AOAC Stakeholder Panel on Dietary Supplements: Stakeholder ... · Chung Hyum, Amway (Nutrilite)...

AOAC Stakeholder Panel on Dietary Supplements: Stakeholder Panel Meeting

Meeting Minutes Friday, September 25, 2015; 8:00 a.m. – 5:00 p.m. PT

Attendees

Panel Members (Present during all or part of the meeting):

Darryl Sullivan, Covance (Chair) Brian Schaneberg, Starbucks (Vice Chair) Asim Ali, Biocell Technology Karen Andrews, USDA John Austad, Covance Jon Bantz, Covance Brad Barrett, Gerstel USA Joseph Betz, NIH/ODS Jim Brown, Sigma-Aldrich Anton Bzhelyansky, USP Teresa Cain, FDA Bob Clifford, Shimadzu Jean-Luc Deborde, SCL Laboratories Steven Dentali, Herbalife Linda Dodd, PB Leiner USA Milda Embuscado, McCormick Heather Figore, Healthy Directions Gabriel Giancaspro, USP Jana Hildreth, Synutra Pure Chung Hyum, Amway (Nutrilite) Prashant Ingle, Herbalife Suhail Ishaq, Biocell Technology Greg Jaudzems, Nestlé David Ji, Analytical Lab Anaheim Holly Johnson, Alkemist Labs Ron Johnson, bioMérieux George Joseph, AsureQuality David Kennedy, Phenomenex Mary Krogull, Eurofins Scientific Adam Kuszak, NIH John Lee, Agilent Katerina Mastovska, Covance Mary McBride, Agilent

Michael McGuffin, AHPA Linda Messick, Covance Deepali Mohindra, Thermo Elizabeth Mudge, BCIT Brian Musselman, IonSense Rick Myers, Kemin Maria Ofitserova, Pickering Labs Melissa Phillips, NIST Curtis Phinney, Consultant Al Pohland, AOAC (Retired) Lars Reimann, Eurofins Scientific Kunal Rehani, Sigma-Aldrich Lanette Richards, Tampa Bay Analytical Catherine Rimmer, NIST Leila Saldanha, NIH Olga Shimelis, Sigma-Aldrich Jules Skamarak, Eurofins Scientific Aniko Solyom, GAAS Analytical John Spzylka, Mérieux NutriSciences Lynn Stephenson, Sigma-Aldrich Kathleen Stenerson, Sigma-Aldrich M John Travis, NSF International Denise Walters, Pfizer Tyler White, Tampa Bay Analytical Laura Wood, NIST David Woollard, RJ Hill Laboratories Jason Wubben, ADM Jinchaun Yang, Waters Weiguo Zhang, Synutra Pure Yanjun Zhang, Herbalife Joseph Zhou, Sunshineville Garrett Zielinski, Covance Jerry Zweigenbaum, Agilent

AOAC Staff (Present during all or part of the meeting): Saliha Argubie, Jim Bradford, Scott Coates, Christopher Dent, Arlene Fox, Dawn Frazier, Deborah McKenzie, La’Kia Phillips, Robert Rathbone

AOAC Stakeholder Panel on Dietary SupplementsMeeting Minutes - September 25, 2015

Meeting Minutes

I. Welcome and Introductions Bradford led introductions and Sullivan opened the meeting of the Stakeholder Panel on Dietary Supplements (SPDS) at approximately 8:15 a.m. PT.

II. Policies and Procedures Sullivan advised that relevant AOAC policies, procedures and guidance can be found in the meeting eBook.

III. Ingredient Updates

Set 1: Anthocyanins, Chondroitin, and PDE5 Inhibitors

Schaneberg informed the group that the Expert Review Panel (ERP) for Set 1 Ingredients had been held in August, 2015. Out of a total twelve (12) methods submitted, two (2) were chosen to move to First Action Official MethodsSM Status: one for Chondroitin and one for PDE5 Inhibitors. No Anthocyanins methods met the current SMPR, so the ERP recommended that the SMPR be revisited.

Set 2: Ashwagandha, Cinnamon, Folin C, and Kratom

Sullivan advised that the Call for Methods for Set 2 Ingredients, along with the Call for Experts on these ingredients, is now open but will be closing in mid-October, 2015. Sullivan recommended that those interested in either submitting a method for consideration, or in applying to be on the ERP, visit the AOAC website (http://www.aoac.org) and select the appropriate tab on the homepage.

Set 3: SMPRs for Approval (Aloin, Tea and Vitamin D) Tea Yanjun Zhang took the floor to give a presentation1 on the work of the SPDS Tea Working Group and the draft SMPR they have developed. The working group has developed one SMPR for quantitative determination of catechins, methyl xanthines, theaflavins, and theanine in tea dietary supplements. After some discussion and minor changes to the SMPR in real time, the document was put to a vote. MOTION to approve the SMPR as amended at this meeting (Mudge/Richards) 19 in favor, 0 opposed, 1 abstain (Bzhelyansky). The motion passed. Aloin Ingle took the floor to give a presentation2 on the work of the SPDS Aloin Working Group and the draft SMPR they have developed. The working group developed one draft SMPR for quantitative determination of Aloin A and Aloin B in Dietary Supplement Products and Raw Materials.

