2018 Sugar ERP - Method Review Book

STAKEHOLDER PANEL ON STRATEGIC FOOD ANALYTICAL METHODS

EXPERT REVIEW PANEL FOR SUGARS / FRUCTANS

SHERATON CENTRE TORONTO 123 Queen Street W, Toronto, ON, Canada CONFERENCE ROOM: Willow, Centre & West

8:30am – 12:00pm Eastern Time e -Registration Opens at 7:30am

CONTACT: SPSFAM@AOAC.ORG

STAKEHOLDER PANEL ON STRATEGIC FOOD ANALYTICAL METHODS

EXPERT REVIEW PANEL FOR SUGARS / FRUCTANS

SHERATON CENTRE TORONTO 123 Queen Street W, Toronto, ON, Canada CONFERENCE ROOM: Willow, Centre & West

8:30am – 12:00pm Eastern Time e -Registration Opens at 7:30am

CONTACT: SPSFAM@AOAC.ORG

AOAC SPSFAM Sugar/Fructans Expert Review Panel  August 29, 2018 

Full Name 

Organization 

George Joseph (ERP Chair) 

AsureQuality, New Zealand 

Nicole Burke 

Kellogg Company 

Philip Andrew Haselberger 

Abbott Nutrition 

Ankur Kumar 

Government of India 

Sookwang Lee 

FDA 

Roberto Molteni 

Italian Ministry of Agriculture 

Hari Narayanan 

Metrohm USA Inc 

Jack Stevens 

Medallion Labs / General Mills, Inc. 

John Szpylka 

Mérieux NutriSciences 

Nancy Thiex 

AAFCO Headquarters Office 

Martine Van Gool 

FrieslandCampina Innovation Centre 

Tom Vennard 

Eurofins Food Integrity and Innovations 

12 OMB Vetted Members of the AOAC SPSFAM Sugar / Fructans Expert Review Panel 

AT LEAST 8 ERP MEMBERS MUST BE PRESENT TO CONSITUTE A QUORUM

AOAC Stakeholder Panel on Strategic Food Analytical Methods Sugar and Fructans Expert Review Panel

Wednesday, August 29, 2019 ; 8 :30 a.m. – 12 :00 p.m. Sheraton Centre Toronto Hotel, Room Willow Centre and West

A G E N D A

1. Welcome and Introductions George Joseph, AssureQuality New Zealand (ERP Chair)

2. Review of AOAC Volunteer Policies & ERP Proccess Overview and Guidelines Deborah McKenzie, AOAC INTERNATIONAL

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. SUG-01

a. Burke Review b. Molteni Review c. Discussion and Vote

B. SUG-02

a. Szpylka Review b. Lee Review c. Discussion and Vote

C. SUG-03

a. Haselberger Review b. Narayanan Review c. Discussion and Vote

D. SUG-04

a. Vennard Review b. Kumar Review c. Discussion and Vote

4. Final Action Requirements for Approved Method(s) 5. Adjourn

SPSFAM Sugar and Fructan ERP 08/01/2018 – v1.0

AOAC Expert Review Panel  Meetings An Orientation

Deborah McKenzie רב Sr. Dir., Standards Development AOAC INTERNATIONAL Sr. Dir., AOAC Research Institute Staff Liaison ‐ Official Methods Board

AOAC Policies & Procedures

Policy on Use of  Association Name,  Identifying Insignia,  Letterhead, Business  Cards

Policy on Volunteer  Conflict of Interest

Policy on Antitrust

Expert Review Panel  Policies and Procedures

OMA Appendix G

1

Policies and Procedures for Adoption of  Official Methods of Analysis 

• OMA, Appendix G: Procedures and Guidelines for the Use of AOAC Voluntary Consensus Standards to Evaluate Characteristics of a Method of Analysis – Expert Review Panels, Official Methods Board, First and Final Action Official Methods – First Action to Final Action Methods: Guidance for AOAC Expert Review Panels • Expert Review Panels – Policies and Procedures • Appendix F: Guidelines for Standard Method Performance Requirements • OMA, About the AOAC Official Methods SM Program

Road to First Action OMA Status

Three modes of entry  and (program  administration)

Expert Review Panels will  review all methods for all  three modes of entry.

2

ERP Meetings

Quorum

Presence of 7  vetted ERP  members 

Presence of  2/3 vetted  ERP members

OR

WHICHEVER IS GREATER IF NO QUORUM, NO OFFICIAL MEETING

Candidate Method Reviews

 In your judgment, does the method sufficiently meet the Standard Method Performance Requirements (SMPR) or community‐based guidance?

