D-1011
Determination of Ginsenosides by HPLC-UV
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1.0 Purpose The purpose of this procedure is to define the method for the determination of ginsenosides in raw materials and finished products by HPLC-UV. 2.0 Scope This procedure applies to the determination of ginsenosides in raw materials in the QC Laboratory at Ion Labs. 3.0 Responsibility 3.1 ‘It is the responsibility of QC Chemists to follow this procedure. 3.2 It is the responsibility of QC Laboratory Management to ensure that this procedure is being followed. 3.3. It is the responsibility of QC Laboratory Management and/or Analytical Development to keep this procedure aligned with current practices. 4.0 Definitions 4.1 QC — Quality Control 4.2. HPLC-UV — High Performance Liquid Chromatography with Ultraviolet Detection 4.3 H2O- Water 44 ACN- Acetonitrile 5.0 References 5.1 PRTCL-22-0060, Protocol, Validation of an Analytical Method for the Determination of Ginsenosides by HPLC-UV [SOP D-101 | Page 2 of 8] Standard Operating Procedure SOP No Rev Page Determination of Ginsenosides by HPLC-UV D-1011 2 of 8 6.0 Supplies 6.1 Chemicals 6.1.1 Ginsenoside Rb! Reference Standard 6.1.2 Ginsenoside Rg1 Reference Standard 6.1.3 USP Powdered Asian Ginseng Extract Reference Standard 6.1.4 Acetonitrile (HPLC grade) 6.1.5 Methanol (HPLC grade) 6.1.6 H20 18 MOercm) 6.2 Glassware 6.2.1 Volumetric glassware as required for standard and sample preparations 6.2.2 Luer-lock syringe and 0.22um nylon filter 6.2.3 Adjustable pipette and tips 6.2.4 1.5-mL or 2-mL Eppendorf centrifuge tubes 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 with a minimum weight of no less than 40 mg 6.3.3. Microbalance with a minimum weight of no less than 3 mg 6.3.4 Wrist action shaker 6.3.5 Centrifuge 7.0 Mobile Phase 7.1 Mobile Phase A — H2O 7.2 Mobile Phase B — ACN 7.3 Diluent — Methanol:H20 (70:30), equilibrate to room temperature before use [SOP D-101 | Page 3 of 8] Standard Operating Procedure SOPNo | Rev Page Determination of Ginsenosides by HPLC-UV D-1011 0 3 of 8 8.0 Working Standard 8.1 Accurately weigh and transfer about 3.75 mg of ginsenoside Rb1 reference standard to a 25-mL volumetric flask. 8.2 Accurately weigh and transfer about 3.75 mg of ginsenoside Rg1 reference standard to the same 25-mL volumetric flask 8.3 Dissolve in and dilute to volume with Diluent. 9.0 System Suitability Solution 9.1 Accurately weigh and transfer about 100 mg of USP Powdered Asian Ginseng Extract reference standard to a 10-mL volumetric flask. oe Add 7.0 mL of methanol and swirl to disperse the powder. 9.3 Slowly dilute to volume with H20 while gently swirling. Do not shake or invert. 9.4 Equilibrate to room temperature for at least 10 min. 9.5 Adjust to volume using H20. 9.6 Sonicate for 10 min with occasional shaking. 9.7 Pass through a 0.22 um syringe filter discarding the first 2-3 mL before collecting a sample for analysis. 10.0 Sample Solution 10.1 Accurately weigh and transfer about 100 mg of raw material sample to a 10-mL volumetric flask. 10.2 Add 7.0 mL of methanol and swirl to disperse the powder. 10.3 Slowly dilute to volume with H2O while gently swirling. Do not shake or invert. 10.4 Equilibrate to room temperature for at least 10 min. 10.5 Adjust to volume using H20. 10.6 Sonicate for 10 min with occasional shaking. [SOP D-101 | Page 4 of 8] Standard Operating Procedure SOP No Rev Page Determination of Ginsenosides by HPLC-UV D-1011 4 of 8 10.7 Pass through a 0.22 um syringe filter discarding the first 2-3 mL before collecting a sample for analysis. 