This standard specifies the determination method of thiamine in various foods. This standard is applicable to the determination of thiamine in various foods. The detection limit of this method is 0.05kg, and the linear range is 0.2kg-10kg. GB/T 5009.84-2003 Determination of thiamine (vitamin B1) in food GB/T5009.84-2003 Standard download decompression password: www.bzxz.net

ICS 67.040
National Standard of the People's Republic of China
GB/T5009.84—2003
Replaces GB/T12390—1990
Determination of thiamine (vitamin B, ) in foods2003-08-11Promulgated
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
Implemented on 2004-01-01
GB/T5009.84—2003
This standard corresponds to AOAC45.1.07 "Fluorometric determination of thiamine in foods" (1995 edition). The degree of consistency between this standard and AOAC45.1.07 is non-equivalent. This standard replaces GB/T12390-1990 "Determination of Thiamine (Vitamin B1) in Food". Compared with GB/T12390-1990, this standard has the following major revisions: the Chinese name of the standard has been revised and changed to "Determination of Thiamine (Vitamin B) in Food"; the structure of the original standard has been revised in accordance with GB/T20001.4--2001 "Standard Preparation Rules Part 4: Chemical Analysis Methods".
This standard is proposed and managed by the Ministry of Health of the People's Republic of China. The drafting unit of this standard: Institute of Nutrition and Food Hygiene, Chinese Academy of Preventive Medicine. The main drafters of this standard: Wang Guangya, Zhang Hongwei, Yang Xiaoli, Men Jianhua, Yang Yuexin. The original standard was first issued in 1990, and this is the first revision. 610
1 Scope
Determination of Thiamine (Vitamin B) in Food This standard specifies the determination method of thiamine in various foods. This standard is applicable to the determination of thiamine in various foods. The detection limit of this method is 0.05μg, and the linear range is 0.2μg～10ug. 2 Principle
GB/T5009.84--2003
Thiamine is oxidized to thiamethoxam in alkaline potassium ferrocyanide solution, and thiamethoxam emits fluorescence under ultraviolet irradiation. Under given conditions and without interference from other fluorescent substances, the intensity of this fluorescence is proportional to the amount of thiamethoxam, that is, proportional to the amount of thiamine in the solution. If the sample contains too many impurities, it should be treated with an ion exchanger to separate thiamine from the impurities, and then the resulting solution should be used for determination. 3 Reagents
3.1 n-Butanol: must be used after redistillation. 3.2 Anhydrous sodium sulfate.
3.3 Amylase and protease.
3.40.1mol/L hydrochloric acid: 8.5mL concentrated hydrochloric acid (relative density 1.19 or 1.20) diluted with water to 1000mL. 3.50.3mol/L hydrochloric acid: 25.5mL concentrated hydrochloric acid diluted with water to 1000mL. 3.62mol/L sodium acetate solution: 164g anhydrous sodium acetate dissolved in water and diluted to 1000mL3.7 Potassium chloride solution (250g/L): 250g potassium chloride dissolved in water and diluted to 1000mL. Acidic potassium chloride solution (250g/L): 8.5mL concentrated hydrochloric acid diluted with 25% potassium chloride solution to 1000mL. 3.8
3.9 Sodium hydroxide solution (150g/L): 15g sodium hydroxide dissolved in water and diluted to 100mL. 3.10
1% potassium ferrocyanide solution (10g/L): 1g potassium ferrocyanide is dissolved in water and diluted to 100mL, and stored in a brown bottle. 3.11 Alkaline potassium ferrocyanide solution: Take 4mL 10g/L potassium ferrocyanide solution and dilute it to 60mL with 150g/L sodium hydroxide solution. Prepare it before use and use it in a dark place. www.bzxz.net
3.12 Acetic acid solution: Dilute 30mL glacial acetic acid with water to 1000mL. 3.13 Activated artificial pumice: Weigh 200g 40-60 mesh artificial pumice, stir and wash it twice with 10 times its volume of hot acetic acid solution (3.12), each time for 10min; then stir and wash it with 5 times its volume of 250g/L hot potassium chloride solution (3.7) for 15min; then stir and wash it with dilute acetic acid solution (3.12) for 10min: finally wash it with hot distilled water until there is no chloride ion. Store it in distilled water. 3.14 Thiamine standard stock solution (0.1mg/mL): Accurately weigh 100mg of thiamine dried with calcium chloride for 24h, dissolve in 0.01mol/L hydrochloric acid, and dilute to 1000mL. Store in a refrigerator away from light. 3.15 Thiamine standard intermediate solution (10μg/mL): Dilute the thiamine standard stock solution 10 times with 0.01mol/L hydrochloric acid, and store in a refrigerator away from light.
