This standard specifies the method for the determination of vitamin K1 by liquid chromatography. This standard is applicable to the determination of vitamin K1 in infant formula and milk powder. GB/T 5413.10-1997 Determination of vitamin K1 in infant formula and milk powder GB/T5413.10-1997 Standard download decompression password: www.bzxz.net

GB/T5413.10-1997
The content of vitamin K1 in infant formula and milk powder is low and is easily decomposed under alkaline conditions. The reversed-phase high-pressure liquid chromatography method given in this standard is determined after repeated experiments based on a large number of domestic and foreign literature, which solves the above problems and is fast and accurate.
This series of standards will replace GB5413-85 from the date of implementation. This standard is proposed by the China Light Industry Federation.
This standard is under the jurisdiction of the National Dairy Standardization Center. The responsible drafting unit of this standard: National Dairy Quality Supervision and Inspection Center. Participating drafting units of this standard: Food Hygiene Supervision and Inspection Institute of the Ministry of Health, Zhejiang Light Industry Research Institute, Harbin Morinaga Dairy Co., Ltd., Nestlé (China) Investment Services Co., Ltd. The main drafters of this standard: Yang Jinbao, Wang Yun, Wang Xinxiang. 259
National Standard of the People's Republic of China
Infant formula and milk powder
Determination of vitamin K
Milk powder and formula foods for infant and young children-Determination of vitamin K, contentScope
This standard specifies the method for the determination of vitamin K, by liquid chromatography. This standard applies to the determination of vitamin K, in infant formula and milk powder. 2 Method Summary
GB/T 5413. 10--- 1997
Replaces GB5413-85
After the sample is hydrolyzed by enzyme, it is extracted with petroleum ether, the solvent is evaporated, and the concentrated sample is subjected to high pressure liquid chromatography, with acetonitrile-methanol-water as the mobile phase and UV detector at a wavelength of 270nm to quantify vitamin K1. 3 Reagents
All reagents, if the specifications are not specified, are analytically pure; all experimental water, if no other requirements are specified, refers to grade tertiary water. 3.1 Taka-diastase. 3.2 Phosphate buffer solution, pH=7.8.
Dissolve 2.78g sodium dihydrogen phosphate and 25.6g disodium nitrogen phosphate in 1L water. 3.3 Lipase: not less than 2300 units per mg protein. :3.4 Sodium hydroxide solution: c(NaOH) is 10mol/L. Dissolve 400g sodium hydroxide granules in 1L water. 3.5 Sodium chloride solution: saturated.
3.6 Ethanol: 95% by volume.
3.7 Petroleum ether.
3.8 Anhydrous sodium sulfate.
3.9 Hexane: chromatographically pure.
Acetonitrile: chromatographically pure.
Methyl acetate: chromatographically pure.
3.12 Water: chromatographically pure.
3.13 Standard solution
3.13.1 Standard stock solution: concentration is 2.5 mg/mL. Accurately weigh 25 mg of vitamin K, put it in a 10 mL volumetric flask, and make up to volume with n-hexane. 3.13.2 Standard working solution, concentration is 0.25 mg/mL. Pipette 1 mL of standard stock solution into a 10 mL volumetric flask, and make up to volume with n-hexane. Approved by the State Administration of Technical Supervision on May 28, 1997 260
Implemented on September 1, 1998
4 Instruments
Common laboratory instruments and:
4.1 Separating funnel: 500 mL. bZxz.net
4.2 Rotary evaporator.
4.3 Constant temperature water bath oscillator.
4.4 High pressure liquid chromatograph: with UV detector. GB/T 5413.10-1997
4.5 Chromatographic column: 25cm×4.6mm, Cg or chromatographic column with equivalent performance. 5 Operating steps
5.1 Sample treatment
5.1.1 Samples containing starch
Accurately weigh 10g sample, put it into a 500mL flat-bottom flask, add 1g Taka amylase (3.1), and then add 50mL 45-50℃ distilled water, mix well, remove the air in the bottle with nitrogen, cover the bottle with a stopper, and place it in a 45℃ oven for 30min. 5.1.2 Samples without starch
Accurately weigh 10g sample, put it into a 500mL conical flask, add 75mL water, and homogenize. 5.2 Preparation of the test solution
5.2.1 Add 50 mL of phosphate buffer solution (3.2) and 1.5-2.0 g of lipase (3.3) to the treated sample solution, and shake continuously in a 37°C water bath for 1 h.
5.2.2 Take out and add 5 mL of sodium hydroxide solution (3.4). 5.2.3 Quantitatively transfer the above solution to a 500 mL separatory funnel, add 25 mL of saturated sodium fluoride solution (3.5) and 50 mL of ethanol (3.6), and mix thoroughly.
5.2.4 Add 100 mL of petroleum ether (3.7), stopper and shake for 2 min. Carefully remove the stopper, add another 50 mL of ethanol (3.6), and stand to separate the layers. The upper layer is the organic phase, the middle layer is the fatty acid layer, and the lower layer is the aqueous phase. 5.2.5 Discharge the aqueous phase and the fatty acid layer into another 500 mL separatory funnel. Extract again with 100 mL of petroleum marrow (3.7), let stand to separate, discard the water phase, and combine the ether phase.
5.2.6 Wash the petroleum ether phase with distilled water until the ether phase is neutral. 5.2.7 Pass the petroleum aldehyde through anhydrous sodium sulfate (3.8) into a 500 mL flat-bottom flask, and evaporate the petroleum ether on a rotary evaporator at 50°C until it is almost dry.
5.2.8 Transfer the residue with petroleum ether (3.7) to a 10 mL test tube and blow it with nitrogen. Accurately add 1 mL of n-hexane, shake thoroughly to dissolve the residue, and obtain the test solution.
1 When the fat content in the sample is less than 4 g, use this amount of lipase; if it is greater than 4 g, add more lipase to completely hydrolyze the fat. 2 The final prepared sample must be effectively cooled in a refrigerator to precipitate the fat. 3 After separation, the water phase and fatty acid layer must be carefully released, otherwise it will increase the interfering substances in the sample. 5.3 Chromatographic analysis conditions
Mobile phase: acetonitrile: methanol: water 70:22:8. Flow rate: 2.0mL/min.
Column temperature: 40℃.
Detector wavelength: 270nm.
Recording paper speed: 0.5cm/min.
Injection volume: 25μL.
Description of analysis results
GB/T 5413.10—1997
Content of vitamin K in sample (mg/100g) = (Hs/H)×cBms xVs
Wherein: Hs is the peak height of vitamin K in sample, HB is the peak height of vitamin K in standard working solution; Cg is the mass concentration of vitamin K in standard working solution, mg/mL; Vs is the sample volume, mL;
ms is the mass of sample, g.
Allowable difference
The maximum deviation of the two measured results shall not exceed 10% of the content.
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