WO2023088166A1 - Mass spectrometry method for perfluoroalkyl ether carboxylic acid and use thereof - Google Patents

Abstract

The present invention relates to a mass spectrometry method for perfluoroalkyl ether carboxylic acid (PFECA) and the use thereof. In the present invention, [CF₃(CF₂)_nO] or [M-H-CO₂] is selected as a parent ion, other fluorinated alkoxy and alkyl fragments are used as daughter ions, new ion pairs are constructed, and ultra-sensitive detection of PFECA in various samples is achieved by optimizing cone voltage and collision energy.

Description

A mass spectrometry method for perfluoroalkyl ether carboxylic acids and its application

The present invention claims the priority of the Chinese patent application submitted to the China Patent Office on November 22, 2021, with the application number 202111385897.0 and the title of the invention "A Mass Spectrometry Method for Perfluoroalkyl Ether Carboxylic Acids and Its Application" , the entire contents of which are incorporated herein by reference.

technical field

The invention belongs to the technical field of mass spectrometry detection, and in particular relates to a method for ultrasensitive detection of perfluoroalkyl ether carboxylic acids based on ultra-high performance liquid chromatography-tandem mass spectrometry and an application thereof.

Background technique

The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the main technique for the analysis of PFASs (per- and polyfluorinated compounds). The detection technology about PFECA (perfluoroalkyl ether carboxylic acid) in the prior art is usually based on taking [MH] ^- as the parent ion, and other characteristic ion fragments are product ions to form multiple reaction monitoring (MRM) ion pairs, but for some For the detection of PFECA, the detection limit of this method is relatively high, which cannot meet the needs of actual detection. Typically, PFASs generate [MH] ^-precursor ions in electrospray (ESI) negative mode, then fragmentation occurs in a collision cell, and product ions are detected in MRM mode. However, PFECA (perfluoroalkylether carboxylic acids) may undergo in-source cleavage and loss of _CO2 neutrality when ionized by an ESI source, manifested as the cleavage of ether oxygen bonds and loss of carboxyl groups in the PFECA backbone. For the above reasons, the abundance of conventional [MH] ^-precursor ions is not high, and some in-source ions and [MH-CO ₂ ] fragment ions with the same retention time as the target analytes will be produced, which affects the ultra-sensitivity of PFECA detection, the mass spectrometry response of PFECA is far inferior to traditional PFASs.

Contents of the invention

Aiming at the low abundance of [MH] ^- due to the breakage of PFECA ether oxygen bonds and the neutral loss of CO ₂ in ESI negative ion mode, the present invention provides a mass spectrometry method for PFECA and its application, selecting [CF ₃ (CF ₂ ) _n O] or [MH-CO ₂ ] as parent ions, other fluorinated alkoxy and alkyl fragments as product ions, construct new ion pairs, and realize the pair by optimizing the cone voltage and collision energy Ultrasensitive detection of PFECA in various samples.

In order to solve the above technical problems, the technical solution of the present invention is:

In the first aspect, the application provides a mass spectrometry method for ultra-sensitive detection of PFECA, the steps comprising: (1) preparing the product into a sample to be tested; (2) performing UPLC-MS/MS detection on the sample to be tested; In ESI negative ion mode, perform a full scan on PFECA to obtain [MH-CO ₂ ] or [CF ₃ (CF ₂ ) _n O] fragments due to the neutral loss of CO ₂ or the breakage of ether oxygen bonds; secondly, select the appropriate [MH -CO ₂ ] or [CF ₃ (CF ₂ ) _n O] fragments are used as parent ions, and product ion scanning is performed on them to obtain product ion information; (3) Obtain the content of perfluoroalkyl ether carboxylic acids in the product.

In some embodiments, ultrasensitive detection of two PFECAs. The chemical structures of the two PFECAs are as follows:

(Perfluoro-2-propoxypropionic acid, GenX)

(perfluoro-3,6-dioxaheptanoic acid, PFO ₂ HpA).

Furthermore, the specific steps of the GenX mass spectrometry detection are to perform a full scan of the GenX secondary mass spectrometer to obtain the precursor ion of [MH-CO ₂ ] m/z 285 produced by the neutral loss of CO ₂ . A product ion scan was performed on m/z 285 to obtain the characteristic product ions of m/ z 185, 169, and 119 formed by the breakage of the ether oxygen bond at m/z 285 and the further loss of CF ₂ fragments. The three ion pairs that established GenX are 285/185, 285/169, and 285/119.

