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Chinese Standard: 'GBT17819-2017'
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GB/T 17819-2017
ICS 65.120
B 46
Replacing GB/T 17819-1999
Determination of Vitamin B12 in Additive Premix -
High Performance Liquid Chromatography
Issued by. General Administration of Quality Supervision, Inspection and
Standardization Administration Committee of the People’s
Republic of China.
Table of Contents
Foreword ... 3 
1 Scope ... 4 
2 Normative References ... 4 
3 Principle ... 4 
4 Reagents and Solutions ... 4 
5 Instruments and Equipment ... 6 
6 Sampling and Sample Preparation ... 6 
7 Analytical Procedure ... 6 
8 Result Calculation ... 8 
9 Repeatability ... 8 
Appendix A (Informative) Standard Chromatogram of Vitamin B12 ... 10 
Determination of Vitamin B12 in Additive Premix -
High Performance Liquid Chromatography
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
7.1 Extraction
7.1.1 Extraction of vitamin premix
Weigh-take 2 g ~ 3 g (accurate to 0.001 g) of sample, place it in 50 mL centrifuge tube.
Accurately add 20 mL of water, then, thoroughly shake it for 30 s. Place it in ultrasonic
water bath, then, start ultrasonic extraction for 30 min; shake it twice during the process.
Start centrifugation for 5 min at 5,000 r/min; take the supernatant. If the sample solution
is vitamin premix whose vitamin content exceeds 10 mg/kg, use 0.45 μm microporous
membrane; determine it with high performance liquid chromatography (HPLC). If the
concentration of vitamin B12 in the sample solution is smaller than 2 μg/mL, handle it
Mobile phase. methanol + sodium hexane sulfonate solution (25 + 75);
Flow rate. 1.0 mL/min;
Temperature. room temperature;
Detection wavelength. 546 nm.
7.3.2 Quantitative determination
Adjust the instrument operating parameters in accordance with the instruction manual
of high performance liquid chromatograph. Inje......
Related standard:   GB/T 17813-2018  GB/T 17815-2018
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