Sennosides A and B Determination using UV-Vis Spectroscopy

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1.0 Purpose 
 
 The purpose of this procedure is to define the method for the quantitation and/or identification
 of sennosides A and or B in raw materials and finished products using HPLC and UV/VIS
 
 spectrophotometry. 
 
 2.0 Scope 
 
 This procedure applies to the quantification and identification of the individual or total values of
 
 sennosides A and B in raw materials and finished products. Sennosides A and B are moderate
 
 chromophores and are measured at 360nm. Other wavelengths should not be used without
 justification. 
 
 3.0 Responsibility 
 
 3.1 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 UV/VIS — Ultraviolet and Visible Electromagnetic Spectrums 
 
 4.2 Na2HPO4-12H20 — Dibasic Sodium Phosphate dodecahydrate 
 
 4.3 NaH2POs:2H20 — Monobasic Sodium Phosphate dihydrate 
 
 4.4 BDSAC — Benzyldimethylstearylammonium chloride 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Sennosides A and B Determination by HPLC using | 2-762 3 bs ea 
 
 UV/VIS Spectroscopy 
 
 45 H20 — Deionized water 
 
 4.6 ACN — Acetonitrile 
 
 4.7 QC - Quality Control 
 
 5.0 References 
 
 5.1 MV-LAB-18-068, Protocol, Sennosides A and B Determination using HPLC with
 UV/VIS Spectroscopy 
 
 5.2. USP 41N36 Monograph for Sennosides 
 
 5.3. D-793, SOP, Cryogenic Grinding of Chewable Gels 
 
 6.0 Supplies 
 
 6.1 Chemicals: All reagents are HPLC grade or better. 
 
 6.1.1 H2O @ 18.2 MQ-cm) 
 
 6.1.2 Sodium Acetate 
 
 6.1.3 ACN 
 
 6.1.4 NazHPO4-12H20 
 
 6.1.5 NaH2PO4:2H20 
 
 6.1.6 BDSAC 
 
 6.1.7 Sennoside A and Sennoside B reference standards 
 
 6.2 Glassware 
 
 6.2.1 HPLC vials, 12mm x 32mm with screw cap enclosures with septa
 
 6.2.2 Scintillation vials 
 
 6.2.3 Mobile phase containers 
 
 6.2.4 Volumetric glassware as required by sample and standard preparations
 
 6.3 Disposables 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Sennosides A and B Determination by HPLC using D-762 3 nage 
 3 of9 
 UV/VIS Spectroscopy 
 
 Gel Tips for adjustable pipettes 
 
 6.3.2 Micro-centrifuge tubes 
 
 6.3.3 Disposable plastic Luer lock Syringe - 3mL, 6mL, or 10mL 
 
 6.3.4 Nylon syringe filters, 0.45ym 
 
 6.3.5 Weigh paper 
 
 6.4 Equipment 
 
 6.4.1 Suitable gradient HPLC system consisting of a pump, autosampler, column oven
 and UV detector with a chromatographic data handling system 
 
 6.4.2 Analytical balance 
 
 6.4.3 Micro balance 
 
 6.4.4 Ultrasonic bath 
 
 6.4.5 Vortex 
 
 6.4.6 Stir Plate 
 
 6.4.7 Eppendorf centrifuge 
 
 6.4.8 Adjustable pipettes 
 
 7.0 Preparation of Mobile Phase, Dissolution Buffer, Samples, and Standards
 
 7.1. Mobile Phase A — 200mM Phosphate Buffer pH5.0:ACN:BDSAC (500:500:5)
 
 71d Transfer 0.062 g of NaxHPO4:12H20 to a 1000-mL mobile phase bottle.
 
 7.1.2 Add 1.53 g of NaH2PO4:2H20. 
 
 7.1.3 Add 5 g of BDSAC. 
 
 7.1.4 Add 500 mL of H20. 
 
 7.1.5 Add 500 mL of ACN, and mix to dissolve. 
 
 7.1.6 Filter through a 0.45 um nylon membrane. 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Sennosides A and B Determination by HPLC using | 9-762 3 Tae 
 
 UV/VIS Spectroscopy 
 
 7.2 Mobile Phase B — 100% ACN 
 
 7.3 Diluent — 1% Sodium Acetate (aq) 
 7.3.1 Transfer 10 g sodium acetate to a 1000-mL mobile phase bottle.
 
 7.3.2 Add 1000 mL H20, and mix to dissolve. 
 
 7.4 Stock Standard Preparation 
 
 7.4.1 Use a micro-balance with a minimum weight of no less than 5 mg to prepare the
 Stock Standards. 
 
 7.4.2 Prepare individual stock solutions of Sennosides A and B. 
 
 7.4.3 Accurately weigh and transfer about 5 mg of each reference standard into separate
 
 50-mL volumetric flasks. 
 
