DRAFT UGANDA STANDARD  
     DUS 2035  
First Edition  
2018 -mm-dd 
  
 
 Reference number  
DUS 2035: 2018  
© UNBS 2018   
  
Steviol glycosides  — Specification  
 DUS 2035 : 2018  
ii © UNBS 2017  – All rights reserved  
 Compliance with this standard does not, of itself confer immunity from legal obligations  
A Uganda Standard does not purport to include all necessary provisions of a contract. Users are 
responsible for its correct application  
© UNB S 2017  
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilised in any 
form or by any means, electronic or mechanical, including photocopying and microfilm, without prior written 
permission from UNBS.  
Requests for permission to reproduce this document should be addressed to  
The Executive Director  
Uganda National Bureau of Standards  
P.O. Box 6329  
Kampala  
Uganda  
Tel: +256 41 7 333 250/1/2  
Fax: +256 414 286 123  
E-mail: info@unbs.go.ug  
Web: www.unbs.go.ug  DUS 2035:2018  
© UNBS 2017  – All rights reserved  iii 
 Contents  Page 
Foreword  ................................ ................................ ................................ ................................ ...................  iv 
Introduction  ................................ ................................ ................................ ................................ ................  v 
1 Scope  ................................ ................................ ................................ ................................ .............  1 
2 Normative references  ................................ ................................ ................................ ...................  1 
3 Terms and definitions  ................................ ................................ ................................ ..................  1 
4 Requirements  ................................ ................................ ................................ ................................  2 
4.1 General requirements  ................................ ................................ ................................ ..................  2 
4.2.2   Microbiological  requirements  ................................ ................................ ................................ .... 2 
5 Contaminants  ................................ ................................ ................................ ................................  2 
6 Pesticide residues  ................................ ................................ ................................ ........................  3 
7 Packaging  ................................ ................................ ................................ ................................ ...... 3 
8 Weights and Meas ures ................................ ................................ ................................ .................  3 
9 Labelling  ................................ ................................ ................................ ................................ ........  3 
10 Sampling  ................................ ................................ ................................ ................................ ....... 3 
Annex  A (normative)  Test for solubility  ................................ ................................ ................................ . 4 
A.1 Procedure  ................................ ................................ ................................ ................................ ...... 4 
Annex  B (normati ve)  Total steviol glycoside content – HPLC chromatographic profile  ..................  5 
B.1 Principle  ................................ ................................ ................................ ................................ ........  5 
B.2 Method A: Determination of major steviol glycosides by HPLC  ................................ .............  5 
B.3 Method B: Determination of minor steviol glycosides by HPLC -MS ................................ ...... 6 
Annex  C  (normative)   Determi nation of pH (Potentiometric Method)  ................................ ................  9 
C.1 Requirements  ................................ ................................ ................................ ................................  9 
C.2 Procedure  ................................ ................................ ................................ ................................ ...... 9 
Annex  D (normative)  Loss on drying  ................................ ................................ ................................ ... 10 
D.1 Requirements  ................................ ................................ ................................ ..............................  10 
D.2 Procedure  ................................ ................................ ................................ ................................ .... 10 
D.2.1  Sample preparation  ................................ ................................ ................................ ....................  10 
D.2.2  Drying  ................................ ................................ ................................ ................................ ..........  10 
Annex  E (normative)  Residual solvents – Methanol and Ethanol  ................................ .....................  11 
E.1 Reagent  ................................ ................................ ................................ ................................ ....... 11 
E.1.1  Method I (Determination carried out in water)  ................................ ................................ .........  11 
E.1.2  Method II (Determination carried out in methanol)  ................................ ................................ . 11 
E.2 Procedure  ................................ ................................ ................................ ................................ .... 11 
Bibliography  ................................ ................................ ................................ ................................ .............  13 
 DUS 2035 : 2018  
iv © UNBS 2017  – All rights reserved  
 Foreword  
Uganda National Bureau of Standards (UNBS) is a parastatal under the Ministry of Trade , Industry and 
Cooperatives established under Cap 327 , of the Laws of Uganda , as amended .  UNBS is mandated to co -
ordinate the elaboration of standards and is  
(a) a member of International Organisation for Sta ndardisation (ISO) and  
(b) a contact point for the WHO/FAO Codex Alimentarius Commission on Food Standards, and  
(c) the National Enquiry Point on TBT  Agreement  of the World Trade Organisation (WTO).  
The work of preparing Uganda Standards is carried out through T echnical Committees. A Technical Committee 
is established to deliberate on standards in a given field or area and consists of key stakeholders including 
government, academia, consumer groups, private sector and other interested parties.  
Draft Uganda Stand ards adopted by the Technical Committee are widely circulated to stakeholders and the 
general public for comments. The committee reviews the comments before recommending the draft standards 
for approval and declaration as Uganda Standards by the National S tandards Council.  
The committee responsible for this document is UNBS/TC 2 [ Food and Agriculture standards ], Subcommittee 
SC 6, [ Food Additives and contaminants ]. DUS 2035:2018  
© UNBS 2017  – All rights reserved  v 
 Introduction  
Stevia rebaudiana  Bertoni  is a perennial shrub in South America and Asia. In the two regions, water extracts of 
crushed leaves of the stevia plant are widely used as a sweetener/sugar replacer. Their use is expanding in 
water -based beverages and dietetic food products as a natural  sugar replacer.   
Steviol glycosides consist of a mixture of compounds containing a steviol backbone conjugated to any number 
or combination o f the principal sugar moieties ( glucose, rhamnose, xylose, fructose, arabin ose, galactose and 
deoxyglucose)  in any  of the orientations occurring in the leaves of Stevia rebaudiana  Bertoni, from which they 
are obtained. The leaves are extracted with hot water and the aqueous extract is passed through an adsorption 
resin to trap and concentrate th e component steviol gly cosides. The resin is washed with a solvent alcohol to 
release the glycosides and the product is recrystallized from methanol or aqueous ethanol. Ion exchange resins 
may be u sed in the purification process and the final product may be spray -dried.  
Stevial glycosides or extracts are reported to be 200 – 300 times sweeter than sucrose.  Codex established  the 
Acceptable Daily Intake for steviol glycosides at 0 – 4 mg/kg body weight  and according to CODEX STAN 192 -
1995 – General Standard for Food Additives, t hey are approved for use in a variety of food products  as INS  
960. 
This standard  has been developed to regulat e the trade and use  of steviol glycosides in food  products . DRAFT UGANDA STANDARD  DUS 2035: 2018  
 
