Determination of Salicylic Acid and its Organic Impurities by HPLC-UV

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1.0 Purpose 
 
 The purpose of this procedure is to define the method for the quantification and identification of
 Salicylic Acid and its Organic Impurities in raw materials and finished products by HPLC/UV.
 
 2.0 Scope 
 
 This procedure applies to the quantification and identification of Salicylic Acid in raw materials
 
 and finished products by HPLC/UV. This procedure applies to the quantification and
 
 identification of the Organic Impurities of Salicylic Acid in raw materials by HPLC/UV. This
 procedure is intended for use in the QC laboratory. 
 
 3.0 Responsibility 
 
 31 It is the responsibility of QC Chemists to follow this procedure. 
 
 3.2‘ It is the responsibility of QC Laboratory Management to ensure that this procedure is
 
 being followed. 
 
 3.3 It is the responsibility of QC Laboratory Management and/or Analytical Development to
 
 keep this procedure aligned with current practices. 
 
 4.0 Definitions 
 
 4.1 HPLC/UV — High Performance Liquid Chromatography with Ultraviolet Detection
 
 4.2 QC — Quality control 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Salicylic Acid and its Organic D-796 1 | Page 2 of 11 
 Impurities by HPLC/UV 
 
 4.3 AD-— Analytical development 
 
 44 SAL - Salicylic acid 
 
 4.5 RCA- Salicylic acid related compound A (4-hydroxybenzoic acid) 
 
 4.6 | RCB-— Salicylic acid related compound B (4-hydroxyisophthalic acid)
 
 4.7 HO - Deionized water 
 
 5.0 References 
 
 dl 
 PRTCL-20-0075, Protocol, Verification of an Analytical Method for the Determination
 of Salicylic Acid and its Organic Impurities by HPLC 
 
 Si D-793, SOP, Cryogenic Grinding of Chewable Gels 
 
 6.0 Supplies 
 
 6.1 Chemicals: All reagents are HPLC grade or better 
 
 6.1.1 SAL reference standard 
 
 6.1.2 RCA reference standard 
 
 6.1.3. RCB reference standard 
 
 6.1.4 Phenol reference standard 
 
 6.1.5 MeOH 
 
 6.1.6 Glacial acetic acid 
 
 6.2 Glassware 
 
 6.2.1 Volumetric glassware as required for standard and sample preparations
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Salicylic Acid and its Organic D-796 | 1 | Page 3 of 11 
 Impurities by HPLC/UV 
 
 6.3. Equipment 
 
 63.1 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 
 
 O:3.0 Wrist action shaker 
 
 6.3.4 Centrifuge 
 
 6.3.5 0.45um luer-lock syringe filter and syringe barrel 
 
 6.3.6 Adjustable pipette and tips 
 
 7.0 Assay of Salicylic Acid 
 
 Tes Mobile Phase Preparation 
 
 7.1.1 Mobile Phase 
 
 7.1.1.1. Transfer 400 mL of methanol to a 1 L media bottle. 
 
 7.1.1.2 Add 10.0 mL of glacial acetic acid. 
 
 7.1.1.3 Add 600 mL of H20. 
 
 7.1.1.4 Mix well. 
 
 Teled Diluent A 
 
 7.1.2.1. Use Mobile Phase. 
 
 7.2 Standard Preparation 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Salicylic Acid and its Organic D-796 | 1 | Page 4 of 11 
 Impurities by HPLC/UV 
 
 7.2.1 Accurately weigh and transfer about 25 mg of SAL reference standard to a 50-
 mL volumetric flask. 
 
 7.2.2 Dissolve in and dilute to volume with Diluent. 
 
 7.3. Sample Preparation 
 
 7.3.1 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 below.
 
 7.3.2 The linear range for SAL is 0.35 mg/mL — 0.65 mg/mL. 
 
 7.3.3. For raw materials: weigh no less than 25 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 65% of the calculated
 
 volume with Diluent A and shake mechanically for 20 minutes. Sonication for
 up to 10 minutes can also be used to assist dissolution. If sonication is used,
 
 allow the sample to equilibrate to room temperature before bringing to final
 
 volume. Bring to final volume using Diluent A. 
 
