Method for determining the amino grafting rate of a solid substrate surface

Abstract

The application belongs to the field of medical instrument analysis, and particularly relates to a method for determining the amino grafting rate of a solid substrate surface. The method comprises detecting the amino grafting rate of the solid substrate surface by using a TNBS method; and the buffer solution used in the TNBS method is a tertiary amine type Good's buffer solution containing EDTA, SDS and DMF. The tertiary amine type Good's buffer solution containing EDTA, SDS and DMF with specific concentrations is used as a reaction medium, and the pretreatment step of crushing the solid substrate to a specific particle size can effectively eliminate the interference of the structures of components other than primary amines in the solid substrate, so that highly specific quantitative detection is realized.

Description

Technical Field

[0001] This invention belongs to the field of medical device analysis, specifically relating to a method for determining the amino grafting rate on the surface of a solid matrix. Background Technology

[0002] Amino-modified polystyrene (PS) microspheres are functional polymer materials with amino (-NH2) active groups introduced into the surface of polystyrene-based spheres. Their core structure includes a hydrophobic polystyrene framework and an amino layer on the surface for coupling biomolecules. They are widely used in bioseparation, immunoassay, and drug delivery. Amino-modified polystyrene microspheres specifically designed for hemoperfusion are structurally more complex. They are not simply modified with amino groups on a smooth, solid sphere surface, but rather are porous microspheres prepared using a styrene-divinylbenzene (St-DVB) copolymer system. These microspheres possess a large specific surface area and abundant pores. Furthermore, the amino groups are often introduced into the framework through special comonomers (such as 3-aminopropylene) rather than simple post-surface modification, thus ensuring high adsorption capacity and excellent mechanical strength while providing coupling sites. In hemoperfusion applications, these amino groups can serve as active arms for directly adsorbing pathogenic substances such as bilirubin and endotoxins, or as intermediate bridges for further coupling with specific ligands such as heparin, polyethyleneimine, or antibodies. Therefore, accurately measuring the amino grafting rate (mmol / g) on ​​the surface of microspheres is crucial for strictly controlling the batch-to-batch quality stability of adsorbents, evaluating ligand immobilization efficiency, and predicting the adsorption performance of the final product.

[0003] While numerous existing methods for amino group detection exist, they all have limitations for solid matrices, especially for amino-modified polystyrene microspheres used in hemoperfusion adsorbents. The ninhydrin method produces false positives due to its reaction with secondary amines, and the product easily adsorbs onto the microsphere surface under heating conditions, interfering with accurate quantification. The Fmoc-Cl method, while specific for primary amines, suffers from systematically low values ​​for high-surface-area microspheres due to pore mass transfer effects during whole-sphere determination, and requires an additional deprotection step, making the operation cumbersome. The OPA method has high sensitivity, but its fluorescent product is easily adsorbed by PS powder and retained by a 0.45 μm filter membrane, leading to systematic underestimation of the results. The fluorescent amine method also faces interference from solid adsorption, and the product stability is poor, affecting reproducibility. Although some literature reports the use of the TNBS method for quantifying amino groups in solid matrices, practical trials have shown that this method cannot achieve accurate and stable determination of amino groups in hemoperfusion adsorbent microspheres. Therefore, it is necessary to develop a method for accurately quantifying amino groups in hemoperfusion adsorbent microspheres to achieve quality control. Summary of the Invention

[0004] To address the above problems, the present invention provides a method for determining the amino grafting rate on the surface of a solid matrix, characterized in that it includes using the TNBS method to detect the amino grafting rate on the surface of the solid matrix; The buffer solution used in the TNBS method is a tertiary amine type Good's buffer solution containing EDTA, SDS and DMF.

[0005] Furthermore, each 100 mL of the tertiary amine Good's buffer contains 1 mL of 0.1-1 M EDTA solution, 2 mL of 1% SDS solution, and 10 mL of DMF; the concentration of the tertiary amine compound in the tertiary amine Good's buffer is 0.05-0.2 M, and the pH value is 8.5-9.0.

[0006] Furthermore, the tertiary amine type Good's buffer includes TAPS buffer, TAPSO buffer, or TABS buffer.

[0007] Furthermore, when the specific surface area of ​​the solid matrix does not exceed 500 m² / g, it is freeze-pulverized to a particle size of <60 μm during testing; when the specific surface area is above 500 m² / g, it is freeze-pulverized to a particle size of <30 μm during testing.

[0008] Furthermore, the solid matrix comprises amino-modified polystyrene microspheres; the amino-modified polystyrene microspheres include those used in hemoperfusion adsorbents.

