AOAC SPSFAM PAC ERP

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SPSFAM EXPERT REVIEW PANEL

FOR

PROANTHOCYANIDIN METHODS

ERP Chair: Holly Johnson, Alkemist Labs

DECEMBER 15, 2017

AOAC HEADQUARTERS 2275 RESEARCH BLVD., STE 300 ROCKVILLE,MARYLAND, 20850 UNITED STATES

contact: spsfam@aoac.org

SPSFAM PAC ERP 12/15/2017 – v1.0 Draft meeting agenda subject to change without notice *Items requiring a vote by ERP Expert Review Panel for SPSFAM Proanthocyanidins in Cranberries Methods Friday, December 15, 2017 12:00 pm – 5:00 pm ET AOAC INTERNATIONAL Headquarters 2275 Research Boulevard, Suite 300 Rockville, Maryland 20871, USA Main Conference Room A G E N D A 1. Welcome and Introductions Holly Johnson, Alkemist Labs (ERP Chair) and Dawn Frazier, AOAC INTERNATIONAL 2. Review of AOAC Volunteer Policies & ERP Process Overview and Guidelines Deborah McKenzie, AOAC INTERNATIONAL 3. Review of Methods For each method, the assigned ERP members will present a review of the methods and manuscripts, after which the ERP will discuss the methods and reach consensus on the status for each method. A. Method PAC-001* a. PAC-001 Reviews led by Rimmer and You, and followed by ERP Discussion and Consensus B. Method PAC-002* a. PAC-002 Reviews led by Tuzumski and Es Safi Review, and followed by ERP Discussion and Consensus C. Method PAC-003* a. PAC-003 Reviews led by Jahromi and Reed, and followed by ERP Discussion and Consensus D. Method PAC-004* a. PAC-004 Reviews led by Mudge and Yadlapalli, and followed by ERP Discussion and Consensus E. Method PAC-005* a. PAC-005 Reviews led by Krueger and Rimmer, and followed by ERP Discussion and Consensus 4. Final Action Requirements for Adopted Methods (if applicable) 5. Adjourn ITINERARY 11:00pm – 12:00pm – Lunch Served 2:30pm – 2:45pm – Afternoon Break

Proanthocyanidins Expert Review Panel, December 15, 2017 1. Holly Johnson, Alkemist Labs (Chair) 2. Nour Eddine ES-SAFI, Mohammed V University, Rabat 3. Ramin Jahromi, Eurofins 4. Christian Krueger, Complete Phytochemical Solutions 5. Elizabeth Mudge, BCIT 6. Jess Reed, University of Wisconsin 7. Kate Rimmer, NIST 8. Tomasz Tuzimski, Medical University of Lublin 9. Sudhakar Yadlapalli, First Source Laboratory Solutions 10. Hong You, Eurofins Scientific

AT LEAST 7 MEMBERS OF THIS ERP MUST BE PRESENT TO MAKE DECISIONS

AOAC SPSFAM PROANTHOCYANIDINS EXPERT REVIEW PANEL

METHODS AND SMPR ACCESS

• AOAC SMPR 2017.003 (Quantitative) • AOAC SMPR 2017.004 (Identification) • METHOD ACCESS (ERP ONLY – PASSWORD REQUIRED)

Proanthocyanidins in cranberry Expert Review Panel

Candidate Method

PAC-001

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PRIMARY REVIEWER: Kate Rimmer, NIST SECONDARY REVIEWER: Hong You, Eurofins

AOAC DECEMBER 2017 ERPs - METHOD REVIEW FORM

Submission Date

2017-12-11 16:18:41

AOAC Expert Review Panel: Method Review

Reviewer Information Required to validate your review.

