Pterostilbene Determination by HPLC with UV-Vis Spectroscopy

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1.0 Purpose 
 
 This document describes the analytical procedure for the determination of Pterostilbene (PSB)
 
 in raw materials and finished products. 
 
 2.0 Scope 
 
 This procedure applies to the identification and quantification of PSB in raw materials and
 
 finished products. 
 
 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 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. AD-— Analytical Development 
 
 4.3 PSB — Pterostilbene 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Pterostilbene Determination by HPLC with UV/Vis D-798 ' Page 2 of 9 
 Spectroscopy 
 
 4.4 ACN -— Acetonitrile 
 
 4.5 MeOH — Methanol 
 
 4.6 H3P04 — Phosphoric Acid 
 
 4.7 ACS-— American Chemical Society 
 
 4.8 HPLC — High Performance Liquid Chromatography 
 
 4.9 UV/Vis — Ultraviolet & Visible Electromagnetic Spectra 
 
 5.0 References 
 
 5.1 PRTCL-20-0043, Protocol, Pterostilbene Determination by HPLC Using UV/Vis
 
 Spectroscopy 
 
 5.2 RPT-20-0036, Report, Pterostilbene Determination by HPLC Using UV/Vis
 
 Spectroscopy 
 
 5.3 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 H3PO04 
 
 6.1.4 MeOH 
 
 6.1.5 PSB Reference Standard 
 
 
 

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 Pterostilbene Determination by HPLC with UV/Vis D-798 I Page 3 of 9 
 Spectroscopy 
 
 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 0.45u 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 Wrist Action Shaker 
 
 6.3.4 | Sonicator Bath 
 
 7.0 Procedure 
 
 dl Mobile Phase & Diluent Preparation 
 
 71.1 Mobile Phase 
 
 7.1.1.1. Mobile Phase A: Add 1000 uL of H3P04 to 1000 mL of water and
 
 mix well. 
 
 7.1.1.2 Mobile Phase B: ACN 
 
 7.1.2 Extraction Solvent = Diluent = MeOH 
 
 7.1.3 Preparations may be scaled as necessary 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 
 Pterostilbene Determination by HPLC with UV/Vis D-798 1 Page 4 of9 
 Spectroscopy 
 
 7.2 Standard Prep 
 
 7.2.1. Accurately weigh and transfer about 25 mg of PSB reference standard into a 100
 mL volumetric flask. Add ~50 mL of Diluent and sonicate for five minutes.
 
 7.2.2 Cool to ambient then dilute to volume with Diluent and mix well — this is the
 
 PSB Stock. Dilute the PSB Stock 2:100 with Diluent — this is the PSB Working
 Standard. 
 
 7.3 Sample Preparation 
 
 7.3.1 Specific sample testing details are provided in each products profile. If a specific
 
 testing details section is not available, then follow preparation procedure as
 described below, maintaining concentration within the linear range listed below.
 
 7.3.2 The validated range for the analytical method is 0.00246 — 0.0123 mg/mL.
 
 1.1.1 For raw materials: weigh no less than 25 mg into a suitably sized volumetric
 
 flask of no less than 100 mL volume. Fill the flask to about 50% of the flask
 
 volume with Diluent and shake mechanically for 15 minutes. Sonicate for 5
 minutes, cool to ambient then dilute to volume with Diluent.
 
 1.1.2 For solid and liquid dose finished products: Combine and homogenize no less
 than ten dosage units. Based on the label claim and fill weight (capsules),
 
 serving size (powders and liquids) or tablet weight per dose, weigh no less than
 100 mg of the pooled dosages into a suitably sized volumetric flask of no less
 
 than 100 mL. Fill the flask to about 50% of the flask volume with Diluent and
 
 shake mechanically for 15 minutes. Sonicate for 5 minutes, cool to ambient then
 dilute to volume with Diluent. 
 
 1.1.3. For chewable gels (gummies), homogenize at least 10 dosage units according to
 the procedure outlined in D-793 Cryogenic Grinding of Chewable Gels. Quickly
 
 weigh no less than 200 mg of the pooled and homogenized dosages into a
 
 
 

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 Pterostilbene Determination by HPLC with UV/Vis D-798 1 Page 5 of 9 
 Spectroscopy 
 
 suitably sized beaker. Add a volume of Diluent equivalent to 50% of the desired
 
 flask volume, add a stir bar, and stir until dissolved. Transfer the solution to a
 
 volumetric flask of the desired size. Use several small portions of Diluent to
 rinse any remaining residue from the beaker into the volumetric flask ensuring
 
 complete transfer, and dilute to volume using Diluent. 
 