© UNBS 2017 – All rights  reserved  1 
 Steviol glycosides  — Specification  
1 Scope  
This Draft Uganda  Standard specifies requirements , sampling and test methods for steviol glycoside s from  
Stevia rebaudiana  Bertoni  intended for human consumption . 
2 Normative references  
The following referenced documents are referred to in the text in such a way that some or all their content 
constitutes requirements of this document. For dated references, only the edition cited applies. For undated 
references, the latest edition of the referenced document (including any amendments) applies.  
AOAC 972.25 , Lead in food. Atomi c absorption spectrophotometric method  
AOAC 952.13,  Arsenic in food. Silver diethyldithiocarbamate  
US 277, General standard for the labelling of food additives when sold as such  
US CAC/GL 50, General guidelines on sampling  
US ISO 4833 -1, Microbiology of the food chain — Horizontal method for the enumeration of microorganisms — 
Part 1: Colony count at 30 ºC by the pour plate technique  
US ISO 4833 -2, Microbiology of the food chain — Horizontal method for the enumeration of microorganisms — 
Part 2: Colony count at 30 ºC by the surface plating technique  
ISO 7251, Microbiology of food and animal feeding stuffs — Horizontal method for the detection and 
enumeration of presumptive Escherichia coli — Most probable number technique  
US ISO 6579 –1, Microbiology of the food chain — Horizontal method for the detection, enumeration and 
serotyping of Salmonella — Part 1: Detection of Salmonella spp.  
US ISO/TS 6579 –2, Microbiology of food and animal feed — Horizontal method for the detection, enumeration 
and serotyping of Salmonella — Part 2: Enumeration by a miniaturized most probable number technique  
US ISO 21527 -2, Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of 
yeasts and moulds — Part 2: Colony count technique in  products with water activity less than or equal to 0.95  
3 Terms and definitions  
For the purposes of this document, the following terms and definitions apply.   
ISO and IEC maintain terminological databases for use in standardization at the following addre ss:  
— ISO Online browsing platform: available at http://www.iso.org/obp   
food grade material  
material made of substances which are safe and suitable for their intended use and which will not impart any 
toxic substanc e or undesirable odour or flavour to the product  CDUS 2035: 2018  
2 © UNBS 2017  – All rights reserved  
 4 Requirements  
4.1 General  requirements  
Steviol glycosides shall be:  
a) white to light yellow , odourless or having a slight  odour characteristic of the stevia extracts; and  
b) freely soluble in a mixture of water and ethanol  (50:50)  when tested in accordance with the procedure 
elaborated in Annex A.  
4.2 Specific requirements  
4.2.1  Physicochemical requirements  
Steviol glycosides shall com ply with the physicochemical requirements given in Table 1  when tested in 
accordance with the test methods  specified therein . 
Table 1 — Physicochemical requirements for steviol glycosides  
S/N Characteristic  Requirement  Test method  
i) Total steviol glycoside content  (dry basis), min.  95 Annex B 
ii) pH (1% solution)  4.5 – 7.0 Annex C 
iii) Loss on drying, % m/m , max.  6 Annex D 
iv) Total ash, % (dry basis) , max.  1 AOAC 923.03  
            Residual solvents  
v) Methanol (mg/kg) , max.  200 
Annex E 
vi) Ethanol (mg/kg) , max.  5 000 
4.2.2  Microbiological  requirements  
Steviol glycosides shall conform to  the microbiological requirements given in Table 2 when tested in accordance 
with the methods specified therein.  
Table 2  — Microbiologica l requirements for steviol glycosides  
S/N Characteristic  Requirement  Test method  
i) Total (aerobic) plate count , CFU/g , max.     1 000  US ISO 4833 -1 / 
US ISO 4833 -2 
ii) Escherichia coli, CFU/g  Not detected  ISO 7251  
iii) Salmonella , CFU/ 25 g Not detected  US ISO 6579 –1 / 
US ISO/TS 6579 –2 
iv) Yeasts and moulds , CFU/g, max .  200 US ISO 21527 –2 
5 Contaminants  
Steviol glycosides shall conform to the contaminant limits specified in Table 3 when tested in accordance with 
the methods specified therein.  CDUS 2035: 2018  
© UNBS 2017  – All rights reserved  3 
 Table 3 — Limits for contaminants for steviol glycosides  
S/N Characteristic  Requirement  Test method  
i) Arsenic (mg/kg) , max.   1 AOAC 952.13  
ii) Lead  (mg/kg) , max.   1 AOAC 972.25  
 
