D-1026

Total Saponins Assay

Section D — Laboratory Operations and Specifications 4 pages

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1.0 Purpose

The purpose of this procedure is to define the process used for the determination of Total

Saponins using UV/Vis.

2.0 Scope

This procedure applies to the determination of Total Saponins as percentage for powdered

samples. This procedure follows “Improving the Vanillin-Sulphuric Acid Method for
Quantifying Total Saponins” from MDPI Technologies, Method <2.2.1> The Usual Vanillin-

Sulphuric Acid TSC Assay.

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 implement this procedure and
to ensure that the procedure is being followed.
3.3 It is the responsibility of QC Laboratory to keep this procedure current with latest Ion
Labs Practice.

4.0 Definitions

4.1 QC — Quality Control

5.0 References

5.1 MDPI Technologies, 2018, 6, 84, Method <2.2.1>

6.0 Procedure

6.1 Equipment

6.1.1 UV/VIS Spectrophotometer

[SOP D-102 | Page 2 of 4]

Standard Operating Procedure SOP No Rev
D-1026 Page 2 of 4
Total Saponins Assay (Vanillin-Sulfuric Acid TSC)

Geka Analytical Balance
6.1.3 Thermometer

6.1.4 Sonicator

6.1.5 Auto Pipette
6.1.6 0.45-um nylon membrane syringe filter

6.1.7 Scintillation Vials

6.1.8 Hot water bath

6.2 Reagents

6.2.1 DI Water — Fresh

6.2.2 Saponin (Sapogenin content 20-35%), Sigma-Aldrich (Quillaja sp.)

6.2.3 Vanillin
6.2.4 Ethyl Alcohol, Sigma-Aldrich, Anhydrous Reagent grade

6.2.5 Sulfuric Acid, Sigma-Aldrich, ACS grade

6.3 Solution Preparation

6.3.1 Vanillin Reagent Preparation
6.3.1.1 Toa10-mL Volumetric flask add approximately 1-gram of Vanillin.

6.3.1.2 |Add 5-7 mL of Ethanol and handshake until all the solids dissolve.

6.3.1.3. Dilute to volume with Ethanol and mix well.
6.3.1.4 Prepare fresh.

6.3.2 72% Sulfuric Acid

6.3.2.1 Ina beaker containing 6.6 mL of DI H20 slowly add and mix 18.4
mL of H2SQOs..
6.3.2.2 Manually stir for at least 30-seconds or until the solution is uniformly

dissolved.
6.3.2.3. Prepare fresh.

[SOP D-102 | Page 3 of 4]

Standard Operating Procedure SOP No Rev See
Total Saponins Assay (Vanillin-Sulfuric Acid TSC) D-1026 0 5

6.3.3 Standard Preparation
6.3.3.1 Approximately weigh 0.1l-gram of Saponin (containing 30%

Sapogenin) into a 50-mL volumetric flask.
6.3.3.2. Dissolve and dilute to volume with Ethanol.

6.3.3.3. Sonicate for 20-minutes.

6.3.3.4 Filter with 0.45-um syringe filter.
mg, WeightofSaponin(mg) Saponin Purity
Pandan Cone an Dilution volume (mL) 100
d —)=
6.3.4 Sample Preparation
6.3.4.1 Approximately weigh 0.1 gram of Sample into a 50 mL volumetric

flask.
6.3.4.2 Dissolve and dilute to volume with Ethanol.

6.3.4.3 Sonicate for 20-minutes.

6.3.4.4 — Filter with 0.45-um nylon membrane syringe filter.
mg, Weight of Sample (mg)
Sample Conc i) =
Dilution volume (mL)
64 Testing

6.4.1 Three separate scintillation vials are taken and labelled Blank, Standard, and
Sample respectively.

6.4.2 Into each vial with an auto pipette add solutions as per the table listed below;

Component Reagent Blank (mL) | Standard (mL) | Sample (mL)

Extraction Solvent 0.25 - -

Standards in Extraction
- 0.25 -
Solvent
Sample in Extraction
- - 0.25
Solvent
Vanillin 8% (w/v) in
0.25 0.25 0.25
Ethanol
72% Sulfuric Acid
2.5 ee 2.5
(v/v) in H2O
6.4.3 Handshake the above solutions for 20-seconds to mix.

[SOP D-102 | Page 4 of 4]

Standard Operating Procedure SOP No Rev
D-1026 Page 4 of 4
Total Saponins Assay (Vanillin-Sulfuric Acid TSC)

6.4.4 The solutions are kept on shaking hot water bath for 15-minutes at 60°C.
6.4.5 Cool to room temperature.

6.4.6 | Measure and record the absorbance of the solutions at 560-nm using a UV/VIS
Spectrophotometer.

6.5 Calculation

Standard Conc Sample Absorbance
%Total Saponins = x x
Sample Conc Standard Absorbance

7.0 Revision History

Revision Date Description of Changes CCR # By
0) 9/17/24 New Procedure N/A Naseem M.