Determination of Levodopa 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/or
 
 identification of Levodopa (L-DOPA) in raw materials and finished products by HPLC using
 
 UV-Vis Spectroscopy. 
 
 2.0 Scope 
 
 This procedure applies to the identification and quantification of L-DOPA in raw materials.
 This method was validated under Protocol MV-LAB-19-154. 
 
 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 AD-— Analytical Development 
 
 4.3. TFA -— Trifluoroacetic Acid 
 
 4.4 MeOH — Methanol 
 
 4.5 L-DOPA — Levodopa 
 
 
 

[SOP D-788 | Page 2 of 7]

 Standard Operating Procedure SOP No Rev 
 Determination of Levodopa by HPLC using UV/VIS D-788 
 Page 2 of 7 
 Spectroscopy 
 4.6 HPLC — High Performance Liquid Chromatography 
 
 4.7 UV/Vis — Ultraviolet & Visible Electromagnetic Spectra 
 
 5.0 References 
 
 =a MV-LAB-19-154, Protocol, Levodopa Determination by HPLC Using UV/Vis
 
 Spectroscopy 
 
 6.0 Supplies 
 
 6.1 Chemicals — All reagents are ACS grade or better 
 
 6.1.1 Milli-Q Water 
 
 6.1.2 MeOH 
 
 6.1.3 TFA 
 
 6.1.4 L-DOPA 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 17mm x 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 
 
 6.3.4 Wrist Action Shaker 
 
 7.0 Procedure 
 
 7.1 Mobile Phase, Extraction Solvent & Diluent Preparation 
 
 

[SOP D-788 | Page 3 of 7]

 Standard Operating Procedure SOP No | Rev 
 Determination of Levodopa by HPLC using UV/VIS D-788 i Page 3 of 7 
 Spectroscopy 
 
 7.1.1. Mobile Phase A— 0.1% TFA 
 
 7.1.1.1. Combine 1 mL of TFA with 1000 mL of Milli-Q Water. Mix well.
 
 TAZ Mobile Phase B - MeOH 
 
 113 Extraction Solvent = Diluent — 90:10 Water / MeOH w/ 0.1% TFA
 
 7.1.3.1 Combine 100ml of MeOH and Iml of TFA with 900 mL of Milli-Q
 Water. Mix well. 
 
 7.1.4 Preparations may be scaled as necessary 
 
 TZ Standard Prep 
 7.2.1. Accurately weigh and transfer about 25 mg of L-DOPA reference standard into
 
 a 50-mL volumetric flask. Add 25mL of Extraction Solvent. Sonicate for 5min,
 
 cool, then QS to volume with Extraction Solvent. 
 
 7.2.2 Dilute 1:10 w/ Diluent for a Working Standard concentration of 50 ug/ml.
 
 7.3 Sample Preparation 
 
 7.3.1 The validated range for the analytical method is 79.9 — 1278.4 ng on column.
 
 Tedud Prepare raw materials like standards. Be sure to consult the specification for
 expected potency, as raw material samples may not be 100%. 
 
 7.3.3 Samples can be extracted in Extraction Solvent at any volume starting from
 50mL. The volume chosen must be in the solubility range of L-DOPA
 
 (validated at 0.5 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 bring the analyte concentration into the linear range.
 
 7.3.4 Fill the flask to about 50% of the calculated volume with Extraction Solvent and
 shake mechanically for 10 minutes. Dilute to volume with Extraction Solvent.
 
 Filter a portion for use in subsequent dilutions / injections.
 
 yRes Perform further dilutions as required using Diluent. 
 
 1:36 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,
 
 

[SOP D-788 | Page 4 of 7]

 Standard Operating Procedure SOP No | Rev 
 Determination of Levodopa by HPLC using UV/VIS D-788 I Page 4 of 7 
 Spectroscopy 
 
 baseline separation of peaks, and extraction efficiency as a part of system
 suitability before data can be reported using this method. 
 
 7.4 HPLC Parameters 
 
 741 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: 1 mL/min 
 
 7.4.4 Wavelength: 282 nm 
 
 7.4.5 Injection Volume: 5 wL 
 
 7.4.6 Run Time: 10 minutes. 
 
 7.4.7 3-D Spectral Range (for Identification) - 210nm to 350nm 
 
 7.4.8 Mobile Phase Gradient 
 
 Time, min %A %B 
 0-3 90 10 
 3-6 80 20 
 6.1 90 10 
 6.1-10 90 10 
 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 or at the end of a run. 
 
 7.6 System Suitability Requirements 
 
 1.034 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%. 
 
 1.6.3 If present, any interference in the diluent should be subtracted out of the sample
 
 and standard peak areas. 
 
 

[SOP D-788 | Page 5 of 7]

 Standard Operating Procedure SOP No | Rev 
 Determination of Levodopa by HPLC using UV/VIS D-788 I Page 5 of 7 
 Spectroscopy 
 
 7.7 Example calculations for determining finished product % label or raw material % purity
 
 TIA WL, =DOPA = & SWteereqXdP ye o E e S V SRE oy 1.00 
 Rs Vsta SA LA 
 Sample peak area 
 
 Mean (n=5) standard peak area 
 
 Weight of the reference standard in mg (corr. for water if applicable)
 
 Volume of the standard preparation accounting for dilutions in mL
 
 Purity of the reference standard in decimal format 
 
 Sample amount in mg (solids) or mL (liquids) 
 
 Volume of the sample preparation accounting for dilutions in mL
 
 Serving size: Average weight of ten dosage units in mg for tablets,
 
 fill weight for capsules, mass of a single serving in mg for powders,
 volume of a single serving from the theoretical formula in mL for
 
 liquids, or 1 for raw materials. 
 
 LA Label amount in mg of analyte (use | for raw materials)
 
 7.8 Column Wash and Storage 
 
 7.8.1 Wash and store the column in 75:25 ACN / Milli-Q Water. 
 
 

[SOP D-788 | Page 6 of 7]

 Standard Operating Procedure SOP No | Rev 
 Determination of Levodopa by HPLC using UV/VIS D-788 I Page 6 of 7 
 Spectroscopy 
 
 8.0 Chromatograms 
 
 8.1 Typical Diluent Chromatogram 
 
 | 
 fi‘ 
 
 e;j Aili T AMA; | Aa L | L H pi ta " al in i 
 
 8.2. Typical Working Standard Chromatogram 
 
 

[SOP D-788 | Page 7 of 7]

 Standard Operating Procedure SOP No Rev 
 Determination of Levodopa by HPLC using UV/VIS D-788 I Page 7 of 7 
 Spectroscopy 
 
 8.3. Typical Sample Chromatogram 
 
 "Yi oo 1 L-DOPA- 2.082 = 
 
 I | 
 | 
 chee 4, ene Nn a 
 
 9.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 09/11/19 | New procedure. N/A C. Perry 07/21/22 Scheduled review: updated logo and format. CC-22-0290 K. Burris | - | - |