Expert Review Panel for Dietary Supplements

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SPDS EXPERT REVIEW PANELS AOAC INTERNATIONAL Expert Review Panel for: Stakeholder Panel on Dietary Supplements (AOAC SPDS) Topics: Folin-C, Free Amino Acids, Ginger, Kratom, and Protein ERP Chair: Brian Schaneberg, Starbucks Corporation

DECEMBER 14, 2017 AOAC HEADQUARTERS 2275 RESEARCH BLVD., STE 300 ROCKVILLE,MARYLAND, 20850 UNITED STATES

Expert Review Panels for SPDS Methods Thursday, December 14, 2017 8:30 am – 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

SPDS Various Ingredients ERPs 12/14/2017 – v1.0 Draft meeting agenda subject to change without notice *Items requiring a vote by ERP 1. Welcome and Introductions (8:30am – 8:45am) Brian Schaneberg, Starbucks (ERP Chair) and Dawn Frazier, AOAC INTERNATIONAL 2. Review of AOAC Volunteer Policies & ERP Process Overview and Guidelines (8:45am – 8:50am) Deborah McKenzie, AOAC INTERNATIONAL 3. Review of Methods (8:50am – 4:40pm) For each method of each ERP, the assigned ERP members will present a review of the methods and manuscripts, after which the ERP will discuss the method and reach consensus on the status for each method. A. Free Amino Acids - FAA-001* (8:50am – 9:50am) a. FAA-001 Reviews led by Zielinski and Johnson and followed by ERP Discussion and Consensus b. Final Action Requirements for Adopted Method (if applicable) B. Protein – PRO-001* and PRO-002* (10:00am – 12:15pm) a. PRO-001 Reviews led by Bhandari and Reimann and followed by ERP Discussion and Consensus b. PRO-002 Reviews led by You and Haselberger and followed by ERP Discussion and Consensus c. Final Action Requirements for Adopted Methods (if applicable) C. Folin C – FOL-001* (1:15pm – 2:15pm) a. FOL-001 Reviews led by Rimmer and Phillips and followed by ERP Discussion and Consensus b. Final Action Requirements for Adopted Method (if applicable) D. Ginger – GIN-001* (2:30pm – 3:30pm) a. GIN-001 Reviews led by Solyom and Brown and followed by ERP Discussion and Consensus b. Final Action Requirements for Adopted Method (if applicable) E. Kratom – KRA-002* (3:40pm – 4:40pm) a. KRA-002 Reviews led by Szpylka and Casey and followed by ERP Discussion and Consensus b. Final Action Requirements for Adopted Method (if applicable) 4. Next Steps (4:40pm – 4:50pm) 5. Adjourn ITINERARY Breakfast served: 8:30am 10:15am – 10:30am - Morning Break 12:15pm – 1:15pm – Lunch Served 2:15pm – 2:30pm – Afternoon Break

Free Amino Acids:

1. Brian Schaneberg, Starbucks 2. Quanyin Gao, Herbalife 3. David Ji, Covance 4. Holly Johnson, Alkemimst

5. George Joseph, AsureQuality 6. Philip Koerner, Phenomenex 7. Kate Rimmer, NIST 8. Jinchuan Yang, Waters 9. Hong You, Eurofins 10. Kurt Young, GNC/NutraManufacturing 11. Garrett Zielinski, Covance

Protein:

1. Brian Schaneberg, Starbucks 2. Sneh Bhandari, Mérieux NutriSciences 3. Quanyin Gao, Herbalife 4. Philiip Haselverger, Abbott 5. Marcus Lowenthal, NIST 6. Curtis Phinney, Curtis S. Phinney, CNS 7. Lars Reimann, Eurofins 8. Kate Rimmer, NIST 9. John Szpylka, Mérieux NutriSciences 10. Tomasz Tuzimski, Medical University of Lublin 11. Jinchuan Yang, Waters 12. Hong You, Eurofins 13. Garrett Zielinski, Covance

Folin-C:

