Online solid-phase extraction-liquid chromatography combined detection method

Abstract

The invention relates to the technical field of analytical chemistry detection, and discloses an on-line solid phase extraction-liquid chromatography combined detection method, which comprises the following steps: adsorbing a target analyte by using polyacrylonitrile-hydrophilic lipophilic equilibrium copolymer composite nanofiber as a solid phase adsorbent; eluting the target analyte on the solid-phase adsorbent by using an eluent, so as to obtain an eluent; transferring the eluent into an analytical column of a liquid chromatograph for analysis to obtain a chromatogram of the target analyte; under the condition that a high-concentration organic phase does not need to be used as an eluent, a target analyte can be separated out at a speed matched with liquid chromatography analysis, so that on-line combination of solid-phase extraction and liquid chromatography can be realized.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical chemistry detection, and particularly to a detection method for on-line solid phase extraction - liquid chromatography coupling. Background Art

[0002] A liquid chromatograph is an instrument that first separates and then analyzes and identifies a mixture by utilizing the difference in the distribution ratio of the mixture between a liquid - solid or two immiscible liquids. Due to the advantages of high resolution, high sensitivity, fast speed, reusable chromatographic column, and easy collection of eluted components, the high - performance liquid chromatograph is widely used in various fields such as biochemistry, pharmaceutical research, environmental analysis, inorganic analysis, food analysis, etc., and is one of the most commonly used instruments in biochemical analysis.

[0003] For a high - performance liquid chromatograph, its detection advantages of high resolution and high sensitivity largely depend on the sample pretreatment process; and solid - phase extraction (SPE) is the most commonly used sample pretreatment technology at present. SPE uses a solid - phase adsorbent as the stationary phase. When a liquid sample flows through it, the target analyte is retained by the solid - phase adsorbent, and then a suitable solvent is selected to elute the analyte, so as to achieve the purpose of purifying, separating, and enriching the target analyte.

[0004] Traditional SPE techniques mostly rely on off - line manual operations, which have the disadvantages of time - consuming, labor - intensive, and easy to generate loss errors; coupling the SPE technique with high - performance liquid chromatography on - line to realize on - line integrated operation of sample pretreatment and liquid chromatography analysis is an effective means to solve the above - mentioned deficiencies.

[0005] As mentioned above, solid - phase extraction realizes the adsorption and enrichment of the target analyte through a solid - phase adsorbent and then through an elution process; currently, the solid - phase extraction process often uses a hydrophilic - lipophilic balance (HLB) copolymer as the solid - phase extraction adsorbent. Although this HLB has high selectivity for target molecules, the elution process requires high - concentration organic solvents such as pure acetonitrile and methanol as eluents. When it is coupled with liquid chromatography on - line, due to the too - high concentration of the organic solvent used as the eluent, each component will be separated in too short a time, resulting in the inability to detect the target component by the liquid chromatograph. Therefore, the existing solid - phase extraction technology is difficult to realize on - line coupling with liquid chromatography, and cannot perform on - line automatic processing and detection, which limits the popularization and application of on - line solid - phase extraction - liquid chromatography coupling. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a detection method combining online solid-phase extraction and liquid chromatography, including the following process: using polyacrylonitrile-hydrophilic-lipophilic balance copolymer composite nanofibers as a solid-phase adsorbent to adsorb the target analyte, and then using an eluent to elute the target analyte on the solid-phase adsorbent to obtain an eluate; transferring the eluate to an analytical column of a liquid chromatograph for analysis to obtain a chromatogram of the target analyte; The target analyte is one of hydrophilic components, hydrophobic components, and amphoteric components; When the target analyte is a hydrophilic component, the eluent is an organic-phase aqueous solution; When the target analyte is a hydrophobic component, the eluent is the mobile phase of the chromatographic analytical column; When the target analyte is an amphoteric component, the eluent is selected from one or a combination of water, organic-phase aqueous solution, and acid-base regulator.

[0007] Further, the polyacrylonitrile-hydrophilic-lipophilic balance copolymer composite nanofibers are prepared according to the following method: dissolving polyacrylonitrile and hydrophilic-lipophilic balance copolymer in a solvent, stirring to form a spinning solution; preparing composite nanofibers from the spinning solution by electrospinning technology to obtain polyacrylonitrile-hydrophilic-lipophilic balance copolymer composite nanofibers.

[0008] Further, the mass ratio of polyacrylonitrile to the hydrophilic-lipophilic balance copolymer is 10:(6 - 7).

[0009] Further, the specific surface area of the polyacrylonitrile-hydrophilic-lipophilic balance copolymer composite nanofibers is 150 - 200 m 2 / g.

[0010] Further, the average pore diameter of the polyacrylonitrile-hydrophilic-lipophilic balance copolymer composite nanofibers is 3.0 - 3.5 nm.

[0011] Further, the hydrophilic components are selected from at least one of vitamin B2, vitamin B 12 , vitamin B9, 5-methyltetrahydrofolic acid, 5-formyltetrahydrofolic acid, 5,10-dimethyltetrahydrofolic acid, S-adenosyl-L-methionine, and S-adenosyl-L-homocysteine.

[0012] Further, the hydrophobic components include phthalate substances.

[0013] Further, the phthalate is selected from at least one of diethyl phthalate, di-n-propyl phthalate, butyl benzyl phthalate, di-n-butyl phthalate, di-n-pentyl phthalate, dicyclohexyl phthalate, di-n-hexyl phthalate, diisooctyl phthalate, and di-n-octyl phthalate.

[0014] Further, the amphoteric component includes quinolone antibiotics.

[0015] Further, the quinolone antibiotics are selected from at least one of ofloxacin, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, and sarafloxacin.