Further, the specific steps of mass spectrometry detection of PFO ₂ HpA are to perform full-scanning of PFO ₂ HpA by MS/MS to obtain [M-CF ₂ COOH] ^- m/z 201 parent ion generated by the breakage of ether oxygen bond. A product ion scan was performed on m/z 201 to obtain the characteristic product ion m/z 85 formed by the breakage of the ether oxygen bond. The transition for PFO ₂ HpA was established as 201/85.

In some embodiments, step (1) specifically includes: grinding or crushing the sample to be tested, adding an organic solvent, using the QuEChERS method to extract and purify the sample to be tested, and performing ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS /MS) detection.

In some embodiments, the organic solvent is acetonitrile, ethyl acetate, dichloromethane. In some embodiments, when the sample to be tested is a biological sample, it is necessary to add acid to the biological sample for protein precipitation or high-speed centrifugation to obtain the sample to be tested.

In some embodiments, chromatographic detection conditions: chromatographic column: Waters Acquity BEH C18 column (1.7 μ m, 2.1 * 50mm); Mobile phase: 2.5mmol/L ammonium acetate aqueous solution/methanol=95/5 (A), methanol/2.5 mmol/L ammonium acetate aqueous solution=95/5(B); flow rate is 0.3mL/min; gradient elution program: 0～0.2min, 95%～5%A; 0.2～3min, 5%A; 3～5min, 5%～95%A; injection volume: 1μL.

In some embodiments, mass spectrometry detection conditions: monitoring mode: multiple reaction monitoring; ion source: ESI; scanning mode: negative ion scanning; source temperature: 150°C; cone voltage: 10V; capillary voltage: 2.5kV; desolvation temperature : 450°C; Desolvent gas flow: 1000L/hr; Cone gas flow: 150L/hr; Atomizer pressure: 7.0bar.

In a second aspect, the present invention provides the application of the above-mentioned mass spectrometry method to detect PFECA in products.

In some embodiments, the products include fruits, environmental water samples, food, food contact materials, aquatic products and the like. Preferably, the environmental water samples include drinking water, surface water, ground water, seawater, and industrial wastewater. Food includes fruits and vegetables, meat, eggs, milk and dairy products, among which fruits and vegetables include apples, watermelons, grapes, blueberries, kiwis, lettuces, radishes, Chinese cabbage cucumbers, tomatoes, cabbage and beans, etc.; meat mainly There are chicken, beef, pork and mutton; eggs mainly include eggs, duck eggs and some egg products; milk and dairy products mainly include fresh milk, milk powder and human breast milk. Food contact materials mainly include plastic bags, food packaging bags and plastic wrap. Aquatic products mainly include various edible freshwater fish, marine fish, shellfish, kelp, etc.

In some embodiments, PFASs include carboxylic acids, sulfonic acids, and phosphoric acid perfluorinated compounds with different carbon chain lengths, as well as chlorine-substituted or hydrogen-substituted fluorine-containing compounds with oxygen atoms inserted into the carbon chain, such as: PFOA, Perfluorooctadecanoic acid, perfluorohexadecanoic acid, PFOS, perfluorobutanesulfonic acid, perfluoropentanoic acid, perfluorohexylphosphonic acid, sodium dodecafluoro-3H-4,8-dioxononanoate, 1-Chloropolyfluoroalkyl ether sulfonic acid, GenX and PFO ₂ HpA, etc. Further, PFASs are GenX or PFO ₂ HpA.

Beneficial effects of the present invention:

The invention discloses an ultrasensitive detection method of PFECA. The method uses UPLC-MS/MS technology, using electrospray negative ion tandem mass spectrometry detection mode (ESI ^- -MRM), using [CF ₃ (CF ₂ ) _n O] or [MH-CO ₂ ] as the parent ion, and other secondary Fluorinated alkoxy and alkyl fragments are used as product ions to form a series of characteristic MRM ion pairs, which improves the mass spectrometry sensitivity of PFECA and increases the response of mass spectrometry to it by 4 to 5 orders of magnitude.

The PFECA ultrasensitive detection method of the present invention has high sensitivity and low detection limit, and is obviously superior to the methods reported in literature.