1. Brian Schaneberg, Starbucks 2. Nour Eddine ES-SAFI, Mohammed V University, Rabat

3. Martha Jennens, Covance 4. Holly Johnson, Alkemist Labs 5. Philip Koerner, Phenomenex 6. Dana Krueger, Krueger Food Laboratories 7. Tom Phillips, State of Maryland 8. Kate Rimmer, NIST 9. Aniko Solyom, GAAS Analytical 10. Darryl Sullivan, Covance 11. John Szpylka, Mérieux NutriSciences 12. Hong You, Eurofins 13. Joseph Zhou, Sunshineville Health Products

Ginger:

1. Brian Schaneberg, Starbucks 2. Paula Brown, BCIT 3. Anton Bzhelyansky, USP 4. Congmei Cao, Herbalife 5. Nour Eddine ES-SAFI

6. Holly Johnson, Alkemist Labs 7. Philip Koerner, Phenomenex 8. Dana Krueger, Krueger Food Laboratories, Inc. 9. Aniko Solyom, GAAS Analytical 10. Jinchuan Yang, Waters 11. Kurt Young, GNC/Nutra Manufacturing 12. Yanjun Zhang, Herbalife

Kratom:

1. Brian Schaneberg, Starbucks 2. Joseph Betz, NIH-ODS

3. Paula Brown, BCIT 4. Christine Casey, FDA 5. Nour Eddine ES-SAFI, Mohammed V University, Rabat 6. Ajai Gupta, CSIR-IIIM 7. Holly Johnson, Alkemist 8. Philip Koerner, Phenomenex 9. Charles Metcalfe, Custom Analytics 10. David Pereira, 11. Tom Phillips, MD Department of Agriculture 12. Darryl Sullivan, Covance 13. John Szpylka, Mérieux NutriSciences 14. Yan Hong Wang, University of Mississippi 15. Hong You, Eurofins Scientific

AOAC SPDS EXPERT REVIEW PANEL

METHODS AND SMPR ACCESS

• ALL SPDS SMPRs • METHODACCESS (ERP ONLY – PASSWORD REQUIRED)

AOAC CANDIDATE METHOD #FAA-001

Submitter Name Submitter Email Organization Method Type Method Name

Philip Haselberger

philip.haselberger@abbott.com

Abbott Nutrition Free Amino Acids

Free Amino Acid Profile and Absence Verification Method by UPLC

Method Author(s)

Philip Haselberger

Method Applicability:

Validated for quantitative determination of: ALA, ARG, ASP, ASN, GLN, GLU, GLY, ILE, LEU, LYS, PHE, PRO, SER, TYR, VAL, CYS, HIS, MET THR, TRP, TAU. Method applicability could be further extended to include: theanine, ornithine, norvaline, GABA, AABA, cysteic acid, beta alanine, carboxylmetylcysteine, pyridethylcysteine, methionine sulfone, hydroxy proline, hydroxy lysine, citruline, carnosine, etc. (see also supporting documentation for additional separation capabilities).

Primary Reviewer: Garrett Zielinski, Covance Secondary Reviewer: Holly Johnson, Alkemist

AOAC DECEMBER 2017 ERPs - METHOD REVIEW FORM

Submission Date

2017-12-07 16:31:32

AOAC Expert Review Panel: Method Review

Reviewer Information Required to validate your review.

Name

Garrett Zielinski

E-mail

Garrett.Zielinski@covance.com

Organization

Covance Laboratories

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

Free Amino Acid Profile and Absence Verification Method by UPLC

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

FAA-001

Applicable SMPR

2017.011

I. Summary of the Method

Samples are dissolved in a mixture of water:6M HCl and internal standard is added. An aliquot is diluted with purified water and derivitized with AQC reagent. Analytical separation is performed via UPLC with a C18 column. Detection and quantitation are performed via absorbance at 260nm and comparison to standards of known concentrations.

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). 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.

It appears that a few compounds from Table 1 in the SMPR are missing accuracy/precision data. Beta-Alanine, Cysteine or Cystine, Hydroxyproline seem to be excluded.

