Quantification of Procyanidins (Acid Butanol Method) by Visible Light Spectroscopy

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1.0 Purpose 
 
 This document describes the analytical procedure for the quantification of procyanidins in raw
 materials and finished products. 
 
 2.0 Scope 
 
 This procedure applies to the quantification of procyanidins in raw materials and finished
 products. The Acid Butanol method involves the HCl catalyzed depolymerization of condensed
 
 tannin in butanol to yield a red anthocyanin product that can be detected
 spectrophotometrically. The method is by nature non-specific, and expresses procyanidin
 
 content as equivalents USP Maritime Pine Extract. This method was validated under Protocol
 
 MV-LAB-19-183. 
 
 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
 
 Personnel to keep this procedure current with the associated monographs and laboratory
 practices. 
 
 4.0 Definitions 
 
 4.1 QC — Quality Control 
 
 4.2 USP -— United States Pharmacopeia 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 Quantification of Procyanidins (Acid Butanol Method) D-790 
 Page 2 of 6 
 by Visible Light Spectroscopy 
 
 4.3 MPE — Maritime Pine Extract 
 
 44 FAS-~- Ferric Ammonium Sulfate Dodecahydrate 
 
 5.0 References 
 
 5.1 MV-LAB-19-183, Protocol, Quantification of Procyanidins (Acid Butanol Method) by
 
 Visible Light Spectroscopy 
 
 5.2. USP Monograph: Maritime Pine Extract, Current Edition. 
 
 6.0 Supplies 
 
 6.1 Chemicals: (Use reagent grade or better.) 
 
 6.1.1 Milli-Q Water 
 
 6.1.2 HCl 
 
 6.1.5 Butanol 
 
 6.1.4 Methanol 
 
 6.1.5 FAS 
 
 6.1.6 USP MPE 
 
 6.2 Supplies and Glassware 
 
 6.2.1 Polystyrene (PS) Cuvettes (Micro or Semi-Micro) 
 
 6.2.2 Volumetric Glassware 
 
 6.2.3 Adjustable Pipettes & Tips 
 
 6.2.4 Weigh Paper & Boats 
 
 6.2.5 Graduated Cylinders 
 
 6.2.6 Beakers 
 
 6.2.7 15 mL Polypropylene Centrifuge Tubes 
 
 6.2.8 40 mL Glass Vials w/ Bonded Septum Caps 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Quantification of Procyanidins (Acid Butanol Method) D-790 1 Page 3 of 6 
 by Visible Light Spectroscopy 
 
 6.3. Equipment 
 
 6.3.1 UV/Vis Spectrophotometer 
 
 6.3.2 Analytical Balance 
 
 6.3.3 Sonicator Bath 
 
 6.3.4 Wrist Action Shaker 
 
 6.3.5 Vortex Mixer 
 
 6.3.6 Heated Water Bath 
 
 6.3.7 Thermometer 
 
 6.3.8 Timer 
 
 6.3.9 Centrifuge 
 
 7.0 Procedure 
 
 7.1 Instrument, Water Baths & Reaction Solution Preparations 
 
 7.1.1 Turn on the spectrophotometer and allow it to initialize. Open the PerkinElmer
 WinLab software and log in. Click on Instruments in the Folder List, then click
 
 Manual Control in the uppermost navigation ribbon. In Settings, enter 551 nm in
 the Wavelength dialog box. Leave the remaining settings unchanged at the
 
 default values. Click the Apply button and let the instrument warm up for
 
 ~30min. 
 
 7.1.2 Select two beakers, each being large enough to accommodate the number of
 40mL vials required to contain the blank, standards and samples. Fill ~1/3 full
 
 with DI water. Cover one with aluminum foil and place in a freezer. Retrieve the
 beaker in the freezer occasionally and break up the surface in order to obtain an
 
 ice bath. Place the other in a 95°C water bath. 
 
 7.1.3 Reagent Solution A — Add 5 mL HCl to 95 mL Butanol and mix well.
 (Preparations may be scaled as necessary. Make fresh daily.) 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 Quantification of Procyanidins (Acid Butanol Method) D-790 | Page 4 of 6 
 
 by Visible Light Spectroscopy 
 
 7.1.4 Reagent Solution B — Dissolve 2g FAS in a mixture of 100 mL water + 17.5 mL
 HCL and mix well. (Preparations may be scaled as necessary. Solution is good
 
 for 15 days.) 
 