 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 65% of the calculated volume with Diluent
 A and shake mechanically for 20 minutes. Sonication for up to 10 minutes can
 
 also be used to assist dissolution. If sonication is used, allow the sample to
 
 equilibrate to room temperature before bringing to final volume. Bring to final
 volume using Diluent A. 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Salicylic Acid and its Organic D-796 | 1 | Page 5 of 11 
 Impurities by HPLC/UV 
 
 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 200 mg of the pooled and homogenized dosages into a
 suitably sized beaker. Add a volume of Diluent A equivalent to 50% of the
 
 desired flask volume, add a stir bar, and stir until dissolved. Transfer the
 
 solution to a volumetric flask of size suitable to generate an analyte
 concentration that is within the validated linear range. Use several small portions
 
 of Diluent A to rinse any remaining residue from the beaker into the volumetric
 flask ensuring complete transfer, and dilute to volume using Diluent A.
 
 To manage large volumes, the sample can be initially dissolved in a smaller
 volume and a portion further diluted using Diluent A to bring the analyte
 
 concentration into the linear range. Dilutions can be made using volumetric
 
 glassware and/or adjustable pipettes. Dilutions can be prepared in HPLC vials.
 
 7.3.6 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.
 
 74 HPLC Parameters 
 
 7.4.1 Column: Restek Roc C18, 5um, 4.6 mm x 100 mm 
 
 7.4.2 Column Temperature: 30 °C 
 
 7.4.3 Flow rate: 1.0 mL/min 
 
 7.4.4 Wavelength: 270 nm 
 
 7.4.5 Injection Volume: 10 pL 
 
 7.4.6 Run Time: 10 minutes 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Salicylic Acid and its Organic D-796 | 1 | Page 6 of 11 
 Impurities by HPLC/UV 
 
 7.4.7 Recommended Spectral Range (for Identification)- 220 nm to 400 nm
 
 Fa) Retention Time 
 
 7.5.1 The retention time of SAL is about 7.2 minutes. 
 
 7.6 Recommended Sequence 
 
 7.6.1. Make at least 2 injections of the diluent. 
 
 7.6.2 Make five injections of Standard Solution. 
 
 7.6.3 Make a single injection of each Sample Preparation. 
 
 Ld System Suitability Requirements 
 
 7.7.1 The %RSD of five consecutive standard injections is NMT 0.73%.
 
 7.7.2 The USP tailing factor is NMT 2.5 
 
 7.713 No significant (>0.5%) interference are present in the diluent injection.
 
 7.8 Column Wash and Storage 
 
 7.8.1. Store the column in Methanol / H20 (50/50) 
 
 8.0 Organic Impurities of Salicylic Acid 
 
 8.1 Mobile Phase Preparation 
 
 8.1.1. Prepare as directed in Section 6.1. 
 
 8.2 Diluent B 
 
 8.2.1 Transfer 350 mL of methanol to a media bottle. 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Salicylic Acid and its Organic D-796 1 | Page 7 of 11 
 Impurities by HPLC/UV 
 
 8.2.2 Add 20 mL of glacial acetic acid. 
 
 8.2.3. Add 150 mL of H20. 
 
 8.2.4 Mix well. 
 
 8.3 Phenol Solution Preparation 
 
 8.3.1 Accurately weigh and transfer about 25 mg of phenol reference standard to a 25-
 mL volumetric flask. 
 
 8.3.2 Dissolve in and dilute to volume with Diluent B. 
 
 8.4 Peak ID Solution 
 
 8.4.1 Transfer 0.1 mL of Phenol Solution to a 10-mL volumetric flask.
 
 8.4.2 Dilute to volume with Diluent B. 
 
 8.5 Stock Standard Preparation 
 
 8.5.1 Accurately weigh and transfer about 50 mg of RCA to a 100-mL volumetric
 
 flask. 
 
 8.5.2 Accurately weigh and transfer about 25 mg of RCB to the same 100-mL
 
 volumetric flask. 
 
 8.5.3. Transfer 10.0 mL of Phenol Solution to the same 100-mL volumetric flask.
 
 8.5.4 Dissolve in and dilute to volume with Diluent B. 
 
 8.6 Working Standard Preparation 
 
 8.6.1 Use the actual purity from the reference standard CofA in your calculations.
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Salicylic Acid and its Organic D-796 | 1 | Page 8 of 11 
 Impurities by HPLC/UV 
 
 8.6.2 Accurately weigh and transfer about 25 mg of SAL reference standard to a 50-
 
 mL volumetric flask. 
 
 8.6.3 Transfer 5.0 mL of Stock Standard to the same 50-mL volumetric flask.
 
 8.6.4 Dissolve in and dilute to volume with Diluent B. 
 
 8.7. Sample Preparation 
 
 8.7.1 Accurately weigh and transfer about 2.5 g of raw material sample to a 50-mL
 
 volumetric flask. 
 
 bt ee Add about 45 mL of Diluent B. 
 