6 Pesticide residues  
Steviol glycosides shall comply with the maximum pesticide residue limits established by the Codex Alimentarius 
Commission.   
7 Packaging  
The product shall be securely packaged in containers made of food grade materials. The packaging  shall be 
able to safeguard  the product from contamination and protect its properti es throughout the shelf life  period .  
8 Weights and Measures  
The weight of th e package s shall comply with the  Weights and Measures (Sales and  Labelling of goods) 
Regulation s of Uganda . 
9 Labelling  
The product shall be labelled in accordance with US 277.  
10 Sampling  
Representative samples of the product shall be drawn in accordance with US CAC/GL 50.  CDUS 2035: 2018  
4 © UNBS 2017  – All rights reserved  
 Annex  A 
(normative)  
 
Test for solubility  
A.1 Procedure  
Transfer 1 g of sample into a  calibrated 10 -ml glass -stoppered cylinder g raduated in 0.1 -ml subdivisions. Slowly 
and in small portions, add a 50:50 mixture of distilled water and ethanol  (95%). Maintain the  temperature at 20  
°C.  
A clear s olution should be obtained . CDUS 2035: 2018  
© UNBS 2017  – All rights reserved  5 
 Annex  B 
(normative)  
 
Total steviol glycoside content – HPLC chromatographic profile  
B.1 Principle   
Determine the percentages of major steviol glycosides (those with ana lytical standards) using  HPLC  (Method 
A). Confirm the presence of each minor steviol glycoside (compounds where analytical standards are not 
available) using HPLC -MS (Method B ). Calculate the concentration of the minor compounds using respective 
molecular mass corrected UV peak area against the rebaudioside A UV standard curve. Calculate their sum 
and express the content on the dried basis.  
B.2 Method A: Deter mination of major steviol g lycosides by HPLC  
B.2.1 Reagents  
Acetonitrile: HPLC grade with transmittance more than 95% at 210 nm.  
- Deionized water: HPLC grade  
-  Standards (Reference and Quality Control Standards):  Stevioside, rebaudioside A, rebaudioside B,   
       rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside M , rebaudioside N,   
       rebaudioside O, dulcoside A, rubusoside and steviolbioside.  
Note: Standards of other steviol glycosides may also be included.  
B.2.2 Apparatus  
HPLC consisting of : a high precision binary pump and an auto sampler (capable of opera ting at 2 °C – 8 °C); 
Diode -Array detector @ UV at 210 nm; and Mass Spect rometric Detector (Electrospray Negative Ionisation over 
a mass range from 50 to 1500 m/z using a unit mass resolution , For use in Method B below) connected in series. 
Agilent 1200 wi th Waters Quattro or equivalent:  
- Column: Luna 5μ C18 (2), 100A, (150 mm x 4.6 mm, 5μm, Phenomenex) or Capcell pak C18 MG II  
       (250 mm x 4.6 mm, 5μm, Shiseido Co. Ltd) or equiv.  
- Column temperature: 50 °C  
- Auto sampler temperature: 2 °C – 8 °C 
- Injection volume: 10 μl  
- Mobile phase A: Deionised or LC -MS grade water (0.2 μm filtered)  
- Mobile phase B: LC -MS grade Acetonitrile (0.2 μm filtered)  
B.2.3 Preparation of s tevio l glycosides standard solutions  
- Prepare individual stock standard solutions (1.5 mg/ml) in water:acetonitrile (7:3);  
- Prepare mixed standard solution (115 μg/ml) by mixing 1.0 ml each individual stock standard solutions;  
- Prepare Peak Identification Standard Solutions (0.1 mg/ml) from individual stock standard solutions in  
       water:acetonitrile (7:3).  CDUS 2035: 2018  