1 Attachment 1: Zhang Presentation 2 Attachment 2: Ingle Presentation


http://www.aoac.org/

After some discussion and minor changes to the draft SMPR, including the removal of the words “raw materials” and replacement with “ingredients,” and an action for AOAC to update the RSDR and RSDR with the draft SMPR was put to a vote. MOTION to approve the SMPR as amended at this meeting (Szpylka/Richards) 20 in favor, 0 opposed, 0 abstain. The motion passed. Vitamin D Austad took the floor to give a presentation3 on the work of the SPDS Vitamin D Working Group and the draft SMPR they have developed. The working group developed one draft SMPR for quantitative determination of Vitamin D in Dietary Supplement Finished Products and Raw Materials. The group changed “raw materials” to “ingredients” as per the discussion of the same on the Aloin SMPR. The analytical range for finished products was raised from 338 (units?) to 12,500 (units?), the same high end as is set for ingredients. Information on NIST reference materials was added – AOAC took the action to clarify these and also include the USP reference material. The draft SMPR was then put to a vote. MOTION to approve the SMPR for “Determination of Vitamin D in Dietary Supplement Finished Products and Ingredients” as amended at this meeting (Austad/Reimann) Sullivan thanked the Set 3 Working Group Chairs and members and advised that the SMPRs will be cleaned up and sent made ready to be published in the Official Methods of Analysis of AOAC INTERNATIONAL4 and also be made available on the AOAC SPDS Website5.

IV. Set 4: New Working Group Launches: Collagen, Lutein, and Turmeric Turmeric Sullivan introduced Solyom, Chair of the new SPDS Turmeric Working Group. Solyom took the floor and gave a presentation6 on the background of Turmeric, the analytical needs, challenges, existing methodologies, and a proposed fitness for purpose statement to launch the new working group. The fitness for purpose stated: “The method will be able to quantify total curcuminoid content, calculated as the sum of curcumin, demethoxycurcumin, and bis-demethyoxycurcumin, in turmeric [Curcuma longa Linn.] powdered botanical raw materials, extracts, and dietary supplement finished products containing turmeric extract, alone or in combination with other herbs such as Piper nigrum and Boswellia serrata. The method must be able to quantify curcuminoid content at levels up to 95% w/w in test materials. The lower limit of quantitation will be determined by the Expert Review Panel.” MOTION by to accept the proposed Fitness for Purpose statement for Turmeric and launch the Turmeric Working Group (Solyom/Reimann). 20 in favor, 0 opposed, 0 abstain. The motion passed.

3 Attachment 3: Austad Presentation 4 http://www.eoma.aoac.org/ 5 http://www.aoac.org/iMIS15_Prod/AOAC/SD/SPDS/AOAC_Member/SH/SPDSCF/SPDSM.aspx?hkey=b8cbd524-33d1-4e51-8cc0-4e2028c367f2 6 Attachment 4: Solyom Presentation


Lutein Myers, Chair of the new AOAC SPDS Lutein Working Group, took the floor to give a presentation7 on the background of lutein, esters, congeners, and metabolites. Myers proposed limiting the scope of this working group to only principal isomers of the most relevant, co-chromatographing compounds: Lutein, 3’-Epilutein, Zeaxanthin, and β-Cryptoxanthin. He reviewed representative existing methods and the background on these analytes. The group agreed to remove the last sentence in the fitness for purpose statement regarding the analytical range and the change the words “raw materials” to “ingredients.” After reviewing some questions with the group, Myers proposed the following fitness for purpose statement: Quantitative measurement of lutein, 3’-epilutein, zeaxanthin and β-cryptoxanthin in both ingredients from which dosage forms are formulated; and in finished products (e.g., softgels, beadlets). MOTION by to accept the proposed Fitness for Purpose statement for Lutein and to launch the Lutein Working Group (Zweigenbaum/Travis). 18 in favor, 0 opposed, 2 abstain (Betz/Johnson). The motion passed. Collagen Ishaq, Chair of the new AOAC SPDS Collagen Working Group, took the floor to give a presentation8 on collagen, including the background of the analyte and its different types, its health significance, the general analytical needs, existing methodologies, challenges and common adulterants. After reviewing some questions to refine the fitness for purpose, Ishaq proposed the following: Quantify total native (undenatured) and hydrolyzed collagen type I, II & III in the raw materials and final finished dosage forms including but not limited to dry powders, tablets, capsules, softgels and liquids . Individually separate and quantify native (undenatured) and hydrolyzed collagen type I, II & III if blended together. MOTION by to accept the proposed Fitness for Purpose statement for Collagen and to launch the Collagen Working Group (Zweigenbaum/Walters). 19 in favor, 0 opposed, 1 abstain (Betz). The motion passed.

V. SPDS Advisory Panel Update Sullivan advised that the SPDS Advisory Panel will meet in December, 2015 to agree on the next six ingredients for SPDS standards development. He asked that anyone with suggestions please submit them to Dawn Frazier ([email protected]) and Christopher Dent ([email protected]) for Advisory Panel consideration.

VI. In Person Working Group Schedule

7 Attachment 5 - Myers Presentation 8 Attachment 6 – Ishaq Presentation


mailto:[email protected]

mailto:[email protected]

Schaneberg announced that the working groups will meet in person on Saturday, September 26 at the following times:

• Collagen: 9:00 a.m. • Lutein: 11:15 a.m. • Collagen: 2:30 p.m.

