1 Scope
This standard specifies the determination method of aflatoxin M1 and aflatoxin M2 (hereinafter referred to as AFT M1 and AFT M2) in foods.
Method I is isotope dilution-liquid chromatography-tandem mass spectrometry, it is applicable to the determination of AFT M1 and AFT M2 in milk, milk products and milk-containing foods for special dietary supplies.
Method II is high performance liquid chromatography, its application scope is the same with Method I.
Method III is enzyme linked immunosorbent assay screening method, it is applicable to the screening determination of AFT M1 in milk, milk products and milk-containing foods for special dietary supplies.
Method I Isotope Dilution-Liquid Chromatography-Tandem Mass Spectrometry
2 Principle
After AFT M1 and AFT M2 in the sample extracted by methanol-water solution and dilute the supernatant with water or phosphate buffer solution, purified and enriched by immunoaffinity column, scale the volume and filter, the filtrate can be separated by liquid chromatography, tested by tandem mass spectrometry and quantified by isotope internal standard method.
3 Reagents and Materials
Unless otherwise specified, reagents used in this method are all analytically pure, and water is the Grade 1 water as specified in GB/T 6682.
3.1 Reagents
3.1.1 Acetonitrile (CH3CN): chromatographically pure.
3.1.2 Methanol (CH3OH): chromatographically pure.
3.1.3 Ammonium acetate (CH3COONH4).
3.1.4 Sodium chloride (NaCl).
3.1.5 Disodium hydrogen phosphate (Na2HPO4).
3.1.6 Sodium dihydrogen phosphate (KH2PO4).
3.1.7 Potassium chloride (KCl).
3.1.8 Hydrochloric acid (HCl).
3.1.9 Petroleum ether (CnH2n+2): boiling range of petroleum ether is 30℃ to 60℃.
3.2 Reagent preparation
3.2.1 Ammonium acetate solution (5mmol/L): weigh 0.39g ammonium acetate and dissolve it in 1000mL water, mix well.
3.2.2 Acetonitrile-water solution (25+75): measure 250mL acetonitrile and add it into 750mL water, mix well.
3.2.3 Acetonitrile-methanol solution (50+50): measure 500mL acetonitrile and add it into 500mL methanol, mix well.
3.2.4 Phosphate buffer solution (hereinafter referred to as PBS): weigh 8.00g sodium chloride, 1.20g disodium hydrogen phosphate (or 2.92g sodium acid phosphate dodecahydrate), 0.20g potassium dihydrogen phosphate and 0.20g potassium chloride, dissolve in 900mL water, adjust the pH value to 7.4 with hydrochloric acid, and dilute to 1000mL with water.
3.3 Standards
3.3.1 AFT M1 standard (C17H12O7, CAS: 6795-23-9): purity ≥98%, or other standard reference materials approved and awarded with reference material certificate by the nation.
3.3.2 AFT M2 standard (C17H14O7, CAS: 6885-57-0): purity ≥98%, or other standard reference materials approved and awarded with reference material certificate by the nation.
3.3.3 13C17-AFT M1 isotope solution (C17H14O7): 0.5μg/mL
3.4 Preparation of standard solution
3.4.1 Standard stock solution (10μg/mL): respectively weigh 1mg (to the nearest 0.01mg) of AFT M1 and AFT M2 , respectively dissolve in acetonitrile and scale the volume to 100mL. Transfer this solution to brown reagent bottle, sealed and preserved in a dark place. Perform concentration calibration prior to use (see Appendix A for calibration method).
3.4.2 Mixed standard stock solution (1.0μg/mL): Accurately pipet respectively 1.00mL AFT M1 and AFT M2 standard stock solution (10μg/mL) in a 10mL volumetric flask, dilute to the scale with acetonitrile to prepare 1.0μg/mL mixed standard solution. This solution shall be sealed and preserved in a dark place at 4℃, the validity period is 3 months.
3.4.3 Mixed standard working solution (100ng/mL): Accurately pipet 1.00mL mixed standard stock solution (1.0μg/mL) and transfer to a 10mL volumetric flask, scale the volume with acetonitrile. This solution shall be sealed and preserved in a dark place at 4℃, the validity period is 3 months.
3.4.4 50ng/mL isotope internal standard working solution 1 (13C17-AFT M1): measure 1mL AFT M1 isotope internal standard (0.5μg/mL) and dilute to 10mL with acetonitrile. It shall be preserved at -20℃ and to be used for the determination of liquid sample. Validity period is 3 months.