6 © UNBS 2017  – All rights reserved  
 - Prepare mixed Committee  standard solutions in the range of 20 – 100 μg/ml by following appropriate 
dilution of mixed standard solution (b) with water:acetonitrile (7:3).  
- Prepare quali ty control and system suitability individual stock standard solutions (1.5 mg/ml) as well as 
mixed standard solution (115 μg/ml) using standards from a different batch /manufacturer (if available).  
- Prepare quality control mixed Committee standard solutions (40 and 80 μg/m l) and system suitability 
standard (52 μg/m l) by following appropriate dilutions of mixed standard solution  
 
B.2.4 Preparation  of sample solution  
Accurately weigh 50 mg (wsample ) of sample and quan titatively transfer into a 50 -ml volumetric flask. Add about 
20 ml  of water:acetonitrile (7:3), sonicate and shake well to dissolve the sample and make up to volume.  
B.2.5 Procedure  
Inject peak iden tification standard solutions , identify peaks and calculate relative retention times (RRT) wit h 
respect to rebaudioside A . 
Inject committee  mixed standard solutions  and construct standard curves for each steviol glycoside. Inject 
quality control and system su itability standard solutions  to ensure a satisfactory committee  system.  
Inject prepared sam ples. Dilute sample solution, if required, to bring the concentration of each analyte within 
the standard curve range. Make duplicate injections. Deduce concentration of each steviol glycoside from its 
corresponding standard curve and obtain average concen tration in sample solution  as Csample  (μg/ml ). 
Calculate the concentration of each steviol glycoside in the sample solution using the following formula:  
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛  (% 𝑤𝑤⁄ )=𝐶𝑠𝑎𝑚𝑝𝑙𝑒  𝑋 100
𝑤𝑠𝑎𝑚𝑝𝑙𝑒 
where,  
𝐶𝑠𝑎𝑚𝑝𝑙𝑒  = average concentration in micrograms per millilitre of steviol glycosides  in the sample solution  
𝑤𝑠𝑎𝑚𝑝𝑙𝑒  = mass of sample in micrograms per millilitre in 1 ml of sample solution (~1000 μg/ml ) 
Note: Above calculation will change if additional dilutions were done prior to LC injection. Analyst shall account such dilutions 
in the calculation.  
Calculate the percentage of major steviol glycosides in the sample by  summation of percentage concentrations  
of individual stev iol glycosides in the sample  (A). 
Note: If the concentration of major stevi ol glycosides in the sample is less than 95%, the  analyst should perform Method B.  
B.3 Method B: Determination of m inor steviol glycosides by HPLC -MS 
B.3.1 Apparatus  
HPLC -MS conditions may vary based on the man ufacturer and model of the system used. Analyst should set 
the conditions following the manufacturer’s instructions.  
The mass spectrometer is connected to the HPLC -UV system used in method A. Analyse the mass spectral 
data of the minor peaks (major stevio l glycoside peaks are identified from RRT in method A). Confirm the 
presence of each minor steviol glycoside from the observed molecular mass ion  and one or more of the followin g 
mass spectral diagnostic ions in Table 5 below.  CDUS 2035: 2018  
© UNBS 2017  – All rights reserved  7 
 Table 5 — Mass spectral diagn ostic ions observed during in -source fragmentation of steviol 
glycosides .  
[Fragment -H] -m/z Identity    
317 Steviol    
427 Related Steviol glycoside #3    
479 Steviol -GLC   
625 Steviol -2GLC [M -16]   
641 Steviol -2GLC    
787 Steviol -3GLC deoxyglucose  [M-16]   
803 Steviol -3GLC    
819   —   
965 Steviol -4GLC    
 
