Determination of Chrysin & 3,3-Diindolymethane by HPLC-UV

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1.0 Purpose 
 
 This document describes the analytical procedure for the determination of Chrysin and 3,3-
 Diindolylmethane in raw materials and finished products. 
 
 2.0 Scope 
 
 This procedure applies to the identification and quantification of Chrysin and 3,3-
 Diindolylmethane in raw materials and finished products. This method was validated under
 
 Protocol MV-LAB-19-124. 
 
 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 QC Laboratory Management to implement this procedure and
 to ensure that the procedure is being followed. 
 
 3.3 It is the responsibility of QC Laboratory Management and/or Analytical Development
 
 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 H3PQ.4 — Phosphoric Acid 
 4.4 MeOH — Methanol 
 
 4.5 CHSN — Chrysin 
 
 4.6 DilM — 3,3-Diindolylmethane 
 
 
 

[SOP D-774 | Page 2 of 7]

 Standard Operating Procedure SOP No Rev 
 Determination of Chrysin & 3,3-Diindolylmethane by D-774 
 Page 2 of 7 
 HPLC/UV 
 4.7 HPLC — High Performance Liquid Chromatography 
 
 4.8 UV/Vis — Ultraviolet & Visible Electromagnetic Spectra 
 
 5.0 References 
 
 5.1 MV-LAB-19-124, Protocol, Chrysin & 3,3-Diindolylmethane Determination Using
 
 HPLC with UV/Vis Spectroscopy 
 
 6.0 Reagents, Supplies, Glassware, and Equipment 
 
 6.1 Chemicals — All reagents are ACS grade or better 
 
 6.1.1 Milli-Q Water 
 
 6.1.2 MeOH 
 
 6.1.3 H3PO4 
 
 6.1.4 CHSN Reference Standard 
 
 6.1.5 DilM 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 or funnels 
 
 6.2.4 10ml Syringes with 17mm x 0.45u 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 Balance 
 
 6.3.3. Sonicator bath 
 
 6.3.4 Wrist Action Shaker 
 
 
 

[SOP D-774 | Page 3 of 7]

 Standard Operating Procedure SOP No | Rev 
 Determination of Chrysin & 3,3-Diindolylmethane by D-774 I Page 3 of 7 
 HPLC/UV 
 
 7.0 Procedure 
 
 7.1 Mobile Phase, Extraction Solvent & Diluent Preparation 
 
 7.1.1 Mobile Phase A — 0.1% H3PO4 
 
 7.1.1.1 Combine 1 mL of H3P04 with 1000 mL of Milli-Q Water. Mix well.
 
 7.1.2 Mobile Phase B - MeOH 
 
 7.1.3. Extraction Solvent - MeOH 
 
 7.1.4 Diluent -MeOH 
 
 7.1.5 Preparations may be scaled as necessary 
 
 7.2 Standard Prep 
 
 7.2.1. Accurately weigh and transfer about 25 mg of CHSN and DiIM reference
 
 standards into a 50-mL volumetric flask. Add 35mL of Extraction Solvent.
 Sonicate for Smin, cool, then QS to volume with Extraction Solvent.
 
 LLL Dilute 1:50 w/ Diluent for a Working Standard concentration of 10 pg/ml.
 
 7.3 Sample Preparation 
 
 7.3.1 The validated range for the analytical method is 12.8 — 255.4 ng CHSN and 13.0
 — 259.4 ng DilIM on column. 
 
 132 For finished products, at least 10 dosage units are pooled and ground by mortar
 
 and pestle. Based on the fill weight (for capsules) or tablet weight per dose and
 the label claim, weigh no less than 716 mg of the pooled dosages into a suitably
 
 sized volumetric flask to generate an analyte concentration that is within the
 
 validated linearity range for the analyte being tested. 
 
 7.3.3 Prepare raw materials like standards. (However, be sure to consult the
 specification for expected potency, as raw material samples may not be 100%.)
 
 7.3.4 Samples can be extracted in Extraction Solvent at any volume starting from
 
 25mL. The volume chosen must be in the solubility range of CHSN & DiIM
 (validated at 0.01 mg/ml). To manage large volumes, the sample can be initially
 
 
 

[SOP D-774 | Page 4 of 7]

 Standard Operating Procedure SOP No | Rev 
 Determination of Chrysin & 3,3-Diindolylmethane by D-774 i Page 4 of 7 
 HPLC/UV 
 
 dissolved in a smaller volume that is within the solubility range and a portion
 
 further diluted to bring the analyte concentration into the linear range.
 
