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Method for simultaneously determining vitamins K1 and K2 in milk powder and application

A vitamin and milk powder technology, applied in the field of analytical chemistry, can solve the problems of incomplete impurity removal, low measurement results, late peak time, etc., and achieve the effects of ensuring accuracy and reproducibility, simplifying operation steps, and shortening time.

Pending Publication Date: 2022-04-12
GUANGZHOU INST FOR FOOD INSPECTION(GUANGZHOU INSPECTION CENT FOR WINE & SPIRITS)
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Vitamin K 1 The detection technology is relatively mature, and most of them use the national standard method-high performance liquid chromatography-fluorescence method, but the detection method of a single target object is established, and the detection and screening throughput is low; with vitamin K 2 The safety of use as a food nutrition fortifier has been recognized, so it is necessary to establish a method that can simultaneously measure vitamin K 1 with K 2 Methods
At present, some literature authors try to use high performance liquid chromatography-fluorescence method to detect vitamin K 1 with K 2 , but there are still many problems: (1) At present, few scholars have studied vitamin K in infant formula milk powder. 2 (2) in the pretreatment of the existing method, the solid lipase added to the sample was not dissolved sufficiently, which easily caused vitamin K 1 with K 2 The measured value is on the low side, and adding ethanol and potassium carbonate solid will easily lead to exothermic phenomenon when it dissolves, which is harmful to vitamin K 1 affect the stability of
(3) Vitamin K 1 , Vitamin K 2 (menaquinone (MK-4), menadione (MK-7), menadione (MK-9)) simultaneous determination of the pre-treatment takes too long, including enzymatic hydrolysis, extraction, Concentration, redissolution and other steps involve multiple extractions and evaporation and dissolution. The operation is cumbersome and time-consuming, and it is easy to cause the loss of the components to be measured, resulting in low measurement results and greatly affecting the analysis efficiency of the sample; (4) the previous method During the processing, the removal of impurities is not complete, and it is easy to interfere with vitamin K 2 Determination of
(5) When the sample is reconstituted, it is easy to bring in water, and the water is easy to damage the zinc powder reduction column; (6) The mobile phase system used for the determination of vitamin K by fluorescence method at this stage is prone to salting-out reaction, and gradient elution cannot be used for vitamin K (7) When liquid chromatography-fluorescence method is used for detection, the peak time of MK-9 is relatively late, and liquid phase detection takes a long time

Method used

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  • Method for simultaneously determining vitamins K1 and K2 in milk powder and application
  • Method for simultaneously determining vitamins K1 and K2 in milk powder and application
  • Method for simultaneously determining vitamins K1 and K2 in milk powder and application

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preparation example Construction

[0082] Preparation of the sample solution to be tested:

[0083] Weigh 1g of milk powder sample, ①enzyme hydrolysis: add 10mL water, a certain amount of lipase aqueous solution, vortex for 2-3min, place in a constant temperature water bath at 37±2°C for 2-3 hours;

[0084] ②Constant volume: Add 10mL of anhydrous isopropanol and 1mL of 5mol / L KOH aqueous solution (or 1.2mL of 14.65mol / L phosphoric acid) to the sample enzymatic hydrolysis solution, dilute to 25mL with 50wt.% isopropanol aqueous solution, and take 2mL Pass through a 0.45 μm filter membrane and wait for analysis on the machine.

Embodiment 1

[0087] This embodiment has measured lipase adding concentration to vitamin K 1 with K 2 The influence of extraction recovery rate, the specific process is: prepare lipase aqueous solutions with different concentrations of 0-0.40g / mL, dissolve lipase at 30°C for 15 minutes, and wait for no particles at the bottom, add 3mL to the sample to be tested, and enzymatically hydrolyze overnight, and the vitamin K in the sample solution after overnight enzymatic digestion 1 and vitamin K 2 content was tested.

[0088] figure 1 is the concentration of lipase added to vitamin K 1 and vitamin K 2 The influence diagram of extraction recovery rate, as shown in the figure, under enzymatic hydrolysis conditions, when the concentration of lipase added is 0.13g / mL~0.16g / mL, vitamin K 1 and vitamin K 2 The recovery rate is the highest; when the concentration of lipase added is lower than 0.13g / mL, the fat of milk powder is not completely enzymatically hydrolyzed, which affects the release ...

Embodiment 2

[0090] This embodiment has measured the effect of lipase enzymolysis time on vitamin K 1 with K 2 The influence of extraction recovery rate, the specific process is: add 3mL of 0.13g / mL lipase solution to the sample solution, and observe the influence of milk powder enzymatic hydrolysis time on the recovery rate of four kinds of vitamin K. Select the range of 1h ~ 8h to measure the recovery rate of vitamin K.

[0091] Such as figure 2 As shown, the recovery rate of vitamin K gradually increased from enzymatic hydrolysis for 0h to 2h; the recovery rate of vitamin K was the highest after enzymatic hydrolysis for 2h to 3h; and unstable. Experiments show that the enzymatic hydrolysis time of lipase is too short and the enzymatic hydrolysis is incomplete, which will affect the subsequent vitamin K 1 and vitamin K 2 It was determined that the extraction efficiency tended to be stable when the enzymolysis time was more than 2 hours. If the enzymatic hydrolysis time exceeds 3 h...

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Abstract

The invention provides a method for simultaneously determining vitamin K1 and vitamin K2 in milk powder and application, and the method comprises the following steps: 1) sample pretreatment: weighing a proper amount of uniformly mixed milk powder sample, adding water and lipase, and carrying out fat hydrolysis at a constant temperature; adding isopropanol and a KOH aqueous solution (or phosphoric acid) for constant volume; and (2) analyzing the contents of the vitamins K1 and K2 in the enzymatic hydrolysate through online solid-phase extraction-liquid chromatography combination. The method has the advantages that the sample pretreatment operation steps are simplified, the solvent consumption is low, the pretreatment time is greatly shortened, the manpower is saved, the automatic online purification detection is realized, the sensitivity is improved, and the accuracy and the stability of the test result are ensured. The method can be used for detecting vitamins in foods and medicines.

Description

technical field [0001] The invention belongs to the technical field of analytical chemistry, in particular to a method for simultaneously determining vitamin K in milk powder 1 and K 2 methods and applications. Background technique [0002] Vitamins are important compounds that participate in the regulation of human metabolic processes, among which vitamin K is not only involved in blood coagulation and anticoagulation, but also participates in physiological and pathological processes such as bone metabolism regulation, cell growth and proliferation, oxidative stress response, and inflammatory response. Vitamin K has K 1 、K 2 、K 3 、K 4 and other forms, where K 1 It is extracted from green plants, K 2 Synthesized by gut bacteria, K 1 and K 2 is a natural fat-soluble vitamin, and K 3 and K 4 It is a water-soluble vitamin that is artificially synthesized. Vitamin K 1 is a key factor in blood clotting, and vitamin K 2 It has the physiological functions of participa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/40G01N30/74G01N21/64
Inventor 蔡伟谊黄景初王宇陈悦铭陈嘉欣苏燕瑜刘佳钱振杰何淑明
Owner GUANGZHOU INST FOR FOOD INSPECTION(GUANGZHOU INSPECTION CENT FOR WINE & SPIRITS)
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