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AOAC Stakeholder Panel on Strategic Food Analytical Methods: Stakeholder Panel Meeting Meeting Minutes Monday, March 13, 2017, 1:00 p.m. – 6:00 p.m. ET

I. Attendees

Panel Members (Present during all or part of the meeting): Erik Konings, Nestlé (Chair) Susan Audino, Audino & Associates Charles Barber, NIST DeAnn Benesh, 3M Sneh Bhandari, Mérieux Nutrisciences Amy Brown, Fl. Dept. of Agriculture Bob Clifford, Shimadzu Jo Marie Cook, Fl. Dept. of Agriculture Nisha Corrigan, Hygena Hans Cruijsen, Freisland Campina David Cunningham, Ocean Spray Cranberries Ken Davenport, 3M Jennifer Donelson, VUV Analytics Robert Donofrio, Neogen Nyla Dubiel, Grain Millers Peter Gibson, GW Research Ltd. Cathy Halverson, TTB Evan Henke, 3M Norma Hill, AOAC Past President, TTB (Ret.) Steve Hoelzer, Hygena Greg Jaudzems, Nestlé George Joseph, AsureQuality New Zealand Sookwang Lee, FDA Haiyan Liu, Ocean Spray Cranberries Kate Mastovska, Covance Josh Messerly, Eurofins

Bill Mindak, FDA Armen Mirzoian, TTB Jenny Nelson, Agilent Mary O’Meara, Grain Millers Inc. Edwin Phifer, FDA Melissa Phillips, NIST Curtis Phinney, Consultant Rick Reba, Nestlé Amanda Rigdon, Emerald Scientific Catherine Rimmer, NIST Brian Schaneberg, Starbucks Tom Seipelt, Abbott Nutrition Tina Shaffer, Hygena Kevin Shores, Valspar Darryl Sullivan, Covance Laboratories Alan Sutton, GW Research Ltd. John Szpylka, Mérieux Nutrisciences Joe Thompson, Abbott Nutrition Paul Valder, ACG Wayne Wargo, Abbott Nutrition Laura Wood, NIST Jason Wubben, ADM William Xue, Catalent Sudhakar Yadlapalli, First Source Laboratory Solutions Joyce Zhu, Jamieson Labs

AOAC Staff (Present during all or part of the meeting)::

Delia Boyd, Scott Coates, Christopher Dent, Jennifer Diatz, Dawn Frazier, Jonathan Goodwin, Deborah McKenzie, Nora Marshall, Tien Milor, La’Kia Phillips, Robert Rathbone, Joyce Schumacher

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II. Meeting Minutes

1. Welcome and Introductions

All attendees were introduced and the meeting commenced at 1:00 pm ET.

2. SPSFAM Update Konings provided a presentation1 regarding SPSFAM’s activities since the last stakeholder panel meeting, including updates on recent expert review panels (ERPs) and the successes of the AOAC Working Group Initiative. Szpylka provided an update on the AOAC Expert Review Panel for SPSFAM for Select Food Allergen Methods (ERP), which had met that morning. ERP chair, Szpylka, advised that the submitted food allergens method would require more work before approval.

3. SMPR Approval Presentations and Consensus

a. SMPR Approval Presentation: Proanthocyanidins in Cranberries (PAC) Schaneberg took the floor to give a presentation2 on the work done by the AOAC Working Group on Proanthocyanidins in Cranberries (PAC Working Group). He explained that the working group had met several times by webinar to develop two sets of Standard Method Performance Requirements (SMPRs):

• SMPRs for Quantitation of proanthocyanidins content in cranberry fruit, juice, beverage, dried cranberry, cranberry sauce, ingredients (concentrations, extracts and powders) and dietary supplement formulationsi

• Identification of Type-A Proanthocyanidin in Cranberry-Based Foods and Dietary Supplementsii

After reviewing the draft standards, Schaneberg made the motions to approve the SMPRs.

MOTION to approve the SMPR for Quantitation of proanthocyanidins content in cranberry fruit, juice, beverage, dried cranberry, cranberry sauce, ingredients (concentrations, extracts and powders) and dietary supplement formulations as presented. (Schaneberg / Cunningham)

21 in favor, 0 opposed, 0 abstained. The motion carried.

MOTION to approve the SMPR for Identification of Type-A Proanthodanidin in Cranberry Based Foods and Dietary Supplements as presented or as revised. (Schaneberg / Cunningham)

21 in favor, 0 opposed, 0 abstained. The motion carried.

b. SMPR Approval Presentation: Cannabis Quantitation

1 Attachment 1 - SPSFAM Update Presentation 2 Attachment 2 – PAC SMPR Approval Presentation

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Audino then took the floor to provide a presentation3 on the work done by the Cannabis Working Group. She reviewed the original fitness for purpose and explained that the group decided to make this first set of SMPRs a bit simpler by doing simple quantitation of cannabinoids in plant material, and another SMPR for quantitation of cannabinoids in concentrates. SPSFAM members recommended a minor modification to the SMPR for cannabis concentrates – the word “each” was removed from the applicability statement. MOTION to approve the SMPRs for Quantitation of cannabinoids in dried plant materials as presented.iii (Audino / Donelson) 20 in favor, 0 opposed, 1 abstention (Nestlé). The motion carried. MOTION to approve the SMPRs for Quantitation of cannabinoids in cannabis concentrates as amended.iv (Audino / Clifford) 20 in favor, 0 opposed, 1 abstention (Nestlé). The motion carried.

4. Launch of New SMPR Working Groups

Konings announced that new initiatives would be launched and invited Sullivan, Chair of the SPSFAM Working Group on Bisphenol A (BPA) in Beverages & Water, to take the floor.

a. Working Group Launch: BPA in Beverages and Water Sullivan provided a presentation4 with which he explained the background, significance, regulatory guidance, challenges, and existing methods for BPA in beverages and water. He then proposed the following fitness for purpose for the working group: Analytical method for the determination of Bisphenol A (BPA) in water and non-alcoholic beverages. MOTION to accept the proposed fitness for purpose for BPA in Beverages and Water. (Mindak / Griswold) Discussion followed this motion. There was a question as to whether alcohol is included in this SMPR. Some stakeholders were concerned that alcoholic beverages would cause packaging to shed BPA faster than non-alcoholic beverages. After further consideration, the group revised the fitness for purpose statement to read: Analytical method for the determination of Bisphenol A (BPA) in commercially packaged ready to consume carbonated and non-carbonated water and non-alcoholic beverages. Konings then asked for a vote on the revised fitness for purpose statement: 19 in favor, 0 opposed, 0 abstentions. The motion carried. The motion passed and the SPSFAM Working Group on BPA in Beverages and Water was formally launched.

3 Attachment 3 – Cannabis SMPR Approval Presentation 4 Attachment 4 – BPA Working Group Launch Presentation

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b. Working Group Launch: Cannabis in Food Products

Audino returned to the floor. She explained that her last presentation was to approve the work done since the last SPSFAM meeting, and this presentation will launch new topics in the Cannabis working group with new objectives to be completed before the next SPSFAM meeting. With that she provided the stakeholders with a presentation5 to move the SPSFAM Working Group on Cannabis Potency into new initiatives, including detection of cannabinoids in foods and detection of pesticide residues in cannabis. She explained that the SPSFAM Advisory Panel for Cannabis made this decision at their meeting in December 2016. She then reviewed the background of cannabis in food products, the significance and implications, and proposed the following fitness for purpose statement: Standard Methods Performance Requirements (SMPRs) for quantitative methods to identify and quantify select cannabinoids in select food matrices. She then reviewed the background of the pesticides issue and proposed a second fitness for purpose, which would lead into the development of a separate SMPR for pesticide detection: Standard Methods Performance Requirements (SMPRs) for quantitative methods to identify and quantify various pesticide residues in dried cannabis materials. Audino then asked for a motion to approve the fitness for purpose statements and formally re-launch the SPSFAM Cannabis Working Group. MOTION to accept the proposed fitness for purpose statements for cannabis in foods in cannabis products. (Clifford / Mindak) 19 in favor, 0 opposed, 1 abstention (Nestlé). The motion carried. MOTION to accept the proposed fitness for purpose statement for pesticides in cannabis products. (Cook / Donelson) 19 in favor, 0 opposed, 1 abstention (Nestlé). The motion carried.

5. Future Working Group

a. Veterinary Drug Residues

Delatour provided the panel background for a future SPSFAM working group that is scheduled to launch in September, 2017. Delatour reviewed a presentation6 explaining the background, methodologies, analytes, and current technologies. He summarized the presentation with the following points: • Uncontrolled occurrence of veterinary drugs in food is a health concern, particularly with

regard to antimicrobial resistance. • Multiresidue analysis is needed for an effective control. • Mass spectrometry is needed for full compliance testing.

5 Attachment 5 – Cannabis Working Group Re-Launch Presentation 6 Attachment 6 – Veterinary Drug Residues Presentation (Delatour)

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• A single LC-MS-based method capable to demonstrate full compliance of veterinary drugs in food does not exist so far.

• Matrix scope should represent current practices in terms of trade and business. • Method performance should fit with throughput and positive rate for as low cost as

possible analytes.

Konings thanked Delatour for his presentation and invited the SPSFAM Veterinary Drugs Residue Working Group Chair, Joe Boison, to take the floor. Boison also gave a presentation7 on veterinary drug residues where he provided further background and defined the goals of the project – to develop a series of SMPRs that will provide acceptance requirements for methods capable of accurately and reliably measuring a wide variety of veterinary drugs in selected matrices, leading to four or more Official Methods of Analysis that comply with the SMPRs. The SMPRs would be developed one after another by the same working group. Konings concluded the subject by stating that the group will be launched in September, but more animal health organizations must to get involved in SPSFAM and more support for this group will be needed.

6. Adjourn Konings provided a final update, that AOAC has not been able to find the resources to launch a sugar working group yet. He then adjourned the meeting at approximately 6:00 p.m. ET.

