Determination of Isoflavones in Raw Materials and Finished Products by HPLC-UV

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1.0 Purpose 
 This document describes the analytical procedure for the determination of Isoflavones in raw
 
 materials and finished products. 
 2.0 Scope 
 
 This procedure applies to the identification and quantification of Isoflavones in raw materials
 and finished products. Total Isoflavones is calculated as the sum of Daidzein, Genistein,
 
 Formononetin and Biochanin A. This method was validated under protocol PRTCL-21-0049.
 3.0 Responsibility 
 
 3.1 ‘It is the responsibility of QC and Analytical chemists who have verified their ability to
 execute this procedure to follow this procedure. 
 
 3.2 ‘It is the responsibility of the QC Laboratory Management to implement this procedure
 and to ensure that the procedure is being followed. 
 
 3.3. ‘It is the responsibility of the QC Laboratory Management and AD Personnel to keep this
 procedure current with the associated monographs and laboratory practices.
 
 4.0 Definitions 
 
 4.1 QC — Quality Control 
 
 4.2 AD-— Analytical Development 
 4.3 ACN -— Acetonitrile 
 
 4.4 DMSO — Dimethylsulfoxide 
 
 4.5 | MeOH — Methanol 
 4.6 TFA — Trifluoroacetic Acid 
 
 4.7 ACS — American Chemical Society 
 
 4.8 | HPLC — High Performance Liquid Chromatography 
 49 UV-—Ultraviolet (Detection) 
 
 
 

[SOP D-100 | Page 2 of 8]

 Standard Operating Procedure 
 SOP No Rev 
 Determination of Isoflavones in Raw Materials and Page 2 of 8 
 D-1001 
 Finished Products by HPLC-UV 
 5.0 References 
 5.1 PRTCL-21-0049, Protocol, Validation of an Analytical Method for the Determination of
 
 Isoflavones in Raw Materials & Finished Products by HPLC-UV 
 5.2. D-793, SOP, Cryogenic Grinding of Chewable Gels 
 
 6.0 Supplies 
 
 6.1 Chemicals — All reagents are ACS grade or better. 
 
 6.1.1 Milli-Q Water 
 6.1.2 ACN 
 
 6.1.3 MeOH 
 
 6.1.4 DMSO 
 6.1.5 TFA 
 
 6.1.6 Daidzein Reference Standard 
 
 6.1.7 Genistein Reference Standard 
 6.1.8 Formononetin Reference Standard 
 
 6.1.9 Biochanin A Reference Standard 
 
 6.2 Supplies and Glassware 
 6.2.1 HPLC vials, 12mm X 32mm with screw cap enclosures w/ septa 
 
 6.2.2 Volumetric glassware and/or adjustable pipettes and tips 
 
 6.2.3. Weigh paper and/or funnels 
 6.2.4 Syringes with 0.45 Nylon Syringe Filters 
 
 6.3. Equipment 
 
 6.3.1 Suitable gradient HPLC system consisting of a pump, autosampler, column oven
 and UV detector with a chromatographic data handling system 
 
 6.3.2 Analytical and/or Top Loading Balance 
 6.3.3. Analytical Micro Balance 
 
 6.3.4 Sonicator Bath 
 
 7.0 Procedure 
 
 7.1 Mobile Phase & Diluent Preparation 
 7.1.1. Mobile Phase 
 
 

[SOP D-100 | Page 3 of 8]

 Standard Operating Procedure 
 SOP No Rev 
 Determination of Isoflavones in Raw Materials and D-1001 0 Page 3 of8 
 Finished Products by HPLC-UV 
 
 7.1.1.1 Mobile Phase A: Combine 250 mL ACN + 750 mL Water + 500 uL of
 TFA and mix well. 
 
 7.1.1.2 Mobile Phase B: Combine 1000 mL ACN + 500 uL of TFA and mix
 well. 
 
 7.1.2 Extraction Solvent / Diluent 
 7.1.2.1 Combine equal volumes of MeOH and DMSO and mix well. Allow the
 resulting solution to equilibrate to room temperature prior to use.
 
