Method for simultaneously and rapidly pre-treating phthalic acid ester and metabolites thereof in biological sample

Abstract

The invention discloses a method for simultaneously and quickly pre-treating phthalic acid ester and metabolites thereof in a biological sample, which comprises the following steps: (1) adding diluted hydrochloric acid into the sample according to a ratio of 0.5 g: 3mL, and vortex for 1-3 minutes; (2) adding acetonitrile into the mixed solution obtained in the step (1) according to a ratio of the sample to acetonitrile of 0.5 g: 3mL, carrying out vortex treatment for 1-3min, and then carrying out ultrasonic treatment for 8-15min; (3) adding an extraction salt bag into the mixed solution obtained in the step (2) according to the mass ratio of the sample to the extraction salt bag being 1: 4, violently shaking to enable the salt bag to be fully and uniformly mixed, and then vortex for 5-8 minutes; and finally, centrifuging for 5-6 minutes at the speed of 3000rpm, and taking supernate, so as to obtain a solution for detecting the phthalate and the metabolites thereof.

Description

Technical Field

[0001] The invention relates to a method for simultaneously and quickly pre-treating phthalate esters and metabolites thereof in a biological sample. Background Art

[0002] Phthalate esters (PAEs) are a typical class of endocrine disruptors, which can be metabolized in vivo to form mono-lipid benzoic acid metabolites (mPAEs). PAEs and mPAEs have been widely detected in biological samples such as animals and plants in my country.

[0003] The pretreatment steps for detecting PAEs and mPAEs in biological samples usually include extraction and purification steps of the target. Currently, the most common pretreatment method for PAEs in biological samples is QuEChERS technology, which has the advantages of simple operation, low solvent usage, and high recovery rate.

[0004] For example, China's published patent document CN105203661A discloses the use of QuEChERS pretreatment to extract and purify multiple PAEs in vegetables, fruits and edible fungi, including the following steps: 1) add acetonitrile of 1 to 2 times the weight of the sample to the sample, and vortex quickly, 2) then add 0.5 to 1.5 times the weight of the sample by weight of anhydrous sodium sulfate and 0.1 to 0.5 times the weight of sodium chloride, mix well and centrifuge, 3) then take the supernatant into a stoppered glass centrifuge tube containing purification filler, vortex, centrifuge and take the supernatant. The above method can pre-treat 23 PAEs in the sample at the same time, but it is not suitable for mPAEs, and cannot achieve simultaneous pre-treatment of PAEs and mPAEs.

[0005] For example, Míriam Hidalgo-Serrano, et al. "Simple method for determining phthalate diesters and their metabolites in seafood species using QuEChERS extraction and liquid chromatography-high resolution mass spectrometry." Food Chemistry 336 (2020): 127722, discloses a method for simultaneous pretreatment of PAEs and mPAEs in aquatic products: 1g of sample is mixed with 10mL of acid water, then 10mL of acetonitrile is added to mix, and then a salt bag is added to separate the layers. After centrifugation, the acetonitrile layer is taken for purification; then, the acetonitrile layer is divided into two parts, and solid phase extraction columns with different purification fillers are used to purify PAEs and mPAEs respectively. Since PAEs and mPAEs have different polarities, the above method needs to use different purification steps to purify PAEs and mPAEs respectively after the biological sample is extracted, resulting in a long pretreatment time. The pretreatment time for one sample takes about 6 hours. Summary of the invention

[0006] The object of the present invention is to provide a method for simultaneously and rapidly pre-treating phthalates and their metabolites in biological samples.

[0007] To achieve the above objectives, the present invention adopts the following technical solutions.

[0008] A method for simultaneously and rapidly pre-treating phthalates and their metabolites in biological samples comprises the following steps:

[0009] (1) Add dilute hydrochloric acid to the sample at a ratio of 0.5 g:3 mL and vortex for 1 to 3 min;

[0010] (2) adding acetonitrile to the mixed solution obtained in step (1) at a ratio of 0.5 g sample to 3 mL acetonitrile, vortexing for 1 to 3 min, and then sonicating for 8 to 15 min;

[0011] (3) Add the extraction salt package to the mixed solution obtained in step (2) at a mass ratio of 1:4 between the sample and the extraction salt package, shake vigorously to mix the salt package thoroughly, and then vortex for 5 to 8 minutes; finally, centrifuge at 3000 rpm for 5 to 6 minutes, take the supernatant, and obtain a solution for detecting phthalates and their metabolites.

