Professor Yang Guangqi, a nutritionist in China, put forward that the safe intake of selenium per person per day is
400μg, in order to control human selenium intake, China's Ministry of Health has also formulated a hygienic standard for selenium in food.
GB13105-91,and the corresponding national standard method GB 12399-90- Fluorescence determination of selenium in food was established. pass by
The operation of this method is complicated, and the reagent 2,2-diaminonaphthalene (2,3-diaminonaphthalene, abbreviated as DAN) used for Ky is highly toxic.
And it needs to be imported. 1 & gt； In the second revision, hydride generation atomic fluorescence spectrometry was proposed, which is rapid, simple, accurate and precise.
In order to make up for the deficiency of GB 12399-90, the selenium in various foods was determined by river spectrometry. In view of atomic fluorescence intensity meter
It has not been popularized in China, so it is the second method to supplement this standard.
1 range
This standard specifies the methods for the determination of selenium in food by fluorescence method and hydride generation atomic fluorescence spectrometry.
This standard is applicable to the determination of selenium in various foods.
First fluorescence method
Principle 2
After the sample was digested with mixed acids, selenium compounds were oxidized to tetravalent inorganic selenium (Se4+) and 2.3- diaminonaphthalene.
(2,3-diaminonaphthalene, abbreviated as DNA) to produce 4,5-benzoselenol,
Under certain conditions, its fluorescence intensity is directly proportional to the concentration of selenium. After extraction with hexane, at the excitation wavelength of 376nm,
The fluorescence intensity was measured at the emission wavelength of 520nm and compared with the drawn standard curve quantitatively. The detection limit of this method is 3ng.
3 reagent
3. 1 cyclohexane.
3.2 nitric acid.
3.3 Perchloric acid.
3.4 hydrochloric acid.
3.5 Hydrobromic acid.
3.6 1+9 hydrochloric acid solution: take 10mL hydrochloric acid and add 90mL water.
3.7 1+ 1 ammonia.
3.8 5+95 selenium-removing sulfuric acid: take 5mL selenium-removing sulfuric acid and add it to 95mL water.
Selenium-removing sulfuric acid: take 200mL sulfuric acid, add 200mL water, add 30mL hydrobromic acid, mix well and put in sand.
Heat the bath to evaporate selenium and water until thick white smoke appears, and the volume should be 200mL.
3.9 0.2mol/LEDTA: Weigh 37g EDTA disodium salt, add water, heat and dissolve it, and dilute it to 500mL after cooling.
3. 10 10% hydroxylamine hydrochloride: dissolve 10g hydroxylamine hydrochloride in water and dilute it to 100mL.
3. 1 1 Mixed acid: nitric acid+perchloric acid (2+ 1).
3.120.1%2,3-diaminonaphthalene (purity 95% ~ 98%) needs to be prepared in a dark room. Weigh 200 mg of Dan in the belt cover 3.
Add 200ml of 0. 1 mol/l hydrochloric acid to the flask and shake it for about15min to completely dissolve it. Adde about 40 ml of cyclohexane,
Continue shaking for 5 minutes, transfer this solution to a separatory funnel, and after the solution is layered, discard the cyclohexane layer and collect DAN.
Layer solution. The purity of cyclohexane used in this way varies, and it usually needs to be purified for about 3 ~ 4 times). Dissolve purified Dan
The solution was stored in a brown bottle, and cyclohexane with a thickness of about 65438±0cm was added to cover the surface of the solution. Put it in the refrigerator. When necessary.
Purify once.
3. 13 selenium standard solution
3. 13. 1 selenium standard stock solution (100μg/mL): accurately weigh 100.0mg elemental selenium (spectrally pure) and dissolve it in a small amount of nitric acid.
Add 2mL of perchloric acid, heat in boiling water bath for 3-4hr, add 8.4mL of hydrochloric acid after cooling, and then put in boiling water bath.
