CN115541788A

CN115541788A - Method for rapidly detecting four perfluorinated compounds in dairy product

Info

Publication number: CN115541788A
Application number: CN202211259093.0A
Authority: CN
Inventors: 秦燕燕; 蹇斌; 牛茂; 洪华嫦
Original assignee: Shenzhen Polytechnic
Current assignee: Shenzhen Polytechnic
Priority date: 2022-10-14
Filing date: 2022-10-14
Publication date: 2022-12-30

Abstract

The invention provides a method for rapidly detecting four perfluorinated compounds in a dairy product, which comprises the following steps: step S1, carrying out centrifugal treatment on a sample to be detected, taking supernatant, adding a treating agent, uniformly mixing, and purifying by a phospholipid removing plate to obtain a purified sample, wherein the treating agent is a 1~5% methanoic acid solution; and S2, determining the contents of perfluorooctanoic acid, perfluorooctane sulfonic acid, perfluorononanoic acid and perfluorodecanoic acid in the purified sample by a liquid chromatography-mass spectrometry method. According to the technical scheme, the concentration level of PFCs in the dairy product is detected by adopting a phospholipid-removing plate extraction-liquid chromatography-mass spectrometry method, and compared with a C18 adsorbent separation solid phase extraction method, the method has the advantages that the precision, the recovery rate and the sample treatment efficiency of PFCs in the dairy product are obviously improved, and the experiment time is effectively saved; and the method has the advantages of good reproducibility, strong sensitivity, high recovery rate and simple operation, and is suitable for rapidly determining four PFCs in the dairy products.

Description

Method for rapidly detecting four perfluorinated compounds in dairy product

Technical Field

The invention belongs to the technical field of dairy product detection, and particularly relates to a rapid detection method for four perfluorinated compounds in a dairy product.

Background

Perfluorinated compounds (PFCs) are a class of common artificially synthesized organic compounds, including perfluorooctanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA) and the like, have the characteristics of water resistance, oil resistance, good stability and the like, and are widely applied to industrial products and daily necessities such as textiles, cosmetics, non-stick coating materials and the like. The perfluorinated compounds can exist in the environment for a long time, enter organisms through ways of breathing, diet and the like, are accumulated and amplified in the organisms, and can influence the health of the reproductive system, endocrine and nervous system of a human body after PFCs enter the human body. Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) are the two most frequently used perfluorinated compounds. In 2009, PFOS was formally recognized as a persistent organic pollutant. Clearly, research to develop detection of perfluorinated compounds (PFCs) is imminent.

Monitoring studies of human PFCs have been widely reported. Body fluids such as blood, urine and breast milk are common media in the monitoring of human pollutants. At present, the blood and urine detection method is relatively mature, dairy products such as breast milk serve as main food sources of infants, and compared with the blood and urine, the PFCs pollution in the dairy products can directly affect the health of the infants. Therefore, the development of a detection method of PFCs in the dairy products has important significance for blocking the transfer of PFCs between mothers and infants and protecting the physical and mental health of infants.

Aiming at human tissue samples, phospholipid substances and matrix effects can seriously influence detection data of UPLC-MS/MS due to the high content of protein and phospholipid in blood and dairy products, and the existing research methods cannot well solve the problems.

Disclosure of Invention

Aiming at the technical problems, the invention discloses a method for rapidly detecting four perfluorinated compounds in a dairy product, which greatly improves the precision and the repeatability of detection.

In contrast, the technical scheme adopted by the invention is as follows:

a method for rapidly detecting four perfluorinated compounds in a dairy product comprises the following steps:

step S1, carrying out centrifugal treatment on a sample to be detected, taking supernatant, adding a treating agent, uniformly mixing, and purifying by a phospholipid removing plate to obtain a purified sample, wherein the treating agent is 1-5% (volume percentage) of formic acid methanol solution;

and S2, determining the contents of perfluorooctanoic acid, perfluorooctane sulfonic acid, perfluorononanoic acid and perfluorodecanoic acid in the purified sample by using a liquid chromatography-mass spectrometry method.

The technical scheme adopts a phospholipid removing method, so that the protein phospholipid in the dairy product can be effectively eliminated, and the precision and the repeatability of the method are greatly improved.

As a further improvement of the invention, in step S1, the treating agent is a 1% (volume percentage) formic acid methanol solution.

