CN102520090A - Method for determining selenium form in selenium-rich health product - Google Patents
Method for determining selenium form in selenium-rich health product Download PDFInfo
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Abstract
The invention discloses a method for determining a selenium form in a selenium-rich health product, which is characterized by comprising the following steps: 1) pretreating the selenium-rich health product, using an extraction buffer Tris-HCl for extracting a sample of the selenium-rich health product, successively adding protease K and protease XIV with an effective amount after the extraction treatment for hydrolysis extraction to obtain a sample solution to be measured; 2) using combination of hydride generation-atomic fluorescence spectroscopy and high performance liquid chromatography for respectively determining a solution with a standard selenium form and the sample solution to be measured. The method of the invention is simple and easy to operate, the repeatability is good, and the used reagent has the advantages of no toxicity and no pollution.
Description
Technical field
The present invention relates to a kind of assay method of selenium-enriched health care article selenium form, particularly relate to the assay method that a kind of selenium-enriched health care article carry out carrying out after the pre-treatment selenium form.
Background technology
Selenium is one of trace element of needed by human, is the constituent of glutathione peroxidase (GSH-Px), have anti-oxidant, anticancer, anti-aging, improve effect (Rotruck J T such as body immunity, protection cardiovascular and cerebrovascular; Pope A L; Ganther H E, et al Selenium:biochemicalrole as a component of glutathione peroxidase.Science, 1973; 179,588-590.).China has 2/3 area to lack selenium, wherein has 1/3 area to be the serious selenium area that lacks.The humans and animals body lacks selenium can cause Keshan disease; Kaschin-Beck disease, the white muscle disease of animal etc., certain cancers in addition; Also closely related (the Ip C.Lessons frombasic research in selenium and cancer prevention.Journal Nutrition of disease such as region thyroid gland obstacle with low selenium environment; 1998,128,1845-1854.).Selenium in people's diet comprises inorganic selenium and organic selenium, and wherein inorganic selenium toxicity is bigger, and the boundary of dosis toxica and requirement is very approaching, and absorbs and utilize rate variance, and biological effectiveness is low, thereby is limited its use amount by strictness.Organic selenium is more conducive to the absorption of human body utilization, and biologically active is strong, the low (Lu Lan of toxicity; Lu Bing; Wang Chune. the analysis of organic selenium and inorganic selenium is inquired in the health food. Chinese sanitary inspection magazine .2010,20 (4): 767-769.), be that more efficient, safe human body is mended the selenium form.The performance of the biological function of selenium mainly depends on it and has form, particularly the form of organic selenium (seleno-amino acids).Therefore, the form of check and analysis seleno-amino acids is significant accurately, can not only consumption guidance person's science mends selenium, can also further understand biochemical characteristic and the selenium of the selenium rule in the nature migration and variation.
At present; The method that China measures organic selenium content is mainly minusing Qin Fang. the mensuration of organic selenium content in the plant. and Wuxi light industry college journal .1998; 17 (4): 74-77.); The principle of minusing is to utilize different reagent that the organic selenium in the testing sample is separated with inorganic selenium earlier, and is because of inorganic selenium mainly exists with selenate radical (SeO42-) and selenite radical (SeO32-) anionic form, soluble in water.Organic selenium mainly be with protein, cellulose and carbohydrates in C, N etc. combine; Exist with macromolecular organic form; So organic solvent extractings such as cyclohexane capable of using or toluene take out organic selenium, through measuring the content of total selenium and inorganic selenium, difference subtracts the content that obtains organic selenium again.The method complex operation step, poor repeatability can only obtain the total amount of organic selenium, and for concrete organic selenium form and unclear, and there is certain toxicity in organic solvents such as used cyclohexane, toluene.The selenium morphological research belongs to forward position research, and the home and abroad does not have the standard of selenium morphological research method at present.The present invention utilizes gentle extracting mode; Extract selenium-containing compound; Utilize liquid chromatography effectively to separate again after utilizing the enzyme hydrolysis selenium-containing compound, utilize highly sensitive atomic fluorescence (AFS) to carry out the mensuration of different shape selenium at last, obtain the content of organic selenium at last.The advantage of the method is to measure the Se content and the organic selenium content of different shape in the selenium-enriched plant, can further understand the transformation rule of plants enriched absorption selenium, and the method is simple to operation, good reproducibility, and degree of accuracy is high, and agents useful for same is nontoxic, pollution-free.