3.4.5 5ng/mL isotope internal standard working solution 2 (13C17-AFT M1): measure 100μL AFT M1 isotope internal standard (0.5μg/mL) and dilute to 10mL with acetonitrile.It shall be preserved at -20℃ and to be used for the determination of solid sample. Validity period is 3 months.
3.4.6 Standard series working solution: Accurately transfer respectively 5μL, 10μL, 50μL, 100μL, 200μL and 500μL standard working solution into 10mL volumetric flask, add 100μL isotope internal standard working solution (50ng/mL), scale the volume to the scale with initial mobile phase to prepare standard solution series with the AFT M1 and AFT M2 concentration of 0.05ng/mL, 0.1ng/mL, 0.5ng/mL, 1.0ng/mL, 2.0ng/mL, and 5.0ng/mL.
4 Apparatus and Equipment
4.1 Balance: the sensibility is 0.01g, 0.001g and 0.00001g.
4.2 Water bath: temperature control at 50℃±2℃.
4.3 Vortex mixer.
4.4 Ultrasonic cleaner.
4.5 Centrifuge: ≥6000 r/min.
4.6 Rotary evaporator.
4.7 Solid-phase extraction device (with vacuum pump).
4.8 Nitrogen blower.
4.9 LC-MS-MS: equipped with ESI source.
4.10 Circular screen: in pore diameter of 1mm to 2mm.
4.11 Glass fibre filter: fast, high load, particles under 1.6μm are reserved in the liquid.
4.12 Disposable microporous filter head: with 0.22μm microporous filter membrane (the selected filter membrane shall be tested with standard solution, and may be used after confirm that no adsorption phenomenon).
4.13 Immunoaffinity column: column capacity ≥100ng (see Appendix B for column capacity, recovery rate and verification method of column recovery rate).
Note: The quality verification of each batch of affinity columns shall be carried out prior to use.
5 Analysis Procedure
The application of immunoaffinity columns from different manufacturers may be slightly different in the operation of sample injection, spray washing and elution, all the operation shall be in accordance with the requirements of operating instructions supplied by the manufacturer.
Warning: The whole analysis procedure shall be performed within specified area. Such area shall be protected from light (direct sunlight), with relatively independent operating desk and waste storage devices. During the whole experiment, the operator shall take the corresponding protective measures according to the requirements of the exposed to toxic substances.
5.1 Extraction of sample
5.1.1 Liquid milk, yoghurt
Weigh 4g (to the nearest 0.001g) well-mixed sample in 50mL centrifuge tube, add 100μL 13C17-AFT M1 internal standard solution (5ng/mL), mix well by oscillating and standing 30min; add 10mL methanol, mix well by 3min vortex. At 4℃, centrifuging the solution for 10min at 6000r/min or filter it with glass fibre filter, take proper amount of supernatant or filtrate and transfer to a beaker, diluted with 40mL water or PBS, reserved.
5.1.2 Milk powder, foods for special dietary supplies
Weigh 1g (to the nearest 0.001g) well-mixed sample in 50mL centrifuge tube, add 100μL 13C17-AFT M1 internal standard solution (5ng/mL), mix well by oscillating and standing 30min; add 4mL hot water, mix well by vortex. If milk powder cannot be fully dissolved, place the centrifuge tube into 50℃ water bath and take it out after milk powder is fully dissolved. After the sample solution cool down to 20℃, add 10mL methanol, mix well by 3min vortex. At 4℃, centrifuging the solution for 10min at 6000r/min or filter it with glass fibre filter, take proper amount of supernatant or filtrate and transfer to a beaker, diluted with 40mL water or PBS, reserved.
5.1.3 Cream
Weigh 1g (to the nearest 0.001g) well-mixed sample in 50mL centrifuge tube, add 100μL 13C17-AFT M1 internal standard solution (5ng/mL), mix well by oscillating and standing 30min; add 8mL petroleum ether, and then add 9mL water and 11mL methanol after the sample dissolved, oscillating for 30min and transfer all the liquid to a separating funnel. Add 0.3g sodium chloride and fully shaking to dissolve it, stand until layering, transfer the lower layer to a round flask and evaporate to below 10mL by rotary evaporator, dilute to 30mL with PBS.
5.1.4 Cheese
Weigh 1g (to the nearest 0.001g) minced, well-mixed sample (filter with circular screen with hole diameter of 1mm to 2mm) in 50mL centrifuge tube, add 100μL 13C17-AFT M1 internal standard solution (5ng/mL), mix well by oscillating and standing 30min; add 1mL water and 8mL methanol, oscillating for 30min. At 4℃, centrifuging the solution for 10min at 6000r/min or filter it with glass fibre filter, take proper amount of supernatant or filtrate and transfer to a round flask, evaporate to below 2mL by rotary evaporator, dilute to 30mL with PBS.