As a further improvement of the invention, in step S2, the chromatographic conditions of the liquid chromatography-mass spectrometry are: the chromatographic column adopts a C18 column; the mobile phase consists of a phase A and a phase B, wherein the phase A is methanol or acetonitrile, and the phase B is 0-0.1% (volume percentage) formic acid aqueous solution added with 1-50 mmol/mL ammonium acetate; gradient elution is adopted;

the mass spectrum conditions of the liquid chromatography-mass spectrometry method are as follows: ionization in negative ion electrospray (ESI-) mode, multiple Reaction Monitoring (MRM) mode, capillary voltage 2800V, CID gas 1.5mTorr, dry gas temperature of 350 deg.C, ion transport tube temperature of 350 deg.C, sheath gas flow of 55Arb, and auxiliary gas flow of 12Arb.

As a further improvement of the invention, in step S2, the chromatographic column is an ACQUITY UPLC BEH C ₁₈ 。

As a further improvement of the invention, in step S2, phase A is methanol and phase B is 0.1% (volume percent) formic acid aqueous solution added with 1mmol/mL ammonium acetate.

The ion transfer tube temperature was 350 ℃, the sheath gas flow was 55Arb, and the auxiliary gas flow was 12Arb.

As a further improvement of the present invention, the procedure of gradient elution is: 0-0.5min, 10% of phase A; increasing the phase A to 95% by weight after 0.5-10 min; 10-13min, 10% of phase A. Wherein the percentages are volume percentages.

As a further improvement of the present invention, in step S2, the mass spectrum parameters are as follows:

compared with the prior art, the invention has the beneficial effects that:

according to the technical scheme, the concentration level of PFCs in the dairy product is detected by adopting a phospholipid-removing plate extraction-ultra-high efficiency liquid chromatography-mass spectrometry method, and compared with a C18 adsorbent separation solid phase extraction method, the method remarkably improves the precision, recovery rate and sample processing efficiency of PFCs detection in the dairy product, and effectively saves the experiment time. The method has the advantages of good reproducibility, strong sensitivity, high recovery rate and simple operation, and is suitable for rapidly measuring four PFCs in the dairy products.

Drawings

FIG. 1 is a mixed standard solution chromatogram of 4 PFCs according to an example of the present invention.

Detailed Description

Preferred embodiments of the present invention are described in further detail below.

Example 1

A method for rapidly detecting four perfluorinated compounds in breast milk comprises the following steps:

step S1, carrying out centrifugal treatment on a sample to be detected, taking supernatant, adding 1% (volume percentage, the same below) formic acid methanol solution serving as a treating agent, uniformly mixing, and purifying by a phospholipid removing plate to obtain a purified sample;

and S2, determining the contents of the four perfluorinated compounds in the purified sample by using a liquid chromatography-mass spectrometry method.

In step S2, the chromatographic conditions of the liquid chromatography-mass spectrometry are: the chromatographic column adopts ACQUITY UPLC BEH C ₁₈ A column; the mobile phase consists of a phase A and a phase B, wherein the phase A is methanol, and the phase B is 0.1% (volume percentage, the same below) formic acid aqueous solution added with 1mmol/mL ammonium acetate; gradient elution is adopted; the procedure for the gradient elution was: 0-0.5min, 10% of phase A; 0.5-10min, gradually increasing the phase A to 95 percent; 10-13min, 10% of phase A.

The following description will be given with reference to specific examples.

(1) Sample preparation:

the sample adopts a volunteering donation mode, donors live in Shenzhen for more than 5 years, are 22-30 years old, do not have the working experience (without professional exposure) of heavily polluted enterprises such as chemical plants, paint plants and the like during the live period, and collect 15 breast milk samples in total. The breast milk samples were collected manually, 50mL of each breast milk was collected and placed in a glass container cleaned with methanol, and the samples were immediately stored in a refrigerator at-20 ℃. All donors were informed of the purpose of the study and signed informed consent.

(2) Instruments and reagents:

a triple quadrupole mass spectrometer TSQ-Quantum Ultra (Thermo Fisher, USA) is adopted, a liquid chromatography system is provided with an automatic sample injector for Dionex U3000 liquid chromatography, and liquid phase is controlled by a Chromeleon workstation. The analytical column was Hypersil GOLD (100 mm. Times.2.1mm, 3 μm, thermo Fisher Co., U.S.A.), ACQUITY UPLC BEH C ₁₈ (100 mm. Times.2.1 mm,1.7 μm, waters Corp., U.S.A.), eclipse Plus C ₁₈ RRHD (50 mm. Times.2.1mm, 1.8 μm, agilent, USA), ACQUITY UPLC HSS T3 (50 mm. Times.2.1mm, 1.8 μm, waters, USA).

