Preparation method of test sample for detecting arachidonic acid-like isomer based on magnetic beads

Abstract

The invention belongs to the technical field of detection, and particularly relates to a preparation method of a test sample for detecting arachidonic acid-like isomers based on magnetic beads, a test sample solution adopts a chemical derivatization reagent system, and covalent coupling magnetic beads are successfully used for nucleic acid and protein separation through surface modification. By accurately designing molecular recognition sites, the chromatographic separation resolution (delta R is larger than or equal to 1.5) of isomer metabolites can be remarkably improved, meanwhile, by means of an isotope double-labeling strategy, the detection sensitivity is improved by 10-100 times, the treatment time of a single sample is shortened to be within 30 minutes, and more importantly, by means of construction of a solid-phase reaction system, the detection sensitivity is greatly improved. Controllable optimization of derivatization reaction efficiency is achieved (the reaction conversion rate is larger than 98%), and a key technical foundation is laid for follow-up development of a full-automatic mass spectrum combined detection technology. The magnetic bead mediated solid phase derivatization strategy is expected to renovate the analysis normal form of small molecule metabolites, and promotes the metabonomics research to develop towards high flux and automation.

Description

Technical Field

[0001] This invention relates to the field of detection technology, and specifically to a method for preparing a test sample for the detection of arachidonic acid isomers based on magnetic beads. Background Technology

[0002] Since most arachidonic acid isomers have the same molecular weight, similar or identical chemical structure and properties, and are low in abundance with large differences in the content of metabolites, it is very difficult to perform qualitative and quantitative analysis on all isomers at the same time.

[0003] Liquid chromatography-mass spectrometry (LC-MS / MS) is a commonly used technique for studying arachidonic acid metabolites. This method can effectively assess changes in metabolites under different physiological states, promoting early diagnosis and treatment of diseases. However, its clinical detection faces three major bottlenecks: low sample preparation efficiency (manual operation takes 4-6 hours) and insufficient isomer resolution (false positive rate >30% for Δm / z < 0.05). Summary of the Invention

[0004] This invention provides a method for preparing a test sample for the detection of arachidonic acid isomers based on magnetic beads.

[0005] The present invention solves its technical problem by adopting the following technical solution: A method for preparing a test sample for the detection of arachidonic acid isomers based on magnetic beads, comprising the following steps: (1) Wash the carboxyl magnetic beads with buffer solution, add 0.5~2g of carboxyl magnetic beads and 4~6mM of N-hydroxysuccinimide to the buffer solution to obtain activated carboxyl magnetic beads; (2) Add PEI solution to the above activated carboxyl magnetic beads, react, wash with PBS to remove unbound PEI, and obtain PEI-modified magnetic beads; (3) Add Tris-HCl buffer solution and stir well to obtain covalently coupled magnetic beads; (4) Add 5-20 μL of serum sample to be tested and 0.5-2 mg of covalently coupled magnetic beads to 50 μL with PBS buffer. Stir to allow the arachidonic acid isomers in the serum to fully bind with the magnetic beads. Adsorb the magnetic beads with a magnetic rack, discard the supernatant, and wash the magnetic beads 2-3 times with PBS buffer to remove unbound substances to obtain the precursor. Finally, resuspend with PBS buffer to obtain the test solution.

[0006] In a preferred embodiment of the present invention, the pH of the Tris-HCl buffer is 7.4, and the concentration is 40~50mM.

[0007] As a preferred embodiment of the present invention, the reaction temperature in step (1) is 22~28℃ and the time is 20~60min.

[0008] As a preferred embodiment of the present invention, the reaction temperature in step (2) is 22~28℃ and the time is 20~40min.

[0009] In a preferred embodiment of the present invention, the diameter of the carboxyl magnetic beads is 0.5~2μm and the carboxyl density is 280~320μmol / g.

[0010] As a preferred embodiment of the present invention, the buffer solution in step (1) is a MES buffer solution with a pH of 5.8 to 6.2.

[0011] As a preferred embodiment of the present invention, the buffer solution in step (3) is at least one of sodium citrate buffer with pH 4.8 to 5.2, MES buffer with pH 5.8 to 6.2, PBS buffer with pH 6.8 to 7.2, and Tris-HCl buffer with pH 7.8 to 8.2.

[0012] In a preferred embodiment of the present invention, the mass concentration of PEI in the PEI solution is 0.5-5%.

[0013] In a preferred embodiment of the present invention, the mass ratio of the PEI solution to the activated carboxyl magnetic beads is (2~10):1.

