Hydroxycitric Acid Determination 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 analysis and/or identification of
 
 Hydroxycitric Acid in raw materials and dietary supplements using HPLC and UV/VIS
 spectrophotometry. 
 
 2.0 Scope 
 
 This procedure applies to the identification of Hydroxycitric Acid forms in raw materials and
 finished products using spectral analysis and quantitation of Hydroxycitric Acid using a traceable
 
 Calcium (-)-Hydroxycitrate reference standard. 
 
 3.0 Responsibility 
 
 3.1 It is the responsibility of QC Laboratory personnel to follow this procedure.
 
 3.2 It is the responsibility of QC Laboratory Management to implement this procedure and
 
 to ensure that this procedure is being followed. 
 
 3.3 It is the responsibility of QC Laboratory and Analytical Development Management to
 keep this procedure aligned with current Ion Labs practices. 
 
 4.0 Definitions 
 
 4.1 UV/VIS — Ultraviolet and Visible Electromagnetic Spectrums 
 
 4.2 HCA -— Hydroxycitric Acid 
 
 4.3. H3PO«4-— Phosphoric Acid 
 
 4.4 KH2PO4-— Potassium dihydrogen phosphate (potassium phosphate monobasic)
 
 4.5 NaOH — Sodium Hydroxide 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Hydroxycitric Acid Determination by HPLC using D-748 4 : ae 
 
 UV/VIS Spectroscopy 
 
 4.6 CofA — Certificate of Analysis 
 
 4.7 H20O- Water 
 
 48 QC - Quality Control 
 
 5.0 References 
 
 5.1 MV-LAB-14-029, Protocol, Hydroxycitric Acid Determination by HPLC and UV/Vis
 Spectroscopy 
 
 5.2. Garcinia Hydroxycitrate USP Monograph 
 
 5.33. D-793, SOP, Cryogenic Grinding of Chewable Gels 
 6.0 Supplies 
 
 6.1 Chemicals: All reagents are HPLC grade or better. 
 
 6.1.1 H20 © 18.2 MQ-cm) 
 
 6.1.2 KH2PO4 
 
 6.1.3 H3PO, 
 
 6.1.4 NaOH 
 
 6.1.5 Calcium (-)-hydroxycitrate reference standard 
 
 6.2 Glassware 
 
 6.2.1 HPLC vials, 12mm x 32mm with screw cap enclosures with septa
 
 6.2.2 Scintillation Vials 
 
 6.2.3. 1L Mobile Phase Container 
 
 6.2.4 Volumetric glassware as required by standard and sample preparations
 
 6.3 Disposables 
 
 6.3.1 Tips for Adjustable Pipettes 
 
 6.3.2 Microfuge tubes 
 
 6.3.3 16mL Test Tubes 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Hydroxycitric Acid Determination by HPLC using D-748 4 x fae 
 UV/VIS Spectroscopy 
 
 6.3.4 Disposable Plastic Luer Lock Syringe —- 3mL, 6mL, or 10mL 
 
 6.3.5 Nylon Syringe Filters, 0.45um 
 
 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 
 
 6.4.3 Vortex 
 
 6.4.4 Stir Plate 
 
 6.4.5 Eppendorf Centrifuge 
 
 6.4.6 Adjustable Pipettes 
 
 7.0 Preparation of Mobile Phase, Dissolution Buffer, Samples, and Standards
 
 Fel Mobile Phase (1.36g/L aqueous KH2PO4 pH 2.5) 
 
 7.1.1. Transfer 1.36g of KH2P04 to a 1000-mL mobile phase bottle. 
 
 7.1.2 Add 900mL of water. 
 
 7.1.3 Adjust with H3POs, to a pH of 2.5. 
 
 7.1.4 Transfer the resulting solution to a 1000-mL volumetric flask.
 
 7.1.5 Dilute to volume with H20, and mix well. 
 
 Ve Diluent (3% H3PO4 in H20) 
 
 7.2.1 Transfer 970ml of H20 to a 1000-mL mobile phase bottle. 
 
 7.2.2 Slowly add 30mL of H3PO4, and mix well. 
 
 7.3 Standard Preparation 
 
 7.3.1 The linear range of the method is 0.1 mg/mL — 1.0 mg/mL. All standard and
 
 sample preparations must be within the linear range. 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Hydroxycitric Acid Determination by HPLC using D-748 4 ae 
 UV/VIS Spectroscopy 
 
 7.3.2 The standard is prepared by weighing no less than the minimum allowed on the
 
 scale at the time of use. Accurately weigh and transfer the reference standard to
 
 an appropriately sized volumetric flask, dissolve in Diluent, and dilute to volume
 using Diluent. 
 
 7.3.3 Dilutions are prepared using Diluent. Dilutions can be made using volumetric
 
 glassware and/or adjustable pipettes. Specific standard concentrations will
 approximate the concentration expected to be found in the product being tested
 
 based on the sample dilution and calculated from the label. Dilutions can be
 
 prepared in HPLC vials. 
 
 7.4 Sample Preparation 
 
 7.4.1 Specific sample testing details are provided in each products profile. If a specific
 testing details section is not available, then follow preparation procedure as
 
 described below, maintaining concentration within the linear range listed above.
 
 7.4.2 For raw materials: weigh no less than 20 mg into a suitably sized volumetric flask
 of no less than 25 mL volume to generate an analyte concentration that is within
 
 the validated linearity range. Dilute to volume with Diluent, and sonicate for 10
 
 min. 
 
