GB8270-1999 --- Stevia Testing Method in China

Measurement of purity of stevia sugar

1、Preparation of standardized solution

Put standardized stevia into a drying oven at 105 centigrade, and stay for 2 hours, then 4 samples (120 mg, 90 mg, 60 mg, and 30 mg) are drawn from the oven, and all are dissolved in acetonitrile / water solution by the ratio 80: 20 respectively, and is diluted to 25 ml respectively.

2、Treatment of sample solutions

120 mg sample is procured from the drying oven, and is dissolved in acetonitrile / water solution by the ratio 80:20, and is also diluted to 25 ml respectively, shaking it until it is well-distributed.

3、Conditions of the liquid phase chromatographic analysis

  • column—NH2
  • 2. lowing solution—acetonireile/water=80:20
  • flow rate: 1.2ml/min
  • checking : violet ray inspector, wavelength 210nm
  • sample injection : 15ul

Firstly measure the 4 standardized stevia solutions and draw a standardized curve (peak area A---concentration curve). Then test the stevia sample solution to record peak area Aa, Ac, As, Ad and Ad' of rebaudioside A, rebaudioside C, stevioside, dulcoside A and steviolbioside respectively, and times 1.20, 1.18, 1.00, 0.98. 0.80, their values are Aa' Ac', As', Ad' and Abb. Then the slope of the standardized cure K=A/C, and calculate the content of the above five compositions Pi (%).

4、Description of the analytical result.

  • Pi=A/K* 25/M * 100
  • "i" refers to " a, c, s d, b"
  • "m" refers to the quality of the sample, g.
  • By calculations, Pa, Pc, Ps, Pd and Pb are work out. And the total content of the stevia sugar is that Pa+Pc+Ps+Pd+Pb.

Measurement of sweetness

Firstly prepare 2% sucrose/water solution. In the meanwhile 2ml stevia sugar is mixed with 100 ml water. Then taste the two solutions and make the comparison. When the sweetness of 2% sucrose/water is equivalent to the stevia solution, then n value is the sweetness of stevia sugar.

Measurement of specific optical rotation


Optical rotation tester, 25 ml glass vessel with ruler, beaker (25 ml)

2、Testing method

  0.25 g sample is put into a beaker, and a little water is added. Then it will be moved into a 500 ml glass vessel, and measure it by 370 nm using optical gauge.
  Description of analytical report. See the following formula.
  Specific absorbance E=a/CH
  H---refers to height of solution, cm;
  a---measured absorbance
  C---sample quality per 100ml solution.

Measurement of moisture

Prepare a glass-made flat cup and put it into a drying oven at 95-100 centigrade, the lid of the cup is aside, and heats it for half an hour, weigh the cup. Do the above continuously so as to get a fixed value.

Prepare 2-10 g stevia sample, and put it into a glass vessel, the thickness of the sample is 5 mm.Put the lid onto the vessel, precisely weigh it and place it into drying oven at 95-105 centigrade, and the lid is aside, dry for 2-4 hours, take it out while the lid is placed right, then place it into a dryer for cooling half an hour, weigh it. Then do the above job again. The mean error between the first job and second job shall not exceed 2 mg, then it is regarded a fixed value.

  X1=(M1-M2)/(M1-M3)* 100
  M1—moisture volume in the sample. %
  M2---quality of the cup and the sample after drying. g
  M3--- quality of the vessel.

Testing Method of Ash

Prepare a suitable porcelain crucible and place it into a high temperature stove, stay for half an hour at 600 centigrade, then cool it till 200 centigrade and take it out; Put it into a dryer for cooling till room temperature, then weigh it. Do the job again till a fixed value comes out.

Add 2-3 g solid sample or 5-10 g liquid sample to the crucible and accurately weigh it.

Liquid sample should be steamed in boiling water. Solid sample or steamed sample shall be heated by little fire till the sample is fully carbonized without smoke, then place it into the high temperature stove, and burn it at 550-600 centigrade till carbon particle is not available, that is called full ash. And cool it at 200 centigrade, take it out, put it into the dryer for cooling, weigh it. Do the job once more till the mean error between the first job and the second job shall not exceed 0.5mg.
  X=(ml-m2) / (m3-m2) * 100
  X—ash content in the stevia sample, %
  m1—quality of porcelain crucible and ash, g.
  m2--- quality of the porcelain crucible, g.
  m3--- quality of the porcelain crucible and stevia sample, g.

Measurement of Heavy Metals

After measurement of ash, 0.5 ml nitric acid is added in the ash, then steams it till dry, and cools it until NO 2 comes out. Hydrochloride acid 2 ml is added, and is then placed on the boiling water for drying, and deionized water 15 ml is added, seven drops of NH3OH is added until the display solvent shows neutral, and is heated and placed into a Na color comparation tube, then deionized water is added and made 25 ml solution, and 1 ml mol/1 l CH3COOH and 10 ml saturated H2S solution are also added, and be kept for 10 minutes. Its color shall not be darker than standardized solution.

Take out Pb solution 10 ml and is diluted to 100 ml, then take out 2 ml from it, do the same job as the above mentioned.

Measurement of Arsenium


Put 5 ml hydrochloride acid in the ash, and pour it into arsenium testing bottle, and 5 ml 16.5% KI solution plus 5 drops of acidic SnCl are added, then 3 g zinc particle is added, then the lid is fixed tightly and is placed on the boiling water at 25-40 centigrade, one hour later, please make comparison with the standardized solution, the color of arsenium shall not be darker than the standardized solution.

2、Standardized solution

Take out 0.001 mg/ml 1 ml as standardized solution, then 5 ml 16.5% KI plus 5 drops of Sync solution are added, and 5 ml hydrochloride acid is added, 3 g zinc particle is added too, then the lid is fixed tightly as done to the sample.