Determination of EGCG by HPLC-UV

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1.0 Purpose 
 
 The purpose of this procedure is to define the method for the quantitation and/or identification
 of (-)Epigallocatechin-3-O-gallate (EGCG) in raw materials and finished product dietary
 
 supplements using HPLC-UV. 
 
 2.0 Scope 
 
 This procedure applies to the quantification and identification of EGCG in raw materials and
 
 finished products in the QC laboratory at lon 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 HPLC-UV — High Performance Liquid Chromatography with Ultraviolet Detection
 
 4.2 QC — Quality Control 
 
 4.3. H3PQO«a-— Phosphoric Acid 
 
 44 ACN-— Acetonitrile 
 
 4.5. HeO- Water 
 
 4.6 PVDF — Polyvinylidene fluoride 
 
 4.7 EGCG —-(-)Epigallocatechin-3-O-gallate 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev Page 
 Determination by EGCG by HPLC-UV D-757 s 2 of 8 
 
 5.0 References 
 
 5.1 PRTCL-23-0001, Protocol, Validation of an Analytical Method for the Determination of
 EGCG by HPLC-UV 
 
 6.0 Supplies 
 
 6.1 Chemicals: All reagents are HPLC grade or better. 
 
 6.1.1 H20 
 
 6.1.2 ACN 
 
 6.1.3. H3PO4 (85%) 
 
 6.1.4 Citric Acid 
 
 6.1.5 EGCG reference standard 
 
 6.2 Glassware 
 
 6.2.1 HPLC vials, 12mm x 32mm with screw cap enclosures with septa 
 
 6.2.2 Volumetric glassware as required for standard and sample preparations
 
 6.2.3 Mobile Phase Container 
 
 6.3 Disposables 
 
 6.3.1 Pipet Tips 
 
 6.3.2 Centrifuge tubes 
 
 6.3.3. Disposable Plastic Luer Lock Syringe and 0.45 pm PVDF Syringe Filters
 
 6.3.4 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 
 
 6.4.3 Ultrasonic bath 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev Page 
 Determination by EGCG by HPLC-UV D-757 a 3 of8 
 
 6.4.4 Eppendorf Centrifuge 
 
 6.4.5 Adjustable Pipet 
 
 7.0 Preparation of Mobile Phase, Dissolution Buffer, Samples, and Standards 
 
 7.1 Mobile Phase A — 0.1% H3PQ4 (aq) 
 
 7.1.1. Transfer 1000 mL of H20 to a 1-L bottle. 
 
 7.1.2 Add 1.0 mL of H3POs, and mix well. 
 
 7.2. Mobile Phase B— ACN 
 
 7.2.1. Transfer 1000 mL of ACN to a 1-L bottle. 
 
 7.3 Diluent — 0.1% H3POq (aq) 
 
 7.3.1. Use Mobile Phase A. 
 
 7.4 Extraction Solution — Ethanol / H20 / Citric Acid / H3PO4 (70/30/0.1/0.25)
 
 7.4.1. Transfer 700 mL of ethanol to a 1-L bottle. 
 
 7.4.2 Add 300 mL of H20. 
 
 7.4.3 Add 1.0 g of citric acid. 
 
 7.4.4 Add 2.5 mL of phosphoric acid, and mix until dissolved. 
 
 8.0 Preparation of Standard and Sample Solutions 
 
 8.1 Stock Standard 
 
 8.1.1 Accurately weigh and transfer about 25 mg of reference standard into a 100-mL
 volumetric flask. 
 
 8.1.2 Dissolve in and dilute to volume using Extraction Solution. 
 
 8.2 Working Standard 
 
 8.2.1 3.0 mL of the Stock Standard into a 25-mL volumetric flask. 
 
 8.2.2 Dilute to volume using Diluent. 
 
 8.3. Stock Sample 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev Page 
 Determination by EGCG by HPLC-UV D-757 4 of 8 
 
 Specific sample testing details are provided in each products profile. Ifa
8.3.1 specific testing details section is not available, then follow preparation

 procedure as described below, maintaining concentration within the linear range
 of this method. 
 
 The linear range of the method is 8 — 252 mcg/mL, all working standard and
8.3.2 sample preparations must be within the linear range.

 Accurately weigh and transfer a quantity of sample containing about 25 mg of
8.3.3 EGCG into a 100-mL volumetric flask.

 8.3.3.1 For finished products: 
 
 : Dosage Unit (mg) 
 Sample Wt (mg) = 25mg X Label Claim (mg) 
 
 8.3.3.2 Forraw materials: 
 
 100 
 Sample WW t (mg) == 25mg x Raw Material Potency (%) 
 
 8.3.4 Dilute to volume with Extraction Solution. 
 
 8.3.5 Sonicate for 20 minutes. 
 
 8.3.6 Equilibrate to room temperature before further dilution. 
 
 8.4 Working Sample Solution 
 
 8.4.1 Transfer 3.0 mL of Stock Sample to a 25-mL volumetric flask. 
 
 8.4.2 Dilute to volume using Diluent. 
 
 8.4.3 Pass through a 0.45 um PVDF syringe filter discarding the first 2— 3 mL before
 
 collecting a sample for analysis. Do not use nylon syringe filters for EGCG
 since they will absorb the analyte resulting in an inaccurate result.
 
