CN-121994965-A - Pretreatment method for serum perfluorinated compound detection based on acidified protein precipitation and liquid-liquid extraction

Abstract

The application relates to the technical field of biological analysis chemistry, in particular to a serum perfluorinated compound detection pretreatment method based on acidified protein precipitation and liquid-liquid extraction, which comprises the steps of acidified acetonitrile protein precipitation, nitrogen blowing concentration, liquid-liquid extraction of a compound extractant and re-dissolution filtration. The method adopts a methyl tertiary butyl ether and ethyl acetate compound extraction system, combines a concentrating-before-extracting process, realizes the pretreatment effects of high recovery rate, low matrix effect and high enrichment factor in a single centrifuge tube, and is suitable for high-throughput, low-cost and green detection of C4-C14 perfluorinated compounds in trace serum.

Inventors

• ZHU MINQI
• ZHANG YAQIONG
• ZHANG YUZHU
• Gu wanhong

Assignees

• 台州市中心医院（台州学院附属医院）

Dates

Publication Date

20260508

Application Date

20260228

Claims (10)

1. 1. The serum perfluorinated compound detection pretreatment method based on acidified protein precipitation and liquid-liquid extraction is characterized by comprising the following steps of: s10, placing 500 mu L of serum sample into a 1.5 ml centrifuge tube, adding 200 mu L of standard substance in perfluoro compound, adding 500-1000 mu L of acetonitrile solution containing 0.1% -1.0% formic acid, mixing for 30 seconds to 2min by vortex, centrifuging at 12000-15000 r/min for 5-10 min at 4 ℃, discarding the precipitate, and reserving the supernatant; s20, transferring the supernatant obtained in the step S10 into a new 1.5ml centrifuge tube, and concentrating to 200-300 mu L under nitrogen blowing at 37 ℃ under nitrogen flow; s30, adding 500 mu L of a compound extractant formed by mixing methyl tertiary butyl ether and ethyl acetate according to a volume ratio of 3:1-1:1 into the supernatant concentrated in the step S20, carrying out vortex oscillation for 1-3 min, centrifuging for 5-8 min at 12000-15000 r/min at 4 ℃, and separating an upper organic phase; and S40, blowing nitrogen to near dryness of the organic phase obtained in the step S30 under the nitrogen flow at the temperature of 37 ℃, re-dissolving the organic phase by using 65 mu L of methanol aqueous solution, wherein the volume ratio of methanol to water in the methanol aqueous solution is 3:2, uniformly mixing the mixture for 0.5-3 min by vortex, and centrifuging the mixture for 5-8 min at the temperature of 4 ℃ at the speed of 12000-15000 r/min, wherein the obtained supernatant is used for liquid chromatography-tandem mass spectrometry analysis.
2. 2. The pretreatment method for serum perfluoro compound detection based on acidified protein precipitation and liquid-liquid extraction according to claim 1, wherein in step S10, the concentration of formic acid in acetonitrile solution is 0.1% -0.5%, and the volume ratio of acetonitrile to serum is 1:1-3:1.
3. 3. The method for pretreatment of serum perfluoro compound detection based on acidified protein precipitation and liquid-liquid extraction according to claim 1, wherein in step S10, the vortex mixing time is 1min, and the centrifugation conditions are 4 ℃, 14000r/min, 8min.
4. 4. The pretreatment method for serum perfluorinated compounds based on acidified protein precipitation and liquid-liquid extraction according to claim 1, wherein in step S20, the concentration end point volume by nitrogen blowing is 250 μl, the flow rate of nitrogen is 1.5-2.0L per minute, and the temperature is 37 ℃.
5. 5. The method for pretreatment of serum perfluoro compound detection based on acidified protein precipitation and liquid-liquid extraction of claim 1, wherein the volume ratio of methyl t-butyl ether to ethyl acetate in step S30 is 1:1.
6. 6. The method for pretreatment of serum perfluoro compound detection based on acidified protein precipitation and liquid-liquid extraction according to claim 1, wherein in step S30, the vortex oscillation time is 2min, and the centrifugation conditions are 4 ℃, 14000r/min, and 6min.
7. 7. The method for pretreatment of serum perfluoro compound detection based on acidified protein precipitation and liquid-liquid extraction according to claim 1, wherein in step S40, the volume ratio of methanol to water in the aqueous methanol solution used for reconstitution is 3:2, and the reconstitution volume is 65 μl.
8. 8. The method for pretreatment of serum perfluoro compound detection based on acidified protein precipitation and liquid-liquid extraction of claim 1, wherein in step S40, the nitrogen blow drying endpoint is visual observation without obvious droplet residue but with trace amounts of wetting.
9. 9. The pretreatment method for serum perfluoro compound detection based on acidified protein precipitation and liquid-liquid extraction according to claim 1, wherein the complex extractant consists of methyl tertiary butyl ether and ethyl acetate in a volume ratio of 3:1 to 1:1 for removing phospholipids, triglycerides and cholesterol and enriching perfluorocarboxylic acid, perfluorosulfonic acid and precursor compounds thereof.
10. 10. The method of claim 1, wherein the perfluorinated compounds include, but are not limited to, perfluorobutyric acid, perfluorovaleric acid, perfluorocaproic acid, perfluoroheptanoic acid, perfluorocaprylic acid, perfluorononanoic acid, perfluorocapric acid, perfluoroundecanoic acid, perfluorododecanoic acid, perfluorobutanesulfonic acid, perfluorohexanesulfonic acid, perfluorooctanesulfonic acid, and chlorinated or hydrogenated precursors thereof.

