Method for removing phospholipid in food and biological sample matrix

Abstract

The invention discloses a method for removing phospholipids in food and biological sample matrixes, and belongs to the technical field of analysis and detection. According to the method, in the pretreatment process of food and biological sample matrixes, an improved inorganic zirconium filler is utilized to perform pretreatment on a sample, phospholipid in the sample can be simply and efficiently removed, and accurate analysis or quantification of subsequent liquid chromatography-mass spectrometry / mass spectrometry is facilitated. By using the method, the lipid can be removed more cleanly, and the thorough lipid removal can be realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical detection, and particularly relates to a method for removing phospholipids from food and biological sample matrices. Background Art

[0002] Phospholipids are the main components of all cell membranes and are widely present in all animal-derived foods and biological samples (including serum, plasma, and whole blood). Even extremely trace amounts of phospholipids can cause ion suppression due to competitive ionization with the target analyte, thereby inhibiting the response of the target substance, which has become a major problem in LC-MS analysis.

[0003] For animal-derived foods, veterinary drugs are widely used in animal-derived foods in the food production industry to prevent animal diseases or as growth promoters. These drugs will accumulate in animal tissues, and improper use will even cause drug residues to continuously accumulate in edible tissues, posing a threat to human health. With the increasing attention to food safety, almost every country has introduced regulations restricting the use of drugs in food animal products.

[0004] For biological samples (such as biological tissues, blood, plasma, etc.), in clinical tests or drug metabolism analysis, when detecting extremely trace metabolites, it is necessary to remove interfering substances in the sample, especially lipid interferents, otherwise it will seriously interfere with the detection.

[0005] In the prior art, the n-hexane extraction method is used: which belongs to the solvent extraction method. The sample is mixed with n-hexane, and then through steps such as stirring, standing, and filtering, the lipids in the sample are finally removed. The main principle of this method is based on chemical and physical interactions. In oil and n-hexane, due to the different polarities of the two substances, it is easy to form two different-phase systems. By utilizing the difference in this different-phase coefficient, the lipids can be extracted from the sample, thereby achieving the effect of removing lipids from the sample. This method belongs to a simple liquid-liquid extraction method and cannot completely remove lipids. The lipid co-extracts will be retained in the sample extract together with the target substance, resulting in abnormal chromatographic peaks, decreased data precision and accuracy, and affecting the data analysis quality of the target substance. The presence of lipids will not only interfere with the analysis of the target substance, but also shorten the service life of the chromatographic column and increase the maintenance requirements such as instrument loss. In addition, liquid-liquid extraction is time-consuming, uses a large amount of solvent, and is not environmentally friendly.

[0006] In the prior art, there are also QuEChERS and organic SPE degreasing methods: by using the chemical bond interaction of organic fillers to adsorb the target substances in the sample, so as to achieve the purpose of removing lipids. In this method, the sample pretreatment usually uses an acetonitrile: water mixture for extraction, and adsorbents such as HLB or C18 are used to adsorb the target compounds to achieve the purpose of removing lipids. However, its lipid removal ability is limited, and there is still lipid interference in the purified sample, which cannot achieve the ideal effect; the presence of lipids will not only interfere with the analysis of the target substances, but also shorten the service life of the chromatographic column and increase the maintenance requirements such as instrument loss. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for removing phospholipids from food and biological sample matrices. In the pretreatment process of food and biological sample matrices, a modified inorganic zirconium filler is used to pretreat the samples, which can simply and efficiently remove phospholipids from the samples, and is more conducive to accurate analysis or quantification by subsequent liquid chromatography-mass spectrometry / mass spectrometry.

[0008] The present invention is achieved through the following technical solutions:

[0009] A method for removing phospholipids from food and biological sample matrices, which is processed by the through-type purification method, specifically including the following steps:

[0010] Weigh a certain amount of the sample, first add water, vortex and oscillate, then add acetonitrile and vortex and oscillate, perform ultrasonic extraction, add an extraction salt packet and vortex, and centrifuge at 4°C to obtain an extract;

[0011] Take the upper-layer acetonitrile extract, mix it with pure water, and directly pass it through the phospholipid removal material to obtain a finished product of food and biological sample matrices that can be directly detected by a liquid chromatography-mass spectrometry analyzer.