[0009] Furthermore, it includes the following steps: 1) Draw the standard curve Take TNBS solution and dilute it with tertiary amine Good's buffer to obtain a series of concentrations for the standard curve working solution; after reacting the standard curve working solution in the dark, add HCl solution to terminate the reaction, and measure A in the solution after the reaction. 355 Values, with the concentration of the standard curve working solution as the x-axis and A as the y-axis. 355 Use the value as the ordinate to plot a standard curve; 2) Detection a. Take amino-modified polystyrene microspheres and unmodified polystyrene microspheres from the same batch, freeze-crush and dry them to obtain the test sample and blank control. b. Take the test sample and blank control separately, disperse them in tertiary amine-type Good's buffer, then add TNBS solution and mix well. After the reaction is complete in the dark, add HCl solution to terminate the reaction. Finally, take the reaction solution to measure A. 355 value; 3) Calculation of amino grafting rate A of the blank control and the test sample 355Substitute the values ​​into the standard curve to calculate the initial and remaining TNBS amounts, then calculate the grafting rate using the following formula: Amino grafting rate on solid surface (mmol / g) = (initial amount of TNBS - remaining amount of TNBS) / mass of microspheres.

[0010] Furthermore, in step 1), the volume ratio of the working solution for the series of concentration standard curves to the HCl solution is 6 ml: 100 μL; the concentration of the working solution for the series of concentration standard curves is 0~10 μmol / mL.

[0011] Furthermore, the cryogenic pulverization in step a is performed by grinding or homogenizing in a liquid nitrogen environment; the grinding time is 2–5 min, and the homogenization is performed twice, each time for 30 s; the drying is performed by vacuum drying at a temperature of 60 ℃ for 6 h.

[0012] Furthermore, in step b, the mass-to-volume ratio of the test sample or blank control to the tertiary amine-type Good's buffer, TNBS solution, and HCl solution is 10 mg: 3 mL: 3 mL: 100 μL; the dispersion is performed by ultrasonic dispersion or vortex dispersion for 30 s.

[0013] Furthermore, the concentration of the TNBS solution is 0.1~0.2%; the concentration of the HCl solution is 0.1 M; and the reaction conditions are: protection from light, temperature 25 ℃, reciprocating shaking at 120 rpm, and reaction time 15~30 min.

[0014] The high specific surface area PS-NH2 microspheres of the present invention are amino-modified polystyrene microspheres with a specific surface area of ​​more than 500 m² / g; the low specific surface area PS-NH2 microspheres are amino-modified polystyrene microspheres with a specific surface area of ​​no more than 500 m² / g.

[0015] This invention discloses a method for determining the amino grafting rate on a solid matrix surface. The method employs the TNBS approach, using a tertiary amine-type Good's buffer solution with specific concentrations of EDTA, SDS, and DMF as the reaction medium. Combined with a pretreatment step of pulverizing the solid matrix to a specific particle size, this effectively eliminates interference from components other than primary amines in the solid matrix, achieving highly specific quantitative detection. Method validation results show that this method has extremely high precision, with a repeatability RSD ≤ 0.6%, fully meeting the stringent quality control requirements for pharmaceutical and medical device intermediates. The reaction conditions are mild and rapid, requiring only 15 minutes at 25-30 °C, reducing the time by 66% compared to classic methods and significantly lowering energy consumption. Simultaneously, it exhibits excellent sensitivity, with a limit of detection (LOD) as low as 0.014 μmol, a limit of quantitation (LOQ) of 0.048 μmol, and a spiked recovery rate as high as 99.03%. All indicators meet the regulatory standards for quality control of pharmaceutical / medical device intermediates.

[0016] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0017] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0018] Figure 1 Optical photograph of PS-NH2-1 microspheres; Figure 2 Electron microscopy image of PS-NH2-1 microspheres; Figure 3 The TNBS standard curve and linear range. Detailed Implementation

[0019] The reagents and equipment used in the specific embodiments of this invention were all purchased commercially. Among them, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), 3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (AMPSO), borate buffer, Bicine buffer, Tris buffer, and 5% (w / v) 2,4,6-trinitrobenzenesulfonic acid (TNBS) solution were all purchased from Sigma-Aldrich.

