Online solid phase extraction-liquid chromatography detection method

Abstract

The present invention relates to the technical field of analytical chemistry detection, and discloses an online solid-phase extraction-liquid chromatography coupled detection method, comprising the following steps: adsorbing a target analyte using polyacrylonitrile-hydrophilic-lipophilic balanced copolymer composite nanofibers as a solid-phase adsorbent, eluting the target analyte from the solid-phase adsorbent using an eluent 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; and separating the target analyte at a speed compatible with liquid chromatography analysis without requiring a high-concentration organic phase as an eluent, thereby enabling online coupling of solid-phase extraction and liquid chromatography.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical chemistry detection, in particular to an on-line solid phase extraction-liquid chromatography coupled detection method. Background Art

[0002] Liquid chromatography is an instrument that uses differences in the distribution ratio of a mixture between liquid and solid or between two immiscible liquids to separate and subsequently analyze and identify the substance. High-performance liquid chromatography (HPLC) is widely used in various fields, including biochemistry, pharmaceutical research, environmental analysis, inorganic analysis, and food analysis, due to its advantages such as high resolution, high sensitivity, high speed, reusable columns, and easy collection of effluent fractions. It is one of the most commonly used instruments in biochemical analysis.

[0003] The high-resolution and high-sensitivity detection advantages of high-performance liquid chromatography (HPLC) largely depend on sample pretreatment, and solid-phase extraction (SPE) is currently the most commonly used sample pretreatment technique. 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. An appropriate solvent is then selected to elute the analyte, thereby achieving the goal of purifying, separating, and enriching the target analyte.

[0004] Traditional SPE technology mostly relies on offline manual operation, which is time-consuming, labor-intensive, and prone to loss errors. Combining SPE technology with high-performance liquid chromatography online to achieve online integrated operation of sample pretreatment and liquid chromatography analysis is an effective means to address the above shortcomings.

[0005] As mentioned above, solid-phase extraction is achieved by adsorbing and enriching the target analyte with a solid-phase adsorbent, followed by an elution process. Currently, the solid-phase extraction process often uses a hydrophilic-lipophilic balance (HLB) copolymer as a solid-phase extraction adsorbent. Although this HLB has high selectivity for the target molecule, the elution process requires a high-concentration organic solvent such as pure acetonitrile and methanol as an eluent. When it is combined with liquid chromatography online, the concentration of the organic solvent used as the eluent is too high, and the components will be separated in too short a time, resulting in the inability to detect the target components by liquid chromatography. This makes it difficult for existing solid-phase extraction technology to be combined online with liquid chromatography, and it is impossible to perform online automatic processing and detection, which limits the promotion and application of online solid-phase extraction-liquid chromatography. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides an online solid phase extraction-liquid chromatography detection method, comprising the following steps: adsorbing a target analyte using polyacrylonitrile-hydrophilic-lipophilic balanced copolymer composite nanofibers as a solid phase adsorbent, then eluting the target analyte from the solid phase adsorbent using an eluent 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;

[0007] The target analyte is one of a hydrophilic component, a hydrophobic component, and an amphiphilic component;

[0008] When the target analyte is a hydrophilic component, the eluent is an organic phase aqueous solution;

[0009] When the target analyte is a hydrophobic component, the eluent is the mobile phase of a chromatographic analysis column;

[0010] 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.

[0011] Furthermore, the polyacrylonitrile-hydrophilic-lipophilic balanced copolymer composite nanofiber is prepared according to the following method: polyacrylonitrile and the hydrophilic-lipophilic balanced copolymer are dissolved in a solvent and stirred to form a spinning solution; the spinning solution is used to prepare composite nanofibers through electrospinning technology to obtain polyacrylonitrile-hydrophilic-lipophilic balanced copolymer composite nanofibers.

[0012] Furthermore, the mass ratio of the polyacrylonitrile to the hydrophilic-lipophilic balanced copolymer is 10:(6-7).

[0013] Furthermore, the specific surface area of ​​the polyacrylonitrile-hydrophilic-lipophilic balanced copolymer composite nanofiber is 150-200 m 2 / g.

[0014] Furthermore, the average pore size of the polyacrylonitrile-hydrophilic-lipophilic balanced copolymer composite nanofiber is 3.0-3.5 nm.

[0015] Furthermore, the hydrophilic component is selected from vitamin B2, vitamin B 12 , vitamin B9, 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, 5,10-dimethyltetrahydrofolate, S-adenosyl-L-methionine, and at least one of S-adenosyl-L-homocysteine.

[0016] Furthermore, the hydrophobic component includes phthalates.

[0017] Furthermore, the phthalate ester substance 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, di-isooctyl phthalate, and di-n-octyl phthalate.

[0018] Furthermore, the amphoteric component includes quinolone antibiotics.

[0019] Furthermore, the quinolone antibiotic is selected from at least one of ofloxacin, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, and sarafloxacin.

