Ascorbic Acid Determination by HPLC with UV-Vis Spectroscopy

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1.0 Purpose 
 
 This document describes the analytical procedure for the determination of Ascorbic Acid (AA)
 
 in raw materials and finished products. 
 
 2.0 Scope 
 
 This procedure applies to the identification and quantification of AA in raw materials and
 
 finished products. This method was validated under Protocol PRTCL-20-0002.
 
 3.0 Responsibility 
 
 It is the responsibility of QC and Analytical chemists who have verified their ability to
3.1 execute this procedure to follow this procedure.

 It is the responsibility of QC Laboratory Management to implement this procedure and
 32 
 to ensure that the procedure is being followed. 
 
 It is the responsibility of QC Laboratory Management and AD Personnel to keep this
3.3 procedure current with the associated monographs and laboratory practices.

 4.0 Definitions 
 
 4.1 QC — Quality Control 
 
 4.2 AD — Analytical Development 
 
 4.3 AA — Ascorbic Acid 
 
 4.4 ACN — Acetonitrile 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Ascorbic Acid Determination by HPLC with UV/Vis D-701 1 | Page 2 of 9 
 
 Spectroscopy 
 
 45 TFA-—Trifluoroacetic Acid 
 
 46 EDTA -— Ethylenediaminetetraacetic Acid 
 
 4.7 ACS— American Chemical Society 
 
 48 HPLC — High Performance Liquid Chromatography 
 
 49 UV/Vis — Ultraviolet & Visible Electromagnetic Spectra 
 
 5.0 References 
 
 PRTCL-20-0002, Protocol, Ascorbic Acid Determination by HPLC using UV/Vis
5.1 Spectroscopy

 5.2 D-793, SOP, Cryogenic Grinding of Chewable Gels 
 
 6.0 Supplies 
 
 6.1 Chemicals — All reagents are ACS grade or better. 
 
 6.1.1 Milli-Q Water 
 
 6.1.2 ACN 
 
 6.1.3 TFA 
 
 6.1.4 Ascorbic Acid Reference Standard 
 
 6.1.5 EDTA Disodium Dihydrate 
 
 6.1.6 Sodium Phosphate Monobasic Dihydrate 
 
 6.1.7 Phosphoric Acid 
 
 6.2 Supplies and Glassware 
 
 6.2.1 HPLC vials, 12mm X 32mm with screw cap enclosures w/ septa 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 
 Ascorbic Acid Determination by HPLC with UV/Vis D-701 | 1 | Page 3 of 9 
 Spectroscopy 
 
 6.2.2 Volumetric glassware and/or adjustable pipettes and tips 
 
 6.2.3 Weigh paper or funnels 
 
 6.2.4 10ml Syringes with 17mm x 0.45u Nylon or Glass Fiber Syringe Filters (Note:
 
 It is recommended that chewable gel samples be filtered using glass fiber syringe
 
 filters.) 
 
 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 
 
 6.3.3. Wrist Action Shaker 
 
 7.0 Procedure 
 
 7.1 Mobile Phase & Diluent Preparation 
 
 7.1.1. Mobile Phase 
 
 7.1.1.1 Mobile Phase A: Add 200 uL of TFA to 1000 mL of water and mix
 
 well. 
 
 7.1.1.2 Mobile Phase B: ACN 
 
 7.1.2 Extraction Solvent / Diluent 
 
 7.1.2.1 Dissolve 2.34g Sodium Phosphate Monobasic Dihydrate and 0.56g
 ETDA Disodium Dihydrate in 1000ml water then pH to 3.0 with
 
 Phosphoric Acid. 
 
 7.1.3 Preparations may be scaled as necessary 
 
 7.2 Standard Prep 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 
 Ascorbic Acid Determination by HPLC with UV/Vis D-701 1 | Page 4 of 9 
 Spectroscopy 
 
 7.2.1. Accurately weigh and transfer about 45 mg of AA reference standard into a 100-
 
 mL volumetric flask. Add 50mL of Diluent and briefly sonicate until all AA is
 
 dissolved. (Caution: Perform this step quickly as AA is air sensitive.)
 
 792 Dilute to volume with Diluent and mix well — this is the AA Stock. Dilute the
 
 AA Stock 1:10 with Diluent — this is the AA Working Standard. 
 
 7.2.3. Alternative standard preparations are acceptable as long as the preparations are
 
 within the linear range of this method, 0.01856 — 0.09280 mg/mL
 
 7.3. Sample Preparation 
 
 7.3.1 Specific sample testing details are provided in each products profile. Ifa specific
 testing details section is not available, then follow preparation procedure as
 
 described below, maintaining concentration within the linear range of this
 
 method. 
 
 7.3.2 The validated range for the analytical method is 0.01856 — 0.09280 mg/mL.
 
 73.3 For finished products, extract sufficient sample with Diluent in order to generate
 an AA concentration that is within the validated linearity range. (Caution: Some
 
 finished products contain alkaline excipients in quantities capable of
 overwhelming the buffer capacity of the Diluent. Make sure that extraction
 
 sample solutions never exceed a pH of 3.0.) When analyzing gummies, powder
 and dissolve dosage forms as detailed in D-793 Cryogenic Grinding of Chewable
 
 Gels. 
 