[0012] The present invention has determined during experiments that by adding dilute hydrochloric acid and acetonitrile to the sample in a ratio of 0.5 g:3 mL, the pretreatment of phthalates and their metabolites can be completed only through the extraction step, without the need to purify them using purification fillers.

[0013] Among them, after adding dilute hydrochloric acid and acetonitrile to the sample in a ratio of 0.5g:3mL, the mixed solution of step (2) is in a suspension state, and the mixed solution after adding the extraction salt bag and mixing is viscous and turbid, which is very different from the phenomenon extracted in the existing QuEChERS technology. Under normal operation, those skilled in the art will increase the volume of the solvent to stratify the mixed solution to achieve the purpose of extraction when encountering the above phenomenon, but the present invention does not continue to increase the volume of the solvent, and unexpectedly found that after centrifuging the mixed solution, the mixed solution is divided into three layers, from bottom to top, a clear colorless aqueous phase, a colored solid emulsion layer, and a clear colorless organic phase, and the pigment is almost completely in the solid emulsion layer, so the method of the present invention can separate a clear colorless organic phase; the clear colorless organic phase can be used for detection without purification, and experiments have shown that its effect is the same as after purification, and it meets the volume requirements of detection, and can also obtain a relatively ideal recovery rate.

[0014] Specifically, the present invention finds that if the volume of dilute hydrochloric acid is too small, it will not be able to penetrate the solid sample, and if the volume is too large, more water-soluble impurities in the sample will be dissolved, thereby requiring an additional purification step and increasing the difficulty of purification; and if the volume of acetonitrile is too small, the organic phase and the aqueous phase cannot be separated, and if the volume is too large, the concentration of the target compound in the acetonitrile layer is low and a concentration step is required, which increases the pretreatment time. In addition, during the concentration process, the matrix concentration is also concentrated. Therefore, in order to avoid high-concentration matrix contamination of the chromatographic column or affecting the accurate quantification of the target object, further purification is required to reduce the concentration of the matrix in the injection solution.

[0015] Furthermore, the invention adds ultrasonic treatment to improve the extraction efficiency and recovery rate of phthalates and their metabolites.

[0016] Preferably, the sample in step (1) is homogenized and freeze-dried, and passed through a 60-mesh sieve.

[0017] Preferably, the pH value of the dilute hydrochloric acid is 2.

[0018] Preferably, the solution is manually shaken up and down 1 to 2 times during the ultrasonic process to increase the contact time between the solid sample and the solution, thereby improving the extraction efficiency of the target object.

[0019] Preferably, the extraction salt package contains anhydrous magnesium sulfate, sodium chloride, disodium citrate sesquihydrate, and trisodium citrate dihydrate; the mass ratio of the anhydrous magnesium sulfate, sodium chloride, disodium citrate sesquihydrate, and trisodium citrate dihydrate is 8:2:1:2. Before adding the extraction salt package, the extracting liquid system in the mixture is an acidic acetonitrile-water solution, at which time the aqueous phase and the organic phase are mutually soluble; after adding the extraction salt package, anhydrous MgSO4 absorbs impurities such as moisture and fatty acids; NaCl has a salting-out effect, which helps the stratification of the aqueous phase and the organic phase, and promotes the weak polarity target (PAEs) and the medium polarity target (mPAEs) to enter the organic phase; disodium citrate sesquihydrate and trisodium citrate dihydrate can adjust the pH value of the solution and promote the transfer of the acidic medium polarity target (mPAEs) from the aqueous phase to the organic phase.

[0020] Preferably, the present invention uses glass sample bottles instead of commonly used glass centrifuge tubes for sample pretreatment. The price of glass sample bottles is much lower than that of glass centrifuge tubes, and the former is only 2% of the latter, which can reduce the detection cost; in addition, because the price of flat-bottomed glass bottles is low, they can be used as disposables, thereby avoiding background value contamination caused by repeated use.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention improves the existing QuEChERS method, specifically by controlling the volume of dilute hydrochloric acid and acetonitrile, effectively reducing the dissolution of impurities, and then using the adsorption function of the extraction salt bag on impurities to achieve the separation of the target PAEs and mPAEs from impurities; therefore, the present invention can not only achieve simultaneous pre-treatment of PAEs and mPAEs, but also does not require complex column purification steps after the sample is extracted, thereby greatly shortening the pre-treatment time, so that the pre-treatment time of one sample is shortened from 6h to within 0.5h, greatly improving the efficiency of the pre-treatment. At the same time, the method of the present invention has strong universality for biological sample matrices, and can be applied to animal samples (fish, meat, eggs, milk, etc.) and plant samples (cereals, beans, etc.) at the same time. DETAILED DESCRIPTION

[0023] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] The raw materials and reagents used in the following examples were purchased from the market. The biological samples used in the present invention were homogenized and freeze-dried, and passed through a 60-mesh sieve, that is, the sample mass was dry weight.

