阅读说明：本技术 分析奶粉中氧化多环芳烃和氯代多环芳烃的含量的方法 (Method for analyzing content of oxidized polycyclic aromatic hydrocarbon and chlorinated polycyclic aromatic hydrocarbon in milk powder ) 是由 吴时敏 严凯 李玮 张立敏 于 2021-08-05 设计创作，主要内容包括：本发明公开了一种分析奶粉中氧化多环芳烃和氯代多环芳烃的含量的方法,包括以下步骤：A1：制定氧化多环芳烃和氯代多环芳烃的标准曲线；A2：利用超声辅助萃取法从奶粉溶液中萃取目标分析物；A3：采用SPE方法净化目标分析物；A4：净化后的样品经浓缩,经气相色谱-三重四级杆串联质谱检测,外标法定量,得到奶粉中氧化多环芳烃和氯代多环芳烃的含量；其中,所述的氧化多环芳烃包括蒽醌和9-芴酮,所述的氯代多环芳烃包括6-氯苯并芘和7-氯苯并蒽。本发明所建立的GC–QQQ–MS方法中,包括色谱柱的选择和升温条件的优化,在较短时间内实现了四种目标分析物9–芴酮、蒽醌、6–氯苯并芘和7–氯苯并蒽的良好分离和色谱响应,兼顾了定量分析的精确性和高效性。(The invention discloses a method for analyzing the content of oxidized polycyclic aromatic hydrocarbon and chlorinated polycyclic aromatic hydrocarbon in milk powder, which comprises the following steps: a1: preparing a standard curve of the oxidized polycyclic aromatic hydrocarbon and the chlorinated polycyclic aromatic hydrocarbon; a2: extracting a target analyte from a milk powder solution by using an ultrasonic-assisted extraction method; a3: purifying the target analyte by SPE method; a4: concentrating the purified sample, detecting by gas chromatography-triple quadrupole tandem mass spectrometry, and quantifying by an external standard method to obtain the contents of oxidized polycyclic aromatic hydrocarbon and chlorinated polycyclic aromatic hydrocarbon in the milk powder; wherein, the oxidized polycyclic aromatic hydrocarbon comprises anthraquinone and 9-fluorenone, and the chlorinated polycyclic aromatic hydrocarbon comprises 6-chlorobenzpyrene and 7-chlorobenzanthracene. The GC-QQQ-MS method established by the invention comprises the selection of a chromatographic column and the optimization of a heating condition, realizes the good separation and chromatographic response of four target analytes, namely 9-fluorenone, anthraquinone, 6-chlorobenzpyrene and 7-chlorobenzanthracene, in a short time, and gives consideration to the accuracy and the high efficiency of quantitative analysis.)

1. A method for analyzing the content of oxidized polycyclic aromatic hydrocarbon and chlorinated polycyclic aromatic hydrocarbon in milk powder is characterized by comprising the following steps:

a1: preparing a standard curve of the oxidized polycyclic aromatic hydrocarbon and the chlorinated polycyclic aromatic hydrocarbon;

a2: extracting a target analyte from a milk powder solution by using an ultrasonic-assisted extraction method;

a3: purifying the target analyte by SPE method;

a4: concentrating the purified sample, detecting by gas chromatography-triple quadrupole tandem mass spectrometry, and quantifying by an external standard method to obtain the contents of oxidized polycyclic aromatic hydrocarbon and chlorinated polycyclic aromatic hydrocarbon in the milk powder;

wherein, the oxidized polycyclic aromatic hydrocarbon comprises anthraquinone and 9-fluorenone, and the chlorinated polycyclic aromatic hydrocarbon comprises 6-chlorobenzpyrene and 7-chlorobenzanthracene.