 8.4.3.1 Alternatively, centrifuge at 6,000 rpm for 5 minutes to remove
 
 particulates. 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev Page 
 Determination by EGCG by HPLC-UV D-757 5 of 8 
 
 9.0 Test Conditions 
 
 9.1 Column — Agilent InfinityLab Poroshell 120 EC-C18, 2.7 um, 4.6 mm x 100 mm, or
 
 equivalent 
 9.2 Flow Rate — 1.0 mL/min 
 
 9.3 UV Detection —-278nm 
 
 9.4 Recommended Spectral Range — 200 nm — 400 nm 
 
 9.5 Injection Volume — 5uL 
 
 9.6 Column Temperature — 35 °C 
 
 9.7 Gradient 
 
 Time (min) % A % B 
 0.0 92 8 
 10.0 84 16 
 10.1 0 100 
 13.0 0 100 
 13.1 92 8 
 17.0 92 8 
 
 9.8 Recommended Sequence 
 
 9.8.1 Make at least two injections of Diluent. 
 
 9.8.2 Make five injections of Working Standard. 
 
 9.8.3 Makea single injection of each Working Sample. 
 
 9.8.4 Makea single injection of the Working Standard after every six samples and/or
 
 at the end of the run. 
 
 9.9 System Suitability Requirements 
 
 9.9.1 No significant (>0.5%) interfering peaks are present in the blank (Diluent)
 
 injection. 
 
 9.9.2 The %RSD of five consecutive injections of Working Standard is NMT 2.0%.
 
 9.9.3 The %RSD of all injection of Working Standard is NMT 2%. 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev Page 
 Determination by EGCG by HPLC-UV D-757 6 of 8 
 
 9.10 Column Wash and Storage 
 
 9.10.1 Wash and store the column with H20/ACN (50/50). 
 
 9.11 Example calculations for determining finished product % label or raw material % purity
 % assay = a x Vera x <2 y = x 100 
911.1 Ry Sample peak area
 
 R, Mean standard peak area 
 
 Wtsiqg Weight of reference standard in mg 
 
 V sta Volume of the standard preparation accounting for dilutions in mL
 
 P Purity of the reference standard in decimal format 
 
 SA Sample amount in mg (solids) or mL (liquids) 
 
 Veni Volume of the sample preparation accounting for dilutions in mL
 
 eye Serving size: Weight of a single dosage unit in mg for tablets and
 
 capsules, volume of a single serving from the theoretical formula in mL
 for liquids, or 1 for raw materials. 
 
 LA Label amount in mg per dose or 1 for raw materials 
 
 10.0 Example Chromatography 
 
 10.1 Blank 
 
 DILUENT 
 40- 
 35+ 
 30+ 
 25- 
 = 20- 
 ¢ 
 15- 
 10+ 
 ! Some Gum 
 1 2 3 5 6 7 8 9 10 14 #=12 #13 «14 ©«6©15~=€«16~~«T 
 Time [min] 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev Page 
 D-757 2 7 of 8 
 Determination by EGCG by HPLC-UV 
 
 10.2. Working Standard 
 2Z2AS059 
 40 cael 
 oO 
 35-7 oo i 
 307 
 25- 
 z 20- 
 & 
 15> 
 10- 
 * IL 
 -57 1 2 i 3 Ls 4 bi 5 fi 6j 7 8 9 10 1kj1 °&«12 1 t 3 1 ' 4 1 bi 5 16 17
 Time [min] 
 10.3 Raw Material Sample 
 R47287 
 40- 
 35" 
 307 
 257 
 20- UAm 
 
 1 > ; 
645.8 618.8 GCGE

 15 
 10- 
 0 SNR 
 -5" € ' + : t a 
 1 2 3 4 5 6 7 8 9 10211 12 13. 14 #18 «416 «17 
 Time {min} 
 10.4 Finished Product Sample 
 220164 
 40- cooS ©) 
 Sa 
 35> 
 30+ 
 254 
 20+ 
 15+ 
 10- ‘wo 
 © 
 5 
 “54 T t T “¥ T T t T T t T T T T r T T 
 1 2 3 4 5 6 7 8 9 10 1 #12 #13 «14 «©16©~=©=6©16006«(17
 Time [min] 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev Page 
 Determination by EGCG by HPLC-UV D-757 2 8 of 8 
 
 11.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 01/02/19 | New N/A J. Maignan Update to reflect current practices, minor edits for clarity, add reference to USP monograph, fix concentration at 0.1mg/mL since ] 04/11/22 method is not validated, add recommended sequence, add system CC-22-0174 S. Sassman suitability section, add column wash and storage, add example chromatography. | - | - |
| 7 | 01/30/23 | A new method was developed and validated according to PRTCL- CC- 23-0001. This revision has been updated to reflect the new method. | 23-0050 | S. Sassman |