 In your judgment, is the method scientifically sound and can be followed?  In your judgment, what are the strengths and weaknesses of the method?  In your judgment, how do the weaknesses weigh in your recommendation for the method?  In your judgment, will the method serve well the stakeholder community that will use the method?  In your judgment, what additional information may be needed to further support the method meeting the SMPR or community‐based guidance?  Members of both Committee on Safety and Committee on Statistics serve as  advisory resources for all ERPs

3

ERP Meetings – Review for First Action  ERP CHAIR & MEMBERS:    Present reviews and discuss the method,  supporting data, and any resulting issues or questions on the method,  review and agree upon final draft of method proposed for decision, and  chair calls for ERP decision in accordance to procedures.

CONSENSUS:   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.    Abstentions do not count towards vote; in case of multiple abstentions the results will  need to be evaluated.  Staff will monitor  and record consensus voting.

STAFF:   Will organize and coordinate meeting,  record  ERP  actions and decisions, draft ERP report and distribute after  chair approval,  work with chair and OMB liaison to complete  checklist and assemble recommendation package  for OMB.

Consensus – First Action to Final Action

 The ERP may then reach consensus on any additional information that it needs to review to be able to make a recommendation for Final Action Official Methods status.

 This is a separate motion.

4

Road to Final Action OMA  Status

Method reproducibility must be  demonstrated before Final Action  consideration. 

ERP determines if sufficient  evidence merits a  recommendation for Final Action  status or repeal. • Only the OMB promotes a  method to “Final Action” status or repeal the method. • Methods that did not meet the  bar would be repealed. • Same for all method submissions

ERP Methods Review & Approval

Methods should be scientifically sound with demonstrating  that it will meet the needs of those using the method  (evidenced by meeting the standard, or other acceptance  criteria) 

ERPs have approved methods with evidence of high potential  to First Action and request additional work or support be  submitted for review prior to ERP convening to recommend an  action to OMB

OMB requires a justification or rationale for methods that are  deemed acceptable and adopted but may not fully meet the  standard set or acceptance criteria.

5

OMB Expectations for First Action

• Safety review needed prior to First Action status

• SLV type of supporting information available per the SMPR – Applicability, Method Performance Requirements Table, System Suitability, Reference Materials, and Validation Guidance • Comparison to SMPR – Documented method performance versus a SMPR – Document reasons for acceptability if method does not meet the SMPR

Publication of First Action Methods

 Any approved method(s) along with supporting manuscript(s) and  documentation sent to AOAC Publications after themeeting.

1. Method incorporating ERP revisions (preferably in AOAC Format) 2. Method Manuscript incorporating specified ERP revisions (in AOAC  Format) 3. Signed AOAC Copyright Authorization form

NO OMA NUMBER ASSIGNED  UNTIL ALL DOCUMENTATION SUBMITTED

 Method and method manuscript prepared for publication  in the Official Methods of Analysis of AOAC  INTERNATIONAL and in Journal of AOAC INTERNATIONAL

 Updates on methods approved or status changes are  published in the Inside  Laboratory Management magazine  and on the AOAC website

6

ERP Meetings – Method Tracking METHOD AUTHOR:    present any method feedback obtained  and any resulting changes to the method, any reproducibility  information, any implemented ERP recommendations, final  draft of method proposed for decision ERP MEMBERS:    present any method feedback obtained and 

discuss any resulting changes to the method, any  reproducibility information, any implemented ERP  recommendations, review and agree upon final draft of  method proposed for decision, and make a recommendation  to OMB. CONSENSUS:    2/3 vote in favor of a motion.    Abstentions do not count towards vote; in case of  multiple abstentions.  Staff will monitor  and record  consensus voting.

STAFF:   Will organize and coordinate meeting,  record   ERP actions and decisions, draft ERP report and  distribute after chair approval,  work with chair and  OMB liaison to complete checklist and assemble  recommendation package  for OMB.