11.0 HPLC Parameters 11.1 Column: Agilent EclipsePlus C18 RRHD 1.8um, 2.1 mm x 50 mm, or equivalent 11.2 Column Temperature: 40 °C 11.3 Flow rate: 0.4 mL/min (do not increase above 0.4 mL/min) 11.4 Wavelength: 203 nm 11.5 Injection Volume: 5 uL 11.6 Run Time: 45 minutes 11.7 Gradient Time (min) % A %B 0.0 84 16 15.0 80 20 15.1 73 27 35.0 68 32 35.1 5 95 40.0 5 95 40.1 84 16 45.0 84 16 11.8 Spectral Range (for Identification)- 200 nm to 400 nm 12.0 Recommended Sequence 12.1 Make two injections of Diluent 12.2 Make five injections of Working Standard 12.3 Make a single injection of System Suitability Solution 12.4 Make a single injection of each Sample Preparation LZ) Make a single injection of Working Standard after every six samples and/or at the end of the run. [SOP D-101 | Page 5 of 8] Standard Operating Procedure SOPNo | Rev Page Determination of Ginsenosides by HPLC-UV D-1011 0 5 of 8 13.0 System Suitability Requirements 13.1 No significant (>0.5%) interfering peaks are present in the blank (Diluent) injection. 13.2 The %RSD of the Working Standard is NMT 2.0% for ginsenosides Rg1 and Rb. 13.3 The USP resolution between ginsenoside Rg1 and Re in the injection of System Suitability Solution is NLT 1.3. 13.4 The USP resolution between ginsenoside Ro and Rb1 in the injection of System Suitability Solution is NLT 1.5. 13.5 The USP resolution between ginsenoside Rd and the peak before it in the injection of System Suitability Solution is NLT 1.3. 13.6 The tailing factors for ginsenosides Rg] and Rb1 in the injections of Working Standard is NMT 2.0. 13.7 For materials being analyzed for the first time using this method, non-conforming results may trigger execution of a product specific method optimization per D-126/D-103. 14.0 Retention Times Note: Retention times are approximate. Peak identification should be accomplished by comparison of the chromatogram obtained from the System Suitability Solution to the chromatogram provided with the USP Powdered Asian Ginseng Extract reference standard. 14.1 Ginsenoside Rgl 15.6 14.2 Ginsenoside Re 16.3 14.3 Ginsenoside Ro 26.4 14.4 Ginsenoside Rb1 26.9 14.5 Ginsenoside Re 28.7 14.6 Malonyl ginsenoside Re 29.3 14.7 Ginsenoside Rb2 30.8 14.8 Malonyl ginsenoside Rb2 = 31.6 [SOP D-101 | Page 6 of 8] Standard Operating Procedure SOP No | Rev Page Determination of Ginsenosides by HPLC-UV D-1011 0 6 of 8 14.9 Ginsenoside Rd 34.9 15.0 Relative Response Factors Analyte RRF Ref Std Ginsenoside Rgl 1.00 Rel Ginsenoside Re 1.00 Rgl Ginsenoside Rb1 1.00 Rb1 Ginsenoside Rc 0.96 Rb1 Ginsenoside Rb2 0.96 Rb1 Ginsenoside Rd 0.80 Rb1 16.0 Example Chromatograms 16.1. Blank DILUENT 100} 80° > 60; z 40- I. : : : : ; ; : " ; . : ; : ; a : \ ee 207 A 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 Time [min] 16.2. Working Standard WSTD (LIN-4) 1007 - 80- ; e > 60 : 3 © z [: 3 40- - 6 % ill | | ¥ S*a 207) [= 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 Time [min] [SOP D-101 | Page 7 of 8] Standard Operating Procedure SOP No | Rev Page Determination of Ginsenosides by HPLC-UV D-1011 0 7 of 8 16.3 System Suitability Solution 22AS076 USP ASIAN 100: 5 Be o 2 8 a 20+ l| = © ae, aie ae ee a ie 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 Time [min] 16.4 Raw Material Sample R46082-1 100 - x 80 3 > 604 3 : ec 2 : 20- 26 Z 2 § la 0-4 7 a a i i dl 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 Time [min] 17.0 Example Calculations R, Wtstgqg XP A V. SS Assay (%) = 2x —StA 2 y Distock y _Tspl_y °° RRF x 100 Rs V stock Veta Splwt LA Ry Peak area for each gingerol present in the injection of Sample Solution Rg Mean Working Standard peak area ratio (5 injections) Wteta Weight of reference standard used to prepare the Stock Standard (mg) P Purity of reference standard (decimal) Verock Volume of the Stock Standard (mL) AGstock Aliquot of the Stock Standard used to prepare the Working Standard (mL) Veta Volume of the Working Standard (mL) Vspl Volume of Sample Solution (mL) [SOP D-101 | Page 8 of 8] Standard Operating Procedure SOP No Rev Page Determination of Ginsenosides by HPLC-UV D-1011 0 8 of 8 Splwt Sample weight (mg) SS Serving size: Average weight of ten dosage units in mg for tablets and capsules, weight of a single serving from the theoretical formula in mg for liquids, or 1 for raw materials LA Label amount in mg (use 1 for raw materials) RRF Relative response factor for the individual ginsenoside 18.0 Revision History | Rev | Date | Description of Changes | CCR # | By | |-----|----------|------------------------|-------|----| | 0 | 11/09/22 | New procedure. N/A S. Sassman | - | - |