Description of drawings

The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention, and the schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations to the present invention. Embodiments of the present invention are described in detail below in conjunction with the accompanying drawings, wherein:

Fig. 1 a and b are the secondary mass spectrogram and product ion scanning figure of GenX under the negative ion mode of embodiment 1;

Fig. 2 a and b are the secondary mass spectrogram and product ion scanning figure of PFO ₂ HpA under the negative ion mode of embodiment 2;

Fig. 3 a and b are the PFECA (1ng/mL) UPLC-MS/MS figure under the condition of table 2 and table 1 respectively of embodiment 3;

Fig. 4 is the UPLC-MS/MS figure of GenX and PFO ₂ HpA in embodiment 4 rape;

Fig. 5 is a UPLC-MS/MS graph of GenX and PFO ₂ HpA in the industrial wastewater of Example 6.

Detailed ways

It should be noted that the following detailed description is exemplary and intended to provide further explanation of the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It should be noted that the terminology used here is only for describing specific implementations, and is not intended to limit the exemplary implementations according to the present application. As used herein, unless the context clearly dictates otherwise, the singular is intended to include the plural, and it should also be understood that when the terms "comprising" and/or "comprising" are used in this specification, they mean There are features, steps, operations, means, components and/or combinations thereof.

As introduced in the background technology, some MRM quantitative methods of PFECA in the prior art using [MH] ^- as the parent ion have low mass spectrometry response and cannot meet the needs of actual detection. In order to solve the above technical problems, this application proposes a new mass spectrometry method based on ion pair optimization, using [CF ₃ (CF ₂ ) _n O] or [MH-CO ₂ ] as the parent ion, and other secondary fluorinated alkanes Oxygen and alkyl fragments are used as product ions to form a series of characteristic MRM ion pairs, which realizes the trace detection of PFECA in various actual samples.

Below in conjunction with embodiment the present invention is further described.

Example 1, the [MH-CO ₂ ] m/z 285 parent ion generated due to the neutral loss of CO ₂ was obtained by performing a full scan of the second-order mass spectrometer on GenX. The product ion scan was performed on m/z 285 to obtain the characteristic product ions of m/ z 185, 169, and 119 formed by the further breakage of the ether oxygen bond of the parent ion and the further loss of the CF ₂ fragment. The three ion pairs that established GenX are 285/185, 285/169, and 285/119 (Table 1). Figure 1a is the MS/MS spectrum of GenX in negative ion mode, and Figure 1b is the product ion scan of GenX.

In Example 2, the [M-CF ₂ COOH] ^- m/z 201 parent ion generated due to the breakage of the ether oxygen bond was obtained by performing a full scan of the MS/MS on PFO ₂ HpA. A product ion scan was performed on m/z 201 to obtain the characteristic product ion m/z 85 formed by further breaking the ether oxygen bond of the parent ion. The transition for PFO ₂ HpA was established as 201/85 (Table 1). Figure 2a is the secondary mass spectrum of PFO ₂ HpA in negative ion mode, and Figure 2b is the product ion scan of PFO ₂ HpA.

Example 3, combined with the MRM mass spectrometry conditions of GenX and PFO ₂ HpA in the literature (Table 2), UPLC-MS/MS comparative detection of 1 ng/mL PFECA was performed. Compared with the conventional MRM with [MH] ^- as the parent ion, the MRM conditions established in this paper, such as [CF ₃ (CF ₂ ) _n O] and [MH-CO ₂ ], can improve the mass spectral response of PFECA by 4 to 5 orders of magnitude , showing that the invention is more conducive to the trace detection of PFECA. Figure 3a is the UPLC-MS/MS diagram of PFECA (1ng/mL) under the conditions of Table 2, and Figure 3b is the UPLC-MS/MS diagram of PFECA (1ng/mL) under the conditions of Table 1.

Table 1 MRM detection conditions of PFECA after optimization

Table 2 PFECA routine MRM testing conditions

Embodiment 4, for the detection of fruit and vegetable samples, in this embodiment, rapeseed is taken as an example to illustrate the detection effect.

Weigh 7.5 g of rapeseed and pulverize it into a 50 mL polypropylene centrifuge tube, add 15 mL of acetonitrile and vortex for 5 min. Add 6.0 g of anhydrous magnesium sulfate and 1.5 g of sodium chloride, shake quickly to mix, shake for 15 min, and centrifuge at 10,000 r/min for 5 min. Take the above 6mL supernatant in a 15mL high-speed centrifuge tube, add 1.8g of anhydrous magnesium sulfate, vortex for 5min, centrifuge at 10000r/min for 5min, take 4mL of the supernatant to pass through a 0.22μm filter membrane, and transfer to a glass bottle. Evaporate to dryness under nitrogen flow at 40°C, reconstitute with 100 μL methanol, and perform UPLC-MS/MS analysis.