Documentation Needed

Method Safety Evaluation

Reference Materials

Evidence of Single Laboratory Validation or equivalent 

Evidence of Reproducibility Assessment 

Published First Action OMA

Method Performance versus SMPR or acceptance criteria

Final draft of First Action OMA to be considered for status update

Rationale or Justification for Repeal or Continuance of First Action OMA

7

Documentation and Communication • AOAC carefully documents the actions of Stakeholder Panel and the Working Groups • AOAC will prepare summaries of the meetings – Communicate summaries to the stakeholders – Publish summaries in the Referee section of AOAC’s  Inside Laboratory Management • AOAC publishes its voluntary consensus standards and Official Methods – Official Methods of Analysis of AOAC INTERNATIONAL – Journal of AOAC INTERNATIONAL • AOAC publishes the status of standards and methods in the Referee section of AOAC’s  Inside Laboratory Management General Expectations for ERPs • You can expect to have a minimum of three weeks to review methods prior to ERP meeting. – You are requested to submit written reviews by specified deadline.  Please alert staff if you are not able to complete on time. – You may have individually assigned methods to review or all of the methods to review.  Please be prepared to discuss these methods during meeting. – You may use the OMA appendices as guidance for types of validation work that can be expected.  If additional information is needed, please ask staff. • ERP Meeting Quorum – If there is no quorum, there is no official meeting.  Please alert staff as early as possible if you are not able to attend a meeting. • ERP Consensus – ERP consensus may not reflect your own personal view – There may be times when a method may not meet all of the criteria exactly; however, the ERP can adopt the method.

8

Ethical Expectations of AOAC Expert  Review Panel Members • Respect for your peer ERP members and chair – Each member has been vetted for expertise relevant to the review of the method(s) in the ERP • Be considerate of each others perspectives and points of view • Be considerate of the ERP’s consensus even if you disagree – Inform staff as early as possible if you cannot attend the scheduled ERP meeting • Be considerate in that your absence can impact the quorum of the ERP and its ability to have an official meeting to make decisions – Notify staff and/or disclose in the ERP meeting if you have a direct or perceived conflict of interest for a specific method • Please review AOAC’s policy on Volunteer Conflict of Interest Ethical Expectations of Expert Review Panel  Members  (con’t) • Respect for Method Authors and Intellectual Property – Each Method Author is encouraged to attend the ERP meeting – Each candidate methods (not yet adopted or published as Official Methods of Analysis of AOAC INTERNATIONAL ) are still the intellectual property of the method author.  Therefore, the information is shared only with the vetted ERP members and is available during the meetings.  Please do not distribute the information without expressed written permission from an appropriate AOAC staff liaison. – Be clear about and justify how additional recommended work is a requirement for First Action, a requirement for Final Action consideration, or something recommended, but not necessary. – Keep your focus on the science

9

ERP Chair Responsibilities

Before Meeting

During Meeting

Moderate discussions based  on agenda

Work with staff on meeting  coordination

Engage staff to encourage  members to reach decision  points

Review submitted and/or  assigned methods

Engage staff on procedural  questions

Review method reviews if  applicable

Engage discussion on feedback  mechanism

Review SMPR(s) and/or  relevant guidance and criteria

ERP Chair Responsibilities

Other Efforts and  Recognitions Can nominate methods for  OMB Award

After Meeting Review Meeting Report  and Approve Final Version

Can nominate ERP members  for OMB Award

Assist with any follow up on  methods

Can assist in identifying  methods for review

Assist in Publication  Reviews

Can serve as a guest editor for  the Journal

10

Roles and Responsibilities

AOAC Official Methods Board Vet and approve stakeholder panel chair & voting members Vet and approve ERP membership and AOAC Experts Render decisions on status of First Action methods (Final Action,  repeal, etc…) Assign a liaison to each stakeholder panel and ERP Coordinate OMB Awards AOAC Expert Review Panels Review methods and meet in person to render decisions on  methods for First Action Official Methods SM status. Track First Action Official Methods SM and modify, if necessary Recommend First Action methods after 2 years or less to OMB  for Final Action, continuance, or Repeal Participate in Consulting Service and PTM reviews for OMA and  harmonized PTM and harmonized OMA method studies AOAC Experts Review and approve PTM validationtesting protocol documentation Peer review of PTM validation manuscript and supporting  documentation AOAC Research Institute ‐ PTM Expert Reviewers Peer Review of PTM validationmanuscripts and supporting  documentation

AOAC Research Institute Independent Laboratories Conduct independent evaluation of candidate method using AOAC  approved testing protocols AOAC Stakeholder Panels Develop  voluntary consensus standards  Assign working groups to  draft standards method performance  requirements Voting members demonstrate  consensus on behalf of  stakeholders AOAC Staff Coordinate method reviews and method approval activities Coordinate OMB meetings Provide trainings and orientations Maintain website and communication Document and publish actions and decisions Coordinate standards development activities Publish standards and methods AOAC Research Institute Technical Consultants Draft validation protocols in Consulting Service for assigned methods

Facilitate PTM evaluation of assigned candidate methods Facilitate comments/responses for assigned OMA reviews

Questions?