 7.4.3 For solid and liquid dose finished products: Combine and homogenize no less
 than ten dosage units. Based on the label claim and fill weight (capsules), serving
 
 size (powders) 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 linear range. Dilute to volume
 
 with Diluent, and sonicate for 10 min. 
 
 7.4.4 For chewable gels (gummies), homogenize at least 10 dosage units according to
 the procedure outlined in D-793 Cryogenic Grinding of Chewable Gels. Quickly
 
 weigh a portion of the pooled and homogenized dosages into a beaker. Use several
 small portions of Diluent to completely transfer the sample into a suitably sized
 
 volumetric flask to generate an analyte concentration that is within the validated
 linear range. Dilute to volume, and sonicate for 10 min. 
 
 
 

[SOP 

 Standard Operating Procedure SOPNo | Rev 
 Hydroxycitric Acid Determination by HPLC using D-748 ‘ Ph me 
 
 UV/VIS Spectroscopy 
 
 7.4.5 To manage large volumes, the standard can be initially prepared at a higher
 
 concentration and further diluted into the linear range using Diluent. Equilibrate
 to room temperature prior to performing further dilution. Dilutions can be
 
 made using volumetric glassware and/or adjustable pipettes. Dilutions can be
 
 prepared in HPLC vials 
 
 7.4.6 If particulates remain in the final sample preparation, a portion may be centrifuged
 at 10,000 rpm for 5 min prior to HPLC analysis. Alternatively, the sample may
 
 be filtered through a 0.45 um membrane discarding the first 3 — 4 mL.
 
 8.0 Test Conditions 
 
 8.1 Gradient — Isocratic 
 
 Time %A %B Gradient Type 
 
 0.00 100 0 0 
 
 10.00 100 0 0 
 
 8.2 Column — XB C18, 5um, 100A, LC column, 250mm x 4.6mm 
 
 8.3. Flow Rate — 1.0mL/min 
 
 8.4 UV Detection —215nm 
 
 8.5 Injection Volume — 20nL 
 8.6 Column Temperature — 28°C 
 
 8.7 3-D Spectral Range — 200nm to 300nm 
 
 8.8 | Recommended Sequence 
 
 8.8.1 Make at least 2 injections of a Blank (Diluent). 
 8.8.2 Make five injections of the Working Standard. 
 
 8.8.3. Make a single injection of each Sample Preparation. 
 8.8.4 Make a single injection of the Working Standard after every six samples and at
 
 the end of the run. 
 8.9 System Suitability 
 
 8.9.1 The %RSD of five consecutive injections of Working Standard is NMT 5.0%.
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Hydroxycitric Acid Determination by HPLC using D-748 4 és ee 
 UV/VIS Spectroscopy 
 
 8.9.2 The %RSD of all standard injections is NMT 5%. 
 
 8.10 Column Wash and Storage 
 
 8.10.1 Rinse the column with H2O / methanol (90/10) at 1 mL/min for at least 15 min.
 
 8.10.2 Rinse the column with H2O / methanol (50/50) at 1 mL/min for at least 10 min.
 
 8.10.3 Store the column with H2O / methanol (50/50). 
 
 9.0 Example Calculations 
 
 Re Wteta xP Vspt SS 
 9% assay =—R. xX Ves x SA xX<—A X 100 
 
 Ry Sample peak area 
 
 Rs Mean standard peak area 
 
 Wt..q Weight of 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 (solids) or mL (liquids) 
 
 Veni Volume of the sample preparation accounting for dilutions in mL 
 
 SS 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 Diluent 
 
 Blank 
 84 
 7 
 a 
 5 
 72 : 
 24 P 3 ron 
 o| 
 - eo ee ee OS en ee ae eo ee oe ee 
 Time [min] 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Hydroxycitric Acid Determination by HPLC using D-748 4 een 
 7 of 7 
 UV/VIS Spectroscopy 
 
 10.2. Working Standard 
 
 21AS171 
 25 i < 
 22.5+ 1S 
 20+ 
17.54 15

 212.5 3 
7.54 2.5;4 ye

 05 1.5 2 2.5 3 3.5 4 45 5 5.5 6 6.5 7 
 Time [min] 
 10.3. Sample 
 
 200190 
 2 s 
 25- 
 22.54 Rae 
 20+ 
17.55 154

 212.5) 2 
 107 = 
 7.5 5 4 } Bg 
 2.55 Z\ a.Oo xoOX 
 () 
 0 0.5 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 
 Time [min] 
 11.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 10/16/14 | New | 14-0822 | X. Shao |
| 1 | 02/24/17 | Biennial review: fixed typographical errors. Changed stability requirements to 100% | 16-1138 | J. Maignan |
| 2 | 07/20/20 | Scheduled Review: Removed requirements, updated CC- responsibilities. Updated scope. Update for consistency with current methods. Add reference to | 20-0514 | J. Maignan |
| 3 | 06/01/22 | method validation. Add recommended sequence section. Replace CC- requirements section with system suitability. Add example chromatography. Remove unnecessary information and align with current SOP | 22-0252 | © Sacman |
| 4 | 05/02/23 | format, add instruction to follow test details containing product CC- specific sample preparation, add specific sample prep instructions for different dosage forms. Changed logo. | 23-0226 | & Sasaman |