Cook for 2 minutes. Dilute it accurately to 1000mL, and its hydrochloric acid concentration is 0.1mol/L. The concentration of the stock solution is 100 μ g/ml.
3. 13.2 selenium standard solution (0. 05μg/mL): dilute 3. 13. 1 solution with 0. 1mol/L hydrochloric acid to make the selenium content 0.05.
Microgram/ml. Put it in the refrigerator.
3. 14 0.02% cresol red indicator: dissolve 50mg of cresol red in water, and add 1+ 1 drop of ammonia water until the titration of cresol red is completed.
After completely dissolving, add water to dilute to 250 ml.
3. 15 EDTA mixed solution: take 50m l(3.9) and (3. 10) respectively, mix well, add 5ml(2. 14) solution and dilute with water.
To1l.
4 Instruments and equipment
4. 1 general laboratory equipment.
4.2 fluorescence spectrophotometer.
5 analysis steps
5. 1 sample treatment and digestion
5. 1. 1 granule: the sample was washed with water for three times, dried at 60℃, ground into powder with stainless steel, stored in plastic bottles and put into small ones.
Wrap camphor essence, cover it tightly and keep it for later use.
5. 1.2 vegetable and other plant foods: take the edible part, rinse it with clear water for three times, then use gauze to absorb water droplets, and use a stainless steel knife.
Chopping, drying the evenly mixed sample at 60℃, weighing and pulverizing for later use.
5. 1.3 Weigh 0.5-2.0g sample (selenium content is 0.0 1-0.5μ g) and put it in a grinding triangular flask, and < S 10mL5+95 will remove selenium.
Acid, after the sample is wetted, add 20mL of mixed acid and leave it overnight. The next day, it was gradually heated on the sand bath. When it was intense,
After it should happen (the solution becomes colorless), continue heating until white smoke is produced, and the solution gradually turns pale yellow, reaching the end point. some
Some vegetable samples are often turbid after digestion, so it is difficult to determine the end point, so we should observe carefully, and some of them have high selenium content.
Vegetables contain a lot of Se6+. After digestion and cooling, it is necessary to add 10mL 1+9 hydrochloric acid to continue heating.
Se6+ is reduced to Se4+.
Determine the end point as described above.
5.2 Decision
Add 20 ml of EDTA mixed solution to the digestive juice of the sample, and adjust it to red-orange with ammonia (1+ 1) or hydrochloric acid (pH 1). 5)
~2.0)。 The following steps are carried out in a dark room: add 3 ml of Dan reagent, mix well, and cook in boiling water bath for 5 minutes, h! Immediately cool, add 3 ml of cyclohexane, shake well for 4 minutes, transfer all the solutions to a separatory funnel, discard the water layer after layering, and transfer the cyclohexane layer to a test tube with a cover, taking care not to mix water droplets with cyclohexane and emit light at the wavelength of 376nm.
The fluorescence intensity of selenium brain was measured at 376nm, and the wavelength of emitted light was 520nm.
5.3 Drawing selenium standard curve: accurately absorb 0, 0.2, 1.0, 2.0 and 4.0mL selenium standard solution, and add water to 5mL.
Simultaneous determination is carried out according to the sample determination steps. When the selenium content is lower than 0.5μg, the fluorescence intensity is linearly related to the selenium content.
In the routine determination of samples, a standard tube (in duplicate) is only needed to be made with a reagent blank close to the selenium content of samples at a time.
Six results
6. 1 calculation
C-B 1
X= - × S × -
A-B·m
In the formula, x refers to the content of selenium in the sample Lv Zhong, μg/g；/g; /g；
A— Yingjiang reading of standard pipe;
B fluorescence reading of blank tube;
C- fluorescence reading of the sample tube;
S —— Selenium content in standard tube, μ g;
M—— sample quality, g.
6.2 Allowable differences in results
The absolute value of relative deviation of parallel determination or repeated determination results in the same laboratory is ≤ 10%.