[0020] All PS-NH2 microspheres with different specific surface areas were prepared in-house according to the following method: I. Low specific surface area PS-NH2 microspheres Low specific surface area PS microspheres: purchased from Dusheng Resin, USA, BET 80 m 2 / g, particle size 300-1000 μm, the synthesized amino microspheres are designated as PS-NH2-1; (1) Chloroacetylation of PS microspheres Microsphere pretreatment: Take 60 g of low specific surface area PS microspheres and place them in a 500 mL beaker. Wash them three times alternately with deionized water and anhydrous ethanol (200 mL of washing solution each time, stir for 15 min and then filter). Then replace the water with anhydrous methanol (wash twice, 150 mL each time). Transfer the microspheres to a vacuum drying oven and dry them at 50 ℃ for 8 h until constant weight.

[0021] Pre-swelling treatment: Add the dried PS-1 microspheres to a 1000 mL three-necked flask, add 600 mL of 1,2-dichloroethane (swelling solvent amount 10 mL / g PS), seal and place in a constant temperature environment of 25 ℃ for 12 h to allow the microsphere channels to fully swell.

[0022] Friedel-Crafts acylation reaction: Nitrogen gas (flow rate 50 mL / min) was introduced into the above swollen system, and the mixture was stirred and dispersed under an inert atmosphere (stirring speed 200 r / min); 45 mmol of chloroacetyl chloride, the acylation reagent, and 41 mmol of Lewis acid catalyst FeCl3 were added sequentially, and the reaction was carried out at a constant temperature of 25 °C for 8 h.

[0023] Post-processing purification: After the reaction was completed, nitrogen flow was stopped, the reaction solution was filtered, and the solid microspheres were collected. The microspheres were washed twice with 1 mol / L dilute hydrochloric acid (200 mL) (to remove Fe salt), then washed with deionized water (300 mL) until the filtrate was neutral, and then washed three times with anhydrous methanol (200 mL) (to remove residual acylation reagents, acidic byproducts, and low molecular weight impurities). The microspheres were placed in a Soxhlet extractor and extracted by reflux with acetone for 6 h, followed by vacuum drying at 60 °C for 10 h to constant weight, to obtain the chloroacetylated PS intermediate (denoted as PS-CO-CH2-Cl-1).

[0024] (2) Carbonyl reduction of chloroacetylated PS microspheres Reduction reaction: Take 50 g of PS-CO-CH2-Cl-1 intermediate prepared in (1), add 500 mL of anhydrous methanol (solvent amount 10 mL / g intermediate), place in a constant temperature reactor at 25 ℃ and stir to disperse for 30 min; slowly add 12 g (about 317 mmol) of reducing agent NaBH4 in batches, control the system temperature to not exceed 30 ℃, and continue to stir the reaction at a constant temperature for 6 h after the addition is completed. A hydrogen collection device is installed during the process to collect hydrogen in time.

[0025] Post-processing: After the reaction was completed, a small amount of deionized water was slowly added dropwise to the system to quench excess NaBH4, and the mixture was stirred for 30 min. The microspheres were collected by filtration and washed three times with anhydrous methanol (200 mL) and twice with deionized water (250 mL) to remove borate byproducts and unreacted reducing agents. The microspheres were transferred to a vacuum drying oven and dried at 55 °C for 8 h to constant weight to obtain a hydroxyl-halogen bifunctional PS intermediate (denoted as PS-CH(OH)-CH2-Cl-1).

[0026] (3) Preparation of amino-grafted PS microspheres Take 40 g of the PS-CH(OH)-CH2-Cl-1 intermediate prepared in (2), add 350 mL of ethanol-acetonitrile mixed solvent (volume ratio 3:1), and stir to disperse evenly; add 80 mL of excess ethylenediamine (5.0 equivalent relative to the molar ratio of halogen groups), heat to 65 ℃ and reflux for 12 h to allow the chlorine atoms on the surface of the microspheres to undergo nucleophilic substitution reaction with ethylenediamine, while the hydroxyl groups are retained in the side chain; after the reaction is completed, filter, and wash successively with ethanol (200 mL) and deionized water (300 mL) until the filtrate is neutral, and dry under vacuum at 60 ℃ for 10 h to obtain hydroxyl-modified aminated PS-1 microspheres (denoted as PS-NH2-1 microspheres). Figures 1-2 ).

[0027] The density of free amino groups was determined to be 0.577 mmol / g by the Fmoc-Cl method.

[0028] II. High specific surface area PS-NH2 microspheres High specific surface area PS microspheres: purchased from Dusheng Resin, USA, BET 610 m 2 / g, particle size 300-1000 μm, the synthesized amino microspheres are designated as PS-NH2-2; The only difference between the preparation steps of PS-NH2-2 microspheres and those of PS-NH2-1 is that high specific surface area PS microspheres are used.

[0029] The density of free amino groups was determined to be 0.985 mmol / g by the Fmoc-Cl method.

