D-1021
Determination of Rhaponticin and Desoxyrhaponticin by HPLC-UV
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1.0 Purpose This document describes the analytical procedure for the determination of Rhaponticin & Desoxyrhaponticin in raw materials and finished products. 2.0 Scope This procedure applies to the identification and quantification of Rhaponticin & Desoxyrhaponticin in raw materials and finished products. This method was validated under protocol PRTCL-23-0069. 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 ACN - Acetonitrile [SOP D-102 | Page 2 of 7] Standard Operating Procedure SOP No Rev Determination of Rhaponticin & Desoxyrhaponticin by D-1021 Page 2 of 7 HPLC-UV 4.3. Hs3PO4- Phosphoric Acid 44 | ACS— American Chemical Society 4.5 HPLC - High Performance Liquid Chromatography 46 UV-—Ultraviolet (Detection) 5.0 References 5.1 PRTCL-23-0069, Protocol, Validation of an Analytical Method for the Determination of Rhaponticin & Desoxyrhaponticin by HPLC-UV 6.0 Supplies 6.1 Chemicals — All reagents are ACS grade or better 6.1.1 ACN 6.1.2 Acetone 6.1.3 H3PO4 6.1.4 Rhaponticin Reference Standard 6.1.5 Desoxyrhaponticin 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 6.2.3. Weigh paper 6.2.4. Syringes with 0.451 nylon syringe filters [SOP D-102 | Page 3 of 7] Standard Operating Procedure SOP No Rev Determination of Rhaponticin & Desoxyrhaponticin by | D-1021 Page 3 of 7 HPLC-UV 6.3. Equipment 6.3.1 Suitable gradient HPLC system consisting of a pump, autosampler, column compartment and UV-Vis detector with a chromatographic data handling system 6.3.2 Analytical Balance 6.3.3 Micro Analytical Balance 6.3.4 Wrist Action Shaker 7.0 Procedure 7.1 Mobile Phase & Extraction Solvent / Diluent Preparation 7.1.1 Mobile Phase 7.1.1.1. Mobile Phase A: Combine 850mL Milli-Q water + 150mL ACN + 500u1 H3PO4 and mix well. 7.11.2 Mobile Phase B: Combine 200mL Milli-Q water + 800mL ACN + 5001 H3POs4 and mix well. 7.1.2 Extraction Solvent / Diluent 7.1.2.1 Combine equal volumes of Milli-Q water and Acetone and mix well. 7.2 Standard Preparation 7.2.1 Prepare standard stock at ~0.03 - 0.06 mg/mL analyte in Extraction Solvent. Sonicate stock for 15 minutes at ~80% volume, cool to ambient then QS to volume. Shake vigorously then filter a SmL aliquot for analysis, discarding the first 3-4mL of filtrate. 7.3 Sample Preparation [SOP D-102 | Page 4 of 7] Standard Operating Procedure SOP No | Rev Determination of Rhaponticin & Desoxyrhaponticin by | D-102! 0 | Page 4 of 7 HPLC-UV 7.3.1 Specific sample testing details are provided in each product profile. If a specific testing details section is not available, follow preparation procedure as described below, maintaining concentration within the linear range of this method. 7.3.2 The validated linear range for the analytical method is 0.0243 — 0.0849 mg/mL Rhaponticin and 0.0246 — 0.0861 mg/mL Desoxyrhaponticin. 7.3.3 Pool at least 10 dosage units and homogenize as appropriate (e.g. grind tablets / capsule fill / powders / stick pack contents by mortar and pestle, cryogenically powder and dissolve gummies, etc.) Extract sufficient sample (based on the raw material manufacturer assay value / finished product profile) with Extraction Solvent in order to generate a sample stock that is ~0.3 — 0.6 mg/mL analyte. Sonicate stock for 15 minutes at ~80% volume, cool to ambient then QS to volume. Shake vigorously then dilute stock 1:10 with Diluent. Shake vigorously then filter a 5mL aliquot for analysis, discarding the first 3-4mL of filtrate. 7.4 HPLC Parameters 7.4.1 Column: Phenomenex Luna Cis(2), 100A, 5um, 4.6 x 250mm 7.4.2 Column Temperature: 30°C 7.4.3 Flow rate: 1.5 mL/min Time, min A %B 7.4.4 Run Time: 20 minutes 0 100 0 75 25 75 7.4.5 Mobile Phase Gradient 8.5 0 100 15 0 100 7.4.6 Wavelength: 310 nm 15.1 100 0 20 100 0 7.4.7 Injection Volume: 10 wL 7.4.8 Suggested 3-D Spectral Range (for Identification): 210nm - 400nm [SOP D-102 | Page 5 of 7] Standard Operating Procedure SOP No Rev Determination of Rhaponticin & Desoxyrhaponticin by D-1021 Page 5 of 7 HPLC-UV 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 Working Standard. 7.5.3. Makea 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 the 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%. 7.7 Example calculations for determining % Assay: 77.1 % = Ra x Mw x os % 10x SS0/LA Rs Veta KR. Sample peak area R, Mean (All) standard peak area Wtyg Weight of the reference standard, mg Vord Volume of the standard preparation accounting for dilutions, mL P Purity of the reference standard in decimal format SA Sample amount, mg a Volume of the sample preparation accounting for dilutions, mL SS Serving Size, mg (Use “1” for raw materials.) [SOP D-102 | Page 6 of 7] Standard Operating Procedure SOP No | Rev Determination of Rhaponticin & Desoxyrhaponticin by | D-1021 0 | Page 6 of 7 HPLC-UV LA Label Amount, mg (Use “1” for raw materials.) 7.8 System Wash, Column Wash and Column Storage 7.8.1 Wash system and column with 90% ACN. Store column on 50% ACN. 8.0 Chromatograms 8.1 Typical Diluent Chromatogram Poe | 12232 Divert UWS_.1WM.310nm ‘0 +0] 104 124 X04 ay a cal Da Vv r——/ "00 70 20 30 40 0 60 70 80 go ‘do +o 120 80 +80 170 +80 +80 ni2n0 8.2 Typiqcal Standard Chromatogram sen, i RrepDesaRny f10h12e23p46 vs US. W310nm aT) a 3-DRep-6587 coal 41-Fhap-5:260 xo zl zo} +0) 0} Ea pL 2-853 _ ios ET 00 10 20 30 40 60 60 70 80 0 700 110 120 +80 *60 170 780 +80 timn0 [SOP D-102 | Page 7 of 7] Standard Operating Procedure SOP No | Rev Determination of Rhaponticin & Desoxyrhaponticin by | P-1021 0 | Page 7 of 7 HPLC-UV 8.3. Typical Raw Material Chromatogram on, RepDesayhepRn 0-122318 RVI ULYS.IWM310nM mtu 2-Rrep-5233 By Kt BD 3-DRep-650 v.00, cst 2] i AIG via 00 40 20 30 40 50 60 70 80 80 wo 10 to to 40 “0 G0 1720 | ee 9.0 Revision History | Rev | Date | Description of Changes | CCR # | By | |-----|----------|------------------------|-------|----| | 0 | 11/10/23 | New procedure. N/A C. Perry | - | - |