[0016] The embodiments of the present invention have the following technical effects: The detection method of online solid-phase extraction-liquid chromatography provided by this application uses PAN-HLB composite nanofibers as the solid-phase adsorbent. Without the need for a high-concentration organic phase as the eluent, the target analyte can be separated at a speed compatible with liquid chromatography analysis, enabling the online coupling of solid-phase extraction and liquid chromatography, integrating online processing and detection. While ensuring the sensitivity and accuracy of liquid chromatography detection, it simplifies the operation process, saves manpower, reduces manual operation errors, and reduces time consumption. Description of the Drawings

[0017] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0018] Figure 1 It is the chromatogram (b) of the standard solution of hydrophobic phthalates detected by online solid-phase extraction using PAN-HLB composite nanofibers and the chromatogram (a) of direct injection analysis provided by the present invention; Figure 2 It is the chromatogram (b) of the standard solution of hydrophobic phthalates detected by online solid-phase extraction using PAN-HLB composite nanofibers and the chromatogram (a) of HLB online extraction column detection provided by the present invention; Figure 3 It is the chromatogram (b) of the standard solution of hydrophobic phthalates detected by online solid-phase extraction using PAN-HLB composite nanofibers and the chromatogram (a) of C18 online extraction column detection provided by the present invention; Figure 4It is the chromatogram (b) of the standard solution of hydrophilic vitamin substances in the present invention detected by on-line solid-phase extraction with PAN-HLB composite nanofibers and the chromatogram (a) of direct injection analysis; Figure 5 It is the chromatogram (b) of the standard solution of hydrophilic vitamin substances in the present invention detected by on-line solid-phase extraction with PAN-HLB composite nanofibers and the chromatogram (a) of on-line extraction column with HLB; Figure 6 It is the chromatogram (b) of the standard solution of hydrophilic vitamin substances in the present invention detected by on-line solid-phase extraction with PAN-HLB composite nanofibers and the chromatogram (a) of on-line extraction column with C18; Figure 7 It is the chromatogram (b) of amphoteric quinolone substances in the present invention detected by on-line solid-phase extraction with PAN-HLB composite nanofibers and the chromatogram (a) of direct injection analysis; Figure 8 It is the chromatogram (b) of amphoteric quinolone substances in the present invention detected by on-line solid-phase extraction with PAN-HLB composite nanofibers and the chromatogram (a) of on-line extraction column with HLB; Figure 9 It is the chromatogram (a) of amphoteric quinolone substances in the present invention detected by on-line solid-phase extraction with PAN-HLB composite nanofibers and the chromatogram (b) of on-line extraction column with C18. Detailed implementation mode

[0019] To make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the present invention.

[0020] To solve the problem that the existing solid-phase extraction technology is difficult to realize on-line coupling with liquid chromatography, the present application provides a detection method of on-line solid-phase extraction-liquid chromatography coupling. The detection method includes the following process: using polyacrylonitrile-hydrophilic lipophilic balance copolymer (PAN-HLB) composite nanofibers as a solid-phase adsorbent to adsorb the target analyte, and then using an eluent to elute the target analyte on the solid-phase adsorbent to obtain an eluate; transferring the eluate to the analytical column of a liquid chromatograph for analysis to obtain the chromatogram of the target analyte; wherein, based on the special structure and performance of the PAN-HLB composite nanofibers in the present application, the solid-phase adsorbent can have excellent adsorption capacity for hydrophilic components, hydrophobic components and amphoteric components at the same time, that is, the target analyte in the present application is one of hydrophilic components, hydrophobic components and amphoteric components.

[0021] For the detection method of on-line solid-phase extraction-liquid chromatography, eluting and separating the target analyte on the solid-phase adsorbent with an eluent is also a key step to ensure the detection sensitivity and accuracy; to achieve the selective extraction of target analytes with different polarities, the eluent is preferably different according to the polarity of the target analyte to be processed; specifically, when the target analyte is a hydrophilic component, the eluent is preferably an aqueous organic solution, and more preferably a low-concentration aqueous organic solution; when the target analyte is a hydrophobic component, the eluent is preferably the mobile phase of the analytical chromatographic column; when the target analyte is an amphoteric component, the eluent is preferably one or a combination of water, aqueous organic solution, and acid-base regulator.

[0022] The detection method of on-line solid-phase extraction-liquid chromatography can be specifically carried out according to the following process: 1) Loading of the on-line solid-phase extraction column: Use a 10 mm×2.1 mm stainless steel column tube with open ends at both ends and detachable as the on-line solid-phase extraction column. Take an appropriate amount of PAN-HLB composite nanofibers and load them into the bottom of the column tube sealed with a sieve plate at one end in batches, compact them with a stainless steel fine rod with a diameter of 0.5 mm, seal the other end with a sieve plate, put it into the outer sleeve, and connect it to the high-performance liquid chromatography system for standby; the amount of nanofibers loaded each time is 8-10 mg, and compact it 5 times with a stainless steel fine rod with a diameter of 0.5 mm.

[0023] 2) Sample treatment: Carry out corresponding treatment for different types of samples: If it is an environmental water sample, filter it through a 0.45 μm filter membrane and use it as the sample to be detected; If it is a food sample (such as milk), weigh 0.1 g, precipitate the protein by adding acid, take the supernatant, filter it through a 0.45 μm filter membrane, and dilute it to 4 mL with ultrapure water as the sample to be detected; If it is a biological sample (such as plasma, urine), take 0.1 mL of the urine sample, add an antioxidant and dilute it to 4 mL with ultrapure water; take 0.1 mL of the plasma sample, add an antioxidant, then add the same volume of acid to precipitate the protein, take the supernatant, filter it through a 0.45 μm filter membrane, and dilute it to 4 mL with ultrapure water as the sample to be detected.

[0024] The sample also includes other similar complex matrix samples.

[0025] 3) On-line solid-phase extraction and detection analysis: Appropriately pipette an aliquot of the treated sample solution to be extracted into a 5 mL injection vial, run the chromatographic program, inject 1000 μL into the on-line solid-phase extraction column. Use ultrapure water or a mixed solvent of organic solvent / water in a certain proportion as the mobile phase of the left pump. The enrichment and purification time is 0.5 - 3 min. Switch the valve path, connect the on-line solid-phase extraction column to the right pump. Use the eluent to elute the target substance adsorbed on the on-line solid-phase extraction column and transfer it to the analytical column. Then switch the valve path back to the original state. At this time, the left pump reactivates the on-line solid-phase extraction column for the next injection treatment, and the right pump connects to the mobile phase for separation and detection. The eluent varies according to the polarity of the components being processed. For hydrophobic components, use the mobile phase of the corresponding analytical chromatographic column; for amphoteric components, use water / organic phase / acid-base regulator; for polar components, use an aqueous solution of a low proportion of organic phase. The enrichment and purification time can be adjusted within the range of 0.5 - 3 min according to the content and properties of the target components in the sample.