 7.3.4 Prepare raw materials like standards. (However, be sure to consult the
 specification for expected potency, as raw material samples may not be 100%.)
 
 7.3.5 Samples can be dissolved in Diluent at any volume starting from 100mL. The
 volume chosen must be in the solubility range of AA (validated at approximately
 0.4 mg/ml). To manage large volumes, the sample can be initially dissolved in a
 
 smaller volume that is within the solubility range and a portion further diluted to
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 Ascorbic Acid Determination by HPLC with UV/Vis D-701 Page 5 of 9 
 Spectroscopy 
 
 bring the AA concentration into the linear range. 
 
 736. Fill the flask to about 50% of the calculated volume with Diluent and shake
 
 mechanically for 10 minutes. (Note: finished products that are liquids, along
 with chewable gel samples that have been previously powdered and dissolved in
 
 Diluent as per D-793 Cryogenic Grinding of Chewable Gels do not require
 mechanical shaking.) QS to volume with Diluent. 
 
 7.3.7. Perform further dilutions as required using Diluent. Filter a Sml aliquot for
 
 analysis, discarding the first 3-4ml of filtrate. 
 
 7.4 HPLC Parameters 
 
 7.4.1. Column: Phenomenex Kinetex XB-C18, 4.6 x 150mm, 2.6m (Or Equivalent)
 
 7.4.2 Column Temperature: 30°C 
 
 7.4.3. Flow rate: 0.7 mL/min 
 
 7.4.4 Mobile Phase Gradient: 
 
 Time, min %A %B 
 0.0 100 0 
 1.0 100 0 
 6.0 95 5 
 6.1 100 0 
 12.0 100 0 
 
 7.4.5 Wavelength: 254 nm 
 
 7.4.6 Injection Volume: 20 wL 
 
 7.4.7 Run Time: 12 minutes (Note: Some finished products may require intermittent
 
 blank or flush injections in order to remove more highly retentive formulation
 constituents that could interfere with subsequent injections.)
 
 7.4.8 Recommended 3-D Spectral Range (for Identification) - 210nm to 350nm
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 
 Ascorbic Acid Determination by HPLC with UV/Vis D-701 1 | Page 6 of 9 
 Spectroscopy 
 
 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 AA Working Standard. 
 
 7.5.3. Make a single injection of each Sample Preparation. 
 
 7.5.4. Make a single injection of the Standard Solution after every ten (10) sample
 
 injections and/or at the end of a 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%. 
 
 7.6.3 If present, any interference in the diluent should be subtracted out of the sample
 
 and standard peak areas. 
 
 7.7 Example calculations for determining finished product % label claim or raw material %
 
 assay: 
 
 7.7.1 0 % AA — R a u x Wt V s e t a a xP X \ S x S Y V a sp e l 100
 
 Ry Sample peak area 
 
 R, Mean (n=5) standard peak area 
 
 Wteta Weight of the reference standard (mg) 
 
 Veta Volume of the standard preparation accounting for dilutions in mL
 
 P Purity of the reference standard in decimal format 
 
 SA Sample amount (mg) 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Ascorbic Acid Determination by HPLC with UV/Vis D-701 1 | Page 7 of 9 
 Spectroscopy 
 
 Veni Volume of the sample preparation accounting for dilutions in mL
 
 SS Serving size: Average weight of ten dosage units for tablets and
 
 chewable gels, fill weight for capsules, mass of a single serving for
 powders, volume of a single serving from the theoretical formula in
 
 mL for liquids, or 1 for raw materials. 
 
 LA Label amount of AA (use 1 for raw materials) 
 
 7.8 System Wash, Column Wash and Column Storage 
 
 7.8.1. Wash and store the column in 50:50 ACN / Water. 
 
 8.0 Chromatograms 
 
 8.1 Typical Diluent Chromatogram 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Ascorbic Acid Determination by HPLC with UV/Vis D-701 1 | Page 8 of 9 
 
 Spectroscopy 
 
 8.2 Typical Working Standard Chromatogram 
 
 sn { - AscoArad b- 3i.3c50 
 
 | 
 ae 4 J| \ 2 3578 
 
 8.3. Typical Raw Material Chromatogram 
 
 “| may 
 1 - AscoAcrid b- 3i.3c57 
 
 vane 3 
 
 ek Ok ie ae ae eee aT a ee 0 #2. 3h. 6 3 6 ae ei ae 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 
 Ascorbic Acid Determination by HPLC with UV/Vis D-701 1 | Page 9 of 9 
 Spectroscopy 
 
 8.4 Typical Finished Product Chromatogram 
 
 =e 
 | {- Ascorbic Aid - 3.55 
 
 3) 
 
 9.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 02/12/20 | New N/A C. Perry | - | - |
| 1 | 12/20/22 | Add sample test details information. Minor edits. CC- | 22-0475 | J. Sassman |