1 DRAFT AOAC SMPR 2018.XXX; Version 7; January 5, 2018. 2 3 Method Name: Determination of Select Flavonoids From Skullcap 4 5 Approved by: Stakeholder Panel on Dietary Supplements (SPDS) 6 7 Intended Use: For quality assurance and compliance to current good manufacturing practices. 8 9 1. Purpose 10 11 AOAC SMPRs describe the minimum recommended performance characteristics to be used 12 during the evaluation of a method. The evaluation may be an on-site verification, a single- 13 laboratory validation, or a multi-site collaborative study. SMPRs are written and adopted by 14 AOAC Stakeholder Panels composed of representatives from the industry, regulatory 15 organizations, contract laboratories, test kit manufacturers, and academic institutions. 16 AOAC SMPRs are used by AOAC Expert Review Panels in their evaluation of validation study 17 data for method being considered for Performance Tested Methods or AOAC Official 18 Methods of Analysis, and can be used as acceptance criteria for verification at user 19 laboratories. [Refer to Appendix F: Guidelines for Standard Method Performance 20 Requirements, Official Methods of Analysis of AOAC INTERNATIONAL (2012) 20th Ed., AOAC 21 INTERNATIONAL, Gaithersburg, MD, USA.] 22 23 2. Applicability: 24 Identification and quantitation of baicalin from skullcap in plant material, dietary 25 ingredients and dietary supplements (see table 1). Determination of chrysin, 26 baicalein, wogonin, wogonoside, scutellarein and scutellarin is also desirable. See 27 Table 2 for additional information on analytes and figure 1 for molecular structures. 28 29 3. Analytical Technique: 30 Any analytical technique that meets the following method performance requirements is 31 acceptable. 32 33 4. Definitions: 34 35 Analytical range – Includes all steps of the analytical procedure including sample 36 preparation and further dilutions. 37 38 Dietary ingredient.— A vitamin; a mineral; an herb or other botanical; an amino acid; a 39 dietary substance for use by man to supplement the diet by increasing total dietary intake; 40 or a concentrate, metabolite, constituent, extract, or combination of any of the above 41 dietary ingredients. {United States Federal Food Drug and Cosmetic Act §201(ff) [U.S.C. 321 42 (ff)]} 43 44 Dietary supplement.— A product intended for ingestion that contains a “dietary ingredient” 45 intended to add further nutritional value to (supplement) the diet. Dietary supplements may 46 be found in many forms such as tablets, capsules, softgels, gelcaps, liquids, or powders. 47 48 Limit of Quantitation (LOQ) .— The minimum concentration or mass of analyte in a given 49 matrix that can be reported as a quantitative result 50 51 Plant Materials – Fresh, dried or cut plant materials 52 53 Repeatability .— Variation arising when all efforts are made to keep conditions constant by 54 using the same instrument and operator and repeating during a short time period. 55 Expressed as the repeatability standard deviation (SDr); or % repeatability relative standard 56 deviation (%RSDr). 57 58 Reproducibility.— The standard deviation or relative standard deviation calculated from 59 among-laboratory data. Expressed as the reproducibility relative standard deviation (SDR); 60 or % reproducibility relative standard deviation (% RSDR). 61 62 Recovery.— The fraction or percentage of spiked analyte that is recovered when the test 63 sample is analyzed using the entire method. 64 65 Skullcap.