[0030] The PS-NH2 microspheres prepared according to the above method are the PS-NH2 microspheres used as blood perfusion adsorbents.

[0031] Example 1: Determination of amino grafting rate on the surface of PS-NH2 microspheres I. Solution Preparation TNBS solution: Take 5% TNBS solution and dilute it with ultrapure water under light-protected conditions to prepare a 0.1% (w / v) TNBS solution; TAPS buffer: Dissolve TAPS in ultrapure water to prepare a 0.1 M solution, and adjust the pH to 8.5 with 0.1 M NaOH. EDTA solution: Dissolve EDTA in ultrapure water to prepare a 0.1 M EDTA solution; SDS solution: Dissolve SDS in ultrapure water to prepare a 1% (w / v) SDS solution; TAPS buffer solution: Take 80 mL of TAPS buffer solution, add 1 mL of EDTA solution, 2 mL of SDS solution and 10 mL of DMF in sequence, then adjust the volume to 100 mL with TAPS buffer solution, mix well, retest the pH, and adjust with 0.1 M NaOH solution to ensure that the final pH is 8.5. II. Drawing the Standard Curve 1) Preparation of working solutions for a series of concentration standard curves: Take 0.1% TNBS solution and dilute it with TAPS comprehensive buffer to prepare solutions with concentrations of 0, 1, 2, 3, 5, 8, and 10 μmol / mL. 2) Take 6 mL of the working solution for the series concentration standard curve, and react with shaking at 25 ℃ and 120 rpm in the dark for 15 min. Finally, add 100 μL of 0.1 M HCl solution, shake well, and react with shaking at 25 ℃ and 120 rpm in the dark for 5 min. Centrifuge at 10,000g for 5 min, and take 200 μL of the supernatant. Add it to a 96-well UV plate; measure A. 355 ; Plot the concentration of the standard curve working solution on the x-axis and A on the y-axis. 355 Use the value as the ordinate to plot a standard curve; III. TNBS Method for Determination 3) Sample pretreatment: Take 500 mg of low specific surface area aminated PS microspheres and 500 mg of the same batch of unmodified low specific surface area PS microspheres, add liquid nitrogen, grind in an ice bath for 2–5 min, sieve to obtain powder with a particle size <60 μm, and vacuum dry at 60 ℃ for 6 h to obtain test sample and blank control. 4) Take 10.0 mg of the test sample and blank control, add 3 mL of TAPS buffer, vortex for 30 s, then add 3 mL of 0.1% TNBS solution and mix well. Incubate at 25 ℃ and 120 rpm in the dark for 15 min with shaking. Finally, add 100 μL of 0.1M HCl solution, shake well, and incubate at 25 ℃ and 120 rpm in the dark for 5 min with shaking. Centrifuge at 10,000 g for 5 min, and take 200 μL of the supernatant. Add to a 96-well UV plate; measure A. 355 ; IV. Calculation A of the blank control and the test sample 355 Substitute the values ​​into the standard curve to calculate the initial and remaining TNBS amounts, then calculate the grafting rate using the following formula: Amino grafting rate on solid surface (mmol / g) = (initial amount of TNBS - remaining amount of TNBS) / mass of microspheres.