Thank you 

11

AOAC STAKEHOLDER PANEL ON STRATEGIC FOOD ANALYTICAL METHODS:

SUGAR EXPERT REVIEW PANEL – METHOD AND SMPR ACCESS

• Method Access [ERP MEMBERS ONLY]

• AOAC SMPR 2018.001 : Sugars in Animal Feed, Pet Food, and Human Food

• AOAC SMPR 2018.002 : Flavanols in Food and Beverages

AOAC SPSFAM Sugar/Fructans Expert Review Panel:  Method Review Assignments 

AOAC  Candidate  Method  Number 

Primary  Reviewer 

Method Title 

Method Authors 

Secondary Reviewer 

Analysis of the Labeling Sugars in Feed, Food and  Ingredients 

SUG‐001 

Jack Stevens 

Nicole Burke 

Roberto Molteni 

Single Lab Validation of Megazyme’s Fructan  Assay Kit (K‐FRUC) Method for the Determination  of Fructan (Inulin, FOS, Levan and Branched  Fructan) in Animal Food (Animal Feed, Pet Food,  and Ingredients) 

Barry V. McCleary, Lucie M. J.  Charmier, Vincent A. McKie, Ciara  McLoughlin and Artur Rogowski. 

John Szpylka 

Sookwang Lee 

SUG‐002 

Sugar Profile by High Performance Anion  Exchange Chromatography with Pulsed  Amperometric Detection 

Tom Vennard, Andrew Ruosch,  David Ellingson 

Philip  Haselberger 

Hariharasubramanian  Narayanan 

SUG‐003 

Quantitation of Six Common Food Sugars and  Inositol by HPLC‐MS 

Thomas  Vennard 

SUG‐004 

Eurofins 

Ankur Kumar

AOAC Candidate Method SUG-001

Submitter Name Submitter Email Organization Method Type Method Name

Jack Stevens

Jack.Stevens@genmills.com

General Mills

Sugar

Analysis of the Labeling Sugars in Feed, Food and Ingredients

Method Author(s)

Jack Stevens

Method Applicability

This method determines the quantity of the nutritional leveling sugars fructose, glucose, galactose, sucrose, maltose, and lactose in feed, foods and ingredients by RI-HPLC utilizing normal phase chromatography.

Primary Reviewer: Nicole Burke, Kellogg Company Secondary Reviewer: Roberto Molteni, Italian Ministry of Agriculture

AOAC SMPR ERPs - 2018 METHOD REVIEW FORM

Submission ID

4096451708597282986

Submission Date

2018-08-09 12:32:50

Name

Nicole Burke

E-mail

nicole.burke@kellogg.com

Organization

Kellogg

Title of Method

Analysis of the Labeling Sugars in Feed, Food, and Ingredients

AOAC Candidate Method Number (e.g. ALN-01)

SUG-001

Applicable SMPR

2018.001 Standard Method Performance Requirements for Sugars in Animal Feed, Pet Food, and Human Food

I. Summary of the Method

This method utilizes normal phase high performance liquid chromatography with refractive index detection to determine the quantities of six sugar analytes used in nutritional labeling: fructose, glucose, galactose, sucrose, maltose, and lactose. The method is capable of quantifying these sugars at levels above 0.1% in animal feed, pet food, and human food. Sugar is extracted from these sample matrices using water as an extraction solvent. The resulting extracts are diluted with acetonitrile, filtered with a 0.2 µm membrane filter, and then separated on an amino column prior to detection by refractive index. Quantitation is calculated on the basis of sample response relative to a ribose internal standard and linear regressions for standards of each sugar analyte. The applicability of the method does not fully support the applicability of the SMPR. The SMPR states that the method must be able to individually measure fructose, galactose, glucose, sucrose, maltose, and lactose; however, the method does not fully demonstrate the ability to individually measure galactose up to the standards of the SMPR. Additionally, while the SMPR states that the method must be able to perform these analyses in selected ingredients and foods consumed by animals, pets, and humans, the method in question only demonstrates analysis in a pet food matrix for sucrose and not all of the sugars specified in the SMPR. Lastly, the SMPR states that the method must account for a variety of potential inferences. While the method provides a table of retention times for a variety of potential inferences, it does not provide any further evidence demonstrating that the method can account for interferences in quantitation. Additionally, the method only provides this retention time information for a small group of analytes that does not represent all, or even a majority, of the potential interferants specified in Table 2. of the SMPR. Yes, the technique of Refractive Index-High Performance Liquid Chromatography utilizing normal phase chromatography could meet the SMPR if it is able to meet the method performance requirements specified in the SMPR for the six analytes. The ability of the method to meet these performance requirements is discussed below in Section III.