The second method is hydride generation atomic fluorescence spectrometry
Principle 7
After the sample was digested by acid heating, the hexavalent selenium in the sample was reduced to 6mol/L hydrochloric acid (HCl).
In hydrochloric acid medium, tetravalent selenium was reduced with sodium borohydride (NaBH4) or potassium borohydride (KBH4) as reducing agent.
The primary hydrogen selenide (SeH2) is atomized by carrier gas (argon), and the hollow cathode lamp is specially made of selenium.
Under irradiation, selenium atoms in the ground state are excited to high energy state, and when they are inactivated and return to the ground state, they will emit fluorescence with characteristic wavelength.
Light, its fluorescence intensity is proportional to selenium content. Quantitative comparison with standard series.
8 reagent
In this method, unless otherwise specified, the reagent used is analytically pure, and the test water is distilled water or equivalent.
Pure water.
8. 1 nitric acid (excellent purity)
8.2 Perchloric acid (excellent purity)
8.3 hydrochloric acid (high purity)
8.4 Mixed acid: mixed acid of nitric acid and perchloric acid (4+ 1).
8.5 Sodium hydroxide (high purity)
8.6 Sodium borohydride solution (8g/L): Weigh 8.0g sodium borohydride (NaBH4) and dissolve it in sodium hydroxide solution (5g/L).
Then fix the volume to 1000mL.
8.7 Potassium ferricyanide (100g/L): Weigh 10.0g potassium ferricyanide (K3Fe(CN)6), dissolve it in 100mL distilled water, and mix well.
8.8 Selenium standard stock solution: accurately weigh 100.0mg selenium (spectrally pure), dissolve it in a small amount of nitric acid, add 2mL perchloric acid, and fix the volume.
Heating in boiling water bath for 3-4 hours, cooling, adding 8.4mL hydrochloric acid, boiling in boiling water bath for 2 minutes, and accurately diluting.
To 1000mL, and the concentration of hydrochloric acid is 0. 1mol/L, which is equivalent to 100μg selenium /ml.
8.9 Selenium standard application solution: take 100μg/mL selenium standard reserve solution 1.0mL, and fix the volume to 100mL, the concentration of this application solution.
Yes 1 μ g/ml.
9 instrument
9. 1 AFS-2 10 dual-channel atomic fluorescence photometer or similar instrument
9.2 electric heating plate
9.3 Automatic temperature control digestion furnace
10 analysis steps
10. 1 sample treatment and digestion
10. 1 granule: the sample was washed with water for three times, dried at 60℃, ground with stainless steel, and stored in plastic bottles for later use.
10.10.2 vegetable and other plant foods: take the edible parts, wash them with clean water, then use gauze to absorb water drops and homogenize them.
Spare.
10. 1.3 Weigh 0.5-2.0g sample into a high-fever cup, add 10.0mL mixed acid and a small amount of glass beads, and cover it with watch glasses.
Cold digestion overnight. Heat it on the electric heating plate the next day and add mixed acid in time. When the solution becomes clear and colorless
White smoke, and then continue to heat to the remaining volume of about 2mL, must not evaporate. Cooling, adding 5mL6mol/L hydrochloric acid,
Continue heating until the solution becomes clear and colorless with white smoke, so that hexavalent selenium can be completely reduced to tetravalent selenium.
Cool and transfer to a 50 ml volumetric flask. Do blank experiments at the same time.
10. 1.4 suck 10mL sample digestive juice into 15mL centrifuge tube, add 2mL concentrated hydrochloric acid and 1mL potassium ferricyanide solution,
Uniformly mixed, to be tested.
Preparation of 10.2 standard curve: put 0.0, 0. 1, 0.2, 0.3, 0.4, 0. *mL standard application solution in 15mL centrifuge tube, adjust the volume to 10mL with deionized water, and then add 2mL concentrated hydrochloric acid and 65438+ respectively.
Standard working curve.