 7.4.4 Dilute to volume using Diluent, and sonicate for 10 minutes or until the reference
 
 standard is completely dissolved. 
 
 7.4.5 Equilibrate to room temperature before performing further dilutions.
 
 7.5 Working Standard Preparation 
 
 7.5.1 Transfer 5.0 mL of Sennoside A Stock Standard into a 10-mL volumetric flask.
 
 7.5.2 Dilute to volume with Sennoside B Stock Standard, and mix well.
 
 7.6 Sample Preparation 
 
 7.6.1 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.
 
 7.6.2. The linear range of the method is 0.01 — 0.1 mg/mL. The analyte concentrations
 in all standard and sample preparations must be within the linear range of the
 
 method. 
 
 7.6.3 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
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev P 
 
 Sennosides A and B Determination by HPLC using D-762 3 Ero 
 UV/VIS Spectroscopy 
 
 the validated linearity range. Dilute to volume with Diluent, and sonicate for 10
 
 min, 
 
 7.6.4 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 50 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. Dilute
 
 to volume with Diluent, and sonicate for 10 min. 
 
 7.6.5 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 a portion of the pooled and homogenized dosages into a beaker. Use several
 
 small portions of Diluent to completely transfer the sample into a suitably sized
 volumetric flask to generate an analyte concentration that is within the validated
 
 linear range. Dilute to volume, and sonicate for 10 min. 
 
 7.6.6 To manage large volumes, the standard can be initially prepared at a higher
 
 concentration and further diluted into the linear range using Diluent. Equilibrate
 to room temperature prior to performing further dilution. Dilutions can be
 
 made using volumetric glassware and/or adjustable pipettes. Dilutions can be
 prepared in HPLC vials 
 
 7.6.7 If particulates remain in the final sample preparation, a portion may be centrifuged
 
 at 10,000 rpm for 5 min prior to HPLC analysis. Alternatively, the sample may
 
 be filtered through a 0.45 um membrane discarding the first 3-4 mL.
 
 8.0 Test Conditions 
 
 8.1 Gradient 
 
 Time %A %B 
 
 0.00 100 0 
 
 35.00 100 0 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Sennosides A and B Determination by HPLC using D-762 3 Meee 
 6 of9 
 UV/VIS Spectroscopy 
 
 40.00 30 70 
 
 50.00 30 70 
 
 55.00 100 0 
 
 60.00 100 0 
 
 8.2 Column — Kinetex XB-C18, 5um, 100A, LC column, 250mm x 4.6mm, or equivalent
 
 8.3 Flow Rate — 1.0mL/min 
 
 8.4 UV Detection — 360nm 
 
 8.5 Injection Volume - 10uL 
 
 8.6 Column Temperature — 40°C 
 
 8.7 Recommended 3-D Spectral Range — 200 to 400 nm 
 
 8.8 Retention Times 
 
 8.8.1 Sennoside A is about 43 min 
 
 8.8.2 Sennoside B is about 25 min 
 
 8.9 Recommended Sequence 
 
 8.9.1 Make at least 2 injections of a Blank (Diluent). 
 
 8.9.2 Make a single injection of the Sennoside A Stock Standard. 
 
 8.9.3 Make a single injection of the Sennoside B Stock Standard. 
 
 8.9.4 Make five injections of the Working Standard. 
 
 8.9.5 Make a single injection of each Sample Preparation. 
 
 8.9.6 Make a single injection of the Working Standard after every six samples and at
 
 the end of the run. 
 