Schaneberg explained that the in person meetings will be to refine the fitness for purpose and begin work on the SMPR, with follow up work to be conducted by telecon. He advised that individuals who would like to be on the working group rosters should sign up online using the form provided.9

VII. Adjourn Sullivan thanked all attendees and the meeting adjourned at approximately 4:30 PT.

SEPTEMBER 25, 2015 SPDS Meeting: ACTION ITEMS Action Owner Update the RSDR and RSDR in the Aloin SMPR AOAC Staff Clarify the reference materials section in the Vitamin D SMPR AOAC Staff Accept all changes to, clean up, and publish Set 3 SMPRs AOAC Staff Work with WG Chairs to Schedule WG telecons AOAC Staff Sign up for desired working groups using form provided - http://www.jotform.us/form/52526363383154

All

Attachments:

Attachment 1: Zhang Presentation

Attachment 2: Ingle Presentation

Attachment 3: Austad Presentation

Attachment 4: Solyom Presentation

Attachment 5: Myers Presentation

Attachment 6: Ishaq Presentation

9 http://www.jotform.us/form/52526363383154


http://www.jotform.us/form/52526363383154

AOAC INTERNATIONALSTAKEHOLDER PANEL ON DIETARY SUPPLEMENTS

Yanjun Zhang, HerbalifeYanjun Zhang, HerbalifeWorking Group on TeaSeptember 25, 2015

Los Angeles, CA, USA

Fitness for Purpose

Quantitative determination of catechins, methyl xanthines, theaflavins and theanine in tea dietary ingredients and supplements.

AOAC Stakeholder Panel on Dietary Supplements Meeting Minutes - September 25, 2015

TeaWorking Group Members

• Yanjun Zhang

• Joseph Betz• James Griffiths

• Steve Royce• Aniko Solyom

• John Szpylka• Jana Hildreth• Martha Jennens‐Clough• David Ji• George Joseph• David Kennedy• Elizabeth Mudge

• James Traub• Lanette Varesi

• Laura Wood

• Jinchuan Yang

• Seong‐Jae Yoo• Joseph Zhou

• Maria Ofitserova• Melissa Phillips• Tom Phillips• Catherine Rimmer

• Joyce Zhu• Garrett Zielinski

Tea Working Group Work to Date

• One In Person Meeting

• Four Teleconferences (March – June 2015)

• One SMPR Drafted

• Public comment period (December 15, 2014 –January 30 2015)January 30, 2015)

• SMPRs made ready for SPDS review and approval


Background

Current Analytical Techniques

Many analytical techniques have been used in tea components analysis including the following.

1. Chromatographic methods: HPLC‐UV, HPLC‐MS, GC, GC‐MS and HPTLCMS, GC, GC MS and HPTLC

2. Spectroscopic methods: NMR and NIR

Background

Challenges

1. Multiple methods may needed because of the analytes’ properties (e.g. catechins and theanine).

2. Special processing and extraction methods may needed because the contents of analytes in the target products (from microgram to gram amounts).

3. The availability of quantitative standards.


Background

• Purpose: This SMPR describes the minimum recommended performance characteristics to be used during the evaluation of a method or methods for the “Quantitative determination of catechins, methyl xanthines, theaflavins and theanine in tea dietary ingredientsand theanine in tea dietary ingredients and supplements.”

Target Matrices Panel

Dietary Supplements Forms:

TabletsTabletsCapsulesSoftgelsGelcapsGummiesChewablesLiquidsPowders


Target Compounds Panel-1Catechins

Common name IUPAC Nomenclature CAS No.