II. Review of the Method Only 1. Does the applicability of the method support the applicability of the SMPR? If not, please explain what is missing.

2. Does the analytical technique(s) used in the method meet the SMPR? If not, please specify how it differs from what is stated in the SMPR.

3. Are the definitions specified in the SMPR used and applied appropriately in the method? If no, please indicate how the terms are used.

Yes, for the most part, the definitions specified in the SMPR are used and applied appropriately in the method. However, to a large degree, this determination is left to reader inference as the method itself does not clearly define the terms being used. For example, while the method provides recovery data, the authors do not define their usage of percent recovery or establish that the test samples were analyzed using the entire method. Additionally, the four spike levels used for recovery determinations are not provided and the authors do not report the endogenous levels of the various sugars in the spiked matrices, where applicable. Additionally, while the method provides repeatability data, it is never referred to as repeatability data; rather, repeatability data is only referred to as precision data with a reported %RSD (as opposed to %RSDr). No. The method as written provides absolutely no information regarding chemical safety, potential hazards or personal protective equipment. One example of potential wording would be, "See AOAC Official Methods of Analysis (2005), Appendix B: Laboratory Safety. Use appropriate personal protective equipment as necessary, such as lab coats, safety glasses, gloves, and a fume hood. Ensure that all solvents and solutions are disposed of according to federal, state, and local regulations." While most of the definitions specified in the SMPR are used and applied appropriately, the method does not provide clear definitions of the terms used and the reader is left to infer. For example, while the method provides recovery data, the authors do not define their usage of percent recovery or establish that the test samples were analyzed using the entire method. Additionally, the four spike levels used for recovery determinations are not provided and the authors do not report the endogenous levels of the various sugars in the spiked matrices, where applicable. This impacts the method because the reader does not know over which analytical range the samples were tested for recovery. Additionally, while the method provides repeatability data, it is never referred to as repeatability data; rather, repeatability data is only referred to as precision data with a reported %RSD (as opposed to %RSDr). This has the potential to mislead readers of the method as precision data should include both repeatability and reproducibility.

4. Does the method, as written, contain all appropriate precautions and warnings related to the method's reagents, components, instrumentation, or method steps that may be hazardous? If no, please suggest wording or option(s). III. Review of Supporting Information 1. Are the definitions specified in the SMPR used and applied appropriately in the supporting documentation (manuscripts, method studies, etc...)? If not, please explain the differences and if the method is impacted by the difference.

2. Is there information demonstrating that the method meets the SMPR Method Performance Requirements using the Reference Materials stated in the SMPR? If not, then specify what is missing and how this impacts demonstration of performance of the method.

No. The method provides % recovery data and repeatability (%RSDr) data; but, the method does not provide any reproducibility data (%RSDR). Additionally, the recovery data is only provided for fructose, glucose, sucrose, maltose, and lactose. The method states that the accuracy of galactose in the method was ascertained with passing a proficiency testing program; however, no data is provided in support of this claim. Furthermore, for the recovery data, no information is provided to ascertain the analytical range being measured and therefore to ascertain which performance requirement ranges the method should fall under. While the recoveries meet the SMPR Method Performance Requirements for the three food matrices investigated (butter, corn starch, and refried beans) when assuming an analytical range of 0.1-5% reported as individual sugars for fructose, glucose, sucrose, maltose, and lactose, there is no data to indicate that the recovery for galactose meets the SMPR Method Performance Requirements. The repeatability data does report the mean value measured across the fifteen (triplicate, five days) values reported for each sample, so the analytical range is known and it can be determined whether or not the %RSDr SMPR Method Performance Requirements are met. With regards to repeatability, the majority of the matrices studied did not pass the SMPR Method Performance Requirements. It becomes difficult to fully evaluate each matrix studied because each of the six analyte sugars were measured in a variety of different matrices with no single matrix being used to evaluate all six analyte sugars. For repeatability, fructose met requirements for one out of four matrices; glucose met requirements for none of the six matrices; sucrose met requirements for nine out of eleven matrices; maltose met requirements for none of the five matrices; lactose met requirements for five of the seven matrices; and, galactose met requirements for one of the three matrices. No. As currently written, the method has a clear emphasis on fructose, glucose, sucrose, maltose, and lactose, with minimal data being provided for galactose, despite the fact that galactose is clearly included in the SMPR applicability statement. No accuracy/recovery data is provided for galactose. Furthermore, repeatability for galactose was only investigated in dairy products, two yogurts and a dried buttermilk powder, despite that fact that galactose is found in a variety of other food forms in appreciable quantities, such as honey, beets, cherries, celery, oats, etc. Additionally, the method is not able to demonstrate that it performs within the SMPR Method Performance Requirements for repeatability for glucose and maltose for any of matrices studied with those sugars. The only sugars that met the repeatability requirements for a majority of the matrices studied were lactose and sucrose; no sugar met repeatability requirements for all matrices studied. The method is not able to demonstrate that any of sugar analytes meet the SMPR Method Performance Requirements for reproducibility, as no data is provided.

