This standard specifies the method for determination of aflatoxin M1 in milk and milk products.
Method I in this standard is applicable to the determination of aflatoxin M1 in milk and milk products; Method II is applicable to the determination of aflatoxin M1 in milk, milk powder and low fat milk, skimmed milk, low fat milk powder and skimmed milk powder; Method III is applicable to the determination of aflatoxin M1 in milk and milk powder; Method IV is applicable to the determination of aflatoxin M1 in liquid milk and milk powder.
2 Normative References
The documents referenced in this standard are indispensable for the application of this standard. For dated references, only the dated edition of the document is applicable to this standard. For undated references, the latest edition (including all amendments) of the document is applicable to this standard.
Method I Clean-up by Immunoaffinity Chromatography and Determination by Liquid Chromatography-Tandem Mass Spectrometry
3 Principle
The sample liquid or solid-sample extracting liquid shall pass through homogeneity, ultrasonic extracting and centrifugation; then the obtained supernatant shall be purified through the immunoaffinity column; then the eluent shall be dried with nitrogen gas and subsequently be volumetrically scaled to be filtered via the microporous filtering film, separated with liquid chromatography, and ionized via electrospray ionization (ESI), and finally be tested with multiple reaction monitoring (MRM) approach. The matrix shall be quantified via standard addition method and external standard method.
4 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.
4.1 Formic acid (HCOOH).
4.2 Acetonitrile (CH3CN): chromatographically pure.
4.3 Petroleum ether (CnH2n+2): boiling range is 30℃~60℃.
4.4 Trichloromethane (CHCl3).
4.5 Nitrogen: purity ≥99.9%.
4.6 Aflatoxin M1 standard sample: purity ≥98%.
4.7 Acetonitrile-water solution(1+4): add 100mL acetonitrile into 400mL water.
4.8 Acetonitrile-water solution(1+9): add 50mL acetonitrile into 450mL water.
4.9 0.1% formic acid water solution: pipet 1mL formic acid (4.1) and dilute with water to 1,000mL.
4.10 Acetonitrile-methanol solution(50+50): add 500mL methanol into 500mL acetonitrile.
4.11 Sodium hydroxide solution (0.5mol/L): weigh 2g sodium hydroxide and dissolve it into 100mL water.
4.12 Blank matrix solution
Weigh 8 negative samples respectively that have the same matrix with the to-be-tested sample and are free of aflatoxin into the 100mL beaker. Conduct the following operations according to the test solutions extraction (6.1) and purification procedure (6.2). Combine the eight pieces of purification liquid and filter with disposable filtration head (5.23) of 0.22μm microporous filtering film. Discard the first 0.5mL filter liquor and take a small amount of filter liquor for the test of liquid chromatographmass spectrometer.
After obtaining the chromatograph-mass spectrogram, contrast with the diagram A.2 of the Appendix A, the corresponding retention time position shall be free of aflatoxin M1. Transfer the residual filter liquor into the brown bottle and preserve it in th refrigerator at -20℃ for the preparation of standard series solution.
4.13 The stock standard solution of aflatoxin M1: weigh 0.10mg (to the accuracy of 0.01mg) standard aflatoxin M1 respectively, dissolve with trichloromethane (4.4) and volumetrically scale to 10mL. The concentration of this standard solution is 0.01mg/mL. Transfer the solution into a brown bottle and preserve it in the refrigerator at -20℃ for standby.
4.14 Standard series solution of aflatoxin M1: pipet 10μL aflatoxin M1 stock standard solution (4.13) into 10mL volumetric flask and blow the trichloromethane with nitrogen gas until it is closely dry. Volumetrically scale the blank matrix solution (4.12) to scale and obtain the M1 standard intermediate of 10ng concentration. And then, use the blank matrix solution (4.12) to dilute the aflatoxin M1 standard intermediate to such series standard working solutions of 0.5ng/mL, 0.8ng/mL, 1.0ng/mL, 2.0ng/mL, 4.0ng/mL, 6.0ng/mL, and 8.0ng/mL.