[0032] Example 2: Determination of amino grafting rate on the surface of PS-NH2 microspheres I. Solution Preparation TNBS solution: Take 5% TNBS solution and dilute it with ultrapure water under light-protected conditions to prepare a 0.1% (w / v) TNBS solution; AMPSO buffer: Dissolve AMPSO in ultrapure water to prepare a 0.1 M solution, and adjust the pH to 8.8 with 0.1 M NaOH. EDTA solution: Dissolve EDTA in ultrapure water to prepare a 0.1 M EDTA solution; SDS solution: Dissolve SDS in ultrapure water to prepare a 1% (w / v) SDS solution; The preparation method of AMPSO buffer solution is as follows: Take 80 mL of AMPSO buffer solution, add 1 mL of EDTA solution, 2 mL of SDS solution and 10 mL of DMF in sequence, then adjust the volume to 100 mL with AMPSO buffer solution, mix well, retest the pH, and adjust with 0.1 M NaOH solution to ensure that the final pH is 8.8. II. Drawing the Standard Curve 1) Working solutions for a series of concentration standard curves: Take 0.1% TNBS solution and dilute it with AMPSO comprehensive buffer to prepare solutions with concentrations of 0, 1, 2, 3, 5, 8, and 10 μmol / mL. 2) Take 6 mL of the working solution for the series concentration standard curve, and react with shaking at 25 ℃ and 120 rpm in the dark for 15 min. Finally, add 100 μL of 0.1 M HCl solution, shake well, and react with shaking at 25 ℃ and 120 rpm in the dark for 5 min. Centrifuge at 10,000g for 5 min, and take 200 μL of the supernatant. Add it to a 96-well UV plate; measure A. 355 ; Plot the concentration of the standard curve working solution on the x-axis and A on the y-axis. 355 Use the value as the ordinate to plot a standard curve; III. TNBS Method for Determination 3) Sample pretreatment: Take 500 mg of low specific surface area aminated PS microspheres and 500 mg of the same batch of unmodified low surface area PS microspheres, respectively, add liquid nitrogen for pre-cooling, homogenize twice for 30 s each time, sieve to obtain powder with a particle size <60 μm, and vacuum dry at 60℃ for 6 h to obtain test sample and blank control. 4) Take 10 mg of the test sample and blank control, add 3 mL of AMPSO buffer solution, sonicate for 30 s, then add 3 mL of 0.1% TNBS solution and mix well. Incubate at 25 ℃ and 120 rpm in the dark for 15 min with shaking. Finally, add 100 μL of 0.1 M HCl solution, shake well, and incubate at 25 ℃ and 120 rpm in the dark for 5 min. Filter the reaction solution through a 0.45 μm syringe filter, and add 200 μL of the filtrate to a 96-well UV plate; measure A. 355 ; IV. Calculation A of the blank control and the test sample 355 Substitute the values ​​into the standard curve to calculate the initial and remaining TNBS amounts, then calculate the grafting rate using the following formula: Amino grafting rate on solid surface (mmol / g) = (initial amount of TNBS - remaining amount of TNBS) / mass of microspheres.

[0033] Example 3: Determination of amino grafting rate on the surface of PS-NH2 microspheres I. Solution Preparation TNBS solution: Take 5% TNBS solution and dilute it with ultrapure water under light-protected conditions to prepare a 0.1% (w / v) TNBS solution; TAPS buffer: Dissolve TAPS in ultrapure water to prepare a 0.1 M solution, and adjust the pH to 8.5 with 0.1 M NaOH. EDTA solution: Dissolve EDTA in ultrapure water to prepare a 0.1 M EDTA solution; SDS solution: Dissolve SDS in ultrapure water to prepare a 1% (w / v) SDS solution; TAPS buffer solution: Take 80 mL of TAPS buffer solution, add 1 mL of EDTA solution, 2 mL of SDS solution and 10 mL of DMF solution in sequence, then adjust the volume to 100 mL with TAPS buffer solution, mix well, retest the pH, and adjust with 0.1 M NaOH solution to ensure that the final pH is 8.5. II. Drawing the Standard Curve 1) Working solutions for a series of concentration standard curves: Take 0.1% TNBS solution and dilute it with TAPS comprehensive buffer to prepare solutions with concentrations of 0, 1, 2, 3, 5, 8, and 10 μmol / mL. 2) Take 6 mL of the working solution for the series concentration standard curve, and react with shaking at 25 ℃ and 120 rpm in the dark for 15 min. Finally, add 100 μL of 0.1 M HCl solution, shake well, and react with shaking at 25 ℃ and 120 rpm in the dark for 5 min. Centrifuge at 10,000g for 5 min, and take 200 μL of the supernatant. Add it to a 96-well UV plate; measure A. 355 ; Plot the concentration of the standard curve working solution on the x-axis and A on the y-axis. 355 Use the value as the ordinate to plot a standard curve; III. TNBS Method for Determination 3) Sample pretreatment: Take 500 mg of high specific surface area aminated PS microspheres and the same batch of unmodified high surface area PS microspheres respectively, add liquid nitrogen, grind in ice bath for 2–5 min, sieve to obtain powder with particle size <30 μm, vacuum dry at 60 ℃ for 6 h to obtain test sample and blank control. 4) Take 10 mg of the test sample and blank control, add 3 mL of TAPS buffer, vortex for 30 s, then add 3 mL of 0.1% TNBS solution and mix well. Incubate at 25 ℃ and 120 rpm in the dark for 15 min with shaking. Finally, add 100 μL of 0.1 M HCl solution, shake well, and incubate at 25 ℃ and 120 rpm in the dark for 5 min with shaking. Centrifuge at 10,000 g for 5 min, and take 200 μL of the supernatant. Add to a 96-well UV plate; measure A. 355 ; IV. Calculation A of the blank control and the test sample 355 Substitute the values ​​into the standard curve to calculate the initial and remaining TNBS amounts, then calculate the grafting rate using the following formula: Amino grafting rate on solid surface (mmol / g) = (initial amount of TNBS - remaining amount of TNBS) / mass of microspheres.