Attachments

Attachment 1: SPSFAM Update Presentation Attachment 2: Proanthocyanidins in Cranberries SMPR Approval Presentation Attachment 3: Cannabis SMPR Approval Presentation Attachment 4: BPA Working Group Launch Presentation Attachment 5: Cannabis Working Group Re-Launch Presentation Attachment 6: Veterinary Drug Residues Presentation (Delatour) Attachment 7: Veterinary Drug Residues Presentation (Boison) Attachment 8: All Approved SMPRs

i SMPRs for Quantitation of proanthocyanidins content in cranberry fruit, juice, beverage, dried cranberry, cranberry sauce, ingredients (concentrations, extracts and powders) and dietary supplement formulations (as approved) ii SMPR for Identification of Type-A Proanthocyanidin in Cranberry-Based Foods and Dietary Supplements (as approved) iii SMPR for Quantitation of cannabinoids in dried plant materials (as approved) iv SMPRs for Quantitation of cannabinoids in cannabis concentrates (as approved)

7 Attachment 7 – Veterinary Drug Residues Presentation (Boison)

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Stakeholder Panel on Strategic Food Analytical Methods (SPSFAM)

SPSFAM Updates: Working Groups and ERPs

Erik J.M. Konings

Nestlé Research Center, Nestec Ltd.

Lausanne, Switzerland

Working Group (WG) Initiative

• December 2014, AOAC Board of Directors initiatesWG Initiative

– as an a mechanism for AOAC Organizational Affiliatemembers to initiate relevant standard developmentprojects using existing AOAC stakeholder panels• Expressed a need for a consensus standards and scientificallyvalid fit for purpose consensus methodology

• WG supported through AOAC Organizational Affiliates fundedand formed through AOAC staff

• AOAC works with Organizational Affiliates to find additionalOrganizational Affiliates with the same need for scientificallyvalid fit for purpose methodology

– WG will develop SMPR to present to an existingstakeholder panels for review

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Why the new WG Initiative?

• Offers companies the opportunities to solve challenges without waiting on priorities of existing stakeholder panels

– Advisory Panel participation and discussion

• WG’s funded by current OA’s and new companies interested in addressing immediate needs

– for analytical standards/standard method performance requirements; and

– scientifically valid fit for purpose methodology

Success for the SPSFAM Working Group Initiative

• Selected Food Allergens– SCIEX Sponsored Working Group to develop SMPR for Selected Food Allergens.

SMPR 2016.002 was developed and approved in March, 2016

• Ethanol in Kombucha– Sponsored by a consortium of Kombucha manufacturers, this working group

developed SMPR 2016.001 which was approved in March, 2016.

• Cannabis– Sponsored by a consortium of interested companies, this working group has

drafted two SMPRs for approval at this meeting

• Proanthocyanadins in Cranberries– Sponsored by Ocean Spray, this working group has drafted two SMPRs for

approval at this meeting.

• BPA in Beverages and Wager– This new initiative has been sponsored by a consortium of companies and the

working group will be launched at this meeting.

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SPSFAM Expert Review Panel (ERP) Updates

– SPSFAM ERP for Selected Food Allergens• Two methods submitted to meet AOAC SMPR 2016.002, Quantitation of Selected Food Allergens.

• No methods approved at that time; ERP reconvenes on March 13, 2017 (this morning)

– SPSFAM ERP for Ethanol in Kombucha• Five methods submitted to meet AOAC SMPR 2016.001,

Determination of Ethanol in Kombucha

• One method approved at that time. ERP reconvenes in September, 2017

Getting Involved

• Individuals.– Get involved in a working group

– Provide feedback on SMPRs

– Submit methods for consideration

• Companies.– Consider becoming an AOAC OA and spearhead a WG

– Provide feedback on SMPRs and submit methods

– Indentify experts to participate in WG

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AOAC INTERNATIONALSTAKEHOLDER PANEL ON STRATEGIC FOOD

ANALYTICAL METHODSBrian Schaneberg, PhD

Starbucks Coffee Company

Proanthocyanidins in Cranberries (PAC) Working Group SMPR PresentationMarch 13, 2017

Marriott Washingtonian Center, Gaithersburg, Maryland, USA

Fitness for Purpose As Agreed September 18, 2016

The method should be applicable to the analysis of cranberry fruit,

juice, beverage, dried cranberry, cranberry sauce, ingredients

(concentrates, extracts and powders) and dietary supplement

formulations, applicable to two potential purposes: (1)Quantitative

QC method able to quantify total proanthocyanidin content,

preferentially as the total sum of all individual oligomers and

polymers present, or alternatively as the total sum with reference to

a suitable surrogate standard, in samples typically ranging from

0.01% to 55% on a w/w basis; and (2) a Qualitative method to

verify authenticity, able to provide information on the distribution of

proanthocyanidin oligomers and polymers present and confirm

presence of A-type versus B-type.

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SPSFAM PAC Working Group Members

•Brian Schaneberg, Starbucks•Paula Brown, BCIT•Anton Bzhelyansky, USP•Tony Chang, International Chemistry Testing•David Cunningham, Ocean Spray Cranberries•Monica Giusti, Ohio State University•Gunter Haesaerts, Pharmatoka•Amy Howell, Rutgers University•Jin Ji, Brunswick Laboratories•Holly Johnson, Alkemist Labs•Erik Konings, Nestlé•Scott Krepich, Phenomenex•Christian Krueger, Complete Phytochemical Solutions

•Scott Kuzdzal, Shimadzu•Haiyan Liu, Ocean Spray Cranberries•Kate Merkh, Lassonde Pappas•Masayuki Nishimura, Shimadzu•Melissa Phillips, NIST•Jess Reed, University of Wisconsin•Kate Rimmer, NIST•John Szpylka, Mérieux Nutrisciences•Xianli Wu, USDA

PAC Working Group Work to Date

•6 teleconferences (October 2016 – December 2016)

•2 SMPRs Drafted

•Public comment period (January, 2017)

•SMPRs made ready for SPSFAM review and approval

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Background

• Product formulations to deliver effective and consistent concentrations of proanthocyanidins

• Standardize products for clinical studies

• Impact of processing on and the shelf‐life of proanthocyanidins in various forms

SMPR Key Points

• (1) Quantitative QC method to support product manufacture

• (2) Qualitative method to verify authenticity

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SMPR Key Challenges

• Liquid and solid products will require different ranges and system suitability requirements.

• Alignment on definition of PACs.

• Determined methods would only focus on solubles.

• ID method would only focus on A‐type detection.

• Reference materials and standard compounds may be difficult.

• ID specificity my be limited due to methods available.

Comments Submitted

• (1) Quantitation

– "Syrups" have not been separately identified as a dietary supplement matrix in any of the other dietary supplement SMPRs. No change to SMPR proposed.

• (2) Identification

– "Syrups" have not been separately identified as a dietary supplement matrix in any of the other dietary supplement SMPRs. No change to SMPR proposed.

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Quantitation SMPR

• Applicability

– The method will be able to quantify total proanthocyanidin content as the sum of all extractable oligomers (> DP2) and polymers present in cranberry (Vaccinium macrocarpon) fruit, juice, beverage, dried cranberry fruit, cranberry sauce, ingredients (concentrates, extracts, powders, and presscake) or dietary supplements (Table 3).

Quantitation SMPR

• Performance RequirementsParameter Requirement

Limit of Quantitation (LOQ) (%) ≤ 0.01

Analytical Range (%) ≤ 0.03 – 55

Liquids SolidsRanges 0.03 – 15% > 15% ‐ 55% 0.03 – 15 % > 15% ‐ 55%Recovery (%) 97 – 103 97 – 103 90 ‐ 107 97 – 103% RSDr < 10 < 5 < 15 < 10% RSDR < 15 < 8 < 20 < 15

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Identification SMPR

• Applicability

– The method will be able to identify the presence of Type‐A proanthocyanidin in cranberry (Vaccinium macrocarpon) fruit, juice, beverage, dried cranberry fruit, cranberry sauce, ingredients (concentrates, extracts, powders, and presscake); or dietary supplements (listed in table 2).

Identification SMPR

• Performance Requirements

Selectivity Study90% probability of identification with 95% confidence (33 correct identifications out of 33 samples known to contain Type‐A proanthocyanidin).*

*Some aberrations may be acceptable if the aberrations are investigated, and acceptable explanations can be determined and communicated to method users.

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Motion

Move to accept the Standard Method Performance Requirements for:• Quantitation of proanthocyanidin content in cranberry fruit, juice, beverage, dried cranberry, cranberry sauce, ingredients (concentrates, extracts and powders) and dietary supplement formulations.

• Identification of Type‐A Proanthocyanidins in Cranberry Based Foods and Dietary Supplements

as presented.

Discussion?

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AOAC INTERNATIONALSTAKEHOLDER PANEL ON STRATEGIC FOOD

ANALYTICAL METHODS

Susan Audino, Audino & AssociatesCannabis Working Group ‐ SMPR Presentation

March 13, 2017

Marriott Washingtonian Center, Gaithersburg, Maryland, USA

Fitness for Purpose As Agreed September 18, 2016

Standard Methods Performance Requirements (SMPRs) for quantitative methods for various measurements of cannabinoids in raw materials, extracts,

topical applications and foods.

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SPSFAM Cannabis Working Group Members

•Susan Audino, Audino & Associates•Patricia Atkins, SPEX•Roger Brauinger, A2LA•Paula Brown, BCIT•Carolyn Burdette, NIST•Bob Clifford, Shimadzu•Jo Marie Cook, Florida Dept. of Ag•Jennifer Donelson, VUV•Kevin George, Eurofins•Peter Gibson, GW Pharmaceuticals•Thomas Hartlein, Teledyne Tekmar•Dorota Inerowicz, Indiana State Chemist•Holly Johnson, Alkemist•Erik Konings, Nestlé•Julie Kowalski, Phenomenex•Mary Kay Krogull, Eurofins•Julie Kowalski, Restek•Scott Kuzdal, Shimadzu•Robert Lockerman, CEM•Cynthia Ludwig, SAgE

•Kate Mastovska, Covance•Elizabeth Mudge, BCIT•Gary Niehaus, Crystal Diagnostics•Masayuki Nishimuram Shimadzu•Melissa Phillips, NIST•Curtis Phinney, Consultant•Klaus Reif, PhytoLab GmbH•Mitzi Rettinger, Millipore Sigma•Kate Rimmer, NIST•Adam Ross, LGC Standards•Travis Ruthenburg, SC Labs•Alicia Stell, CEM•Alan Sutton, GW Pharma•Aniko Solyom, GAAS Analytical•Kathy Stenerson, MilliporeSigma•John Szpylka, Mérieux Nutrisciences•Jane Weitzel, Consultant•Seth Wong, TEQ Analytical•Josh Wurzer, SC Labs•Hong You, Eurofins

Background

Perspective:

Cannabis Industry Expected to be Worth $50 Billion by 2026 (Bloomberg Markets Report, September 12, 2016)

Estimated size of the legal US industry (MMJ and Recreational): $7.1 Billion in 2016, increased more than 25% from 2015

89% of Americans support legalization of medicinal use. Increased 15% since 2011.