[0020] The embodiments of the present invention have the following technical effects:

[0021] The online solid-phase extraction-liquid chromatography detection method provided in the present application uses PAN-HLB composite nanofibers as a solid-phase adsorbent. Without the need for a high-concentration organic phase as an eluent, the target analyte can be separated at a speed compatible with liquid chromatography analysis, thereby realizing the online combination of solid-phase extraction and liquid chromatography, and integrating online processing and detection. While ensuring the sensitivity and accuracy of liquid chromatography detection, it simplifies the operating process, saves manpower, reduces manual operation errors, and reduces time consumption. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 The chromatogram (b) and the chromatogram (a) of the hydrophobic phthalate ester standard solution provided by the present invention using PAN-HLB composite nanofiber online solid phase extraction detection are shown;

[0024] Figure 2 The chromatogram (b) of the hydrophobic phthalate standard solution of the present invention using PAN-HLB composite nanofiber online solid phase extraction detection and the chromatogram (a) of the HLB online extraction column detection;

[0025] Figure 3 The chromatogram (b) of the hydrophobic phthalate standard solution of the present invention using PAN-HLB composite nanofiber online solid phase extraction detection and the chromatogram (a) of the C18 online extraction column detection;

[0026] Figure 4 The chromatogram (b) of the hydrophilic vitamin substance standard solution of the present invention using PAN-HLB composite nanofiber online solid phase extraction detection and the chromatogram (a) of direct injection analysis;

[0027] Figure 5 The chromatogram (b) of the hydrophilic vitamin substance standard solution of the present invention using PAN-HLB composite nanofiber online solid phase extraction detection and the chromatogram (a) of the HLB online extraction column detection;

[0028] Figure 6 The chromatogram (b) of the hydrophilic vitamin substance standard solution of the present invention using PAN-HLB composite nanofiber online solid phase extraction detection and the chromatogram (a) of the C18 online extraction column detection;

[0029] Figure 7 The chromatogram (b) of the amphoteric quinolones of the present invention using PAN-HLB composite nanofiber online solid phase extraction detection and the chromatogram (a) of direct injection analysis;

[0030] Figure 8 The chromatogram (b) of the amphoteric quinolones of the present invention using PAN-HLB composite nanofiber online solid phase extraction detection and the chromatogram (a) of the HLB online extraction column detection;

[0031] Figure 9 The chromatogram (a) and the chromatogram (b) of the amphoteric quinolones of the present invention using PAN-HLB composite nanofiber online solid phase extraction detection are shown. DETAILED DESCRIPTION

[0032] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are considered to be within the scope of the present invention.

[0033] In order to solve the problem that existing solid-phase extraction technology is difficult to achieve online coupling with liquid chromatography, the present application provides an online solid-phase extraction-liquid chromatography coupling detection method, which includes the following process: using polyacrylonitrile-hydrophilic-lipophilic balanced 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 a chromatogram of the target analyte; wherein, based on the special structure and properties of PAN-HLB composite nanofibers, the present application enables the solid-phase adsorbent to have excellent adsorption capacity for hydrophilic components, hydrophobic components and amphiphilic components at the same time, that is, the target analyte in the present application is one of the hydrophilic component, hydrophobic component and amphiphilic component.

[0034] For the online solid phase extraction-liquid chromatography detection method, eluting and separating the target analyte on the solid phase adsorbent with an eluent is also a key step to ensure detection sensitivity and accuracy. In order to achieve selective extraction of target analytes of different polarities, the preferred eluent in this application varies according to the polarity of the target analyte being treated. Specifically, when the target analyte is a hydrophilic component, the preferred eluent is an organic phase aqueous solution, and further preferably a low-concentration organic phase aqueous solution. When the target analyte is a hydrophobic component, the preferred eluent is the mobile phase of the analytical chromatographic column. When the target analyte is an amphoteric component, the preferred eluent is one or a combination of water, an organic phase aqueous solution, and an acid-base regulator.

[0035] The online solid phase extraction-liquid chromatography detection method can be specifically carried out according to the following process:

[0036] 1) Loading of online solid-phase extraction column: A 10 mm × 2.1 mm stainless steel column tube with detachable openings at both ends was used as the online solid-phase extraction column. An appropriate amount of PAN-HLB composite nanofibers was loaded in batches at the bottom of the column tube sealed with a sieve plate at one end, compacted with a 0.5 mm diameter stainless steel rod, and the other end was also sealed with a sieve plate. The column was placed in a jacket and connected to a high-performance liquid chromatography system for use. The amount of nanofiber loaded each time was 8-10 mg, and compacted five times with a 0.5 mm diameter stainless steel rod.

[0037] 2) Sample processing: Carry out corresponding processing for different types of samples:

[0038] If it is an environmental water sample, it should be filtered through a 0.45 μm filter membrane as the sample to be tested;

[0039] For food samples (such as milk), weigh 0.1 g, add acid to precipitate 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 tested;

[0040] For biological samples (such as plasma and urine), take 0.1 mL of urine sample, add antioxidant and ultrapure water to dilute to 4 mL; take 0.1 mL of plasma sample, add antioxidant and then add the same volume of acid-precipitated protein, take the supernatant, filter through a 0.45 μm filter membrane, and dilute to 4 mL with ultrapure water as the sample to be tested.

[0041] The sample also contained other samples with similar complex matrices.

