GB/T 5009.158-2003 Determination of vitamin K1 in vegetables

This standard specifies the determination method of vitamin K1 in vegetables. This standard is applicable to the determination of vitamin K1 in various vegetables, green plants and their dried products. The limit of this standard method is 0.5Kg, and the linear range is 1Kg/mL-100Kg/mL. GB/T 5009.158-2003 Determination of vitamin K1 in vegetables GB/T5009.158-2003 Standard download decompression password: www.bzxz.net

ICS67.040
National Standard of the People's Republic of China
GB/T5009.158—2003
Determination of vitamin K, in vegetables
Determination of vitamin K, in vegetables2003-08-11 Issued
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
Implementation on 2004-01-01
GB/T5009.158-2003
This standard corresponds to the determination of vitamin D in foods in the vitamins and nutrients section of AOAC.45 (English version of 1995). The consistency between this standard and AOAC.45 is non-equivalent. This standard is proposed and managed by the Ministry of Health of the People's Republic of China. The drafting unit of this standard is: Institute of Nutrition and Food Hygiene, Chinese Academy of Preventive Medicine. The main drafters of this standard are: Wang Zhu, Wang Guangya, Zhou Ruihua, Wang Guodong, and Yang Yuexin. 321
GB/T5009.158—2003
This standard refers to the pretreatment process of AOAC.45 Vitamins and Nutrients Determination of Vitamin D in Food (1995 Edition), and uses phosphate-treated alumina as the chromatographic column and high performance liquid chromatography reverse phase column to conduct qualitative and quantitative analysis of vitamin K in vegetables. 322
1 Scope
Determination of Vitamin K in Vegetables
This standard specifies the determination method of vitamin K in vegetables. This standard is applicable to the determination of vitamin K in various vegetables, green plants and their dried products. The limit of this standard method is 0.5μg, and the linear range is 1μg/mL～100μg/mL. 2 Principle
GB/T5009.158-2003
Vitamin K in vegetables is extracted with petroleum ether and injected into alumina chromatographic columns treated with phosphate for chromatographic separation to remove interferences. Collect the eluent fraction containing vitamin K, concentrate and fix the volume, then inject it into a high-performance liquid chromatography column and measure it at 248nm using a UV detector. Calculate the content of vitamin K in the sample by the external standard method. 3 Reagents and materials
Chromatographic water and organic solvents must be redistilled before use. 3.1 Anhydrous sodium sulfate: Before use, it must be baked in an oven at 150℃ for 4h~8h to remove moisture. 3.2 0.14mol/1. Sodium sulfate solution: Weigh 20g of anhydrous sodium sulfate, dissolve it in distilled water and fix the volume to 1L. 3.3 Propylene glycol.
3.4 ​​Petroleum ether: boiling range 30℃~60℃.
3.5 Ether: does not contain peroxide.
3.5.1 Method for checking peroxide: add 1mL 0.6mol/L potassium iodide solution to 5mL ether, shake for 1min, if there is peroxide, free iodine will be released, and the water layer will be yellow. Or add 4 drops of 5g/L starch solution, the water layer will be blue. Then the ether contains peroxide and needs to be treated before use.
3.5.2 Method for removing peroxide: add a piece of pure iron wire to the bottle during re-distillation, discard the first and last 10% part, and collect the distilled ether. Check the peroxide again, it should meet the requirements.
3.6 Eluent: petroleum ether + ether (97+3). 3.7 Methanol: high-grade pure.
3.8 n-hexane: high-grade pure.