56% Physicians support nationwide legalization of medicinal cannabis.

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Background: States Assume Control

As of March 2, 2017

Background: Matrices

• Edible Products

– Incorporated into recipes/preparations

– Topical

• Transdermal

• Sublingual/Oral

• Inhalation

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Background: Testing

Most states require testing, including:

Chemical• Heavy Metals• Residual Solvents• Content (potencies of select cannabinoids)• Terpenes• Pesticide Residues

Microbiological• Aflatoxin B1, B2, G1, G2

• Ochratoxin A• STEC

Background: Challenges of Testing

To date…

o There are no established action levels or tolerance levels of cannabis contaminants, such as pesticide residues.

o There are no established product label requirements.

o There is no harmonization of testing requirements between/among states.

o Active Pharmaceutical Ingredients (APIs) are not fully characterized in terms of “medicines”

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Background: Challenges of Testing

To date:

There are no consensus or standard test methods for chemical or biological testing.

Background: The Cannabis AP & WG

• Six Organizational Affiliates stepped up to the plate, forming an Advisory Panel to address the testing issue(s).

• Many needs were identified and then prioritized.

• The Working Group focused on the need to determine most interested cannabinoids in:

– Dried Plant Material

– Concentrates

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Cannabis Working Group Work to Date

•1 In Person Meeting (September 2016)

•5 teleconferences (October 2016 – December 2016)

•2 SMPRs Drafted

•Public comment period (January, 2017)

•SMPRs made ready for SPSFAM review and approval

SMPR Key Points

• SMPR: Quantitation of cannabinoids in Dried Plant Materials

• SMPR: Quantitation of cannabinoids in Cannabis Concentrates

• Identify & Quantify:

– Required Cannabinoids: CBD, CBDA, CBN, THC, THCA

– Additional & Desirable: CBC, CBCA, CBDVA, CBG, CBGA, CBDV, 8THC, THCV, THCVA

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SMPR Key Points

Concentrates

ParameterRequirement

THC, THCA, CBDA, CBD, Individually Reported

Requirement (Additional, Desirable

Cannabinoids (Table 1b),

including CBN)

Limit of Quantitation (LOQ) (% w/w)

≤ 0.3 ≤ 0.3

Analytical Range (% w/w)

≤ 0.3 – ca. 100 ≤ 0.3 – ca. 50

Dried Plant Material

Parameters

Ranges (% w/w)

≤ 0.3 – 1 > 1 ‐ 10> 10 – ca.

100

Recovery (% w/w) 95 – 105 97 ‐ 103 98 ‐ 102

% RSDr ≤ 5 ≤ 4 ≤ 2

% RSDR ≤ 7 ≤ 5 ≤ 3

Parameters

Ranges (%)(Pertains to Cannabinoids in Table 1b)

0.1 – 1 > 1 ‐ 25 > 25 – ca. 50

Recovery (%) 95 – 105 97 ‐ 103 98 ‐ 102

% RSDr ≤ 5 ≤ 4 ≤ 2

% RSDR ≤ 7 ≤ 5 ≤ 3

ParameterRequirement

(Pertains to Cannabinoids in Table 1b)


Analytical Range (%) 0.1 – ca. 50*

*Lower concentrations may be acceptable as applicable for cannabinoids listed in Table 1B.

Comments Submitted

5 Comments were received for SMPR –dried materials.

1. The proposed procedures should add (a) Instrument Detection Limit (IDL), and (b) Method Detection Limit (MDL)

Action: WG decided to use LOD which is the equivalent of IDL, which uses blank matrix samples; LOD uses matrix samples. MDL was purposefully removed by the WG.

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Comments Submitted

2. LOQ: The method should be able to quantify at lower levels for the minor cannabinoids because most are below 0.1% in the flowers.

‐ LOQ should be lowered to 0.03‐0.05% to account for the minor cannabinoids

Action: Amended Dried Material Tables 2 and 3 to specify performance requirements are relative to cannabinoids listed in Table 1b.

Comments Submitted

3. If the LOQ isn’t low enough, additional columns should be added for the low contents with expected RSDs.

‐ Additional RSD information should be added. These could be higher RSDs, 8‐10% because of the minor levels and inherent variability of the plants.

Action: RSD information is consistent with the current LOQ. No further action is necessary.

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Comments Submitted

4. There should be some kind of information to help an ERP understand the homogeneity issues that are possible with these plants. 1. They are resinous, cannabinoids are concentrated in the resin, which can clump during grinding. 2. Between flower variation can be high, grinding multiple flowers can impact the homogeneity. 3. Grinding can introduce heat, which will cause degradation of cannabidiolic acids into neutral forms, resulting in less accurate results. Grinding would be the best option for homogeneous samples, but in some cases there are issues with clumped resin, highly variable samples and additional grinding would impact the results and lead to inaccurate data.‐ There should be a comment stating that the method performance should be demonstrated with homogeneous samples, and note that in some cases materials may not meet these criteria due to inherent variation.

Action: Amended Section 8: Validation Guidance

5. Tables 2 and 3 should identify which cannabinoids apply

‐ Propose the following changes to Tables 2 and 3 headers: ”These performance requirements apply only to the cannabinoids in Table 1a”

Action: Amendments per recommendation.

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Motion

Move to accept the Standard Method Performance Requirements for:

• Quantitation of cannabinoids in Dried Plant Materials

• Quantitation of cannabinoids in Cannabis Concentrates

as presented.

Discussion?

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The Determination of Bisphenol A in Beverages”

Darryl Sullivan – Covance LaboratoriesAOAC Mid Year Meeting – Gaithersburg, MDMarch 13, 2017

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p p'

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BPA is safe at the current levels occurring in foods" based on extensive research, including two more studies issued by the agency in early 2014

“EFSA’s experts concluded on each occasion that they could not identify any new evidence which would lead them to revise their opinion that the known level of exposure to BPA is safe

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SUSAN AUDINO, PHD, WORKING GROUP CHAIR

AOAC INTERNATIONAL MID‐YEAR MEETING

GAITHERSBURG, MD

MARCH 13, 2017

Stakeholder Panel Strategic Food Analytical Methods: Background and Fitness for Purpose for ‐Potency of Cannabinoids in Food Products‐Pesticide Residues on Dried Plant Material

Cannabis Advisory PanelSusan Audino, Chair

GW Pharmaceuticals

SC Laboratories

SCIEX

SPEX

Sigma Aldrich

CEM

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Advisory Panel Direction

•Develop 2 SMPRs between April and August 2017

•SMPR: Determine potency of defined cannabinoids in select food matrices

•SMPR: Determine residual pesticides in dried plant materials

Objective I: Potency in Food Products

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Background

•Cannabis may be introduced in any number of ways:◦ Oral/Sublingual (tinctures, concentrates)

◦ Inhalation (smoking, vaporizing)

◦ Transdermal (salves, creams, patches)

Background – Food Products•Cannabinoids are

◦ Fat Soluble◦Must be heated to convert acids to neutrals

•Edible products are becoming a preferred method of dosing◦ Effects are delayed and are longer lasting due to slower absorption

◦ Strength depends on concentrations used

•Bioavailability becomes more complex as matrix becomes more complex

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Background: Food Products•Cannabis dosing by edibles is on the rise.

•Potential reasons:◦ Easier

◦ More options

◦ Stigma on smoking

◦ Use by children

◦ Can be applied to products for human and animal consumption

Cannabis and Food IndustryPrepared Food ‐ Sources◦ Restaurateurs

◦ Cookbooks

◦ Finished Products

Introductions◦ Infused and carried through all or part of the cooking/baking process

◦ Sprayed/dusted/dipped on surface

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Cannabis and Food Industry“This shift in legislation across North America has resulted in the emergence of budding industries providing alternative forms of ingesting marijuana. From marijuana‐infused coffee pods to a plethora of drinks and teas, companies are getting creative in their attempt to satisfy the growing recreational marijuana market. In Colorado, 4,815,650 units of edible marijuana products were sold in the first year of recreational pot sales in 2014. This has contributed to $700 million in annual sales in the state of Colorado alone!” Food Grads blog, November 2, 2016

“It is quite possible that 5% to 7% of food sold in Canada within 10 years may contain cannabis, including ready‐to‐eat, biscuits, desserts, and so on.” ‐‐Sylvain Charlebois, Dean of the Faculty of Management and Professor of Agri‐Food Distribution and Policy at Dalhousie University; By Special to Lift (https://news.lift.co/author/specialtolift/) December 27, 2016

Significance & Implications ‐ LabelingColorado:

Effective 2016: Stores and medical centers may only sell marijuana marked with the universal symbol.

• Retail marijuana specific universal symbol rules:— Every single standardized serving (a serving consists of 10 mg of THC) of an edible retail marijuana product must be individually marked, stamped or imprinted with the new universal symbol.

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Significance & Implications•The determination of Active Pharmaceutical Ingredients, APIs, have not been fully established for this industry.

•There is no federal oversight to ensure “truth in labeling” that does exist with other finished products and commodities.

Significance & Implications•The quality of analytical testing laboratories vary from exceptionally good to …. Not so good.

•One of many questions to answer: when laboratory testing occurs, is it on the finished product or tested throughout the stages of production? Does it make a difference?

•Consumer – Patient – Safety: questionable at best.

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Existing Methods

At present, there are no standard or consensus analytical methods to identify or quantitate

cannabinoids present in foodstuffs.

Challenges

•Matrices range from simple to complex.

•Extraction/Sample Preparation will likely be different for different types of matrices.

•Different states = different regulations.

•Different regulations = different guidelines.

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Proposed Fitness for Purpose

~ Standard Methods Performance Requirements (SMPRs) for quantitative methods to identify and quantify select cannabinoids in select food matrices ~

Objective II:

Determination of Pesticides in Dried Plant Material

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Background: Pesticides•U.S. EPA Definition:

“Any substance applied to a crop with the intended purpose of preventing, destroying, repelling, or mitigating a pest.”

•Classifications:

Insecticides Herbicides Fungicides

Rodenticides Antimicrobials Plant Growth Regulators

Background: EPA•The impact of pesticides on cannabis crops – not fully understood.

•Cannot and does not approve any chemicals used on any outlawed plant.

•Products used in the cannabis industry are not necessarily illegal, however safety profiles do not include inhalation.

•Because it is an illegal product, no “USDA Organic” certification is possible.

