Method for analyzing selenium form of selenium-rich proteoglycan based on HPLC-ICP-MS

A technology of HPLC-ICP-MS and seleno-enriched protein, which is applied in the field of analyzing selenium-enriched proteoglycan selenium, can solve the problems of difficult control of acid extraction conditions, destruction of selenoamino acids, low efficiency, etc., and achieves good method stability, High recovery rate and accurate quantitative effect

Active Publication Date: 2021-04-09
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these two methods are suitable for extracting inorganic selenium and free organic selenium, but the efficiency is very low when extracting protein-bound organic selenium such as selenomethionine (SeMet) and selenocysteine ​​(SeCys) (Zembrzuska, 2014; Bierla, 2008), in addition, acid extraction conditions are difficult to control, often resulting in the destruction of selenoamino acids (Tie, ​​2015)

Method used

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  • Method for analyzing selenium form of selenium-rich proteoglycan based on HPLC-ICP-MS
  • Method for analyzing selenium form of selenium-rich proteoglycan based on HPLC-ICP-MS
  • Method for analyzing selenium form of selenium-rich proteoglycan based on HPLC-ICP-MS

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Experimental program
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Effect test

Embodiment 1

[0032] Embodiment 1 The drawing of standard curve

[0033] Accurately weigh 25.0 mg Na 2 SeO 3 Dissolve in water to make 50 µg / mL Na 2 SeO 3 Standard stock solution: Accurately weigh 20.0 mg of SeCys2, add it into water containing dilute acid to make a 200 µg / mL standard stock solution; accurately weigh 20.0 mg of SeMeCys, add it to water, and make a 200 µg / mL standard stock solution; accurately Weigh 20.0 mg of SeMet, add it into water, and make a standard stock solution of 200 µg / mL. Mix the above standard stocks in proportion so that Na 2 SeO 3 , SeCys2, SeMeCys and SeMet concentration ranges of 5-250 µg / L, 1-25 µg / L, 1-25 µg / L, 4-100 µg / L series of mixed standard working solutions. According to the conditions in Table 1, apply HPLC-IPC-MS analysis, and make a standard curve as shown in Table 2.

Embodiment 2

[0034] Example 2 Na in the sample 2 SeO 3 , SeCys2, SeMeCys and SeMet content determination

[0035]Accurately weigh 5 parts of about 15 mg of selenium-enriched proteoglycan and proteoglycan in a 2 mL centrifuge tube, add 5 mg of proteinase K, and then add 1.5 mL of Tris-HCl buffer (0.1 mol / L, pH=7.48), Shake at a constant temperature at 37°C for 18 hours (200 rpm), then centrifuge the enzymolysis solution for 10 min at 6000 rpm / min, collect the supernatant, and use HPLC-ICP-MS to analyze the selenium compound in the enzymolysis supernatant: Na 2 SeO 3 , SeCys2, SeMeCys and SeMet for analysis. See the hydrolysis diagram of selenium-enriched proteoglycan figure 2 , the content determination results are shown in Table 3.

Embodiment 3

[0036] Na in the sample of Example 3 2 SeO 3 、SeCys 2 , SeMeCys and SeMet recovery assay

[0037] Accurately weigh 3 parts of 15 mg Z0206 proteoglycan into a 2 mL centrifuge tube, add 4 mixed standard substances of low, medium and high according to Table 4, and repeat 5 times. Add 15 mg proteinase K and Tris-HCl buffer (0.1 mol / L, pH=7.48) to a final volume of 1.5 mL, shake at a constant temperature at 37°C for 18 hours (200 rpm), and then centrifuge the enzymatic solution 10min, the rotation speed is 6000 rpm / min, collect the supernatant, and apply HPLC-ICP-MS to the selenium compound in the enzymatic hydrolysis supernatant: Na 2 SeO 3 , SeCys2, SeMeCys and SeMet were analyzed and Na were calculated according to the standard curve 2 SeO 3 , SeCys2, SeMeCys and SeMet content and recovery.

[0038] According to the result and discussion that above embodiment 1-3 obtains:

[0039] Drawing of standard curve

[0040] to Na 2 SeO 3 , SeCys2, SeMeCys and SeMet to make a s...

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Abstract

The invention discloses a method for analyzing the selenium form of selenium-rich proteoglycan based on HPLC-ICP-MS. Protease K and a Tris-HCl buffer solution are added into the selenium-enriched proteoglycan, enzymolysis is performed for 18 hours at the constant temperature of 37 DEG C, and high performance liquid chromatography-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS) is applied to analyze selenium compounds in enzymolysis supernatant: sodium selenite (Na2SeO3), selenocystine (SeCys2), selenium methyl selenocysteine (SeMeCys) and selenomethionine (SeMet). According to the method, only four selenium compounds are obtained through enzymolysis and are highly matched with a standard substance, and quantification is accurate; and the recovery rate is 78.35-111.42%, and the method is high in recovery rate, mild in condition and good in stability.

Description

technical field [0001] The invention relates to a method for analyzing the selenium form of selenoprotein polysaccharide based on HPLC-ICP-MS. Background technique [0002] Selenium is the active center of many enzymes such as glutathione peroxidase (Jagtap, 2016), and at the same time, it has anti-cancer and anti-aging functions (Axley, 1991). However, the range between the required amount of sodium selenite and the toxic amount is very narrow, and the biological function of selenium is closely related to its form (Suhajda, 2000). Inorganic selenium mainly has two forms of selenate and selenite, while organic selenium has three forms of selenoamino acid, seleno small peptide and selenoprotein (Maseko, 2013). Compared with inorganic selenium, organic selenium has higher biological activity and lower toxicity, and is more easily absorbed by the body (Kieliszek, 2013; Kouba, 2014). At present, most studies focus on the determination of total selenium, but this is far from en...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/34G01N30/36G01N30/72G01N30/86
CPCG01N30/02G01N30/06G01N30/34G01N30/36G01N30/7253G01N30/8679
Inventor 王凤芹曹进平汪以真路则庆程远之
Owner ZHEJIANG UNIV
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