2. The method for analyzing the content of the oxidized polycyclic aromatic hydrocarbon and the chlorinated polycyclic aromatic hydrocarbon in the milk powder according to claim 1, wherein a standard solution of seven levels of 9-fluorenone, anthraquinone, 6-chlorobenzpyrene and 7-chlorobenzanthracene is prepared by taking dichloromethane as a solvent, wherein the seven levels are 1, 2, 5, 10, 20, 50 and 100 μ g/kg, the concentrations of the oxidized polycyclic aromatic hydrocarbon and the chlorinated polycyclic aromatic hydrocarbon in the standard solution are used as abscissa, and the response values of the oxidized polycyclic aromatic hydrocarbon and the chlorinated polycyclic aromatic hydrocarbon detected by gas chromatography-triple quadrupole tandem mass spectrometry are used as ordinate to draw a standard curve.

3. The method for analyzing the content of the oxidized polycyclic aromatic hydrocarbon and the chlorinated polycyclic aromatic hydrocarbon in the milk powder according to claim 1 or 2, wherein the detection conditions of the gas chromatography-triple quadrupole tandem mass spectrometry are as follows:

a chromatographic column: an Agilent DB-EUPAH capillary chromatographic column with the specification of 20m multiplied by 0.18mm multiplied by 0.14 mu m;

temperature rising procedure: maintaining at 80 deg.C for 1min, increasing to 220 deg.C at 20 deg.C/min, increasing to 300 deg.C at 5 deg.C/min, and maintaining for 1 min;

the sample introduction temperature, the temperature of the transmission line, the temperature of the ion source and the temperature of the quadrupole rod are respectively 320 ℃, 300 ℃ and 150 ℃;

sample introduction amount: 1 mu L of the solution;

carrier gas: helium (99.999%);

flow rate of carrier gas: 1mL/min, no flow diversion;

an ionization mode: EI +, 70 eV;

the scanning mode is as follows: full scan, 33-300 amu, ion monitoring mode (SIM) was selected.

4. The method for analyzing the content of oxidized polycyclic aromatic hydrocarbon and chlorinated polycyclic aromatic hydrocarbon in the milk powder as claimed in claim 1, wherein the step A2 comprises:

a201: preparing a milk powder solution, wherein the mass ratio of milk powder to distilled water is 1: 7;

a202: extracting oxidized polycyclic aromatic hydrocarbon and chlorinated polycyclic aromatic hydrocarbon by nitrogen-nitrogen dimethyl formamide;

a203: extracting oxidized polycyclic aromatic hydrocarbon and chlorinated polycyclic aromatic hydrocarbon by using n-hexane;

a204: drying and concentrating: the n-hexane phase extract was transferred to a glass tube, dried over anhydrous sodium sulfate and concentrated to 2mL with nitrogen at 20 ℃.

5. The method for analyzing the content of the oxidized polycyclic aromatic hydrocarbon and the chlorinated polycyclic aromatic hydrocarbon in the milk powder according to claim 4, wherein the step A202 is specifically as follows: accurately weighing a milk powder solution in a first centrifugal tube, adding n-hexane and N-dimethyl methyl phthalein amine, fully mixing, carrying out vortex oscillation on the first centrifugal tube on a vortex oscillator, then carrying out ultrasonic centrifugation, wherein a lower layer liquid is a N-dimethyl methyl phthalein amine phase, an upper layer is a n-hexane phase, a N-dimethyl methyl phthalein amine phase is reserved for standby application, the n-hexane phase is continuously extracted once by the N-dimethyl methyl phthalein amine, and the two N-dimethyl methyl phthalein amine phases are combined to obtain a N-dimethyl methyl phthalein amine extracting solution;

wherein, the proportion relation of the milk powder solution, the n-hexane and the nitrogen dimethyl phthalein amine is 5 g: 4mL of: 8 mL.

