a-Mangostin Determination by HPLC with UV-Vis Spectroscopy

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1.0 Purpose 
 
 This document describes the analytical procedure for the determination of a-Mangostin in raw
 materials. 
 
 2.0 Scope 
 
 This procedure applies to the identification and quantification of a-Mangostin in raw materials.
 
 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. 
 
 Dun 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 AD — Analytical Development 
 
 4.3 ACN — Acetonitrile 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 o-Mangostin Determination by HPLC with UV/Vis D-799 1 Page 2 of 8 
 Spectroscopy 
 
 4.4 H3PO4 — Phosphoric Acid 
 
 4.5 ACS -— American Chemical Society 
 
 4.6 HPLC — High Performance Liquid Chromatography 
 
 4.7 UV/Vis — Ultraviolet & Visible Electromagnetic Spectra 
 
 5.0 References 
 
 5.1 PRTCL-20-0083, Protocol, a-Mangostin Determination by HPLC Using UV/Vis
 Spectroscopy 
 
 52 RPT-20-0045, Report, -Mangostin Determination by HPLC Using UV/Vis Spectroscopy
 
 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 a-Mangostin 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 
 
 
 

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 Standard Operating Procedure SOP No | Rev 
 a-Mangostin Determination by HPLC with UV/Vis D-799 1 Page 3 of8 
 Spectroscopy 
 
 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 
 
 7.1. 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 
 
 fh Extraction Solvent = Diluent = ACN 
 
 Pudi Preparations may be scaled as necessary 
 
 7.2 Stock Standard 
 
 Taek Accurately weigh and transfer about 25 mg of a-Mangostin reference standard
 into a 50 mL volumetric flask. 
 
 Tadd Add 30 mL of Diluent, and sonicate for five minutes. 
 
 Tideo Equilibrate to ambient temperature, dilute to volume with Diluent, and mix well.
 
 7.3 Working Standard 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 a-Mangostin Determination by HPLC with UV/Vis D-799 1 Page 4 of 8 
 Spectroscopy 
 
 7.3.1 Transfer 5.0 mL of the Stock Standard to a 25-mL volumetric flask.
 
 7.3.2 Dilute to volume with Diluent, and mix well. 
 
 7.4 Sample Preparation 
 
 7.4.1. The validated range for the analytical method is 0.049 — 0.247 mg/mL.
 
 7.4.2 Extract sufficient sample (based on the manufacturer assay value) with Diluent
 in order to generate a concentration that is within the validated linear range.
 
 7.4.3 Samples can be dissolved in Diluent at any volume starting from 100mL. The
 
 volume chosen must be in the solubility range of a-Mangostin (validated at ~100
 ug/ml). 
 
 7.4.4 To manage large volumes, the sample can be initially dissolved in a smaller
 volume that is within the solubility range and a portion further diluted to bring
 
 the o-Mangostin concentration into the linear range. 
 
 7.4.5 Fill the flask to about 60% of the chosen volume with Diluent and shake
 
 mechanically for 10 minutes. Dilute to volume, then sonicate for 5 minutes.
 Equilibrate to ambient temperature, and perform further dilutions as required
 
 using Diluent. 
 
 7.4.6 Filter a Sml aliquot for analysis, discarding the first 3-4ml of filtrate.
 Alternatively, centrifuge a portion at 10,000 rpm for 5 min to remove
 
 particulates. 
 
 7.5 HPLC Parameters 
 
 7.5.1. Column: Phenomenex Luna Cig (2), 4.6 x 250mm, 5pm (Or Equivalent)
 
 7.5.2 Column Temperature: 35°C 
 
 7.5.3 Flow rate: 1.5 mL/min 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 a-Mangostin Determination by HPLC with UV/Vis D-799 Page 5 of 8 
 Spectroscopy 
 
 7.5.4 Mobile Phase: Gradient 
 
 7.5.4.1. Time (min) %A %B 
 
 0 40 60 
 
 40 60 
 
 30 70 
 
 20 Zo 75 
 
 21 95 
 
 26 95 
 
 28 40 60 
 
 35 40 60 
 
 7.5.5 Wavelength: 318 nm 
 
 7.5.6 Injection Volume: 5 pL 
 
 7.5.7 Run Time: 35 minutes 
 
 7.5.8 | Recommended 3-D Spectral Range (for Identification) - 205nm to 350nm
 
 7.6 Recommended Sequence 
 
 7.6.1 Make at least 2 injections of the Diluent. 
 
 7.6.2 Make at least five (5) injections of a-Mangostin Working Standard.
 
 7.6.3 Make a single injection of each Sample Preparation. 
 
 
 

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 Standard Operating Procedure SOP No | Rev 
 a-Mangostin Determination by HPLC with UV/Vis D-799 1 Page 6 of 8 
 Spectroscopy 
 
 7.6.4 Make a single injection of the Standard Solution after every ten (10) sample
 injections and/or at the end of a run. 
 
 hd System Suitability Requirements 
 
 7.7.1 The %RSD of five (5) consecutive standard injections is NMT 2.0%
 
 7.7.2 The %RSD of all standard injections is NMT 3.0%. 
 
 7.7.3 If present, any interference in the diluent should be subtracted out of the sample
 
 and standard peak areas. 
 
 7.8 Example calculations for determining finished product raw material % assay:
 
 aw Ru 4, WteraXP y 1, Vapi 
 78.1 “%H = “— x Vent i ae Ka 100 
 
 Ru Sample peak area 
 
 R, Mean (n=5) standard peak area 
 
 Wtsta 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 mg 
 
 Vspi Volume of the sample preparation accounting for dilutions in mL
 
 L2 System Wash, Column Wash and Column Storage 
 
 7.9.1 Wash and store the column in 60:40 ACN / Water. 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 a-Mangostin Determination by HPLC with UV/Vis D-799 1 Page 7 of8 
 Spectroscopy 
 
 8.0 Example Chromatograms 
 
 8.1 Typical Diluent Chromatogram 
 
 8p nr ah Samra 
 
 7" 
 
 8.2. Typical Working Standard Chromatogram 
 
 | 
 
 8.3. Typical Raw Material Chromatogram 
 
 } 
 | 
 | 
 I 
 
 
 

[SOP 

 Standard Operating Procedure SOPNo | Rev 
 a-Mangostin Determination by HPLC with UV/Vis D-799 1 Page 8 of 8 
 Spectroscopy 
 
 9.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 10/08/20 | New N/A C. Perry | - | - |
| 1 | 04/03/24 | Scheduled review: edit for consistency with current methods CC- | 24-0130 | S. Sassman |