All experimental waters were obtained from Milli-Q silicon ultra pure water system (Millipore, usa, 18.2M Ω cm); 100-1000. Mu.L and 20-200. Mu.L pipettes were purchased from Eppendorf, 1-10 mL neck dispensers were purchased from Sartorius, germany, 96 Kong Guxiang extraction devices were purchased from Waters, ostro 96 well phospholipid plates were purchased from Waters, bench top high speed centrifuges 1-14 were purchased from Sigma, germany, and 50mL polypropylene centrifuge tubes were purchased from Thermo Fisher, america.

100 μ g/mL perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluorooctanoic acid (PFOA)Alkanesulfonic acid (PFOS) standards were purchased from Sigma-Aldrich, america, merck, for laboratory methanol (chromatographically pure), merck, germany, ammonium acetate, formic acid (chromatographically pure), tedia, america, anhydrous MgSO ₄ Both (analytical purity) and NaCl (analytical purity) were purchased from national pharmaceutical group chemical reagents, inc., C ₁₈ The adsorbent was purchased from Agilent technologies, inc.

(3) Preparation of Standard solutions

Accurately sucking 100 mu g/mL of standard solution, preparing 5 gradient standard concentration mixed standard solutions by using the initial proportion solution of the mobile phase for preparing a working curve, wherein the concentration of the mixed standard solutions is 0.05, 0.1, 1, 10 and 50ng/mL respectively, and sealing and storing in a refrigerator at 4 ℃. Adding different amounts of mixed standard solutions into the blank breast milk to obtain 3 quality control solutions with different concentrations, wherein the concentrations are respectively 0.5 ng/mL, 5 ng/mL and 50ng/mL, and the quality control solutions are used for low concentration, medium concentration and high concentration quality control and are used for calculating the recovery rate, the intra-day precision and the inter-day precision.

(4) Control of pre-processing

4 samples with very low PFCs content (signal-to-noise ratio less than 3) were used as breast blanks, and the concentrations were used as baseline values for blank control. In order to avoid interference introduced in the pretreatment process, all containers used in the experiment are glass products.

(5) Sample pretreatment

After 1mL breast milk sample is centrifuged at high speed, 0.4mL supernatant is taken, 0.6mL 1% (volume percentage) methanoic acid solution is added, the mixture is uniformly mixed and passes through a 96 Kong Qulin lipid plate, and the pressure of a vacuum pump is controlled to enable the sample to flow out in 4 min. The purified sample was transferred to a 2mL sample bottle for qualitative analysis.

(6) Conditions of liquid chromatography

Ultra-high performance liquid chromatography column: ACQUITY UPLC BEH C ₁₈ (ii) a Column temperature: 40 ℃; mobile phase: the phase A is methanol, and the phase B is 0.1 percent (volume percentage) of formic acid water and 1mmol/mL of ammonium acetate; the gradient elution procedure is shown in table 1; the sample volume of the automatic sample introduction system is 10 mu L.

TABLE 1 gradient elution procedure

(7) Conditions of Mass Spectrometry

Ionization in negative ion electrospray (ESI-) mode, multiple Reaction Monitoring (MRM) mode; capillary voltage 2800V, CID gas 1.5mTorr, dry gas temperature of 350 deg.C, ion transport tube temperature of 350 deg.C, sheath gas flow of 55Arb, and auxiliary gas flow of 12Arb. The mass spectral parameters are shown in table 2.

TABLE 2 Mass Spectrometry parameters

Comparative example 1

This comparative example employed C on the basis of example 1 in step S1 ₁₈ Solid phase adsorbent separation extraction (see, e.g., lankova D, lacina O, pulkrabova J, et al, the determination of perfluorinated substrates, brominated flame retardants and the example of the synthesis of human breast milk and intake for milk [ J]The samples were treated with Talanta,2013, 117 (Complete): 318-325).

Namely, different pretreatment methods are used for 4 PFCs in the sample breast milk, and a 96-hole phospholipid removing plate and C adopted by Lankova and the like are respectively adopted ₁₈ The adsorbent separation solid phase extraction method was used for comparison, and the measurement results are shown in table 3.

As can be seen from the results in Table 3, the recovery of 4 PFCs was better than that of C after the breast samples were extracted on 96-well phospholipid-removing plates ₁₈ Separating solid phase by adsorbent and extracting. Meanwhile, the 96-hole phospholipid removing plate greatly shortens the extraction time and saves the pretreatment time when samples are treated in large batches.