Name

Catherine Rimmer

E-mail

catherine.rimmer@nist.gov

Organization

NIST

Remember, the main purpose of your review is to ensure the method conforms to the applicable SMPR. View and download SMPRs here:

SPDS SMPRs SPSFAM SMPRs

Method Review

Title of Method

Deconvolution of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identification of A-type proanthocyanidins in cranberry based foods and dietary supplements

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

PAC-001

Applicable SMPR

2017.004

I. Summary of the Method

Proanthocyanidins are extracted with 70% acetone in an ultrasonic bath for 15 min (X3) then filtered and centrifuged. The solution was then loaded on a Sephadex LH-20 column and eluted with ethanol, then ethanol/methanol (1:1), then 80% acetone and the 80% acetone eluent was reserved to isolate the PACs from the other phenolics. The eluent was evaporated and solubilized in methanol. The methanol solution was then spotted on a stainless steel target followed by the addition of DHB. A specific technique was used to assure that the sample and matrix would co-crystallize evenly (no coffee ring effect). Calibrants including procyanidin A2 mixed with procyanidin B2 were mixed by mass and the spectra were collected. The overlapping isotopic distribution patterns were studied and understood and a deconvolution model was developed allowing for the determination of the ratio of A-type to B-type linkages.

II. Review of the Method Only:

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. 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).

The method investigated frozen CPC powder alone and mixed with apple products. The applicability statement calls for one or more of the following: fruit, juice, beverage, dried cranberry fruit, cranberry sauce, ingredients, and/or dietary supplements. It might be nice to see how this performs in a different matrix, however, technically it meets the requirements of the SMPR and I was impressed by the addition of testing a material with no A-type linkages.

Yes.

I did not notice safety precautions, however, there are very few that are likely to be necessary.

III. Review of Information in Support of the Method

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

Calibrants and matrix based materials were mixed and extracted to allow for a chemometric deconvolution of the data. Listed reference materials were not used, however, for the work done, the reference materials were not a necessity (yet)

This is a more qualitative SMPR with selectivity requirements for a POI. The authors included information typically found in a quantitative SMPR but not in this one. The method performance requirement is 90% probability of identification with 95% confidence (33 correct identifications out of 33 samples know to contain A- type PAC). The results of the 21 samples were not presented as POI, so that calculation should be performed

IV. General Submission Package

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?

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?

Yes

Yes. The authors also cited related references, it may be nice to have additional information in the method rather than going to multiple sources.

This method provides a semiquantitative ratio of A-type to B-type linkages in cranberry with a specific analytical test.

None

7. Any general comments about the method?

None

Recommendation for the Method

I would be more comfortable recommending the method if a wider variety of matrices from the SMPR were used but I liked the inclusion of the study with apple that has no A- type PACs. The method should be considered for AOAC first action status.

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.

AOAC DECEMBER 2017 ERPs - METHOD REVIEW FORM

Submission Date

2017-12-11 09:08:36

AOAC Expert Review Panel: Method Review

Reviewer Information Required to validate your review.

Name

Hong You

E-mail

hongyou@eurofinsus.com

Organization

Eurofins Scientific, Inc.

Remember, the main purpose of your review is to ensure the method conforms to the applicable SMPR. View and download SMPRs here:

SPDS SMPRs SPSFAM SMPRs

Method Review

Title of Method

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

PAC-001

Applicable SMPR

2017.004

I. Summary of the Method

This method submission is a composite of 3 previous publications which include 2 journal papers and 1 conference poster. This MALDI-TOF Mass Spectrometry method showed that it can accurately determine the ratios of A- to B-type interflavan bonds in both purified procyanidins standard mixture and the proanthocyanidins that were isolated from the fresh fruit powder and press cake of cranberry and apple.

II. Review of the Method Only:

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.

No.

This method is able to determine A-type to B-type interflavan bonds in cranberry proanthocyanidins in cranberry fresh fruit powder, which is a matrix that was listed in Table 1 of SMPR. However, AOAC SMPR is seeking an identification method that can returns a binary, YES/NO test result about if cranberry Type-A proanthocyanidins present in an unknown sample. Validating the method based on the probability of identification model is a necessary step according to the AOAC validation guidance appendix K.