catechin(2R,3S)‐2‐(3,4‐dihydroxyphenyl)‐3,4‐dihydro‐2H‐

chromene‐3,5,7‐triol154‐23‐4

epicatechin(2R,3R)‐2‐(3,4‐dihydroxyphenyl)‐3,4‐dihydro‐2H‐

490 46 0epicatechinchromene‐3,5,7‐triol

490‐46‐0

epigallocatechin(2R,3R)‐2‐(3,4,5‐trihydroxyphenyl)‐3,4‐dihydro‐2H‐

chromene‐3,5,7‐triol970‐74‐1

catechingallate[(2R,3R)‐2‐(3,4‐dihydroxyphenyl)‐5,7‐dihydroxy‐3,4‐

dihydro‐2H‐chromen‐3‐yl] 3,4,5‐trihydroxybenzoate130405‐40‐2

epicatechingallate

(2R, 3R)‐ 2‐ (3, 4‐ dihydroxyphenyl)‐ 3, 4‐ dihydro‐ 5, 7‐

dihydroxy‐ 2H‐ 1‐ benzopyran‐ 3‐ yl‐ ester‐ 3, 4, 5‐

trihydroxy‐ benzoic acid

1257‐08‐5

epigallocatechin

(−)‐cis‐3,3′,4′,5,5′,7‐hexahydroxyflavane, (−)‐cis‐2‐

(3,4,5‐trihydroxyphenyl)‐3,4‐dihydro‐1(2H)‐

benzopyran‐3,5,7‐triol

970‐74‐1

gallate 3,4,5‐trihydroxybenzoic acid 149‐91‐7

gallocatechin(2S,3R)‐2‐(3,4,5‐trihydroxyphenyl)‐3,4‐dihydro‐

1(2H)‐benzopyran‐3,5,7‐triol3371‐27‐5

Target Compounds Panel-1Catechins

Catechins structures


Target Compounds Panel-2Methyl xanthines


caffeine 1,3,7‐trimethylpurine‐2,6‐dione 58‐08‐2

theobromine 3,7‐dimethyl‐1H‐purine‐2,6‐dione 83‐67‐0

theophylline 1,3‐dimethyl‐7H‐purine‐2,6‐dione 58‐55‐9

Target Compounds Panel-2Methyl xanthines

Caffeine Theobromine Theophylline


Target Compounds Panel-3Theaflavins


theaflavin

3,4,5‐trihydroxy‐1,8‐bis[(2R,3R)‐3,5,7‐

trihydroxy‐3,4‐dihydro‐2H‐chromen‐2‐

yl]‐6H‐benzo[7]annulen‐6‐one

4670‐05‐7

[(2R 3R) 5 7 dih d 2 [3 4 5

theaflavin‐3‐gallate

[(2R,3R)‐5,7‐dihydroxy‐2‐[3,4,5‐

trihydroxy‐6‐oxo‐8‐[(2R,3R)‐3,5,7‐

trihydroxychroman‐2‐

yl]benzo[7]annulen‐1‐yl]chroman‐3‐yl]

3,4,5‐trihydroxybenzoate

30462‐34‐1

theaflavin‐3'‐gallate

[(2R,3R)‐5,7‐dihydroxy‐2‐[3,4,5‐

trihydroxy‐6‐oxo‐8‐[(2R,3R)‐3,5,7‐


yl]benzo[7]annulen 1 yl]chroman 3 yl]

28543‐07‐9

yl]benzo[7]annulen‐1‐yl]chroman‐3‐yl]

3,4,5‐trihydroxybenzoate

theaflavin‐3‐3'‐

digallate

[1‐[(2R,3R)‐3,5‐dihydroxy‐7‐(3,4,5‐

trihydroxybenzoyl)oxychroman‐2‐yl]‐

3,5‐dihydroxy‐6‐oxo‐8‐[(3R)‐3,5,7‐


yl]benzo[7]annulen‐4‐yl] 3,4,5‐

trihydroxybenzoate

33377‐72‐9

Target Compounds Panel-3Theaflavins


Target Compounds Panel-4Theanine

N-ethyl-L-glutamine; (2S)-2-ammonio-5-(ethylamino)-5-oxopentanoate. CAS registry number: 3081-61-6

Analytical Range and LOQ RequirementsAll Analytes

Component catechinsmethyl

xanthinestheaflavins theanine

Analytical

range (ppm)

10 –

500,000

10 –

500,000

10 –

100,000

10 –

100,000

LOQ (ppm) 5LOQ (ppm) 5


Recovery, Repeatability, and Recovery for All Analytes

Ranges (ppm)

10 – 50 51 – 500 501 – 4,000 4,001 – 20,000 > 20,000

Recovery (%)

80 – 110 90 – 107 95 – 105 97 – 103 98 – 102

RSDr (%) ≤ 7 5 4 2 2

RSDR (%) ≤ 10 8 6 3 3RSDR (%) ≤ 10 8 6 3 3

SMPR Key Points

• Complex of dietary supplement matrix – need specialized extraction and sample preparation.

• Wide range of contents of analytes – need precise analytical techniques.

• Four classes of analytes‐tea catechins (8 ), methylFour classes of analytes tea catechins (8 ), methyl xanthines (3), theaflavins (4), and theanine (1) –need quantitative standards.


Comments Submitted (if any)

Motion

• Move to accept the Standard Method Performance Requirements for the “Determination of Catechins, Methyl Xanthines, Theaflavins, and Theanine in Tea Dietary Ingredients and Supplements” as presented.


Discussion?



Prashant Ingle HerbalifePrashant Ingle, HerbalifeWorking Group on Aloin

September 25, 2015


Aloin in AloeWorking Group Members

Prashant Ingle (Chair) Brad Barrett Joseph Betz

Darryl Sullivan James Traub Lanette Varesi

Clyde Don Nour Eddine Es-Safi James Griffiths George Joseph David Kennedy Elizabeth Mudge Melissa Phillips

Yanjun Zhang Garrett Zielinski

Tom Phillips Catherine Rimmer Brian Schaneberg Aniko Solyom

AOAC Working group on Aloin Prashant Ingle 2September 25th, 2015


Aloin Working Group Work to Date

1 In Person Meeting

3 teleconferences (March – June 2015)( )

1 SMPR Drafted

Public comment period (July 2nd 2015 – August 7th, 2015)

SMPRs made ready for SPDS review and approval


Applicability

The method must be able to quantitate Aloin A and Aloin B separately in:separately in:

Raw materials

Dietary supplement finished products in liquid, gel, powder, tablet, and soft gel matrices; and aloe vera leaf juice and dry juice ingredients



Definitions

• Aloin A 1,8-Dihydroxy-10-(β-D-glucopyranosyl)-3-(hydroxymethyl)-9(10H)-anthracenone. Also known as barbaloin, CAS No.: 1415-73-2. 22

• Aloin B (10R)-1,8-dihydroxy-3-(hydroxymethyl)-10-[(2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-25 hydroxymethyl)oxan-2-yl]-10H-anthracen-9-one, Also known as beta-D-isomer barbaloin 26 or isobarbaloin CAS No : 28371-

O

OH O OH

OHH H

OH

OHOH

HO

Anthraquinone skelton

Glycoside portion

known as beta D isomer barbaloin 26 or isobarbaloin. CAS No.: 2837116-6


O

OH O OH

OHH H

OH

OHOH

HO

Analytical Range and LOQ Requirements

Following analytical range and LOQ parameters were discussed since March 2015:

Parameter Finished Products Raw Material

Analytical range (ppm) 0.01 to 100 0.01 to 12,500

LOQ (ppm) 0.005 0.005

Aloin in Aloe (Aloin A or Aloin B):



Recovery, Repeatability, and Recovery for Aloin A and Aloin B

Ranges (ppm)

Performance Parameters for Aloin A or B:

Parameter

g (pp )

Finish Product and Raw ingredients Raw ingredients

0.01 - 1 >1 - 10 >10 - 30 >30 - 100 >100 - 1000 >1000 -12,500

% Repeatability (RSDr)

21 11 7 6 5 3

Recovery (%) 60-115 80-110 90-107 95-105

% Reproducibility% Reproducibility (RSDR) 32 16 11 9 7 4


Motion

• Move to accept the Standard Method Performance Requirements for Aloin in Aloe as presented.





