Determination of Cynaropicrin by HPLC using UV-Vis Spectroscopy

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1.0 Purpose 
 
 The purpose of this procedure is to define the method for the quantification and identification
 
 of Cynaropicrin in raw materials and finished products by HPLC using UV-Vis Spectroscopy.
 
 2.0 Scope 
 
 This procedure applies to the identification and quantification of Cynaropicrin, using
 
 Cynaroside and/or Chlorogenic Acid as a standard in raw materials and finished products by
 HPLC/UV. 
 
 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
 
 Personnel to keep this procedure aligned with current practices. 
 
 4.0 Definitions 
 
 4.1 HPLC/UV — High Pressure Liquid Chromatography with Ultraviolet Detection
 
 4.2 QC — Quality Control 
 
 4.3 CofA — Certificate of Analysis 
 
 4.4 CNP — Cynaropicrin 
 4.5 CNS — Cynaroside 
 
 4.6 CGA — Chlorogenic Acid 
 
 4.7 ACN — Acetonitrile 
 
 4.8 MeOH — Methanol 
 
 

[SOP D-782 | Page 2 of 6]

 Standard Operating Procedure SOP No | Rev 
 Determination of Cynaropicrin by HPLC using D-782 I aE 
 UV/VIS Spectroscopy 
 
 4.9 TFA — Trifluoroacetic Acid 
 
 4.10 He2O- Deionized water 
 
 5.0 References 
 
 521 MV-LAB-19-033, Protocol, Cynaroside Determination using HPLC with UV/VIS
 
 Spectroscopy 
 
 6.0 Supplies 
 
 6.1 Chemicals: All reagents are HPLC grade or better 
 
 Gal CGA Reference Standard 
 
 6.162 ACN 
 
 6.1.3 MeOH 
 
 6.1.4 TFA 
 
 6.2 Glassware 
 
 6.2.1 Volumetric glassware as required for standard and sample preparations
 
 6.3. Disposables (as required for standard and sample preparations) 
 
 6.3.1 10mL Pipette Tips 
 
 6.3.2 ImL Pipette Tips 
 
 6.3.3 200uL Pipette Tips 
 
 6.3.4 Microcentrifuge tubes 
 
 6.3.5 16mL Test Tubes 
 
 6.3.6 Weigh paper 
 
 6.4 Equipment 
 
 6.4.1 Suitable gradient HPLC system consisting of a pump, autosampler, column oven
 and UV detector with a chromatographic data handling system 
 
 6.4.2 Analytical Balance 
 
 

[SOP D-782 | Page 3 of 6]

 Standard Operating Procedure SOP No | Rev 
 Determination of Cynaropicrin by HPLC using D-782 I : se 
 UV/VIS Spectroscopy 
 
 6.4.3 Centrifuge 
 
 6.4.4 Adjustable Pipette 
 
 7.0 Procedure 
 
 7.1. Mobile Phase Preparation 
 
 7.1.1 Mobile Phase A 
 
 7.1.1.1 Combine 1000 mL of H20 with 0.1 mL of TFA 
 
 7.1.2 Mobile Phase B 
 
 7.1.2.1 Combine 500 mL of ACN with 0.05 mL of TFA 
 
 7.1.3 Diluent 
 
 7.1.3.1 Combine 250 mL of H20 with 250 mL of MeOH 
 
 7.2. Stock Standard Preparation 
 
 7.2.1 Accurately weigh and transfer about 50 mg of CGA reference standard to a
 
 100-mL volumetric flask. 
 
 7.2.2 Dissolve in and dilute to volume with Diluent. 
 
 7.3. Working Standard Preparation 
 
 7.3.1 Transfer 3.0 mL of the Stock Standard to a 25-mL volumetric flask.
 
 7.3.2 Dilute to volume with Diluent. 
 
 7.3.3. The standard preparation may be scaled as required. 
 
 7.4 Sample Preparation 
 
 7.4.1 The linear range of the method is 10 — 100 ug/mL with a 10 pL injection
 
 volume. The sample concentration must be within the linear range of the
 method: 
 
 7.4.2 For raw materials: weigh no less than 30 mg into a suitably sized volumetric
 
 flask of no less than 25 mL volume to generate an analyte concentration that is
 
 

[SOP D-782 | Page 4 of 6]

 Standard Operating Procedure SOP No | Rev 
 Determination of Cynaropicrin by HPLC using D-782 I ray 
 UV/VIS Spectroscopy 
 
 within the validated linearity range. Dissolve in and dilute to volume with
 
 diluent. Perform further dilutions as required using diluent.
 