Yes

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

Would be ideal to have sources for reference grade amino acids.

It appears that a Beta Alanine, Cysteine or Cystine (not sure what CYS refers to), and Hydroxyproline are missing. It does not appear that the different product types are identified with type. Per the SMPR, powders, tablets, liquids, and capsules should be tested. Accuracy was determined by comparison to another validated method for profile levels. We should discuss if this is an acceptable approach as the SMPR references % recovery which should be based on spiking a known amount of analyte(s).

IV. General Submission Package

It would be good to see accuracy and precision data for the missing compounds.

Yes. There is system suitability but not necessarily control material assessment.

Data not provided.

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

Method is well-written and complete. It uses basic equipment and instruments. Sample preparation procedure and derivitization is fairly simple. The attention to detail regarding derivitization of samples (particularly the sample color) is a plus.

Does not appear to have complete data for all matrices. One question would be how the different matrices would potentially negatively impact the derivitization.

7. Any general comments about the method?

Overall, it seems like a very good method. The only gaps are sources for reference standards, missing a few compounds, and data for all matrices in SMPR.

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.

Not at this point due to cons/weaknesses specified previously.

AOAC CANDIDATE METHOD # PRO-001

Submitter Name

Aihua Liu

Submitter Email

aliu@genysislabs.com

Method Name

Identification of Pea, Rice and Soy Proteins in Raw Materials and Finished Goods using ESI HPLC-MS/MS

Method Author(s)

Genysis Labs

Method Type Proteins Primary Reviewer: Sneh Bhandari, Merieux Nutrisciences Secondary Reviewer(s): Lars Reimann, Eurofins

AOAC DECEMBER 2017 ERPs - METHOD REVIEW FORM

Submission Date

2017-12-12 13:01:17

AOAC Expert Review Panel: Method Review

Reviewer Information Required to validate your review.

Name

SNEH BHANDARI

E-mail

sneh.bhandari@mxns.com

Organization

Merieux NutriSciences

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

Identification of Pea, Rice and Soy Proteins in Raw Materials and ------------------ --HPLC-MS/MS

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

PRO-001

Applicable SMPR

AOAC SMPR 2016.016

I. Summary of the Method

The samplle is extracted in 5 mM Urea Tris buffer followed by reduction with DTT and alkylation with iodoacetamide. The extract is digested with trypsin at 37C overnight abd quenched by formic acid.The extract is filtered and diluted with 0.1% formic acid and internal std (Beta-Casomorphin 1-4 peptide) and analyzed by LC-MS/MS. Pea, rice and soy protein identifications were based on the comparison with MRL sample, which is made from raw materials identified using PCR.

II. Review of the Method Only:

Yes, with scope of Pea, Rice and Soy proteins in raw and finished dietary supplement materials

Yes. Pea, rice and soy protein identifications in the method based on the comparison with MRL sample, which is made from raw materials identified using PCR. The identification is LC-MS/MS based by analysis of corresponding specific peptides in the samples following tryptic digestion of the extracted proteins.

Yes,

No distinctly. The authors may add a separate paragraph about safety emphasizing toxicity of reagents like iodoacetamide and other chemicals

III. Review of Information in Support of the Method

Yes.

Yes, The method describes and is based on the use of "LCS" and "MRL" which are in house Reference materials for analysis of relevant proteins (pea, rice and soy). The uthors may provide more details how these materials are prepared for method opeartion.

Yes in most parts. Two minor differences/exceptions. 1. The SMPR requires analysis of false-positive rate of the method with samples containing non-protein ingredients at 10% target concetration. The validation study has performed analysis of false-positive rate of the method with samples containing non-protein ingredients at 0.42-7.85% target concetration. 6 out of 8 non-protein ingredient were evaluated under 2% traget concentration. 2. The method did not target any peptides (molecular weight <10,000 daltons) for false positive identification nor anay nontarget proteins (molecular weight >10,000 daltons) in the mathod validation.