[0034] The following experimental examples further illustrate the beneficial effects of the present invention: Experimental Example 1: Study on the TNBS method for determining the amino grafting rate on the surface of a solid matrix Amino-modified PS microspheres are important intermediates in hemoperfusion adsorbents, and the accurate determination of their surface amino grafting rate is directly related to the accurate prediction of adsorbent performance. In previous studies, the TNBS method was determined based on literature review and laboratory conditions. However, actual testing revealed that the reported reaction system of this method suffers from poor stability and low accuracy when applied to PS-based solid matrices, making it difficult to meet the measurement requirements. To address this issue, an experiment was conducted using a tertiary amine-type Good's buffer solution containing ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulfate (SDS), and a polar organic solvent as the reaction medium, which significantly improved the stability of the reaction system and the accuracy of the results. Based on this, further optimization studies were conducted on the reaction conditions to enable the optimized TNBS method to cover a wider range of PS-based solid matrix types and meet the method validation requirements.

[0035] I. Determination of Amino Grafting Rate of Macroporous, Low Specific Surface Area PS-NH2 Microspheres with Different Particle Sizes TAPS buffer solution was used as the reaction medium for the TNBS method to detect amino-grafted PS microspheres PS-NH2-1 (BET 80 m). 2 The amino grafting rate (with a theoretical amino density of approximately 0.577 mmol / g) is as follows: (1) Solution preparation: ①TAPS buffer solution: Take 80 mL of 0.1 M TAPS buffer solution (adjust pH to 8.5 with 0.1 M NaOH), add 1 mL of 0.1 M EDTA, 2 mL of 1% SDS, and 10 mL of N,N-dimethylformamide (DMF), and then adjust the volume to 100 mL with 0.1 M TAPS buffer solution. Mix well and check the pH to 8.5. ②TNBS solution: Take 5% TNBS solution, dissolve it in ultrapure water under light-protected conditions, and prepare a 0.1% (w / v) TNBS solution. Prepare fresh before use.

[0036] (2) Sample pretreatment: Take 500 mg of PS-NH2-1 microspheres, add liquid nitrogen, grind in an ice bath for 2–5 min, pass through a pharmacopoeia sieve to classify into four different particle sizes: <15 μm, 15–30 μm, 30–60 μm, and >60 μm, and vacuum dry at 60 ℃ for 6 h.

[0037] (3) TNBS reaction: Weigh 10 mg of powder of different particle sizes into 10 mL centrifuge tubes, add 3 mL of TAPS buffer solution, and vortex for 30 s. Add 3 mL of 0.1% TNBS solution and gently shake to mix. In the dark, at 25 ℃ and 120 rpm (reciprocating), the reaction was carried out for 15 min.

[0038] (4) Termination and separation: Add 100 μL of 0.1 M HCl, shake well, and incubate at 25 ℃ and 120 rpm (reciprocating) in the dark for 5 min. Centrifuge at 10,000 g for 5 min, take 200 μL of the supernatant, and add it to a 96-well UV plate; measure A. 355 .

[0039] (5) Blank is “The same batch of ungrafted amino PS microspheres are repeated according to steps (2) to (4).

[0040] (6) Standard curve: Take 0.1% (w / v) TNBS buffer (3.41 mM), dilute with TAPS comprehensive buffer to prepare solutions with concentrations of 0, 1, 2, 3, 5, 8, and 10 μmol / mL, respectively. Take 6 mL of each concentration solution and react for 15 min under light-protected, 25 ℃, and 120 rpm reciprocating shaking conditions. Add 100 μL of 0.1 M HCl, shake well, and shake at 25 ℃ and 120 rpm (reciprocating) under light-protected conditions for 5 min. Centrifuge at 10,000 g for 5 min, take 200 μL of the supernatant, and add it to a 96-well UV plate; measure A. 355 Finally, the standard curve y = 0.3943x + 0.0669 (R² = 0.9994) was obtained.

[0041] (7) Calculation of grafting rate A blank determination 355 Values ​​and A measured in pretreated samples 355 Substitute the values ​​into the standard curve to calculate the initial amount and remaining amount of TNBS, and then calculate the grafting rate according to the following formula. The specific results are shown in Table 1.

[0042] Amino grafting rate on solid surface (mmol / g) = (Initial amount of TNBS - Remaining amount of TNBS) / Mass of microspheres Table 1 Grafting rate of amino groups in PS-NH2-1 Note: Deviation from theoretical value (%) = (Theoretical value - Measured value) / Theoretical value 100%.