[0012] Preferably, the acetonitrile aqueous solution is prepared from acetonitrile and water in a mass ratio of 8:2.

[0013] Preferably, the phospholipid removal material is a modified inorganic zirconium filler.

[0014] Preferably, the method for the phospholipid removal material to remove phospholipids is as follows:

[0015] S1. Weigh a certain amount of the sample, add a mixed internal standard use liquid and pure water, vortex and mix evenly, then add a formic acid-acetonitrile solution and vortex for 1 min, add an extraction salt packet and vortex for 1 min, and centrifuge at 4°C at 10000 r / min to obtain an extract;

[0016] S2. Take the upper-layer acetonitrile extract, mix it with pure water, directly pass it through the extraction column for purification, let the liquid nitrogen flow out, dry it by blowing, dissolve it with an acetonitrile solution, pass it through a 0.2 - 0.22 μm microporous filter membrane, and directly use a liquid chromatography-mass spectrometry analyzer for determination.

[0017] Preferably, the concentration of the mixed internal standard working solution is 100 μg / L.

[0018] Preferably, the volume ratio of the mixed internal standard working solution, pure water, and formic acid - acetonitrile solution is 125 μL: 3 mL: 10 mL.

[0019] Preferably, the mass concentration of the formic acid - acetonitrile solution is 5 - 7%.

[0020] Preferably, in S2, the volume ratio of the supernatant to pure water is 4 mL: 1 mL.

[0021] Preferably, the extraction column is composed of stacked Anavo HMR - Lipid extraction materials.

[0022] The above Anavo HMR - Lipid extraction material is a modified inorganic zirconium filler high - efficiency lipid - adsorbing material. This material has a high specific surface area: the unique synthetic inorganic pore structure provides a large specific surface area. (Fats with a molecular weight below 10,000 Da will enter the pores and be retained). Specific adsorption: Functional groups with specific selective adsorption for phospholipids are densely modified on the inorganic framework, which can selectively adsorb and remove phospholipids from complex matrices efficiently. Thorough removal of phospholipids: It has an affinity adsorption only for phospholipids and can theoretically remove 100% of phospholipids without adsorption for small - molecule compounds (such as acidic, neutral, and basic ions).

[0023] Preferably, when the acetonitrile solution is made up to volume, the mass concentration of the acetonitrile solution is 30 - 40 wt%.

[0024] Compared with the prior art, the present invention has at least the following technical effects:

[0025] (1) The present invention provides a method for removing phospholipids from food and biological sample matrices. During the pretreatment process of food and biological sample matrices, a modified inorganic zirconium filler is used to pretreat the samples, which can simply and efficiently remove phospholipids from the samples, and is more conducive to accurate analysis or quantification by subsequent liquid chromatography - mass spectrometry / mass spectrometry.

[0026] (2) Using the above method, lipids can be removed more cleanly, and thorough lipid removal can be achieved. Description of the Drawings

[0027] Figure 1 It is the total ion current chromatogram of 18 compounds and internal standards in Test Example 1. Detailed Embodiments

[0028] The implementation scheme of the present invention will be described in detail below in conjunction with embodiments. However, those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For the specific conditions not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0029] Example 1:

[0030] A method for removing phospholipids from food and biological sample matrices, which is processed by the through-type purification method, specifically includes the following steps:

[0031] Weigh a certain amount of the sample, first add water, vortex and oscillate, then add acetonitrile and vortex and oscillate, perform ultrasonic extraction, add an extraction salt packet and vortex, and centrifuge at 4°C to obtain an extract;

[0032] Take the upper-layer acetonitrile extract, mix it with pure water, and directly pass it through the phospholipid removal material to obtain a finished product of food and biological sample matrices that can be directly used for detection by a liquid chromatography-mass spectrometry analyzer.

[0033] The acetonitrile aqueous solution is prepared from acetonitrile and water in a mass ratio of 8:2.