IV. General Submission Package

The method does not describe a distinct system suitability tests or controls but entire method is based on use of LCS and MRL. The authors may adda paragraph defing their role also in system suitability.

Yes

Method may add some clarifications about the procedure guideles in preparation of LCS and MRL samples. They may also calify how the selection of the materials is made about the samples to meet scope of the method.

The method is straight forward and uses advance technology available at this time.

The authors have not proved uniqueness/specificty of target peptided selected for the idenitification of the respective proteins.

7. Any general comments about the method?

The method is suitable for the pupose

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.

The methods meets most of the criteria of SMPR. The authors may complete the gaps metioned (fals positive determination at target conc. of 10% and also with non-target peptdes and proetisn). The method may be moved to First Action status.

AOAC CANDIDATE METHOD # PRO-002

Submitter Name

Aihua Liu

Submitter Email

aliu@genysislabs.com

Method Name

Identification of Milk Proteins in Raw Materials and Finished Goods using ESI HPLC-MS/MS

Method Author(s)

Genysis Labs

Method Type

Proteins

Primary Reviewer: Hong You, Eurofins Secondary Reviewer: Philip Haselberger, Abbott Nutrition

AOAC DECEMBER 2017 ERPs - METHOD REVIEW FORM

Submission Date

2017-12-08 04:08:02

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

Identification of Milk Proteins in Raw Materials and Finished Goods using ESI HPLC-MS/MS

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

PRO-002

Applicable SMPR

2016.015

I. Summary of the Method

This method is applicable for the identification of milk proteins (a-S1-Caein and b-Lactoglobulin) in dietary supplement raw materials and finished products. The proteins in sample were treated with urea and fractionated by trypsin into specific peptides, which are then detected by HPLC-triple quad mass spectrometry. Three peptides were chosen as target analytes for the identification of each protein.

II. Review of the Method Only:

Yes. Authors tested one type of casein protein and one type of whey protein, which are listed in the Table 1 of SMPR. Authors also showed this method can distinguish target analytes from potential adulterants in two types of dietary supplements (pre workout finished goods and premixed vitamin).

Yes.

No. Authors should demonstrate the safety of handling reagents and instruments when using this method.

III. Review of Information in Support of the Method

Yes.

Authors did not provide clear information on the reference materials that they used for establishing LCMS/MS transition parameters.

Yes and no. The POI evaluation of the validation studies has met the minimum acceptance criteria in the method performance requirement table. However, melamine, surfactants, peptides, and nontarget proteins were not evaluated as potential adulterants in the selectivity study.

IV. General Submission Package

Authors did not provide any supporting information for demonstrating the safety of using this method.

The method doesn't contain system suitability tests. System suitability is recommended to use to evaluate if other brand or model of HPLC-triple quad mass detector can be suitable for running this method. Mass accuracy and relative responses of pseudomolecular ion isotopes may be tested to evaluate system suitability. The method used control material for each run. However, authors need to provide the detailed formulation and source of this control material. Also, the quality control criteria of using the control material needs to be clarified.

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 doesn't contain system suitability tests.

Yes. The method contains blank check samples, lowest point check standard (MRL), and midrange point check standard (LCS) for each run sequence.

Authors need to disclose the formulation information of control material, mixture A, and mixture B.

The method is clearly written and generally easy to follow. Most validation experiments were designed based on the SMPR and most SMPR requirements were successfully met.

Authors are recommended to demonstrate the rationales behind the selection of target peptides. Normally, the bottom up shotgun protein identification needs to use software and database for data processing and interpretation. The whole peptide profile is used instead of picking 3 specific peptides.

Authors are recommended to provide more supporting information such as retention time, MS chromatogram, and mass spectrum for each target peptide.

7. Any general comments about the method?

Despite minor flaws that listed before, this method generally met SMPR 2016.015 requirements.

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.

Yes. Despite minor flaws that listed before, this method generally met SMPR 2016.015 requirements and therefore is recommended to be adopted as a First Action Official Method after all recommended supporting information can be provided by authors.

