AOAC SMPR® 2018.005 Standard Method Performance Requirements

Home , Desmethoxyyangonin, Flavokavain, Flavokavain A, Flavokavain B, Methysticin

AOAC SMPR® 2018.005 Recovery.—The fraction or percentage of spiked analyte that is recovered when the test sample is analyzed using the entire method. Repeatability.—Variation arising when all efforts are made Standard Method Performance Requirements to keep conditions constant by using the same instrument and (SMPRs®) for Determination of Kavalactones and/or Flavokavains from Kava (Piper methysticum) operator and repeating during a short time period. Expressed as the

repeatability standard deviation (SDr); or % repeatability relative

standard deviation (%RSDr). Intended Use: Quality Assurance and Compliance to Reproducibility.—The standard deviation or relative standard Current Good Manufacturing Practices deviation calculated from among-laboratory data. Expressed as

1 Purpose the reproducibility standard deviation (SDR); or % reproducibility

AOAC SMPRs describe the minimum recommended relative standard deviation (%RSDR). performance characteristics to be used during the evaluation of a 5 Method Performance Requirements method. The evaluation may be an on-site verification, a single- laboratory validation, or a multi-site collaborative study. SMPRs See Tables 3 and 4. are written and adopted by AOAC stakeholder panels composed 6 System Suitability Tests and/or Analytical Quality Control of representatives from the industry, regulatory organizations, contract laboratories, test kit manufacturers, and academic Suitable methods will include blank check samples, and check institutions. AOAC SMPRs are used by AOAC expert review standards at the lowest point and midrange point of the analytical panels in their evaluation of validation study data for methods being considered for Performance Tested MethodsSM or AOAC Official range. A control sample must be included. SM Methods of Analysis , and can be used as acceptance criteria for 7 Reference Material(s) verification at user laboratories. [Refer to Appendix F: Guidelines for Standard Method Performance Requirements, Official Methods See Table 5 for sources of kavalactone and flavokavains of Analysis of AOAC INTERNATIONAL (2016) 20th Ed., AOAC materials, and Table 6 for sources of plant materials. INTERNATIONAL, Rockville, MD, USA.] Refer to Annex F: Development and Use of In-House Reference 2 Applicability Materials in Appendix F: Guidelines for Standard Method Identification and quantitation of the six major kavalactones Performance Requirements, 20th Ed. of the Official Methods of (desmethoxyyangonin, dihydrokavain, yangonin, kavain, Analysis of AOAC INTERNATIONAL (2016). Available at: http:// dihydromethysticin, and methysticin) and/or flavokavains A, B, and C (see Table 1 for more detailed information on analytes and www.eoma.aoac.org/app_f.pdf. Figure 1 for molecular structures) derived from the underground 8 Validation Guidance portions of kava (Piper methysticum) in plant material, dietary ingredients, and dietary supplements as listed Table 2. Methods All target analytes and all matrices listed in Table 1 claimed will be accepted that identify and quantify either flavokavains and/ by the method submitter shall be evaluated. Data from different or kavalactones. matrixes may be pooled together to determine overall analytical 3 Analytical Technique range, LOQ, recovery, RSDr, and RSDR. However, all target Any analytical technique that meets the following method analytes and claimed matrices must be represented in the complete performance requirements is acceptable. evaluation. 4 Definitions Appendix D: Guidelines for Collaborative Study Procedures to Analytical range.—Includes all steps of the analytical procedure, Validate Characteristics of a Method of Analysis, 20th Ed. of the including sample preparation and further dilutions. Official Methods of Analysis of AOAC INTERNATIONAL (2016). Dietary ingredient.—A vitamin; a mineral; an herb or other Available at: http://www.eoma.aoac.org/app_d.pdf. botanical; an amino acid; a dietary substance for use by man to supplement the diet by increasing total dietary intake; or a Appendix K: Guidelines for Dietary Supplements and concentrate, metabolite, constituent, extract, or combination of any Botanicals, 20th Ed. of the Official Methods of Analysis of AOAC of the above dietary ingredients. {United States Federal Food Drug INTERNATIONAL (2016). Also at: J. AOAC Int. 95, 268(2012); and Cosmetic Act §201(ff) [U.S.C. 321 (ff)]} DOI: 10.5740/jaoacint.11-447 and available at: http://www.eoma. Dietary supplement.—A product intended for ingestion that contains a “dietary ingredient” intended to add further nutritional aoac.org/app_k.pdf. value to (supplement) the diet. Dietary supplements may be found 9 Maximum Time-to-Determination in many forms, such as tablets, capsules, softgels, gelcaps, liquids, or powders. No maximum time. Limit of quantitation (LOQ).—The minimum concentration or mass of analyte in a given matrix that can be reported as a Approved by the AOAC Stakeholder Panel on Dietary Supplements quantitative result. (SPDS). Final Version Date: March 16, 2018.

