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This standard replaces "Determination of Benzo(a)pyrene in Foods" (GB/T 5009.27-2003), "Animal and Vegetable Fats and Oils - Determination of Benzo(a)pyrene - Reverse-phase High Performance Liquid Chromatography Method" (GB/T 22509-2008), "Determination of Benzo(a)pyrene in Aquatic Products - High Performance Liquid Chromatography" (SC/T 3041-2008) and "Determination of Benzo[a]pyrene in Meat Products - High Performance Liquid Chromatography Method" (NY/T 1666-2008).
Compared with the above standards, this standard has the following main changes:
——The standard name is modified to "National Food Safety Standard-determination of benzo(a)pyrene in foods";
—— The application scope of methods is modified;
——The pretreatment procedure of sample is modified;
——The fluorescence spectrophotometry and visual colorimetry are deleted.
National Food Safety Standard - Determination of Benzo(a)Pyrene in Foods
1 Scope
This standard specifies the method for determination of benzo(a)pyrene in foods.
This standard is applicable to determination of benzo(a)pyrene in grains and their products (rough rice, brown rice, rice, wheat, wheat flour, corn, corn meal, corn pulp and corn flakes), meat and meat products (smoked, roasted and broiled meat), aquatic animals and their products (Smoked and broiled aquatic products), fats and oils and their products.
2 Principle
The specimens are extracted by organic solvent, purified by neutral alumina or molecular engram columella, concentrated to be dry, dissolved by acetonitrile, separated by reversed-phase liquid chromatography, inspected by fluorescence detector, and quantified by external standard method (with the nature being determined according to remain time of chromatographic peak).
3 Reagents and Materials
Unless otherwise specified, analytically pure reagents and Grade 1 water specified in GB/T 6682 are used in this method.
3.1 Reagents
3.1.1 Methyl benzene (C7H8): chromatographically pure.
3.1.2 Acetonitrile (CH3CN): chromatographically pure.
3.1.3 N-hexane (C6H14): chromatographically pure.
3.1.4 Dichloromethane (CH2Cl2): chromatographically pure.
3.2 Standard
Benzo(a)pyrene standard (C20H12, CAS No.: 50-32-8): standard substance with purity of 99.99% or a reference material certificate authorized and granted by the country.
Warning——benzo(a) pyrene is a kind of known carcinogenic substance, so the safety protection shall be paid special attention to during determination. The determination shall be carried out in the fuming cupboard: wear gloves and try to reduce exposure. In case that skin is already polluted, it shall be soaked and scrubbed by 10% aqueous sodium hypochlorite solution; observe the skin for bluish violet spot under UV-light and, if any, wash the spot till it disappears.
3.3 Preparation of standard solution
3.3.1 Benzo(a)pyrene standard stock solution (100 μg/mL): accurately weigh 1mg (accurate to 0.01mg) of benzo(a)pyrene and place at 10mL-volumetric flask, dissolve with methylbenzene and scale the volume. Protect from light at 0℃~5℃ refrigerator for 1 year.
3.3.2 Benzo(a)pyrene standard intermediate solution (1.0μg/mL): suck 0.10mL of benzo(a)pyrene standard stock solution (100μg/mL) and scale the volume to 10mL with acetonitrile. Protect from light at 0℃~5℃ refrigerator for 1 month.
3.3.3 Benzo(a)pyrene standard working solution: dilute the benzo(a)pyrene standard intermediate solution (1.0μg/mL) with acetonitrile to obtain calibration curve solution with concentration of 0.5 ng/mL, 1.0 ng/mL, 5.0 ng/mL, 10.0 ng/mL and 20.0 ng/mL; prepare it immediately before use.
3.4 Materials
3.4.1 Neutral alumina column: particle size of filler 75μm~150μm, 22g, 60mL.
Note: moisture in the air significantly affects property of neutral alumina column, thus it shall be immediately used or sealed and protected from light once its packaging is opened. Due to the difference in the activity of different brands of aluminum oxide, the control sample is recommended to be tested or subject to standard addition recovery test to verify if its activity meets the requirements of recovery rate.
3.4.2 Benzo(a)pyrene molecular engram column: 500 mg, 6mL.
Note: Due to the difference in the quality of different molecular engram columns, the quality control sample is recommended to be tested or subject to standard addition recovery test to verify if it meets the requirements.
3.4.3 Microfiltration membrane: 0.45μm.
4 Apparatus and Equipment
4.1 Liquid chromatograph: with fluorescence detector.
4.2 Analytical balance: with sensibility of 0.01mg and 1mg respectively.
4.3 Grinder.
4.4 Tissue homogenizer.
4.5 Centrifuger: rotation speed≥4,000r/min.
4.6 Vortex oscillator.
4.7 Ultrasonic vibrator.
4.8 Rotary evaporator and nitrogen blower.
4.9 Solid-phase extraction device.
5 Analysis Procedure
5.1 Sample preparation, extraction and purification
5.1.1 Grains and their products
Pretreatment: remove impurity, grind to uniform sample, store in clean sample bottle and indicate with mark, and preserve for standby use under room temperature or according to preservation conditions required by product package.
Extraction: weigh 1g (accurate to 0.001g) of sample, add into 5mL of n-hexane, conduct 0.5min of vortex mixing, 10 min of ultrasonic extraction under 40℃ and 5min of centrifugation(4000 r/min), and transfer supernatant. Add into 5mL n-hexane again and extract it once again. Combine the supernatant and purify it by either of the following 2 methods.
Purification method 1: neutral alumina column: activate it with 30mL of n-hexane, close bottom cock when the liquid level declines to column bed. Transfer to-be-purified solution to the column, open the cock, collect purified solution to eggplant-shaped bottle at the rate of 1mL/min, transfer into 50mL n-hexane and elute, and continue to collect purified solution. Rotate and evaporate the purified solution under 40℃ to about 1mL, transfer to chromatograph sample injection bottle, and concentrate to be nearly dry under 40℃ nitrogen flow. Clean eggplant-shaped bottle with 1mL of n-hexane, and re-transfer washing solution to chromatograph sample injection bottle and concentrate to be nearly dry. Accurately pipet 1mL of acetonitrile to the sample injection bottle, conduct 0.5 min of vortex re-dissolution for determination of liquid chromatogram after passing through microporous membrane.
Purification method 2: benzo(a)pyrene molecular engram column: activate it with 5mL of methylene chloride and 5mL of n-hexane successively. Transfer to-be-purified solution to the column and, when the liquid level declines to column bed, elute it with 6mL of n-hexane and discard effluent. Elute with 6mL of methylene chloride, and collect the purified solution to test tube. Blow the purified solution with nitrogen under 40℃; accurately pipet 1mL of acetonitrile to conduct 0.5min of vortex re-dissolution for determination of liquid chromatogram after passing through microporous membrane.
5.1.2 Smoked, roasted and broiled meat and smoked and broiled aquatic products
Pretreatment: remove bone for meat and fish, and shell for shellfish; grind edible part uniformly, store at clean sample bottle, indicate with mark and preserve for standby use at -16℃~-18℃ refrigerator.
Foreword i
1 Scope
2 Principle
3 Reagents and Materials
4 Apparatus and Equipment
5 Analysis Procedure
6 Expression of Analysis Results
7 Precision
8 Others
Appendix A Liquid chromatogram for Benzo(a)pyrene Standard Solution