The Morphometric pre-treating method of selenium also there is report abroad, Selenium species in leavesof chicory, dandelion; Lamb ' s lettuce and parsley (Darja Mazej, VekoslavaStibilj, et al.Food Chemistry; 2008; 107:75-83.) a kind of method of utilizing the protease hydrolytic sample to carry out the test of selenium form is then separately disclosed, the ratio of this method proteinase consumption and sample is 1: 2～1: 5, cost is very high.
So, develop a kind of to being that the sample pre-treatments and the method for testing of main evaluating objects, low-cost high-efficiency extremely is necessary with seleno-amino acids.
Summary of the invention
The objective of the invention is to overcome the deficiency that existing selenium somatometry of physique technology particularly exists in the extractive technique, provide a kind of easy and simple to handle, extraction efficiency is high, good reproducibility, the assay method of selenium form in the low selenium-enriched health care article of cost.
In order to solve these problems of the prior art, technical scheme provided by the invention is following:
The assay method of selenium form in a kind of selenium-enriched health care article is characterized in that said method comprising the steps of:
(1) pre-service of selenium-enriched health care article: use extraction damping fluid Tris-HCl that selenium-enriched health care article sample is extracted, Proteinase K, the proteinase XIV that adds effective dose after extraction is handled successively is hydrolyzed to extract and obtains testing sample solution;
(2) adopting hydride to take place---atomic fluorescence spectrometry and high performance liquid chromatography coupling are measured standard selenium form solution and testing sample solution respectively; Carry out quantitatively with peak height or peak area; With the external standard method in the data processing; Preserve behind the drawing standard working curve; With sample peak integral processing, utilize external standard method to proofread and correct then, can calculate the content of selenocystine in the testing sample solution (Se-Cys2), selenium methyl halfcystine (SeMeCys), Se (IV), selenomethionine (SeMet) according to formula (1);
X＝C×V/m (1)
Wherein: X is the content of test substance in the sample, and unit is every kilogram of microgram (μ g/kg); C is the concentration of this material in the liquid to be measured, and unit is every liter of microgram (μ g/L); V is for measuring total liquid volume, and unit is a milliliter (mL); F is an extension rate; M is for claiming appearance quality (volume), and unit is gram (g/mL).
Preferably; The concentration of extracting damping fluid Tris-HCl in the said method is 100mmol/L; PH7.5, the ratio of health-care liquid article sample and extract (V: V) be the ratio (m: V) be 1: 25～1: 100 of 1: 5～1: 10 or solid health food sample and extract.
Preferably, method for distilling adopts ultrasonic Extraction in the said method, and extraction time is 10～30min.
Preferably, (the m: V) be the ratio (m: m) be 1: 20～1: 100 of 1: 100～1: 2000 or Proteinase K and solid health food sample of the ratio of Proteinase K and health-care liquid article sample in the said method; Hydrolysis temperature is controlled at 40～55 ℃.
Preferably, (the m: V) be 1: 100～1: 2000, proteinase XIV and the fixing ratio of health products sample (m: m) be 1: 20～1: 100 of the ratio of proteinase XIV and health-care liquid article sample in the said method; Hydrolysis temperature is controlled at 30～45 ℃.
Preferably, sample solution is under 0～10 ℃ after the hydrolysis process in the said method, and supernatant is crossed the filter membrane of 0.2～0.5 μ m after the centrifugal treating, makes supernatant to be measured.