41

3/9/2017

10

Background: Circular Reasoning

Monitor PesticidesMonitor Pesticides

EPA Approved Pesticides

EPA Approved Pesticides

No Approved Pesticides for Cann

No Approved Pesticides for Cann

•Pesticide use is generally overseen by the federal government.

•Individual states are now tasked with this burden◦ Little to no consistency between/among states

Background: Pesticide Regulation

•Hot Topic in the industry

•In general, • ~ 67 compounds

• One group conducted a cursory review and isolated 50—60 repeated pesticides

• Chemist in CA tested cannabis for 4 common pesticides (bifenthrin, diazinon, paclobutrazol, permethrin) and found nearly 70% residues were found in smoke.

• Health Canada – unexpectedly found pyrethrin, myclobutanil

42

3/9/2017

11

Background: Regulations?•Past two years:• OR – 2 recalls due to pesticide contaminated cannabis

• CA – established safety regulations to go into effect in 2018• CO – multiple recalls due to pesticide contamination

Effects of Pesticides on ConsumersMedicinal Population: ◦ Already immuno‐compromised

◦ Inhaling cannabis ‐> pesticides are directly deposited in lungs

◦ Oral cannabis ‐> organs will facilitate some pesticide decomposition.

43

3/9/2017

12

Background: Summary•Pesticides are clearly a problem• United States• International

•No federal oversight

•Little to no state oversight

•What little oversight that does exist, is not necessarily based on empirical science

Task?Advisory Panel Goals:◦ Assess: what is the industry seeing? What pesticides are most prominently used?

◦ Divide pesticides into meaningful groups or lists

◦ Identify those that are most commonly seen and most measurable

◦AOAC International to take a leadership position◦ Without federal regulation, states would benefit from a panel of relevant pesticide‐detection methods

44

3/9/2017

13

Proposed Fitness for Purpose

~ Standard Methods Performance Requirements (SMPRs) for quantitative methods to identify and quantify various

pesticide residues in dried cannabis materials ~

QUESTIONS & DISCUSSION

SUSAN AUDINO, PHDCONSULTANT

[email protected]

45

Lucie RACAULT, Thomas BESSAIRE, Aurélien DESMARCHELIER & Thierry DELATOUR*

Nestlé Research Centre, Lausanne, Switzerland

*Member of Chemical Contaminants and Residues in Food Community*Chair of Subgroup Environmental & Emerging Contaminants

March 13, 2017

AOAC Midyear Meeting, Rockville, MD, March 13‐17, 2017

Stakeholder Panel on Strategic Food Analytical Methods

Working Group Launch: Veterinary Drug Residues

j

Antimicrobial Resistance

“The overlap of critical lists for human and veterinary medicine can provide further information, allowing an appropriate balance to be struck between animal health

needs and public health considerations”

● Aminoglycosides

● Carbapenems & other penems

● Cephalosporins

● Cyclic esters

● Fluoro‐ and other quinolones

● Glycopeptides

● Glycylcyclines

● Lipopetides

● Macrolides & ketolides

● Monobactams

● Oxazolidinones

● Penicillins

● Polymyxins

● Rifamycins

● Drugs used solely to treat tuberculosisor other mycobacterial disease

List of antimicrobials of veterinary importance

46

Farm

Raw materialcollection center

Factory raw materialwarehouse

Factory lineFinished product

warehouse

Arrival at factory

RM specification

& CoA

External supplier

j

Quality Testing along the Supply Chain & Manufacturing

Farm

Raw materialcollection center

Factory raw materialwarehouse

Factory lineFinished product

warehouse

Arrival at factory

RM specification

& CoA

External supplier

Rapid methods for effective releaseConfirmatory methods

for full compliance

j

Quality Testing along the Supply Chain & Manufacturing

47

j

Liquid Chromatography-Mass Spectrometry

Search: March 2, 2017

… about

5’730’000hits!

Market and Specific Needs

Supply constraints

Operator skills

Specific regulation

Restricted importation

Corporate Requirements

Analytical Volume

Internal vs External Approach

Agricultural Services (Corporate and Zones)

Field information

Fraud scenarios

Training

Regulatory

Local regulation (e.g. EU, US, China etc…)

Codex

Early Warning/Chemical Contaminants Experts/Corporate

Quality

Assess likelihood of ocurrence

Anticipate and mitigate incidents

Alignment on Official National Control Plan

Alignment with authorities control plan in global monitoring program

Team of experts to define an integrated approach

Analytical Development

Agricultural

Early Warning

Local Needs

RegulatoryCorporate

Requirements

OfficialControl

Alert System

Internal Alerts System

Early Warning, Positive findings data capture system

External Alerts

Consumers, Suppliers, Contaminants network

j

Integrated Approach for Analytical Development

48

100 Analytes

50 Analytes

5 Analytes

j

How Wide the Multi-residue Method is?

100 Analytes

50 Analytes

5 Analytes

Antibiotics

Antiparasitics

Tranquilizers

Anti‐inflammatories

n = 102n = 102n = 102

n = 40n = 40n = 40

n = 5

n = 3

j

Bioactives

49

100 Analytes

50 Analytes

5 Analytes

Antibiotics

Tranquilizers


n = 102n = 102n = 102

n = 40

n = 5

n = 3

‐Lactams (23)Sulfonamides (21)Quinolones (18)Aminoglycosides (12)Tetracyclines (10)Macrolides (7)Amphenicols (3)Diaminopyrimidines (2)Lincosamides (2)Rifamycins (2)Aminocoumarins (1)Streptogramins (1)

Antiparasitics

j

Substances

100 Analytes

50 Analytes

5 Analytes

Antibiotics

Tranquilizers


n = 102

n = 40n = 40n = 40

n = 5

n = 3

Antiparasitics

Benzimidazoles (14)Coccidiostats (11)Avermectins (6)Salicylanilides (4)Halogenated phenols (1)Imidazothiazoles (1)Organophosphorous comp. (1)PraziquantelPyrantel

j

Substances

50

100 Analytes

50 Analytes

5 Analytes

Antibiotics

Antiparasitics

Tranquilizers


n = 102

n = 40

n = 5

n = 3

CarprofenDiclofenacFlunixinMeloxicamPhenylbutazone

j

Substances

100 Analytes

50 Analytes

5 Analytes

Antibiotics

Antiparasitics

Tranquilizers


n = 102

n = 40

n = 5

n = 3

CarazololChlorpromazineXylazine

j

Substances

51

j

Relevant Food Commodities

An approach including raw materials, semi‐finished and finished products

Meat/Seafood‐based productsMilk‐based products

• Raw milk

The «USUALLY‐SHOWN» matrices

• Fresh or cooked meat, fish and seafood

An approach including raw materials, semi‐finished and finished products

Meat/Seafood‐based productsMilk‐based products

Need for «Quick Easy Cheap Rugged and Safe» like methods

• Raw milk

The «USUALLY‐SHOWN» matrices

• Fresh or cooked meat, fish and seafood

• Babyfood in jars and pots (based on vegetables, meat/fish, pasta, cereals, vegetable oil etc..)

• Milk fractions(e.g. Skimmed milk powder, whey protein concentrate/hydrolysate, lactose etc...)

• Infant Cereals with milk

• Infant Cereals with meat tissues

• Meat, fish and seafood powder(e.g. Shrimp, duck, meat, pork, lamb, beef, chicken, veal etc...)

The «FORGOTTEN» matrices

• Formulae with milk(e.g. Infant, follow‐on, grow‐up formulae; hydrolysed formulae; adult formulae etc...)

j

Relevant Food Commodities

52

j

Beyond Raw Milk …

Whole milk powder

Skim milk powder

Vegetable oils

Soybean

Sugar

Cotton

Wheat

Beef

Other coarse grains

Maize

Poultry

Other oilseeds

Cheese

Butter

Rice

Pork

Biodiesel

Ethanol

Roots & tubers

0 10 20 30 40 50 60 70 Share of exports (%) in total production (2013‐2015)

Source: OECD/FAO, 2016, OECD‐FAO Agricultural Outlook 2016‐2025, OECD Publishing, Parishttp://dx.doi.org/10.1787/agr_outlook‐2016‐en

Distribution of animal drugs amongcurd, whey, and milk protein

fractions in spikedskim milk and whey

Shappell et al. 2017J. Agric. Food Chem., 65, 938‐949

245 articles in peer‐reviewed scientific journals (1997‐2016)

Publication year

Number of publications

2016

2011

2006

2001

199610 20 30 40

j

Literature Available for Veterinary Drugs by LC-MS(/MS)

53

j


1996

2001

2006

2011

2016

0 50 100 150 200

245 articles in peer‐reviewed scientific journals (1997‐2016)

Publication year

Number of analytes

1996

2001

2006

2011

2016

0 50 100 150 200

… only 15 methods capable to analyze 120 analytes and more

Publication year

Number of analytes

120 analytes

j

What About Fitness-for-Purpose?

54

j


1996

2001

2006

2011

2016

0 50 100 150 200

… and only 2 methods applicable to at least three matrices!

Publication year

Number of analytes

120 analytes

Chen et al., 2016, J. Chromatogr. B

LC‐QqQ – Egg, Milk, Muscle

Robert et al., 2013, Food Addit. Contam. A

LC‐QqQ – Egg, Honey, Milk, Muscle

j


245 articles in peer‐reviewed scientific journals

Period: 1997‐2016

55

j


245 articles in peer‐reviewed scientific journals

Period: 1997‐2016

177 methodsdescribed for

a single food matrix

j


Chen et al., 2016, J. Chromatogr. BLC‐QqQ – Egg, Milk, Muscle

Screening or quantitative?

A clear purpose accompanied with a set of comprehensive validation data is needed.