Discussion?




John Austad, CovanceJohn Austad, CovanceWorking Group on Vitamin D

September 25, 2015


Fitness for Purpose

The method will separate and accurately p yquantitate vitamin D2 (ergocalciferol), vitamin D3 (cholecalciferol), and their previtamin d and hydroxy forms in dietary supplement consumer products and the raw materials used to formulate these productsmaterials used to formulate these products.


Vitamin DWorking Group Members

• John Austad• W. Barrett• Joseph Betz

• David Kennedy• Adam Kuszak• Elizabeth Mudge

• John Szpylka• James Traub• Lanette Varesi

• Carolyn Burdette• Clyde Don• Andy Erickson• James Griffiths• Jana Hildreth• Prashant Ingle

• Matthew Noestheden

• Melissa Phillips• Curtis Phinney• Catherine Rimmer

• Nandakumara(nandu kumara)

• Denise Walters

• JP Woods

• Jinchuan Yang

• Seong‐Jae Yoo• Troy Zhang• Weiguo Zhang

• Martha Jennens‐Clough

• David Ji• George Joseph

(nandu kumara) Sarma

• Brian Schaneberg• Aniko Solyom

• Yanjun Zhang

• Joyce Zhu• Garrett Zielinski

Vitamin D Working Group Work to Date

•1 In Person Meeting

•2 teleconferences (April – June 2015)

•1 SMPR Drafted

•Public comment period (July 7, 2015 – August 7 2015)7, 2015)

•SMPRs made ready for SPDS review and approval


SMPR Key Points

1. Method Performance Requirements: Table 1: Analytical Range & LOQ Based on Matrix

Parameter Finished Products Raw Materials

Analytical range ppm* 0.5 – 338 1,250 ‐ 12,500

Limit of Quantitation ppm* 0.4 1,000

Table 2: Method Performance Requirements as a Function of Range

Parameter Ranges (µg/g)*

< 10 ‐ 15 >15 ‐ 50 >50 – 500 >500 – 4,000 >4000 – 12,500

Recovery (%) 80 ‐ 110 90 ‐ 107 95 – 105 95 – 105 97 – 103

% R t bilit% Repeatability (RSDr)

8 7 5 4 3

% Reproducibility (RSDR)

12 10 8 6 4

* Measured as individual forms of Vitamin D and pre‐Vitamin D


• One Comment Submitted

– “The two certified reference materials I mentioned were vitamin D2 and vitamin D3. We have other vitamin D standards in the attached but they are the metabolites.”

• Cerilliant Vitamin D2V‐024 Vitamin D3V‐025– 1 mg/ml CRM in Ethanol

St k h ld D dd t SMPR– Stakeholders: Do we add to SMPR as a requirement to use?


Motion

• Move to accept the Standard Method Performance Requirements for Vitamin D as presented.

Discussion?


STAKEHOLDER PANEL ON DIETARY SUPPLEMENTSDIETARY SUPPLEMENTS

Background & Fitness for PurposeTURMERIC

Chair: AnikoM Solyom GAAS AnalyticalChair: Aniko M. Solyom, GAAS Analytical

Westin Bonaventure Hotel, Los Angeles, California

September 25, 2015

SPDS Advisory Panel Priorities (Set 4)

Factors:Factors: • Research interests • economic importance• availability of methods & standards

Scopes: h d f dPrimary: Quantitative methods for curcuminoids

Secondary: Reduced forms


Turmeric (Curcuma longa L.)Common names: turmeric, turmeric root, Indian saffronMember of the ginger family, Zingiberaceae

Turmeric rhizoma

UsesCulinary:

fl i d l i t• flavoring and coloring agent• main spice in curryTraditional Chinese and Ayurvedic medicine:• topical application for eczema and wound healing• aid digestion and liver function• relieve arthritis pain• regulate menstruation

Source: NCCIH Dietary Supplement Database (https://nccih.nih.gov/health/turmeric/ataglance.html

• regulate menstruation

Current research:• osteoarthritis, Alzheimer disease, eye inflammation, • colorectal cancer, Crohn’s disease, diabetes, stomach upset• gingivitis, stomach ulcer, irritable bowel syndrome, RA and more…


Turmeric (Curcuma longa L.)