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. 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.

Yes.

No. This method doesn't contain safety information regarding handling reagents, components, and instrumentations.

III. Review of Information in Support of the Method

Yes.

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. 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? 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. 3. Is there information demonstrating that the

No.

Authors were not able to validate the method selectivity using the probability of identification model. In the response letter to the CSO of AOAC, authors mentioned their observed percentages were within 3.6% of predicted percentages. However, the 3.6% percentage in method discussion section 3.3 refers to the amount ratio percentage between A-type bond and B-type bond procyanidin standard mixture. This percentage concept is irrelevant to the percentage used in the probability of identification model.

No.

The SMPR requires to evaluate at least 33 samples known to contain Type-A proanthocyanidin and at least 33 samples that contain non Type-A proanthocyanidin. In the submitted method, authors were only able to test cranberry press cake sample in 14 replicates.

IV. General Submission Package

Yes. The safety precautions in operating MALDI-TOP MS might be worth mentioning.

No. This information about the system suitability test was not clearly written in the method. The method did not include the requirement of using a quality control material.

No.

No system suitability tests and controls were used in this method.

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?

No. The method was not easy to follow and does not contain important method information such as instrument operation parameters.

This method is articulate and robust on estimating ratios of A- to B-type bonds in cranberry proanthocyanidin.

This method was not written and validated in a format that is required by the AOAC SMPR 2017.004. The method did not test all types of matrices listed in Tier 1 of Table 3 in the SMPR. Authors did not validate this method based on the probability of identification model that is required according to the AOAC validation guidance appendix K. The method has the potential to be used to identify the presence of Type-A proanthocyanidin in cranberry. However, because of above-mentioned flaws, the current method is not recommended to be adopted as an AOAC First Action Official Method.

7. Any general comments about the method?

Recommendation for 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.

No.

I do not recommend this method to be adopted as a First Action because it does not have enough data to meet SMPR validation guidance and method performance requirements.

Proanthocyanidins in cranberry Expert Review Panel

Candidate Method

PAC-002

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PRIMARY REVIEWER: TOMASZ TUZIMSKI, MEDICAL UNIVERSITY OF LUBLIN

SECONDARY REVIEWER: NOUR EDDINE ES-SAFI, MOHAMMAD V UNIVERSITY, RABAT

AOAC DECEMBER 2017 ERPs - METHOD REVIEW FORM

Submission Date

2017-12-05 17:57:30

AOAC Expert Review Panel: Method Review

Reviewer Information Required to validate your review.

Name

TOMASZ TUZIMSKI

E-mail

tomasz.tuzimski@umlub.pl

Organization

MEDICAL UNIVERSITY OF LUBLIN

Remember, the main purpose of your review is to ensure the method conforms to the applicable SMPR. View and download SMPRs here:

SPDS SMPRs SPSFAM SMPRs

Method Review

Title of Method

4-(dimethylamino)cinnamaldehyde assay using cranberry proanthocyanidin standard for quantification of soluble proanthocyanidins in cranberry foods and dietary supplements

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

PAC-002

Applicable SMPR

SMPR 2017.003 (and 2017.004)

I. Summary of the Method

The method described is applicable for the detection and quantitation of proanthocyanidin (PAC) content in cranberry products. The 4-(dimethylamino)cinnamaldehyde (DMAC) assay is currently used to quantify proanthocyanidin (PAC) content in cranberry products. However, this method suffers from issues of accuracy and precision in the analysis and comparison of PAC levels across a broad range of cranberry products. In a multi-operator/multi-day study design, a cranberry proanthocyanidin (c-PAC) standard was compared to procyanidin A2 (ProA2) dimer for accurate quantification of PAC in commercial cranberry juices, lab generated cranberry blends and cranberry powders. The c-PAC standard reflects the structural heterogeneity of cranberry PAC degree of polymerization, hydroxylation pattern and ratios of ‘A-type’ to ‘B-type’ interflavanyl bonds. Use of the c-PAC standard to quantify PAC content in cranberry samples resulted in values that were higher than those determined by procyanidin A2 (ProA2), which were inaccurate. YES: The applicability of the method is partially adequate to the applicability of the SMPR. My remarks are follows - Authors should add: - Values of recoveries for analytes (and SD or RSD); according with: 'Determined from spiked blanks or samples with at least seven independent analyses per concentration level at a minimum of three concentration levels covering the analytical range. Independent means at least at different times. If no confirmed (natural) blank is available, the average inherent (naturally containing) level of the analyte should be determined on at least seven independent replicates.' - Values of LOD and LOQ for all analytes.

