GB/T 5009.189-2003 Determination of fumonisic acid in Tremella fuciformis
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ICS67.040bZxz.net
National Standard of the People's Republic of China
GB/T5009.189—2003
Partially replaces GB11675-1989
Determination of bongkrekic acid in tremella fuciformis berk
Promulgated on August 11, 2003
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
Implemented on January 1, 2004
This standard replaces the determination of bongkrekic acid in 5.1 of GB11675—1989 "Sanitary Standard of Tremella fuciformis". This standard is proposed and managed by the Ministry of Health of the People's Republic of China. GB/T5009.189—2003
This standard was drafted by Henan Food Hygiene Supervision and Inspection Institute and Nanyang District Health and Epidemic Prevention Station of Henan Province. The main drafters of this standard are Ren Zhongshan, Ma Jun, Zhao Guoqing, Zhang Ding and Wei Xiting. 501
GB/T5009.189—2003
Bongkreki acid (BA), also known as flavotoxin A, is a toxin produced by Pseudomonas cocovenenans that can cause food poisoning. The system name is 3-carboxymethyl-17-methoxy-618,21-trimethyl-docosahexa-2,4,8,12,14,18,20-heptaenedioic acid. 502
1 Scope
Determination of fumonisic acid in Tremella
This standard specifies the determination method of fumonisic acid in Tremella. This standard is applicable to the determination of fumonisic acid in Tremella. Thin layer chromatography method
2 Principle
GB/T5009.189—2003
After the fumonisic acid in the sample is extracted, purified and concentrated, the content is determined according to the minimum detection amount of the color point displayed on the short-wave ultraviolet light GF254 silica gel thin layer chromatography.
3 Instrument
3.1 Chromatography tank (inner diameter length × width × height, 25cm×6cm×4cm). 3.2 GF2s silica gel thin layer 50mm×200mm×0.3mm), homemade, activated at 110℃~115℃ for 2h~3h, placed in a desiccator and can be stored for one week.
3.3 Ultraviolet lamp (wavelength 254nm).
3.4 Ultraviolet spectrophotometer.
4 Reagents
Unless otherwise specified, the reagents used in this standard are all analytically pure. Water is distilled water or water of equivalent purity, and the solution is an aqueous solution. 4.1 Silica gel: GF254 for chromatography.
4.2 Thin layer chromatography developing solvent: petroleum ether-anhydrous ether-glacial acetic acid [60+40+1.5]. 4.3 Fumaric acid standard solution: Weigh the fumaric acid standard, prepare it with methanol to contain 20pg of fumaric acid per ml for determination, and store it in a refrigerator at 4℃ away from light.
4.4 Methanol.
4.5 Glacial acetic acid.
4.6 Petroleum ether, boiling range 30℃～60℃.
4.7 Anhydrous ether.
Triazine.
85g/100mL phosphoric acid.
40g/L sodium bicarbonate aqueous solution.
4.116mol/L hydrochloric acid.
5 Analysis steps
5.1 Extraction of fumonisic acid
After the dried Tremella sample is crushed and passed through a 40-mesh sieve, 20g is weighed and placed in a stoppered conical flask, 20mL of methanol is added, and it is soaked at room temperature in the dark for 1h, then 80mL of trichloromethane and 0.2mL of 85g/100mL phosphoric acid are added. After the fresh Tremella sample is cut, ground and mixed, 10g is weighed, 16mL of methanol is added, and it is soaked at room temperature in the dark for 1h, then 64mL of chloroform and 0.16mL of 85g/100ml. phosphoric acid are added; shake for 30min, filter, and take 50mL of filtrate from the dried Tremella and 40mL of filtrate from the fresh Tremella. 503
GB/T5009.189-2003
Place the above filtrate into a separatory funnel, add an aqueous sodium bicarbonate solution of the same volume as the filtrate, shake for 2 minutes, let stand to separate, use a pipette with an automatic control ball to suck out the upper layer into another separatory funnel, repeat the extraction twice with an aqueous sodium bicarbonate solution, 10 mL each time, shake gently, let stand, combine the three sodium bicarbonate water layers, add 25 ml of trichloromethane, shake for 2 minutes, let stand to separate, discard the trichloromethane layer, slowly drip 6 mol/L hydrochloric acid into the separatory funnel to adjust the pH of the solution to 2-3, add petroleum ether (boiling range 30℃~60℃) 50mL (add 40mL to fresh Tremella sample), shake for 3min, let stand to separate, take out the petroleum ether layer and put it into a pear-shaped bottle, repeat the extraction with 30mL and 20mL of petroleum ether respectively (20mL for fresh Tremella sample twice), put the petroleum ether layers into the same bottle, concentrate to dryness in a 40℃ water bath with reduced pressure blowing, transfer to a concentrator tube with 1mL scale with methanol, concentrate to less than 0.2mL at 40℃ with reduced pressure method, wash the tube wall with a little methanol, continue to concentrate to dryness, add 0.125mL of methanol, dissolve the dry matter, mix well, and use as the sample solution for thin layer chromatography determination. 5.2 Thin layer chromatography determination
Use the short side of the thin layer plate as the bottom side, and use a micro syringe to drop 8μL and 10μL of 20ug/ml standard solution at two points on the baseline 3cm away from the bottom side, and two points of sample solution, 10μL per point (add 20μL to fresh Tremella sample), and then drop 10μL of 20μg/mL standard solution at one point of the sample solution. Use a developing agent to develop 16cm, and take it out and evaporate it. The results observed under 254nm ultraviolet light are as follows: a) A black dot should appear at the standard point;
b) If no bright-colored dot appears at the corresponding position of the sample solution, the content of fumonisin in the sample is below the sensitivity of the determination method of 0.25pg/g; if there is a bright-colored dot at the corresponding position, and the sample solution overlaps with the standard solution dot at another point, it is positive. According to the intensity of the black dot of the sample solution, reduce the number of microliters to be added, or add different numbers of microliters after dilution until the intensity and area of the sample solution and the standard color dot are consistent. The transfer value of fumonisin is 0.22. 5.3 Result calculation
See formula (1).