[0034] The phospholipid removal material is a modified inorganic zirconium filler.

[0035] The method for the phospholipid removal material to remove phospholipids is as follows:

[0036] S1. Weigh a certain amount of the sample, add a mixed internal standard use liquid and pure water, vortex and mix evenly, then add a formic acid-acetonitrile solution and vortex for 1 min, add an extraction salt packet and vortex for 1 min, and centrifuge at 4°C at 10000 r / min to obtain an extract;

[0037] S2. Take the upper-layer acetonitrile extract, mix it with pure water, directly pass it through the extraction column for purification, let the liquid nitrogen flow out, dry it by blowing, dissolve it with an acetonitrile solution, pass it through a 0.2 - 0.22 μm microporous filter membrane, and directly use a liquid chromatography-mass spectrometry analyzer for determination.

[0038] The concentration of the mixed internal standard use liquid is 100 μg / L.

[0039] The volume ratio of the mixed internal standard use liquid, pure water, and formic acid-acetonitrile solution is 125 μL: 3 mL: 10 mL.

[0040] The mass concentration of the formic acid-acetonitrile solution is 5 - 7%.

[0041] In S2, the volume ratio of the supernatant to pure water is 4 mL: 1 mL.

[0042] The extraction column is composed of stacked Anavo HMR-Lipid extraction materials.

[0043] When making up the volume of the acetonitrile solution, the mass concentration of the acetonitrile solution is 30-40 wt%.

[0044] Experimental Example 1:

[0045] Referring to the "GB 31658.22-2022 National Food Safety Standard - Determination of β-agonist residues in animal foods - Liquid chromatography-tandem mass spectrometry", a detection method for determining 18 β-agonists in animal foods by UPLC-MS / MS was established: Using 5% formic acid acetonitrile as the extraction solvent, after adding the Anavo Que Extraction Salt Tube extraction salt packet, it was purified using the Anavo HMR-Lipid extraction column, concentrated by nitrogen blowing and re-dissolved, separated and detected using the Anavo PFP / C18 chromatographic column, and quantified by the isotope internal standard method.

[0046] Figure 1 It is the total ion current chromatogram of 18 compounds and internal standards in GB 31658.22-2022.

[0047] The pretreatment of this method is simple. It adopts the purification mode of removing impurities by HMR-lipid in passing, omitting the cumbersome processes such as activation, equilibration, and elution of the cationic solid-phase extraction column, and the treatment solution can be directly loaded for purification. The quantification limit is 0.4 μg / kg, and the recovery rate is between 86.0% and 115.4%.

[0048] Note: In the GB 31658.22-2022 standard, the purification mode of retaining target compounds using a mixed cation exchange solid-phase extraction column is used, and this purification mode is cumbersome.

[0049] Adopting the purification mode of removing impurities: greatly simplifies the purification process, also saves a large amount of solvent consumption; greatly shortens the time required for sample pretreatment. According to the national standard, it takes more than 3 hours for one sample, and the optimized method can be loaded onto the machine for detection in less than 1 hour.

[0050] Experimental Example 2:

[0051] Determination of Eugenol Residues in Aquatic Products in GB 31656.6-2021

[0052] To standardize the use of fishery anesthetics and ensure food safety, "GB 31656.6-2021 - Determination of Eugenol Residues in Aquatic Products" has been officially implemented in China since February 1, 2022.

[0053] With reference to "Determination of Eugenol Residue in Aquatic Products - GB 31656.6 - 2021" and "2023 National Food Contamination Monitoring SOP Manual", a detection method for eugenol and ethyl 3 - aminobenzoate methyl sulfonate by UPLC - MS / MS was established. The sample was extracted with acetonitrile / water, purified by Anavo HMR solid - phase extraction cartridge, re - dissolved by nitrogen blowing, separated on Anavo PFP / C18 chromatographic column, and quantified by isotope internal standard method.

[0054] The pretreatment of this method is simple. It adopts the purification mode of removing impurities by passing HMR - lipid, eliminating the cumbersome processes such as activation, equilibration, and elution of the C18 extraction column, and the processing solution can be directly loaded for purification. The limit of quantification is 0.45 μg / kg, and the recovery rate is between 80.6% - 110.1%.