II. Review of the Method Only:

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.

YES: The analytical techniques in the method are adequate and meet the SMPR.

YES: Definitions, which are specified in the SMPR, were used and applied appropriately in the method.

Yes: The method contains all appropriate precautions and warnings related to the method’s reagents, components, instrumentation, or method steps that may be hazardous.

III. Review of Information in Support of the Method

YES: The definitions specified in the SMPR were used and applied appropriately in the supporting documentation (manuscripts, method studies, etc.).

YES: There is a piece of information demonstrating that the method meets the SMPR Method Performance Requirements using the Reference Material stated in the SMPR.

YES: There is a piece of information demonstrating that the method performs within the SMPR Method Performance Requirements table specifications for all analyses in the SPMR applicability statement.

IV. General Submission Package

In my opinion, there is no need to implement any additional steps in the method evaluated.

YES: Yes, there are.

YES: There is a piece of information demonstrating that the method system suitability tests and control as specified in the SMPR worked appropriately and expected.

The method is well described and substantively prepared. The concepts, analyses, and methodology are adequately developed. The method proposed is well integrated and well-reasoned.

In my opinion the pros/strengths of the method can be following: 1. The internal standard used for quantification analyse of analyte should, as little as possible, differ from the analyte determined. Use of the c-PAC standard to quantify PAC content in cranberry samples resulted in values that were higher than those determined by procyanidin A2. Increased accuracy is critical for estimating PAC content in relationship to research on authenticity, efficacy, and bioactivity (also especially in designing clinical trials for determination of putative health benefits). 2. The total of seventeen (n=17), six-point standard reference curves, were generated by the operators (n=3) using both c-PAC and ProA2 reference standards. The cons/weakness of the method may be connect with: Specificity is one of most important analytical parameter. In my opinion, Authors may/should showed matrix effect: that - there were are or no - interferences from the matrices. The following aspects of the method (summary, presentation of the topic, definition of research objectives, presentation of research methodology and detailed presentation of method) are described in a satisfactory manner.

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

7. Any general comments about the method?

Recommendation for 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.

In my opinion, the AOAC Candidate Method (#PAC-002) described by Christian Krueger et al. can be futher refined and next adopted in its modified form as a First Action and recommended for publication in the Official Methods of Analysis of AOAC INTERNATIONAL.

AOAC DECEMBER 2017 ERPs - METHOD REVIEW FORM

Submission Date

2017-12-07 13:26:57

AOAC Expert Review Panel: Method Review

Reviewer Information Required to validate your review.

Name

Nour Eddine ES-SAFI

E-mail

nouressafi@yahoo.fr

Organization

Mohammed V University in Rabat

Remember, the main purpose of your review is to ensure the method conforms to the applicable SMPR. View and download SMPRs here:

SPDS SMPRs SPSFAM SMPRs

Method Review

Title of Method

4-(dimethylamino)cinnamaldehyde assay using cranberry proanthocyanidin standard for quantification of of soluble proanthocyanidins in cranberry foods and dietary supplements

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

PAC-002

Applicable SMPR

SMPR 2017.003

I. Summary of the Method

The method entitled "4-(dimethylamino)cinnamaldehyde assay using cranberry proanthocyanidin standard for quantification of soluble proanthocyanidins in cranberry foods and dietary supplements" presents results dealing with the quantitative analysis of proanthocyanidins in 4 cranberry powders, 4 laboratory cranberry juices and 4 commercially available cranberry juices. The proposed method used the DMAC assay with a cranberry proanthocyanidin standard and the obtained results were compared to those obtained with A2 proanthocyanidin.