 8.7.3 Sonicate until completely dissolved. 
 
 8.7.4 Equilibrate to room temperature. 
 
 8.7.5 Dilute to volume with Diluent B. 
 
 8.8 HPLC Parameters 
 
 8.8.1 Column: Kinetex XB-C18, 5um, 4.6 mm x 150 mm 
 
 8.8.2 Column Temperature: 30 °C 
 
 8.8.3 Flow rate: 0.75 mL/min 
 
 8.8.4 Wavelength: 270 nm 
 
 8.8.5 Injection volume: 2 wL 
 
 8.8.6 Run Time: 12 minutes 
 
8.8.7 Recommended Spectral Range (for Identification)- 220 nm to 400 nm

 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 
 Determination of Salicylic Acid and its Organic D-796 | 1 | Page 9 of 11 
 Impurities by HPLC/UV 
 
 8.9 Retention Times 
 
 Note: The elution order of RCB and Phenol may switch. Use the Peak ID Solution to evaluate
 
 the retention time of these compounds. 
 
 8.9.1 RCA*=3.1 minutes 
 
 8.9.2 Phenol ~ 4.7 minutes 
 
 8.9.3 RCB*#=5.7 minutes 
 
 8.9.4 SAL =9.0 minutes 
 
 8.10 Recommended Sequence 
 
 8.10.1 Make at least 2 injections of Diluent B. 
 
 8.10.2 Make five injections of the Working Standard. 
 
 8.10.3 Make a single injection of the Peak ID Solution. 
 
 8.10.4 Make a single injection of each Sample Solution. 
 
 8.11 System Suitability Requirements 
 
 8.11.1 The %RSD of six consecutive standard injections of the Working Standard is
 
 NMT 5.0%. 
 
 8.11.2 The USP resolution between phenol and RC-B is NLT 2.0. 
 
 8.11.3 No significant (>0.5%) interference are present in the inj ection of Diluent B.
 
 8.12 Column Wash and Storage 
 
 8.12.1 Store the column in Methanol / H20 (50/50) 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Salicylic Acid and its Organic D-796 | 1 | Page 10 of 11
 Impurities by HPLC/UV 
 
 9.0 Example calculation 
 
 9.1.1 %assay = " x oe a x “4 x 100 
 
 Ry Sample peak area 
 
 R, Mean standard peak area 
 
 Wtetg Weight of reference standard in mg 
 
 Vsta Volume of the standard preparation accounting for dilutions in mL
 
 P Purity of the reference standard in decimal format 
 
 SS Serving size: Average weight of ten dosage units in mg for tablets and
 
 capsules, weight of a single serving from the theoretical formula in mg
 
 for liquids, or 1 for raw materials. 
 
 Splwet Sample weight in mg 
 
 Vspl Volume of the sample preparation accounting for dilutions in mL
 
 LA Label amount in mg (use 1 for raw materials) 
 
 10.0 Representative Chromatograms 
 
 10.1 Assay of Salicylic Acid 
 
 WSsTD 
 a2 
 aa 
 1004 
 1) 
 € 50) 
 25 
 T 
 0 
 06 od tel oe oe. 8 BS. 4 A. Se 86 BB FS ee 
 Time [min] 
 
 

[SOP 

 Standard Operating Procedure 
 SOP No | Rev 
 Determination of Salicylic Acid and its Organic D-796 | 1 | Page 11 of 11 
 Impurities by HPLC/UV 
 
 SAMPLE 
 
 5 
 = 504 
 257 
 ty) OT Gs 2 ae es Be ae Ae 8. eS 65 7 75 8 85 9 
 Time [min] 
 10.2 Organic Impurities 
 
 3 WSTD wy off = a} oaFe [vay b a a 3 
 o+ro 
 2 204 
 E 
 5 OST 1 a 8 2 25 8 35 4 45 A 5 5S 6 OBS TF 75 8 B85 9 95 10 105 11 115 12
 Time [min] 
 
 SAMPLE 
 ar a 3% 
 301 4 3% we 
 at 
 2 
 E 
 10: 
 0 OS 1 15 2 25 3 35 4 45 A 5 55 6 SE 7 75 8 8S 9 95 10105 11 115 12 
 Time [min] 
 
 11.0 Revision History CCR # By 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 08/18/20 | New | - | - |
| 1 | 04/03/24 | A av d a d il a i b n l s e t , r u a c d t d io s n p t e o c i f f o i l c l s o a w m p p r l o e d u p c r t e p s p i e n c s i t f r i u c c t t i e o s n t s d e f t o a r i l g s u m if mies, CC- correct calculation error, edit for consistency with current methods. | 24-0132 | €- Suscman |