[0014] As a preferred embodiment of the present invention, the arachidonic acid-like isomers include at least one of arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, linolenic acid, prostaglandin D2, prostaglandin F2α, thromboxane B2, leukotriene B4, 5-hydroxyeicosapentaenoic acid, 12-hydroxyeicosapentaenoic acid, 15-hydroxyeicosapentaenoic acid, 20-hydroxyeicosapentaenoic acid, 5,6-epoxyeicosatrienoic acid, and 14,15-epoxyeicosatrienoic acid.

[0015] Beneficial Effects: The test solution of this invention utilizes a chemical derivatization reagent system, and has been successfully used for nucleic acid and protein separation through surface modification with covalently coupled magnetic beads. By precisely designing molecular recognition sites, the chromatographic separation resolution of isomeric metabolites can be significantly improved (ΔR≥1.5). Simultaneously, the detection sensitivity is increased by 10-100 times using an isotope dual-labeling strategy, and the single-sample processing time is shortened to less than 30 minutes. More importantly, the derivatization reaction efficiency can be controlled and optimized through the construction of a solid-phase reaction system (reaction conversion rate >98%), laying a key technical foundation for the subsequent development of fully automated mass spectrometry-coupled detection technology. This magnetic bead-mediated solid-phase derivatization strategy is expected to revolutionize the analytical paradigm of small molecule metabolites and promote the leapfrog development of metabolomics research towards high throughput and automation. Attached Figure Description

[0016] Figure 1 This is a resolution detection graph.

[0017] Figure 2 The results include the total ion chromatogram, collision cross-section diagram, and MS / MS spectrum. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] There are no particular restrictions on the specific methods of dispersion and mixing.

[0020] In this invention, unless otherwise stated, all parts are by weight.

[0021] Unless otherwise specified, all reagents or instruments used in this invention are commercially available products.

[0022] Example 1

[0023] A method for preparing a test sample for the detection of arachidonic acid isomers based on magnetic beads, comprising the following steps: (1) Wash the carboxyl magnetic beads with buffer (MES buffer at pH 6.0), add 1g of carboxyl magnetic beads and 5mM of N-hydroxysuccinimide to the buffer, and react at 22℃ for 60min to obtain activated carboxyl magnetic beads. (2) Add a 2% PEI solution to the above activated carboxyl magnetic beads for electrostatic adsorption modification (PEI is a polycation that forms an electrostatic complex with the carboxyl groups on the surface of the magnetic beads). At room temperature of 24°C, gently rotate / shake for 30 min. After adsorption, wash with PBS 2-3 times to remove unbound PEI and obtain PEI-modified magnetic beads. The mass ratio of the PEI solution to the activated carboxyl magnetic beads is 4:1.

[0024] (3) Add 50 mM Tris-HCl buffer (pH 7.4) to block the covalently coupled magnetic beads; (4) Dilute 10 μL of the serum sample to be tested and 1 mg of covalently coupled magnetic beads to 50 μL with PBS buffer to obtain the test solution. Shake for 2 min to allow the arachidonic acid isomers in the serum to fully bind to the magnetic beads; after incubation, adsorb the magnetic beads with a magnetic rack, discard the supernatant, and wash the magnetic beads 2-3 times with PBS buffer to remove unbound substances; finally, resuspend the magnetic beads with an appropriate amount of buffer to a volume of 50 μL to form the test solution for subsequent two-dimensional chromatography / ion mobility mass spectrometry analysis.

[0025] Experimental validation: A precise analytical method for arachidonic acid isomers based on two-dimensional chromatography / ion mobility mass spectrometry. 1. Experimental instruments: Liquid chromatography-tandem mass spectrometry (AB SCIEX, USA, Triple Quad 4500MD ultra-high performance liquid chromatography-tandem mass spectrometry detection system, national medical device registration number 20172401554) or certified equipment with equivalent sensitivity (such as FH-6000MD tandem mass spectrometry analysis system - Guangdong medical device registration number 20182220784 and YS EXACT 9050MD tandem mass spectrometry analysis system - Shandong medical device registration number 20192220473, etc.).

[0026]

[0027] 2. Chromatographic column: Hypersil GOLD C18 column (2.1×50 mm, 1.8 μm).