II. Review of the Method Only:

Yes. The SMPRs indicated that the method will be able to quantify proanthocyanidin present in cranberry in ONE or MORE of the following: fruit, juice, beverage, dried cranberry fruit, cranberry sauice, ingredients, or dietary supplements. The proposed method was reported to be applied to cranberry powders and juices. I think that it will be of interest to apply the method to the other matrices indicated in the SMPRs.

Yes

No safety information is included.

III. Review of Information in Support of the Method

No. The authors did not use "Reproducibility", "Repeatability" and "Recovery".

No. No reference material was used in the validation.

1- LOD and LOQ values were investigated only for A2 proanthocyanidin.

2- "Operator effect" and "Day effect" for both cranberry and A2 proanthocyanidin standards were investigated and their corresponding parameters are reported in the proposed method.

3- Parameters concerning recovery were not investigated.

IV. General Submission Package

Yes, recovery performance of the method is to be done.

the method did not include blank check sample.

Yes

As indicated by the authors, the proposed method refers to two previously published publications (Feliciano et al., 2012 & Krueger et al., 2016). I think that the proposed method need to be reformatted from existing publications and supplemental information into a single document. This will enhance the quality of the method and makes easier its reading and understanding.

1- Well known colorimetric assay 2- Procedure well described 3- Relatively simple method 4- Easy to conduct

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

1- There are data for only powder and juices. Not all dietary ingredients indicated in the SMPR 2- No recovery data are given. 3- No LOQ and LOD data for cranberry proanthocyanidin standard 4- Need to be reformatted in a new complete form 5- The phytochemical composition of the cranberry proanthocyanidin standard is not well known

7. Any general comments about the method?

A simple and promising method but may need more data

Recommendation for 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.

I think that the proposed method could be a promising candidate for the quantification of cranberry proanthocyanidins and then would support this method for First Action status, but would recommend additional work, more supporting data and investigation of the remaining points listed in SMPR (LOQ and LOD of c-PAC standard, use of other cranberry dietary ingredients, recovery data, blanck check sample, ...). I also think that the proposed method should be rewritten and proposed in a more complete form making it easy to read and understand.

Proanthocyanidins in cranberry Expert Review Panel

PAC-003

Candidate Method

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PRIMARY REVIEWER: RAMIN JAHROMI, Eurofins SECONDARY REVIEWER: JESS REED, University of Wisconsin

AOAC DECEMBER 2017 ERPs - METHOD REVIEW FORM

Submission Date

2017-12-01 16:25:05

AOAC Expert Review Panel: Method Review

Reviewer Information Required to validate your review.

Name

Ramin Jahromi

E-mail

raminjahromi@eurofinsus.com

Organization

Eurofins

Remember, the main purpose of your review is to ensure the method conforms to the applicable SMPR. View and download SMPRs here:

SPDS SMPRs SPSFAM SMPRs

Method Review

Title of Method

Proanthocyanidins in Cranberry

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

PAC-003

Applicable SMPR

SMPR 2017.003

I. Summary of the Method

Cranberry extract powder, cranberry juice concentrate, or cranberry capsules are extracted with methanol, reacted with DMAC and quantified against a standard curve of Procyanidin A2 using UV/VIS with kinetic reaction at 610 nm.

II. Review of the Method Only:

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.

Based on the reported data the method was performed on extract, juice concentrate, and capsules. The method was not tested on other types of the cranberry supplements. However, the method seems to be applicable to the stated matrices but there is no indication how it compares to LC-MS or HPLC data.

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.

UV/VIS spectrophotometer capable of kinetic measurements at 610 nm is acceptable for the method.

