GB/T 17819-2017Determination of vitamin B12 in additive premix—High performance liquid chromatography (English PDF)

1 Scope

This Standard specifies the method of high performance liquid chromatography in the determination of vitamin B12 in additive premix.

This Standard is applicable to the determination of vitamin B12 in compound premix feed and vitamin premix feed.

The detection limit of the method in this Standard is 0.1 mg/kg; the quantitative limit is 0.5 mg/kg.

2 Normative References

The following documents are indispensable to the application of this Standard. In terms of references with a specified date, only versions with a specified date are applicable to this Standard. The latest version (including all the modifications) of references without a specified date is also applicable to this Standard.

GB/T 6682 Water for Analytical Laboratory Use - Specification and Test Methods

GB/T 14699.1 Feeding Stuffs - Sampling

GB/T 20195 Animal Feeding Stuffs - Preparation of Test Samples

3 Principle

Use water to extract vitamin B12 in sample. After SPE purification and enrichment, adopt high performance liquid chromatograph for separated detection. Adopt external standard method to quantify it.

4 Reagents and Solutions

Unless it is otherwise indicated, all the reagents adopted in this Standard are of analytical purity. Water used for chromatography shall satisfy the requirements of Grade-1 water in GB/T 6682. Water used for experiments shall satisfy the requirements of Grade-3 water in GB/T 6682.

4.1 Acetonitrile. chromatographic purity.

4.10 1% phosphate solution. take 1 mL of phosphate, then, add it to 1,000 mL of water; start ultrasonic degassing.

5 Instruments and Equipment

5.1 Commonly used instruments and equipment in the laboratory.

5.2 Electronic balance. division value. 0.0001 g; division value. 0.001 g.

5.3 Centrifuge. it shall be able to reach 5,000 r/min (relative centrifugal force. 2,988 g).

5.4 Ultrasonic water bath.

5.5 Solid phase extraction device.

5.6 High performance liquid chromatograph. equipped with UV detector with adjustable wavelength (or diode matrix detector).

5.7 Nitrogen blowing device.

5.8 UV spectrophotometer.

5.9 C18 solid phase extraction column. 500 mg/6 mL or solid phase extraction column with equivalent performance.

6 Sampling and Sample Preparation

Take representative feed sample in accordance with GB/T 14699.1; cut down the sampling by quartering. Prepare the sample in accordance with GB/T 20195, grind it; filter all of it through 0.25 mm screen hole. Mix it up, place it in a sealed container. Keep it away from light; reserve for later usage.

7 Analytical Procedure

8 Result Calculation

Vitamin B12 content in the sample shall be calculated by the mass fraction X. The numerical value shall be expressed in mg/kg and calculated in accordance with Formula (2). Where, P1---peak area value of sample solution; V---diluted volume, expressed in mL; C---concentration of vitamin B12 standard working fluid, expressed in µg/mL;

P2---peak area value of vitamin B12 standard working fluid; m---sample mass, expressed in g.

The determination result shall be expressed in the arithmetic mean value obtained through parallel determination. The result shall retain 3 significant figures.

9 Repeatability

In terms of additive premix feed whose vitamin B12 content is smaller than or equals to 50 mg/kg, the difference between two independent determination results obtained under repeatability conditions and the arithmetic mean value shall be not more than 15% of the arithmetic mean value of the two determined values. […]