[0026] The detection method of on-line solid-phase extraction-liquid chromatography coupling provided by this application uses PAN-HLB composite nanofibers as the solid-phase adsorbent. Without the need for a high-concentration organic phase as the eluent, the target analyte can be separated at a speed compatible with liquid chromatography analysis, thus enabling the on-line coupling of solid-phase extraction and liquid chromatography, integrating on-line processing and detection. While ensuring the detection sensitivity and accuracy of liquid chromatography, it simplifies the operation process, saves manpower, reduces manual operation errors, and reduces time consumption.

[0027] Moreover, compared with traditional methods such as liquid-liquid extraction, the detection method provided by this application reduces the usage amount of organic solvents, which conforms to the concept of green chemistry. The on-line solid-phase extraction column of PAN-HLB composite nanofibers is easy to operate, time-saving and labor-saving, and can be recycled more than 400 times, with obvious advantages. The operation error is small, and the precision of parallel operations is very high.

[0028] In addition, as described above, during the solid-phase extraction process, the solid-phase extraction agent is usually placed in a solid-phase extraction column to adsorb and enrich the target analyte, and then the target analyte is separated by a corresponding eluent. Since components with different polarities bind to the solid-phase extraction agent in different ways to achieve the enrichment of the target component, generally, the existing same solid-phase extraction column can only enrich the target analyte with a certain polarity or within a specific polarity range, resulting in a narrow application range of the solid-phase extraction column. If the solid-phase extraction process is combined with a liquid chromatograph to form an on-line integrated structure, during the working process, different solid-phase extraction columns need to be frequently replaced according to the different polarities of the target analyte, which to a certain extent limits the application of the on-line solid-phase extraction-liquid chromatography combined detection method. In this application, by using PAN-HLB composite nanofibers as the solid-phase adsorbent and combining with a specific eluent, different polar target analytes can be efficiently enriched by one solid-phase extraction column, and the target analyte can be separated at a suitable rate during the elution process. Compared with the traditional method, the enrichment factor can be increased by 10 - 50 times, significantly improving the detection sensitivity, and trace levels of different polar components can be detected. This method has a wide applicability and can process target components with different polarities such as hydrophilic, hydrophobic, and amphoteric without replacing the solid-phase extraction column, and is applicable to a variety of complex sample systems, such as the detection of different polar pollutants in water samples in environmental monitoring, the analysis of different polar drugs and metabolites in biological samples in biomedicine, the determination of different polar additives and pollutants in food detection, etc., which helps to promote the application of on-line solid-phase extraction-liquid chromatography combination.

[0029] In this application, the PAN-HLB composite nanofibers are preferably prepared by the following method: Dissolve polyacrylonitrile (PAN) and a hydrophilic-lipophilic balance copolymer (HLB) in a solvent, preferably the solvent is N,N-dimethylformamide (DMF), and stir, preferably stir for 24 hours, to form a spinning solution; Prepare composite nanofibers from the spinning solution by electrospinning technology to obtain PAN-HLB composite nanofibers; preferably, the process parameters of the electrospinning process are as follows: voltage 18 kV, advancing rate 2.0 mL / h, needle-receiving plate distance 15 cm.

[0030] In the PAN-HLB composite nanofibers prepared in this application, there are generated oxazole groups. The oxazole groups have certain basicity and a special electron cloud structure, which enables them to form specific weak interactions with the target substance, such as hydrogen bonds, π-π stacking, etc. At the same time, HLB is embedded in the three-dimensional structure of PAN fibers. While endowing the composite fibers with good structural stability and appropriate deformability, this structure also changes the elution kinetics, so that the PAN-HLB composite nanofibers can combine with substances with different polarities in different ways and achieve the separation of the target analyte through the corresponding eluent.

[0031] For better understanding, the structure, adsorption, and elution processes of the PAN-HLB composite nanofibers will be specifically analyzed as follows: 1) Structural stability and dynamic regulation of oxazolyl groups: The three-dimensional structure of PAN fibers endows the composite fibers with good structural stability and appropriate deformability. During the elution process, the flow and penetration of a low proportion of organic solvents will affect the material structure. The PAN-HLB composite nanofibers can maintain a relatively stable structure under such circumstances, and their deformability helps the eluent penetrate better around the HLB action sites. The oxazolyl groups can dynamically regulate their interaction with the target substance with the slight deformation of the structure. When the eluent enters, the distance and angle between the oxazolyl groups and the target substance change, further promoting desorption, so that desorption can be completed without the need for high-concentration organic solvents. In contrast, the structure of the pure HLB particle material is relatively rigid, making it difficult for the eluent to penetrate, and lacking this dynamic regulation mechanism of oxazolyl groups, it is difficult to adapt to the elution process through structural adjustment, resulting in difficulty in eluting the target substance.

[0032] 2) Regulation of the elution equilibrium by oxazolyl groups: In the PAN-HLB composite nanofibers, the newly formed oxazolyl groups play a unique role. The oxazolyl groups have certain basicity and a special electron cloud structure, which enables them to form specific weak interactions with the target substance, such as hydrogen bonds, π-π stacking, etc. During the elution stage, a low proportion of organic solvents can change the microenvironment around the oxazolyl groups and weaken their interaction with the target substance. For hydrophilic related substances, such as folic acid, the nitrogen atom on the oxazolyl group can form hydrogen bonds with carboxyl, hydroxyl, etc. in the hydrophilic substance molecule. In the presence of a low proportion of organic solvents, the organic solvent molecules will compete for the hydrogen bond sites, breaking the hydrogen bond connection between the oxazolyl group and the hydrophilic substance, and promoting the desorption of the target substance.