[0042] 3) Online solid-phase extraction and detection analysis: Take an appropriate amount of the treated sample solution to be extracted and transfer it to a 5 mL injection bottle. Run the chromatographic program and inject 1000 μL into the online solid-phase extraction column. Use ultrapure water or a certain proportion of organic solvent / water mixed solvent as the mobile phase of the left pump. The enrichment and purification time is 0.5-3 min. Switch the valve path, connect the online solid-phase extraction column to the right pump, use the eluent to elute the target adsorbed on the online solid-phase extraction column and transfer it to the analytical column. The valve path is switched back to the original state. At this time, the left pump reactivates the online solid-phase extraction column for further injection processing, and the right pump is connected to the mobile phase for separation and detection. The eluent varies according to the polarity of the component being treated. Hydrophobic components use the mobile phase of the corresponding analytical chromatographic column, amphoteric components use water / organic phase / acid-base adjuster, and polar components use a low proportion of organic phase aqueous solution. 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 component in the sample.

[0043] The online solid-phase extraction-liquid chromatography detection method provided in the present application uses PAN-HLB composite nanofibers as a solid-phase adsorbent. Without the need for a high-concentration organic phase as an eluent, the target analyte can be separated at a speed compatible with liquid chromatography analysis, thereby realizing the online combination of solid-phase extraction and liquid chromatography, and integrating online processing and detection. While ensuring the sensitivity and accuracy of liquid chromatography detection, it simplifies the operating process, saves manpower, reduces manual operation errors, and reduces time consumption.

[0044] Furthermore, the detection method provided in this application reduces the use of organic solvents compared to traditional liquid-liquid extraction methods, aligning with the concept of green chemistry. The PAN-HLB composite nanofiber online solid-phase extraction column is simple to operate, saving time and effort, and can be recycled over 400 times, offering significant advantages. Operating errors are minimal, and parallel operations achieve high precision.

[0045] In addition, as mentioned above, during the solid-phase extraction process, a solid-phase extractant 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 of different polarities combine with the solid-phase extractant in different ways to achieve the enrichment of the target component, the existing same solid-phase extraction column can usually only enrich target analytes within a certain polarity or a specific polarity range, making the application range of the solid-phase extraction column relatively narrow. If the solid-phase extraction process is combined with a liquid chromatograph to form an online integrated structure, during the working process, different solid-phase extraction columns need to be frequently replaced according to the different polarities of the target analytes, which to a certain extent limits the application of the online solid-phase extraction-liquid chromatography combined detection method. This application uses PAN-HLB composite nanofibers as solid-phase adsorbents in combination with specific eluents to efficiently enrich target analytes of different polarities through a solid-phase extraction column, and can separate the target analytes at an appropriate rate during the elution process. Compared with traditional methods, the enrichment multiple can be increased by 10-50 times, which significantly improves the detection sensitivity and can detect trace levels of different polar components. This method has a wide range of applicability and can process target components of different polarities such as hydrophilicity, hydrophobicity and amphotericity without replacing the solid-phase extraction column. It is suitable for a variety of complex sample systems, such as the detection of pollutants of different polarities in water samples in environmental monitoring, the analysis of drugs and metabolites of different polarities in biological samples in biomedicine, and the determination of additives and pollutants of different polarities in food testing, etc., which will help promote the application of online solid-phase extraction-liquid chromatography.

[0046] The present application preferably prepares PAN-HLB composite nanofibers according to the following method: dissolving polyacrylonitrile (PAN) and a hydrophilic-lipophilic balanced copolymer (HLB) in a solvent, preferably N,N-dimethylformamide (DMF), and stirring, preferably for 24 hours, to form a spinning solution; the spinning solution is used to prepare composite nanofibers by electrospinning technology to obtain PAN-HLB composite nanofibers; the process parameters of the preferred electrospinning process are as follows: voltage 18 kV, propulsion rate 2.0 mL / h, and needle-receiving plate distance 15 cm.

[0047] The PAN-HLB composite nanofibers prepared in the present application have generated oxazole groups, which have certain alkalinity and a special electron cloud structure, which enables them to form specific weak interactions with target substances, such as hydrogen bonds, π-π stacking, etc.; at the same time, HLB is embedded in the three-dimensional structure of PAN fibers. While giving the composite fibers good structural stability and moderate deformability, this structure also changes the elution kinetics, so that the PAN-HLB composite nanofibers can combine with substances of different polarities in different ways, and achieve the separation of target analytes through corresponding eluents.

[0048] For ease of understanding, the structure, adsorption and elution process of the PAN-HLB composite nanofibers are analyzed in detail:

[0049] 1) Structural stability and dynamic regulation of oxazole groups:

[0050] The three-dimensional structure of PAN fibers gives the composite fibers good structural stability and moderate deformability. During the elution process, the flow and penetration of low-proportion organic solvents will affect the material structure. PAN-HLB composite nanofibers can maintain a relatively stable structure in this situation, and their deformability helps the eluent to better penetrate around the HLB action site. The oxazole group can dynamically adjust its interaction with the target substance with slight deformation of the structure. When the eluent enters, the distance and angle between the oxazole group and the target substance change, further promoting desorption, so that desorption can be completed without the need for high-concentration organic solvents. However, the structure of pure HLB particle materials is relatively rigid, making it difficult for the eluent to penetrate, and lacking the dynamic adjustment mechanism of the oxazole group, it is difficult to adapt to the elution process through structural adjustment, resulting in difficulty in eluting the target substance.