3. Is there information demonstrating that the

method performs within the SMPR Method Performance REquirements table specifications for all analytes in the SMPR applicability statement? If not, please specify what is missing and whether or not the method's applicability should be modified.

IV. General Submission Package 1. Based on the supporting information, were there any additional steps in the evaluation of the method that indicated the need for any additional precautionary statements in the method?

Yes, as with any analytical chemistry method, this method should have a safety section at the beginning calling attention to potential hazards and the precautions that can be taken to minimize risk. As the method provides no precautionary statement, there should be an addition added. From a quality aspect, the method should add a precautionary statement presenting clear criteria for determining the mobile phase concentration. The method states that the mobile phase concentration varies to achieve good separation of analytes, with the concentration ranging from 75% Acetonitrile at the beginning of column lifetime to 82% Acetonitrile at the end of the column's lifetime. However, the actual concentration used is left to the complete discretion of the user with no criteria provided as to what constitutes "achieving [sic] good separation of analytes." Without providing clear criteria for this determination, there is significant risk of the method not meeting performance criteria for reproducibility as the mobile phase concentration is determined at the sole discretion of individual operators. The SMPR states that the method should include blanks and appropriate check standards as system suitability tests and/or analytical quality control. The method does not contain either blanks or check standards as specified by the SMPR. While one of the sugar standards can act as a internal standard blank, as it contains only the internal standard ribose and 0% of each of the sugar analytes, no data is shown for such a blank. No data is shown for running blanks between samples to demonstrate that there is no carryover between sample injections. The method does not provide any information regarding how often the set of five sugar standards should be run to act as a quality check. Ideally, the method should specify that the set of five sugar standards be run at the beginning and end of the batch of samples, so that the linear regression can be performed with bracketed standards. Additionally, an appropriate check standard should be injected every X number of samples as a quality check, with X dependent on method repeatability/drift. There is clearly a need for these tests and controls since the method did not clearly demonstrate that it met the method performance requirements for repeatability. Not applicable. As the method does not include any data with regards to system suitability tests and controls there is no demonstration whether or not they work appropriately or as expected. While the method is written concisely, there are possible opportunities for improving method clarity. As discussed above in IV.1. the authors should provide more clarity regarding the mobile phase concentration and the criteria used to determine it. Under step 5 of the procedure, additional detail should be provided regarding the microwave instructions. In step 18 of the procedure, additional detail should be provided for set-up of the chromatographic run, ie. how often to inject the standards, how many samples can be analyzed in a batch, how often a check sample should be injected, parameters for the strong and weak needle and seal wash, etc. The method utilizes equipment that common in most analytical laboratories that analyze food, and no additional instrumentation, such as Ion Chromatography, is required. Likewise, the method uses sample separation and analyses work-flows familiar to trained operators in the field.

2. Does the method contain system suitability tests or controls as specified by the SMPR? If not, please indicate if there is a need for such tests or controls and which ones.

3. Is there information demonstrating that the method system suitability tests and controls as specified in the SMPR worked appropriately and as expected? If no, please specify. 4. Based on the supporting information, is the method written clearly and concisely? If no, please specify the needed revisions.