Table 3 results of recovery normalized for two pretreatment methods (n = 5)

Example 2

In this example, the treating agent of step S1 was prepared by using pure methanol solution and 5% formic acid methanol solution, respectively, and compared with example 1, and the results are shown in Table 4. The results show that the treatment with 1% formic acid methanol solution and 5% formic acid methanol solution has a recovery rate significantly better than that of the pure methanol solution, and the treatment with 1% formic acid methanol solution has the best effect.

Table 4 results of recovery normalized by addition of different solvent pretreatment methods (n = 5)

Example 3

In this embodiment, based on embodiment 1, hypersil GOLD, eclipse Plus C are selected in step S2 ₁₈ Several columns RRHD, ACQUITY UPLC HSST 3 were compared with example 1, the mobile phase being methanol: adding 0.1% formic acid into 1mmol/L ammonium acetate, continuously injecting 4 kinds of mixed standard samples with PFCs concentration of 10ng/mL, and comprehensively evaluating the abundance, half-peak width, peak shape, signal-to-noise ratio and other indexes of the 4 compounds in different chromatographic columns by comparing.

Compared with Hypersil GOLD and Eclipse Plus C ₁₈ RRHD, ACQUITY UPLC HSS T3 chromatographic columns, PFOA, PFNA, PFDA in ACQUITY UPLC BEH C ₁₈ The column is obviously superior in performance, the abundance of 3 substances is higher than that of other 3 chromatographic columns, the signal-to-noise ratio is also the highest of 4 chromatographic columns, but the chromatographic behavior of PFOS on the 4 chromatographic columns is not greatly different, and the mixed standard chromatogram of 4 PFCs is shown in figure 1.

Example 4

In this embodiment, an ACQUITY UPLC BEH C is used in step S2 based on embodiment 1 ₁₈ The column was compared with example 1 using several different mobile phase systems, the conditions of which are shown in Table 5. The chromatographic behavior of PFCs in different mobile phases is slightly different, but can basically meet the detection requirements. The following is a specific analysis. When the organic phase is methanol, the abundance of the 4 PFCs is highest in the case of adding 1mmol/L ammonium acetate into 0.1% formic acid and 50mmol/L ammonium acetate solution in the case of the aqueous phase mobile phase, and is highest in the case of 10mmol/L acetic acidAmmonium added 0.1% formic acid water and 50mmol/L ammonium acetate added 0.1% formic acid water were slightly less abundant. However, the signal-to-noise ratios of 1mmol/L ammonium acetate added with 0.1% formic acid and 50mmol/L ammonium acetate solution are slightly different, wherein the signal-to-noise ratio of PFOA in 50mmol/L ammonium acetate solution is slightly higher than that of PFOA in 1mmol/L ammonium acetate added with 0.1% formic acid, the signal-to-noise ratios of PFOS in two aqueous phase mobile phases are not greatly different, the signal-to-noise ratios of PFNA and PFDA in 1mmol/L ammonium acetate added with 0.1% formic acid are obviously higher than that of 50mmol/L ammonium acetate solution, and the reproducibility of 4 PFCs in low-concentration ammonium acetate is better. Under the condition of taking acetonitrile as an organic phase, 4 PFCs only have changed retention time, do not improve the signal-to-noise ratio and the peak pattern, and have reduced abundance.

TABLE 4 optimization of mobile phase systems

Example 5

The method of example 1 was tested for linear range and detection limit, and specifically included:

preparing a series of mixed standard solutions of 4 PFCs with the mass concentration of 0.05-50 ng/mL, continuously injecting samples with each concentration for 5 times, drawing a standard curve by taking the mean value, and finally obtaining a correlation coefficient, a linear interval and a linear regression equation. LOD (detection limit) was calculated based on 3-fold signal-to-noise ratio, and LOQ (quantitative limit) was calculated based on 10-fold signal-to-noise ratio as shown in Table 5.

TABLE 5 Linear regression equation, linear Range, LOD and LOQ for PFCs

The method of example 1 was analyzed for recovery rate, day-to-day precision and day-to-day precision, and specifically included:

accurately sucking 100 mu g/mL of standard mixed solution, adding the standard mixed solution into blank breast milk, preparing samples with the concentration of 0.5, 5 and 50ng/mL at 3 levels respectively to be detected, extracting the prepared samples to be detected through a 96-hole phospholipid removing plate respectively, repeatedly measuring the samples with each concentration for 5 times, sealing the samples after sample injection and storing the samples in a low-temperature refrigerator, and setting the sample injection time for 1, 7, 15, 30 and 45 days in sequence to obtain the precision in the daytime and in the day. The data show that the average recovery rate of the PFCs with low, medium and high concentrations is 88.7-110.1%, the precision in the day is 3.8-8.9%, and the precision in the day is 7.4-10.8%, which indicates that the method has good accuracy and precision.