Preferably, high performance liquid chromatography and hydride take place in the said method---and liquid chromatography and atomic fluorescence condition are during the atomic fluorescence spectrometry coupling:
Moving phase (40mmol/L (NH
4)
2HPO
4, pH 6.0); Flow velocity 1mL/min; Sampling volume 100 μ L; Peristaltic pump rotating speed 65rpm; Lamp current/auxilliary negative electrode: 100mA, 45mA; Photomultiplier negative high voltage: 300V; Flow rate of carrier gas: 300mL/min; Shield gas flow speed: 700mL/min.
Preferably, high performance liquid chromatography and hydride take place in the said method---and the hydride occurrence condition is during the atomic fluorescence spectrometry coupling:
Reductive agent composition: 0.35%NaOH+1.2%NaBH
4, flow velocity: 6mL/min; Oxidizer composition: 0.35%NaOH+0.8%H
2O
2, flow velocity: 6mL/min; Current-carrying: 10%HCl, flow velocity: 6mL/min.
Preferably; Said method start back is provided with liquid chromatography and atomic fluorescence, treat instrument stabilizer after, do typical curve earlier; Measure the sample solution of handling well then; The retention time of selenocystine (Se-Cys2), selenium methyl halfcystine (SeMeCys), Se (IV), selenomethionine (SeMet) is followed successively by under these conditions: 2.6min, 3.2min, 4.0min; 5.0min the chromatographic peak retention time of the selenocystine in the testing sample (Se-Cys2), selenium methyl halfcystine (SeMeCys), Se (IV), selenomethionine (SeMet) is compared variation range and within ± 5%, is promptly thought and be this material to be determined with standard solution.
The present invention relates to the assay method of selenium form in a kind of selenium-enriched health care article, is after utilizing ultrasonic Extraction, combines gentle enzyme hydrolysis again, makes the abundant hydrolysis of selenium-containing compound in the selenium-enriched health care article be beneficial to the mensuration of selenium form.Described is the pre-treating method of main selenium morphological index with seleno-amino acids, and its step is described below:
(1) pipette mixing or take by weighing earlier and grind the selenium-enriched health care article sieve in plastic centrifuge tube, add extract damping fluid Tris-HCl (100mmol/L, pH7.5), mixing.The ratio of liquid selenium-enriched health care article and extract (V: V) be 1: 5～1: 10, the ratio of solid health food and extract (m: V) be 1: 25～1: 100;
(2) mixed liquor is put into ultrasonic cleaning machine ultrasonic Extraction 10～30min;
(3) add Proteinase K again, constant-temperature shaking under 50 ℃ of conditions (rotating speed 150～200rpm can fully vibrate the liquid in the plastic centrifuge tube) is extracted 4～12h.The ratio of Proteinase K and fluid sample (m: V) be 1: 100～1: 2000, the ratio of Proteinase K and solid sample (m: m) be 1: 20～1: 100;
(4) add proteinase XIV again, 37 ℃, constant-temperature shaking (rotating speed 150～200rpm can fully vibrate the liquid in the plastic centrifuge tube) is extracted 4～12h.The ratio of proteinase XIV and fluid sample (m: V) be 1: 100～1: 2000, the ratio of proteinase XIV and sample (m: m) be 1: 20～1: 100;
(5) with the solution that obtains in 1～4 step under 4 ℃, with 10000 rev/mins of centrifugal 30min of rotating speed, get the filter membrane that supernatant is crossed 0.22 μ m, obtain solution to be measured.
(6) set liquid chromatography and atomic fluorescence condition:
Moving phase (40mmol/L (NH4) 2HPO4, pH 6.0); Flow velocity 1mL/min; Sampling volume 100 μ L; Peristaltic pump rotating speed 65rpm; Lamp current/auxilliary negative electrode: 100mA, 45mA; Photomultiplier negative high voltage: 300V; Flow rate of carrier gas: 300mL/min; Shield gas flow speed: 700mL/min.
The hydride occurrence condition:
Reductive agent composition: 0.35%NaOH+1.2%NaBH4, flow velocity: 6mL/min; Oxidizer composition: 0.35%NaOH+0.8%H2O2, flow velocity: 6mL/min; Current-carrying: 10%HCl, flow velocity: 6mL/min.