56

j


Robert et al., 2013, Food Addit. Contam. ALC‐QqQ – Egg, Honey, Milk, Muscle

Muscle

Milk

Egg

Honey

122

140

153 compounds

131

133 Validated compounds

Tetracycline epimers not included

j

Antimicrobial Agents of Veterinary Importance

AminoglycosidesAmphenicols‐LactamsMacrolidesQuinolonesSulfonamidesDiaminopyrimidinesTetracyclines

VCIACritically important

RifamycinsArsenical‐LactamsCoccidiostatsLincosamidesPhosphonic acidPleuromutilinsPolypeptides

VHIAHighly important

VIA

Important

84 27 8Number ofcompounds

Aminocoumarins, Quinoxalines, Strep

togram

ins…

May 2015

57

j

Antimicrobial Agents of Veterinary Importance

VCIACritically important

VHIAHighly important

VIA

Important

May 2015

Robert et al., 2013, Food Addit. Contam. ARobert et al., 2013, Food Addit. Contam. A

Our approachOur approach

VCIA VHIA

VIA

j

Regulatory Framework

EU, USA, Canada, China, Codex Alimentarius

58

with 198 compoundsregulated


j


j


with 198 compoundsregulated165

152685211


59

j

Regulation of -Lactams in Milk

Cefacetril

Cefalexin

Ceftiofur

Cefapirin 1

Cefazolin

Cefoperazone

Cloxacillin

Dicloxacilin

Nafcillin

Oxacillin

Cefalonium

Cefquinome

Amoxicillin

Ampicillin

Penicillin‐G

Cefadroxil

Cefuroxime

Penicillin‐V

Sulbactam

Tazobactam

Aspoxicillin

Piperacillin

25 50 75 100Banned

Regulatory limits (µg/kg)

1251 as a sum with desacetylcefapirin

Limits

European Union

j


Cefacetril

Cefalexin

Ceftiofur

Cefapirin 1

Cefazolin

Cefoperazone

Cloxacillin

Dicloxacilin

Nafcillin

Oxacillin

Cefalonium

Cefquinome

Amoxicillin

Ampicillin

Penicillin‐G

Cefadroxil

Cefuroxime

Penicillin‐V

Sulbactam

Tazobactam

Aspoxicillin

Piperacillin

25 50 75 100Banned


1251 as a sum with desacetylcefapirin

Limits

Screening Target Concentration

European Union

60

j


Cefacetril

Cefalexin

Ceftiofur

Cefapirin 1

Cefazolin

Cefoperazone

Cloxacillin

Dicloxacilin

Nafcillin

Oxacillin

Cefalonium

Cefquinome

Amoxicillin

Ampicillin

Penicillin‐G

Cefadroxil

Cefuroxime

Penicillin‐V

Sulbactam

Tazobactam

Aspoxicillin

Piperacillin

25 50 75 100Banned


1251 as a sum with desacetylcefapirin (EU)

Limits


European Union

United States of America

Canada

China

Codex Alimentarius

j

Regulation of Quinolones in Meat

Flumequine

Difloxacin

Danofloxacin

Marbofloxacin

Oxolinic acid

Ciprofloxacin 1

Enrofloxacin 1

Sarafloxacin

Cinoxacin

Enoxacin

Fleroxacin

Lomefloxacin

Nalidixic acid

Norfloxacin

Ofloxacin

Pefloxacin

Pipemidic acid

Piromidic acid

100 200 300 400Banned


5001 as a sum in the EU and China

Limits


European Union


Canada

China

Codex Alimentarius

61

j

Regulation of Benzimidazoles in Meat

Limits


European Union


Canada

China

Codex Alimentarius

Albendazole

Albendazole sulfone

Albendazole sulfoxide

Albendazole‐2‐aminosulfone

5‐Hydroxythiabendazole

Febantel

Febendazole

Flubendazole

Mebendazole

Netobimin

Oxfendazole

Oxibendazole

Thiabendazole

Triclabendazole

100 200 300 400Banned


500

Unspiked test portion

Water + 0.1% formic acid added to acetonitrile

Spiked test portion

Na2SO4 + NaCl + citrate salts added

Sample shaked and centrifuged

Clean‐up ‘Most‐of‐them’

6‐mL Supernatant collected

d‐SPE with Na2SO4/PSA/C18

Fraction evaporated to dryness

Residue reconstituted in methanol/water 15/85

Extract analyzed by LC‐MS/MS Run A

Clean‐up ‘Avermectins’

8‐mL Supernatant collected

d‐SPE with MgSO4/PSA/C18

Fraction evaporated to dryness

Residue reconstituted in methanol/water 80/20

Extract analyzed by LC‐MS/MS Run B

j

Stream ‘Multi-class’ Procedure

62

j

Quinolones in Beef Powder Extract at 1x STC

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

2.7e6

Inte

nsi

ty, c

ps

12

3

4,5,6

78

9

10

11

12

13

14

15

16

17

18

N° Compound Name STC µg/kg

1 Pipemidic Acid 10

2 Marbofloxacin 10

3 Fleroxacin 10

4 Ofloxacin 10

5 Pefloxacin 10

6 Enoxacin 10

7 Norfloxacin 10

8 Ciprofloxacin 10

9 Enrofloxacin 10

10 Danofloxacin 10

11 Lomefloxacin 10

12 Difloxacin 10

13 Sarafloxacin 10

14 Cinoxacin 10

15 Oxolinic acid 10

16 Nalidixic acid 10

17 Flumequine 10

18 Piromidic acid 10

j

Benzimidazoles in Chicken Fat Extract at 1x STC

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0Time, min

1.5e6

Inte

nsi

ty, c

ps

1

2

3

45

6

7

8

9

10

11

12

13

14


1 5‐Hydroxythiabendazole 10

2 Albendazole‐2‐amino sulfone 10

3 Thiabendazole 10

4 Albendazole sulfoxide 10

5 Albendazole Sulfone 10

6 Netobimin 10

7 Oxibendazole 10

8 Oxfendazole 10

9 Mebendazole 10

10 Albendazole 10

11 Flubendazole 10

12 Fenbendazole 10

13 Febantel 10

14 Triclabendazole 10

63

j

Avermectins in Salmon Extract at 1x STC

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0Time, min

9.0e4

Inte

nsi

ty, c

ps

1

2

3

4

5

6

j

Chloramphenicol at 1x STC

4.00 4.10 4.20 4.30 4.40 4.50 4.60 4.70 4.80 4.90Time, min

6000

Inte

nsi

ty, c

ps

1

2

3

4

5

64

j

Stream ‘-Lactams’ Procedure


Phosphate buffer added to acetonitrile

Spiked test portion

Na2SO4 + NaCl added


d‐SPE with Na2SO4/C18

Fraction evaporated down to 500 µL

Extract analyzed by LC‐MS/MS

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5Time, min

3.4e5

Inte

nsi

ty, c

ps

1

2,3

4,5

6,7

8

9

10

11

12,13,14

15

16

17

18

19,20

2122,23


1 Desacetylcefapirin 20

2 Amoxicillin 4

3 Tazobactam 25

4 Cefadroxil 50

5 Sulbactam 25

6 Aspoxicillin 25

7 Cefapirin 10

8 Cefquinome 10

9 Cefacetril 50

10 Cefalonium 10

11 Cefalexin 50

12 Ampicillin 4

13 Cefuroxime 50

14 Cefazolin 50

15 Cefoperazone 50

16 Ceftiofur 50

17 Piperacillin 10

18 Penicillin‐G 4

19 Oxacillin 10

20 Penicillin‐V 10

21 Cloxacillin 10

22 Dicloxacillin 10

23 Nafcillin 5

j

-Lactams in Skimmed Milk Powder at 1x STC

65

j

Stream ‘Tetracyclines’ Procedure


EDTA 0.1 M in water + acetonitrile added to the test portion

Spiked test portion

Sample stored at ‐ 20ᵒC for 1 hour


Acetonitrile supernatant defatted with hexane

Sample evaporated to dryness


Residue reconstituted in aqueous oxalic acid/methanol 70/30

j

Tetracyclines in Meat-based Babyfood Extract at 1x STC

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5Time, min

7.9e5

Inte

nsi

ty, c

ps

1 1’

2

2’

3

3’

4

4’

5

5’

66

j

Stream ‘Aminoglycosides’ Procedure


EDTA 0.5% + TCA 2% in water added to the test portion

Spiked test portion

Supernatant 1+1 diluted with ammonium carbonate buffer

Clean‐up with Molecularly Imprinted Polymer (MIP) SPE


Compounds eluted with 30 mM HFBA in acetonitrile/water 25/75


j

Aminoglycosides in Egg Powder Extract at 1x STC

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5Time, min

2.5e4

Inte

nsi

ty, c

ps

1

2

3

4

6

5

7, 8

9

10, 11

14

12, 13

67

j

Monitoring Data for Veterinary Drug Residues

A total of fifteen confirmed positive milksamples were identified out of the 1’912total samples.

0.78%

In 2012, there were just over 1’000 non‐compliant samples from over 425’000total samples.

0.24%

j

Method Validation …

… based on ‘Guidelines for the validation of screening methods for residuesof veterinary medicines’ ‐ Community Reference Laboratories (CRLs)20/1/2010.

Aim: Check if samples are above or below an established threshold, called

the Screening Target Concentration (STC).

Results: Expressed either as < STC or Suspect.

Response: Relative comparison of the peak areas between the unspiked and the

spiked samples.

●

●

●

Samples

Milk‐based ingred./prod.

Meat/seafood ingred./prod.

● Milk fractions (n=16)

● Infant formulae & powders (n=15)

● Infant cereals (n=5)

● Meat/seafood powders (n=10)

● Fresh/cookedmeat/seafood (n=10)

● Meat‐based babyfoods (n=10)

Design

Fortified at 0, 1x & 2x STC

Three analysts involved

Over a period of 15 days

Criteria

Cut‐off level

False suspect rate < 10%

False negative rate < 5%

Retention time < 0.2 min

Identification with 2 transitions

68

j

Cut-off Level

0%

20%

40%

60%

80%

100%

0 10 20 30 40 50 60 70

Response

(in %)

Number of Samples (n=67)

Response in Blank Samples Response in Spiked Samples (1 STC)

Cut‐off at 20%

Data collected for albendazole

Cergy, Vittel (France)

Nunspeet (The Netherlands)

Orbe (Switzerland)

Padova (Italy)

Rzeszow (Poland)

Weiding (Germany)

York (United Kingdom)

Shoham

Zhukovsky

ClayvilleMulgrave

Tianjin

CabuyaoDubai

Moga

Navanakorn

Shah Alam

Singapore

Abidjan

Santiago São Paulo

Antigua

Toluca

St LouisDublin

j

Quality Control in the Operations

69

Take-Home Message

Uncontrolled occurrence of veterinary drugs in food is a health concern, particularlywith regard to antimicrobial resistance.

Multiresidue analysis is needed for an effective control.

Mass spectrometry is needed for full compliance testing.

A single LC‐MS‐based method capable to demonstrate full compliance of veterinarydrugs in food does not exist so far.

Matrix scope should represent current practices in terms of trade and business.

Method performance should fit with throughput and postive rate for a as‐low‐cost‐as‐possible analysis.

