GB/T 18872-2017Determination of vitamin K3 in feeds—High performance liquid chromatography (English PDF)

Foreword

This Standard was drafted in accordance with the rules given in GB/T 1.1-2009.

This Standard replaces GB/T 18872-2002 "Determination of Vitamin K3 in feeds-High-performance liquid chromatography". Compared with GB/T 18872-2002, the main technical changes in this Standard are as follows.

- Supplemented the concentrate supplement in the scope of application; Added the varieties of feed additive vitamin K3, that is, this method is also applicable to vitamin K3 added in the form of nicotinamide bisulfite menadione and dimethylpyrimidinol menadione sulfite;

- In the scope, it specifies that the content of vitamin K3 is calculated as menadione; Revised the measurement range above 2mg/kg to the limit of quantification 0.4mg/kg;

- Improved the sample extraction methods from ammonia water hydrolysis to sodium carbonate solution hydrolysis;

- Added the conversion coefficient between menadione sodium bisulfite, nicotinamide bisulfite menadione, dimethylpyrimidinol menadione sulfite and menadione.

1 Scope

This Standard specifies the high performance liquid chromatography for the determination of vitamin K3 (menadione sodium bisulfite, nicotinamide bisulfite menadione, dimethylpyrimidinol menadione sulfite, as menadione) in feeds.

This Standard applies to the determination of vitamin K3 in compound feeds, concentrated feeds, additive premixed feeds and concentrate supplements. The limit of quantification of the method is 0.4mg/kg.

2 Normative references

The following referenced documents are indispensable for the application 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.

GB/T 603, Chemical reagent - Preparations of reagent solutions for use in test methods

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

The specimen is extracted with chloroform and sodium carbonate solution and converted into free menadione. It is separated by reversed-phase C18 column, detected by UV detector, and quantified by external standard method.

4 Reagents and materials

Unless otherwise stated, all reagents in this Standard are analytically pure. The test water is in line with the requirements for grade three water in GB/T 6682.

4.1 Trichloromethane.

4.2 Methanol. chromatographically pure.

4.5 Anhydrous sodium sulfate.

4.6 Diatomaceous earth.

4.7 Diatomaceous earth and anhydrous sodium sulfate mixture. Weigh 3g of diatomaceous earth and mix with 20g of anhydrous sodium sulfate.

4.8 Menaquinone standard. content is >= 96%.

4.10 Menadione standard working solution. Accurately pipette 1.00mL of menadione standard stock solution (4.9) into a 100mL brown volumetric flask. Use methanol to dissolve. Dilute to the scale. Mix well. The concentration of the working solution is about 5µg/mL. Store at -18°C. It is valid for 3 months.

5 Instruments and equipment

5.1 Commonly used laboratory equipment.

5.2 Balance. resolution is 0.001g.

5.3 Balance. resolution is 0.0001g.

5.4 Balance. resolution is 0.00001g.

5.8 High performance liquid chromatograph. with UV tunable wavelength detector (or diode matrix detector).

6 Sampling and specimen preparation

Take representative feed samples according to GB/T 14699.1. Conduct down-sampling by quartering.

7 Analysis steps

WARNING -- Because vitamin K3 is sensitive to air and ultraviolet light, and the chloroform solution used as an extractant has certain toxicity, all operations should be performed in a fume hood away from light.

8 Result calculation

The content X of menaquinone in the specimen, expressed as the mass fraction of menaquinone in the sample, in milligrams per kilogram (mg/kg), is calculated according to formula (1).

9 Repeatability

For feeds with vitamin K3 content less than 100mg/kg, the difference between the two independent determination results obtained under repeatability and their arithmetic mean is not more than 20% of the arithmetic mean of the two determinations.

Annex A (informative) Example of labeled amount of feed sample, weighed sample amount and dilution ratio of menadione extract

The example of the labeled amount, weighed sample amount and the dilution ratio of the menadione extract of the feed sample are shown in Table A.1.

Annex B (informative) Chromatogram of menadione standard solution

The chromatogram of the standard solution of menadione is shown in Figure B.1. […]