6. The method for analyzing the content of the oxidized polycyclic aromatic hydrocarbon and the chlorinated polycyclic aromatic hydrocarbon in the milk powder according to claim 4, wherein the step A203 is specifically as follows: adding a sodium chloride solution with the mass fraction of 4% into a nitrogen-nitrogen dimethyl phthalein amine extracting solution, uniformly mixing, adding 8mL of n-hexane, sufficiently shaking, uniformly mixing, standing, taking an upper layer liquid as an n-hexane phase and a lower layer liquid as a water phase, reserving the n-hexane phase for later use, continuously extracting the water phase once with the n-hexane, and combining the two n-hexane phases to obtain an n-hexane phase extracting solution;

wherein, the proportion relation of the nitrogen-nitrogen dimethyl phthalein amine extract, the sodium chloride solution and the n-hexane is 16 mL: 50mL of: 8 mL.

7. The method for analyzing the content of the oxidized polycyclic aromatic hydrocarbon and the chlorinated polycyclic aromatic hydrocarbon in the milk powder as claimed in claim 1, wherein the step A3 is specifically as follows: purifying by using a Supelclean Florisil solid phase extraction cartridge, firstly, carrying out activation balance on the solid phase extraction cartridge, then loading, after loading is finished, leaching non-target substances in an extraction solution, eluting the target substances by using an eluent, collecting the eluent, concentrating the eluent under reduced pressure until the eluent is dry, and dissolving by using an upper machine solvent.

8. The method for analyzing the contents of oxidized polycyclic aromatic hydrocarbon and chlorinated polycyclic aromatic hydrocarbon in the milk powder as claimed in claim 7, wherein the activating equilibrium of the solid phase extraction column is performed by activating with dichloromethane and balancing with n-hexane.

9. The method for analyzing the content of the oxidized polycyclic aromatic hydrocarbon and the chlorinated polycyclic aromatic hydrocarbon in the milk powder as claimed in claim 7, wherein the leacheate is a mixture of the following materials in a volume ratio of 95:5, and the eluent is a mixed solution of n-hexane and dichloromethane, and the volume ratio of the eluent is 1:2, and the upper machine solvent is dichloromethane.

Technical Field

The invention relates to a method for analyzing polycyclic aromatic hydrocarbon derivatives in the field of food safety, in particular to a method for analyzing the contents of oxidized polycyclic aromatic hydrocarbon and chlorinated polycyclic aromatic hydrocarbon in milk powder.

Background

Polycyclic Aromatic Hydrocarbons (PAHs for short) refer to a class of persistent organic pollutants that structurally contain two or more fused benzene rings, are lipophilic, are easily accumulated in adipose tissues, and can further harm human health through the enrichment and amplification of food chains. Oxidized Polycyclic Aromatic Hydrocarbons (OPAHs) are widely present in various matrixes, have stronger redox characteristics than the matrixes, are easier to accumulate in organisms and induce stronger teratogenic and mutagenic toxic effects. Chlorinated polycyclic aromatic hydrocarbons (ClPAHs) are also a class of polycyclic aromatic hydrocarbon derivatives with carcinogenic properties, which can be generated through photochemical reactions and the like in the processes of disinfection, combustion and the like of tap water, and exhibit toxicity equivalent similar to dioxin and even higher. It has been reported in the literature to have greater toxicity than the parent PAHs.

At present, a great deal of research at home and abroad finds that OPAHs and ClPAHs have strong carcinogenicity, and a great amount of residues in environmental samples can enter a food chain along with atmospheric migration, and the hydrophobic property enables the OPAHs and the ClPAHs to be accumulated in fat-rich food, thereby further threatening food safety. Milk powder as a fat-containing food, the European Union has specified that benzo [ a ] pyrene, one of the PAHs in infant milk powder, should not exceed 1 μ g/kg. Given the higher toxicity of OPAHs and ClPAHs, the safety limits may be prohibitive. Therefore, establishing two typical methods for simultaneously and quantitatively detecting OPAHs (9-fluorenone and anthraquinone) and ClPAHs (6-chlorobenzpyrene and 7-chlorobenzanthracene) in milk powder is very important for the safety and quality guarantee of the milk powder.

