D-771

Ribose Determination by UV-Vis Spectroscopy

Section D — Laboratory Operations and Specifications Revision 3 7 pages

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1.0 Purpose

The purpose of this procedure is to define the method for the quantitation of Ribose content in

raw materials and finished products using UV/VIS spectrophotometry.

2.0 Scope

This procedure applies to the quantification of ribose 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

personnel to keep this procedure aligned with current practices.

4.0 Definitions

41 UV/VIS — Ultraviolet and Visible Electromagnetic Spectrums

4.2 H2SO4- Sulfuric Acid

4.3 CofA — Certificate of Analysis

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4.4 RT—Room Temperature

4.5 H2O- Millipore Water

4.6 STD — Standard

4.7 eGMP-— Current Good Manufacturing Practices

4.8 Ribose — a pentose class sugar used in nature in nucleosides, vitamins and enzymes

4.9 QS — Quantity Sufficient

5.0 References

5.1 Journal of Applied Pharmaceutical Science 01 (03); 2011; 93-95

6 BO 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5.4 Manufacturers Internal test method (unpublished)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 H2SOz

6.1.4 Ribose

6.2 Glassware

6.2.1 GC headspace vials, with crimp cap enclosures

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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 45 Spectrophotometer, Perkin Elmer, with Win-Lab Software

6.4.2 Crimper

6.4.3 Decrimper

6.4.4 Analytical Balance

6.4.5 Hot water bath

6.4.6 Ultrasonic bath

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6.4.7. Vortex

6.4.8 Stir Plate

6.4.9 Eppendorf Centrifuge

6.4.10 10mL Pipette

6.4.11 ImL 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 ribose

reference material for calculations. The stock standard preparation reflects 100%

content for the analyte assayed.

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

H2O. 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.

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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, 20uL, 40uL, 80uL, 120uL, and
160uL into separate GC headspace vials (or other appropriate vials)

and dilute each to 400uL with DI H20. Add 200uL of the 5% phenol
solution to each vial.

8.1.1.2 In a fume hood, carefully add 1mL of concentrated H2SO4 to each
vial. Be careful with H2SQs as it is corrosive and when mixed with

water will make the vials very hot.

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 400uL of the solution into a GC headspace vial

(or other appropriate vial) and add 200uL of the 5% phenol solution to the vial.

8.2.2 In a fume hood, carefully add ImL of concentrated H2SO4. Be careful with

H2SQq as it is corrosive and when mixed with water will make the vials very

hot.

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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 (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.

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 ribose in the sample can be determined by the equation

% content = [C]sam * D/ SW * 100

[C]sam = Concentration of sample from step 10.1.2 (mg/mL)

D = Sample dilution volume (mL)

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SW = Sample weight (mg)

11.0 Revision History

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 01/18/19 | New. N/A J. Maignan 03/16/23 Minor edits for clarity. CC-23-0142 S.Sassman | - | - |