The results show that the GenX content in rapeseed obtained by the method established by the present invention is 0.02ng/g, and PFO ₂ HpA is not detected. Fig. 4 is a UPLC-MS/MS diagram of GenX and PFO ₂ HpA in rapeseed.

Embodiment 5, for the detection of meat samples, in this embodiment, pork is taken as an example to illustrate the detection effect.

Weigh 7.5 g of pork and pulverize it into a 50 mL polypropylene centrifuge tube, add 15 mL of acetonitrile and vortex for 5 min. Add 6.0 g of anhydrous magnesium sulfate and 1.5 g of sodium chloride, shake quickly to mix, shake for 15 min, and centrifuge at 10,000 r/min for 5 min. Take the above 6mL supernatant in a 15mL high-speed centrifuge tube, add 0.5mL trifluoroacetic acid and 1.8g anhydrous magnesium sulfate, vortex for 5min, centrifuge at 10000r/min for 5min, take 4mL supernatant and pass through a 0.22μm filter membrane, Transfer to a glass bottle. Evaporate to dryness under nitrogen flow at 40°C, reconstitute with 100 μL methanol, and perform UPLC-MS/MS analysis.

The results show that the content of GenX and PFO ₂ HpA in pork obtained by the method established by the present invention is 0.3ng/g and 0.2ng/g.

Embodiment 6, aiming at the detection of aquatic product samples, in this embodiment, hairtail is taken as an example to illustrate the detection effect.

Skinned and deboned hairtail, obtained edible part, weighed 7.5g and pulverized into a 50mL polypropylene centrifuge tube, added 15mL acetonitrile and vortexed for 5min. Add 6.0 g of anhydrous magnesium sulfate and 1.5 g of sodium chloride, shake quickly to mix, shake for 15 min, and centrifuge at 10,000 r/min for 5 min. Take the above 6mL supernatant in a 15mL high-speed centrifuge tube, add 0.5mL trifluoroacetic acid and 1.8g anhydrous magnesium sulfate, vortex for 5min, centrifuge at 10000r/min for 5min, take 4mL supernatant and pass through a 0.22μm filter membrane, Transfer to a glass bottle. Evaporate to dryness under nitrogen flow at 40°C, reconstitute with 100 μL methanol, and perform UPLC-MS/MS analysis.

The results show that the GenX content in the hairtail obtained by the method established by the present invention is 1.5ng/g, and the PFO ₂ HpA content is 0.7ng/g.

Embodiment 6, aiming at the detection of environmental water samples, in this embodiment, industrial wastewater is taken as an example to illustrate the detection effect.

Pipette 7.5mL water sample, put it in a 50mL centrifuge tube, add 15mL acetonitrile, vortex for 5min, let it stand for 5min, take the above 6mL organic layer in a centrifuge tube, add 25mg PSA and 25mg GCB for purification, and centrifuge at 10000r/min for 5min Finally, 4 mL of the supernatant was passed through a 0.22 μm filter membrane and transferred to a glass bottle. Evaporate to dryness under nitrogen flow at 40°C, reconstitute with 100 μL methanol, and perform UPLC-MS/MS analysis.

The results show that the GenX content in the industrial wastewater obtained by the method established by the present invention is 8.3ng/g, and the PFO ₂ HpA content is 2.6ng/g. Fig. 5 is a UPLC-MS/MS diagram of GenX and PFO ₂ HpA in industrial wastewater.

Embodiment 7, for the detection of milk samples, in this embodiment, milk is taken as an example to illustrate the detection effect.

Take 7.5g of milk in a 50mL polypropylene centrifuge tube, add 15mL of acetonitrile and 0.5mL of trifluoroacetic acid, vortex for 5min, let it stand for 5min, take the above 6mL organic layer in a centrifuge tube, add 25mg of PSA and 25mg of GCB to purify, and use 10000r After centrifugation for 5 min at 1/min, 4 mL of the supernatant was passed through a 0.22 μm filter membrane and transferred to a glass bottle. Evaporate to dryness under nitrogen flow at 40°C, reconstitute with 100 μL methanol, and perform UPLC-MS/MS analysis.

The results showed that GenX and PFO ₂ HpA were not detected in milk by the method established by the present invention.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (10)

1. A mass spectrometry method for ultrasensitive detection of perfluoroalkyl ether carboxylic acids, characterized in that it comprises the following steps: (1) preparing the product into a sample to be tested; (2) subjecting the sample to be tested to an ultra-high performance liquid phase Chromatography-tandem mass spectrometry detection; in ESI negative ion mode, perform a full scan on perfluoroalkyl ether carboxylic acids to obtain [MH-CO ₂ ] or [CF _{3 (} CF ₂ ) _n O] fragment; secondly, select the appropriate [MH-CO ₂ ] or [CF ₃ (CF ₂ ) _n O] fragment as the precursor ion, and perform product ion scanning on it to obtain product ion information; (3) get Content of perfluoroalkyl ether carboxylic acids in the product.
2. The mass spectrometry method according to claim 1, wherein the perfluoroalkyl ether carboxylic acid is GenX or PFO ₂ HpA.
3. The mass spectrometry method according to claim 2, characterized in that, GenX is carried out to a full scan of the secondary mass spectrometer to obtain the [MH- _{CO 2 ]} m/z 285 precursor ion produced by the neutral loss of CO ; to m/z 285 Product ion scanning to obtain m/z 185, 169, and 119 characteristic product ions formed by ether oxygen bond breakage at m/z 285 and further loss of CF ₂ fragments; three ion pairs of GenX were established as 285/185, 285/169 , 285/119.
4. The mass spectrometry method according to claim 2, characterized in that, PFO ₂ HpA is subjected to a full scan of the secondary mass spectrum to obtain the [M-CF ₂ COOH] ^-m /z 201 parent ion produced by the breakage of the ether oxygen bond; Z 201 carried out product ion scanning to obtain the characteristic product ion m/z 85 formed by the breakage of ether oxygen bond; the ion pair of PFO ₂ HpA was established as 201/85.
5. The mass spectrometry method according to claim 1, wherein the step (1) specifically includes: grinding or crushing the sample to be tested, adding an organic solvent, using the QuEChERS method to extract and purify the sample to be tested, and perform ultra-efficient Liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) detection.
6. The mass spectrometry method according to claim 5, characterized in that, the organic solvent is acetonitrile, ethyl acetate, dichloromethane; preferably, when the sample to be tested is a biological sample, it is necessary to add acid to the biological sample for protein extraction. The sample to be tested is obtained after precipitation or high-speed centrifugation.
7. The mass spectrometry method according to claim 1, characterized in that chromatographic detection conditions: chromatographic column: Waters Acquity BEH C18 column, 1.7 μm, 2.1 × 50mm; mobile phase: 2.5mmol/L ammonium acetate aqueous solution/methanol=95/5 (A), methanol/2.5mmol/L ammonium acetate aqueous solution=95/5 (B); flow rate is 0.3mL/min; gradient elution program: 0～0.2min, 95%～5%A; 0.2～3min, 5% %A; 3~5min, 5%~95%A; injection volume: 1μL.
8. The mass spectrometry method according to claim 1, characterized in that, mass spectrometry detection conditions: monitoring mode: multiple reaction monitoring (MRM); ion source: ESI; scanning mode: negative ion scanning; source temperature: 150 ° C; cone voltage: 10V ; Capillary voltage: 2.5kV; Desolvation temperature: 450°C; Desolvation flow rate: 1000L/hr; Cone gas flow rate: 150L/hr;
9. The application of the mass spectrometry method according to any one of the above claims in the detection of perfluoroalkyl ether carboxylic acids in products.
10. The application according to claim 9, wherein the product includes environmental water samples, food, food contact materials or aquatic products; preferably, the environmental water samples include drinking water, surface water, groundwater, seawater or industrial wastewater; Food includes fruits and vegetables, meat, eggs and milk or dairy products; food contact materials include plastic bags, food packaging bags or plastic wrap; aquatic products include freshwater fish, marine fish, shellfish or kelp; the perfluoroalkyl Ether carboxylic acids include carboxylic acids of different carbon chain lengths, sulfonic acids, phosphoric acid perfluorinated compounds, and chlorine-substituted or hydrogen-substituted fluorine-containing compounds; preferably, including PFOA, perfluorooctadecanoic acid, perfluorinated Hexadecanoic acid, PFOS, perfluorobutanesulfonic acid, perfluoropentanoic acid, perfluorohexylphosphonic acid, sodium dodecafluoro-3H-4,8-dioxononanoate, 1-chloropolyfluoroalkyl ether sulfonate acid, GenX, and _PFO2HpA ; more preferably, GenX or _PFO2HpA .

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