After confirming the presence of a minor steviol glycoside, correct its mean peak area (obtained from the UV 
chromatogram) as described below.  
B.3.2 Calculation of minor steviol  glycosides content  
Calculate the molecular mass corrected peak area abundance for each minor steviol glycoside as:  
𝑀𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟  𝑚𝑎𝑠𝑠  𝑐𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑  𝑝𝑒𝑎𝑘  𝑎𝑟𝑒𝑎 = M𝑥 x MPA
M𝑅𝑒𝑏 A 
where,  
Mx = molecular mass of the minor steviol  glycoside  
MRebA = molecular mas s of Rebaudioside A (967 amu)  
MPA = mean peak area  
Deduce the concentration of each minor steviol glycoside  in micrograms per millilitre from  the UV standard curve 
of rebaudioside A. Calculate the concentration of each minor  steviol glycoside in the sample solution using the 
following formula:  
Minor steviol glycoside concentration (%w/w) = 𝐶𝑜𝑛𝑐 𝑠𝑎𝑚𝑝𝑙𝑒  ×100
𝑊𝑒𝑖𝑔 ℎ𝑡𝑠𝑎𝑚𝑝𝑙𝑒 
Note: The above calculation will change if additional dilutions were done prior to LC inj ection. Analyst shall account for such 
dilutions in the calculation.  
Calculate the percentage of minor steviol glycosides in the sample by summation of percentages of individual 
minor steviol glycosides in the sample (B).  
Determine the total  steviol glycos ides content using the following formula:  
TSG = (𝐴+𝐵) ×100
100 −𝑀    CDUS 2035: 2018  
8 © UNBS 2017  – All rights reserved  
 where:  
   TSG is the t otal steviol glycosides content in %w/w, on the dried basis;  
   A is the percentage major steviol glycosides;  
B is the percent minor steviol glycosides; and  
M is the percentage loss on drying  
 
 
 
  CDUS 2035: 2018  
© UNBS 2017  – All rights reserved  9 
 Annex  C  
(normative )  
 