X = 0.2 >
Wherein:
X- content of fumonisin, in micrograms per gram (ug/g): 0.2- minimum detectable amount of fumonisin, in micrograms (μg); V, the volume of methanol solution added, in milliliters (mL); V2-the volume of sample solution added when the minimum detectable amount appears, in milliliters (mL); D-the total dilution multiple of the sample solution;
-the mass of the equivalent sample when dissolved in methanol, in grams (g). 5.4 Confirmation test
For samples with high content, further confirmation test can be carried out. After the remaining positive sample solution and the blank methanol solution are separated by thin layer, the chromatographic band corresponding to the fumonisin standard and the blank silica gel are scraped and collected. Add 4 mL of methanol to each, soak at room temperature for 1 to 2 hours, mix well, centrifuge, aspirate the supernatant, use blank silica gel methanol eluent as a control, and measure with a UV spectrophotometer. There should be two highest absorption peaks of fumonisin at 267 nm and 236 nm.
High-pressure liquid chromatography determination method
.6 Principle
After the fumonisin in the sample is extracted, purified and concentrated, the content is determined according to the peak area on the high-pressure liquid chromatography. 7 Reagents
7.1 High-pressure liquid chromatography eluent: methanol-water-glacial acetic acid [75+27+1.7]. Before preparation, all reagents and water used must be re-steamed separately. 7.2 Other reagents are the same as 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 4.10, 4.11. 504
8 Apparatus
8.1 High pressure liquid chromatograph.
8.2 20μL sample loop.
8.3 UV detector, wavelength adjusted to 267nm. 8.4 Integrator.
8.5 Precolumn 4.6mm (ID) × 5cm, filled with Cis silica gel, particle size diameter of 10μm. GB/T5009.189—2003
8.6 Analytical column AltexUItrasphereTM-ODS, particle size diameter of 5μm, 4.6mm (ID) X25cm. 9 Analysis steps
9.1 Extraction of fumonisin
Same as 5.1, but without using methanol for transfer at the end, add 0.5 mL of methanol directly into the bottle to dissolve the dry matter, mix, take out the supernatant, centrifuge it, put it into a small test tube, and use it as the sample solution for high-pressure liquid chromatography determination. 9.2 High-pressure liquid chromatography determination
High-pressure liquid chromatography operating conditions: flow rate 1.1 mL/min, paper speed 0.5cm/min, the sensitivity of the detector is to adjust 20μL of 10μg/mL standard solution to 70% to 90% of the full scale. When doing high pressure liquid chromatography, first use the eluent to balance the analytical column, and then enter 20μL of standard solution and sample solution of different concentrations from the injection port device. Within the linear range of the standard peak area of fumonisin, compare the peak area of the sample solution with that of the standard to calculate the content of fumonisin in the sample. The retention time of fumonisin is 17min. 9.3 Result calculation
See formula (2).
Wherein:
×VxDx1
X--the content of fumonisin, in micrograms per gram (μg/g): m
-the concentration of fumonisin standard solution, in micrograms per milliliter (ug/mL); A,-the peak area of the sample solution
A-the peak area of fumonisin standard solution; V
-the volume of methanol solution, in milliliters (mL); D the total dilution multiple of the sample solution;
9.4 Confirmation
The mass of the equivalent sample when dissolved in methanol, in grams (g). If the sample is positive, it is also necessary to confirm it by the method of overlapping the sample solution and the standard solution in thin layer chromatography. (2)
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