(7) the start back is provided with liquid chromatography and atomic fluorescence by above-mentioned condition; After treating instrument stabilizer; Do typical curve earlier, measure the sample solution of handling well then, the retention time of selenocystine (Se-Cys2), selenium methyl halfcystine (SeMeCys), Se (IV), selenomethionine (SeMet) is followed successively by under these conditions: 2.6min; 3.2min; 4.0min, 5.0min, the chromatographic peak retention time of the selenocystine in the testing sample (Se-Cys2), selenium methyl halfcystine (SeMeCys), Se (IV), selenomethionine (SeMet) is compared variation range and within ± 5%, is promptly thought and be this material to be determined with standard solution.
(8) carry out quantitatively with peak height or peak area; With the external standard method in the data processing; Preserve behind the drawing standard working curve; With sample peak integral processing, utilize external standard to proofread and correct then, can obtain the concentration of selenocystine in the solution to be measured (Se-Cys2), selenium methyl halfcystine (SeMeCys), Se (IV), selenomethionine (SeMet).
(9) can calculate the concentration of selenocystine in the testing sample (Se-Cys2), selenium methyl halfcystine (SeMeCys), Se (IV), selenomethionine (SeMet) according to formula (1).
X＝C×V/m ..................(1)
In the formula:
The content of test substance in the X-sample, unit are every kilogram of microgram (μ g/kg);
The concentration of this material in the C-liquid to be measured, unit is every liter of microgram (μ g/L);
V-mensuration total liquid volume, unit are milliliter (mL);
The F-extension rate;
M-claims a kind quality (volume), and unit is gram (g/mL).
The present invention utilizes gentle extracting mode, behind the ultrasonic Extraction selenium-containing compound again plurality of enzymes be used in combination and make the thorough hydrolysis of selenium-containing compound, so not only the enzyme dosage of use is few, and is with low cost and hydrolysis effect good.The advantage of the method is simple to operation, and extraction efficiency is high, good reproducibility, and cost is low, and agents useful for same is nontoxic, pollution-free.
Compare prior art, the present invention has following beneficial effect:
1, selects the nearly neutral Tris-HCl of gentle extraction reagent: pH, make selenium-containing compound form in leaching process keep stable.
2, adopt ultrasonic Extraction in the leaching process, selenium-containing compound is more dissolved in the extract, improved extraction efficiency.
3, ultrasonic Extraction is before enzyme-added, the one, the structure of destructive enzyme not influences the activity of enzyme, the 2nd, make selenium-containing compound more dissolve in extract after, be more conducive to the hydrolysis of enzyme.
4, use 2 kinds of protease hydrolytics, the selenium-containing compound hydrolysis is more thorough, compare extraction efficiency with a kind of protease hydrolytic and improve more than 40%, and extraction ratio all can reach more than 80%, the amount of the proteinase of required use still less, cost savings are more than 10 times.
5, the inventive method is simple to operation, good reproducibility, and agents useful for same is nontoxic, pollution-free.
Description of drawings
Below in conjunction with accompanying drawing and embodiment the present invention is further described:
Fig. 1 is that selenium form standard is separated spectrogram; Peak sequence successively, retention time and the concentration gradient of selenocystine (Se-Cys2), selenium methyl halfcystine (SeMeCys), Se (IV), selenomethionine (SeMet) have been disclosed in the spectrogram.
Fig. 2 is the mensuration figure as a result of the selenium-enriched health care article of the embodiment of the invention 1 certain famous brand name; Can know that by Fig. 2 there are 4 kinds of selenium forms in the selenium-enriched health care article that adopt extraction conditions of the present invention and utilize highly sensitive morphological analysis instrument can measure this brand, can obtain the content of each form and account for the ratio of total selenium according to calculated by peak area.
Fig. 3 is the mensuration figure as a result of the embodiment of the invention 2 certain brand selenium yeast sheet; Can know by Fig. 3, adopt extraction conditions of the present invention and utilize highly sensitive morphological analysis instrument can measure in this brand selenium yeast sheet only to have two kinds of selenoaminoacid forms, according to the content of each form of calculated by peak area and account for the ratio of total selenium.