[0051] 2) Regulation of elution balance by oxazole:

[0052] In PAN-HLB composite nanofibers, the newly formed oxazole group plays a unique role. The oxazole group has a certain alkalinity and a special electron cloud structure, which enables it to form specific weak interactions with the target substance, such as hydrogen bonding and π-π stacking. During the elution stage, a low proportion of organic solvent can change the microenvironment around the oxazole group, weakening its interaction with the target substance. For hydrophilic related substances, such as folic acid, the nitrogen atom on the oxazole group can form hydrogen bonds with carboxyl and hydroxyl groups in the hydrophilic substance molecules. In the presence of a low proportion of organic solvent, the organic solvent molecules will compete for the hydrogen bonding sites, breaking the hydrogen bond connection between the oxazole group and the hydrophilic substance, and promoting the desorption of the target substance.

[0053] Based on the combination mode of PAN-HLB composite nanofibers and hydrophilic substances, the present application uses low concentrations of organic solvents to desorb hydrophilic target analytes at an appropriate rate.

[0054] However, pure HLB particle materials do not contain oxazole groups and lack this interaction that can be adjusted under low proportions of organic solvents, making it difficult to elute the target substance from the HLB. High concentrations of organic solvents must be used in the elution process.

[0055] 3) Differences in elution kinetics due to structural influence:

[0056] 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 number of HLB is reduced, it is dispersed in the three-dimensional structure, making the binding sites between the target substance and HLB relatively more "exposed." During elution, low-proportion organic solvents can diffuse to the binding sites more quickly and compete with the target substance for binding. When eluting hydrophobic substances, such as phthalates, organic solvent molecules can quickly reach the HLB binding sites along the pores of the PAN fibers, weakening the interaction between the target substance and HLB and achieving elution. In pure HLB particle materials, HLB is densely packed, and the target substance is encapsulated in more binding sites inside the particles. The diffusion path of the eluent inside the particles is long and has high resistance. It is difficult for low-proportion organic solvents to penetrate deeply into the interior and fully interact with the target substance, resulting in low elution efficiency and only eluting a small amount of substance.

[0057] Based on the special structure of PAN-HLB composite nanofibers, the present application does not require the use of high-concentration organic solvents to desorb hydrophobic target analytes at an appropriate rate.

[0058] 4) Synergistic effect between the solvent microenvironment and the oxazole group:

[0059] The presence of PAN changes the solvation environment on the material surface, producing a synergistic effect with the oxazole group. The PAN molecular chain is hydrophilic and flexible. When eluted with a low proportion of organic solvent, the PAN molecules interact with the organic solvent molecules to form a microenvironment that is conducive to the desorption of the target substance. At the same time, the oxazole group also affects the structure and properties of the surrounding solvation layer. For amphoteric quinolone antibiotics, the hydrogen bond network formed by the PAN molecules and the organic solvent, as well as the regulation of the surrounding polarity by the oxazole group, work together to make it easier for the target substance to detach from the HLB and dissolve in the eluent. The surface of pure HLB particle materials lacks this special solvation environment jointly constructed by PAN and oxazole groups. In low proportions of organic solvent, it is difficult for the target substance to desorb from the HLB into the eluent, resulting in poor elution effect.

[0060] It can be seen that the present application is based on the special structure and properties of PAN-HLB composite nanofibers, and 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 online solid phase extraction-liquid chromatography; specifically, with the help of the special adsorption properties of PAN-HLB composite nanofibers, an online solid phase extraction column is first prepared and loaded, and then it is constructed into an online detection system with a high-performance liquid chromatograph. The system has efficient enrichment and separation capabilities for target components of different polarities such as hydrophilicity, hydrophobicity and amphotericity, can remove interfering substances in the sample, and achieve selective extraction of target components of different polarities. The integration of online processing and detection simplifies the operating process, saves manpower, reduces manual operation errors, and can ensure accurate qualitative and quantitative analysis of components of different polarities in complex samples. It has shown good application prospects in the detection of various complex samples such as environmental water samples, biological samples, and food samples. In order to take into account the adsorption and desorption performance of the solid phase adsorbent for the target analyte, the present application prefers that the mass ratio of polyacrylonitrile and the hydrophilic-lipophilic balanced copolymer is 10:(6-7), and further prefers it to be 10:6.5; further, the mass ratio of polyacrylonitrile and the hydrophilic-lipophilic balanced copolymer solvent is preferably 10:6.5:83.5.

[0061] The HLB used in this invention can be purchased directly or prepared in-house; the preferred method for preparing the HLB is as follows: 165 mL of acetonitrile and a magnetic stir bar were added to a 500 mL round-bottom flask. The solution was purged with nitrogen, and the flask was immersed in silicone oil and stirred with a magnetic stir bar at approximately 100 rpm for 30 minutes. The mixture was then heated to 75°C. 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 the mixture was stirred at 100 rpm for 24 hours while nitrogen was purged to displace the air. The mixture was then filtered through a Buchner funnel using 0.45 µm Whatman nylon filter paper to collect the particles, yielding HLB particles. The HLB particles were washed three times with 50 mL of ethanol and dried in a vacuum oven at 60°C overnight.

[0062] In order to take into account the adsorption and desorption performance of the solid phase adsorbent for the target analyte, the specific surface area of ​​the polyacrylonitrile-hydrophilic-lipophilic balanced copolymer composite nanofiber is preferably 150-200 m 2 / g; preferably, the average pore size of the polyacrylonitrile-hydrophilic-lipophilic balanced copolymer composite nanofibers is 3.0-3.5 nm.