[0025] Example 1: Recovery of phthalates and their metabolites

[0026] (1) Weigh 0.5 g of anhydrous sodium sulfate instead of biological sample, place it in the corresponding 8 mL glass sample bottle, and add phthalate esters and their metabolite standards; then add 3 mL of dilute hydrochloric acid with a pH value of 2 and vortex for 1 min;

[0027] (2) Add 3 mL of acetonitrile to the mixture obtained in step (1), vortex for 1 min, and then sonicate for 10 min, shaking the sample up and down manually once during the process to mix the sample;

[0028] (3) Add 2 g of extraction salt bag, the mass ratio of anhydrous magnesium sulfate, sodium chloride, disodium citrate sesquihydrate, and trisodium citrate dihydrate in the extraction salt bag is 8:2:1:2; then shake the glass sample bottle vigorously by hand and vortex for 5 minutes; finally centrifuge at 3000 rpm for 5 minutes, and take 1 mL of supernatant for the detection of phthalates and their metabolites;

[0029] (4) The contents of phthalates and their metabolites were determined by liquid chromatography-high resolution mass spectrometry, and the recovery rates were calculated.

[0030] (5) Recovery rate results: The recovery rates of dibutyl phthalate, di-2-ethylhexyl phthalate, and diisobutyl phthalate were 77±8%, 97±10%, and 88±3%, respectively; the recovery rates of monobutyl phthalate, mono-(2-ethylhexyl) phthalate, and monoisobutyl phthalate were 92±8%, 94±6%, and 93±7%, respectively.

[0031] Example 2: Determination of the content of phthalates and their metabolites in fish

[0032] (1) Weigh 0.5 g of fish sample into the corresponding 8 mL glass sample bottle, add 3 mL of dilute hydrochloric acid with a pH value of 2, and vortex for 1 min;

[0033] (2) Add 3 mL of acetonitrile to the mixture obtained in step (1), vortex for 1 min, and then sonicate for 8 min, shaking the sample up and down manually once during the process to mix the sample.

[0034] (3) Add 2 g of extraction salt bag, the mass ratio of anhydrous magnesium sulfate, sodium chloride, disodium citrate sesquihydrate, and trisodium citrate dihydrate in the extraction salt bag is 8:2:1:2; then shake the glass sample bottle vigorously by hand and vortex for 5 minutes; finally centrifuge at 3000 rpm for 5 minutes, and take 1 mL of supernatant for the detection of phthalates and their metabolites;

[0035] (4) weighing the fish sample obtained in step (1) again, pre-treating it (extracting and purifying) it using the method proposed by Míriam Hidalgo-Serrano, and testing the solution obtained by the pre-treatment for phthalates and their metabolites;

[0036] (5) The content of phthalates and their metabolites was determined by liquid chromatography-high resolution mass spectrometry.

[0037] (6) The test results are shown in Table 1.

[0038] Table 1 Comparison of the test results after pretreatment by the method of the present invention and the method of Míriam Hidalgo-Serrano (unit: ng / g dry weight)

[0039]

[0040] The results in Table 1 show that there is no significant difference in the test results obtained by pre-treating fish samples using the method of the present invention and the existing method proposed by Míriam Hidalgo-Serrano, which indicates that the method of the present invention can achieve simultaneous pre-treatment of PAEs and mPAEs without complex purification steps.

[0041] Example 3: Determination of the content of phthalates and their metabolites in pork

[0042] (1) Weigh 0.5 g of pork sample into the corresponding 8 mL glass sample bottle, add 3 mL of dilute hydrochloric acid with a pH value of 2, and vortex for 2 min;

[0043] (2) Add 3 mL of acetonitrile to the mixed solution obtained in step (1), vortex for 1 min, and then sonicate for 10 min, shaking the sample up and down manually twice to mix the sample;

[0044] (3) Add 2 g of extraction salt bag, the mass ratio of anhydrous magnesium sulfate, sodium chloride, disodium citrate sesquihydrate, and trisodium citrate dihydrate in the extraction salt bag is 8:2:1:2; then shake the glass sample bottle vigorously by hand and vortex for 8 minutes; finally centrifuge at 3000 rpm for 6 minutes, and take 1 mL of supernatant for the detection of phthalates and their metabolites;

[0045] (4) The content of phthalates and their metabolites was determined by liquid chromatography-high resolution mass spectrometry.