5. Based on the supporting information, what are the pros/strengths of the method?

6. Based on the supporting information, what are the cons/weaknesses of the method?

The method, as currently written, does not demonstrate that it meets the SMPR Method Performance Requirements. The method also introduces ambiguities and potential reproducibility concerns by leaving the mobile phase concentration up to operator discretion. The method does not achieve adequate resolution of galactose and glucose, which necessitates the use of peak height instead of peak area for concentration determinations. - It is deeply upsetting that the method makes no mention of chemical safety. - Despite being based on RI-HPLC methods that have been in use for decades, the method lists no references/citations - Potential carry-over between samples, particularly those from high fat or high protein matrices, is not addressed - Sample preparation of difficult to grind samples is not addressed, ie. fruit gummies - The method provides no requirements for calibration curve performance such as R2 or residuals criteria - No information is provided regarding using check samples for quality control - Not enough information is provided to insure that the method is free from interferences - It would be nice to have provided standard and sample stability data to support the storage conditions provided in the method - Solutions of 100% water are described as stable for six months stored at room temperature with no concern for microbial growth - It would be preferable for the method to measure all of the analyte sugars over the same analytical range and for all of the analyte sugars to have the same number of calibration standards. Because galactose has only three standard levels, there are potential implications on the accuracy of the resulting calibration curve. - The method repeatability could likely be improved by utilizing the column and temperature control features on the Acquity UPLC system instead of relying on ambient temperatures - It would be beneficial for the method to show a sample chromatogram near the top of the calibration range to properly evaluate the resolution of the galactose and glucose peaks. In the chromatogram that is shown at the lowest calibration standard, there is not baseline separation of the two peaks. - The method would benefit from providing additional data regarding how data was generated for the various aspects of the validation - While galactose is inherently present in dairy products, it is also naturally present in a wide variety of other matrices and it would be beneficial to evaluate its repeatability in other, non-dairy, matrices - In foot-note number eleven, it is noted that repeatability results for the Saco dried buttermilk sample are not reported for lactose because the sample contained lactose at >50% and was not reanalyzed by dilution of the samples to be within linear range of the calibration curve, which raises questions as to why not. As currently written, I do not recommend this method to be adopted as a First Action and published in the Official Methods of Analysis of AOAC INTERNATIONAL because the method does not demonstrate that the SMPR Method Performance Requirements are met. If the method is revised such that sufficient information is provided to demonstrate that the SMPR Method Performance Requirements are met, then I would be willing to reconsider the method for adoption.

7. Any general comments about the method?

V. Final Recommendation Do you recommend this method be adopted as a First Action and published in the Official Methods of Analysis of AOAC INTERNATIONAL? Please specify rationale.

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AOAC Candidate Method SUG-002

Submitter Name

Barry McCleary

Submitter Email

barry@megazyme.com

Organization

Megazyme

Method Type

Fructans

Method Name

Single Lab Validation of Megazyme’s Fructan Assay Kit (K- FRUC) Method for the Determination of Fructan (Inulin, FOS, Levan and Branched Fructan) in Animal Food (Animal Feed, Pet Food, and Ingredients)

Method Author(s)

Barry V. McCleary, Lucie M. J. Charmier, Vincent A. McKie, Ciara McLoughlin and Artur Rogowski.

Primary Reviewer: John Szpylka, Mérieux Nutrisciences Secondary Reviewer: Sookwang Lee, FDA

AOAC SMPR ERPs - 2018 METHOD REVIEW FORM

Submission ID

4099965788519339504

Submission Date

2018-08-13 14:09:38

Name

John Szpylka

E-mail

john.szpylka@mxns.com

Organization

Merieux NutriSciences

Title of Method

Single Lab Validation of Megazyme's Fructan Assay Kit (K-FRUC) Method for the Determination of Fructan (Inulin, FOS, Levan and Branched Fructan) in Animal Food (Animal Feed, Pet Food, and Ingredients)

AOAC Candidate Method Number (e.g. ALN-01)

SUG-002

Applicable SMPR

2018.002

I. Summary of the Method

The forms of fructans listed in SMPR are extracted from pet foods, feeds, and ingredients into boiling water. Sucrose, starches, and maltodextrin are concurrently enzymatically hydrolyzed to glucose and fructose. All reducing sugars are then converted to sugar alcohols, thus removing them as potential interferences. The method states native fructans and non-reducung FOS are not affected by these reactions. The SMPR-listed fructans are hydrolyzed to fructose and glucose using a mixture of three enzymes to cover the different possible fructan internal sugar linkages. Quantitation of the total amount of reducing sugars (fructan-derived fructose and glucose) is performed using the PAHBAH reducing sugar method. A single point calibration of absorbance /54.5 ug fructose is used. The total dietary fructan amount of inulin, FOS, levan, and branched fructan is measured by this method. The method removed interferences of starches, sucrose, and other reducing sugars.

II. Review of the Method Only 1. Does the applicability of the method support the applicability of the SMPR? If not, please explain what is missing. 2. Does the analytical technique(s) used in the method meet the SMPR? If not, please specify how it differs from what is stated in the SMPR. 3. Are the definitions specified in the SMPR used and applied appropriately in the method? If no, please indicate how the terms are used.

The analytical technique does measure the total amount of ditetary fructans.

All definitions are used correctly except for reproducibility. The reported RSDR study was performed on 4 separate occasions by 2 independent analysts. While these results should remain in the report as an intermediate precision study, a broader number of variables (different locations, equipment, reagents, and analysts) needs to be performed to fully estimate method reproducibility (RSDR).