►

►

►

►

►

►

Thanks for your attention!

Thierry DELATOURNestlé Research CentreVers‐chez‐les‐Blanc1000 Lausanne 26, Switzerland

Phone: +41.21.785.9220e‐Mail: [email protected]

70

DR. JOE BOISON

CHAIR

Veterinary Drugs ‐ Uses Are applied or administered to any food‐producing animal for

therapeutic,

prophylactic or diagnostic purposes,

Sometimes for modification of physiological functions or behavior.

Veterinary drugs can be used as

antimicrobials

antiparasitic agents

71

Veterinary Drug Use in Food Animal Production The incorrect use of veterinary drugs in animal production has led to residues in various edible animal tissues, and microbial resistance is now pointed as major concern for public health.

Organizations such as Codex Alimentarius, European Commission or the US Food & Drug Administration have established maximum residue limits (MRLs)/tolerances for many veterinary medicinal products in foodstuffs from animal origin, while

banned/prohibited from use in food animal production.

Need for Methods Therefore, the need to have methods capable to screen and confirm the possible presence of a broad range of substances for veterinary use in food becomes of paramount importance to ensure that regulatory requirements are met, and ultimately to protect consumers and facilitate global trade.

72

Proposal

Create an AOAC working group on veterinary drugs under the Stakeholder Panel on Strategic Food Analytical Methods (SPSFAM).

Develop a series of SMPRs for LC‐MS/MS methods for screening and confirmation of one hundred and fifty one (151) veterinary drug residues

Proposal

One hundred and fifty one (151) veterinary drug residues including amphenicols, aminoglycosides, avermectins, ‐lactams, coccidiostats, macrolides, quinolones, sulfonamides, and tetracylines,

73

Proposal

in raw materials (milk, meat, fish, seafood and egg),

semi‐finished ‐ skimmed milk powder,

whey protein concentrate/hydrolysate,

lactose,

meat powder and related materials and

finished products infant formula,

milk‐ and meat‐based infant cereals,

baby food

Project outcome Review submitted LC‐MS/MS methods for consideration as AOAC INTERNATIONAL Official Methods of Analysis (OMA).

74

Project Goals

A series of SMPRs that will provide acceptance requirements for methods capable of accurately and reliably measuring a wide variety of veterinary drugs in selected matrices.

Four or more Official Methods of Analysis that comply with the SMPRs.

Time Table Summer 2017: AOAC will identify renowned subject matter experts to participate in the working group and a working group chair.

September 2017: The working group chair will present a ‘launch presentation’ to the stakeholders.

October 2017 to February 2018: The working group will meet by teleconferences to draft SMPRs.

75

Time Table March 2018: The working group chair will present draft SMPRs for approval by SPSFAM.

April 2018 – August 2018: The working group will meet by teleconferences to draft SMPRs.

September 2018: The working group chair will present draft SMPRs for approval by SPSFAM.

Call for Methods and Call for Experts.

AOAC Expert Review Panel to review methods.

Benefits to You

If you are a method developer, SMPRs will provide a standard for veterinary drug methods that you can use to develop methods that will be used by AOAC Expert Review Panels to evaluate candidate methods.

If you are a food manufacturer or food distributor, you can participate in development of standards for veterinary drug residue methods. These standards will then be used by method developers to develop methods, and by AOAC for method evaluation so that OMA adopted methods best meet your analytical needs.

76

AOAC Expectations

AOAC has estimated that this project will take a year to develop SMPRs for methods to measure 151 different veterinary drugs in selected food ingredient and food matrices.

AOAC is seeking partners to co‐fund the project.

Nestlé has provided seed funding for the project to initiate the working group for $60,000, which will fund half of the project. AOAC is looking for 6 or more additional Organizational Affiliate funders to contribute $10K to form an SPSFAM working group to develop SMPRs on veterinary drugs in selected food ingredient and food matrixes, to start at the AOAC 2017 Annual Meeting in Atlanta, Georgia, USA.

AOAC Expectations

If you are not currently an Organizational Affiliate – Support the SPSFAM working group by becoming an Organizational Affiliate(OA). As an OA, your contribution and participation provides you with a voice in the sound, science‐based solution to restoring public trust in a company’s products and controlling risk exposure to a company’s brand and business.

If you are already an OA ‐ contribute $10K to develop SMPRS for the veterinary drugs in selected food ingredient and food matrixes.

As a contributor you would have a standing working group of experts that would develop standards as needed and as a contributing member, you would have a voice in the prioritization of standards to be developed.

77

Time frame

The deadline for this initial contribution of $10K is May 31, 2017.

If you are ready to contribute and be a part of this initiative, see DAWN after/during the meeting.

78

DRAFT AOAC SMPR 2017.XXX; Version 12; March 13, 2017 1 2 Method Name: Quantitation of cannabinoids in dried plant materials. 3

4 Intended Use: Consensus-based Reference method. 5

6 1. Purpose: AOAC SMPRs describe the minimum recommended performance characteristics to be used 7

during the evaluation of a method. The evaluation may be an on-site verification, a single-laboratory 8 validation, or a multi-site collaborative study. SMPRs are written and adopted by AOAC Stakeholder 9 Panels composed of representatives from the industry, regulatory organizations, contract 10 laboratories, test kit manufacturers, and academic institutions. AOAC SMPRs are used by AOAC Expert 11 Review Panels in their evaluation of validation study data for method being considered for 12 Performance Tested Methods or AOAC Official Methods of Analysis, and can be used as acceptance 13 criteria for verification at user laboratories. 14

15 2. Applicability: 16

The method will be able to identify, and quantify each individual cannabinoids (as listed in Table 1a 17 and Table 1b) in dried plant materials. 18

19 3. Analytical Technique: 20

Any analytical technique(s) that measures the analytes of interest and meets the following method 21 performance requirements is/are acceptable. 22

23 4. Definitions: 24

25 Limit of Quantitation (LOQ) 26 The minimum concentration or mass of analyte in a given matrix that can be reported as a 27 quantitative result. 28

29 Quantitative method 30 Method of analysis which response is the amount of the analyte measured either directly 31 (enumeration in a mass or a volume), or indirectly (color, absorbance, impedance, etc.) in a certain 32 amount of sample. 33

34 Repeatability 35 Variation arising when all efforts are made to keep conditions constant by using the same 36 instrument and operator and repeating during a short time period. Expressed as the repeatability 37 standard deviation (SDr); or % repeatability relative standard deviation (%RSDr). 38

39 40

79

Reproducibility 41 The standard deviation or relative standard deviation calculated from among-laboratory data. 42 Expressed as the reproducibility standard deviation (SDR); or % reproducibility relative standard 43 deviation (% RSDR). 44

45 Recovery 46 The fraction or percentage of spiked analyte that is recovered when the test sample is analyzed 47 using the entire method. 48 49 Dried Plant Materials 50 Dried whole or milled flower plant material from Cannabis sativa and its hybrids. 51

52 5. Method Performance Requirements: 53

54 See table 2 and 3. 55

56 6. System suitability tests and/or analytical quality control: 57

Suitable methods will include blank check samples, and check standards at the lowest point and 58 midrange point of the analytical range. 59

60 7. Reference Material(s): 61 62

See tables 1A and 1B for sources of reference materials. 63 64

Refer to Annex F: Development and Use of In-House Reference Materials in Appendix F: Guidelines 65 for Standard Method Performance Requirements, 19th Edition of the AOAC INTERNATIONAL Official 66 Methods of Analysis (2012). Available at: http://www.eoma.aoac.org/app_f.pdf 67 68

8. Validation Guidance: 69 70 Method performance should be demonstrated with homogeneous samples. Inherent variation in 71 the plant may preclude or limit homogeneity for the following reasons: (a) they are resinous, 72 cannabinoids are concentrated in the resin, which can clump during grinding; (b) between flower 73 variation can be high, grinding multiple flowers can impact the homogeneity; (c) grinding can 74 introduce heat, which will cause degradation of cannabidiolic acids into neutral forms, resulting in 75 less accurate results. Grinding would be the best option for homogeneous samples, but in some 76 cases there are issues with clumped resin, highly variable samples and additional grinding would 77 impact the results and lead to inaccurate data. 78 79 Appendix D: Guidelines for Collaborative Study Procedures To Validate Characteristics of a Method 80 of Analysis; 19th Edition of the AOAC INTERNATIONAL Official Methods of Analysis (2012). Available 81 at: http://www.eoma.aoac.org/app_d.pdf 82 83 Appendix F: Guidelines for Standard Method Performance Requirements; 19th Edition of the AOAC 84 INTERNATIONAL Official Methods of Analysis (2012). Available at: 85 http://www.eoma.aoac.org/app_f.pdf 86 87 Appendix K: Guidelines for Dietary Supplements and Botanicals; 19th Edition of the AOAC 88 INTERNATIONAL Official Methods of Analysis (2012). Available on line at: 89 http://www.eoma.aoac.org/app_k.pdf 90 91

80

http://www.eoma.aoac.org/app_f.pdf


http://www.eoma.aoac.org/app_d.pdf


http://www.eoma.aoac.org/app_k.pdf


92 9. Maximum Time-To-Result: None 93 94 95

81

Table 1A: Required Cannabinoids 96 Common Name

Abbrev-iation

IUPAC Name CAS Number

Molecular Structure

Reference Material

Cannabidiol CBD 2-[(1R,6R)-6-isopropenyl-3-methylcyclohex-2-en-1-yl]-5-pentylbenzene-1,3-diol

13956-29-1

Restek Cerilliant Sigma-Aldrich API Standards Echo Pharm Lipomed AG

Cannabidiolic Acid

CBDA 2,4-dihydroxy-3-[(1R,6R)-3-methyl-6-prop-1-en-2-ylcyclohex-2-en-1-yl]-6-pentylbenzoic acid [SGC: name corrected]

1244-58-2

Cerilliant USP Restek Lipomed AG Echo Pharmaceutical

Cannabinol CBN 6,6,9-Trimethyl-3-pentyl-benzo[c]chromen-1-ol

521-35-7

Cerilliant Restek

Tetrahydro-cannabinol

THC (−)-(6aR,10aR)-6,6,9-Trimethyl-3-pentyl-6a,7,8,10a-tetrahydro-6H-benzo[c]chromen-1-ol

1972-08-3

Cerilliant USP Echo Pharmaceuticals

Tetrahydro-cannabinolic acid

THCA (6aR,10aR)-1-hydroxy-6,6,9-trimethyl-3-pentyl-6a,7,8,10a-tetrahydro-6h-benzo[c]chromene-2-carboxylic acid