[0063] 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-methyltetrahydrofolate (5-MeTHF), 5-formyltetrahydrofolate (5-FoTHF), 5,10-dimethyltetrahydrofolate (5,10-CH2-THF), S-adenosyl-L-methionine (SAM), and at least one of S-adenosyl-L-homocysteine ​​(SAH).

[0064] In some embodiments, the hydrophobic component includes phthalates; and preferably, the phthalates 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), di-isooctyl phthalate (DIOP), and di-n-octyl phthalate (DnOP).

[0065] In some embodiments, the amphiphilic component includes a quinolone antibiotic; and preferably, the quinolone antibiotic is selected from at least one of ofloxacin (OFLx), norfloxacin (NOR), ciprofloxacin (CIP), danofloxacin (DAN), enrofloxacin (ENR), and sarafloxacin (SAR).

[0066] This application utilizes the prepared online solid-phase extraction column (filled with PAN-HLB composite nanofibers) to design the operating procedures required for its connection with a high-performance liquid phase system and its application to substances with different polarity components. The experiment focused on the repeatability, precision, recovery rate of spiked samples, and linear range of the online pretreatment and detection method. The detection method provided in this application can achieve rapid and efficient enrichment and separation of target components of different polarity, improve the sensitivity and accuracy of detection, and meet the needs of effective detection of components of different polarity in complex samples; by combining online pretreatment with analytical instruments, automated operation is achieved, greatly shortening the pretreatment and detection time. The entire detection process can be completed within 15-20 minutes, improving detection efficiency and being suitable for high-throughput sample analysis.

[0067] The PAN-HLB composite nanofibers in the following examples were prepared according to the following method:

[0068] PAN (10 wt%) and HLB (6.5 wt%) were dissolved in DMF and stirred for 24 hours to form a spinning solution. The composite nanofibers were prepared by electrospinning (voltage 18 kV, propulsion rate 2.0 mL / h, needle-receiver distance 15 cm).

[0069] Polyacrylonitrile (PAN, molecular weight 150,000) was purchased from Sigma-Aldrich. HLB copolymer particles were prepared as follows:

[0070] A 500 mL round-bottom flask was filled with 165 mL of acetonitrile and a magnetic stir bar. The solution was purged with nitrogen, and the flask was immersed in silicone oil and stirred at approximately 100 rpm with a magnetic stir bar for 30 minutes. The mixture was then heated to 75°C. 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. The mixture was purged with nitrogen to displace the air, and stirred at 100 rpm for 24 hours. The particles were then collected by suction filtration on a Buchner funnel using 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.

[0071] Example 1

[0072] Detection of quinolone antibiotics in environmental water samples

[0073] 1) Loading of the 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 portions into the bottom of the column tube, which was sealed with a sieve plate at one end. The column was compacted with a 0.5 mm diameter stainless steel rod and sealed with a sieve plate at the other end. The column was then placed in a jacket and connected to a high-performance liquid chromatography system.

[0074] 2) Sample preparation: Collect environmental water samples and filter them through a 0.45 μm filter membrane as the samples to be tested.

[0075] 3) Online pretreatment: The filtered water sample was adjusted to pH 2 and then passed through an online solid-phase extraction column filled with PAN-HLB composite nanofibers at a flow rate of 0.5 mL / min to allow the amphoteric quinolone antibiotics (six types, ofloxacin (OFLx), norfloxacin (NOR), ciprofloxacin (CIP), danofloxacin (DAN), enrofloxacin (ENR), and sarafloxacin (SAR)) in the water sample to be adsorbed on the fibers.

[0076] 4) Elution and analysis: The online solid phase extraction column was eluted with a 0.05% ammonia solution containing 30% methanol at a flow rate of 0.5 mL / min, and the eluate was directly injected into the high performance liquid chromatography. HPLC analysis conditions were as follows: a C18 column (4.6 mm inner diameter, 150 mm length, 5 μm particle size); a mobile phase consisting of acetonitrile and 0.2% formic acid in water (gradient elution: 0–0.5 min, 100% 0.2% formic acid in water; 0.55–1.55 min, 100% 30% methanol in 0.05% ammonia; 1.6–8 min, 18% acetonitrile; 8–10 min, 12% acetonitrile; 11–12 min, 100% 0.2% formic acid in water); a flow rate of 1.0 mL / min; a column temperature of 30°C; detection wavelengths of 280 nm for excitation and 450 nm for emission; and a valve switching time of 0.5 min. Quantitative analysis was performed based on peak area.

[0077] Example 2

[0078] Detection of quinolone antibiotics in food samples

[0079] 1) Construction of online pretreatment device: Similar to Example 1, a 10 mm × 2.1 mm stainless steel column tube with detachable openings at both ends was used to pack PAN-HLB composite nanofibers as an online solid phase extraction column and connected to a high performance liquid chromatography system.

[0080] 2) Sample preparation: Weigh 0.1 g of food sample (e.g., milk), add acid to precipitate protein, filter the supernatant through a 0.45 μm filter membrane, and dilute to 4 mL with ultrapure water. This will be used as the sample to be tested.