[0043] As can be seen from the results in Table 1, for low specific surface area microspheres, the 30–60 μm range is close to the true value with a deviation of less than 5%, while the >60 μm range is slightly underestimated. This indicates that under the experimental conditions, the macroporous structure allows TNBS to permeate without excessive grinding. As long as the microsphere powder particle size is <60 μm, the grafting rate of amino groups can be accurately determined.

[0044] II. Determination of Amino Grafting Rate in High Specific Surface Area PS-NH2 Microsphere Powders with Different Particle Sizes TAPS buffer solution was used as the reaction medium for the TNBS method to detect amino-grafted PS microspheres PS-NH2-2 (BET 610 m). 2 The amino grafting rate (with a theoretical amino density of approximately 0.985 mmol / g) is as follows: (1) Solution preparation: ①TAPS buffer solution: Take 80 mL of 0.1 M TAPS buffer solution (adjust pH to 8.5 with 0.1 M NaOH), add 1 mL of 0.1 M EDTA, 2 mL of 1% SDS, and 10 mL of N,N-dimethylformamide (DMF), and then adjust the volume to 100 mL with 0.1 M TAPS buffer solution. Mix well and check the pH to 8.5. ②TNBS solution: Take 5% TNBS solution, dissolve it in ultrapure water under light-protected conditions, and prepare a 0.1% (w / v) TNBS solution. Prepare fresh before use.

[0045] (2) Sample pretreatment: Take 500 mg of PS-NH2-2, add liquid nitrogen, pre-cool and homogenize for 30 s × 2 times, pass through the pharmacopoeia sieve to classify into four different particle sizes: <15 μm, 15–30 μm, 30–60 μm and >60 μm, and vacuum dry at 60 ℃ for 6 h.

[0046] (3) TNBS reaction: Weigh 10 mg of powder of different particle sizes into 10 mL centrifuge tubes, add 3 mL of TAPS buffer solution, and sonicate for 30 s. Add 3 mL of 0.1% TNBS solution and gently shake to mix. In the dark, at 25 ℃ and 120 rpm (reciprocating), the reaction was carried out for 15 min.

[0047] (4) Termination and separation: Add 100 μL of 0.1 M HCl, shake well, and shake at 25 ℃ and 120 rpm (reciprocating) in the dark for 5 min. Take the reaction solution directly, filter it through a 0.45 μm syringe filter, take 200 μL of the solution, and add it to a 96-well UV plate; measure A. 355 .

[0048] (5) Blank is “The same batch of ungrafted amino PS microspheres are repeated according to steps (2) to (4).

[0049] (6) Standard curve: Take 0.1% TNBS solution and dilute it with TAPS buffer to prepare solutions with concentrations of 0, 1, 2, 3, 5, 8, and 10 μmol / mL. Take 6 mL of each concentration solution and react for 15 min under light-protected conditions, at 25 ℃ and 120 rpm with reciprocating shaking. Add 100 μL of 0.1 M HCl, shake well, and shake at 25 ℃ and 120 rpm (reciprocating shaking) under light-protected conditions for 5 min. Directly take the reaction solution, filter it through a 0.45 μm syringe filter, and take 200 μL of the solution. Add it to a 96-well UV plate; measure A. 355 Finally, the standard curve was plotted.

[0050] (7) Calculation of grafting rate A blank determination 355 Values ​​and A measured in pretreated samples 355 Substitute the values ​​into the standard curve to calculate the initial amount and remaining amount of TNBS, and then calculate the grafting rate according to the following formula. The specific results are shown in Table 2.

[0051] Amino grafting rate on solid surface (mmol / g) = (Initial amount of TNBS - Remaining amount of TNBS) / Mass of microspheres Table 2 Grafting rate of amino groups in PS-NH2-2 Note: Deviation from theoretical value (%) = (Theoretical value - Measured value) / Theoretical value 100%.

[0052] As shown in Table 2, the highest amino grafting rate (0.976 mmol / g) was obtained when PS microspheres were ground to <15 μm, with a deviation of 0.91% from the theoretical value of 0.985 mmol / g by the TNBS method. The deviation from the theoretical value was also within 5% when the powder was ground to 15-30 μm. The measured value decreased with increasing particle size, indicating that PS microspheres need to be ground finely enough to allow the TNBS method to fully contact the internal amino sites, thus avoiding underestimation due to mass transfer limitations during whole-sphere measurements. This result confirms the necessity of particle size fractionation for high specific surface area microspheres.