23978-85-0


97 98 Table 1B: Additional, Desirable Cannabinoids 99

Name Abbreviation

IUPAC Name CAS Number Molecular Structure Reference Material

Cannabichromene CBC 2-Methyl-2-(4-methylpent-3-enyl)-7-pentyl-5-chromenol

20675-51-8

Cerilliant Sigma Aldrich Echo Pharmaceuticals

Cannabichromenicacid

CBCA 5-Hydroxy-2-methyl-2-(4-methyl-3-penten-1-yl)-7-pentyl-2H-chromene-6-carboxylic acid

20408-52-0

no reference material

Cannabidivarinic acid

CBDVA 2,4-dihydroxy-3-[(1R,6R)-3-methyl-6-prop-1-en-2-ylcyclohex-2-en-1-yl]-6-propylbenzoic acid

31932-13-5

Cerilliant

Cannabigerol CBG 2-[(2E)-3,7-dimethylocta-2,6-dienyl]-5-pentyl-benzene-1,3-diol

25654-31-3

Cerilliant Lipomed AG

82

NIST: 1,3-Benzenediol, 2-(3,7-dimethyl-2,6-octadienyl)-5-pentyl-

NIST: 2808-33-5

Echo Pharmaceuticals SPEX Certiprep Tocris (UK)

Cannabigerolic - acid

CBGA 3-[(2E)-3,7-dimethylocta-2,6-dienyl]-2,4-dihydroxy-6-pentylbenzoic acid

25555-57-1

Cerilliant Echo Pharmaceuticals SPEX Certiprep

Cannabidivarin CBDV 2-((1S,6S)-3-methyl-6-(prop-1-en-2-yl) cyclohex-2-enyl)-5-propylbenzene-1,3-diol

24274-48-4

Cerilliant SPEX Certiprep

Δ8 Tetrahydro-cannabinol

Δ8 THC 6,6,9-trimethyl-3-pentyl-6a,7,10,10a-tetrahydrobenzo[c]chromen-1-ol

5957-75-5


Tetrahydro-cannabivarin

THCV 6,6,9-Trimethyl-3-propyl-6a,7,8,10a-tetrahydro-6H-benzo[c]chromen-1-ol

28172-17-0

Cerilliant USP

Tetrahydrocannabivarin - acid

THCVA 28172-17-0

No reference material

100 101 102

Table 2: Method performance requirements (part 1) for cannabinoids in Table 1b. 103

Parameter Requirement


Analytical Range (%) 0.1 – ca. 50*

*Lower concentrations may be acceptable as applicable for cannabinoids listed in Table 1B.

104 105 106

Table 3: Method performance requirements (part 2) for cannabinoids in Table 1b. Parameters Ranges (%)

83

0.1 – 1 > 1 - 25 > 25 – ca. 50

Recovery (%) 95 – 105 97 - 103 98 - 102

% RSDr ≤ 5 ≤ 4 ≤ 2

% RSDR ≤ 7 ≤ 5 ≤ 3

84

DRAFT AOAC SMPR 2016.XXX; Version 3; March 13, 2017 1 2 Method Name: Quantitation of cannabinoids in cannabis concentrates 3 4 Intended Use: Consensus-based reference method. 5 6 1. Purpose: AOAC SMPRs describe the minimum recommended performance characteristics to be used 7



The method will be able to identify, and quantify individual cannabinoids (as listed in Table 1a and 17 Table 1b) present in cannabis concentrates. 18 19

3. Analytical Technique: 20 Any analytical technique(s) that measures the analytes of interest and meets the following method 21 performance requirements is/are acceptable. 22


25 Cannabis Concentrates 26 A product resulting from chemical or physical processing of cannabis sativa or any of its hybrids, 27 largely free of solvents with cannabinoid content higher than the starting material. 28 29 Limit of Quantitation (LOQ) 30 The minimum concentration or mass of analyte in a given matrix that can be reported as a 31 quantitative result. 32 33

Quantitative method 34 Method of analysis which response is the amount of the analyte measured either directly 35 (enumeration in a mass or a volume), or indirectly (color, absorbance, impedance, etc.) in a certain 36 amount of sample. 37

38 Repeatability 39 Variation arising when all efforts are made to keep conditions constant by using the same 40 instrument and operator and repeating during a short time period. Expressed as the repeatability 41 standard deviation (SDr); or % repeatability relative standard deviation (%RSDr). 42 43

44

85

Reproducibility 45 The standard deviation or relative standard deviation calculated from among-laboratory data. 46 Expressed as the reproducibility standard deviation (SDR); or % reproducibility relative standard 47 deviation (% RSDR). 48

49 Recovery 50 The fraction or percentage of spiked analyte that is recovered when the test sample is analyzed 51 using the entire method. 52 53

5. Method Performance Requirements: 54 55 See table 2 and 3. 56



61 7. Reference Material(s): 62 63

See tables 1A and 1B for sources of reference materials. 64 65 Refer to Annex F: Development and Use of In-House Reference Materials in Appendix F: Guidelines 66 for Standard Method Performance Requirements, 19th Edition of the AOAC INTERNATIONAL Official 67 Methods of Analysis (2012). Available at: http://www.eoma.aoac.org/app_f.pdf 68 69

8. Validation Guidance: 70 71 Appendix D: Guidelines for Collaborative Study Procedures To Validate Characteristics of a Method 72 of Analysis; 19th Edition of the AOAC INTERNATIONAL Official Methods of Analysis (2012). Available 73 at: http://www.eoma.aoac.org/app_d.pdf 74 75 Appendix F: Guidelines for Standard Method Performance Requirements; 19th Edition of the AOAC 76 INTERNATIONAL Official Methods of Analysis (2012). Available at: 77 http://www.eoma.aoac.org/app_f.pdf 78 79 Appendix K: Guidelines for Dietary Supplements and Botanicals; 19th Edition of the AOAC 80 INTERNATIONAL Official Methods of Analysis (2012). Available on line at: 81 http://www.eoma.aoac.org/app_k.pdf 82 83 84

9. Maximum Time-To-Result: None 85 86 Table 1A: Required Cannabinoids 87

Common Name

Abbrev-iation

IUPAC Name CAS Number

Molecular Structure

Reference Material

Cannabidiol CBD 2-[(1R,6R)-6-isopropenyl-3-methylcyclohex-2-en-1-yl]-5-pentylbenzene-1,3-diol

13956-29-1

Restek Cerilliant Sigma-Aldrich API Standards Echo Pharm Lipomed AG

86



http://www.eoma.aoac.org/app_d.pdf




Cannabidiolic Acid

CBDA 2,4-dihydroxy-3-[(1R,6R)-3-methyl-6-prop-1-en-2-ylcyclohex-2-en-1-yl]-6-pentylbenzoic acid [SGC: name corrected]

1244-58-2

Cerilliant USP Restek Lipomed AG Echo Pharmaceutical

Cannabinol CBN 6,6,9-Trimethyl-3-pentyl-benzo[c]chromen-1-ol

521-35-7

Cerilliant Restek

Tetrahydro-cannabinol

THC (−)-(6aR,10aR)-6,6,9-Trimethyl-3-pentyl-6a,7,8,10a-tetrahydro-6H-benzo[c]chromen-1-ol

1972-08-3


Tetrahydro-cannabinolic acid

THCA (6aR,10aR)-1-hydroxy-6,6,9-trimethyl-3-pentyl-6a,7,8,10a-tetrahydro-6h-benzo[c]chromene-2-carboxylic acid

23978-85-0


88 89 Table 1B: Additional, Desirable Cannabinoids 90

Name Abbreviation

IUPAC Name CAS Number Molecular Structure Reference Material

Cannabichromene CBC 2-Methyl-2-(4-methylpent-3-enyl)-7-pentyl-5-chromenol

20675-51-8

Cerilliant Sigma Aldrich Echo Pharmaceuticals

Cannabichromenicacid

CBCA 5-Hydroxy-2-methyl-2-(4-methyl-3-penten-1-yl)-7-pentyl-2H-chromene-6-carboxylic acid

20408-52-0

no reference material

Cannabidivarinic acid

CBDVA 2,4-dihydroxy-3-[(1R,6R)-3-methyl-6-prop-1-en-2-ylcyclohex-2-en-1-yl]-6-propylbenzoic acid

31932-13-5

Cerilliant

Cannabigerol CBG 2-[(2E)-3,7-dimethylocta-2,6-dienyl]-5-pentyl-benzene-1,3-diol

NIST: 1,3-Benzenediol, 2-(3,7-dimethyl-2,6-octadienyl)-5-pentyl-

25654-31-3 NIST: 2808-33-5

Cerilliant Lipomed AG Echo Pharmaceuticals SPEX Certiprep Tocris (UK)

87

Cannabigerolic - acid

CBGA 3-[(2E)-3,7-dimethylocta-2,6-dienyl]-2,4-dihydroxy-6-pentylbenzoic acid

25555-57-1

Cerilliant Echo Pharmaceuticals SPEX Certiprep

Cannabidivarin CBDV 2-((1S,6S)-3-methyl-6-(prop-1-en-2-yl) cyclohex-2-enyl)-5-propylbenzene-1,3-diol

24274-48-4


Δ8 Tetrahydro-cannabinol

Δ8 THC 6,6,9-trimethyl-3-pentyl-6a,7,10,10a-tetrahydrobenzo[c]chromen-1-ol

5957-75-5


Tetrahydro-cannabivarin

THCV 6,6,9-Trimethyl-3-propyl-6a,7,8,10a-tetrahydro-6H-benzo[c]chromen-1-ol

28172-17-0

Cerilliant USP

Tetrahydrocannabivarin - acid

THCVA 28172-17-0

No reference material

91 92

88

93 Table 2: Method performance requirements (part 1). 94


THC, THCA, CBDA, CBD, Individually Reported

Requirement (Additional, Desirable Cannabinoids (Table 1b), including

CBN)

Limit of Quantitation (LOQ) (% w/w) ≤ 0.3 ≤ 0.3

Analytical Range (% w/w) ≤ 0.3 – ca. 100 ≤ 0.3 – ca. 50

*Reported as individual cannabinoids 95 96 97 98 99 100 101 102 103 104 105 106

Table 3: Method performance requirements (part 2).