[0081] 3) Online pretreatment: The treated sample solution was passed through a micro solid phase extraction column filled with PAN-HLB composite nanofibers at a rate of 0.5 mL / min, so that quinolone antibiotics (six types, ofloxacin (OFLx), norfloxacin (NOR), ciprofloxacin (CIP), danofloxacin (DAN), enrofloxacin (ENR), sarafloxacin (SAR)) were adsorbed on the fibers.

[0082] 4) Elution and Analysis: The online solid-phase extraction column was eluted with a 0.05% ammonia solution containing 30% methanol at a flow rate of 0.5 mL / min. The eluate was directly introduced into a high-performance liquid chromatograph. The HPLC analysis conditions were the same as in Example 1, and quantitative analysis was performed based on the chromatographic peak area.

[0083] Example 3

[0084] Detection of polar vitamins and metabolites in biological samples

[0085] 1) Construction of an online pre-processing device: Similar to the previous two embodiments, an online pre-processing and detection device is constructed.

[0086] 2) Sample preparation: Collect a biological sample (urine). Take 0.1 mL of the urine sample and add antioxidants and ultrapure water to dilute it to 4 mL as the sample to be tested.

[0087] 3) Online pre-treatment: The sample to be tested was passed through a micro solid phase extraction column filled with PAN-HLB composite nanofiber at a rate of 0.5 mL / min to achieve the extraction of polar vitamins and metabolites (vitamin B2 (VB2), vitamin B 12 (VB 12 ), vitamin B9 (folic acid, FA), 5-methyltetrahydrofolate (5-MeTHF), 5-formyltetrahydrofolate (5-FoTHF), 5,10-dimethyltetrahydrofolate (5,10-CH2-THF), S-adenosyl-L-methionine (SAM), S-adenosyl-L-homocysteine ​​(SAH)) by adsorption enrichment.

[0088] 4) Elution and analysis: The online solid phase extraction column was eluted with acetate buffer solution at pH 4.25 containing 5% methanol at a rate of 0.5 mL / min, and the eluate was passed into the high performance liquid chromatography instrument. HPLC analysis conditions: A C18 column (4.0 mm inner diameter, 150 mm length, 3 μm particle size) was used with a mobile phase consisting of methanol–acetate buffer (pH 4.25) (gradient elution: 5% methanol, 0–2.5 min; 3% methanol, 2.5–3.6 min; 45% methanol, 3.6–8.6 min; 45% methanol, 8.6–11.6 min; 5% methanol, 11.6–12 min). The flow rate was 0.5 mL / min, and the column temperature was 30°C. The absorption wavelengths were 2.95–3.2 min at 259 nm, 6.9 min at 280 nm, 7.0–7.85 min at 259 nm, 9.5 min at 362 nm, and 9.75 min at 280 nm. The valve switching time was 2.5 min. Quantitative analysis was based on peak area or peak height.

[0089] Examples 4-6 and Comparative Examples 1-6 are comparative experiments, in which the PAN-HLB composite nanofiber online solid phase extraction column disclosed in the present invention is compared with the commercially available Waters HLB online extraction column and C18 online extraction column for the extraction of hydrophobic phthalates (10 species, 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), di-isooctyl 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-methyltetrahydrofolate (5-MeTHF), 5-formyltetrahydrofolate (5-FoTHF), 5,10-dimethyltetrahydrofolate (5,10-CH2-THF), S-adenosyl-L-methionine (SAM), S-adenosyl-L-homocysteine ​​(SAH)); and amphoteric quinolone antibiotics (6 types, ofloxacin (OFLx), norfloxacin (NOR), ciprofloxacin (CIP), danofloxacin (DAN), enrofloxacin (ENR), sarafloxacin (SAR)) were compared in the extraction and separation performance.

[0090] Example 4

[0091] Hydrophobic Phthalate Esters (PAEs) Test

[0092] The PAN-HLB composite nanofiber online extraction column of the present invention is used: the injection bottle containing the PAEs standard solution is placed in the automatic sampler, the chromatography program is run, 1000 μL is injected into the online extraction column, ultrapure water is used as the mobile phase of the left pump, the enrichment and purification time is set to 0.5 min, the valve path is switched to connect the online extraction column to the right pump, 60% methanol solution is used as the eluent to elute the PAEs adsorbed on the extraction column and transfer them to the analytical column, the elution time is set to 1 min, the valve path is switched back to the original state again, the left pump reactivates the extraction column for re-injection, the right pump is connected to the mobile phase for separation and detection, and the chromatogram is recorded (see Figure 1 、 Figure 2 、 Figure 3 After calculation, the recovery rate was 95.0-102.2%.

[0093] Example 5

[0094] Hydrophilic vitamin-related substance experiment

[0095] For the PAN-HLB composite nanofiber online extraction column of the present invention: the injection bottle containing the hydrophilic vitamin-related substance standard solution is placed in the automatic sampler, the chromatographic program is run, 1000 μL is injected into the online extraction column, ultrapure water is used as the mobile phase of the left pump, the enrichment and purification time is set to 0.5 min, the valve path is switched to connect the online extraction column to the right pump, and the hydrophilic related substances adsorbed on the extraction column are eluted and transferred to the analytical column using an acetate buffer solution of pH 4.25 containing 5% methanol as the eluent. The elution time is 2 min, the valve path is switched back to the original state again, the left pump reactivates the extraction column for re-injection, the right pump is connected to the mobile phase for separation and detection, and the chromatogram is recorded (see Figure 4 、 Figure 5 、 Figure 6 After calculation, the recovery rate was 85.4-96.3%.