3.9 Neutral alumina: for chromatography, 100 mesh to 200 mesh. 3.9.1 Treatment of alumina: Take 250g of neutral alumina, 20g of disodium hydrogen phosphate, and 1.6L of water, put them into a 2L conical flask, place it in a boiling water bath for 30min, and shake it from time to time. Cool, pour off the upper liquid (including suspended fine particles), and then filter it with a Buchner funnel. Transfer the residue to a flat-bottomed glass dish and bake it in a 150℃ drying oven for 3h~5h until the difference between two weighings is less than 3g. Stir from time to time during the baking process to avoid caking, and store it in a desiccator after cooling. 3.9.2 Deactivation: Add the phosphate-treated alumina (3.9.1) into a stoppered conical flask. Add deionized water at a ratio of 9.0mL water per 100g of alumina, cover the bottle tightly, heat it in a steam bath or 80℃ drying oven for 3min~5min, and shake the conical flask vigorously so that the alumina can flow freely without caking. Cool and let it stand for 30min to make the water distribution even. 3.9.3 Inspection of aluminum oxide: Take 1.0 mL of the standard working solution, blow dry with nitrogen (3.11), and then dissolve and dilute to 1.0 mL with petroleum ether (3.4). Then install the column according to 5.2.2.1, add the standard solution to the column, and perform column chromatography according to the chromatographic separation steps in 5.2.2.2. Collect the eluted fraction containing vitamin K in a rotary evaporator (4.5.1), concentrate, blow dry with nitrogen, and dilute to 1.0 mL with n-hexane (3.8). Inject, measure by HPLC, and record the peak area or peak height (A). Take another standard application solution and measure directly, and record the peak area or peak height (A,). Compare A with A. If the value is between 0.97 and 1.03 (A/A,), it means that the column efficiency is good and the aluminum oxide treatment is qualified. Otherwise, it needs to be deactivated again. If the ratio cannot reach 0.97 after verification again, alumina needs to be prepared again. 3.10 Vitamin K, (purity>98%) standard solution. 3.10.1 Vitamin K, standard stock solution: Accurately weigh 50.0 mg of pure vitamin K, put it in a 50 mL brown volumetric flask, dissolve it with n-hexane and dilute it to the mark, that is, the concentration of the stock solution is 1.0 mg/mL. Divide the stock solution into bottles and store it in the freezer. 3.10.2 Vitamin K, standard working solution: Accurately pipette vitamin K, standard stock solution (3.10.1), accurately dilute it with n-hexane at a ratio of 1:50, that is, the concentration of the vitamin K standard working solution is 20 μg/mL. 3.10.3 Calibration of standard working solution: Take vitamin K, standard working solution, measure the ultraviolet absorbance value at a wavelength of 248 nm, the thickness of the cuvette is 1 cm, use n-hexane as a blank, measure 3 times, take the average value, and calculate the concentration of the standard working solution according to formula (1). X
×m×10%
Wherein:
—Vitamin K, standard working solution concentration, unit is microgram per milliliter (μg/mL); X-
A-—average ultraviolet absorbance value of standard working solution; E——molar extinction coefficient 20000;
m——Vitamin K, molecular weight 450.7;
10——conversion factor from grams per liter (g/L) to micrograms per milliliter (μg/mL). 3.11 High-purity nitrogen 99.9%.
4 Instruments and equipment
4.1 Common laboratory equipment.
4.2 Crusher.
4.3 Constant temperature drying oven.
4.4 Chromatographic column, a 0.8cm×30cm glass column. The bottom end shrinks and becomes thinner, equipped with a piston. There is a glass screen plate about 1 cm above the piston, and the pore size of the screen plate is 16μm~30μm. The upper end of the column expands into a liquid storage tank with a volume of about 30mL. It needs to be dried before use. 4.5 Rotary evaporator.
Rotary evaporation bottle matched with rotary evaporator: with stopper, round bottom, and a volume of 150mL. 4.6 Constant temperature water bath.
4.7 High-speed centrifuge.
Small centrifuge tube matched with high-speed centrifuge: with stopper, and a volume of 1.5mL~3.0mL. 4.8 Ultraviolet spectrophotometer.
4.9 High-performance liquid chromatograph with ultraviolet detector. Analytical steps (all operations must be performed in the dark) 5
5.1 Sample processing
5.1.1 Fresh sample: remove debris, wash the edible part, wipe off the surface moisture, chop, and prepare a homogenate with a crusher. Store in the dark and low temperature.