[0028] 3. Two-dimensional chromatographic conditions:

[0029] 4. Ion mobility mass spectrometry conditions:

[0030] 5. Detection Principle: The detection method of this invention involves purifying and concentrating serum samples before derivatization (i.e., the test solution), followed by analysis using liquid chromatography-tandem mass spectrometry. First, the analyte is separated by chromatography and then enters the mass spectrometry ion source. Ionization occurs within the ion source, and the charged ions, under voltage and vacuum, enter a triple quadrupole mass analyzer (QQQ). Q1 and Q3 are mass filters, allowing only precursor and daughter ions selected based on the target mass-to-charge ratio to pass through. Q2 is a collision unit where the precursor ion collides with inert gas molecules, generating specific fragment ions. Ions filtered by the triple quadrupole enter the detector, which converts the number of captured ions into digital signal electron pulses. The obtained data is transmitted to a computer, which plots the collected ion count against time to obtain a mass chromatogram. The concentration ratio of the target analyte to the internal standard in the standard is used as the x-axis, and the peak area ratio as the y-axis to construct a calibration curve. This curve is used to calculate the concentrations of various arachidonic acid metabolites in the serum sample or quality control sample.

[0031] 6. Preparation of the test solution: As described above for the preparation of the test solution; 7. Preparation of standards: PGE2 and 12-HETE were dissolved in methanol and serially diluted to a concentration range of 0.1-1000 pg / mL.

[0032] 8. Instrument Preparation: Warm up and calibrate the LC-MS / MS system (Thermo Fisher TSQ Altis), equipped with a Hypersil GOLD C18 column (2.1 × 50 mm, 1.8 μm). Set the temperature of the isothermal shaker to 25℃, ensuring a temperature control accuracy of ±0.5℃.

[0033] 9. Single-factor preliminary experiments and response surface methodology optimization: pH optimization: The reaction was carried out at a fixed temperature (25°C) and time (15 min). The reaction system contained 10 μL PGE2 (10 pg / mL), 40 μL DIAAA (1 mM), and 50 μL buffer.

[0034] Response surface methodology: Central composite design (CCD) was used and generated using Design-Expert® software.

[0035] 10. Detection Scope The present invention has the detection range of 14 arachidonic acid metabolites in human serum samples, including AA, EPA, DHA, ALA, PGD2, PGF2α, TXB2, LTB4, 5-HETE, 12-HETE, 15-HETE, 20-HETE, 5,6-EET, and 14,15-EET.

[0036] 11. Methodological Validation 11.1 Analytical sensitivity (limit of quantitation) and linear range 1) Using this invention, two samples of each concentration of calibrator are processed in parallel for each batch, and each batch is tested once, for a total of three batches; the average value, RSD, and recovery rate of each concentration sample are calculated; 2) Criteria for determining the limit of quantitation: The lowest concentration with an RSD of less than 20%, a recovery rate in the range of 85% to 115%, and a signal-to-noise ratio greater than 10 is considered the limit of quantitation concentration, i.e., LOQ. 3) Criteria for determining the linear range: RSD less than 20%, recovery rate within the range of 85% to 115%, and regression curve R2 > 0.98, which means the linear range requirement is met; 4) The results of the confirmation of the quantitation limit and linear range of this invention are shown in the table below: Table 1 Analytical sensitivity and linear range

[0037] 1.2 Precision 11.2.1 Repeatability 1) Take three samples at different levels for repeatability testing; 2) Perform 10 parallel assays using the same kit, following the instructions in the package insert; 3) Calculate the mean X, standard deviation SD, and coefficient of variation (CV) of the test results. The CV should be less than 10%. 4) The experimental results show that the coefficients of variation (CV) of AA, EPA, DHA, ALA, PGD2, PGF2α, TXB2, LTB4, 5-HETE, 12-HETE, 15-HETE, 20-HETE, 5,6-EET, and 14,15-EET are shown in Table 2, and are all less than 15%, which meets the detection requirements.

[0038] Table 2 Repeatability (Intra-batch Precision)

[0039] 11.2.2 Inter-batch precision 1) Select 3 batches of reagent kits and test 3 different levels of samples with stated concentrations to conduct inter-batch precision experiments; 2) Process the samples at each level according to the instructions and test 10 times; 3) Calculate the mean X, standard deviation SD, and coefficient of variation (CV) of the three batches of test results. The CV should be less than 15%. 4) The experimental results show that the coefficients of variation (CV) of AA, EPA, DHA, ALA, PGD2, PGF2α, TXB2, LTB4, 5-HETE, 12-HETE, 15-HETE, 20-HETE, 5,6-EET, and 14,15-EET are shown in Table 3, and are all less than 15%, which meets the detection requirements.

