Determination of a-Lipoic Acid by HPLC-UV

Original Document

Scanned document (image-only PDF)

Extracted Text

Searchable text extracted from PDF

1.0 Purpose 
 
 This document describes the analytical procedure for the determination of a-Lipoic Acid in raw
 
 materials and finished products. 
 
 2.0 Scope 
 
 This procedure applies to the identification and quantification of a-Lipoic Acid in raw materials
 
 and finished products. This method was validated under Protocol MV-LAB-19-125.
 
 3.0 Responsibility 
 
 3.1. ‘It is the responsibility of QC and Analytical chemists who have verified their ability to
 
 execute this procedure to follow this procedure. 
 
 3.2 It is the responsibility of QC Laboratory Management to implement this procedure and
 to ensure that the procedure is being followed. 
 
 3.3. It is the responsibility of QC Laboratory Management and/or Analytical Development
 
 Personnel to keep this procedure current with the associated monographs and laboratory
 
 practices. 
 
 4.0 Definitions 
 
 4.1 QC — Quality Control 
 
 4.2. H3P04 — Phosphoric Acid 
 
 4.3 b — Potassium Phosphate, Monobasic 
 
 4.4 MeOH — Methanol 
 
 4.5 ACN — Acetonitrile 
 
 4.6 H2O — Deionized Water 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 Determination of o-Lipoic Acid by HPLC/UV D-797 Page 2 of 7 
 
 4.7 ALA-—a-Lipoic Acid 
 
 4.8 HPLC — High Performance Liquid Chromatography 
 
 4.9 UV/Vis — Ultraviolet & Visible Electromagnetic Spectra 
 
 5.0 References 
 
 5.1 MV-LAB-19-125 — a-Lipoic Acid Determination Using HPLC with UV/Vis
 
 Spectroscopy 
 
 6.0 Supplies 
 
 6.1 Chemicals — All reagents are ACS grade or better 
 
 6.1.1 HO 
 
 6.1.2 MeOH 
 
 6.1.3. ACN 
 
 6.1.4 KPhos Mono 
 
 6.1.5 H3PQO4 
 
 6.1.6 ALA Reference Standard 
 
 6.2 Supplies and Glassware 
 
 6.2.1. HPLC vials, 12mm X 32mm with screw cap enclosures w/ septa 
 
 6.2.2 Volumetric glassware and/or adjustable pipettes and tips 
 
 6.2.3 Weigh paper or funnels 
 
 6.2.4 10ml Syringes with 0.45u Nylon 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. Sonicator bath 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of a-Lipoic Acid by HPLC/UV D797 | 1 | Bagesok7 
 
 6.3.4 Wrist Action Shaker 
 
 7.0 Procedure 
 
 Tal Mobile Phase, Extraction Solvent & Diluent Preparation 
 
 Felal Buffer — 0.68 g/L KPhos Mono 
 
 7.1.1.1 Combine 0.68g of KPhos Mono with 1000 mL of H2O. Mix well and
 
 filter/degas at 0.45y. 
 
 pe: 8.3% H3PO4 Solution 
 
 7.1.2.1 Dilute 8.3 mL of H3POq to 100 mL with H2O. Mix well. 
 
 7.1.3 Mobile Phase — 58:46:9 MeOH/ 0.68g per L KPhos Mono/ ACN, pH 3.0 — 3.1
 
 7.1.3.1 Combine 580 mL MeOH, 460 mL Buffer, and 90 mL ACN. Mix well.
 
 7.1.3.2. pH adjust mobile phase to 3.0 — 3.1 using the 8.3% H3POs.
 
 7.1.4 Extraction Solvent - MeOH 
 
 719 Diluent — 60:40 MeOH / H20 
 
 7.1.6 Preparations may be scaled as necessary 
 
 Te Standard Prep 
 
 Accurately weigh and transfer about 25 mg of ALA reference standard into a 50-
7.2.1 mL volumetric flask. Add 25mL of Extraction Solvent. Sonicate for Smin,

 equilibrate to room temperature, then QS to volume with Extraction Solvent.
 
 Dilute 1:5 w/ Diluent for a Working Standard concentration of 100 pg/ml.
 Tidak 
 
 7.3 Sample Preparation 
 Specific sample testing details are provided in each products profile. If a specific
 Tdel 
 testing details section is not available, then follow preparation procedure as
 described below, maintaining concentration within the linear range listed below.
 
 The validated range for the analytical method is 25 — 500 mcg/mL.
7.3.2 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of o-Lipoic Acid by HPLC/UV D-797 E ca eageeeh? 
 