[0033] Based on the binding mode of the PAN-HLB composite nanofibers with hydrophilic substances, the present application can desorb hydrophilic target analytes at an appropriate rate using low-concentration organic solvents.

[0034] However, there are no oxazolyl groups in the pure HLB particle material, lacking this adjustable interaction at a low proportion of organic solvents, resulting in difficulty in eluting the target substance from the HLB, and high-concentration organic solvents must be used during the elution process.

[0035] 3) Differences in elution kinetics under the influence of structure: HLB is embedded in the three-dimensional structure of PAN fibers, and this structure changes the elution kinetics. In PAN-HLB composite nanofibers, although the amount of HLB decreases, it is dispersed in the three-dimensional structure, making the binding sites of the target substance with HLB relatively more "exposed". During elution, a low proportion of organic solvent can diffuse to the binding sites more quickly and compete with the target substance for binding. When eluting hydrophobic substances such as phthalate esters, organic solvent molecules can quickly reach the HLB binding sites along the pores of PAN fibers, weakening the interaction between the target substance and HLB and achieving elution. In pure HLB particulate materials, HLB is closely packed, and there are more binding sites where the target substance is wrapped inside the particles. The diffusion path of the eluent inside the particles is long and the resistance is large. A low proportion of organic solvent is difficult to penetrate deep inside to fully interact with the target substance, resulting in low elution efficiency and only a small amount of substances can be eluted.

[0036] Based on the special structure of PAN-HLB composite nanofibers, this application can desorb hydrophobic target analytes at an appropriate rate without using high-concentration organic solvents.

[0037] 4) Synergistic effect of solvation microenvironment and oxazolyl group: The presence of PAN changes the solvation environment on the material surface and produces a synergistic effect with the oxazolyl group. The PAN molecular chain has hydrophilicity and flexibility. When eluting with a low proportion of organic solvent, PAN molecules interact with organic solvent molecules to form a microenvironment conducive to the desorption of the target substance. At the same time, the oxazolyl group also affects the structure and properties of the surrounding solvation layer. For amphoteric quinolone antibiotics, the hydrogen bond network formed by PAN molecules and organic solvents and the regulation of the surrounding polarity by the oxazolyl group work together to make the target substance more likely to detach from HLB and dissolve in the eluent. The surface of pure HLB particulate materials lacks this special solvation environment jointly constructed by PAN and the oxazolyl group. In a low proportion of organic solvent, it is difficult for the target substance to desorb from HLB and enter the eluent, and the elution effect is not good.

[0038] It can be seen that based on the special structure and properties of the PAN-HLB composite nanofibers, this application uses them in solid-phase extraction technology, which can not only achieve efficient enrichment of hydrophilic, hydrophobic, and amphoteric substances, but also complete the desorption of target analytes without the need for high-concentration organic solvents, thereby realizing on-line solid-phase extraction-high performance liquid chromatography coupling; specifically, by virtue of the special adsorption properties of the PAN-HLB composite nanofibers, an on-line solid-phase extraction column is first prepared and packed, and then it is constructed into an on-line detection system with a high-performance liquid chromatograph. This system has high-efficiency enrichment and separation capabilities for different polar target components such as hydrophilic, hydrophobic, and amphoteric, can remove interfering substances in the sample, and achieve selective extraction of different polar target components. The integration of on-line processing and detection simplifies the operation process, saves manpower, reduces manual operation errors, and can ensure the accurate qualitative and quantitative analysis of different polar components in complex samples. It shows good application prospects in the detection of various complex samples such as environmental water samples, biological samples, and food samples. To balance the adsorption and desorption properties of the solid-phase adsorbent for target analytes, the mass ratio of polyacrylonitrile to the hydrophilic-lipophilic balance copolymer in this application is preferably 10:(6-7), and further preferably 10:6.5; further, the mass ratio of polyacrylonitrile, the solvent of the hydrophilic-lipophilic balance copolymer is preferably 10:6.5:83.5.

[0039] The HLB in the present invention can be directly purchased or prepared by oneself; this application preferably prepares HLB according to the following method: In a 500 mL round-bottom flask, add 165 mL of acetonitrile and a magnetic stir bar. Purge the solution with nitrogen, immerse the flask in silicone oil, stir the magnetic stir bar at about 100 rpm for 30 minutes, heat to 75 °C, and then add 4 ml of divinylbenzene (DVB) cross-linking agent (80%, 20% styrene v / v, Sigma-Aldrich) and 1.5 mL of n-vinylpyrrolidone (N-VP) (99%, Sigma-Aldrich) monomer. Finally, add 200 mg of azobisisobutyronitrile (AIBN) as an initiator, introduce N2 to displace air, and stir at 100 rpm for 24 hours. Then filter with a 0.45 µm Whatman nylon filter paper on a Buchner funnel, collect the particles, and obtain HLB particles. Wash the HLB particles 3 times with 50 mL of ethanol and dry them overnight in a 60 °C vacuum oven.

[0040] To balance the adsorption and desorption properties of the solid-phase adsorbent for target analytes, the specific surface area of the polyacrylonitrile-hydrophilic-lipophilic balance copolymer composite nanofibers in this application is preferably 150-200 m 2 / g; the average pore diameter of the polyacrylonitrile-hydrophilic-lipophilic balance copolymer composite nanofibers is preferably 3.0-3.5 nm.

[0041] In some embodiments, the hydrophilic component includes water-soluble vitamins and their metabolites. Specifically, the hydrophilic component is preferably selected from vitamin B2 (VB2), vitamin B 12 (VB 12 ), vitamin B9 (folic acid, FA), 5-methyltetrahydrofolic acid (5-MeTHF), 5-formyltetrahydrofolic acid (5-FoTHF), 5,10-dimethyltetrahydrofolic acid (5,10-CH2-THF), S-adenosyl-L-methionine (SAM), S-adenosyl-L-homocysteine (SAH), or at least one of them.

[0042] In some embodiments, the hydrophobic component includes phthalate substances; and preferably the phthalate substances are selected from at least one of dimethyl phthalate (DMP), diethyl phthalate (DEP), di-n-propyl phthalate (DPrP), butyl benzyl phthalate (BBP), di-n-butyl phthalate (DnBP), di-n-pentyl phthalate (DAP), dicyclohexyl phthalate (DCHP), di-n-hexyl phthalate (DnHP), diisooctyl phthalate (DIOP), and di-n-octyl phthalate (DnOP).