The results of the tests performed on 5 breast milk samples by the method of example 1 are shown in table 6, and it can be seen that PFOA is detected completely, the detection rate of PFOS is 80%, the detection rates of the 2 PFCs are high, the detection rates of PFNA and PFDA are 20% and 0%, respectively, and the detection rates of the two PFCs are low.

TABLE 6 content of 4 PFCs in 5 parts of breast milk

According to the experimental results, the precision and reproducibility of the ultra-high performance liquid chromatography-mass spectrometry are easily affected by the matrix effect, particularly for breast milk samples, phospholipid substances are one of main factors interfering with the reproducibility and precision of the ultra-high performance liquid chromatography-mass spectrometry, the influence of the protein phospholipid can be eliminated by the phospholipid plate pre-treatment method, the reproducibility and precision of the determination method can be improved, and the determination method can achieve convenient and rapid effects. And C ₁₈ Compared with the adsorbent solid phase extraction method, the dephosphorized lipid plate pretreatment method used in the method has certain advantages, not only reduces complex and tedious pretreatment processes, realizes the treatment requirement of large-scale samples, reduces pretreatment time, and greatly improves recovery rate, but also can well reduce background noise and remarkably improve signal-to-noise ratio due to the good elimination effect of the protein and phospholipid components in the breast milk, thereby being good for helping to reduce the minimum detection limit and the quantitative detection limit, and providing more choices for the detection of trace PFCs.

The foregoing is a further detailed description of the invention in connection with specific preferred embodiments and it is not intended to limit the invention to the specific embodiments described. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

Claims

1. A method for rapidly detecting four perfluorinated compounds in a dairy product is characterized by comprising the following steps:

step S1, carrying out centrifugal treatment on a sample to be detected, taking supernatant, adding a treating agent, uniformly mixing, and purifying by a phospholipid removing plate to obtain a purified sample; wherein the treating agent is 1-5% methanoic acid solution;

and S2, determining the contents of perfluorooctanoic acid, perfluorooctane sulfonic acid, perfluorononanoic acid and perfluorodecanoic acid in the purified sample by a liquid chromatography-mass spectrometry method.

2. The method for rapidly detecting four perfluorinated compounds in the dairy product according to claim 1, which is characterized in that: in step S1, the treating agent is a 1% methanoic acid solution.

3. The method for rapidly detecting four perfluorinated compounds in the dairy product according to claim 1, which is characterized in that: in step S2, the chromatographic conditions of the liquid chromatography-mass spectrometry are: the chromatographic column adopts a C18 column; the mobile phase consists of a phase A and a phase B, wherein the phase A is methanol or acetonitrile, and the phase B is 0-0.1% formic acid aqueous solution added with 1-50 mmol/mL ammonium acetate; gradient elution is adopted;

the mass spectrum conditions of the liquid chromatography-mass spectrometry method are as follows: ionization in a negative ion electrospray mode, a multi-reaction monitoring mode, capillary voltage 2800V, CID gas 1.5mTorr, dry gas temperature of 350 ℃, ion transport tube temperature of 350 ℃, sheath gas flow of 55Arb, and auxiliary gas flow of 12Arb are adopted.

4. Four perfluorocompounds in a dairy product according to claim 3The rapid detection method is characterized in that: in step S2, the chromatographic column adopts ACQUITY UPLC BEH C ₁₈ 。

5. The method for rapidly detecting four perfluorinated compounds in the dairy product according to claim 4, wherein the method comprises the following steps: in step S2, phase A was methanol and phase B was 0.1% aqueous formic acid to which 1mmol/mL ammonium acetate was added.

6. The method for rapidly detecting four perfluorinated compounds in the dairy product according to claim 5, wherein the method comprises the following steps: the procedure for the gradient elution was: 0-0.5min, 10% of phase A; 0.5-10min, gradually increasing the phase A to 95 percent; 10-13min, 10% of phase A.

7. The method for rapidly detecting four perfluorinated compounds in the dairy product according to claim 6, wherein the method comprises the following steps: in step S2, the mass spectrum parameters are as follows: