Total Polysaccharide Determination by UV-VIS Spectroscopy

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1.0 Purpose 
 
 The purpose of this procedure is to define the method for the quantitation of total
 polysaccharide content in raw materials using UV/VIS spectrophotometry.
 
 2.0 Scope 
 
 This procedure applies to the quantification of polysaccharides in raw materials and straight fill
 finished products. This method is not appropriate as an identification for individual sugars and
 
 all sugars will react to form a chromaphore. Therefore, care must be taken in the use of this
 method to quantify one sugar in the presence of others as all other present sugars will be
 
 quantitated. 
 
 3.0 Responsibility 
 
 3.1 It is the responsibility of QC and Analytical 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 HeSOs-— Sulfuric Acid 
 
 
 

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 4.3. CofA — Certificate of Analysis 
 
 4.4 RT — Room Temperature 
 
 4.5 H20O- Millipore Water 
 
 4.6 STD - Standard 
 
 4.7 cGMP — Current Good Manufacturing Practices 
 
 4.8 Polysaccharide — a polymeric carbohydrate molecule comprised of long chains of
 
 monosaccharide units 
 
 4.9 QS — Quantity Sufficient 
 
 4.10 QC -— Quality Control 
 
 5.0 References 
 
 5.1 Journal of Applied Pharmaceutical Science 01 (03); 2011; 93-95 
 
 5.2 Int. J. Modern Biol. Med. 4 (3); 2013; 204-215 
 
 5.3 Analytical Chemistry 28 (3); 1956; 350-356 
 
 5.4 Manufacturers Internal test method (unpublished) 
 
 6.0 Supplies 
 
 6.1 Chemicals: All reagents are ACS grade or better. 
 
 6.1.1 Millipore Water 
 
 6.1.2 Phenol 
 
 6.1.3 H2SOg 
 
 
 

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 6.1.4 Sucrose 
 
 6.2 Glassware 
 
 6.2.1 GC headspace vials, with crimp cap enclosures 
 
 6.2.2 Scintillation Vials 
 
 6.2.3. 50mL Volumetric Flask 
 
 6.2.4 100mL Volumetric Flask 
 
 6.3 Disposables 
 
 6.3.1 10mL Pipette Tips 
 
 6.3.2 ImL Pipette Tips 
 
 6.3.3. 200uL Pipette Tips 
 
 6.3.4 1.5mL microfuge tubes 
 
 6.3.5 16mL Test Tubes 
 
 6.3.6 Disposable Plastic Luer Lock Syringe — 3mL, 6mL, or 10mL 
 
 6.3.7 Nylon Syringe Filters, 0.2um 
 
 6.3.8 Weigh paper 
 
 6.4 Equipment 
 
 6.4.1 Lambda 365 Spectrophotometer, Perkin Elmer, with Win-Lab Software
 
 6.4.2 Crimper 
 
 6.4.3. Decrimper 
 
 
 

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 6.4.4 Analytical Balance 
 
 6.4.5 Hot water bath 
 
 6.4.6 Ultrasonic bath 
 
 6.4.7. Vortex 
 
 6.4.8 Stir Plate 
 
 6.4.9 Eppendorf Centrifuge 
 
 6.4.10 10mL Pipette 
 
 6.4.11 1ImL Pipette 
 
 6.4.12 200uL Pipette 
 
 7.0 Preparation of Reagents, Samples, and Standards 
 
 7.1 5% Phenol Solution 
 
 Prepared by mixing phenol stock and water to a concentration of 5% (v/v)
 
 7.2 Standard Preparation 
 
 7.2.1 Use the actual purity from the CofA or the standard certification for sucrose
 reference material for calculations. The stock standard preparation reflects 100%
 
 content for the analyte assayed. 
 
 Example: Sucrose, 99% purity 
 
 Prepare 50mL of a 1mg/mL solution 
 
 Img/mL x50mL = 50mg 
 
 50mg/0.99 = 50.5mg 
 
 
 

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 Dissolve 50.5mg up to 50mL = 1.0mg/mL 
 
 7.2.2. Accurately weigh about 100mg of Ref standard into a 100mL volumetric and
 
 QS with DI H20 and dissolving fully to make a 1mg/mL solution.
 
 7.2.3. Pipet 10.0mL of the above solution into a 100mL volumetric and QS to with DI
 
 H20. This is the standard stock. 
 
 7.3 Sample Preparation 
 
 7.3.1. Accurately weigh about 50mg of sample into a 50mL volumetric and QS with
 
 DI H20. 
 
 7.3.2 Pipet 1.0mL of the above solution into a 50mL volumetric and QS with DI H20.
 
 This is the sample stock. 
 
 7.3.3. Sample preparation amounts and dilutions may need to be adjusted to ensure
 
 sample concentration is within the standard calibration curve.
 
 $8.0 Test Procedure 
 
 8.1 Make a calibration curve 
 
 8.1.1 A calibration curve should include a blank and 3-5 points. An example standard
 curve is listed below. 
 
 8.1.1.1 Using the standard stock, pipet 0.0, ImL, 2mL, 4mL, 6mL, and 8mL
 into separate GC headspace vials (or other appropriate vials) and dilute
 
 each to 20mL with DI H2O. Transfer 800uL of each of the standard
 
 stock into separate glass vials and add 400uL of the 5% phenol
 solution to each vial. 
 
 8.1.1.2 Ina fume hood, carefully add 2mL of concentrated H2SOs to each vial.
 Be careful with H2SQOxq as it is corrosive and when mixed with water
 
 will make the vials very hot. 
 
 
 

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 8.1.1.3 Carefully and gently swirl the vials and crimp to seal. Heat in a water
 bath at 80°C for 30min. 
 
 8.2 Preparing Sample 
 
 8.2.1 Using the sample stock, pipet 800uL of the solution into a GC headspace vial
 
 (or other appropriate vial) and add 400uL of the 5% phenol solution to the vial.
 
 8.2.2 In a fume hood, carefully add 2mL of concentrated H2SOq. Be careful with
 H2SOs as it is corrosive and when mixed with water will make the vials very
 
 hot. 
 
 8.2.3 Carefully and gently swirl the vial and crimp to seal. Heat in a water bath at
 
 80°C for 30min. 
 
 9.0 Measurement 
 
 9.1 After the 30 minutes of heating standard and samples is complete, allow to cool for
 
 15min. 
 
 9.2. De-crimp vials and transfer to cuvettes for analysis. 
 
 9.3 Measure each standard and sample vial at 490nm being sure to use the blank to zero the
 spectrophotometer and write the values obtained in a laboratory notebook.
 
 10.0 Calculations 
 
 10.1 Calculations for Determining Quantity 
 
 10.1.1 Plot the concentration (ppm) (Y-axis) vs absorbance (X-axis) for the standard
 
 points. 
 
 10.1.2 Use the slope of the standard line to obtain the concentration (Y) of the sample
 
 solution from the absorbance (X) from the equation y = mx + b.
 
 
 

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 10.1.2.1 Y = value on the y-axis which is concentration of the sample
 
 10.1.2.2 X = value on the x-axis which is the absorbance of the sample
 
 10.1.2.3 b = y-intercept 
 
 10.1.2.4 m=slope of the line obtained from standard calibration curve
 
 10.1.3 The % content of polysaccharide in the sample can be determined by the
 
 equation 
 
 % content = [C]sam * [ Dilution amount (mL) / Sample Weight (mg) | * 100
 
 [C]sam= Concentration of sample in mg/mL. 
 
 11.0 System Suitability 
 
 11.1. The coefficient of determination (R”) for the calibration is NLT 0.98.
 
 12.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 01/18/19 | New. N/A J. Maignan | - | - |
| 1 | 04/11/23 | Change instrument to Lambda 365 (new instrument), edit calculation CC- for clarity, change requirements section to system suitability. Increased the volume used to make the standards for the standard 7 og/og/24 curve in order to utilize glass pipettes. Doubled standard and sample CC-24-0352 W. Davis test volumes to fill more of the cuvettes. Specified concentration units for the graph. | 23-0184 | S. Sassman |