[0043] In some embodiments, the amphoteric component includes quinolone antibiotics; and preferably the quinolone antibiotics are selected from at least one of ofloxacin (OFLx), norfloxacin (NOR), ciprofloxacin (CIP), danofloxacin (DAN), enrofloxacin (ENR), and sarafloxacin (SAR).

[0044] In this application, an online solid-phase extraction column (packed with PAN-HLB composite nanofibers) is used to design its connection with a high-performance liquid system and the operating procedures required for different polar component substances. In the experiment, the repeatability, precision, recovery rate of spiked samples, linear range and other methodological indicators of this online pretreatment and detection method were mainly investigated. The detection method provided in this application can achieve rapid and efficient enrichment and separation of different polar target components, improve the sensitivity and accuracy of detection, and meet the needs of effectively detecting different polar components in complex samples; through the combination of online pretreatment and analytical instruments, automated operation is realized, greatly shortening the pretreatment and detection time. The entire detection process can be completed within 15-20 minutes, improving the detection efficiency and being applicable to high-throughput sample analysis.

[0045] The PAN-HLB composite nanofibers in the following examples are prepared according to the following method: PAN (10 wt%) and HLB (6.5 wt%) were dissolved in DMF and stirred for 24 hours to form a spinning solution. Composite nanofibers were prepared by electrospinning technology (voltage 18 kV, propulsion rate 2.0 mL / h, needle-receiving plate distance 15 cm) to obtain PAN-HLB composite nanofibers.

[0046] Polyacrylonitrile (PAN, molecular weight 150,000) in this application was purchased from Sigma-Aldrich. HLB copolymer particles were prepared as follows: In a 500 mL round-bottom flask, 165 mL of acetonitrile and a magnetic stir bar were added. The solution was purged with nitrogen, and the flask was immersed in silicone oil and stirred with a magnetic stir bar at about 100 rpm for 30 minutes, heated to 75°C, and then 4 ml of divinylbenzene (DVB) crosslinker (80%, 20% styrene v / v, Sigma-Aldrich) and 1.5 mL of n-vinylpyrrolidone (N-VP) (99%, Sigma-Aldrich) monomer were added. Finally, 200 mg of azobisisobutyronitrile (AIBN) was added as an initiator, and N2 was introduced to replace the air, and stirred at 100 rpm for 24 hours. Then, the particles were collected by suction filtration on a Buchner funnel with 0.45 µm Whatman nylon filter paper to obtain HLB particles. The HLB particles were washed three times with 50 mL of ethanol and dried in a vacuum oven at 60°C overnight.

[0047] Example 1

[0048] Detection of quinolone antibiotics in environmental water samples 1) Filling of online solid phase extraction column: A 10 mm × 2.1 mm stainless steel column tube with detachable openings at both ends was used. The prepared PAN-HLB composite nanofiber material was loaded in batches into the bottom of the column tube sealed with a sieve plate at one end, compacted with a stainless steel rod with a diameter of 0.5 mm, and sealed with a sieve plate at the other end. After being loaded into a jacket, it was connected to the high performance liquid chromatography system.

[0049] 2) Sample preparation: Collect environmental water samples and filter them through a 0.45 μm filter membrane as samples to be tested.

[0050] 3) Online pretreatment: After adjusting the pH of the filtered water sample to 2, it was passed through an online solid phase extraction column filled with PAN-HLB composite nanofibers at a flow rate of 0.5 mL / min to adsorb the amphoteric quinolone antibiotics (six types, ofloxacin (OFLx), norfloxacin (NOR), ciprofloxacin (CIP), danofloxacin (DAN), enrofloxacin (ENR), and sarafloxacin (SAR)) in the water sample on the fiber.

[0051] 4) Elution and analysis: The on-line solid-phase extraction column was eluted with an ammonia water (0.05%) solution containing 30% methanol at a flow rate of 0.5 mL / min, and the eluate directly entered the high-performance liquid chromatograph. The analysis conditions of the high-performance liquid chromatograph were set as follows: C18 chromatographic column (inner diameter 4.6 mm, length 150 mm, particle size 5 μm); the mobile phase was acetonitrile–0.2% formic acid aqueous solution (gradient elution, 0 - 0.5 min, 100% 0.2% formic acid aqueous solution; 0.55–1.55 min, 100% ammonia water (0.05%) solution containing 30% methanol; 1.6–8 min, 18% acetonitrile; 8–10 min, 12% acetonitrile; 11–12 min, 100% 0.2% formic acid aqueous solution); the flow rate was 1.0 mL / min; the column temperature was 30 °C; the detection wavelength excitation wavelength was 280 nm, and the emission wavelength was 450 nm; the valve switching time was 0.5 min. Quantitative analysis was performed according to the chromatographic peak area.

[0052] Example 2

[0053] Detection of quinolone antibiotics in food samples 1) Construction of on-line pretreatment device: Similar to Example 1, a 10 mm×2.1 mm stainless steel column tube with open ends at both ends and detachable was filled with PAN-HLB composite nanofibers as the on-line solid-phase extraction column, and the high-performance liquid chromatography system was connected.

[0054] 2) Sample preparation: Weigh 0.1 g of food sample (such as milk), after protein precipitation with acid addition, take the supernatant and filter it through a 0.45 μm filter membrane, and then dilute it to 4 mL with ultrapure water as the sample to be detected.

[0055] 3) On-line pretreatment: The processed sample solution was passed through the micro solid-phase extraction column filled with PAN-HLB composite nanofibers at a flow rate of 0.5 mL / min, so that the quinolone antibiotics (6 kinds, ofloxacin (OFLx), norfloxacin (NOR), ciprofloxacin (CIP), danofloxacin (DAN), enrofloxacin (ENR), sarafloxacin (SAR)) were adsorbed on the fibers.