 8.10 System Suitability 
 
 8.10.1 The %RSD of five consecutive injections of Working Standard is NMT 5.0%.
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Sennosides A and B Determination by HPLC using D-762 3 Z a 
 
 UV/VIS Spectroscopy 
 
 8.10.2 The %RSD of all Working Standard injections is NMT 5%. 
 
 8.11 Column Wash and Storage 
 
 8.11.1 Rinse the column with H20 / ACN (90/10) at 1 mL/min for at least 15 min.
 
 8.11.2 Rinse the column with H20 / ACN (50/50) at 1 mL/min for at least 10 min.
 
 8.11.3 Store the column with H2O / ACN (50/50). 
 
 9.0 Example Calculation 
 
 Ry Wtsta XP Vspr SS 
 %s iaessany = ——t hX ee e x sa x—“txa X* 110 
 
 Ry Sample peak area 
 
 R, Mean standard peak area 
 
 Wtsta Weight of reference standard in mg 
 
 Veta Volume of the standard preparation accounting for dilutions in mL
 
 P Purity of the reference standard in decimal format 
 
 SA Sample amount in mg 
 
 Vspt Volume of the sample preparation accounting for dilutions in mL
 
 SS Serving size: Weight of a single dosage unit in mg or | for raw materials.
 
 LA Label amount in mg per dose or 1 for raw materials 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Page 
 Sennosides A and B Determination by HPLC using D-762 3 
 8 of 9 
 UV/VIS Spectroscopy 
 10.0 Example Chromatography 
 
 10.1. Blank 
 DAD1A,Sig=360.4 Ref=off 
 56 
 50-4 
 48+ 
 40+ 
 384 
 2 2380-74 
 20+ 
 154 
 104 
 5-4 
 274.”5 
 a 
217.5 0

 50.24-4 
 14N 
 278.24y 
 4 T 6 r 8 + 1 T 0 : 1 T 2 1 u 4 y # T 1 6 #1 t 8 #2 T 0 2 y 2 24 26 28 30 32 34 36 38 40 42 44 46 48 50 62 54 56 68 60
 Time [min] 
 10.2 Sennoside A Stock Standard 
 DAD1A,Sig=360,4 Ref=off 
 454 
 404 
 38> 
 2 on 
 184 
 10+ 630.2 232 2 615 
 » 
 522,34 
 A 
 edisonneS 
 &8 8 
 a id 
 2 4 6 & 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 S50 52 54 86 58 60
 Time [min} 
 10.3. Sennoside B Stock Standard 
 DAD1A.Sig=360.4 Ref=off 
 50+ 
 4s 
 40+ 
 35+ 
 2 23504+ 
 20- 
 18+ 
 10-4 
583.42 B

 edisonneS 
 14183264 884.5 057.5 
 y 
 %0.24ea 
 4N 
 10.4 Working Standard 
 DAD1A,Sig=360,4 Ref=off 
 -$ 24 26 28 30 32 34 36 38 40 42 44 46 48 60 52 54 68 60
 Time [min] 
 so, 
 45-4 
 40+ 
 307 
 2 254 
 154 
 10-4 
 5-4 1 
 530.2 572 073 254 2 
 415.42"= 
 B 
 edisonneS 
 6736 
 2a 
 A edisonneS 
 nra 
 2 4 6 68 10 12 14 16 18 20 22 24. 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 86 68 60
 Time [min) 
 

[SOP 

 Standard Operating Procedure SOP No | Rev z 
 Sennosides A and B Determination by HPLC using 3 LaF 
 9 of 9 
 UV/VIS Spectroscopy 
 10.5 Finished Product Sample 
 
 PS DAD1A,Sig=360.4 Ref=off 
 457} i 4 
 40+ 3 
 
 154 eek sor8 a8 _ 3 =e 
 sl a oe 
 2 4 6 6 10 12 14 16 18 20 22 24 26 oe 34.36 38 40 42 44 46 48 S50 52 4 86 68 60
 
 11.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 01/02/19 | New procedure. N/A J. Maignan Updated for consistency with current methods. Added recommended sequence section. Replaced requirements with ; nee system suitability section. Added column wash and storage. Cede02i8 S Sassnaa Added example chromatography. Updated logo and format. Added instruction to follow test details containing product specific sample preparation. Added specific sample prep | - | - |
| 2 | 06/06/23 | instructions for different dosage forms. Adjusted retentions CC- times. Updated example chromatograms. Updated logo and format. | 23-0266 | S. Sassman |
| 3 | 01/17/24 | Changed NaOAc to sodium acetate since it was not defined. CC- Corrected typo in gradient. | 24-0023 | © Sasenian |