AOAC DECEMBER 2017 ERPs - METHOD REVIEW FORM

Submission Date

2017-12-13 15:36:52

AOAC Expert Review Panel: Method Review

Reviewer Information Required to validate your review.

Name

Philip Haselberger

E-mail

philip.haselberger@abbott.com

Organization

Abbott Nutrition

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

Identification of Milk Proteins in Raw Materials and Finished Goods using ESI HPLC-MS/MS

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

PRO-002

Applicable SMPR

2016.015

I. Summary of the Method

Method is based on an UPLC-MS/MS analysis of peptides after the sample has been through a trypsin digestion. Positive ID of the presence of milk protein at >0.1% is made by comparison relative to a lab prepared MRL sample that is nominally 0.1%. The presence of milk protein is only confirmed if 3 signature peptides from alpha-S1-casein and beta-lactoglobulin are all present. Otherwise the protein is not confirmed.

II. Review of the Method Only:

Partially. Some of the compounds listed in Table 2 of the SMPR were not addressed.

Partially. Depending on interpretation the method submitted may not meet the required number of replicates stated for SLV matrix study under the POI at low concentration section. However all of the POI data presented does meet the SMPR requirements at the relevant levels.

Yes.

No reagent hazards noted. Given that some are mildly hazardous it would likely be helpful to maybe recommend analysts to read over the relevant MSDS as appropriate.

III. Review of Information in Support of the Method

Yes.

Not really applicable, reference materials noted in SMPR just refers to Appendix F.

No, but the SMPR specifies one or more of the proteins specified. The method is specific for what it is stated to be valid for (milk protein), therefore not a problem.

IV. General Submission Package

No.

None stated in SMPR. There is some language in the method that potentially infers a system suitability requirement (around the incubation time), but it is not entirely clear what the intent is for that step.

Not applicable.

4. Based on the supporting information, is the method written clearly and concisely? If no, please specify the needed revisions.

Some clarifications suggested as follows:

C.h. -->Recommend some additional guidance on choice of trypsin. Current lack of specificity could lead to reproducibility problems.

D.i. -->Should nitrogen conversion factors be listed for the sake of consistency? In other words, how is the amount of protein being defined if the intent is to incorporate equal portions? Also, should the total Kjeldahl protein listed in this section be 19.79% instead of 79.16%? Four equal parts with the same average would then equal a total of the same value.

E.d.-->Suggest listing the set temperature here.

E.n.--> Long incubation time, is this truly needed?

F.a. --> Column listed does not match column listed in section B.c. Which is the correct column?

Table 2017.B --> Is metaline supposed to be melamine?

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?

For the samples tested this method appears to exceed the POI limits outlined in the SMPR. The type of prep used in well known in proteomic analysis therefore many labs will be able to execute this method.

Depending on interpretation the data presented may not meet the SMPR requirements for replication.

7. Any general comments about the method?

The method document could benefit from a few clarifications (column choice, trypsin recommendation, etc.).

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 do recommend this method for first action status if the author agrees to fill in the current gaps to the SMPR (did not test against peptides as per Table 2, and potentially insufficient replication relative to Table 3 of the SMPR).

AOAC CANDIDATE METHOD # FOL-04

Submitter Name

Sharon Brunelle

Submitter Email

sbrunelle@aoac.org

Organization

Various

Method Type

Folin-C

Method Name

Determination of Total Phenolic Content Using the Folin-C Assay: Single Laboratory Validation Steve Kupina, Chris Fields, Mark Roman, Sharon Brunelle Applicable to the determination of total polyphenolic content in grape seed, grape skin, coffee, cocoa, and black tea extracts.)

Method Author(s)

Method Applicability:

Primary Reviewer: Kate Rimmer, NIST Secondary Reviewer: Tom Phillips, State of Maryland

AOAC DECEMBER 2017 ERPs - METHOD REVIEW FORM

Submission Date

2017-12-08 15:19:13

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

Determination of Total Phenolic Content Using the Folin C Assay: Single Laboratory Validation

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

FOL-04

Applicable SMPR

2015.009

I. Summary of the Method

An aqueous extraction of a sample matrix is performed then followed by reaction with Folin & Ciocalteu's (Folin-C) reagent. The light absorption is then determined at 765 nm and compared with a gallic acid calibration curve. For this method, the procedure was examined in a series of dietary supplements including: grape seed extract, grape skin extract, black tea extract, green coffee extract and cocoa extract.