Embodiment:
Following examples are used to explain the present invention, but can not be used for limiting scope of the present invention.The implementation condition that adopts among the embodiment can be done further adjustment according to the condition of concrete producer, and unaccounted implementation condition is generally the condition in the normal experiment.
At present; Selenium-enriched health care article kind on the market is various; Because of its selenium source that adds in process of production (inorganic selenium source or organic selenium source) and amount difference; Also there are very big difference in the content of selenium and existence form in the final selenium-enriched health care article, on the market in the selenium-enriched health care article content of selenium and selenium with which type of form exist actually, how the consumer this select safe and effective selenium-enriched health care article to exist much to feel uncertain; Therefore the mensuration of Se content and form in this series products is also had higher requirement, the present invention has selected to account for domestic selenium-enriched health care article market share high product and has measured.
Selenium somatometry of physique in the selenium-enriched health care article (liquid) of embodiment 1 certain famous brand name
Selenium somatometry of physique pre-treating method step to the selenium-enriched health care article (liquid) of certain famous brand name is optimized, and optimizing process and result are following:
Pipette 1mL selenium-enriched health care article in the 15mL plastic centrifuge tube, add 5mL and extract damping fluid Tris-HCl, ultrasonic Extraction 15min adds the 1mg Proteinase K; 50 ℃, constant-temperature shaking culture 8h adds 1mg proteinase XIV again; 37 ℃, under 4 ℃, per minute rotating speed 10000 changes behind the constant-temperature shaking culture 8h; After centrifugal 30min, supernatant cross the filter membrane of 0.22 μ m, the machine for preparing liquid to be measured.Liquid phase chromatogram condition is set to moving phase (40mmol/L (NH4) 2HPO4, pH 6.0); Flow velocity 1mL/min; Sampling volume 100 μ L; Peristaltic pump rotating speed 65rpm; Lamp current/auxilliary negative electrode: 100mA, 45mA; Photomultiplier negative high voltage: 300V; Flow rate of carrier gas: 300mL/min; Shield gas flow speed: 700mL/min.The hydride occurrence condition is set to reductive agent composition: 0.35%NaOH+1.2%NaBH4, flow velocity: 6mL/min; Oxidizer composition: 0.35%NaOH+0.8%H2O2, flow velocity: 6mL/min; Current-carrying: 10%HCl, flow velocity: 6mL/min.
The start back is provided with liquid chromatography and atomic fluorescence by above-mentioned condition; After treating instrument stabilizer; Do typical curve earlier, measure the sample solution of handling well then, the retention time of selenocystine (Se-Cys2), selenium methyl halfcystine (SeMeCys), Se (IV), selenomethionine (SeMet) is followed successively by under these conditions: 2.6min; 3.2min; 4.0min, 5.0min, the chromatographic peak retention time of the selenocystine in the testing sample (Se-Cys2), selenium methyl halfcystine (SeMeCys), Se (IV), selenomethionine (SeMet) is compared variation range and within ± 5%, is promptly thought and be this material to be determined with standard solution.
Carry out quantitatively with peak height or peak area; With the external standard method in the data processing; Preserve behind the drawing standard working curve; With sample peak integral processing, utilize external standard to proofread and correct then, can obtain the concentration of selenocystine in the solution to be measured (Se-Cys2), selenium methyl halfcystine (SeMeCys), Se (IV), selenomethionine (SeMet).And according to formula calculation sample selenium morphological data.
Screening of table 1 optimal conditions and effect
Can know by table 1; Selecting ultrasonic time in selenium-enriched health care article (liquid) the selenium somatometry of physique pre-treating method of certain famous brand name is 15min; Utilize 2 kinds of protease hydrolytics and addition to be respectively 1mg; And the extraction efficiency of incubation time selenium-containing compound when being 8h is very desirable, and extraction efficiency can reach more than 85%, and is higher more than 50% than the extraction ratio of a kind of protease hydrolytic.
