMPR requirement of 98-103% for >10% w/w. Assessed by determination of six replicates in two different samples. 20 tablets composited, weight equivalent to one

Analytical Range:

LOQ:

Accuracy/Recovery

Precision (RSDr)

CHON-002

tablet used. Inter-day Precision was reported at 1.94% RSD and Intra-day precision was reported at 1.53%, which meets the SMPR of less than or equal to 2%. Assessed using the combined %RSD of the analysis of two samples over two days. This was done within the same laboratory rather than a multi-laboratory validation as stated in the SMPR. No system suitability results were reported for the validation nor are there any stated requirements in the method to ensure the system is operating as intended prior to analysis.

Reproducibility (RSDR)

System Suitability

Is the method safe to use as written? Are there any safety concerns with any component, instrument, reagent or instruction of the method? If yes, please clearly state the concerns. Does the method include all necessary safety precautions and/or warning statements? Yes or No. If No, please suggest potential precautions or warnings that can be added to the method for users or recommend safer option(s) if warranted.

Yes

No

There is no mention of any safety precautions in the method.

Recommendations

I do not recommend the method be adopted for First Action status. The method uses instrumentation that is not widely used in commercial testing laboratories. The methodology lacks specificity and robustness. There are far too many parameters that require strict controls thus making this method undesirable for routine QC testing.

Final Recommendation:

CHON-002

Question

Response

General

AOAC Method Number:

CHON-002

Isotachophoretic Determination of GA and CS in Dietary Supplements

Method Title:

Method Author(s):

Vaclavikova, E., et al.

Reviewer Name:

Curtis Phinney

This method is based on (cITP), a capillary electrophoretic technique based on ionic mobility, with conductivity and UV (254 nM) detection.

Summary of Method

Method Scope / Applicability:

Applicable to dietary supplements and raw materials.

The technique is very well described in two related papers. The authors investigated the role of economically significant and common impurities in great detail. The method is described fully, with considerable background and supporting information.

General Comments About the Method:

Method Clarity

Rigorous experimental design Addresses significant quality concerns Addresses economic adulterants Simple sample prep and reagents

Pros/Strengths

Validated using alternate guidelines (ICH '95, '96) No explicit system suitability step No inter lab data, 2-day inter day study Supporting Data Extensive supporting data and discussion of interferences. Supporting data may not address all SMPR Exhaustively optimized method. Raw data is not presented in this paper. Performance Characteristics

Cons/Weaknesses

General Comments

Method Optimization

Analytical Range:

< 1% to 100%

CHON-002

LOQ:

9 mg/L

Accuracy/Recovery

99.7 +/- 2.6%

Precision (RSDr)

< 2%

< 2% reported

Reproducibility (RSDR)

System Suitability

Not explicitly addressed in paper. Safety Review

Is the method safe to use as written? Are there any safety concerns with any component, instrument, reagent or instruction of the method? If yes, please clearly state the concerns. Does the method include all necessary safety precautions and/or warning statements? Yes or No. If No, please suggest potential precautions or warnings that can be added to the method for users or recommend safer option(s) if warranted.

Yes

Not any apparent from paper

High potential iTCE source/leads/connectors?

Recommendations

Final Recommendation:

Qualified approval

CHON-003

Question

Response

General

AOAC Method Number:

CHON-003

Chondroitin by IR and Dimethylene Blue (DMBB) spectrophotometry

Method Title:

Method Author(s):

Not stated

Reviewer Name:

Aniko Solyom

Colorimetric method for the quantification of chondroitin sulfate. According to the method description, the method is applicable for the quantification of chondroitin sulfate in raw materials and finished products, such as tablets.

Summary of Method

Method Scope / Applicability:

General Comments About the Method: Method Clarity

Detailed SOP style

Relatively quick and cheap method for the analysis of raw materials and probably simple finished products. 1. Chondroitin-DMMB complex will aggregate immediately, therefore the reading time should be the same for each sample/standard. Room for significant human error! 2. Not a specific method for complex samples. If anything (including adulterants)in the sample reacts with DMMB and absorbs at 525 nm, the results are not valid.

Pros/Strengths

Cons/Weaknesses

Supporting Data

General Comments Method Optimization

Not Provided Performance Characteristics

Calibration curve: 5-15 microgram chondroitin sulfate (only 3 point calibration curve) Chondroitin sulfate content of the

Analytical Range:

CHON-003

LOQ:

Not provided

Spike recovery: 102.5-103.6% at three spike levels (50, 100 and 150%). The number of repeated spikes is not reported The results of 5 sample preparations in two laboratories were provided. The overall RSDn is 7.11 and that is larger than required in the SMPR

Accuracy/Recovery

Precision (RSDr)

Reproducibility (RSDR) System Suitability

NA

Safety Review

Is the method safe to use as written?

Yes

Are there any safety concerns with any component, instrument, reagent or instruction of the method? If yes, please clearly state the concerns. Does the method include all necessary safety precautions and/or warning statements? Yes or No. If No, please suggest potential precautions or warnings that can be added to the method for users or recommend safer option(s) if warranted.

No

Yes

Recommendations

The method as written is applicable for the analysis of raw materials and simple finished products . The method doesn't meet the Method Performance Requirements stated in the SMPR, but these might be improved after further method development/optimization. Depending on the quality of the other submission this method can be a candidate for further consideration.

Final Recommendation:

CHON-003

Question

Response

General

AOAC Method Number:

CHON-003

Chondroitin by IR and Dimethylmethylene Blue (DMMB) Spectrophotometry

Method Title:

Method Author(s):

Not given

Reviewer Name:

Professor Nour Eddine ES-SAFI

CHON-003 method presents results dealing with the use of UV-visible spectrophotometry for the quantitative analysis of chondroitin sulfate in raw material and finished products after reaction with dimethylmethylene blue reagent.

Summary of Method

Method Scope / Applicability:

Not Specific to CS

The method is simple but critical in terms of time, photometric determination at 525 nm and may be not specific for chondroitinsulphate. Although recent studies have demonstrated the efficiency of DMMB over other assays this method suffers from multiple drawbacks. The reagent used is unstable, interferences with DNA or other negatively charged molecules seriously modify the response, and, in addition, the GAG-dye complex is not stable in solution, and GAG measurements are not accurate because of this instability. Indeed, variations of the time between sample measurements may modify results. Furthermore, accuracy, precision, and linearity are poor and even unacceptable at levels of less than 5mg/ml of GAGs.

General Comments About the Method:

Method Clarity

Poor

Pros/Strengths

Simple

The dimehtylmethylene blue dye binding method used for determination of galactosaminoglycan is not specific to sulfated GAGs. The dye also reacts with negatively charged polymers (e.g. hyaluronic acid and nucleic acids) and anions to form metachromasia. It is important to know that the color yield of metachromasia differs between CS preparations with different structure.

Cons/Weaknesses