4. Does the method, as written, contain all appropriate precautions and warnings related to the method's reagents, components, instrumentation, or method steps that may be hazardous? If no, please suggest wording or option(s). III. Review of Supporting Information 1. Are the definitions specified in the SMPR used and applied appropriately in the supporting documentation (manuscripts, method studies, etc...)? If not, please explain the differences and if the method is impacted by the difference. 2. Is there information demonstrating that the method meets the SMPR Method Performance Requirements using the Reference Materials stated in the SMPR? If not, then specify what is missing and how this impacts demonstration of performance of the method. method performs within the SMPR Method Performance REquirements table specifications for all analytes in the SMPR applicability statement? If not, please specify what is missing and whether or not the method's applicability should be modified. 3. Is there information demonstrating that the

Yes.

All definitions are used correctly except for reproducibility. The reported RSDR study was performed on 4 separate occasions by 2 independent analysts. While these results should remain in the report as an intermediate precision study, a broader number of variables (different locations, equipment, reagents, and analysts) needs to be performed to fully estimate method reproducibility (RSDR).

A Certified Reference Material was listed as not being currently available.

The method measured total fructans as total fructans including inulin, levan, branched fructans, agavins (agave fructans), and FOS as listed in SMPR.

IV. General Submission Package 1. Based on the supporting information, were there any additional steps in the evaluation of the method that indicated the need for any additional precautionary statements in the method?

Potential interferences (sucrose, reducing sugars, starches) were addressed.

Sample preparation watch-outs such resolubilizing possible precipitated fructans during storage (5.7 (a) (iii)); and fading of PAHBAH color complex over time (5.7 (c) (5)) were nicely included in the method procedure. A section on "Indicative Controls" was also included. No additional precautions needed. **Clarification of the (162/180) term in the calculation is needed. The other AOAC Total Fructans Method 997.08 lists this term as [(180+162(n-1))/180n)] to account for different degrees of polymerization. Is this still needed for this new method, especially for branched fructans? **What is unclear in the method procedure is freeze-drying wet samples as described in section 5.6 (a). A freeze dryer is not listed in the equipment, nor is a freeze-drying correction factor listed in the final equation. Also, samples are extracted in water after weighing. QUESTION: Is freeze drying wet samples needed to achieve the lower limit of detection?

2. Does the method contain system suitability tests or controls as specified by the SMPR? If not, please indicate if there is a need for such tests or controls and which ones. 3. Is there information demonstrating that the method system suitability tests and controls as specified in the SMPR worked appropriately and as expected? If no, please specify. 4. Based on the supporting information, is the method written clearly and concisely? If no, please specify the needed revisions. 5. Based on the supporting information, what are the pros/strengths of the method? 6. Based on the supporting information, what are the cons/weaknesses of the method?

Blanks and appropriate check standards are in the method.

Those items are covered in the report.

The method is written clearly.

The method was found to measures the total fructan content in foods, feeds, and ingredients described in SMPR. Simple instruments are used to perform this method. Enzyme solutions can either be created by the laboratory or purchased pre-made.

Method reproducibility is as yet unknown so remains needed for Final Action Status consideration.

7. Any general comments about the method?

No additional comments.

V. Final Recommendation Do you recommend this method be adopted as a First Action and published in the Official Methods of Analysis of AOAC INTERNATIONAL? Please specify rationale.

I recommend the method be adopted as a First Action Method of Analysis upon clarification of Items and 2 below. Item 3 is for progression to Final Action Status.

ITEM 1: Is freeze drying wet samples needed to achieve the lower limit of detection?

ITEM 2: Clarification of the (162/180) term in the calculation is needed. The other AOAC Total Fructans Method 997.08 lists this term as [(180+162(n- 1))/180n)] to account for different degrees of polymerization. Is this still needed for this new method, especially for branched fructans?

ITEM 3: Perform reproducibility study encompassing a broader number of variables (different locations, equipment, reagents, and analysts).

AOAC Candidate Method SUG‐003 

Submitter Name 

Tom Vennard 

Submitter Email 

thomas.vennard@covance.com

Organization 

Covance 

Method Type 

Sugar 

Method Name 

Sugar Profile by High Performance Anion Exchange  Chromatography with Pulsed Amperometric Detection  

Method Author(s) 

Tom Vennard, Andrew Ruosch, David Ellingson 

Method Applicability 

Applicable to determination of galactose, glucose, sucrose,  fructose, lactose, isomaltulose, and maltose in food products,  pet food and animal feed, infant formula, and dietary  supplements. 

Primary Reviewer:  Philip Haselberger, Abbott Nutrition  Secondary Reviewer:  Hari Narayanan, Metrohm USA

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