Measure the result:
The detection limit of table 2 instrument and precision requirement
Table 2 is the requirement of selenium morphological analysis instrument detection limit and precision.Fig. 1 is that selenium form standard is separated spectrogram
Certain famous brand name selenium-enriched health care article of table 3 are measured the result
Annotate: sample size n=6
Fig. 2 is that certain famous brand name selenium-enriched health care article is measured the result.By table 3, Fig. 2 can know, there are 4 kinds of selenium forms in certain well-known selenium-enriched health care article, and the ratio that organic selenium accounts for total selenium is 26.18%～37.58%.
The mensuration result of certain famous brand name selenium-enriched health care article recovery of standard addition of table 4
Can be known that by table 4 we add the recovery of 4 kinds of selenium forms of target 93.7%～109.1%, recovering effect is very desirable, pre-treating method and assay method is described accurately and reliably.
Selenium somatometry of physique in embodiment 2 certain brand selenium yeast sheet
Take by weighing the 0.1g sample in the 15mL plastic centrifuge tube, add 5mL and extract damping fluid Tris-HCl, ultrasonic Extraction 30min adds the 1mg Proteinase K; 50 ℃, constant-temperature shaking culture 12h adds 1mg proteinase XIV again; 37 ℃, under 4 ℃, per minute rotating speed 10000 changes behind the constant-temperature shaking culture 12h; After centrifugal 30min, supernatant crossed the filter membrane of 0.22 μ m, last machine was measured organic selenium content.Identical among other conditions and the embodiment one, mensuration, the mark-on of the screening of optimum extraction conditions and sample reclaimed measure result such as following table.
Screening of table 5 optimal conditions and effect
Can know by table 5; Selecting ultrasonic time in the Se-enriched yeast sheet selenium somatometry of physique pre-treating method of certain brand is 30min; Utilize 2 kinds of protease hydrolytics and addition to be respectively 1mg, and incubation time is 12h, the extraction efficiency of selenium-containing compound is very desirable; Extraction efficiency can reach 85%, improves more than 40% than a kind of protease hydrolytic efficient.
Certain brand Se-enriched yeast sheet of table 6 is measured the result
Annotate: sample size n=3
Fig. 3 is certain brand selenium yeast sheet sample determination result.Can know that by table 6 and Fig. 3 Selenium in Selenium-Yeast mainly exists with the selenomethionine form, the net result of mensuration is that the ratio that selenomethionine accounts for total selenium is more than 65%.
Certain brand selenium yeast sheet mark-on of table 7 reclaims measures the result
The recovery of standard addition that can know selenium yeast sheet sample by table 7 is 88%～113.8%, and recovering effect is very desirable, pre-treating method and assay method is described accurately and reliably.
In sum, the present invention can make the abundant hydrolysis of selenium-containing compound in the selenium-enriched health care article be beneficial to the determination and analysis of selenium form.The accurate check and analysis of selenium form in the selenium-enriched health care article, the determination and analysis that ability consumption guidance person science is mended selenium, particularly organic selenium (seleno-amino acids) form is significant.
Above-mentioned instance only is explanation technical conceive of the present invention and characteristics, and its purpose is to let the people who is familiar with this technology can understand content of the present invention and enforcement according to this, can not limit protection scope of the present invention with this.All equivalent transformations that spirit is done according to the present invention or modification all should be encompassed within protection scope of the present invention.
Claims (9)
1. the assay method of selenium form in the selenium-enriched health care article is characterized in that said method comprising the steps of:
(1) pre-service of selenium-enriched health care article: use extraction damping fluid Tris-HCl that selenium-enriched health care article sample is extracted, Proteinase K, the proteinase XIV that adds effective dose after extraction is handled successively is hydrolyzed to extract and obtains testing sample solution;
(2) adopting hydride to take place---atomic fluorescence spectrometry and high performance liquid chromatography coupling are measured standard selenium form solution and testing sample solution respectively; Carry out quantitatively with peak height or peak area; With the external standard method in the data processing; Preserve behind the drawing standard working curve; With sample peak integral processing, utilize external standard method to proofread and correct then, can calculate the content of selenocystine in the testing sample solution (Se-Cys2), selenium methyl halfcystine (SeMeCys), Se (IV), selenomethionine (SeMet) according to formula (1);
X=C×V/m (1)
Wherein: X is the content of test substance in the sample, and unit is every kilogram of microgram (μ g/kg); C is the concentration of this material in the liquid to be measured, and unit is every liter of microgram (μ g/L); V is for measuring total liquid volume, and unit is a milliliter (mL); F is an extension rate; M is for claiming appearance quality (volume), and unit is gram (g/mL).