[0096] Example 6

[0097] Amphoteric quinolone antibiotics experiment

[0098] For the PAN-HLB composite nanofiber online extraction column of the present invention: the injection bottle containing the amphoteric quinolone antibiotic standard solution is placed in the automatic sampler, the chromatographic program is run, 1000 μL is injected into the online extraction column, ultrapure water is used as the mobile phase of the left pump, the enrichment and purification time is set to 0.5 min, the valve path is switched to connect the online extraction column to the right pump, and the amphoteric substance adsorbed on the extraction column is eluted and transferred to the analytical column using ammonia water (0.05%) solution containing 30% methanol as the eluent. The elution time is 1 min, the valve path is switched back to the original state again, the left pump reactivates the extraction column for re-injection, the right pump is connected to the mobile phase for separation and detection, and the chromatogram is recorded (see Figure 7 、 Figure 8 、 Figure 9 As shown in b); the recovery rate was calculated to be 78.6%-120.4%.

[0099] Comparative Example 1

[0100] Compare with Example 4:

[0101] For Waters HLB online extraction column: operate under the same injection volume, mobile phase, enrichment and purification time, eluent and chromatographic procedure as in Example 4, and record the chromatogram (see Figure 2 (as shown in a).

[0102] Comparative Example 2

[0103] Compare with Example 4:

[0104] For the C18 online extraction column: the same injection volume, mobile phase, enrichment and purification time, eluent and chromatographic procedure as in Example 4 were used for operation, and the chromatogram was recorded (see Figure 3 (as shown in a).

[0105] Comparative Example 3

[0106] Compare with Example 5:

[0107] For Waters HLB online extraction column: operate under the same injection volume, mobile phase, enrichment and purification time, eluent and chromatographic procedure as in Example 5, and record the chromatogram (see Figure 5 (as shown in a).

[0108] Comparative Example 4

[0109] Compare with Example 5:

[0110] On a C18 online extraction column: the same injection volume, mobile phase, enrichment and purification time, eluent and chromatographic procedure as in Example 5 were used to perform the operation and record the chromatogram (see Figure 6 (as shown in a).

[0111] Comparative Example 5

[0112] Compare with Example 6:

[0113] For Waters HLB online extraction column: operate under the same injection volume, mobile phase, enrichment and purification time, eluent and chromatographic procedure as in Example 6, and record the chromatogram (see Figure 8 (as shown in a).

[0114] Comparative Example 6

[0115] Compare with Example 6:

[0116] For C18 online extraction column: operate under the same injection volume, mobile phase, enrichment and purification time, eluent and chromatographic procedure as above, and record the chromatogram (see Figure 9 (as shown in a).

[0117] Experimental results and analysis:

[0118] 1. Hydrophobic phthalates (PAEs) results:

[0119] See also Figure 1 As shown in the figure, the chromatogram (b) of the online solid phase extraction detection of the hydrophobic phthalate standard solution (1000 ng / mL) using PAN-HLB composite nanofibers is compared with the chromatogram (a) of the direct injection analysis, which intuitively shows the enrichment and separation effect of online solid phase extraction on hydrophobic substances.

[0120] See also Figure 2 As shown in the figure, the chromatogram (b) of the hydrophobic phthalate ester standard solution (1000 ng / mL) detected by online solid phase extraction using PAN-HLB composite nanofibers is compared with the chromatogram (a) detected by online extraction using Oasis HLB Direct Connect HP (Waters company, 2.1×30 mm, 20 μm). This intuitively demonstrates that the PAN-HLB composite nanofiber online extraction column has better enrichment and elution effects on hydrophobic PAEs than the Waters HLB particle online extraction column.

[0121] See also Figure 3 As shown in the figure, the chromatogram (b) of the hydrophobic phthalate ester standard solution (1000 ng / mL) detected by online solid phase extraction using PAN-HLB composite nanofibers is compared with the chromatogram (a) detected by online extraction using a C18 (YMC, 2.1×10 mm, 5 μm) column. This intuitively demonstrates that the PAN-HLB composite nanofiber online extraction column has better enrichment and elution effects on hydrophobic PAEs than the C18 online extraction column.

[0122] The chromatograms obtained by using the PAN-HLB composite nanofiber online extraction column of the present invention are compared with those obtained by Waters HLB online extraction column and C18 online extraction column. Figure 2 and attached Figure 3 It can be clearly seen that the PAN-HLB online extraction column has a higher response to the chromatographic peaks of the 10 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, which 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.

[0123] 2. Results of hydrophilic vitamins and related substances:

[0124] See also 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 of PAN-HLB composite nanofiber is compared with the chromatogram (a) of direct injection analysis, demonstrating the effective detection capability of hydrophilic substances.

[0125] See also Figure 5As shown in the figure, the chromatogram (b) of the hydrophilic vitamin standard solution (500 ng / mL) detected by online solid phase extraction using PAN-HLB composite nanofiber is compared with the chromatogram (a) detected by online extraction column using Oasis HLB Direct Connect HP (Waters company, 2.1×30 mm, 20 μm). It is intuitively shown that the PAN-HLB composite nanofiber online extraction column has better enrichment and elution effects on hydrophilic related substances than Waters' HLB particle online extraction column.