 7.4.3 For solid dose finished products: record the weight of ten dosage units, and
 
 calculate the average weight of a single dosage unit. Based on the label claim
 and fill weight (for capsules) or tablet weight per dose, weigh no less than 50
 
 mg of the pooled dosages into a suitably sized volumetric flask of no less than
 
 25 mL to generate an analyte concentration that is within the validated linearity
 range. Dilute the sample to 2/3 of the flask volume with diluent and swirl to
 
 dissolve. Shaking or sonication can also be used to assist dissolution. After the
 sample is dissolved, equilibrate the sample to room temperature (if sonicated),
 
 and dilute to volume using diluent. Perform further dilutions as required using
 
 diluent. 
 
 7.4.4 If particulates remain in the final sample preparation, a portion may be
 centrifuged at 10,000 rpm for 200 seconds prior to HPLC analysis.
 
 7.4.5 For finished products or raw materials being analyzed for the first time using
 
 this method, an in process validation is required to demonstrate spectral purity,
 baseline separation of peaks, and extraction efficiency as a part of system
 
 suitability before data can be reported using this method. 
 
 1 HPLC Parameters 
 
 Pel Column: Phenomenex Luna C18(2), 5um, 4.6 mm x 250 mm 
 
 LZ Column Temperature: 30°C 
 
 fee Flow rate: 1.0 mL/min 
 
 7.5.4 Wavelength: 205 nm 
 
 TD Injection Volume: 10 uL 
 
 7.5.6 Run Time: 60 min for samples, 25 min for standards 
 
 7.5.7 Spectral Range (for Identification)- 210 nm to 360 nm 
 
 

[SOP D-782 | Page 5 of 6]

 Standard Operating Procedure SOP No | Rev 
 Determination of Cynaropicrin by HPLC using D-782 | : sae 
 UV/VIS Spectroscopy 
 
 7.5.8 Gradient Profile: 
 
 Time %MPA %MPB 
 0 95 5 
 4 95 5 
 ZI 78 2) 
 26 78 22 
 44 65 35 
 49 20 80 
 50 95 5 
 60 95 5 
 
 7.6 Recommended Sequence 
 
 7.6.1 Make at least 2 injections of the diluent. 
 
 7.6.2 Make five (5) injections of Standard Solution. 
 
 7.6.3 Make a single injection of each Sample Preparation. . 
 
 7.6.4 Make a single injection of the Standard Solution after every ten (10) sample
 
 injections or at the end of a run. 
 
 7.7. System Suitability Requirements 
 
 7.7.1 The %RSD of the first five (5) standard injections is NMT 5.0%.
 
 7.7.2 The %RSD of all standard injections is NMT 5%. 
 
 7.7.3 If present, any interference in the diluent should be subtracted out of the sample
 
 and standard peak areas. 
 7.8 Retention Time (RT), Relative Retention Time (RRT), Relative Response Factor (RRF)
 
 7.8.1 CGA: RT = 18.6 min, RRT = 1.00, RRF = 1.00 
 
 7.8.2 CNS: RT = 26.8 min, RRT = 1.44, RRF = 0.336 
 
 7.8.3 CNP: RT = 40.9 min, RRT = 2.20, RRF = 0.582 
 
 7.9 Example calculations for determining finished product % label or raw material % purity
 
 7.9.1 % assay = WR * y SWter V g o e ca X Pe V s s i p e l x aAWe x RRF x 100
 
 Ri Sample peak area 
 
 

[SOP D-782 | Page 6 of 6]

 Standard Operating Procedure SOP No | Rev 
 Determination of Cynaropicrin by HPLC using D-782 | page 
 6 of 6 
 UV/VIS Spectroscopy 
 
 R, Mean standard peak area 
 
 Wteta Weight of reference standard in mg (correct for moisture if required)
 
 Vstd Volume of the standard preparation accounting for dilutions in mL
 
 P Purity of the reference standard in decimal format 
 
 SW Sample weight in mg 
 
 Vspl Volume of the sample preparation accounting for dilutions in mL
 
 AW Average weight of a single tablet or capsule in mg (use | for raw
 
 materials) 
 
 LA Label amount in mg per dose (use 1 for raw materials) 
 
 RRF Relative Response Factor from Section 7.8. 
 
 7.10 Column Wash and Storage 
 
 7.10.1 Rinse and store the column with H20 / ACN (50/50) 
 
 8.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 06/26/19 | New i 07/21/22 Scheduled review: updated logo and format. CC-22-0289 K. Burris | - | - |