At present, although the analysis method and residual risk of PAHs have attracted extensive attention at home and abroad, the research on high-toxicity PAHs derivatives ClPAHs (6-chlorobenzpyrene and 7-chlorobenzanthracene) and OPAHs (9-fluorenone and anthraquinone) is few, and the analysis method of PAHs in milk powder is not reported yet; moreover, the complex matrixes such as fat, protein and the like in the milk powder make the accurate determination of the trace PAHs extremely difficult. The present technology aims to address the two challenges described above.

Disclosure of Invention

Aiming at the difficult problems that the residual levels of 9-fluorenone, anthraquinone, 6-chlorobenzpyrene and 7-chlorobenzanthracene in the existing milk powder matrix cannot be quantitatively analyzed, the invention provides a method for analyzing the content of oxidized polycyclic aromatic hydrocarbon and chlorinated polycyclic aromatic hydrocarbon in milk powder.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a method for analyzing the content of oxidized polycyclic aromatic hydrocarbon and chlorinated polycyclic aromatic hydrocarbon in milk powder comprises the following steps:

a1: preparing a standard curve of the oxidized polycyclic aromatic hydrocarbon and the chlorinated polycyclic aromatic hydrocarbon;

a2: extracting a target analyte from a milk powder solution by using an ultrasonic-assisted extraction method;

a3: purifying the target analyte by SPE method;

a4: concentrating the purified sample, detecting by gas chromatography-triple quadrupole tandem mass spectrometry, and quantifying by an external standard method to obtain the contents of oxidized polycyclic aromatic hydrocarbon and chlorinated polycyclic aromatic hydrocarbon in the milk powder;

wherein, the oxidized polycyclic aromatic hydrocarbon comprises anthraquinone and 9-fluorenone, and the chlorinated polycyclic aromatic hydrocarbon comprises 6-chlorobenzpyrene and 7-chlorobenzanthracene.

The specific method for establishing the standard curve of the oxidized polycyclic aromatic hydrocarbon and the chlorinated polycyclic aromatic hydrocarbon is as follows: the method comprises the steps of respectively using dichloromethane as a solvent to prepare seven levels of standard solutions of 9-fluorenone, anthraquinone, 6-chlorobenzpyrene and 7-chlorobenzanthracene, wherein the levels are 1, 2, 5, 10, 20, 50 and 100 mu g/kg, using the concentrations of oxidized polycyclic aromatic hydrocarbon and chlorinated polycyclic aromatic hydrocarbon in the standard solutions as horizontal coordinates, using response values of the oxidized polycyclic aromatic hydrocarbon and the chlorinated polycyclic aromatic hydrocarbon detected by gas chromatography-triple quadrupole tandem mass spectrometry as vertical coordinates, and drawing a standard curve.

The detection conditions of the gas chromatography-triple quadrupole tandem mass spectrometry are as follows:

a chromatographic column: an Agilent DB-EUPAH capillary chromatographic column with the specification of 20m multiplied by 0.18mm multiplied by 0.14 mu m;

temperature rising procedure: maintaining at 80 deg.C for 1min, increasing to 220 deg.C at 20 deg.C/min, increasing to 300 deg.C at 5 deg.C/min, and maintaining for 1 min;

the sample introduction temperature, the temperature of the transmission line, the temperature of the ion source and the temperature of the quadrupole rod are respectively 320 ℃, 300 ℃ and 150 ℃;

sample introduction amount: 1 mu L of the solution;

carrier gas: helium (99.999%);

flow rate of carrier gas: 1mL/min, no flow diversion;

an ionization mode: EI +, 70 eV;

the scanning mode is as follows: full scan, 33-300 amu, ion monitoring mode (SIM) was selected.