3.16 Thiamine standard working solution (0.1ug/mL): Dilute the thiamine standard intermediate solution 100 times with water and prepare it when used. 3.17 Bromocresol green solution (0.4g/L): Weigh 0.1g bromocresol green, put it in a small mortar, add 1.4ml.0.1mol/L sodium hydroxide solution and grind for a while, then add a little water and continue grinding until completely dissolved, and dilute to 250mL with water. 4 Instruments
4.1 Electric constant temperature incubator.
4.2 Fluorescence spectrophotometer.
4.3 Maizel-Gerson reaction bottle: as shown in Figure 1. 611
GB/T5009.84—-2003
4.4 Base exchange tube: as shown in Figure 2.
Figure 1 Maizel-Gerson reaction bottle
5 Analysis steps
5.1 Sample preparation
5.1.1 Sample preparation
Liquid storage tank
Exchange column
Capillary
Figure 2 Base exchange tube
After the sample is collected, use a homogenizer to make a homogenate and store it in a low-temperature refrigerator. Thaw it and mix it before use. The dried sample should be crushed as much as possible and then used.
5.1.2 Extraction
5.1.2.1 Accurately weigh a certain amount of sample (estimated thiamine content is about 10μg~30μg, generally weigh 2g~10g sample), place in a 100mL conical flask, add 50mL 0.1mol/L or 0.3mol/L hydrochloric acid to dissolve it, place in a pressure cooker and heat to hydrolyze at 121℃ for 30 min, and take out after cooling.
5.1.2.2 Adjust the pH value to 4.5 with 2mol/L sodium acetate (with 0.4g/L bromocresol green as external indicator). 5.1.2.3 Add amylase and protease at a ratio of 20mg amylase and 40mg protease per gram of sample. Keep warm in a 45℃~50℃ incubator overnight (about 16h).
5.1.2.4 Cool to room temperature, make up to 100mL, then mix and filter to obtain the extract. 5.1.3 Purification
5.1.3.1 Spread a little absorbent cotton on the bottom of the exchange column of the base exchange tube, add water to remove the bubbles in the cotton fiber, and then add about 1g of active artificial pumice to make it reach one-third of the height of the exchange column. Keep the liquid level in the base exchange tube always higher than the active artificial pumice. 5.1.3.2 Use a pipette to add 20mL~60mL of extract (so that the total amount of thiamine passing through the active artificial pumice is about 2μ~5μg). 5.1.3.3 Add about 10mL of hot distilled water to rinse the exchange column and discard the washing liquid. Repeat this three times. Add 20mL of 250g/L acidic potassium chloride (temperature is about 90℃), collect this solution in a 25mL graduated test tube, cool to room temperature, and dilute to 25mL with 250g/L acidic potassium chloride, which is the sample purification solution. 5.1.3.5 Repeat the above operation, add 20mL of thiamine standard working solution into the base exchange tube to replace the sample extraction solution, and obtain the standard purification solution.
5.1.4 Oxidation
Add 5mL of sample purification solution into the two reaction bottles A and B respectively. 5.1.4.2 Add 3mL 150g/l sodium hydroxide to reaction bottle A and 3mL alkaline potassium ferrocyanide solution to reaction bottle B under light-proof conditions, shake for about 15s, then add 10mL n-butanol; shake both reaction bottles A and B vigorously for 1.5min. 5.1.4.3 Repeat the above operation, replacing the sample purification solution with the standard purification solution. 5.1.4.4 After standing and stratification, remove the lower alkaline solution and add 2g to 3g anhydrous sodium sulfate to dehydrate the solution. 5.2 Determination
5.2.1 Fluorescence measurement conditions:
excitation wavelength 365nm; emission wavelength 435nm; excitation wave slit 5nm, emission wave slit 5nm. 612
: Sequentially measure the following fluorescence intensities:
sample blank fluorescence intensity (sample reaction bottle A); a)
standard blank fluorescence intensity (standard reaction bottle A); c)
sample fluorescence intensity (sample reaction bottle B); standard fluorescence intensity (standard reaction bottle B). 6 Calculation of results
X-(UU)X
Wherein:
Thiamine content in the sample, in milligrams per hundred grams (mg/100g); fluorescence intensity of the sample;
fluorescence intensity of the blank sample;
fluorescence intensity of the standard;
fluorescence intensity of the standard blank;
concentration of the standard working solution of thiamine, in micrograms per milliliter (μg/mL); volume of the standard working solution of thiamine used for purification, in milliliters (mL); volume of the sample after hydrolysis, in milliliters (mL); - volume of the extract used for purification of the sample, in milliliters (mL); mass of the sample, in grams (g);
coefficient for converting the sample content from micrograms per gram (μg/g) to milligrams per hundred grams (mg/100g). The calculation result should retain two significant figures.
7 Precision
GB/T5009.84—2003
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean. 613
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