10.3 determination
10.3. 1 Reference conditions of the instrument:
Negative high voltage: 340 v; Lamp current:100ma; ; Atomization temperature: 800℃; Furnace height: 8 mm; Carrier gas flow
Speed: 500ml/min; Protective gas flow:1000 ml/min; Measurement method: standard curve method; Reading mode: peak area;
Delay time:1s; ; Reading time:15s; ; Liquid adding time: 8s; Sample volume: 2mL.
10.3.2 determination: choose one of the following methods according to the experimental situation.
10.3.2. 1 concentration measurement: after setting the best conditions of the instrument, gradually raise the furnace temperature to the required temperature.
After stabilizing 10 ~ 20 minutes, start measuring. Continuously inject the sample with a standard series of zero tubes, and after the reading is stable, go to.
Measure and draw standard curves in standard series. Transferring to sample determination, respectively determining sample blank and sample digestive juice,
Before measuring different samples, the sampler should be cleaned. Calculate the sample determination result according to the following formula.
10.3.2.2 automatic calculation result measurement of the instrument: set the best conditions of the instrument and input them in the sample parameter interface.
The following parameters: sample mass (g or mL), dilution volume (mL), and select the concentration unit of the result. Gradually increase the furnace temperature.
After reaching the required temperature, stabilize 10 ~ 20 minutes, and then start measuring. Continuously inject the sample with a standard series of zero tubes and wait for the reading.
After stabilization, switch to standard series measurement and draw standard curve. Enter the blank again before transferring to the sample for determination.
Value measurement state, sample with sample blank digestive juice, and let the instrument take its average value as the blank value deducted at the bottom. Then.
Samples can be determined in sequence. After the measurement is completed, select Print Report to automatically print the measurement results.
10.4 result
10.4. 1 calculation
(C -C0)×V× 1000
X= -
M× 1000× 1000
Where: x: the content of selenium in the sample, mg/kg (or mg/L);
C: measured concentration of sample digestive juice, ng/ml;
C0: measured concentration of sample blank digestive juice, ng/ml;
M: sample mass (volume), g (ml);
V: total volume of sample digestive juice, mL.
10.4.2 The detection limit of this standard instrument is 0.5ng/mL.
The linear range of standard curve is 0 ~ 400 ng/ml.
Methods The recovery rate: standard powder: 85.7% ~ 102.3% milk powder: 88.8% ~10/0.2%.
Control results of standard reference materials:
Determination times of reference material (n) Standard value of measured value
Tea (GBW 08505) 7 0.044 0.45438+0 0.05438+00
Pig liver (gbw08551) 70.931.940 0.050
Relative standard deviation: RSD=2.6%(n= 12)
Appendix a (prompt appendix)
Determination of Selenium in Food
Hydride atomic fluorescence spectrometry
1 method evaluation
Determination of selenium in food by hydride generation atomic fluorescence spectrometry is simple, rapid, accurate and sensitive.
Good precision, wide linear range, low toxicity of used reagents and strong practicability.
2 Method development and verification results
┌——————┬——————┬——————┬————————┐
│ Unit │ Beijing Municipal Health │ Food Hygiene of the Ministry of Health │ Beijing Imported Food Hygiene │
Epidemic prevention station, health supervision and inspection office, supervision and inspection office
├——————┼——————┼——————┼————————┤
Instrument model AFS-220xdy-2AFS-210.
├——————┼——————┼——————┼————————┤
│ detection limit │ 0.5 (ng/ml) │ 0.3 (ng/ml) │ 0.2 (ng/ml)
├——————┼——————┼——————┼————————┤
│ recovery rate │ 85.7-112.3 │ 95.4-120.0 │
├——————┼——————┼——————┼————————┤
Name of quality control sample, tea pig liver, milk powder and bovine serum, Gan Lan pig liver.
│ │GBN GBN │GBN GBN │ GBN GBN │
│ │08505 0855 1 │0859 09 13 1 │08504 0855 1 │
│ Analysis result │ 0.044 0.931.209 38.2 │ 0.082 0.934 │
│ standard value │ 0.041.940 │ 0.22 38.9 │ 0.083 0.934 │.