Spectra of turmeric extract

Approx. 5% of the plant is curcumin

CurcuminoidsO OO O

CH3O

OH

CH3O

OH

O O

OH

CH3O

OH

CurcuminMW:368

DemethoxycurcuminMW:338OH OH

O O

OH OH

MW:338

BisdemethoxycurcuminMW:308


Significance

• In the Dietary Supplement Database (DSLD) 1,044 products contained turmeric and/or curcumin(oids) and/or extracts (out of total 42,000 DS)

f h d /• 47% of these products turmeric/curcumin as a component of a blend

Source: Leila G. Saldanha, PhD, RD, Office of Dietary Supplement, NIH. Personal communication

Significance190 clinical trials190 clinical trials between 1996 and 2015(http://clinicaltrials.gov)


Challenges• Nomenclature:• Nomenclature:

– Turmeric, turmeric oil– Curcumin, curcuminoids

– Standardized to x% curcumin

AdulterationI di i d i i f• Indian turmeric trade types curcumin contents ranging from 2.1% to 8.6%, with an average of 4.8%.

• Curcuma longa L. adulterated with wild species: Curcuma zeodaria, Curcuma malabarica – toxicity and poor quality

• Adulterated with artificial colors – metanyl yellow

• Saffron is adulterated with turmeric


Challenges

• Clinical Phase I studies have shown that the blood serum levels of curcumin are in the ng/mL range after oral doses of up to 8 g of curcumin, suggesting very low gastro‐intestinal bioavailability

• The reasons for the low oral bioavailability of curcumin are not yet knowny– chemical instability (degradation products are vanilin, ferulic acid,

feruroyl methane) – rapid metabolism

– poor absorption– accumulation in cells of the gastro‐intestinal tract

Analytical NeedsQ i i h d f i id i• Quantitative method for curcuminoids in– Raw material (plant material without authentication)– Extracts

– Finished products containing only turmeric and/or curcuminoids

– Finished products containing other ingredients (vitamins, other DS, herbs, metanyl yellow)

– Biological fluids (?)Biological fluids (?)

• Quantitative method for curcuminoids in– Capsules – Tablets

– Tinctures

– Softgel capsules


Existing Methods

S iFi d h “ i d lid i ” d “2014 2015”• SciFinder search: “turmeric and validation” and “2014‐2015” yielded 97 references

• Spectrophotometric method for the estimation of curcumin in bulk and pharmaceutical formulation

• 1H‐NMR and PCR for detecting Curcuma longa wild species adulterantsadulterants

• HPLC and LC/MS are widely used analytical techniques

History of turmeric method validation

• February 16, 2010: AOAC Expert Review Panel

• 2009: “Call for Methods” to quantify curcuminoidcontent in dietary supplements and raw materials.

• 38 methods was submitted

evaluates the submitted methods and selects TUR‐36, developed by GAAS Analytical as the method recommended for further consideration.


History of turmeric method validation

• March 22 2012: NIH publishes a Request for Quotes

• October 29, 2012: ChromaDex announces the award f t th d d l t lid ti t t

• March 22, 2012: NIH publishes a Request for Quotes (RFQ) for the “Method Optimization and Single‐Laboratory Validation of an Analytical Method: Total Curcuminoid Content of Turmeric”

of two method development validation contracts. “These contracts will create single laboratory validated methods for the quantitative determination ….. of total curcuminoid content in turmeric.”

Fitness for Purpose (2010)

Turmeric Method Fitness For Purpose Statement (2010)“The method will be able to quantify total curcuminoid content, calculated as the sum of curcumin, demethoxycurcumin, and bis-demethyoxycurcumin, in turmeric [Curcuma longa Linn.] powdered botanical raw materials, extracts, and dietary supplement finished products containing turmeric extract, alone or in combination with other herbs such as Piper nigrum and Boswellia serrata. The method must be able to quantify curcuminoid content at levels up to 95% w/w in test materials. The lower limit of quantitation will be determined by the Expert Review Panel.”


Fitness for Purpose (proposal)

Questions/notes to the stakeholders and member of the working group:

• What happened between 2009 and 2015?– More complex finished products on the market,

l l h b dcontaining multiple herbs and vitamins

– Number of clinical trials increased – need of analytical support

SPDS Advisory Panel Priorities (Set 4)

Factors:Factors: • Research interests • economic importance

• availability of methods & standards

Scopes: Primary: Quantitative methods for curcuminoids

Secondary: Reduced forms


Challenges

• Clinical Phase I studies have shown that the blood serum levels of curcumin are in the ng/mL range after oral doses of up to 8 g of curcumin, suggesting very low gastro‐intestinal bioavailability

• The reasons for the low oral bioavailability of curcumin are not yet knowny– chemical instability (degradation products are vanilin, ferulic acid,

feruroyl methane) – rapid metabolism

– poor absorption– accumulation in cells of the gastro‐intestinal tract

Metabolism of curcumin in Caco‐2 cellsOH OOH O

H3CO

HO

OCH3

OHCUR

OH O

H3CO

HO

OCH3

OHhexahydro-CUR

CUR glucuronide

CUR sulfate

hexahydro-CUR glucuronide

hexahydro-CUR sulfatehexahydro CUR

OH OH

H3CO

HO

OCH3

OHoctahydro-CUR

octahydro-CUR glucuronide

octahydro-CUR sulfate


Analytical Needs• Quantitation of parent compound(s) and metabolites inQuantitation of parent compound(s) and metabolites in

biological fluids to support clinical trials

blood

urine

• Reduced forms in dietary supplement finished products? Probably too expensive.

feces

Challenges

b l b l ll f• Low bioavailability ‐ very small amount of analyte

• Expensive instrumentation

• Radioactive tracing (?)A il bilit f t d d• Availability of standards

• Price of the standards


Available Metabolite Standards

• Dihydrocurcumin:– Used to be available from Indofine. Excerpt from a recent e‐mail: “Unfortunately I

have bad news. We are unable to isolate dihydrocurcumin at this time. We made several attempts and were unsuccessful. For that reason we have to cancel this order. We are continuing to try, so I will keep you informed of our progress”

• Tetrahydrocurcumin (CAS 36062‐04‐1):– Santa Cruz Biotechnology 1 g $240

C t Ch i l C 1 $221 50– Crescent Chemical Company 1 g $221.50– TLC PharmaChem, Cayman Chemical 10 mg $200– Toronto Research Chemicals 100 mg $100, 1 g $120

• Hexahydrocurcumin (CAS 36062‐05‐2)– Crescent Chemical Company 10 mg $526


Need clarification from the stakeholders and advisory board members


QUESTIONS??