2. method according to claim 1; The concentration that it is characterized in that extraction damping fluid Tris-HCl in the said method is 100 mmol/L; PH7.5, the ratio of health-care liquid article sample and extract (V:V) is that the ratio (m:V) of 1:5 ~ 1:10 or solid health food sample and extract is 1:25 ~ 1:100.
3. method according to claim 1 is characterized in that method for distilling adopts ultrasonic Extraction in the said method, and extraction time is 10 ~ 30 min.
4. method according to claim 1, the ratio (m:V) that it is characterized in that Proteinase K and health-care liquid article sample in the said method is 1:20 ~ 1:100 for the ratio (m:m) of 1:100 ~ 1:2000 or Proteinase K and solid health food sample; Hydrolysis temperature is controlled at 40～55 ℃.
5. method according to claim 1 is characterized in that the ratio (m:V) of proteinase XIV and health-care liquid article sample in the said method is 1:100 ~ 1:2000, proteinase XIV and fixedly the ratio of health products sample (m:m) be 1:20 ~ 1:100; Hydrolysis temperature is controlled at 30～45 ℃.
6. method according to claim 1 is characterized in that sample solution is under 0～10 ℃ after the hydrolysis process in the said method, and supernatant is crossed the filter membrane of 0.2～0.5 μ m after the centrifugal treating, makes supernatant to be measured.
7. method according to claim 1 is characterized in that high performance liquid chromatography and hydride take place in the said method---liquid chromatography and atomic fluorescence condition are during the atomic fluorescence spectrometry coupling:
Moving phase (40 mmol/L (NH
4)
2HPO
4, pH 6.0); Flow velocity 1 mL/min; Sampling volume 100 μ L; Peristaltic pump rotating speed 65 rpm; Lamp current/auxilliary negative electrode: 100mA, 45mA; Photomultiplier negative high voltage: 300V; Flow rate of carrier gas: 300mL/min; Shield gas flow speed: 700mL/min.
8. method according to claim 7 is characterized in that high performance liquid chromatography and hydride take place in the said method---the hydride occurrence condition is during the atomic fluorescence spectrometry coupling:
Reductive agent composition: 0.35%NaOH+1.2%NaBH
4, flow velocity: 6 mL/min; Oxidizer composition: 0.35%NaOH+0.8%H
2O
2, flow velocity: 6 mL/min; Current-carrying: 10% HCl, flow velocity: 6 mL/min.
9. method according to claim 8; It is characterized in that said method start back is provided with liquid chromatography and atomic fluorescence; After treating instrument stabilizer; Do typical curve earlier, measure the sample solution of handling well then, the retention time of selenocystine (Se-Cys2), selenium methyl halfcystine (SeMeCys), Se (IV), selenomethionine (SeMet) is followed successively by under these conditions: 2.6 min; 3.2 min; 4.0 min, 5.0 min, the chromatographic peak retention time of the selenocystine in the testing sample (Se-Cys2), selenium methyl halfcystine (SeMeCys), Se (IV), selenomethionine (SeMet) is compared variation range and within ± 5%, is promptly thought and be this material to be determined with standard solution.
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| CN103399117A (en) * | 2013-08-26 | 2013-11-20 | 中国水产科学研究院黄海水产研究所 | Detection method of selenium form in aquatic product |
| CN113325105A (en) * | 2021-05-26 | 2021-08-31 | 陕西科技大学 | Method for detecting selenomethionine in selenium-enriched fish |
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