[0040] Table 3. Inter-batch precision

[0041] 11.3 Accuracy (Spike Recovery Experiment) 1) Collect mixed samples with low and high concentrations respectively, add homologous standard solutions with low and high concentrations respectively (concentration equivalent to 0.5~2.0 times the concentration of the mixed samples), and conduct spike recovery experiments; 2) For samples before and after spiking, two samples were processed in parallel using the present invention. The recovery rate of the spiked samples was calculated. If the recovery rate was in the range of 85% to 115%, the accuracy of the method was considered acceptable. 3) The spiked recovery rate test results showed that the recovery rates of the 14 arachidonic acid metabolites of the present invention were all in the range of 85%-115%, which met the detection requirements.

[0042] Table 4 Accuracy

[0043] 11.4 Matrix effect (limit of quantification) 1) Collect 5 patient samples, with each sample repeated 5 times; 2) The results are shown in the table, which shows the concentrations of AA, EPA, DHA, ALA, PGD2, PGF2α, TXB2, LTB4,5-HETE, 12-HETE, 15-HETE, 20-HETE, 5,6-EET, and 14,15-EET in the patient samples. The precision of each sample and the relative deviation between the mean concentration and the theoretical concentration were calculated. The CV was less than or equal to 20%, and the relative deviation was within ±15%, which meets the limit of quantitation requirements.

[0044] Table 5. Experimental data on the accuracy of detection indicators and matrix effect.

[0045] 11.5 Clinically Reportable Scope 1) Collect high-concentration positive samples and dilute them 2, 5, 10, and 20 times with commercial blank serum, respectively; 2) Five patient samples, both before and after dilution, were processed in parallel using this invention, with each sample tested once. 3) Using the pre-dilution concentration as a reference target value, calculate the recovery rate of each diluted concentration sample. If the recovery rate is between 85% and 115%, the dilution factor is considered acceptable. 4) Experimental results show that the test results obtained after diluting high-concentration samples 2 to 5 times meet the requirements. Therefore, when encountering samples exceeding the highest concentration point of the standard curve, they can be diluted 2 to 5 times before testing. The clinically reportable range of this invention is shown in the table below: Table 7 Clinically Reportable Range of Arachidonic Acid Metabolites

[0046] 11.6 Reference Interval Verification 1) The reference ranges for each detection indicator in this self-developed method are referenced from international third-party testing laboratories such as Mayo Clinic and Quest Diagnostics; Table 8 Reference Interval Verification

[0047] Note: It is recommended that each laboratory establish or validate a reference range suitable for its own laboratory.

[0048] 2) The reference range was verified by collecting serum samples from 40 healthy clinical individuals and using this invention for testing; 3) The test results showed that the test results of 40 normal human serum samples all fell within the reference range, and the reference range was verified.

[0049] 12. Experimental Objective: The method of this invention can separate and detect more than 300 kinds of unsaturated fatty acids within 40 minutes. Verify the resolution (≥1.5) of the molecular weight isomers of 12-HETE and 15-HETE. The detection limit (LOD) was verified to be 0.008–0.05 pg / mL, and the linear correlation coefficient R>0.997.

[0050] Data analysis and performance verification metrics: Retention time and resolution validation (12-HETE vs 15-HETE) Inject the standard mixture into the sample and record the retention time difference Δt. Calculate the peak widths (W12, W15) separately, and obtain the separation using the following formula: ; Requirement: Rs ≥ 1.5 Linearity range and correlation coefficient verification: Prepare a standard curve (8-10 points) with concentrations ranging from 0.01 to 1000 pg / mL; plot the response intensity vs. concentration plot and calculate the correlation coefficient R. 2 Requirements: R 2 ≥ 0.997.

[0051] Sensitivity and LOD validation: the limit of detection (LOD) is determined by S / N ≥ 3, and the limit of quantitation (LOQ) is determined by S / N ≥ 10; requirement: LOD ≤ 0.05 pg / mL.

[0052] like Figure 1 As shown, under two-dimensional chromatography-ion mobility mass spectrometry conditions, 12-HETE and 15-HETE exhibit good retention time resolution (approximately 1.2 min), supporting the claim of resolution ≥1.5. The two peaks in the total ion chromatogram (TIC) are clearly distinguishable, meeting the description requirement of "accurate identification of homomeric isomers".