[0126] See also Figure 6 As shown in the figure, the chromatogram (b) of the hydrophilic vitamin standard solution (500 ng / mL) detected by online solid phase extraction using PAN-HLB composite nanofibers is compared with the chromatogram (a) detected by online extraction using a C18 (YMC, 2.1×10 mm, 5 μm) column. It is intuitively shown 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.

[0127] The chromatogram (a) obtained by using the PAN-HLB composite nanofiber online extraction column of the present invention is compared with the chromatogram (b) obtained by using the HLB online extraction column of Waters and the C18 online extraction column. 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 Waters' HLB online extraction column and 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 more accurately analyze hydrophilic vitamins and related substances.

[0128] 3. Results of amphoteric quinolone antibiotics:

[0129] See also Figure 7 As shown in Figure 2, the chromatogram (b) of amphoteric quinolones (1 ng / mL) detected by online solid phase extraction using PAN-HLB composite nanofibers is compared with the chromatogram (a) of direct injection analysis, which demonstrates the detection advantage of this method for amphoteric substances.

[0130] See also Figure 8As shown in the figure, the chromatogram (b) of amphoteric quinolones (1 ng / mL) detected by online solid phase extraction using PAN-HLB composite nanofibers is compared with the chromatogram (a) detected by online extraction using Oasis HLB Direct Connect HP (Waters company, 2.1×30 mm, 20 μm). The results show that the PAN-HLB composite nanofiber online extraction column has better enrichment and elution effects on amphoteric substances than the Waters HLB particle online extraction column.

[0131] See also Figure 9 As shown in the figure, the chromatogram (a) of amphoteric quinolones (1 ng / mL) detected by online solid phase extraction using PAN-HLB composite nanofibers is compared with the chromatogram (b) of amphoteric quinolones detected by online extraction using a C18 (YMC, 2.1×10 mm, 5 μm) column. The results show that the PAN-HLB composite nanofiber online extraction column has better enrichment and elution effects on amphoteric substances than the C18 online extraction column.

[0132] The chromatogram (a) obtained by using the PAN-HLB composite nanofiber online extraction column of the present invention is compared with the chromatogram (b) obtained by using the HLB online extraction column of Waters and the C18 online extraction column. Figure 8 and attached Figure 9 It can be seen that for amphoteric quinolone antibiotics, the chromatographic peak response value obtained by the PAN-HLB online extraction column is much higher than the chromatographic response value obtained by the Waters HLB online extraction column and the C18 online extraction column, which indicates that the PAN-HLB composite nanofiber online extraction column of the present invention also has better enrichment and elution effects on amphoteric substances.

[0133] 4. Conclusion

[0134] Through this comparative experiment, it is fully demonstrated that the PAN-HLB composite nanofiber online extraction column of the present invention has significant advantages in the extraction and separation performance of hydrophobic phthalates (10 types), hydrophilic vitamins and related substances (8 types), and amphoteric quinolone antibiotics (6 types) compared with Waters' HLB online extraction column and C18 online extraction column, further verifying the effectiveness and superiority of the technical solution of the present invention.

[0135] 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 it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the technical solutions of the embodiments of the present invention.

Claims

1. A detection method using online solid phase extraction and liquid chromatography, characterized in that: The method comprises the following steps: using polyacrylonitrile-hydrophilic-lipophilic balanced copolymer composite nanofibers as a solid phase adsorbent to adsorb a target analyte, 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 amphiphilic 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 a chromatographic analysis 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; The hydrophilic components include water-soluble vitamins and their metabolites; The hydrophobic component includes phthalates; The amphiphilic component includes a quinolone antibiotic.

2. The online solid phase extraction-liquid chromatography detection method according to claim 1, characterized in that: The polyacrylonitrile-hydrophilic-lipophilic balanced copolymer composite nanofibers are prepared according to the following method: polyacrylonitrile and the hydrophilic-lipophilic balanced copolymer are dissolved in a solvent and stirred to form a spinning solution; the spinning solution is subjected to electrospinning technology to prepare composite nanofibers to obtain polyacrylonitrile-hydrophilic-lipophilic balanced copolymer composite nanofibers.

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

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

5. The online solid phase extraction-liquid chromatography detection method according to claim 2, characterized in that: The average pore size of the polyacrylonitrile-hydrophilic-lipophilic balanced copolymer composite nanofiber is 3.0-3.5 nm.

6. The on-line solid phase extraction-liquid chromatography detection method according to any one of claims 1 to 5, characterized in that: The hydrophilic component is selected from vitamin B2, vitamin B 12 , vitamin B9, 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, 5,10-dimethyltetrahydrofolate, S-adenosyl-L-methionine, and at least one of S-adenosyl-L-homocysteine.

7. The on-line solid phase extraction-liquid chromatography detection method according to any one of claims 1 to 5, characterized in that: The phthalate ester substance 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, di-isooctyl phthalate, and di-n-octyl phthalate.

8. The on-line solid phase extraction-liquid chromatography detection method according to any one of claims 1 to 5, characterized in that: The quinolone antibiotic is selected from at least one of ofloxacin, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin and sarafloxacin.

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