II. Review of the Method Only:

The methods tests five product types and the applicability calls for dietary supplement raw materials and finished products. All of the products tested were extracts. It might be good to look at performance in plants and solid oral dosage forms.

Yes. This SMPR specifically required a Folic-C method that meets the method performace requirements.

Yes, the terms are used properly.

Safety precautions are not noted, however, the method does not appear particularly hazardous.

III. Review of Information in Support of the Method

Definittions are applied appropriately

Reference materials suggested in the SMPR were not used

There is only one quantity being tested, the total soluble phenolic content and it appears in the SMPR tables. In the tables it is listed as the mean phenolic content (where the content is from the aqueous extraction).

IV. General Submission Package

Blank and check samples at the lowest and midpoint of the analytical range were required along with evaluation of matrix and interfering compound. The authors performed reasonable calibration tests and investigated the potential interference from sugars. (there are positive results for ascorbic acid, amino acids, and sugars).

The authors used gallic acid calibration and information about the performance of the samples on different days. However there were no reference materials used.

A multifactor analysis was performed

Yes

The method performs well for most of the matrices examined.

The RSDr for Coffee Extract and Cocoa Extracts were out of the range listed on the SMPR. It was noted on the Cocoa Extract was likely below the limit of quantitation.

Recovery fell within the ranges of the performance requirements.

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

This is not a comment on the specific method and instead a comment on the use of method dependent "total phenolic" determinations. As the community is moving away from the use of these methods and the meaning of the results, does the AOAC want to pursue first action methods in this category?

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.

If the AOAC wants to move forward with this type of method (a total phenolic method) then this method should be tested with raw plant materials and solid oral dosages in order to represent the raw materials and final products as stated in applicability. I would also recommend that the method should be tested more with products at the extreme high and low ends and possibly with systematic sample types (varying the matrix effects) to see if there is a pattern to where the method meets the RSDr in the SMPR and where it does not.

AOAC CANDIDATE METHOD #: GIN-001

Submitter Name Submitter Email

Hong You

hongyou@eurofinsus.com

Organization

Eurofins Supplement Analysis Center, Eurofins Scientific, Inc.

Method Type Method Name

Ginger

Determination of Select Nonvolatile Ginger Constituents by HPLC‐DAD Hong You (corresponding author), Bailey Ireland, Michael Moeszinger, Laura Snow, Scott Krepich, Vivian Takagawa This method is for the determination of select nonvolatile ginger constituents by high performance liquid chromatography using a diode‐ array detector (HPLC‐DAD), and was developed by following AOAC Guidelines for Single‐Laboratory Validation. This method is applicable to identify and quantitate [6]‐, [8]‐, and [10]‐gingerols; [6]‐, [8]‐, and [10]‐shogaols; [6]‐paradol, and zingerone in dietary ingredients and supplements that contain ginger, ginger extract, or/and purified nonvolatile ginger constituents.

Method Author(s)

Method Applicability:

Primary Reviewer: Aniko Solyom, GAAS Analytical Secondary Reviewer: Paula Brown, BCIT

AOAC DECEMBER 2017 ERPs - METHOD REVIEW FORM

Submission Date

2017-12-07 20:01:40

AOAC Expert Review Panel: Method Review

Reviewer Information Required to validate your review.

Name

Aniko Solyom

E-mail

asolyom@gaasanalytical.com

Organization

GAAS Analytical

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

Determination of Select Nonvolatile Ginger Constituents by HPLC-DAD g

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

GIN-001

Applicable SMPR

2017_012

I. Summary of the Method

HPLC-DAD method for the identification and quantitation of [6]-, [8]- and [10]- gingerols, [6]-, [8]- and [10]-shogaols, [6]-paradol and zingerone in dietary ingredients and supplements.