5.2 Purification
5.2.1 Preparation of immunoaffinity column
Recover the immunoaffinity column preserved at low temperature to room temperature.
5.2.2 Purification
After discard the liquid within immunoaffinity column, transfer aforesaid sample solution to a 50mL injector cylinder, regulate the drop flow rate of 1mL/min to 3mL/min. At the end of sample solution drip off, add 10mL water into the injector cylinder to spray washing the immunoaffinity column with steady flow rate. At the end of water drip off, swab off the column with vacuum pump. Separate from vacuum system, place a 10mL graduated test tube under the column, remove 50mL injector cylinder and add 2×2mL acetonitrile (or methanol) to elute the column, the drop flow rate controlled at 1mL/min to 3mL/min. Swab off the column with vacuum pump, collect all the eluent into the graduated test tube. Blow, slowly, the eluent to nearly dry with nitrogen at 50℃, scale the volume to 1.0mL with initial mobile phase; dissolve the residues by vortex 30s, filter with 0.22μm filter membrane, collect the collect into sample-injecting bottle for use.
Note: Full automatic (on-line) or semi-automatic (offline) solid phase extraction instrument can be used after optimization of operating parameters. In order to prevent aflatoxin M destroyed, relevant operation shall be performed in a dark place (protect form direct sunlight).
5.3 Reference conditions for liquid chromatography
Reference conditions of liquid chromatography are as follows:
a) Liquid chromatographic column: C18 column (100mm in column length, 2.1mm in inner diameter and 1.7μm in particle size), or equivalent.
b) Temperature of chromatographic column: 40℃.
c) Mobile phase: Phase A, ammonium acetate water solution (5 mmol/L); Phase B, acetonitrile-methanol (50+50). Gradient elution: see Table 1.
d) Flow rate: 0.3mL/min.
e) Sample injection volume: 10μL.
5.4 Reference conditions of mass spectrometry
Reference conditions of mass spectrometry are as follows:
a) Detection mode: multi-ion reaction monitoring (MRM).
b) Control conditions of ion source: see Table 2.
c) Selective parameters of ion: see Table 3;
d) Liquid chromatography-mass spectrogram and daughter ion scanning pattern: see Appendix C.
1 Scope
This standard specifies the determination method of aflatoxin M1 and aflatoxin M2 (hereinafter referred to as AFT M1 and AFT M2) in foods.
Method I is isotope dilution-liquid chromatography-tandem mass spectrometry, it is applicable to the determination of AFT M1 and AFT M2 in milk, milk products and milk-containing foods for special dietary supplies.
Method II is high performance liquid chromatography, its application scope is the same with Method I.
Method III is enzyme linked immunosorbent assay screening method, it is applicable to the screening determination of AFT M1 in milk, milk products and milk-containing foods for special dietary supplies.
Method I Isotope Dilution-Liquid Chromatography-Tandem Mass Spectrometry
2 Principle
After AFT M1 and AFT M2 in the sample extracted by methanol-water solution and dilute the supernatant with water or phosphate buffer solution, purified and enriched by immunoaffinity column, scale the volume and filter, the filtrate can be separated by liquid chromatography, tested by tandem mass spectrometry and quantified by isotope internal standard method.
3 Reagents and Materials
Unless otherwise specified, reagents used in this method are all analytically pure, and water is the Grade 1 water as specified in GB/T 6682.
3.1 Reagents
3.1.1 Acetonitrile (CH3CN): chromatographically pure.
3.1.2 Methanol (CH3OH): chromatographically pure.
3.1.3 Ammonium acetate (CH3COONH4).
3.1.4 Sodium chloride (NaCl).
3.1.5 Disodium hydrogen phosphate (Na2HPO4).
3.1.6 Sodium dihydrogen phosphate (KH2PO4).
3.1.7 Potassium chloride (KCl).
3.1.8 Hydrochloric acid (HCl).
3.1.9 Petroleum ether (CnH2n+2): boiling range of petroleum ether is 30℃ to 60℃.
3.2 Reagent preparation
3.2.1 Ammonium acetate solution (5mmol/L): weigh 0.39g ammonium acetate and dissolve it in 1000mL water, mix well.
3.2.2 Acetonitrile-water solution (25+75): measure 250mL acetonitrile and add it into 750mL water, mix well.
3.2.3 Acetonitrile-methanol solution (50+50): measure 500mL acetonitrile and add it into 500mL methanol, mix well.
3.2.4 Phosphate buffer solution (hereinafter referred to as PBS): weigh 8.00g sodium chloride, 1.20g disodium hydrogen phosphate (or 2.92g sodium acid phosphate dodecahydrate), 0.20g potassium dihydrogen phosphate and 0.20g potassium chloride, dissolve in 900mL water, adjust the pH value to 7.4 with hydrochloric acid, and dilute to 1000mL with water.