 7.1.3. Preparations may be scaled as necessary 
 
 7.2 Standard Prep 
 7.2.1. Solution A: Accurately weigh and transfer about 5 mg of Daidzein reference
 
 standard into a 100 mL volumetric flask. QS to volume with Diluent and sonicate
 until dissolved. 
 
 7.2.2 Solution B: Accurately weigh and transfer about 5 mg of Genistein reference
 standard into a 100 mL volumetric flask. QS to volume with Diluent and sonicate
 until dissolved. 
 
 7.2.3. Working Standard: Accurately weigh and transfer about 10 mg of Formononetin
 reference standard into a 50 mL volumetric flask with the aid of ~15 mL Diluent.
 Accurately weigh and transfer about 10 mg of Biochanin A reference standard
 into the same 50 mL volumetric flask with the aid of ~15 mL Diluent. Using glass
 
 volumetric pipets, transfer 5 mL of Solution A and 5 mL of Solution B into the
 50 mL volumetric flask containing the Formononetin and Biochanin A. QS to
 volume with Diluent and sonicate until dissolved. 
 
 7.3 Sample Preparation 
 7.3.1 The validated linear range for the analytical method is 0.96034 — 4.80168 ug/mL
 
 Daidzein, 0.98529 — 4.92646 ug/mL Genistein, 41.81429 — 209.07144 ug/mL
 Formononetin and 42.46386 — 212.31930 ug/mL Biochanin A. 
 7.3.2 Extract sufficient sample (based on the raw material manufacturer assay value or
 
 finished product label claim) with Extraction Solvent in order to generate a
 concentration that is within the validated linear range. 
 7.3.3. When analyzing powders, fill the flask to 80% of the chosen volume with
 
 Extraction Solvent and sonicate for 10 minutes. Cool to ambient temperature then
 QS to volume. Filter a Sml aliquot for analysis, discarding the first 3-4ml of
 filtrate. 
 
 

[SOP D-100 | Page 4 of 8]

 Standard Operating Procedure 
 SOP No Rev 
 Determination of Isoflavones in Raw Materials and D-1001 0 Page 4 of 8 
 Finished Products by HPLC-UV 
 
 7.3.4 When analyzing gummies, prepare the samples as per D-793 utilizing a 20 minute
 beaker stir time. QS to volume in the volumetric flask and add a 10 minute
 sonication. Filter a Sml aliquot for analysis, discarding the first 3-4ml of filtrate.
 
 7.3.5 For materials being analyzed for the first time using this method, an in process
 validation is required to demonstrate spectral purity and extraction efficiency as
 a part of system suitability before data can be reported using this method.
 
 7.4 HPLC Parameters 
 
 74.1 Column: Supelco Ascentis Express 90A Cs, 4.6 x 100mm, 2.7um SPP (Or
 Equivalent) 
 
 7 A2 Column Temperature: 45°C 
 74.3 Flow rate: 0.5 mL/min 
 
 74.4 Mobile Phase: Gradient 
 
 7.4.4.1 Time,min %B 
 0.00 0 
 
 2.00 Q 
 
 20.00 60 
 20.10 0 
 
 25.00 0 
 
 7.4.5 Wavelength: 254 nm 
 7.4.6 Injection Volume: 5 wL 
 
 7.4.7 Run Time: 25 minutes 
 
 74.8 3-D Spectral Range (for Identification): 210nm - 350nm 
 7.5 | Recommended Sequence 
 
 7.5.1 Make at least 2 injections of the Diluent. 
 
 Tae Make at least five (5) injections of Working Standard. 
 7.5.3 Make a single injection of each Sample Preparation. 
 
 7.6 System Suitability Requirements 
 
 7.6.1 The %RSD of five (5) consecutive standard injections is NMT 2.0%
 7.6.2 If present, any interference in the diluent should be subtracted out of the sample
 and standard peak areas. 
 