Determination of pH (Potentiometric Method ) 
C.1 Requirements  
  - pH meter  
  - Distilled/deionised water  
  - Absorbent tissue  
  - Two standard buffer solutions (High pH and low pH , respectively )  
C.2 Procedure  
Use a calibrated pH meter and follow the manufacturer's instructions. Each time the electrodes are used, rinse 
them with distilled or deionised water and carefully blot them dry with clean absorbent tissue. Form a fresh 
reference electrode liquid junction. Rinse the sample vessel three times with each new solution to be introduced.  
Choose two standard buffers to bracket, if possible, the anticipated pH of the unknown. Warm or cool these 
standards as necessary to match within 2 °C the temperature of the unknown, and initially set the temperature 
compensator to that temperature. Immerse the electrodes in a portion of the first standard buffer, and following 
the manufacturer's instructions adjus t the appropriate standardization control (knob, switch, or button) until the 
pH reading is that of the buffer. Repeat this procedure with fresh portions of the first standard buffer until two 
successive readings are within ± 0.02 pH unit without an adjust ment of the standardization control.  
Rinse the electrodes, blot dry, and immerse them in a portion of the second standard buffer of lower pH. Do not 
change the setting of the standardization control. Following the  manufacturer's instructions, adjust the sl ope 
control (thumbwheel switch, knob, or  temperature compensator) until the exact buffer pH is displayed.  
Repeat the sequence of standardization with both buffers unt il the pH readings are within ± 0.02 pH unit for both 
buffers without any adjustment of ei ther control (the amount of sample  to be used in sample preparation is given 
where applicable in the individual specification ). 
The pH of the unknown solution may then be measured . The difference between the results  of two pH 
determinations when carried ou t simultaneously on in rapid succession by the same analyst, under the same 
conditions,  should not exceed 0.05 pH unit .  
Note: Always re -standardize the instrument after even a short period during which the amplifier is turned off.  
 
 CDUS 2035: 2018  
10 © UNBS 2017  – All rights reserved  
 Annex  D 
(normative)  
 
Loss on drying   
D.1 Requirements  
  - Weighing bottle, with a stopper  
  - Air oven  
  - Desiccator  
  - Weighing scale   
D.2 Procedure  
D.2.1  Sample preparation  
Weigh 1 to 2 g of sample  (M1). Tare a glass -stoppered, shallow weighing bottle that has been dried for 30 
minutes at 105 ºC. Transfer the sample into the bottle, replace the cover, and weigh the bottle and the sample  
(M2). Distribute the sample as evenly as practicable to a depth of about 5 mm , and not over 10 mm.  
D.2.2  Drying  
Place the bottle with its contents in the drying chamber, removing the stopper and leaving it also in the chamber, 
and dry the sample at the 105 ºC for 2 hours.  Upon opening the chamber, close the bottle promptly and al low it 
to come to room temperature in a desiccator . Weigh the cool bottle and its contents (M 3).  
Calculate the loss on drying from the following equation:  
Loss on drying (%w/w) = 𝑀2− 𝑀3
𝑀1 ×100 
where:  
 M1 is the mass of sample in grams;  
 M2 is the mass  of sample and weighing bottle in grams before drying; and  
 M3 is the mass of sample and weighing bottle in grams after drying and cooling in a desiccator.  
If the sample  melts at a temperature lower than 105 ºC, prepare the sample as described above, then place it 
in a vacuum desiccator containing sulfuric acid. Evacuate the desiccator to 130 Pa (1 mm of mercury), maintain 
this vacuum for 24 h, and then weigh the dried sample.  Calculate the loss on drying using the same equation 
above.  CDUS 2035: 2018  
© UNBS 2017  – All rights reserved  11 
 Annex  E 
(normat ive) 
 