[0046] (5) Test results: The main phthalates detected in pork were dibutyl phthalate, di-2-ethylhexyl phthalate, and diisobutyl phthalate, with contents of 22536 ng / g dry weight, 228 ng / g dry weight, and 543 ng / g dry weight, respectively. The main phthalate metabolites detected were monobutyl phthalate, mono(2-ethylhexyl) phthalate, and monoisobutyl phthalate, with contents of 112 ng / g dry weight, 5 ng / g dry weight, and 12 ng / g dry weight, respectively.

[0047] Example 4: Determination of the content of phthalates and their metabolites in eggs

[0048] (1) Weigh 0.5 g of egg sample into the corresponding 8 mL glass sample bottle, add 3 mL of dilute hydrochloric acid with a pH value of 2, and vortex for 3 min;

[0049] (2) Add 3 mL of acetonitrile to the mixed solution obtained in step (1), vortex for 1 min, and then sonicate for 12 min, shaking the sample up and down manually once during the process to mix the sample.

[0050] (3) Add 2 g of extraction salt bag, the mass ratio of anhydrous magnesium sulfate, sodium chloride, disodium citrate sesquihydrate, and trisodium citrate dihydrate in the extraction salt bag is 8:2:1:2; then shake the glass sample bottle vigorously by hand and vortex for 8 minutes; finally centrifuge at 3000 rpm for 6 minutes, and take 1 mL of supernatant for the detection of phthalates and their metabolites;

[0051] (4) The content of phthalates and their metabolites was determined by liquid chromatography-high resolution mass spectrometry.

[0052] (5) Test results: The main phthalates detected in eggs were dibutyl phthalate, di-2-ethylhexyl phthalate, and diisobutyl phthalate, with contents of 3520 ng / g dry weight, 148 ng / g dry weight, and 106 ng / g dry weight, respectively. The main phthalate metabolites detected were monobutyl phthalate, mono(2-ethylhexyl) phthalate, and monoisobutyl phthalate, with contents of 34 ng / g dry weight, 3 ng / g dry weight, and 4 ng / g dry weight, respectively.

[0053] Example 5: Determination of the content of phthalates and their metabolites in milk

[0054] (1) Pipette 3 mL of milk sample into the corresponding 8 mL glass sample bottle, adjust the pH value of the sample to 2 with concentrated hydrochloric acid, and vortex for 1 min;

[0055] (2) Add 3 mL of acetonitrile to the mixed solution obtained in step (1), vortex for 1 min, and then sonicate for 15 min, shaking the sample up and down manually twice during the process to mix the sample;

[0056] (3) Add 2 g of extraction salt bag, the mass ratio of anhydrous magnesium sulfate, sodium chloride, disodium citrate sesquihydrate, and trisodium citrate dihydrate in the extraction salt bag is 8:2:1:2; then shake the glass sample bottle vigorously by hand and vortex for 5 minutes; finally centrifuge at 3000 rpm for 5 minutes, and take 1 mL of supernatant for the detection of phthalates and their metabolites;

[0057] (4) The content of phthalates and their metabolites was determined by liquid chromatography-high resolution mass spectrometry.

[0058] (5) Test results: The main phthalates detected in milk are dibutyl phthalate, di-2-ethylhexyl phthalate, and diisobutyl phthalate, with contents of 54 ng / mL, 9 ng / mL, and 5 ng / mL, respectively. The main phthalate metabolites detected are monobutyl phthalate, mono(2-ethylhexyl) phthalate, and with contents of 5 ng / mL and 1 ng / mL, respectively.

[0059] Example 6: Determination of the content of phthalates and their metabolites in rice

[0060] (1) Weigh 0.5 g of rice sample into a corresponding 8 mL glass sample bottle, add 3 mL of dilute hydrochloric acid with a pH value of 2, and vortex for 1 min;

[0061] (2) Add 3 mL of acetonitrile to the mixed solution obtained in step (1), vortex for 1 min, and then sonicate for 10 min, manually shaking up and down 1 to 2 times during the process to mix the sample;

[0062] (3) Add 2 g of extraction salt bag, the mass ratio of anhydrous magnesium sulfate, sodium chloride, disodium citrate sesquihydrate, and trisodium citrate dihydrate in the extraction salt bag is 8:2:1:2; then shake the glass sample bottle vigorously by hand and vortex for 5 minutes; finally centrifuge at 3000 rpm for 5 minutes, and take 1 mL of supernatant for the detection of phthalates and their metabolites;

[0063] (4) The content of phthalates and their metabolites was determined by liquid chromatography-high resolution mass spectrometry.