3.3 Standards
3.3.1 AFT M1 standard (C17H12O7, CAS: 6795-23-9): purity ≥98%, or other standard reference materials approved and awarded with reference material certificate by the nation.
3.3.2 AFT M2 standard (C17H14O7, CAS: 6885-57-0): purity ≥98%, or other standard reference materials approved and awarded with reference material certificate by the nation.
3.3.3 13C17-AFT M1 isotope solution (C17H14O7): 0.5μg/mL
3.4 Preparation of standard solution
3.4.1 Standard stock solution (10μg/mL): respectively weigh 1mg (to the nearest 0.01mg) of AFT M1 and AFT M2 , respectively dissolve in acetonitrile and scale the volume to 100mL. Transfer this solution to brown reagent bottle, sealed and preserved in a dark place. Perform concentration calibration prior to use (see Appendix A for calibration method).
3.4.2 Mixed standard stock solution (1.0μg/mL): Accurately pipet respectively 1.00mL AFT M1 and AFT M2 standard stock solution (10μg/mL) in a 10mL volumetric flask, dilute to the scale with acetonitrile to prepare 1.0μg/mL mixed standard solution. This solution shall be sealed and preserved in a dark place at 4℃, the validity period is 3 months.
3.4.3 Mixed standard working solution (100ng/mL): Accurately pipet 1.00mL mixed standard stock solution (1.0μg/mL) and transfer to a 10mL volumetric flask, scale the volume with acetonitrile. This solution shall be sealed and preserved in a dark place at 4℃, the validity period is 3 months.
3.4.4 50ng/mL isotope internal standard working solution 1 (13C17-AFT M1): measure 1mL AFT M1 isotope internal standard (0.5μg/mL) and dilute to 10mL with acetonitrile. It shall be preserved at -20℃ and to be used for the determination of liquid sample. Validity period is 3 months.
3.4.5 5ng/mL isotope internal standard working solution 2 (13C17-AFT M1): measure 100μL AFT M1 isotope internal standard (0.5μg/mL) and dilute to 10mL with acetonitrile.It shall be preserved at -20℃ and to be used for the determination of solid sample. Validity period is 3 months.
3.4.6 Standard series working solution: Accurately transfer respectively 5μL, 10μL, 50μL, 100μL, 200μL and 500μL standard working solution into 10mL volumetric flask, add 100μL isotope internal standard working solution (50ng/mL), scale the volume to the scale with initial mobile phase to prepare standard solution series with the AFT M1 and AFT M2 concentration of 0.05ng/mL, 0.1ng/mL, 0.5ng/mL, 1.0ng/mL, 2.0ng/mL, and 5.0ng/mL.
4 Apparatus and Equipment
4.1 Balance: the sensibility is 0.01g, 0.001g and 0.00001g.
4.2 Water bath: temperature control at 50℃±2℃.
4.3 Vortex mixer.
4.4 Ultrasonic cleaner.
4.5 Centrifuge: ≥6000 r/min.
4.6 Rotary evaporator.
4.7 Solid-phase extraction device (with vacuum pump).
4.8 Nitrogen blower.
4.9 LC-MS-MS: equipped with ESI source.
4.10 Circular screen: in pore diameter of 1mm to 2mm.
4.11 Glass fibre filter: fast, high load, particles under 1.6μm are reserved in the liquid.
4.12 Disposable microporous filter head: with 0.22μm microporous filter membrane (the selected filter membrane shall be tested with standard solution, and may be used after confirm that no adsorption phenomenon).
4.13 Immunoaffinity column: column capacity ≥100ng (see Appendix B for column capacity, recovery rate and verification method of column recovery rate).
Note: The quality verification of each batch of affinity columns shall be carried out prior to use.
5 Analysis Procedure
The application of immunoaffinity columns from different manufacturers may be slightly different in the operation of sample injection, spray washing and elution, all the operation shall be in accordance with the requirements of operating instructions supplied by the manufacturer.
Warning: The whole analysis procedure shall be performed within specified area. Such area shall be protected from light (direct sunlight), with relatively independent operating desk and waste storage devices. During the whole experiment, the operator shall take the corresponding protective measures according to the requirements of the exposed to toxic substances.