The specific operation of extracting target analytes from the milk powder solution is as follows:

a201: preparing a milk powder solution, wherein the mass ratio of milk powder to distilled water is 1: 7;

a202: extracting oxidized polycyclic aromatic hydrocarbon and chlorinated polycyclic aromatic hydrocarbon by nitrogen-nitrogen dimethyl formamide: accurately weighing a milk powder solution in a first centrifugal tube, adding n-hexane and N-dimethyl methyl phthalein amine, fully mixing, carrying out vortex oscillation on the first centrifugal tube on a vortex oscillator, then carrying out ultrasonic centrifugation, wherein a lower layer liquid is a N-dimethyl methyl phthalein amine phase, an upper layer is a n-hexane phase, a N-dimethyl methyl phthalein amine phase is reserved for standby application, the n-hexane phase is continuously extracted once by the N-dimethyl methyl phthalein amine, and the two N-dimethyl methyl phthalein amine phases are combined to obtain a N-dimethyl methyl phthalein amine extracting solution;

wherein, the proportion relation of the milk powder solution, the n-hexane and the nitrogen dimethyl phthalein amine is 5 g: 4mL of: 8 mL;

a203: extracting the oxidized polycyclic aromatic hydrocarbon and the chlorinated polycyclic aromatic hydrocarbon by using n-hexane: adding a sodium chloride solution with the mass fraction of 4% into a nitrogen-nitrogen dimethyl phthalein amine extracting solution, uniformly mixing, adding 8mL of n-hexane, sufficiently shaking, uniformly mixing, standing, taking an upper layer liquid as an n-hexane phase and a lower layer liquid as a water phase, reserving the n-hexane phase for later use, continuously extracting the water phase once with the n-hexane, and combining the two n-hexane phases to obtain an n-hexane phase extracting solution;

wherein, the proportion relation of the nitrogen-nitrogen dimethyl phthalein amine extract, the sodium chloride solution and the n-hexane is 16 mL: 50mL of: 8 mL;

a204: drying and concentrating: the n-hexane phase extract was transferred to a glass tube, dried over anhydrous sodium sulfate and concentrated to 2mL with nitrogen at 20 ℃.

The specific method for purifying the target analyte by adopting the SPE method comprises the following steps: purifying by using a Supelclean Florisil solid phase extraction column, firstly, carrying out activation balance on the solid phase extraction column, then loading, after loading is finished, leaching and washing off non-target analytes on the solid phase extraction column, then eluting the target analytes by using an eluent, collecting the eluent, concentrating the eluent under reduced pressure until the eluent is dry, and dissolving by using an organic solvent.

The activation equilibrium of the solid phase extraction column is that dichloromethane is used for activation and n-hexane is used for equilibrium.

The leacheate is prepared from the following components in a volume ratio of 95:5, and the eluent is a mixed solution of n-hexane and dichloromethane, and the volume ratio of the eluent is 1:2, and the upper machine solvent is dichloromethane.

Due to the adoption of the technical scheme, compared with the prior art, the invention has the following advantages and positive effects:

at present, four PAHs derivatives (6-chlorobenzopyrene, 7-chlorobenzanthrene, 9-fluorenone and anthraquinone) are mostly determined by two relatively simple matrixes of atmosphere and water, and the method realizes good separation and chromatographic response of four target analytes (9-fluorenone, anthraquinone, 6-chlorobenzopyrene and 7-chlorobenzanthrene) in a short time (25min) under the interference of complex matrixes such as fat, protein and the like in milk powder, and gives consideration to the accuracy and the high efficiency of quantitative analysis. The invention successfully extracts four PAHs derivatives from milk powder by optimizing the proportional relation of n-hexane and N-dimethyl phthalein amine in the extraction process. Meanwhile, in the aspect of instrument parameters, chromatographic conditions (specification of a chromatographic column, filler, injection port temperature, a temperature rise program and the like) are compared and optimized aiming at the four PAHs derivatives, so that good separation degree is obtained; further optimizing mass spectrum parameters (monitoring ion fragments, ion sources, quadrupole temperature and the like), realizing simultaneous and high-sensitivity analysis of PAHs derivatives with different properties, realizing good separation and mass spectrum response of four target analytes (9-fluorenone, anthraquinone, 6-chlorobenzpyrene and 7-chlorobenzanthracene) in a short time (25min), and considering the accuracy and high efficiency of quantitative analysis.