Residual solvents  – Methanol  and Ethanol  
E.1 Reagent  
 - Blank  solution : sample with very low solvent content  
 - Internal standard  solution : 3-methyl -2-pentanone  
 - Methanol  
 - Distilled water  
E.1.1  Method I (Determination carried out in water)  
Blank solution:  Weigh accurately 0.20 g of the blank into an injection vial. Add 5.0 ml of distilled water and 1.0 
ml of the internal standard solution. Heat at 60 °C for 10 min and shake vigorously for 10 sec.  
Internal standard solution:  Add 50 ml of distilled water to a 50 ml injection vial and seal. Accurately weigh and 
inject 15 µl 3-methyl -2-pentanone through the septum and reweigh to within 0.01 mg.  
Samples:  Weigh accurately 0.20 g sample into an injection vial. Add 5.0 ml water and add 1.0 ml of the internal 
standard solution. Heat at 60 °C for 10 min and shake vigorously for 10 sec.  
Calibration solution:  Weigh accurately 0.20 g of the blank into an injection vial. Add 5.0 ml of the water and 
1.0 ml of the internal standard solution. Weigh the vial accurately to within 0.01 mg. Inject a known volume of 
the component of interest through the septum and again  reweigh the vial. Heat at 60 °C for 10 min and shake 
vigorously for 10 sec.  
E.1.2  Method II (Determination carried out in methanol)  
Internal standard solution:  Add 50.0 ml methanol to a 50.0 ml injection vial and seal. Accurately weigh and 
inject 15 µl 3-methyl -2-pentanone through the septum and reweigh to within 0.01 mg.  
Blank solution:  Weigh accurately 0.20 g of the blank into an injection vial. Add 5.0 ml of methanol and 1.0 ml 
of the internal standard solution. Heat at 60° for 10 min and shake vigorous ly for 10 sec.  
Samples:  Weigh accurately 0.20 g sample into an injection vial. Add 5.0 ml methanol and add 1.0 ml of the 
internal standard solution. Heat at 60 °C for 10 min and shake vigorously for 10 sec.  
Calibration solution:  Solution A: Add 50.0 ml me thanol to a 50.0 ml vial and seal. Accurately weigh, to within 
0.01 mg, the vial and inject 50 µl of the component of interest through the septum. Reweigh the vial. Mix well.  
Weigh into an injection vial, 0.20 g of blank, add 4.9 ml of methanol and 1.0 ml internal standard solution. 
Introduce 0.1 ml of Solution A into the injection vial. Mix well and heat at 60 °C for 10 min and shake vigorously 
for 10 sec.  
E.2 Procedure  
Place the sample, blank and calibration samples in the sa mple tray of the head -space ga s chromatograph – 
FID system. Analyse using the following analytical conditions.  
Column:  Fused silica, length 0.8 m, i.d. 0.53mm, coated  with DB -wax, film thickness 1 µm CDUS 2035: 2018  
12 © UNBS 2017  – All rights reserved  
 Coupled with:  Fused silica, length 30 m, i.d. 0.53 mm, coated with DB -1, film thicknes s 5µm 
Conditions:  
- Carrier gas:  Helium  
- Flow rate:  208 kPa, 5 ml/min  
- Detector:  FID  
- Temperatures  
Injector:  140 °C 
Oven conditions: 35  °C for 5 min, then 5  °C /min to 90  °C, then 6 min at 90  °C 
Detector: 300 °C 
- Head space sampler  
Sample heating temperature: 60 °C 
Sample heating period: 10 min  
Syringe temperature: 70  °C 
Transfer temperature: 80  °C 
Sample gas injection: 1.0 ml in split mode  
Calculation:  
A x B x C / 50 = mg component per injection vial  
where:  
A = relative peak area of the component concerned  
B = mass of internal standard  in milligrams  
C = calibration factor  
Determination of calibration factors  
Method 1:  
C = D x 50 / (E x (F – G)) 
Method 2:  
C = D / (E x (F – G) x 10)  
where:  
D = mass of sample in milligrams  
E = mass of internal standard in milli grams  
F = relative peak area of component for the calibration solution  
G = relative peak area of the same component for the blank solution  CDUS 2035: 2018  
© UNBS 2017  – All rights reserved  13 
 Bibliography   
[1] JECFA  (2017 ), Steviol Glycosides from Stevia rebaudiana  Bertoni  Residue monograph  
 
[2]  JECFA (2006), Analytical methods, test procedures and laboratory solutions used by and  
  referenced in the food additive specifications, Volume 4.CDUS 2035 : 2018  
© UNBS 2017 – All rights  reserved  15 
 Certification marking  
Products that conform to Uganda  standards may be marked with Uganda National Bureau of Standards (UNBS) 
Certification Mark shown in the figure below.  
The use of the UNBS Certification Mark is governed by the Standards Act, and the Regulations made 
thereunder. This mark can be used only  by those licensed under the certification mark scheme operated by the 
Uganda National Bureau of Standards and in conjunction with the relevant Uganda Standard. The presence of 
this mark on a product or in relation to a product is an assurance that the goo ds comply with the requirements 
of that standard under a system of supervision, control and testing in accordance with the certification mark 
scheme of the Uganda National Bureau of Standards. UNBS marked products are continually checked by UNBS 
for confor mity to that standard.  
Further particulars of the terms and conditions of licensing may be obtained from the Director, Uganda National 
Bureau of Standards.  
 
 
CDUS 2035: 2018    
 
ICS 67.220.20  
Price based on nn pages  
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