The method needs a better explanation of PAC A2 standard preparation. Is there a reagent blank and/ or procedure blank?

Should each standard and sample be analyzed by UV/VIS individually? For example: Add 1 mL DMAC to the standard 1 and start UV/VIS. After completion add reagent to std 2.... Method should indicate in the title that is for determining "total" PAC's. Is using PAC type A to determine total PAC adequate or the results will be overestimated?

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

Adding H2SO4 to Methanol needs a safety caution.

III. Review of Information in Support of the Method

The definitions used in the method are appropriate.

There is no reference material or controls listed to ensure accuracy of the results.

Procyanidins A2 standard has not information how it was obtained or CAS number. This information is important for method review.

3. Is there information demonstrating that the

The method performance is within SMPR specifications. What is the LOQ for 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. 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? 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?

IV. General Submission Package

The method abstract and the Introduction contain information that may not be supported by the regulatory agencies in the US and EU when dealing with health claims.

For the method:

It should state that it is to determine "Total PACs as PAC A2".

Method indicates using Proanthocyanidins A2 standard but no CAS Number or supplier is noted.

There is no mention of what control to use and the value to expect.

The linearity, RSD, and recoveries meet the requirement.

Standard preparation needs more explanation. Reagent blanks and Procedure blanks need to be explained. Adding DMAC reagent for the UV/VIS needs more explanation. Standards and reagents stability information is needed.

Method is simple to follow and can be used as a routine quality control procedure in any average laboratory.

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

The method is determining "total PACs as PAC A2" since it is using standard A2 only.

There is no mention of interfering substances and if false positive is possible.

The method should be limited to the matrices used in the study.

7. Any general comments about the method?

The method can be used by any laboratory when access to more sophisticated instruments is not available or there is a need for rapid analysis.

There is not mention of how the data is compared to other methods and that is something I would like to see.

Recommendation for 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.

If the method is re-written according to AOAC format, all standards are defined by supplier or CAS Number, a comparison study is performed to compare the method with data from LC methods, applicable matrix to be defined, any interference to be listed, and method LOQ to be determined I would recommend it for first action. I would like to discuss this method further with colleagues at AOAC meeting. Due to the very short time to review the method my recommendation is only a preliminary evaluation.

AOAC DECEMBER 2017 ERPs - METHOD REVIEW FORM

Submission Date

2017-12-06 10:40:26

AOAC Expert Review Panel: Method Review

Reviewer Information Required to validate your review.

Name

Jess Reed

E-mail

jdreed@wisc.edu

Organization

UW-Madison

Remember, the main purpose of your review is to ensure the method conforms to the applicable SMPR. View and download SMPRs here:

SPDS SMPRs SPSFAM SMPRs

Method Review

Title of Method

Single laboratory validation of total A-type proanthocyanidins in cranberry using 4-dimethylcynnamaldehyde

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

PAC-003

Applicable SMPR

PROATHOCYAnidins

I. Summary of the Method

A modified method for using 4-dimethylcinnamaldyde (DMAC) reagent to quantify A-type PAC in cranberry products.

II. Review of the Method Only:

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.

This method does not support the applicability of the SMPR because:

1. The method is precise button accurate. The method severely underestimates the amount of PAC in cranberries because the A2 dimer is an inappropriate standard because the stoichiometry of reaction of the A2 dimer is significantly different than the mixture of oligomers of higher degree of polymerization that are present in cranberries. 2. The tile of the method is misleading because the DMAC reagent reacts with all proanthocyanidins not just a type PACs. Feliciano et al (2012) showed that although the kinetics of reaction of A2 and B2 dimers were different, there was essentially no difference between the two standards when the absorbance at Tmax was used to estimate PAC concentration and, both standards severely underestimate PAC content. This method does not meet the SMPR because it does not quantify total proanthocyanidin content as the sum of all extractable oligomers and polymers present in cranberries because the method uses an inappropriate standard.

