Determination of Lidocaine and Phenoxyethanol 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 identification
 of Lidocaine and Phenoxyethanol in raw materials and finished products by HPLC/UV.
 
 2.0 Scope 
 
 The procedure applies to the identification and quantification of Lidocaine and Phenoxyethanol
 in raw materials and finished products by HPLC/UV, specifically in drug product DCRO00013.
 
 3.0 Responsibility 
 
 3.1 It is the responsibility of QC Chemists to follow this procedure. 
 
 It is the responsibility of QC Laboratory Management to ensure that this procedure is
3.2 being followed.

 It is the responsibility of QC Laboratory Management and/or Analytical Development
3.3 Personnel to keep this procedure aligned with current practices.

 4.0 Definitions 
 
 HPLC/UV — High Pressure Liquid Chromatography with Ultraviolet Detection
4.1 4.2 QC — Quality Control
 
 4.3 CofA — Certificate of Analysis 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Lidocaine and Phenoxyethanol D-783 1 age 
 2 of7 
 by HPLC using UV/VIS Spectroscopy 
 
 4.4 LDC — Lidocaine 
 
 4.5 POE — 2-Phenoxyethanol 
 
 4.6 DMA -— 2,6-Dimethylaniline HCl 
 
 4.7 Euxyl® PE9010 — A preservative blend containing 90% Phenoxyethanol
 
 4.8 ACN — Acetonitrile 
 
 4.9 H3PO4 — 85% Phosphoric Acid 
 
 4.10 H20 — Deionized water 
 
 5.0 References 
 
 5.1 MV-LAB-19-062 Validation of an Analytical Method for the Determination of
 
 Lidocaine by HPLC/UV 
 
 6.0 Supplies 
 
 6.1 Chemicals: All reagents are HPLC grade or better 
 
 6.1.1 LDC Reference Standard 
 
 6.1.2 POE Reference Standard 
 
 6.1.3 DMA 
 
 6.1.4 ACN 
 
 6.1.5 H3PO4 
 
 6.2 Glassware 
 
 6.2.1 Volumetric glassware as required for standard and sample preparations
 
 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 Centrifuge 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Lidocaine and Phenoxyethanol D-783 : Page 
 3 of 7 
 by HPLC using UV/VIS Spectroscopy 
 
 6.3.4 Adjustable Pipette 
 
 7.0 Procedure 
 
 El Mobile Phase Preparation 
 
 deka Mobile Phase A 
 
 7.1.1.1 Combine 1000 mL of H20 with 1.0 mL of H3PO, 
 
 (a Mobile Phase B 
 
 7.1.2.1 ACN 
 
 Eis Extraction Solvent 
 
 7.1.3.1 Combine 500 mL Water with 500 mL ACN 
 
 7.1.4 Diluent 
 
 7.1.4.1 Combine 900 mL of MP-A with 100 mL of CAN 
 
 7.1.5 Scale all mobile phases, extraction solvents and diluent as needed.
 
 7.2 Standard Preparation 
 
 7.2.1 Standard solutions are stable for two days at room temperature.
 
 eee Dilutions should be prepared using volumetric glassware. 
 
 7.2.3 Standard preparation may be scaled up as required. Scaling down is generally
 not recommended. 
 
 7.2.4 LDC Stock: Transfer 50 mg of LDC reference standard (or 61.6 mg of
 
 LDC*HCl monohydrate reference standard) into a 50-mL volumetric flask.
 
 Dissolve in and dilute to volume with Extraction Solvent. 
 
 T,2ied POE Stock: Only prepare if quantifying POE. Use a pipet to transfer about
 112.5 mg of POE reference standard directly into a 250-mL volumetric flask
 
 taking care to direct the standard to the bottom of the flask without touching the
 side. Dissolve in and dilute to volume with Extraction Solvent.
 
 Fa2® DMA Stock: Transfer 40 mg of DMA to a 25-mL volumetric flask. Dissolve in
 
 and dilute to volume with Extraction Solution. The DMA Stock does not expire
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Lidocaine and Phenoxyethanol D-783 | 
 Page 
 4 of 7 
 by HPLC using UV/VIS Spectroscopy 
 and may be kept in the refrigerator until needed. If additional peaks are observed
 in the chromatogram of the Working Standard, prepare a fresh DMA stock.
 
 7.2.7 Working Standard: Transfer 4.0 mL of LDC Stock, 2.0 mL of POE Stock, and
 
 1.0 mL of DMA Stock into a 100-mL volumetric flask. Dilute to volume with
 Diluent. 
 
 ie Sample Preparation 
 
 7.3.1 LDC is stable in sample preparations at room temperature for one day. Samples
 which require analysis of POE should be analyzed within 24 hours of
 
 preparation. 
 