The analysis method provided by the invention is adopted to analyze 9-fluorenone, anthraquinone, 6-chlorobenzpyrene and 7-chlorobenzanthracene in milk powder, under two addition levels of 5 mu g/kg and 10 mu g/kg, the blank standard recovery rate of each target is 63.38-109.54%, the relative standard deviation is 3.82-12.81%, the detection limit of the method is 0.08-0.78 mu g/kg, the sensitivity is high, the requirement of quantitative analysis is met, and the method has the potential of being popularized to other OPAHs and ClPAHs.

Drawings

FIG. 1 is a chromatogram of 9-fluorenone, anthraquinone, 6-chlorobenzpyrene and 7-chlorobenzanthracene standards;

FIG. 2 is a mass spectrum of 9-fluorenone, anthraquinone, 6-chlorobenzpyrene and 7-chlorobenzanthracene standard.

Detailed Description

The method for analyzing the content of the oxidized polycyclic aromatic hydrocarbon and the chlorinated polycyclic aromatic hydrocarbon in the milk powder provided by the invention is further described in detail by combining the attached drawings and specific examples. Advantages and features of the present invention will become apparent from the following description and from the claims.

In the following examples, all reagents are HPLC grade.

Example 1

A method for analyzing the content of oxidized polycyclic aromatic hydrocarbon and chlorinated polycyclic aromatic hydrocarbon in milk powder adopts an instrument and a reagent: GC-QQQ-MS (7890B-7000D); all reagents used were HPLC grade.

The method comprises the following steps of firstly, preparing a standard curve of oxidized polycyclic aromatic hydrocarbon and chlorinated polycyclic aromatic hydrocarbon, and specifically comprises the following steps:

1.1 configuring seven levels of standard solutions of 9-fluorenone (9-FO), Anthraquinone (ATQ), 6-chlorobenzpyrene (6-BaP) and 7-chlorobenzanthracene (7-BaA) with dichloromethane as a solvent, wherein the seven levels are 1, 2, 5, 10, 20, 50 and 100 mu g/kg, respectively, and drawing a standard curve by taking the concentrations of oxidized polycyclic aromatic hydrocarbon and chlorinated polycyclic aromatic hydrocarbon in the standard solutions as abscissa and the response values of the oxidized polycyclic aromatic hydrocarbon and the chlorinated polycyclic aromatic hydrocarbon detected by gas chromatography-triple quadrupole tandem mass spectrometry as ordinate. The concentration at which the response value of each target analyte was 3 times the noise value (S/N was 3) was used as the detection limit of the method, and the concentration at which S/N was 10 was used as the quantification limit of the method.

1.2 the detection conditions of the gas chromatography-triple quadrupole tandem mass spectrometry are as follows:

a chromatographic column: an Agilent DB-EUPAH capillary chromatographic column with the specification of 20m multiplied by 0.18mm multiplied by 0.14 mu m;

temperature rising procedure: maintaining at 80 deg.C for 1min, increasing to 220 deg.C at 20 deg.C/min, increasing to 300 deg.C at 5 deg.C/min, and maintaining for 1 min;

the sample introduction temperature, the temperature of the transmission line, the temperature of the ion source and the temperature of the quadrupole rod are respectively 320 ℃, 300 ℃ and 150 ℃;

sample introduction amount: 1 mu L of the solution;

carrier gas: helium (99.999%);

flow rate of carrier gas: 1mL/min, no flow diversion;

an ionization mode: EI +, 70 eV;

the scanning mode is as follows: full scan, 33-300 amu, ion monitoring mode (SIM) was selected.

Mass spectrum parameters, standard curve linear correlation (R2), detection Limit (LOD) and quantification Limit (LOQ) of 9-fluorenone, anthraquinone, 6-chlorobenzpyrene and 7-chlorobenzanthracene are shown in the following table 1, and chromatograms and mass spectrograms of 9-fluorenone, anthraquinone, 6-chlorobenzpyrene and 7-chlorobenzanthracene standard are shown in figure 1 and figure 2. As can be seen, in the embodiment, the detection limit of 9-fluorenone, anthraquinone, 6-chlorobenzpyrene and 7-chlorobenzanthracene is 0.08-0.12 mug/kg, the quantitative limit is 0.28-0.41 mug/kg, and the chromatographic separation degree of each substance is high, so that the method meets the requirement of qualitative and quantitative detection of trace substances.