The method does not use the definitions in the SMPR because the SMPR clearly states extractable oligomers greater than DP2. However the authors make no attempt to distinguish between the A2 dimer and the oligomers of higher of higher DP. Most of the proanthocyanidins in cranberries have a DP higher than 2 and also contain more than one A type interflavan bond.

Perhaps some concern regarding H2SO4.

III. Review of Information in Support of the Method

The references are inadequate because the authors ignore the publication of Feliciano et al (2012). This publication clearly shows the problem of using PAC dimers as standards for the DMAC assay. This overbite is a serious concern with the the authors intent for this method and needs to be addressed in the the discussion of the review board. The problem of standards and its interaction with degree of polyerizatio of PAC is well know and needs to be address in any method that we adopt.

The method should have tested accuracy using PACs that were isolated from cranberry products that were described in the SMPR Method Performance Requirements.

See Feliciano et al (2012)

IV. General Submission Package

Accuracy needs to be addressed.

The method needs to be validated against isolated cranberry PACs.

No because the method was not validated against cranberry PACs and therefore there is no way to assess accuracy.

In general the method is written clearly and concisely. However the use of A type PAC is incorrect because the DMAC reagent reacts with all PACs and it is misleading the state that the method is specific to A type PACs from cranberries.

The DMAC method is a rapid method that has great potential for use in standardizing PAC content in products.The use of H2SO4 and methanol appears to be a valid modifications of the original method but needs to validated.

The problem of accuracy and appropriate standards is a serious constraint. The method is non-specific and therefore an alternative method for identification of A type PACs is required as requested in AOAC SMPR® 2017.004

7. Any general comments about the method?

The modified method is worthy of further consideration but the use of appropriate standards has to be addressed.

Recommendation for 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.

No, the authors overlooked and do not discuss the problem of appropriate standards and failed to reference Feliciano et al (2012).

Proanthocyanidins in cranberry Expert Review Pane l

PAC-004

Candidate Method

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PRIMARY REVIEWER: ELIZABETH MUDGE, BCIT SECONDARY REVIEWER: SUDHAKAR YADLAPALLI, FIRST SOURCE LABORATORY SOLUTIONS

AOAC DECEMBER 2017 ERPs - METHOD REVIEW FORM

Submission Date

2017-12-05 23:30:53

AOAC Expert Review Panel: Method Review

Reviewer Information Required to validate your review.

Name

Elizabeth Mudge

E-mail

emudge@bcit.ca

Organization

BCIT

Remember, the main purpose of your review is to ensure the method conforms to the applicable SMPR. View and download SMPRs here:

SPDS SMPRs SPSFAM SMPRs

Method Review

Title of Method

Development of a new thiolosis HPLC methods for the analysis of bioactive procyanidins in cranberry products

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

PAC-004

Applicable SMPR

SMPR 2017_003

I. Summary of the Method

This method is the development of a new thiolysis HPLC-UV method for the quantitation of total procyanidins, ratio of type-A linkages and A-type procyanidin equivalents in cranberry products. In terms of cranberry products evaluated they quantified total procyanidins in cranberry juice, dried cranberries and a dietary supplement (form not described).

II. Review of the Method Only:

Yes. According to the publication, this method is applicable to quantitation of total proanthocyanidins (Procyanidin A2 and Procyanidin B2) after thiolysis of procyanidin B2. It was applied to three different matrices: dried cranberries, cranberry juice and and dietary supplement.

The method used is suitable to meet the SMPR. No specific requirements were made regarding the method used. There is some information that is not easily described within the methodology to understand how thiolysis is performed on the authentic samples.

Yes, the definitions are applied appropriately, with the exception that it would be desirable to collect repeatability data on authentic samples as opposed to spiked samples.

No - hazards are not specified in the method.

III. Review of Information in Support of the Method

Yes, with the exception of repeatability (authentic samples would be desirable). It is difficult to understand the RSDr in relation to total procyanidin calculations in authentic samples.

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