 To manage large volumes, the sample can be initially dissolved in a smaller
 
 volume and a portion further diluted using Diluent to bring the analyte
 concentration into the linear range. Dilutions can be made using volumetric
 
 glassware and/or adjustable pipettes. Dilutions can be prepared in HPLC vials.
 
 7.3.3 Filter through a 0.45 um membrane discarding the first 3 — 4 mL before
 collecting a portion for analysis. Alternatively, centrifuge an aliquot of the final
 
 sample at 10,000 rpm for 5 min to remove particulates. 
 
 7.4 HPLC Parameters 
 
 TAA Column: Restek Raptor ARC-18, 4.6 x 150mm, 2.7m (Or Equivalent)
 
 7.4.2 Column Temperature: 35°C 
 
 7.4.3 Flow rate: 1.0 mL/min 
 
 7.4.4 Mobile Phase: Isocratic 50% A / 50% B 
 
 7.45 Wavelength: 330 nm 
 
 7.4.6 Injection Volume: 5 wL 
 
 7.4.7 Run Time: 10 minutes 
 
 
 

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 Pterostilbene Determination by HPLC with UV/Vis D-798 1 Page 6 of 9 
 Spectroscopy 
 
 7.5 Recommended Sequence 
 
 7.5.1. Make at least 2 injections of the Diluent. 
 
 7.5.2. Make at least five (5) injections of PSB Working Standard. 
 
 7.5.3. Make a single injection of each Sample Preparation. 
 
 7.5.4 Make a single injection of the Standard Solution after every ten (10) sample
 
 injections and/or at the end of a run. 
 
 7.6 System Suitability Requirements 
 
 7.6.1 The %RSD of five (5) consecutive standard injections is NMT 2.0%
 
 7.6.2 The %RSD of all standard injections is NMT 3.0%. 
 
 76.3 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 % LC or raw material % assay:
 
 771 % — RRuu = . x , WtstaxP ., SS x V < s 2 pl x 100 
 Rs Vstd SA 
 
 Ru Sample peak area 
 
 R, Mean (n=5) standard peak area 
 
 Weta Weight of the reference standard in mg 
 
 Veta Volume of the standard preparation accounting for dilutions in mL
 
 P Purity of the reference standard in decimal format 
 
 SA Sample amount in g 
 
 
 

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 Standard Operating Procedure SOP No | Rev 
 
 Pterostilbene Determination by HPLC with UV/Vis D-798 1 Page 7 of9 
 Spectroscopy 
 
 SS. Serving size in g: Average weight of ten dosage units for tablets, fill
 
 weight for capsules, mass of a single serving for powders, volume of
 
 a single serving from the theoretical formula for liquids, or 1 for raw
 materials. 
 
 Veni Volume of the sample preparation accounting for dilutions in mL
 
 LA Label amount of analyte in mg. (Use 1 for raw materials.)
 
 7.8 System Wash, Column Wash and Column Storage 
 
 7.8.1 Wash and store the column in 50:50 ACN / Water. 
 
 8.0 Example Chromatograms 
 
 8.1 Typical Diluent Chromatogram 
 
 min| 
 
 
 

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 Standard Operating Procedure SOP No Rev 
 
 Pterostilbene Determination by HPLC with UV/Vis D-798 Page 8 of 9 
 Spectroscopy 
 
 8.2 Typical Working Standard Chromatogram 
 
 © ff mau 
 
 8.3. Typical Raw Material Chromatogram 
 
 
 

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 Standard Operating Procedure SOP No | Rev 
 
 Pterostilbene Determination by HPLC with UV/Vis D-798 I Page 9 of 9 
 Spectroscopy 
 
 8.4 — Typical Finished Product Chromatogram 
 
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 9.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 09/01/20 | New N/A C. Perry Add instruction to follow product specific test details if available, | - | - |
| 1 | 04/03/24 | add specific sample prep instructions for gummies, edit for CC- consistency with current methods. | 24-0129 | S. Sassman |