5.1.2 Dry sample, grind, pass 60-day sieve, mix, and store in the dark and low temperature. 5.2 Sample pretreatment
5.2.1 Extraction
5.2.1.1 Fresh sample Accurately weigh 2.000g10.000g (vitamin K, content not less than 2μg), add to a stoppered conical flask, add 510 times the volume of acetone (3.3), cover the stopper, shake for 3min~5min, and let it stand for 1min. The following operations are performed according to 5.2.1.3. 5.2.1.2 Drying test sample: Weigh 0.200g~4.000g (vitamin K, content not less than 2g), add to mortar, add 2~4 times amount of anhydrous sodium sulfate, grind evenly, transfer to conical flask, add 25mL acetone, shake for 3min~5min, and let stand for 1min. 324
GB/T5009.158-2003
5.2.1.3 Transfer the supernatant to a separatory funnel containing 50mL 0.14mol/L sodium sulfate solution, wash the residue with 25ml acetone for 2~3 times, and add the supernatant to the separatory funnel. Continue to use 25mL petroleum ether (3.4) Wash the conical flask 3 to 4 times until it is colorless: and put it into the same separatory funnel. Shake for 1 minute, let it stand and separate. Discard the lower aqueous phase, and repeatedly wash the organic phase with 0.14 mol/L sodium sulfate solution until the aqueous phase is clear. Pass the organic phase through a funnel filled with anhydrous sodium sulfate, filter it into a rotary evaporator, wash the separatory funnel and the anhydrous sodium sulfate layer with petroleum ether, and put the washing liquid into the rotary evaporator. 5.2.1.4 Concentration and volume adjustment: Evaporate the above extract on a rotary evaporator under reduced pressure, with a water bath temperature of 80°C, evaporate to about 1 mL, remove the rotary evaporator and blow dry with nitrogen, add 2.0 mL of petroleum ether to dissolve and adjust the volume. Prepare for column chromatography. 5.2.2 Column chromatography
5.2.2.1 Column filling: Take a dry chromatographic column, soak the verified alumina in petroleum ether, wet fill the chromatographic column, let the alumina flow freely and evenly, until the column height is 20cm, add 1cm of anhydrous sodium sulfate at the top. Open the piston and adjust the flow rate to 1 drop per second. 5.2.2.2 Chromatographic separation: When the petroleum ether flows to 0.5cm above the column, add 1ml of sample extract, and when the extract flows to the same level as the top of the column, add 2mL of petroleum ether to rinse the column wall. Add 10mL of petroleum ether twice, wash, and discard the effluent. Then, elute with 30mL of eluent and collect the effluent in a rotary evaporator. 5.2.2.3 Concentration and volume adjustment: Concentrate the above effluent to nearly dryness in a rotary evaporator, remove it and blow it dry with nitrogen, and then adjust the volume to 2mL with n-hexane. bzxz.net
5.3 Determination
Place the fixed volume solution into a small plastic centrifuge tube, centrifuge for 5 minutes (5000r/min), and the supernatant is used for HPLC analysis. 5.3.1 High performance liquid chromatography analysis conditions (reference conditions) Pre-column ultrasphereODS, particle size 10μm, specification 4.0mm×45mm. Analytical column ultrasphereODS, particle size 5μm, specification 4.6mm×250mm. Mobile phase methanol + n-hexane (98+2).
Flow rate 1.5mL/min.
Injection volume 20μL.
Detector UV detector, wavelength 248nm, range 0.01~0.05. 5.3.2 Quantitative determination
5.3.2.1 Instrument stability determination: Adjust the instrument operating parameters and sensitivity (AUFS) according to the manual of the high performance liquid chromatograph. For accurate determination, perform system adaptability test on the analytical column as required. Take the standard working solution and inject it according to the analytical conditions for measurement for 6 times in a row, and calculate the average value, standard deviation and relative standard deviation (RSD) of the peak area or peak height. If RSD < 1%, it means that the instrument is stable and the sample can be measured.
5.3.2.2 Standard curve: Take 0.50, 1.00, 2.00, 4.00, 6.00, 8.00 10.00 ml of vitamin K standard working solution respectively, add them to the separatory funnel, process according to the sample extraction and column chromatography steps, and finally concentrate and make the volume to 1.0 ml, that is, the vitamin K content at each point in the standard working curve is equivalent to 5.0, 10.0, 20.0, 40.0, 60.0, 80.0, 100.0 μg/mL respectively. Measure according to the analytical conditions of 5.3.1 and record the peak area or peak height. Draw a standard curve with the vitamin K content as the horizontal axis and the peak area or peak height as the vertical axis. 5.3.2.3 Sample determination: Take the sample concentrate (5.2.2.3) and inject it for determination according to the analytical conditions of 5.3.1. 6 Result calculation: The content of vitamin K in the sample is calculated according to formula (2). X = cXViXVX100
Wherein:
X--the content of vitamin K in the sample, in micrograms per hundred grams (μg/100g); c--the result of vitamin K found on the standard curve or obtained by regression equation, in micrograms per milliliter (μg/mL); V--the volume of the sample after extraction, in milliliters (mL); · (2)
GB/T5009.158-2003
Vz--the amount of liquid taken when the sample column is chromatographically separated, in milliliters (mL); V3--the volume of the sample after chromatographic separation, in milliliters (mL); m--the mass of the sample, in grams (g)).
The calculation result shall retain three significant figures.
7 Precision
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean. 326
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