And the second step, firstly extracting the polycyclic aromatic hydrocarbon in the milk powder, and the specific steps comprise:

1. preparing a milk powder solution, wherein the mass ratio of milk powder to distilled water is 1: 7;

2. extracting oxidized polycyclic aromatic hydrocarbon and chlorinated polycyclic aromatic hydrocarbon by nitrogen-nitrogen dimethyl formamide: accurately weighing 5g of milk powder solution in a centrifuge tube, adding 4mL of n-hexane and 8mL of N-dimethyl formamide, fully mixing, carrying out vortex oscillation on a first centrifuge tube on a vortex oscillator, then carrying out ultrasonic centrifugation, wherein a lower layer liquid is a N-dimethyl formamide phase, an upper layer is a n-hexane phase, a N-dimethyl formamide phase is reserved for standby application, the n-hexane phase is continuously extracted once with the N-dimethyl formamide, and the two N-dimethyl formamide phases are combined and transferred to a separating funnel;

3. extracting the oxidized polycyclic aromatic hydrocarbon and the chlorinated polycyclic aromatic hydrocarbon by using n-hexane: adding 50mL of sodium chloride solution with the mass fraction of 4% into the N-dimethyl phthalein amine extracting solution in a separating funnel, uniformly mixing, adding 8mL of n-hexane, sufficiently shaking, uniformly mixing, standing, wherein the upper layer liquid is an n-hexane phase, the lower layer liquid is a water phase, reserving the n-hexane phase for later use, continuously adding 8mL of n-hexane into the water phase for extraction once, and combining the two n-hexane phases to obtain an n-hexane phase extracting solution;

3. drying and concentrating: the n-hexane phase extract was transferred to a glass tube, dried over anhydrous sodium sulfate and concentrated to 2mL with nitrogen at 20 ℃.

Thirdly, purifying the target analyte by SPE method

a) Activating and balancing, namely activating twice by 6mL of dichloromethane and balancing twice by 6mL of n-hexane;

b) loading a sample, adding 2mL of solution to be purified into a small column, and discarding an effluent liquid;

c) leaching, namely leaching by using 5mL of n-hexane, leaching the small column by using 5mL of n-hexane/dichloromethane (95:5, v/v), and discarding leaching solution;

d) elution, 5mL of n-hexane: eluting with dichloromethane (1:2, v/v), and collecting eluate;

e) redissolving, concentrating the eluent to 1mL under reduced pressure, adding 500 μ L toluene, keeping blowing to within 20mg, and performing GC-QQQ-MS detection analysis by using dichloromethane to reach a constant volume of 250 μ L.

Fourthly, GC-QQQ-MS detection and analysis

And (4) referring to mass spectrometer parameters of the standard curve formulated in the first step, and analyzing by using instrument analysis software to obtain the contents of the four PAHs in the milk powder.

The method comprises the following steps:

recovery (recovery), in-day precision (day) of the four analytes in a milk powder blank at two spiking levels of 5. mu.g/kg and 10. mu.g/kg^aRSD_r) Day time precision (^bRSD_R) The detection limits and the quantification limits are shown in Table 1.

TABLE 19 Mass Spectrometry parameters and method validation results for fluorenone, anthraquinone, 6-chlorobenzpyrene and 7-chlorobenzanthracene

Note:^aRSD_rrelative standard deviation under the condition of repeated experiments, n is 6;

^bRSD_Rrelative standard deviation under intermediate precision experimental conditions, n is 6.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments. Even if various changes are made to the present invention, it is still within the scope of the present invention if they fall within the scope of the claims of the present invention and their equivalents.