 7.3.3 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. Fill the flask to about 50% of the calculated volume
 with Extraction Solvent and shake mechanically for 10 minutes. Sonicate for 5
 
 minutes, equilibrate to room temperature and bring up to volume with Extraction
 
 Solvent. 
 
 7.3.4 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 and liquids) or tablet weight per dose, weigh no less than 100 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. Fill
 
 the flask to about 50% of the calculated volume with Extraction Solvent and shake
 mechanically for 10 minutes. Sonicate for 5 minutes, equilibrate to room
 
 temperature and bring up to volume with Extraction Solvent. 
 
 7.3.5 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 no less than 400 mg of the pooled and homogenized dosages into a beaker.
 Use several small portions of Extraction Solvent to completely transfer the sample
 
 into a suitably sized volumetric flask to generate an analyte concentration that is
 
 within the validated linear range. Fill the flask to about 50% of the calculated
 volume with Extraction Solvent and shake mechanically for 10 minutes. Sonicate
 
 for 5 minutes, equilibrate to room temperature and bring up to volume with
 
 Extraction Solvent. 
 7.3.6 To manage large volumes, the sample can be initially prepared at a higher
 
 concentration and further diluted into the linear range using Diluent. Dilutions
 
 can be made using volumetric glassware and/or adjustable pipettes. Dilutions can
 be prepared in HPLC vials 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 Determination of a-Lipoic Acid by HPLC/UV D-797 Page 5 of 7 
 
 7.3.7 Centrifuge an aliquot of the final sample at 10,000 rpm for 5 min to remove
 particulates. Alternatively, the sample may be filtered through a 0.45 um
 
 membrane discarding the first 3 - 4 mL before collecting a portion for analysis.
 
 7.4 HPLC Parameters 
 
 7.4.1 Column: Agilent InfinityLab Poroshell 120 EC-C18, 4.6 x 100mm, 2.7u
 
 7.4.2 Column Temperature: 40°C 
 
 7.4.3 Flow rate: 0.9 mL/min 
 
 7.4.4 Wavelength: 215 nm 
 
 7.4.5 Injection Volume: 5 pL 
 
 7.4.6 Run Time: 10 minutes. 
 
 7.4.7 Recommended 3-D Spectral Range (for Identification) - 200nm to 350nm
 
 7.4.8 Mobile Phase Gradient - Isocratic 
 
 7.5 Recommended Sequence 
 
 7.5.1 Make at least 2 injections of the Diluent. 
 
 7.5.2 Make five (5) injections of Working Standard. 
 
 7.5.3 Make a single injection of each Sample Preparation. 
 
 7.5.4 Make a single injection of the Working Standard after every ten (10) sample
 
 injections and at the end of a run. 
 
 7.6 System Suitability Requirements 
 
 7.6.1 The %RSD of the first five (5) 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 or raw material % purity
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of a-Lipoic Acid by HPLC/UV D-797 1 Pasecon7 
 
 % Analyte = “a x — nxe x axe 
 
 Ry Sample peak area 
 
 R, Mean (n=5) standard peak area 
 
 Wtsta Weight of the reference standard in mg (corr. for water if applicable)
 
 Ved Volume of the standard preparation accounting for dilutions in mL
 
 P Purity of the reference standard in decimal format 
 
 SA Sample amount in mg 
 
 Vspl Volume of the sample preparation accounting for dilutions in mL
 
 SS Serving size: Weight of a single dosage unit in mg (use 1 for raw
 materials) 
 
 LA Label amount in mg of analyte (use 1 for raw materials)
 
 7.8 Column Wash and Storage 
 
 7.8.1 Wash and store the column in 75:25 MeOH / Milli-Q Water. 
 
 8.0 Example Chromatograms 
 
 8.1 Typical Diluent Chromatogram 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of o-Lipoic Acid by HPLC/UV D-797 Deo) Eager ek 
 
 8.2 Typical Working Standard Chromatogram 
 
 i ALA. 3.657 
 
 } 
 ! 
 | 
 | 
 || 
 || 
 I | 
 1 
 || 
 
 8.3. Typical Sample Chromatogram 
 
 | 
 | 
 
 ] 
 H| 
 I 
 | \ \ T-ALA-360 
 i Mt i\ 
 
 9.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 08/26/20 | New N/A C. Perry Minor edits for consistency with current methods. Simplified mobile : beiDeie t p e h s a t s d e e t p a r i e l p s. a r A a d ti d o e n. d A sp d e d c e if d i c i n s s a t m r p uc l t e i o p n r e to p f i o n l st l r o u w c t s i a o m n p s l f e o r p r d e i p ff a e r r a e t n i t o n Cer r 2> o 0272" . S.Sassman dosage forms. Updated format and logo. | - | - |