 7.7. Example calculations for determining % assay / label claim: 
 
 

[SOP D-100 | Page 5 of 8]

 Standard Operating Procedure 
 Determination of Isoflavones in Raw Materials and pag + Page 5 of 8 
 Finished Products by HPLC-UV 
 
 7.7.1 % = oe x Maat? x Sx x 100 
 
 Ry Sample peak area 
 
 R, Mean (n=5) standard peak area 
 Wtz.q Weight of the reference standard 
 
 Vista Volume of the standard preparation accounting for dilutions in mL
 
 P Purity of the reference standard in decimal format 
 
 SA Sample amount 
 hh Serving size: Average weight of ten dosage units or theoretical weight
 in mg for tablets / gummies, theoretical fill weight for capsules,
 
 theoretical mass of a single serving in mg for powders, volume of a
 single serving from the theoretical formula in ml for liquids, or use 1 for
 raw materials. 
 
 LA Label amount in mg of analyte (use | for raw materials)
 
 Vent Volume of the sample preparation accounting for dilutions in mL
 
 7.8 System Wash, Column Wash and Column Storage 
 
 7.8.1. Wash and store the column in 75:25 ACN / Water. 
 
 

[SOP D-100 | Page 6 of 8]

 Standard Operating Procedure SOP N R 
 Determination of Isoflavones in Raw Materials and ty a 
 Page 6 of 8 
 Finished Products by HPLC-UV 
 
 8.0 Chromatograms 
 8.1 Typical Diluent Chromatogram 
 
 1 __ = Red Clover Isotiazone Run 10-19-21 #2 Odvent iS 1M 4 
 ae mAU 
 
 $46 
 $254 
 40%4 
 375 
 386 
 323 
 HG 
 2? 
25.0 225

 200 
 459 
 i 
 q| 
 { 
 i}4| 
 g g § 
 { t\ \ © t a - i m f c 2= y at een ee e 8z 9- e , 7 >2. ee ciee — jp apace :a;
 ran 
 27Ca 3 Q ' 12 :3 0 ‘ 1 6 20S0 SS 2 :23 
 8.2 Typiciacall Standdaarr d Chromatogram 
 j Red ¢ ¢ lsofiazone Run 10-18-21 #3 TO Uy_viS_1 
 mAU 
 
 109 a3 r r 5: 2& ii = & nS2 | 8 $=2&8 
 +. 3 A e¢ fF =]i Se. **Oe Bow 
 TLS aN vAa ts a\ e ¥®. beP4 lto.} y 3 T ies t P* TTOrT pAo, raO m twreset
 Tart 
 2 240 3 50 8 10 0t 0 1 1 , 1 18 212To5 2 23 
 
 

[SOP D-100 | Page 7 of 8]

 Standard Operating Procedure 
 SOP No Rev 
 Determination of Isoflavones in Raw Materials and Page 7 of 8 
 D-1001 0 
 Finished Products by HPLC-UV 
 8.3 Typical Raw Material Chromatogram 
 183R4] 7 m R A ed Claovwer isoflavone Run 19-19-21 213 ReA-t EEO SxVc L2H om
 oh 
 149 4 
 SNE> 
 E 
 SE= 
 is4 Ara A A A 
 126 - 
 1194 
 
 Zt096 
 OSLBROt- 
 0© 
 c 
 a 
s 
e668 
 CF 
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 min 
 140 1a6r0 
 
 8.4 Typical Finished Product Chromatogram 
 169 Y Red Ciover isofiavona Run 12-19-21 #1é FP 
 1604 
 “ 
 ) 
 
 _ 
 e 
 r 
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 rat 
 16T0 17T9 1 
 
 

[SOP D-100 | Page 8 of 8]

 Standard Operating Procedure 
 Determination of Isoflavones in Raw Materials and reer = Page 8 of 8 
 Finished Products by HPLC-UV 
 
 9.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 11/05/21 | New N/A C. Perry | - | - |