│ (μg/g) │0.0 10？ 0.05│0.02 ? 2.3 │? 0.008? 0.05 │
├——————┼——————┼——————┼————————┤
│ Relative standard │ 2.6 │ 2. 1 │ 5. 1 │
Deviation (rsd%) │ │ │ │
├——————┼——————┼——————┼————————┤
│ standard curve stage │ r = 0.9999 │ r = 0.9992 │
Relationship number (r) │ │ r=0.9999 │ │.
└——————┴——————┴——————┴————————┘
3 operational considerations and experience introduction
(1) During heating and digestion, the sample in this method must not evaporate to avoid selenium loss.
(2) For undigested samples, if sulfuric acid needs to be added for digestion, selenium needs to be removed from sulfuric acid, and the selenium content in Rr sulfuric acid is.
Higher.
4 The preparer of this standard:
Beijing Health and Epidemic Prevention Station: Tian and Mao Hong
Institute of Food Hygiene Supervision and Inspection of Ministry of Health: Yang Huifen, Huang, Chen Qingchuan.
Beijing imported food hygiene supervision and inspection institute: Yan Guang
-
-
Approved by the Ministry of Health of the People's Republic of China 1996-06- 19, implemented 1996-09-0 1.
Beware of excessive selenium in imported peas.
Zheng Wanli from Xiamen Entry-Exit Inspection and Quarantine Bureau
The General Administration of Quality Supervision, Inspection and Quarantine issued a risk warning, demanding that all problems found in strict inspection be returned.
On the afternoon of April 14, the reporter learned from Xiamen Inspection and Quarantine Bureau that in order to effectively prevent food contaminated by toxic and harmful substances from entering the country, AQSIQ issued a risk warning, requiring local inspection and quarantine institutions to strengthen inspection and quarantine of imported peas and detect selenium content in batches. All imported peas that fail to meet the requirements of China after inspection and quarantine shall be returned.
In order to prevent peas that do not meet China's health and safety requirements from entering the country and avoid unnecessary economic losses for imported enterprises, Xiamen Inspection and Quarantine Bureau authorized a spokesperson to issue a warning to imported enterprises, hoping to attract great attention. Especially when signing a trade contract, we should pay attention to strictly signing the inspection and quarantine clauses, and urge foreign suppliers to test peas for toxic and harmful substances such as selenium and pesticide residues before export. Products that do not meet China's hygiene requirements should not be exported.
It is reported that in February this year, China's quality inspection department detected that the selenium content in yellow peas imported from the United States and peas imported from Canada exceeded China's national standards. According to the Plant Inspection Department of Xiamen Inspection and Quarantine Bureau, the number of peas imported from Xiamen has been rising since the second half of last year due to the strong demand in food and feed industries in Zhangzhou and other places. In 2005, 20 batches of peas were imported at 609 1 ton, and in the first quarter of this year, the import volume has reached 4646 tons in 14 batches. At present, selenium has not exceeded the standard.
[Noun explanation] Selenium is an essential trace element for life. Scientists have found that selenium is related to the prevention and treatment of many diseases. In China, selenium is used to prevent Keshan disease (an endemic disease with myocardial necrosis as the main symptom) and Kaschin-Beck disease. Some people think that selenium can prevent cancer and resist aging.
However, the intake of selenium is not as high as possible. It is found that excessive intake of selenium by livestock will lead to poisoning, stunting, depilation and even death in areas with high selenium content. At present, people attach great importance to selenium deficiency, but the harm of excessive selenium intake is still insufficient. Scientists have found that the selenium content in human body is about15mg based on the weight of a standard person of 70kg. The human body should maintain a general balance between selenium intake and excretion. Of course, selenium deficiency will cause illness, but excessive intake will also cause harm to the human body. The national standard GB 2762-2005 stipulates that the maximum allowable limit of selenium in beans and products is 0.3mg/kg.