InChI key GUOLPTJISA-N VMPITWQZSA-N VMPITWQZSA-N LBPRGKRZSA-N RMKNXTFCSA-N NSHDSACASA-N BQYQJAHWSA-N BQYQJAHWSA-N FWEMWIAWSA-N CMDGGOBGSA-N RSIWXFIBHXYNFM- CGIBCVBDFUTMPT- XLHIYUYCSMZCCC- UXUFMIJZNYXWDX- XEAQIWGXBXCYFX- XEAQIWGXBXCYFX- DKKJNZYHGRUXBS- VOOYTQRREPYRIW- GTEXBOVBADJOQH- QKQLSQLKXBHUSO- UNII code 5L1NI60TGB M832AIJ6HX R970U49V3C W1ES06373M FZ66MQ73GS F2MBQ8QRUN NW8ZGW9XRZ

CAS No. 500-64-1 587-63-3 495-85-2 500-62-9 3155-48-4 1775-97-9 19902-91-1 15345-89-8 37951-13-6 37308-75-1 (1635-33-2) (1952-41-6) (3420-72-2) (alternative) (56798-34-6) - -pyran- ) - 2- H E ) - 2- E 5- yl) ethenyl] - - pyran- 2- one H -pyran-2-one H - pyran- 2- one - pyran- 2- one H H - pyran- 2- one - 2 -2 4- methoxy- 6- [(1 H ) - 2- (4- methoxyphenyl) 2-one E 4- methoxy- 6- [(1 - Pyran- 2- one )-2-phenylethenyl]-2 4- methoxy- 6- (2- phenylethyl) IUPAC name IUPAC H E 4- methoxy- 2 2 ) - 2- (1, 3-Benzodioxol- phenyl-2-propen-1-one ethenyl] -2 E phenylethenyl] phenylethenyl] ) -5, 6- Dihydro- hydroxyphenyl)-2-propen-1-one methoxyphenyl)-2-propen-1-one 5, 6- Dihydro- R )-6-[2-(1,3-Benzodioxol-5-yl)ethyl]-5,6- dihydro-4-methoxy-2 )-1-(2-Hydroxy-4,6-dimethoxyphenyl)-3- S ) -6- [(1 (6 )-1-(2-Hydroxy-4,6-dimethoxyphenyl)-3-(4- )-1-(2-Hydroxy-4,6-dimethoxyphenyl)-3-(4- ) -5, 6- dihydro- E 5, 6- dihydro- 4- Methoxy- 6- [(1 R (6 E E S (2 (6 (2 (2 (6 4-Methoxy-6-[(1 )-(+)- S )-dihydrokavain, S Yangonin d,l-Kavain marindinin) Common name persicochalcone) dihydromethysticin) Kavain ((R)-(+)-kavain) 4-hydroxyflavokawain B) 4-methoxyflavokawain B) Dihydromethysticin (( Flavokavain A (flavokawain A, (flavokawain A Flavokavain Flavokavain B (flavokawain B, Flavokavain C (flavokawain C, Information on kavalactones and flavokavaines of interest Methysticin ((+)-methysticin, kavahin) Dihydrokavain ((+)-( Desmethoxyyangonin (5,6-dehydrokawain) Table 1. No. 1 2 3 4 5 6 7 8 9 10

© 2018 AOAC INTERNATIONAL Table 2. Examples of plant material, dietary supplements, and dietary ingredients Dried plant material Liquid extracts (including tinctures) Soft extracts Dry extracts Tablets Capsules (including softgels)

O OCH3 H H H

H CO H CO H CO 3 3 O 3 E E E

O H O H O H

O O O

Kavain Methysticin Yangonin

O H

H3CO H3CO H3CO O E

O O O H

O O O

Dihydrokavain Dihydromethysticin Desmethoxyyangonin

OCH3 OH

H H3CO OCH3 H3CO OCH3 3CO OH

E E E

OH O OH O OCH3 O

Flavokawain A Flavokawain B Flavokawain C Figure 1. Molecular structure of kavalactones and flavokavains of interest.

Table 3. Analytical range and LOQ based on matrix Parameter Kavalactonesa Flavokavainsa Analytical range, mg/gb 5–750 0.1–25 Limit of quantitation, mg/g ≤5 ≤0.1

a Reported as individual constituents.

b Range may be narrower depending on the analytical matrix.

Table 4. Method performance requirements as a function of range Acceptance criteria Acceptance criteria Acceptance criteria Parameter kavalactonesa lower range flavokavainsa (0.1–5 mg/g) upper range flavokavainsa (>5–25mg/g) Recovery, % 90–110 90–110

RSDr, % ≤7.5 ≤15 ≤7.5

RSDR, % ≤10 ≤20 ≤10 a Reported as individual constituents.

ACC Corp. API0003191 Botanical Liaisons, Inc.

Inc. 8163AH AK Scientific http://www.botanicalliaisons.com/materials.html

1801 Scientific AvaChem AvaChem

41866 80488 52007 75575 51773 Sigma 5790585 6550 4989 1043 1045 1042 American Herbal Pharmacopoeia (Alkemist) Extrasynthese List/AHP-BRM%20List%20Order%208.4.17.pdf http://www.herbal-ahp.org/documents/BRM-CRS%20 89239 89185 89250 89186 89293 89184 83762 83763 83854 PhytoLab B0061 (Alkemist) Extrasynthese USP 1355709 UNII code 3P306S300W BOW48C81XP Yangonin d,l-Kavain Compound root Kavain ((R)-(+)-kavain)

Methysticin ((+)-methysticin, kavahin) Plant Desmethoxyyangonin (5,6-dehydrokawain) Botanical Dihydromethysticin ((S)-(+)-dihydromethysticin) Flavokavain B (flavokawain B, persicochalcone) Sources of kavalactone and flavokavain materials Sources of reference plant materials Dihydrokavain ((+)-(S)-dihydrokavain, marindinin) Rhizome and root reference material Flavokavain C (flavokawain C, 4-hydroxyflavokawain B) Flavokavain A (flavokawain A, 4-methoxyflavokawain B) (flavokawain A Flavokavain 6. Powdered kava extract Piper methysticum Table 5. No. 1 2 3 4 5 6 7 8 9 10 Table No. 1 2 3 4