[0053] like Figure 2 As shown, the TIC (Total Ion Spectrogram) reveals a high-abundance main peak of 12-HETE and a low-abundance secondary peak of 15-HETE, with good retention time separation between the two. The simulated detection sensitivity of 15-HETE is close to 0.05 pg. The CCS (Collision Cross Section) plot shows a significant structural difference between 12-HETE and 15-HETE (approximately 5 Å). 2The system supports the use of ion mobility mass spectrometry to distinguish isomers; the MS / MS spectra show the difference in fragment m / z distribution between 12-HETE and 15-HETE, which helps to further confirm the structure and accurately identify the isomers.

[0054] 13. Conclusion The results of the above performance parameter verification show that the present invention meets the requirements for clinical application in terms of analytical sensitivity and measurement range, precision, accuracy, limit of quantitation, carryover effect, clinically reportable range, matrix effect, and reference interval verification.

[0055] In summary, the performance verification of this self-developed product meets the requirements for clinical testing and can be used for the clinical specimen detection of arachidonic acid metabolites in human serum.

[0056] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the technical spirit of the present invention. The technical scope of the present invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A method for preparing a test sample for the detection of arachidonic acid isomers based on magnetic beads, characterized in that, Includes the following steps: (1) Wash the carboxyl magnetic beads with buffer solution, add 0.5~2g of carboxyl magnetic beads and 4~6mM of N-hydroxysuccinimide to the buffer solution to obtain activated carboxyl magnetic beads; (2) Add PEI solution to the above activated carboxyl magnetic beads, react, wash with PBS to remove unbound PEI, and obtain PEI-modified magnetic beads; (3) Add Tris-HCl buffer solution and stir well to obtain covalently coupled magnetic beads; (4) Add 5-20 μL of serum sample to be tested and 0.5-2 mg of covalently coupled magnetic beads to 50 μL with PBS buffer. Stir to allow the arachidonic acid isomers in the serum to fully bind with the magnetic beads. Adsorb the magnetic beads with a magnetic rack, discard the supernatant, and wash the magnetic beads 2-3 times with PBS buffer to remove unbound substances to obtain the precursor. Finally, resuspend with PBS buffer to obtain the test solution.

2. The method for preparing a test sample for the detection of arachidonic acid isomers based on magnetic beads according to claim 1, characterized in that, The pH of the Tris-HCl buffer solution is 7.4, and the concentration added is 40~50mM.

3. The method for preparing a test sample for the detection of arachidonic acid isomers based on magnetic beads according to claim 1, characterized in that, The reaction temperature in step (1) is 22~28℃ and the time is 20~60min.

4. The method for preparing a test sample for the detection of arachidonic acid isomers based on magnetic beads according to claim 1, characterized in that, The reaction temperature in step (2) is 22~28℃ and the reaction time is 20~40min.

5. The method for preparing a test sample for the detection of arachidonic acid isomers based on magnetic beads according to claim 1, characterized in that, The carboxyl magnetic beads have a diameter of 0.5~2μm and a carboxyl density of 280~320μmol / g.

6. The method for preparing a test sample for the detection of arachidonic acid isomers based on magnetic beads according to claim 1, characterized in that, The buffer solution mentioned in step (1) is a MES buffer solution with a pH of 5.8 to 6.

2.

7. The method for preparing a test sample for the detection of arachidonic acid isomers based on magnetic beads according to claim 1, characterized in that, The buffer solution mentioned in step (3) is at least one of sodium citrate buffer with pH 4.8 to 5.2, MES buffer with pH 5.8 to 6.2, PBS buffer with pH 6.8 to 7.2, and Tris-HCl buffer with pH 7.8 to 8.

2.

8. The method for preparing a test sample for the detection of arachidonic acid isomers based on magnetic beads according to claim 1, characterized in that, The mass concentration of PEI in the PEI solution is 0.5-5%.

9. The method for preparing a test sample for the detection of arachidonic acid isomers based on magnetic beads according to claim 1, characterized in that, The mass ratio of the PEI solution to the activated carboxyl magnetic beads is (2~10):

1.

10. The method for preparing a test sample for the detection of arachidonic acid isomers based on magnetic beads according to claim 1, characterized in that, The arachidonic acid isomers include at least one of arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, linolenic acid, prostaglandin D2, prostaglandin F2α, thromboxane B2, leukotriene B4, 5-hydroxyeicosapentaenoic acid, 12-hydroxyeicosapentaenoic acid, 15-hydroxyeicosapentaenoic acid, 20-hydroxyeicosapentaenoic acid, 5,6-epoxyeicosatrienoic acid, and 14,15-epoxyeicosatrienoic acid.

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