[0056] 4) Elution and analysis: The on-line solid-phase extraction column was eluted with an ammonia water (0.05%) solution containing 30% methanol at a flow rate of 0.5 mL / min, and the eluate directly entered the high-performance liquid chromatograph. The analysis conditions of the high-performance liquid chromatograph were the same as those in Example 1, and quantitative analysis was performed according to the chromatographic peak area.

[0057] Example 3

[0058] Detection of polar vitamins and metabolites in biological samples 1) Construction of the on-line pretreatment device: Similar to the previous two embodiments, an on-line pretreatment and detection device was built.

[0059] 2) Sample preparation: A biological sample (urine) was collected. 0.1 mL of the urine sample was added with an antioxidant and diluted to 4 mL with ultrapure water to obtain the sample to be detected.

[0060] 3) On-line pretreatment: The sample to be detected was passed through a micro-solid phase extraction column packed with PAN-HLB composite nanofibers at a flow rate of 0.5 mL / min to achieve the adsorption and enrichment of polar vitamins and metabolites (vitamin B2 (VB2), vitamin B 12 (VB 12 ), vitamin B9 (folic acid, FA), 5-methyltetrahydrofolic acid (5-MeTHF), 5-formyltetrahydrofolic acid (5-FoTHF), 5,10-dimethyltetrahydrofolic acid (5,10-CH2-THF), S-adenosyl-L-methionine (SAM), S-adenosyl-L-homocysteine (SAH)).

[0061] 4) Elution and analysis: An acetate buffer solution with pH 4.25 containing 5% methanol was used as the eluent to elute the on-line solid phase extraction column at a flow rate of 0.5 mL / min, and the eluate entered a high performance liquid chromatograph. Analysis conditions of the high performance liquid chromatograph: A C18 column (inner diameter 4.0 mm, length 150 mm, particle size 3 μm) was used, and the mobile phase was methanol–acetate buffer solution with pH 4.25 (gradient elution, 0–2.5 min, 5% methanol; 2.5–3.6 min, 3% methanol; 3.6–8.6 min, 45% methanol; 8.6–11.6 min, 45% methanol; 11.6–12 min, 5% methanol); the flow rate was 0.5 mL / min; the column temperature was 30 °C; the absorption wavelengths were 2.95–3.2 min / 259 nm, 6.9 min / 280 nm, 7.0–7.85 min / 259 nm, 9.5 min / 362 nm, 9.75 min / 280 nm. The valve switching time was 2.5 min, and quantitative analysis was performed based on the peak area or peak height.

[0062] Examples 4-6 and Comparative Examples 1-6 were comparative experiments. The PAN-HLB composite nanofiber online solid-phase extraction column disclosed in the present invention was compared with the commercially available Waters HLB online extraction column and C18 online extraction column for hydrophobic phthalate substances (10 kinds, dimethyl phthalate (DMP), diethyl phthalate (DEP), di-n-propyl phthalate (DPrP), butyl benzyl phthalate (BBP), di-n-butyl phthalate (DnBP), di-n-amyl phthalate (DAP), dicyclohexyl phthalate (DCHP), di-n-hexyl phthalate (DnHP), diisooctyl phthalate (DIOP), di-n-octyl phthalate (DnOP)); hydrophilic vitamin-related substances (vitamin B2 (VB2), vitamin B 12 (VB 12 )), vitamin B9 (folic acid, FA), 5-methyltetrahydrofolic acid (5-MeTHF), 5-formyltetrahydrofolic acid (5-FoTHF), 5,10-dimethyltetrahydrofolic acid (5,10-CH2-THF), S-adenosyl-L-methionine (SAM), S-adenosyl-L-homocysteine (SAH)); and the extraction and separation performance of amphoteric quinolone antibiotics (6 kinds, ofloxacin (OFLx), norfloxacin (NOR), ciprofloxacin (CIP), danofloxacin (DAN), enrofloxacin (ENR), sarafloxacin (SAR)) was compared.

[0063] Example 4

[0064] Experiment on hydrophobic phthalate (PAEs) substances Using the PAN-HLB composite nanofiber online extraction column of the present invention: Place the sample vial containing the PAEs standard solution in the autoinjector, run the chromatographic program, inject 1000 μL into the online extraction column, use ultrapure water as the mobile phase of the left pump, set the enrichment and purification time to 0.5 min, switch the valve path to connect the online extraction column to the right pump, use a 60% methanol solution as the eluent to elute the PAEs substances adsorbed on the extraction column and transfer them to the analytical column, set the elution time to 1 min, switch the valve path back to the original state again, reactivate the extraction column with the left pump for another injection, and connect the right pump to the mobile phase for separation and detection, and record the chromatogram (as shown in b in Figure 1 、 Figure 2 、 Figure 3 ); After calculation, the recovery rate is 95.0 - 102.2%.

[0065] Example 5

[0066] Experiment on hydrophilic vitamin-related substances For the PAN-HLB composite nanofiber online extraction column of the present invention: Place the sample vial containing the hydrophilic vitamin-related substance standard solution in the auto-sampler, run the chromatographic program, inject 1000 μL into the online extraction column. Use ultrapure water as the mobile phase for the left pump, set the enrichment and purification time to 0.5 min, switch the valve path to connect the online extraction column to the right pump. Use an acetate buffer solution with 5% methanol and pH 4.25 as the eluent to elute the hydrophilic-related substances adsorbed on the extraction column and transfer them to the analytical column. The elution time is 2 min. Switch the valve path back to the original state again, and the left pump reactivates the extraction column for another injection. The right pump connects to the mobile phase for separation and detection, and record the chromatogram (as shown in b in Figure 4 and Figure 5 and Figure 6 ); After calculation, the recovery rate is 85.4 - 96.3%.

[0067] Example 6

[0068] Experiment on amphoteric quinolone antibiotics For the PAN-HLB composite nanofiber online extraction column of the present invention: Place the sample vial containing the amphoteric quinolone antibiotic standard solution in the auto-sampler, run the chromatographic program, inject 1000 μL into the online extraction column. Use ultrapure water as the mobile phase for the left pump, set the enrichment and purification time to 0.5 min, switch the valve path to connect the online extraction column to the right pump. Use an ammonia water (0.05%) solution with 30% methanol as the eluent to elute the amphoteric substances adsorbed on the extraction column and transfer them to the analytical column. The elution time is 1 min. Switch the valve path back to the original state again, and the left pump reactivates the extraction column for another injection. The right pump connects to the mobile phase for separation and detection, and record the chromatogram (as shown in b in Figure 7 and Figure 8 and Figure 9 ); After calculation, the recovery rate is 78.6% - 120.4%.