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.

The method quantitates all the required components [6]-. [8]- and [10] gingerol and [6]-shogaol. In addition, the method quantifies four of the desirable constituents, [8]- and [10]-shogaol, [6]-paradol and zingerone

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

On page 13 there is a confusion how LOD and LOQ is used. The submission states: "Therefore, the limit of quantitation (LOD) of this method was set at the low limits of the analytical range for each target analyte". Only LOQ was defined in the SMPR, as the "minimum content of analyte in the given matrix that can be reliably and precisely quantified....". LOQ can be calculated from the signal to noise ratio or from the standard deviation of the slope of 5 calibration curves (at low concentration).

That definition used in the submission is more like LLOQ, the lowest level of quantitation.

On page 17 "Intermediate precision" was used instead of "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).

Yes, the submission includes a "Safety" section.

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.

The supporting documents (Appendices) don't have definitions.

The SMPR listed four reference materials, three of them (USP 1291504, NIST 3398 and NIST 3399) were used in the studies.

Analytical range: If the analytical range defined in the SMPR is for the total amount of nonvolatile ginger constituents, the method meets the requirements. If the analytical range is for each individual analyte, than the method doesn't meet the requirements.

Limit of Quantitation: met requirements (also see note for II. 3)

Recovery: Spike recovery studies were performed using Galangal and excipient blend as matrices; the recoveries from these matrices met the SMPR requirements. RSDr: Most of the individual analytes met the SMPR's requirement. The reasons for higher RSDr were low concentration, liquid samples and possibly inadequate separation

RSD(R): The RSD(R) for majority of the zingerone and some of the 6-paradol, 6-shogaol and 10-gingerol quantitation was more than 8%.

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.

The SMPR did not specify system suitability criteria, but the presented system suitability data comply with the requirements of USP General Chapter <621>; RSD less or equal 2.0% calculated from 5 replicate injections of the standard preparation.

See answer to question IV. 2

Yes

- The method quantifies not just the required analytes, but four of the optional analytes too.

- All the matrices listed in the SMPR were tested

- The HPLC method is short, and can be easily converted into LC-MS method

- Different extraction methods were investigated and optimized

- Sample preparation is not complicated

- Large amount of validation data

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

- The major weakness of the method is that baseline separation is not achieved for most of the analytes in the real samples (Appendix 5) and the baseline is not stable in most of the real samples

Sample 063: Only 6-shogaol, 8-shogaol and 10-shogaol peaks are baseline separated

Sample 099: Only zingerone, 6-shogaol, and 8-shogaol peaks are baseline separated

Sample 251: Only 6-shogaol, 6-paradol and 10-shogaol peaks are baseline separated

Sample 420: 10-gingerol is not baseline separated

Sample 580: Only zingerone, 6-shogaol, 8-shogaol and 10-shogaol peaks are baseline separated

Sample 875: Only 6-shogaol and 8-shogaol peaks are baseline separated

Sample 889: Only 6-shogaol, 10-gingerol and 10-shogaol peaks are baseline separated

Sample 986: Only 6-shogaol and 8-shogaol peaks are baseline separated

- I don't agree how the fReference Material Stock (on page 3) were prepared. The concentration of the stock solution is not correct; weighing the standard(s) and add 20 mL methanol doesn't define the final volume of the solution. The correct method to prepare calibration solutions is to weigh the standar(s) into a Class A volumetric flask and dilute to volume. - I have similar problem with the sample preparation (the volume of 60 mg sample + 10 mL diluent is more than 10 mL). In addition, 1 g sample mixed with 10 mL diluent is probably a paste and not a solution.

- There is no description how the softgel capsules were prepared for the analysis

7. Any general comments about the method?

Well written method with large amount of validation 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.

The method has a great potential to be a First Action method, but I am very concerned about the quantitation of the analytes in the real samples. I would suggest to modify the gradient method and/or try different column(s) and mobile phases to achieve baseline separation in the real samples.

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