[0055] The purification mode used in the GB 31656.6 - 2021 standard is to retain the target compound by C18 extraction column, and this purification mode is cumbersome. The optimized method by Anavo adopts the purification mode of removing impurities by HMR, with through - type purification, simple extraction process, and simple and efficient purification steps.

[0056] Less pretreatment time: It takes about 4 - 5 hours for one sample in GB 31656.6 - 2021. For the optimized method, it takes less than 1 hour.

[0057] Better recovery rate: The more pretreatment steps there are, the more factors will affect the recovery rate. For the optimized method, the recovery rate is between 80.6% - 110.1%.

[0058] Less solvent consumption: The consumption of pretreatment reagents is reduced by nearly 3 times, which reduces costs and is more environmentally friendly.

[0059] Note: For veterinary drug residue samples, the main interfering substances are proteins and lipids. Anavo HMR has excellent lipid - removing effect (an innovative inorganic solid - phase extraction material, a unique adsorbent for selectively removing lipids in complex matrices).

[0060] Finally, it should be noted that the above - mentioned are only the preferred embodiments of the present invention and are not used to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for removing phospholipids from food and biological sample matrices, characterized in that, The treatment is carried out by the through-type purification method, which specifically includes the following steps: Weigh a certain amount of the sample, first add water, vortex and oscillate, then add acetonitrile and vortex and oscillate, extract by ultrasound, add an extraction salt packet and vortex, and centrifuge at 4°C to obtain an extract; Take the upper-layer acetonitrile extract, mix it with pure water, and directly pass it through a phospholipid removal material to obtain a finished product of food and biological sample matrix for direct detection using a liquid chromatography-mass spectrometry analyzer.

2. The method for removing phospholipids from food and biological sample matrices according to claim 1, wherein The acetonitrile aqueous solution is prepared by mixing acetonitrile and water in a mass ratio of 8:

2.

3. A method for removing phospholipids from food and biological sample matrices according to claim 1, characterized in that, The phospholipid removal material is a modified inorganic zirconium filler.

4. A method for removing phospholipids from food and biological sample matrices according to claim 3, characterized in that, The method for the phospholipid removal material to remove phospholipids is as follows: S1. Weigh a certain amount of the sample, add a mixed internal standard working solution and pure water, vortex and mix evenly, add a formic acid-acetonitrile solution and vortex for 1 min, then add an extraction salt packet and vortex for 1 min, and centrifuge at 4°C at 10000 r / min to obtain an extract; S2. Take the upper-layer acetonitrile extract, mix it with pure water, directly purify it through an extraction column, flow out liquid nitrogen, dry it by blowing, dissolve it to a constant volume with an acetonitrile solution, pass it through a 0.2 - 0.22 μm microporous filter membrane, and directly use a liquid chromatography-mass spectrometry analyzer to determine.

5. A method for removing phospholipids from food and biological sample matrices according to claim 4, characterized in that, The concentration of the mixed internal standard working solution is 100 μg / L.

6. A method for removing phospholipids from food and biological sample matrices according to claim 4, characterized in that, The volume ratio of the mixed internal standard working solution, pure water, and formic acid-acetonitrile solution is 125 μL: 3 mL: 10 mL.

7. A method for removing phospholipids from food and biological sample matrices according to claim 4, characterized in that, The mass concentration of the formic acid-acetonitrile solution is 5 - 7%.

8. A method for removing phospholipids from food and biological sample matrices according to claim 4, characterized in that, In S2, the volume ratio of the supernatant to pure water is 4 mL: 1 mL.

9. A method for removing phospholipids from food and biological sample matrices according to claim 4, characterized in that, The extraction column is composed of stacked Anavo HMR-Lipid extraction materials.

10. A method for removing phospholipids from food and biological sample matrices according to claim 4, characterized in that, When dissolving to a constant volume with the acetonitrile solution, the mass concentration of the acetonitrile solution is 30 - 40 wt%.

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