 7.3.2 Finished Products: For topical semi-solid dosage forms: based on the label
 claim, transfer no less than 250 mg of the product into a suitably sized
 
 volumetric flask of no less than 25 mL to generate a concentration of LDC that
 
 is about 0.4 mg/mL and/or a concentration of POE that is about 0.09 mg/mL.
 Dilute the sample to 2/3 of the flask volume with Extraction Solvent and shake
 
 mechanically for 30 min or until the sample is completely dispersed. Dilute to
 volume with Extraction Solvent. Transfer a 5.0 mL aliquot of the completely
 
 dispersed sample into a 50-mL volumetric flask and dilute to volume with
 Diluent. Filter a portion through a 0.45 um membrane discarding the first 3 mL.
 
 ExamplTeh:e product contains 4% LDC and 1% Euxyl® PE9010 
 
 Euxyl® PE9010 contains 90% POE 
 
 Prepare 50 mL of a 0.4 mg/mL LDC and 0.09 mg/mL POE solution
 
 0.4 mg/mL LDC + (4%/100%) x 50 mL = 500 mg 
 
 0.09 mg/mL POE + (90%/100%) + (1%/100%) x 50 mL = 500 mg
 
 Dissolve 500 mg of sample in 50 mL of Diluent and filter
 
 7.3.3. Raw Materials: For LDC determination, weigh no less than 40 mg of raw
 material sample into a suitably sized volumetric flask to generate a
 
 concentration of about 0.4 mg/mL Dissolve in and dilute to volume with diluent.
 
 7.3.4 Perform further dilutions as required using diluent. 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 D-783 1 Page 
 Determination of Lidocaine and Phenoxyethanol 
 5 of7 
 by HPLC using UV/VIS Spectroscopy 
 7.3.5 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,
 
 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 
 
 7.4.1 Column: Kinetex XB-C18, 5um, 4.6 mm x 150 mm 
 
 7.4.2 Column Temperature: 30 °C 
 
 74.3 Flow rate: 0.8 mL/min 
 
 TAA Wavelength: 220 nm with 4 nm bandwidth 
 
 7.4.5 Injection Volume: 5 uL 
 
 7.4.6 Run Time: 15 minutes 
 
 7.4.7 Spectral Range (for Identification)- 210 nm to 360 nm 
 
 74.8 Gradient Profile: 
 
 Time “MPA %MPB 
 0 90 10 
 10 10 90 
 10.1 90 10 
 15 90 10 
 
 7.5 Recommended Sequence 
 
 7.5.1 Make at least 2 injections of the diluent. 
 
 hoe Make five (5) injections of Standard Solution. 
 
 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 or 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%. 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Lidocaine and Phenoxyethanol D-783 { 
 Page 
 6 of 7 
 by HPLC using UV/VIS Spectroscopy 
 7.6.3 The average (n=5) tailing factor for LDC is NMT 1.5. 
 
 7.6.4 The average (n=5) resolution of DMA and LDC is NLT 1.8. 
 
 7.6.5 If present, any interference in the diluent should be subtracted out of the sample
 and standard peak areas. 
 
 7.7. _Retention Times 
 
 7.7.1 DMA: RT = 4.23 min, RRT = 0.914 
 
 7.7.2 LDC: RT = 4.63 min, RRT = 1.000 
 
 7.7.3 POE: RT = 6.52 min, RRT = 1.408 
 
 7.8 Example calculations for determining finished product % label or raw material % purity
 
 7.8.1 %assay= 4R x —WtstSq HX=P x —V P x —100 
 Rs Veta Splwt | LA 
 
 Ry Sample peak area 
 
 Rg Mean standard peak area 
 
 Wteta Weight of reference standard in mg (correct for moisture if required)
 
 Vsta Volume of the standard preparation accounting for dilutions in mL
 
 P Purity of the reference standard in percent 
 
 Spl we Sample weight in mg 
 
 Vspl Volume of the sample preparation accounting for dilutions in mL
 
 LA Label amount in percent (use 1 for raw materials) 
 
 7.9 Column Wash and Storage 
 
 7.9.1 Rinse the column with H2O / ACN (80/20) 
 
 7.9.2 Store the column in H20 / ACN (50/50) 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev ° 
 Determination of Lidocaine and Phenoxyethanol D-783 I “ os 
 by HPLC using UV/VIS Spectroscopy 
 
 8.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 06/26/19 | New N/A S. Sassman | - | - |
| 1 | 07/21/22 | Scheduled review: updated logo and format. CC- | 22-0295 | _K. Burris |