[0069] Comparative Example 1 Compare with Example 4: For the HLB online extraction column of Waters: Operate under the same conditions such as injection volume, mobile phase, enrichment and purification time, eluent, and chromatographic program as in Example 4, and record the chromatogram (as shown in a in Figure 2 ).

[0070] Comparative Example 2 Compare with Example 4: For the C18 online extraction column: Similarly operate under the same conditions such as injection volume, mobile phase, enrichment and purification time, eluent, and chromatographic program as in Example 4, and record the chromatogram (as shown in a in Figure 3 ).

[0071] Comparative Example 3 Compare with Example 5: For the Waters HLB online extraction column: Operate under the same conditions as in Example 5, such as injection volume, mobile phase, enrichment and purification time, eluent, and chromatographic program, and record the chromatogram (see Figure 5 as shown in a).

[0072] Comparative Example 4 Compare with Example 5: For the C18 online extraction column: Also operate under the same conditions as in Example 5, such as injection volume, mobile phase, enrichment and purification time, eluent, and chromatographic program, and record the chromatogram (see Figure 6 as shown in a).

[0073] Comparative Example 5 Compare with Example 6: For the Waters HLB online extraction column: Operate under the same conditions as in Example 6, such as injection volume, mobile phase, enrichment and purification time, eluent, and chromatographic program, and record the chromatogram (see Figure 8 as shown in a).

[0074] Comparative Example 6 Compare with Example 6: For the C18 online extraction column: Also operate under the same conditions as above, such as injection volume, mobile phase, enrichment and purification time, eluent, and chromatographic program, and record the chromatogram (see Figure 9 as shown in a).

[0075] Experimental Results and Analysis: 1. Results of hydrophobic phthalate esters (PAEs): See Figure 1 As shown, the chromatogram (b) of the hydrophobic phthalate ester standard solution (1000 ng / mL) detected by PAN-HLB composite nanofiber online solid-phase extraction is compared with the chromatogram (a) of direct injection analysis, which intuitively shows the enrichment and separation effects of online solid-phase extraction on hydrophobic substances.

[0076] See Figure 2As shown in the figure, the chromatogram (b) of the hydrophobic phthalate standard solution (1000 ng / mL) detected by online solid-phase extraction with PAN-HLB composite nanofibers is compared with the chromatogram (a) detected by the Oasis HLB Direct Connect HP (Waters company, 2.1×30 mm, 20 μm) online extraction column, intuitively reflecting that the PAN-HLB composite nanofiber online extraction column has a better enrichment and elution effect on hydrophobic PAEs substances than the Waters HLB particle online extraction column; See Figure 3 As shown in the figure, the chromatogram (b) of the hydrophobic phthalate standard solution (1000 ng / mL) detected by online solid-phase extraction with PAN-HLB composite nanofibers is compared with the chromatogram (a) detected by the C18 (YMC, 2.1×10 mm, 5 μm) online extraction column, intuitively reflecting that the PAN-HLB composite nanofiber online extraction column has a better enrichment and elution effect on hydrophobic PAEs substances than the C18 online extraction column.

[0077] The chromatogram obtained by using the PAN-HLB composite nanofiber online extraction column of the present invention is compared with the chromatograms obtained by the Waters HLB online extraction column and the C18 online extraction column. From the attached Figure 2 and attached Figure 3 It can be clearly seen that the PAN-HLB online extraction column has a higher chromatographic peak response to 10 kinds of PAEs. The Waters HLB online extraction column only has a good enrichment and elution effect on DnHP, DIOP, and DnOP with weaker polarity, while the C18 online extraction column has almost no enrichment and elution effect under the same conditions. This shows that the PAN-HLB composite nanofiber online extraction column of the present invention has a more efficient enrichment and elution ability for hydrophobic PAEs substances and can more accurately separate and detect various PAEs substances.

[0078] 2. Results of hydrophilic vitamins and related substances: See Figure 4 As shown in the figure, the chromatogram (b) of the hydrophilic vitamin standard solution (500 ng / mL) detected by online solid-phase extraction with PAN-HLB composite nanofibers is compared with the chromatogram (a) of direct injection analysis, demonstrating the effective detection ability for hydrophilic substances.

[0079] See Figure 5As shown, the chromatogram (b) of the hydrophilic vitamin standard solution (500 ng / mL) detected by online solid-phase extraction with PAN-HLB composite nanofibers is compared with the chromatogram (a) detected by the Oasis HLB Direct Connect HP (Waters company, 2.1×30 mm, 20 μm) online extraction column, intuitively presenting that the PAN-HLB composite nanofiber online extraction column has better enrichment and elution effects on hydrophilic-related substances than the Waters' HLB particle online extraction column.

[0080] See Figure 6 As shown, the chromatogram (b) of the hydrophilic vitamin standard solution (500 ng / mL) detected by online solid-phase extraction with PAN-HLB composite nanofibers is compared with the chromatogram (a) detected by the C18 (YMC, 2.1×10 mm, 5 μm) online extraction column, intuitively presenting that the PAN-HLB composite nanofiber online extraction column has better enrichment and elution effects on hydrophilic-related substances than the C18 online extraction column.

[0081] The chromatogram (a) obtained by using the PAN-HLB composite nanofiber online extraction column of the present invention is compared with the chromatograms (b) obtained by the Waters' HLB online extraction column and the C18 online extraction column. From the attached Figure 5 and attached Figure 6 It can be seen that for folic acid-related substances such as SAM and 5-CH3-THF, the chromatographic peaks obtained by the PAN-HLB online extraction column are clearer and the response values are higher, while the Waters' HLB online extraction column and the C18 online extraction column only have good enrichment and elution effects on SAM. This shows that the PAN-HLB composite nanofiber online extraction column of the present invention has better enrichment and elution effects on hydrophilic vitamins and related substances, and can analyze hydrophilic vitamins and related substances more accurately.