5.1 Extraction of sample
5.1.1 Liquid milk, yoghurt
Weigh 4g (to the nearest 0.001g) well-mixed sample in 50mL centrifuge tube, add 100μL 13C17-AFT M1 internal standard solution (5ng/mL), mix well by oscillating and standing 30min; add 10mL methanol, mix well by 3min vortex. At 4℃, centrifuging the solution for 10min at 6000r/min or filter it with glass fibre filter, take proper amount of supernatant or filtrate and transfer to a beaker, diluted with 40mL water or PBS, reserved.
5.1.2 Milk powder, foods for special dietary supplies
Weigh 1g (to the nearest 0.001g) well-mixed sample in 50mL centrifuge tube, add 100μL 13C17-AFT M1 internal standard solution (5ng/mL), mix well by oscillating and standing 30min; add 4mL hot water, mix well by vortex. If milk powder cannot be fully dissolved, place the centrifuge tube into 50℃ water bath and take it out after milk powder is fully dissolved. After the sample solution cool down to 20℃, add 10mL methanol, mix well by 3min vortex. At 4℃, centrifuging the solution for 10min at 6000r/min or filter it with glass fibre filter, take proper amount of supernatant or filtrate and transfer to a beaker, diluted with 40mL water or PBS, reserved.
5.1.3 Cream
Weigh 1g (to the nearest 0.001g) well-mixed sample in 50mL centrifuge tube, add 100μL 13C17-AFT M1 internal standard solution (5ng/mL), mix well by oscillating and standing 30min; add 8mL petroleum ether, and then add 9mL water and 11mL methanol after the sample dissolved, oscillating for 30min and transfer all the liquid to a separating funnel. Add 0.3g sodium chloride and fully shaking to dissolve it, stand until layering, transfer the lower layer to a round flask and evaporate to below 10mL by rotary evaporator, dilute to 30mL with PBS.
5.1.4 Cheese
Weigh 1g (to the nearest 0.001g) minced, well-mixed sample (filter with circular screen with hole diameter of 1mm to 2mm) in 50mL centrifuge tube, add 100μL 13C17-AFT M1 internal standard solution (5ng/mL), mix well by oscillating and standing 30min; add 1mL water and 8mL methanol, oscillating for 30min. At 4℃, centrifuging the solution for 10min at 6000r/min or filter it with glass fibre filter, take proper amount of supernatant or filtrate and transfer to a round flask, evaporate to below 2mL by rotary evaporator, dilute to 30mL with PBS.
5.2 Purification
5.2.1 Preparation of immunoaffinity column
Recover the immunoaffinity column preserved at low temperature to room temperature.
5.2.2 Purification
After discard the liquid within immunoaffinity column, transfer aforesaid sample solution to a 50mL injector cylinder, regulate the drop flow rate of 1mL/min to 3mL/min. At the end of sample solution drip off, add 10mL water into the injector cylinder to spray washing the immunoaffinity column with steady flow rate. At the end of water drip off, swab off the column with vacuum pump. Separate from vacuum system, place a 10mL graduated test tube under the column, remove 50mL injector cylinder and add 2×2mL acetonitrile (or methanol) to elute the column, the drop flow rate controlled at 1mL/min to 3mL/min. Swab off the column with vacuum pump, collect all the eluent into the graduated test tube. Blow, slowly, the eluent to nearly dry with nitrogen at 50℃, scale the volume to 1.0mL with initial mobile phase; dissolve the residues by vortex 30s, filter with 0.22μm filter membrane, collect the collect into sample-injecting bottle for use.
Note: Full automatic (on-line) or semi-automatic (offline) solid phase extraction instrument can be used after optimization of operating parameters. In order to prevent aflatoxin M destroyed, relevant operation shall be performed in a dark place (protect form direct sunlight).
5.3 Reference conditions for liquid chromatography
Reference conditions of liquid chromatography are as follows:
a) Liquid chromatographic column: C18 column (100mm in column length, 2.1mm in inner diameter and 1.7μm in particle size), or equivalent.
b) Temperature of chromatographic column: 40℃.
c) Mobile phase: Phase A, ammonium acetate water solution (5 mmol/L); Phase B, acetonitrile-methanol (50+50). Gradient elution: see Table 1.
d) Flow rate: 0.3mL/min.
e) Sample injection volume: 10μL.
5.4 Reference conditions of mass spectrometry
Reference conditions of mass spectrometry are as follows:
a) Detection mode: multi-ion reaction monitoring (MRM).
b) Control conditions of ion source: see Table 2.
c) Selective parameters of ion: see Table 3;
d) Liquid chromatography-mass spectrogram and daughter ion scanning pattern: see Appendix C.