Stakeholder panel on dietary supplementsStakeholder panel on dietary supplementsBackground & Fitness for Purpose

Lutein, esters, congeners& metabolites

Rick Myers, PhD

AOAC Annual Meeting

Log Angeles, CA

25 September 2015

Background on analytes• Carotenoids are a diverse family of botanical pigments• Minimal biosynthesis in animals; so must derive from diet• Botanical function

– Mediate photoinduced electron transfer to chlorophyll– Quench singlet/triplet‐chlorophyll that can damage allied

tissues during very active photosynthesis• Two relevant carotenoid families

– Carotenes: hydrocarbons (orange)Carotenes: hydrocarbons (orange)– Xanthophylls: hydroxylated carotenes (yellow)

• Only xanthophylls of interest here. Dozens exist!• Often exists as fatty acid esters in nascent tissue


Proposal for analytes:

1. Limit to only principal isomers of the four most relevant, co‐chromatographing compoundsa. Luteinb. 3’‐Epilutein (significant epimer loss of lutein)c. Zeaxanthind β‐Cryptoxanthind. β‐Cryptoxanthin

2. Saponify initial extracta. Analyze free compounds only

1. Lutein

o (3R 3’R 6’R) β ε carotene 3 3’ diol; dietaryo (3R,3’R,6’R)‐β,ε‐carotene‐3,3’‐diol; dietaryo Commercial and supplemental roles

• Accumulates throughout human retina• Reportedly rescues AMD• Present in other tissues, relevance under study• Colors white egg yolks yellow• Antioxidant• Colorant (E161b)

o Structure

o Proposed daily dose: 10 mg


2. Zeaxanthino β β carotene 3 3' diol; dietaryo β,β‐carotene‐3,3 ‐diol; dietaryo Zeaxanthin differs from lutein only by placement of single double bond.

o Commercial and supplemental roles• Also accumulates in human retina; predominates in macula lutea

• Reportedly rescues AMD• Colors white egg yolks yellowColors white egg yolks yellow• Colorant (E161h)

o Structure


3. β‐Cryptoxanthin

o (3R 3’R 6’R) β ε carotene 3 3’ diol; dietaryo (3R,3 R,6 R)‐β,ε‐carotene‐3,3 ‐diol; dietaryo Commercial and supplemental roles

• Provitamin in humans; converted to vitamin A• Possible antioxidative DNA protection, bone health, others• Colorant (E161c) in Australia and New Zealand; not in US or EU

o Structure



4. 3’‐Epiluteino (3R,3’S,6’R)‐luteino Not dietary—no biological or commercial roleo Significant epimer product and loss of lutein

o Occurs in aqueous acido Reaction likely proceeds by SN1 and SN2, but mostly SN2 since conversion exceeds 50%

o Structure

General Analytical NeedsMethod shouldMethod should

– Quantitatively de‐esterify all analyte forms

– Separate and accurately quantify relevant free analytes• Lutein

• Zeaxanthin

• β‐Cryptoxanthin• 3’ Epilutein (principal lutein metabolite)• 3 ‐Epilutein (principal lutein metabolite)

– Determine the above in• Raw materials used in dietary supplement formulations

• Finished products?


Challenges

• Quantitative recovery from finished product forms, e.g., beadlets, softgels, etc.

• Dozens of carotenoids co‐extract from their botanical sourcesA l t diffi lt t t f h• Analytes are difficult to separate from one each other, isomers, and metabolites

Existing Methods GeneralExisting Methods ‐ General

• HPLC – UV• U/HPLC – MS/MS


Representative existing methods

• USP FCC 7 monograph• Total carotenoids• HPLC‐UV• Normal phase (silica)• Mobile phases: hexane, EtOAc

• AOAC 2009.04AOAC 2009.04

• Lycopene in dietary supplements and raw materials • Protease digestion and extraction with DCM:EtOH

• HPLC‐UV (alkylamide‐bonded silica)• Mobile phase: IPA:NH4OAc: ACN:MeOH:alkylamine

A complex extract requires both normal and reversed phase chromatography for robust separation

• Khachik et al. (1997) and Khachik et al. (1992)• Carotenoids and their metabolites in human milk and serum, and Carotinoids and their oxidation products in human plasma, respectively

• Two complementary chromatographic methods1. Reversed phase (C18), ACN:MeOH:DCM, UV/MS2. Normal phase (Nitrile‐bonded silica), hexane:DCM:MeOH:

alkylamine, UV/MS

• AOAC 2005.07

• β‐Carotene in dietary supplements and raw materials • Protease digestion and extraction with DCM:EtOH• Two complementary HPLC chromatographic methods

1. Reversed phase (C18), IPA:NH4OAc: ACN:MeOH:alkylamine, UV2. Reversed phase (C30), MeOH:MTBE:alkylamine, UV


Regulatory GuidanceRegulatory Guidance

• Evaluation of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), 2004

• The FDA has confirmed the Joint Expert Committee on Food Additives’ (JECFA) ruling (Sept 8, 2004) that free lutein and

hi f f h izeaxanthin are safe for human consumption.