- For the purposes of this SMPR, the term skullcap refers to the species Scutellaria 66 lateriflora and Scutellaria baicalensis. 67 68 69 5. Method Performance Requirements: 70 71 See table 3 and 4. 72 73 6. System suitability tests and/or analytical quality control: 74 Suitable methods will include blank check samples, and check standards at the lowest point 75 and midrange point of the analytical range. A control sample must be included. 76 77 7. Reference Material(s): 78 79 See table 5 for sources of flavonoids reference materials. See table 6 for sources of 80 Scutellaria species. 81 82 Refer to Annex F: Development and Use of In-House Reference Materials in Appendix F: 83 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 8. Validation Guidance: 88 89 Appendix D: Guidelines for Collaborative Study Procedures To Validate Characteristics of a 90 Method of Analysis; 19th Edition of the AOAC INTERNATIONAL Official Methods of Analysis 91 (2012). Available at: http://www.eoma.aoac.org/app_d.pdf 92 93 Appendix K: Guidelines for Dietary Supplements and Botanicals 19th Edition of the AOAC 94 INTERNATIONAL Official Methods of Analysis (2012). Also at: . AOAC Int. 95, 268(2012); DOI: 95 10.5740/jaoacint.11-447 and available at: http://www.eoma.aoac.org/app_k.pdf 96 97 9. Maximum Time-To-Determination: No maximum time. 98 99 100 101 102 103 Table 1: Examples of Plant Material, Dietary Supplements and Dietary Ingredients 104 105 Dried plant material 106 Liquid extracts (including tinctures) 107 Dry extracts 108 Tablets 109 Capsules 110 Multi-botanical finished product 111 112 113 114 Table 2: Additional Information on Select Flavanoids 115 Compound Name CAS # UNII InChI Key PubChem
5,6,7-Trihydroxy-2-phenyl-4H-1- FXNFHKRTJBSTCS-
Baicalein (Noroxylin) 491-67-8 benzopyran-4-one 49QAH60606 UHFFFAOYSA-N 5281605
5,6-Dihydroxy-4-oxo-2-phenyl-4H-1- Baicalin (Baicalein-7-O- IKIIZLYTISPENI-
benzopyran-7-yl β-D- 21967-41-9 glucuronide) 347Q89U4M5 ZFORQUDYSA-N 64982 glucopyranosiduronic acid
Chrysin (5,7- 5,7-Dihydroxy-2-phenyl-4H-1-benzopyran- RTIXKCRFFJGDFG-
480-40-0 Dihydroxyflavone) 4-one 3CN01F5ZJ5 UHFFFAOYSA-N 5281607
Dihydrobaicalin (5,6,7- (2S)-3,4-Dihydro-5,6-dihydroxy-4-oxo-2- UVNUGBQJLDGZKE-
Trihydroxyflavanone 7-O-β- phenyl-2H-1-benzopyran-7-yl β-D- 56226-98-3 NA XDZPIWCFSA-N 14135325 D-glucuronide) glucopyranosiduronic acid
5,6-Dihydroxy-2-(2-hydroxyphenyl)-4-oxo- ARCCSELFQKSKDR-
Ikonnikoside I 4H-1-benzopyran-7-yl β-D- 119152-49-7 NA ZFORQUDYSA-N 10183148 glucopyranosiduronic acid
Lateriflorein (5,6,7- 5,6,7-Trihydroxy-2-(2-methoxyphenyl)- OHWUYMZEFLQFQB- Trihydroxy-2'- 1329-06-2 NA 10913608 4H-1-benzopyran-4-one UHFFFAOYSA-N methoxyflavone)
Lateriflorin (Lateriflorein 7- 5,6-Dihydroxy-2-(2-methoxyphenyl)-4- 521066-22-8 NA NA NA O-β-D-glucuronide) oxo-4H-1-benzopyran-7-yl
116 117 Table 2: Additional Information on Select Flavanoids (cont’d.) Compound Name CAS # UNII InChI Key PubChem Oroxylin A 7-O-β-D- 5-Hydroxy-6-methoxy-4-oxo-2-phenyl-4H- glucuronide (6- QXIPXNZUEQYPLZ-
1-benzopyran-7-yl β-D- 36948-76-2 Methoxybaicalein 7-O-β-D- O84RM2NAEQ QSUZLTIMSA-N 14655552 glucopyranosiduronic acid glucuronide)
Scutellarein (6- 5,6,7-Trihydroxy-2-(4-hydroxyphenyl)-4H- JVXZRQGOGOXCEC-
529-53-3 Hydroxyapigenin) 1-benzopyran-4-one P460GTI853 UHFFFAOYSA-N 5281697
5,6-Dihydroxy-2-(4-hydroxyphenyl)-4-oxo- Scutellarin (Scutellarein 7-O- 27740-01-8 DJSISFGPUUYILV-
4H-1-benzopyran-7-yl β-D- β-D-glucuronide) (1329-06-2) 16IGP0ML9A ZFORQUDYSA-N 185617 glucopyranosiduronic acid