Description

Pretreatment method for serum perfluorinated compound detection based on acidified protein precipitation and liquid-liquid extraction Technical Field The invention belongs to the technical field of environment and biological analysis chemistry, and particularly relates to a serum perfluorinated compound detection pretreatment method based on acidified protein precipitation and liquid-liquid extraction. Background In the field of pretreatment for detection of perfluoro compounds (Per-and polyfluoroalkyl substances, PFASs) in serum, the prior art has mainly relied on methods such as protein precipitation or Solid Phase Extraction (SPE). The protein precipitation method has the advantages of simple operation and lower cost, but has limited purification capability, usually only can remove protein components, has weak removal effect on coexisting interferents such as phospholipid, salt, endogenous small molecules and the like, and is easy to cause obvious matrix effect in the liquid chromatography-mass spectrometry combined analysis process, thereby influencing the accuracy and precision of quantitative results. In addition, when processing low concentration PFASs samples, the method may reduce detection sensitivity due to sample dilution effects, which is detrimental to trace analysis. On the other hand, although the solid-phase extraction method can realize better purification effect, the process is complicated, a plurality of steps such as activation, sample loading, leaching and elution are involved, the technical requirements on operators are high, and a disposable SPE (solid phase extraction) column is needed, so that the detection cost is increased, and a larger plastic waste burden is brought, which has a certain conflict with the current development trend of green analytical chemistry. Meanwhile, lipid and particulate matters in a serum sample are easy to cause SPE column blockage, so that the stability of recovery rate is affected, and even the column is invalid. In the related patent searched, CN214310339U discloses a rapid sample pretreatment device based on a liquid chromatography-mass spectrometry method, which adopts a filter tube and ultrafiltration tube combined structure for mixing, separating and purifying solid samples. However, the device is mainly designed for solid samples, is not optimized for the characteristics of perfluorinated compounds in complex biological liquid samples such as serum and the like, lacks an effective removing mechanism for lipid and small molecular interferents, and is difficult to meet the pretreatment requirements of PFASs high-sensitivity and high-selectivity detection. Another related patent CN209946091U discloses a special device for pretreatment of pesticide residue detection based on the QuEChERS method, and the pretreatment efficiency is improved by replacing manual operation with mechanical oscillation. Although the QuEChERS method has wide application in pesticide residue detection, the core is mainly salting out and adsorbent purification, and for perfluorinated compounds with strong polarity and high stability in serum matrixes, the conventional QuEChERS adsorbent (such as PSA, C18 and the like) has limited retaining and purifying effects on PFASs, and the device does not integrate an acidification step, cannot effectively promote protein denaturation precipitation and target substance release, and does not combine a liquid-liquid extraction mechanism to further remove lipophilic interferents. In summary, the prior art limits the application of the serum perfluorinated compounds in mass screening due to serious matrix interference caused by insufficient purifying capacity or complicated flow, high cost and large environmental burden. Therefore, there is a need for a pretreatment method that combines efficient deproteinization, effective removal of lipid and small molecule interference, simple operation, and environmental friendliness, so as to improve accuracy, sensitivity, and applicability of perfluorocompound detection. Disclosure of Invention The invention aims to solve the defects in the prior art, and provides a serum perfluorinated compound detection pretreatment method based on acidified protein precipitation and liquid-liquid extraction. In order to achieve the above purpose, the present invention adopts the following technical scheme: The serum perfluorinated compound detection pretreatment method based on acidified protein precipitation and liquid-liquid extraction comprises the following steps: S10, taking 500 mu L of serum sample, placing the serum sample into a 1.5 ml centrifuge tube, adding 1000-1500 mu L of acetonitrile solution containing 0.1% -1.0% formic acid, mixing for 30 seconds to 2 minutes by vortex, centrifuging for 5-10 minutes at 12000-15000 r/min at 4 ℃, discarding the precipitate, and reserving the supernatant; s20, transferring the supernatant obtained in the step S10 into a new 1.5ml centrifuge tube, and concentrating to 20