• Vitamin status in some countries

General Method Requirements1 Q i i i f i1. Quantitative extraction from matrices

a. Raw materials from which dosage forms are formulatedb. Finished products (e.g., softgels, beadlets) including proteolytic or

other matrix release methods

2. Ensure against losses (e.g., oxidation, photolysis) during extraction, workup and analysis

3. Separation from matrix interferent for accurate and reproducible quantitationreproducible quantitation

4. The analytical range of the chosen method must encompass below 1 mg



Quantitative measurement of lutein, 3’‐epilutein, zeaxanthin and β‐cryptoxanthin in both raw materials from which dosage forms are formulated; and in finished products (e.g., softgels, beadlets). Methods must be able to accurately measure in theMethods must be able to accurately measure in the analytical range below 1 mg per dosage unit.

Questions to address• Address extraction from botanical tissues?• Address extraction from botanical tissues?• Esters? Or free compounds only (saponify first)?• Quantify losses:

– To epilutein?– Only all‐trans species (or include cis species)?Oxidized species?– Oxidized species?

• Lutein only, or include:– Zeaxanthin?

– β‐Cryptoxanthin?– Others?


QUESTIONS?


STAKEHOLDER PANEL ON

DIETARY SUPPLEMENTS

COLLAGENCOLLAGEN

OUTLINEk d ll d d ff• Background on collagen and its different types.

• Health Significance.• General Analytical Needs.• General Overview of the Existing Analytical Methods &

Techniques.

• Analytical ChallengesAnalytical Challenges.• Fitness for purpose statement.

• Questions???


BACKGROUND ON COLLAGENAND ITS DIFFERENT TYPES

Collagen is the most abundant protein found in the body ofanimals making up about 25% to 35% of whole body protein.It is fibrous in nature and provides the structural support tothe framework of the body. It connects and supports bodytissues such as skin, bone, tendons and cartilage. It is a largetriple helical molecule in its native formtriple helical molecule in its native form.

Collagen Fibril


So far 28 types of collagen have been identified. Types I, II, III are the most commonly used in dietary supplements.

• Type I Collagen is found in skin, muscle, tendon, ligament and bone tissue. (Most Abundant Type)

• Type II Collagen is found in cartilage.

• Type III Collagen is found alongside with Type I Collagen.



Collagen in Dietary Supplements

Given the large molecular weight of native collagen (~300k) most commercial collagen dietary ingredients are predigested/hydrolyzed (fragmented) to improve bioavailability.

Type I & III Collagen (aka Gelatin) - Bovine, Porcine, Marine Carcass Derived. Economical due to lower production cost and higher yield.

Type II Collagen – Chicken Cartilage Derived. Lower Yield. Higher Cost.

HEALTH SIGNIFICANCE

Th ll l t th t i t i th k t idThe collagen supplements that exist in the market are wide ranging in molecular composition, molecular weight distribution, source (animal species), mechanism of action, and clinical efficacy.

Used by consumers to promote:

• Joint health• Skin health• Connective tissue health• Hair and nail health


GENERAL ANALYTICAL NEEDS

• Method shouldMethod should– Quantify collagen Type I, II & III in raw materials and finished product

matrices.

– Specific enough to individually separate and quantify collagen type I, II & III if blended together.

– Identify the species of the collagen whether it is derived from marine, chicken, bovine or porcine sources.

– Identify the adulterant(s) as other protein in the collagen samples.

GENERAL OVERVIEW OF THE EXISTING METHODS & TECHNIQUES

•Nitrogen based protein analysis methods to calculate the collagen content (unspecific)

•Other total protein analysis methods (unspecific)

•Western blot using specific collagen antibodies. (Not effective in hydrolyzed collagen samples)

•ELISA for specific type of collagen. (Not effective in hydrolyzed collagen samples)

•Hydroxyproline based collagen assay method. (Not type or species specific)


ANALYTICAL CHALLENGES

• Collagen is a complex molecule to purify and analyze• Collagen is a complex molecule to purify and analyze.

• Testing is complicated once the collagen is hydrolyzed and fragmented into peptides, which can widely vary in size, length and molecular weight distribution depending on a host of factors including production method, process time, hydrolysis method, and raw material source.

• Need cost effective, quick turnaround analysis time and simple test method that can be run in an in-house laboratory.

COMMON ADULTERANTS

• Type I & III Collagen / Gelatin products are sometimes adulterated with lower cost proteins.

• Type II Collagen is adulterated with type I & III collagen/gelatin and other lower cost proteinscollagen/gelatin and other lower cost proteins.


FITNESS FOR PURPOSE STATEMENT

The method should be able to:The method should be able to:• Quantify total collagen type I, II & III in the raw materials and

final finished dosage forms including but not limited to dry powders, tablets, capsules, softgels and liquids .

• Individually separate and quantify collagen type I, II & III if blended together. d f h f h ll l h h h• Identify the specie of the collagen samples whether they are derived from bovine, porcine, marine or chicken.

• Identify the adulterants present in the form of other protein within the collagen samples.

QUESTIONS???


AOAC Stakeholder Panel on Dietary Supplements: Stakeholder ... · Chung Hyum, Amway (Nutrilite)...

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