 7.2 Standard Prep 
 
 7.2.1. Transfer ~67 mg USP MPE into a 50 mL volumetric flask. Add ~30 mL of
 
 methanol and swirl to dissolve. Dilute to volume with methanol and mix well.
 This is the USP MPE Stock Solution. 
 
 7.2.2 Transfer 600, 800, 1000, 1200 & 1400 uL aliquots of the USP MPE Stock
 
 Solution into individual 10 mL volumetric flasks. Dilute to volume with
 
 methanol and mix well. 
 
 7.3. Sample Extraction 
 
 7.3.1. The validated range for the analytical method is 0.054 — 0.126 mg/mL.
 
 7.3.2 Raw Materials: Given the (vendor declared) raw material assay value, dissolve
 the sample in methanol at a concentration at or below 2.5 mg/mL, then dilute to
 
 a convenient concentration within the validated range. 
 
 7.3.3 Finished Products: Given the finished product formulation, transfer a calculated
 mass of sample to a 100 mL volumetric flask such that the concentration is at or
 
 below 0.0825 mg/mL. Add 50 mL of methanol and shake mechanically for 15
 
 minutes, then dilute to volume with methanol and mix thoroughly. Sonicate for
 20 minutes, mix thoroughly and allow to cool to ambient temperature. Transfer
 
 ~10 mL to a 15 mL centrifuge tube and spin down at 4000 rpm for 3 minutes.
 Be careful not to disturb the clear supernatant for use in the chromogen
 
 formation reaction. 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 Quantification of Procyanidins (Acid Butanol Method) D-790 1 Page 5 of 6 
 
 by Visible Light Spectroscopy 
 
 7.4 Chromogen Formation Reactions 
 
 7.4.1 Assemble and run the reactions in the 40 mL glass vials as indicated in the table
 
 below: 
 
 Blank Standard Sample 
 Reagent Solution A, mL 6.0 6.0 6.0 
 Reagent Solution B, uL 250 250 250 
 Standard Prep, uL aan 1000 --- 
 Sample Prep, uL aa aa 1000 
 Methanol, uL 1000 
 Cap tightly, vortex mix then submerge in the 95°C bath for 40 minutes.
 Transfer the reaction vials to the ice bath for ~1 minute then vortex mix and allow
 to stabilize at ambient temperature. 
 
 7.5 Absorbance Readings 
 
 7.5.1 Transfer the blank reaction solution to a cuvette. Orient at the 1 cm pathlength
 
 in position 1 of the sample holder. 
 
 7.5.2 Click the Actions tab then select Autozero. Click OK in the UV WinLab dialog
 box when prompted in order to zero the instrument on the blank.
 
 7.5.3. Record a single absorbance reading for each of the standard and sample
 
 preparations. 
 
 7.6 System Suitability Requirements 
 
 7.6.1. Graph the standard concentration versus the absorbance and obtain the
 regression line equation and correlation coefficient (1). 
 
 7.6.2 The assay is considered valid if r? > 0.99. 
 
 7.7 Calculations for Raw Material % Assay 
 
 7.7.1. Sample Concentration, mg/mL = (Sample Mass, mg (Corr. for Water) / Stock
 Volume, mL) * Dilution Factor, mL/mL 
 
 7.7.2. USP MPE Eq’s in Sample, mg/mL = (Absorbance — Intercept)/Slope
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 Quantification of Procyanidins (Acid Butanol Method) D-790 Page 6 of 6 
 
 by Visible Light Spectroscopy 
 
 7.7.3 Content of Procyanidins, % = (USP MPE Eq’s in Sample, mg/mL / Sample
 Concentration, mg/mL) * 100 
 
 7.8 Example Calculation for Finished Product % Label Claim 
 
 7.8.1 Label Claim, % = (% Assay/100 * Serving Size, mg)/ Label Claim, mg) * 100
 
 8.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 11/27/19 | New N/A C. Perry 03/16/23 Scheduled review: updated logo and format. CC-23-0140 K. Burris | - | - |