[0082] 3. Results of amphoteric quinolone antibiotics: See Figure 7 As shown, the chromatogram (b) of the amphoteric quinolone substance (1 ng / mL) detected by online solid-phase extraction with PAN-HLB composite nanofibers is compared with the chromatogram (a) of direct injection analysis, reflecting the detection advantage of this method for amphoteric substances.

[0083] See Figure 8As shown, the chromatogram of the zwitterionic quinolone (1 ng / mL) detected by on-line solid-phase extraction using PAN-HLB composite nanofibers (b) is compared with the chromatogram detected by the Oasis HLB Direct Connect HP (Waters company, 2.1×30 mm, 20 μm) on-line extraction column (a), intuitively showing that the PAN-HLB composite nanofiber on-line extraction column has better enrichment and elution effects on zwitterionic substances than the Waters' HLB particle on-line extraction column.

[0084] See Figure 9 As shown, the chromatogram of the zwitterionic quinolone (1 ng / mL) detected by on-line solid-phase extraction using PAN-HLB composite nanofibers (a) is compared with the chromatogram detected by the C18 (YMC, 2.1×10 mm, 5 μm) on-line extraction column (b), intuitively showing that the PAN-HLB composite nanofiber on-line extraction column has better enrichment and elution effects on zwitterionic substances than the C18 on-line extraction column.

[0085] The chromatogram (a) obtained by using the PAN-HLB composite nanofiber on-line extraction column of the present invention is compared with the chromatograms (b) obtained by the Waters' HLB on-line extraction column and the C18 on-line extraction column. From the attached Figure 8 and attached Figure 9 It can be seen that for zwitterionic quinolone antibiotics, the chromatographic peak response value obtained by the PAN-HLB on-line extraction column is much higher than the chromatographic response values obtained by the Waters' HLB on-line extraction column and the C18 on-line extraction column, indicating that the PAN-HLB composite nanofiber on-line extraction column of the present invention also has better enrichment and elution effects on zwitterionic substances.

[0086] 4. Conclusion Through this comparative experiment, it is fully proved that the PAN-HLB composite nanofiber on-line extraction column of the present invention has significant advantages in the extraction and separation performance of hydrophobic phthalate esters (10 kinds), hydrophilic vitamins and related substances (8 kinds), and zwitterionic quinolone antibiotics (6 kinds) compared with the Waters' HLB on-line extraction column and the C18 on-line extraction column, further verifying the effectiveness and superiority of the technical solution of the present invention.

[0087] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the embodiments of the present invention.

Claims

1. An on-line solid phase extraction-liquid chromatography coupled detection method, characterized in that, It includes the following process: using polyacrylonitrile-hydrophilic-lipophilic balance copolymer composite nanofibers as a solid-phase adsorbent to adsorb the target analyte, and then using an eluent to elute the target analyte on the solid-phase adsorbent to obtain an eluate; transferring the eluate to an analytical column of a liquid chromatograph for analysis to obtain a chromatogram of the target analyte; The target analyte is one of a hydrophilic component, a hydrophobic component, and an amphoteric component; When the target analyte is a hydrophilic component, the eluent is an organic phase aqueous solution; When the target analyte is a hydrophobic component, the eluent is the mobile phase of the chromatographic analytical column; When the target analyte is an amphoteric component, the eluent is selected from one or a combination of water, an organic phase aqueous solution, and an acid-base regulator.

2. The detection method of online solid-phase extraction-liquid chromatography coupling according to claim 1, characterized in that, The polyacrylonitrile-hydrophilic-lipophilic balance copolymer composite nanofibers are prepared according to the following method: dissolving polyacrylonitrile and a hydrophilic-lipophilic balance copolymer in a solvent, stirring to form a spinning solution; preparing composite nanofibers from the spinning solution by electrospinning technology to obtain polyacrylonitrile-hydrophilic-lipophilic balance copolymer composite nanofibers.

3. The detection method of online solid-phase extraction-liquid chromatography coupling according to claim 2, wherein, The mass ratio of the polyacrylonitrile to the hydrophilic-lipophilic balance copolymer is 10:(6 - 7).

4. The detection method of on-line solid phase extraction-liquid chromatography coupling according to claim 2, characterized in that, The specific surface area of the polyacrylonitrile-hydrophilic lipophilic balance copolymer composite nanofiber is 150-200 m 2 / g.

5. The detection method of on-line solid phase extraction-liquid chromatography coupling according to claim 2, wherein The average pore size of the polyacrylonitrile-hydrophilic-lipophilic balance copolymer composite nanofibers is 3.0 - 3.5 nm.

6. The detection method of online solid phase extraction-liquid chromatography coupling according to any one of claims 1-5, characterized in that, The hydrophilic component is selected from at least one of vitamin B2, vitamin B 12 , vitamin B9, 5-methyltetrahydrofolic acid, 5-formyltetrahydrofolic acid, 5,10-dimethyltetrahydrofolic acid, S-adenosyl-L-methionine, and S-adenosyl-L-homocysteine.

7. The detection method of online solid phase extraction-liquid chromatography coupling according to any one of claims 1-5, characterized in that, The hydrophobic component includes phthalate substances.

8. The detection method of online solid phase extraction-liquid chromatography coupling according to claim 7, characterized in that, The phthalate substances are selected from at least one of diethyl phthalate, di-n-propyl phthalate, butyl benzyl phthalate, di-n-butyl phthalate, di-n-pentyl phthalate, dicyclohexyl phthalate, di-n-hexyl phthalate, diisooctyl phthalate, and di-n-octyl phthalate.

9. The detection method of on-line solid phase extraction-liquid chromatography coupling according to any one of claims 1-5, characterized in that, The amphoteric component includes quinolone antibiotics.

10. The detection method of on-line solid phase extraction-liquid chromatography coupling according to claim 9, characterized in that, The quinolone antibiotics are selected from at least one of ofloxacin, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, and sarafloxacin.

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