 7.3.5 Fill the flask to about 70% of the calculated volume with Extraction Solvent and
 
 shake mechanically for 10 minutes. Sonicate for 5 minutes, cool to room
 temperature and bring up to volume with Extraction Solvent. Filter a portion for
 
 use in subsequent dilutions. 
 
 7.3.6 Perform further dilutions as required (if applicable) using Diluent.
 
 7.3.7 For finished products or raw materials being analyzed for the first time using
 
 this method, an in process validation is required to demonstrate spectral purity,
 baseline separation of peaks, and extraction efficiency as a part of system
 
 suitability before data can be reported using this method. 
 
 7.4 HPLC Parameters 
 
 7A Column: Agilent InfinityLab Poroshell 120 EC-C18, 4.6 x 100mm, 2.7u
 
 7.4.2 Column Temperature: 40°C 
 
 7.4.3 Flow rate: 0.9 mL/min 
 
 7.4.4 Wavelength: 284 nm 
 
 7.4.5 Injection Volume: 5 uL 
 
 7.4.6 Run Time: 10 minutes. 
 
 7.4.7 3-D Spectral Range (for Identification) - 200nm to 400nm 
 
 7.4.8 Mobile Phase Gradient - Isocratic 
 
 7.5 Recommended Sequence 
 
 7.5.1 Make at least 2 injections of the Diluent. 
 
 7.5.2 Make five (5) injections of Working Standard. 
 
 7.5.3 Make a single injection of each Sample Preparation. 
 
 7.5.4 Make a single injection of the Working Standard after every ten (10) sample
 
 injections or at the end of a run. 
 
 
 

[SOP D-774 | Page 5 of 7]

 Standard Operating Procedure SOP No Rev 
 Determination of Chrysin & 3,3-Diindolylmethane by D-774 | Page 5 of 7 
 HPLC/UV 
 
 7.6 System Suitability Requirements 
 
 7.6.1 The %RSD of the first five (5) standard injections is NMT 2.0%
 
 7.6.2 The %RSD of all standard injections is NMT 3.0%. 
 
 7.6.3 If present, any interference in the diluent should be subtracted out of the sample
 and standard peak areas. 
 
 7.7 Example calculations for determining finished product % label or raw material % purity
 
 7.7.1 % Analyte = = x an <x 2 x 100 
 
 Ry Sample peak area 
 
 R, Mean (n=5) standard peak area 
 
 Wteta Weight of the reference standard in mg (corr. for water if applicable)
 
 V sta Volume of the standard preparation accounting for dilutions in mL
 
 P Purity of the reference standard in decimal format 
 
 SA Sample amount in mg (solids) or mL (liquids) 
 
 Veni Volume of the sample preparation accounting for dilutions in mL
 
 SS Serving size: Average weight of ten dosage units in mg for tablets,
 fill weight for capsules, mass of a single serving in mg for powders,
 
 volume of a single serving from the theoretical formula in mL for
 
 liquids, or 1 for raw materials. 
 
 LA Label amount in mg of analyte (use 1 for raw materials)
 
 7.8 Column Wash and Storage 
 
 7.8.1 Wash and store the column in 75:25 MeOH / Milli-Q Water. 
 
 
 

[SOP D-774 | Page 6 of 7]

 Standard Operating Procedure SOP No Rev 
 Determination of Chrysin & 3,3-Diindolylmethane by D-774 Page 6 of 7 
 HPLC/UV 
 
 8.0 Chromatograms 
 
 8.1 Typical Diluent Chromatogram 
 
 8.2 Typical Working Standard Chromatogram 
 
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[SOP D-774 | Page 7 of 7]

 Standard Operating Procedure SOP No | Rev 
 Determination of Chrysin & 3,3-Diindolylmethane by D-774 \ Page 7 of 7 
 HPLC/UV 
 
 8.3. Typical Sample Chromatogram 
 
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 9.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 06/26/19 | New N/A C. Perry 06/22/22 Updated logo and format. CC-22-0287 K. Burris | - | - |