[0064] (5) Test results: The main phthalates detected in rice are dibutyl phthalate, di-2-ethylhexyl phthalate, and diisobutyl phthalate, with contents of 288 ng / g dry weight, 404 ng / g dry weight, and 47 ng / g dry weight, respectively. The main phthalate metabolites detected are monobutyl phthalate and mono(2-ethylhexyl) phthalate, with contents of 15 ng / g dry weight and 4 ng / g dry weight, respectively.

[0065] Example 7: Determination of the content of phthalates and their metabolites in soybeans

[0066] (1) Weigh 0.5 g soybean sample into the corresponding 8 mL glass sample bottle, add 3 mL of dilute hydrochloric acid with a pH value of 2, and vortex for 1 min;

[0067] (2) adding 3 mL of acetonitrile to the mixture obtained in step (1), vortexing for 1 min, and then sonicating for 10 min, manually shaking up and down 1 to 2 times during the process to mix the sample;

[0068] (3) Add 2 g of extraction salt package, the mass ratio of anhydrous magnesium sulfate, sodium chloride, disodium citrate sesquihydrate, and trisodium citrate dihydrate in the extraction salt package is 8:2:1:2; shake the glass sample bottle vigorously by hand, vortex for 5 minutes; finally centrifuge at 3000 rpm for 5 minutes, and take 1 mL of supernatant for detection of phthalates and their metabolites;

[0069] (4) The content of phthalates and their metabolites was determined by liquid chromatography-high resolution mass spectrometry.

[0070] (5) Test results: The main phthalates detected in soybeans include dibutyl phthalate, di-2-ethylhexyl phthalate, and diisobutyl phthalate, with contents of 231 ng / g dry weight, 31 ng / g dry weight, and 36 ng / g dry weight, respectively. The main phthalate metabolites detected include monobutyl phthalate, monobenzyl phthalate, and mono(2-ethylhexyl) phthalate, with contents of 58 ng / g dry weight, 607 ng / g dry weight, and 4 ng / g dry weight, respectively.

Claims

1. A method for rapid pretreatment of phthalates and their metabolites in biological samples, characterized in that: The following steps are involved: (1) Add dilute hydrochloric acid to the sample at a ratio of 0.5 g:3 mL and vortex for 1 to 3 min; (2) adding acetonitrile to the mixed solution obtained in step (1) at a ratio of 0.5 g sample to 3 mL acetonitrile, vortexing for 1 to 3 min, and then sonicating for 8 to 15 min; (3) Add the extraction salt package to the mixed solution obtained in step (2) at a mass ratio of 1:4 between the sample and the extraction salt package, shake vigorously to mix the salt package thoroughly, and then vortex for 5 to 8 minutes; finally, centrifuge at 3000 rpm for 5 to 6 minutes, take the supernatant, and obtain a solution for detecting phthalates and their metabolites.

2. The method for rapid simultaneous pretreatment of phthalates and their metabolites in biological samples according to claim 1, characterized in that: The sample in step (1) was homogenized and freeze-dried, and passed through a 60-mesh sieve.

3. The method for rapid simultaneous pretreatment of phthalates and their metabolites in biological samples according to claim 2, characterized in that: The pH value of the dilute hydrochloric acid is 2.

4. The method for rapid simultaneous pretreatment of phthalates and their metabolites in biological samples according to claim 3, characterized in that: Manually shake up and down 1 to 2 times during the ultrasound process.

5. The method for rapid simultaneous pretreatment of phthalates and their metabolites in biological samples according to claim 4, characterized in that: The extraction salt package contains anhydrous magnesium sulfate, sodium chloride, disodium citrate sesquihydrate, and trisodium citrate dihydrate; the mass ratio of the anhydrous magnesium sulfate, sodium chloride, disodium citrate sesquihydrate, and trisodium citrate dihydrate is 8:2:1:

2.

6. The method for rapid simultaneous pretreatment of phthalates and their metabolites in biological samples according to claim 5, characterized in that: Glass sample bottles were used for sample preparation.

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