Parameters Ranges (% w/w)

≤ 0.3 – 1 > 1 - 10 > 10 – ca. 100

Recovery (% w/w) 95 – 105 97 - 103 98 - 102

% RSDr ≤ 5 ≤ 4 ≤ 2

% RSDR ≤ 7 ≤ 5 ≤ 3

89

DRAFT AOAC SMPR 2017.XXX; Version 5; 13 March 2017

Method Name: Identification of Type-A Proanthocyanidins in Cranberry -Based Foods and Dietary Supplements

Intended Use: Consensus-based reference method.

1. Purpose: AOAC SMPRs describe the minimum recommended performance characteristics tobe used during the evaluation of a method. The evaluation may be an on-site verification, asingle-laboratory validation, or a multi-site collaborative study. SMPRs are written andadopted by AOAC Stakeholder Panels composed of representatives from the industry,regulatory organizations, contract laboratories, test kit manufacturers, and academicinstitutions. AOAC SMPRs are used by AOAC Expert Review Panels in their evaluation ofvalidation study data for method being considered for Performance Tested Methods or AOACOfficial Methods of Analysis, and can be used as acceptance criteria for verification at userlaboratories.

2. Applicability:The method will be able to identify the presence of Type-A proanthocyanidin in cranberry(Vaccinium macrocarpon) in one or more of the following: fruit, juice, beverage, driedcranberry fruit, cranberry sauce, ingredients (concentrates, extracts, powders, andpresscake); or dietary supplements (listed in table 2).

3. Analytical Technique:Any analytical technique that detects the analytes of interest and meets the methodperformance requirements is acceptable.

4. Definitions:

Dietary supplementsA product intended for ingestion that contains a "dietary ingredient" intended to addfurther nutritional value to (supplement) the diet. Dietary supplements may be found inmany forms such as tablets, capsules, softgels, gelcaps, liquids, or powders.

IdentificationIdentification is the characterization of the substance being analyzed, including its chemical,mineral, or biological classification, as applicable. In many investigations the identity of theanalyte is assumed and the correctness of the assumption is merely confirmed.

90

5. Method Performance Requirements:

Table 1: Method Performance Requirements

Selectivity Study 90% probability of identification with 95% confidence (33 correct identifications out of 33 samples known to contain Type-A proanthocyanidin).*

*Some aberrations may be acceptable if the aberrations are investigated, and acceptable explanations can be determined and communicated to method users.

6. System suitability tests and/or analytical quality control:

Suitable methods will include blank check samples, and check standards at the lowest point (to be revised)

7. Reference Material(s):

SRM 3281 Cranberry (Fruit)* SRM 3282 Low Calorie Cranberry Juice Cocktail* SRM 3283 Cranberry Extract* SRM 3284 Cranberry-Containing Solid Oral Dosage Form* *Characterized for organic acids, not proanthocyanidins, but provides a standard, homogeneous material. Please contact Dr. Catherine Rimmer, Research Chemist, NIST, for materials. [email protected], (301) 975-3651. Refer to Annex F: Development and Use of In-House Reference Materials in Appendix F: Guidelines for Standard Method Performance Requirements, 19th Edition of the AOAC INTERNATIONAL Official Methods of Analysis (2012). Available at: http://www.eoma.aoac.org/app_f.pdf

8. Validation Guidance: Information on analytical performance for all claimed matrixes must be submitted. Method developers should evaluate at least 33 samples known to contain Type-A proathocyanidin and at least 33 samples that contain non Type-A proanthocyanidin. Validation data must include examples of non Type-A matrices listed in tier 1 of table 3. Additional non Type-A matrices are listed in tier 2 of table 3. Validation test samples should be blind coded, and randomly mixed with respect to presence or absence of Type-A proanthocyanadin. Appendix D: Guidelines for Collaborative Study Procedures To Validate Characteristics of a Method of Analysis; 19th Edition of the AOAC INTERNATIONAL Official Methods of Analysis (2012). Available at: http://www.eoma.aoac.org/app_d.pdf Appendix F: Guidelines for Standard Method Performance Requirements; 19th Edition of the AOAC INTERNATIONAL Official Methods of Analysis (2012). Available at: http://www.eoma.aoac.org/app_f.pdf

91

Appendix K: Guidelines for Dietary Supplements and Botanicals; 19th Edition of the AOAC INTERNATIONAL Official Methods of Analysis (2012). Available on line at: http://www.eoma.aoac.org/app_k.pdf

9. Maximum Time-To-Result: None Table 2: Examples of Dietary Supplements capsules (containing dried cranberry fruit powder, dry extract) extracts gummies liquids powders softgel capsules (oil &/or water-based) tablets (containing dried cranberry fruit powder, dry extract) tinctures

92

Table 3: Sources of Non Type-A Proanthocyanidins

Tier 1 (required)

Apple (Malus domestica Borkh.) Grape skins, grapeseed extract (Vitis vinifera L.) Black chokeberry (Aronia melanocarpa (Michx.) Elliott) or Purple chokeberry (Aronia arbutifolia (L.) Pers.)

Tier 2 (additional)

Ginkgo biloba L. Hawthorn (Crataegus laevigata (Poir.) DC., Crataegus monogyna Jacq.) Dragon’s blood (Croton lechleri Müll.Arg.) Japanese horse chestnut (Aesculus turbinata Blume) Pine bark (Pinus sylvestris L., Pinus pinaster Aiton) Plum (Prunus domestica L.) Other Vaccinium species: huckleberry (V. ovatum Pursh), highbush blueberry (V. corymbosum

L.), lowbush blueberry (V. angustifolium Aiton), lingonberry (V. vitis-idaea L.), European cranberry (V. (Turcz. ex Rupr.) Schmalh.)

Cocoa (Theobroma cacao L.) Barley (Hordeum vulgare L.) Sorghum (Sorghum bicolor (L.) Moench) Blackcurrant (Ribes nigrum L.) Gooseberry (Ribes uva-crispa L.) Common bean (Phaseolus vulgaris L.) Hazelnut (Corylus avellana L.) Pecan (Carya illinoinensis (Wangenh.) K.Koch) Pistachio (Pistacia vera L.)

93

DRAFT AOAC SMPR 2017.XXX; Version 5; March 13, 2017 12

Method Name: Quantitation of proanthocyanidin content in cranberry fruit, 3 juice, beverage, dried cranberry, cranberry sauce, ingredients 4 (concentrates, extracts and powders) and dietary supplement 5 formulations. 6

7Intended Use: Reference method for cGMP compliance. 8

91. Purpose: AOAC SMPRs describe the minimum recommended performance characteristics to be used 10



The method will be able to quantify total proanthocyanidin content as the sum of all extractable 20 oligomers (> DP2) and polymers present in cranberry (Vaccinium macrocarpon) in one or more of 21 the following: fruit, juice, beverage, dried cranberry fruit, cranberry sauce, ingredients 22 (concentrates, extracts, powders, and presscake) or dietary supplements (Table 3). 23

24 3. Analytical Technique: 25

Any analytical technique(s) that measures the analytes of interest and meets the following method 26 performance requirements is/are acceptable. 27


30 Cranberry Proanthocyanadins 31 A mixture of oligomeric and polymeric flavan-3-ols, primarily epicatechin and catechin, of the A and 32 B type. 33

34 Dietary Ingredients 35 A vitamin; a mineral; an herb or other botanical; an amino acid; a dietary substance for use by man 36 to supplement the diet by increasing total dietary intake; or a concentrate, metabolite, constituent, 37 extract, or combination of any of the above dietary ingredients.1 38

39 Dietary supplements 40 A product intended for ingestion that contains a "dietary ingredient" intended to add further 41 nutritional value to (supplement) the diet. Dietary supplements may be found in many forms such as 42 tablets, capsules, softgels, gelcaps, liquids, or powders. 43

44 Limit of Quantitation (LOQ) 45

1 Federal Food Drug and Cosmetic Act §201(ff) [U.S.C. 321 (ff)

94

The minimum concentration or mass of analyte in a given matrix that can be reported as a 46 quantitative result. 47

48 49

Quantitative method 50 Method of analysis which response is the amount of the analyte measured either directly 51 (enumeration in a mass or a volume), or indirectly (color, absorbance, impedance, etc.) in a certain 52 amount of sample. 53

54 Repeatability 55 Variation arising when all efforts are made to keep conditions constant by using the same 56 instrument and operator and repeating during a short time period. Expressed as the repeatability 57 standard deviation (SDr); or % repeatability relative standard deviation (%RSDr). 58

59 Reproducibility 60 The standard deviation or relative standard deviation calculated from among-laboratory data. 61 Expressed as the reproducibility standard deviation (SDR); or % reproducibility relative standard 62 deviation (% RSDR). 63

64 Recovery 65 The fraction or percentage of spiked analyte that is recovered when the test sample is analyzed 66 using the entire method. 67

68 69

5. Method Performance Requirements: 70 See table 1 and 2. 71



76 7. Reference Material(s): 77

78 Refer to Annex F: Development and Use of In-House Reference Materials in Appendix F: Guidelines 79 for Standard Method Performance Requirements, 19th Edition of the AOAC INTERNATIONAL Official 80 Methods of Analysis (2012). Available at: http://www.eoma.aoac.org/app_f.pdf 81

82 8. Validation Guidance: 83

84 Appendix D: Guidelines for Collaborative Study Procedures To Validate Characteristics of a Method 85 of Analysis; 19th Edition of the AOAC INTERNATIONAL Official Methods of Analysis (2012). Available 86 at: http://www.eoma.aoac.org/app_d.pdf 87

88 Appendix F: Guidelines for Standard Method Performance Requirements; 19th Edition of the AOAC 89 INTERNATIONAL Official Methods of Analysis (2012). Available at: 90 http://www.eoma.aoac.org/app_f.pdf 91

92 Appendix K: Guidelines for Dietary Supplements and Botanicals; 19th Edition of the AOAC 93 INTERNATIONAL Official Methods of Analysis (2012). Available on line at: 94 http://www.eoma.aoac.org/app_k.pdf 95

96

95

97 9. Maximum Time-To-Result: None 98

99 Table 1: Method performance requirements (part 1) 100

101



Analytical Range (%) ≤ 0.03 – 55

102 103

Table 2: Method performance requirements (part 2)

Liquids Solids Ranges 0.03 – 15% > 15% - 55% 0.03 – 15 % > 15% - 55% Recovery (%) 97 – 103 97 – 103 90 - 107 97 – 103 % RSDr < 10 < 5 < 15 < 10 % RSDR < 15 < 8 < 20 < 15

Table 3: Examples of Dietary Supplements

capsules extracts liquids powders softgel capsules tablets tinctures gummies

96

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