1 Scope
This standard specifies the isotope dilution liquid chromatography-tandem mass spectrum method for the determination of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) in animal-derived food.
This standard is applicable to the determination of PFOS and PFOA in animal-derived food.
2 Principle
Extract the PFOS and PFOA from the specimen by using acetonitrile, purify the matters with dispersive solid-phase extraction, determine them with liquid chromatography-tandem mass spectrometry, and quantify them with internal standard method.
3 Reagents and Materials
Unless otherwise specified, analytically-pure reagents and Grade I water (defined in GB/T 6682) are adopted for the purposes of this method.
3.1 Methanol (CH3OH): chromatographically pure.
3.2 Acetonitrile (CH3CN): chromatographically pure.
3.3 Hydrochloric acid (HCl).
3.4 Sodium chloride (NaCl).
3.5 Ammonium acetate (CH3COONH4): chromatographically pure.
3.6 Primary-secondary amine solid phase adsorbent (PSA): 40μm~60μm, ProElut filler or equivalent.
3.7 C18Adsorbent: 40μm~60μm, ProElut filler or equivalent.
3.8 Graphitized carbon black adsorbent (GCB): 40μm~60μm,ProElut filler or equivalent.
3.9 Ammonium acetate solution (5mmol/L): Accurately weigh 0.3855g (to the nearest of 0.0001g) of ammonium acetate, dissolve it and scale the volume to 1,000mL with water.
3.10 Standard solution of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA): Accurately weigh 10.00mg (to the nearest of 0.0001g) of PFOA (purity ≥98%; CAS: 335-67-1; CF3(CF2)6COOH) and PFOS (purity ≥99%; CAS: 1763-23-1; C8HF17O3S) respectively, and dilute them to 100mL with methanol to prepare the standard solutions of PFOA and PFOS (the concentration of each solution is 100μg/mL). The solutions are preserved at -4 ℃ and are valid within 6 months.
3.11 PFOA standard stock solution: pipet the PFOA standard product solution and dilute it with methanol to prepare the PFOA standard stock solution with the concentration of 100.0μg/L. PFOS standard stock solution: pipet the PFOS standard product solution and dilute it with methanol to prepare the PFOS standard stock solution with the concentration of 100.0μg/L. Both solutions are preserved at -4℃ and are valid within 4 months.
Note: perfluorooctane sulfonates (PFOS) exists in the solution in a state of perfluorooctane sulfonate ions; sample solution and standard solution are quantified with perfluorooctane sulfonate.
3.12 Series of mixed standard working solutions: pipet some of the standard stock solution and dilute it with methanol to prepare the series of mixed standard working solutions with the concentration of PFOA being 0.05μg/L, 0.1μg/L, 0.5 μg/L, 1.0μg/L, 2.0 μg/L and 10.0μg/L successively and that of the PFOS being 0.1μg/L, 0.2μg/L, 1.0μg/L, 10.0μg/L, 20.0μg/L and 40.0μg/L successively. The solutions are preserved at -4℃ with a validity period of 2 months.
3.13 13C4-PFOA standard product solution: a standard product solution which is prepared by diluting the concentration to 50.0μg/mL with methanol; 1,2,3,4-13C4-PFOS standard product solution: a standard product solution which is prepared by diluting the concentration to 50.0μg/mL with methanol. Both solutions are preserved at -4℃ with a validity period of 6 months.
3.14 Internal standard mixed working solution I: pipet some of the standard product solutions of 13C4-PFOA and 1, 2, 3, 4-13C4-PFOS and dilute them with methanol to prepare the internal standard mixed working solutions of 13C4-PFOA (10.0μg/L) and 1, 2, 3, 4-13C4-PFOS (50.0μg/L). The solutions are preserved at -4℃ with a validity period of 4 months.
3.15 Internal standard mixed working solution II: pipet some of the internal standard mixed working solutions I of 13C4-PFOA and 1, 2, 3,4-13C4-PFOS, and dilute them with methanol to prepare the internal standard mixed working solutions of 13C4-PFOA (1.0μg/L) and 1, 2, 3, 4-13C4-PFOS (5.0μg/L). The solutions are preserved at -4℃ with a validity period of 2 months.
3.16 Mixed series standard working solutions containing 13C isotope as the internal standard substance: pipet some of the standard stock solution and internal standard mixed working solution I and dilute them with methanol to prepare the mixed series standard working solutions containing 13C isotope as the internal standard substance, of which the concentrations of PFOA are 0.05 μg/L, 0.1μg/L, 0.5μg/L1 1.0μg/L, 2.0μg/L, and 10.0μg/L successively, the concentrations of PFOS are 0.1μg/L, 0.2μg/L, 1.0μg/L, 10.0μg/L, 20.0μg/L and 40.0μg/L successively, and the concentrations of 13C4-PFOA and 1, 2, 3, 4-13C4-PFOS are 1.0μg/L and 5.0μg/L respectively. The solutions are preserved at -4℃ with a validity period of 2 months.
3.17 Filter membrane: 0.22μm organic phase filter membrane.
4 Instruments and Apparatus
4.1 Liquid chromatography-tandem mass spectrometry: equipped with electrospray ionization source (ESI).
4.2 Nitrogen evaporator.
4.3 Grinder.
4.4 Centrifuge: rotation speed≥5,000r/min.
4.5 Homogenizer, of which the referred rotation speed is 3,400r/min ~24,000r/min.
4.6 Analytical balance: with sensitivity of 0.1 mg and 0.01 g.
5 Preparation of Samples
Take the edible part (deboned and decladded) of fish, shrimp, crab, shellfish, octopus, chicken, pork and beef, cut them into small pieces and grind them into meat paste with the grinder; remove the shell of egg products and churn the remaining part into homogenate; mix the milk uniformly for use. Place the samples in a glass sample bottle, seal and mark the bottle, then preserve it at a temperature of -18℃.
6 Analysis Steps
6.1 Sample pretreatment
6.1.1 Extraction
Weigh 5g specimen (to the accuracy of 0.01g) (defrost and homogenize the sample prior to use), place it in a 50mL polypropene centrifugal tube and add in 400μL of internal standard mixed working solution; then add in 5mL of water into the tube, eddy for 1 min, add in 10mL acetonitrile (3.2) and 30μL hydrochloric acid (3.3), and vibrate for 10min. Add in 2g sodium chloride (3.4), shake it for 10min and centrifuge it at 5,000r/min for 10min. Transfer the acetonitrile solution of the upper-layer into another test tube, and blow it with nitrogen to approximately 4mL in the water bath of 45℃ for purifying.
6.1.2 Purification
Transfer the above-mentioned solution into 15mL polypropene centrifugal tube containing 100mg PSA (3.6), 40mg C18 (3.7) and 20mg GCB (3.8), shake for 10min and centrifuge at 5,000r/min for 10min; transfer the supernatant solution into another test tube, blow it dry with nitrogen in 45℃ water bath and dissolve it with 1mL of methanol (3.1); suck it in a 1mL injector, and filter it through a 0.22μm organic filter membrane for test.
1 Scope
2 Principle
3 Reagents and Materials
4 Instruments and Apparatus
5 Preparation of Samples
6 Analysis Steps
7 Quality Control
8 Expression of Analysis Result
9 Precision
10 Others
Appendix A Reference Conditions of Mass Spectrum
Appendix B Typical Liquid Chromatography - Tandem Mass Spectrum Multi-response Monitoring (MRM) Chart of PFOA and PFOS and Their Internal Standard