(3S,5S,5'aS,7'S,8'S,8'aR)- 5-(3-Furanyl)-3', 4,5,5',5'a,7',8',8'a-octahydro-8'-hydroxy- AONLJCCUYGGOSW-
Teucrin A 12798-51-5 NA 7'-methyl-spiro[furan-3(2H),6'-[6H] PJERILTQSA-N 159529 naphtho[1,8-bc]furan]-2,2'(4'H)-dione (E)-4-[3-(3,4-Dihydroxyphenyl)-2- propenoate]-2-(3,4-dihydroxyphenyl)ethyl UDHCHDJLZGYDDM-
Teucrioside O-α-L-lyxopyranosyl-(1→2)-O-6-deoxy-α- 115872-98-5 NA JPCQEIDESA-N 14034194 L-mannopyranosyl-(1→3)-β-D- glucopyranoside (E)-4-[3-(3,4-Dihydroxyphenyl)-2- Verbascoside (Acteoside, propenoate]-2-(3,4-dihydroxyphenyl)ethyl FBSKJMQYURKNSU-
Kusaginin, Russetinol, 61276-17-3 O-α-rhamnopyranosyl(1→3)-4-O-caffeoyl- 3TGX09BD5B ZLSOWSIRSA-N 5281800 Stereospermin) β-D glucopyranoside
5,7-Dihydroxy-8-methoxy-2-phenyl-4H-1- XLTFNNCXVBYBSX-
Wogonin 632-85-9 benzopyran-4-one POK93PO28W UHFFFAOYSA-N 5281703
Wogonoside (Oroxinidin, 5-Hydroxy-8-methoxy-4-oxo-2-phenyl-4H- LNOHXHDWGCMVCO-
Glychionide B, Wogonin 7-O- 1-benzopyran-7-yl β-D- 51059-44-0 ETX4944Z3R NTKSAMNMSA-N 3084961 β-D-glucuronide) glucopyranosiduronic acid 118 119 Table 3: Analytical Range & LOQ 120 Parameter Minimum Requirement Analytical range (%, w/w) 1 - 50
Limit of Quantitation (%, w/w) 1 121 122 123 Table 4: Method Performance Requirements 124
Parameter Minimum Requirement
% Recovery 92-105
% RSDr ≤ 5
% RSDR ≤ 8 125 126 127 128 129 130 131 132 Table 5: Sources of Selected Flavanoids 133 Compound Phytolab Extrasynthese Sigma EP USP
Baicalein (Noroxylin) 89577 1171 92081 1048357
Baicalin (Baicalein-7-O-glucuronide) 89322 1280 S 94121 Y0001273 1048368
Chrysin (5,7-Dihydroxyflavone) 80382 1362 S 95082 NA NA
Dihydrobaicalin (5,6,7-Trihydroxyflavanone 7-O-β- NA NA NA NA NA D-glucuronide)
Ikonnikoside I NA NA NA NA NA
Lateriflorein (5,6,7-Trihydroxy-2'-methoxyflavone) NA NA NA NA NA
Lateriflorin (Lateriflorein 7-O-β-D-glucuronide) NA NA NA NA NA
Oroxylin A 7-O-β-D-glucuronide (6- NA NA NA NA NA Methoxybaicalein 7-O-β-D-glucuronide)
Scutellarein (6-Hydroxyapigenin) 83283 1334 S S0327 NA NA
Scutellarin (Scutellarein 7-O-β-D-glucuronide) 89788 NA 73577 NA NA
Teucrin A 89285 NA NA NA NA
NA NA NA NA NA Teucrioside
Verbascoside (Acteoside, Kusaginin, Russetinol, NA Stereospermin) 89289 4994 S v4015 1711455
Wogonin 89825 1304 S 681670 NA NA
Wogonoside (Oroxinidin, Glychionide B, Wogonin NA NA NA 7-O-β-D-glucuronide) 82684 SMB00081 134 135 136 Table 6: Sources of Scutellaria species. 137 138 Skullcap Aerial Parts: American Herbal Pharmacopoeia, Product Code 524. 139 Scutellaria baicalensis, root. American Herbal Pharmacopoeia, Product Code 563474. 140 Scutellaria lateriflora, herb. Alkemist Labs, Product Code 942495. 141 Scutellaria lateriflora, herb. American Herbal Pharmacopoeia, Product Code 533097. 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 Figure 1: Molecular Structures of Selected Flavanoids 161 162 163 O OH O OH
S OH S OH HO S S HO S S
O R O R S OH S OH
O O HO O O O
S
HO HO HO
OH O OH O OH O
Baicalin Baicalein Dihydrobaicalin O OH
S OH HO S S
R O H3CO H3CO S OH
O O HO O
HO HO
OH O OH O
Lateriflorin Lateriflorein
O OCH3 OCH3 O O O HO O
HO S S
S R S HO OH
OH OH O OH O
164 Wogonoside Wogonin 165 166 Figure 1: Cont’d 167 168 O OH
S OH HO S S
R H3CO O O S OH
O O O HO O O O
HO S S
S R S HO OH H3CO OH OH O OH O OH O
2'-Methoxy-chrysin 7-O-Glucoronide Chrysin Oroxylin A 7-O-Glucoronide
O OH O OH
OH OH S S HO S S HO S S
O R OH OH O R HO S OH S OH
O O HO O O O
HO HO HO
OH O OH O OH O
169 Scutellarin Scutellarein Ikonnikoside I