[0053] Based on the influence of the fineness of macroporous PS microspheres with different specific surface areas on the detection results, it can be determined that in the detection system using TAPS comprehensive buffer as the reaction medium of the TNBS method, microspheres with low specific surface area should be selected in the <60 μm range, and macroporous microspheres with high specific surface area should be selected in the <30 μm range, which can balance accuracy and ease of operation.

[0054] III. Other tertiary amine-type Good's buffer as a reaction medium for detecting the amino grafting rate of PS microspheres AMPSO buffer solution was used as the reaction medium for the TNBS method to detect the amino grafting rate of amino-grafted PS microspheres PS-NH2-1. The preparation method of AMPSO buffer solution (pKa 9.0) was as follows: 80 mL of 0.1 M AMPSO buffer solution (adjusted to pH 8.8 with 0.1 M NaOH) was added, along with 1 mL of 0.1 M EDTA, 2 mL of 1% SDS, and 10 mL of DMF. The volume was then adjusted to 100 mL with 0.1 M AMPSO buffer solution, mixed well, and the pH was rechecked to 8.8. The preparation, detection steps, and calculation methods for other solutions were the same as those under the section "I. Detection of Amino Grafting Rate of Microsphere Powder with Different Particle Sizes of Macroporous Low Surface Area PS-NH2 Microspheres". Specific detection results are shown in Table 3.

[0055] Table 3 Grafting rate of amino groups in PS-NH2-1 Note: Deviation from theoretical value (%) = (Theoretical value - Measured value) / Theoretical value 100%.

[0056] As shown in Table 3, when using the TNBS method to detect the amino grafting rate of PS microspheres, the results obtained with the buffer medium constructed using AMPSO were consistent with those obtained with the buffer medium constructed using TAPS. This indicates that under this method, neither buffer produces selective interference to the colorimetric reactions of TNBS and amino groups. Therefore, in practical operation, AMPSO or TAPS can be flexibly selected as the main buffer medium based on the availability or cost of reagents.

[0057] IV. Methodological Validation of the TNBS Method Using PS-NH2-1 microspheres, the reaction conditions were optimized and the methodology was validated based on the method under "I. Detection of Amino Grafting Rate of Microsphere Powder with Different Particle Sizes of Macroporous Low Surface Area PS-NH2 Microspheres". The specific optimization conditions and validation items are shown in Table 4.

[0058] Table 4. TNBS Method Condition Optimization and Validation Framework (1) Optimization of TNBS concentration Except for variations in TNBS concentration, all other detection conditions were fixed. Specific conditions: 10 mg microsphere powder (15-30 μm) + 3 mL 0.1 M TAPS buffer (pH 8.5) → vortex for 30 seconds → add 3 mL of TNBS at different concentrations → gently shake to mix → incubate at 25°C in the dark, 120 rpm for 15 minutes → add 0.1 mL of 0.1 M hydrochloric acid → incubate at 25°C in the dark, 120 rpm for 5 minutes → centrifuge / filter → measure A. 355 The results are shown in Table 5.

[0059] Table 5 Results of TNBS Concentration Optimization As shown in Table 5, 0.1% TNBS in the reaction system is sufficient, and further increasing the concentration has limited effect on improving the measured values ​​(<2%). Furthermore, with increasing TNBS concentration, the reagent blank background increases significantly, and the detection results begin to show large deviations. Therefore, the optimal concentration of TNBS in the TNBS method is 0.1% (w / v).

[0060] (2) Changes in TAPS concentration Except for variations in TAPS concentration, all other detection conditions were fixed. Specific conditions: 10 mg of microsphere powder (15-30 μm) + 3 mL of a combined buffer solution containing different concentrations of TAPS → vortex for 30 seconds → add 3 mL of 0.1% TNBS → gently shake to mix → incubate at 25°C in the dark, 120 rpm for 15 minutes → add 0.1 mL of 0.1 M hydrochloric acid → incubate at 25°C in the dark, 120 rpm for 5 minutes → centrifuge / filter → measure A. 355 The results are shown in Table 6.

[0061] Table 6 Results of TAPS Concentration Optimization Note: Deviation from theoretical value (%) = (Theoretical value - Measured value) / Theoretical value 100%.

[0062] As shown in Table 6, 0.05-0.2 M TAPS in the reaction system provides a stable buffer for the TNBS reaction. Too low a concentration (<0.05 M) results in insufficient buffer capacity, severe pH drift, incomplete reaction, low measured values, and poor precision; too high a concentration (>0.2 M) leads to a significant increase in blank concentration and may cause precipitation. Therefore, TAPS concentrations less than 0.05 M or greater than 0.2 M are unsuitable for quantitative detection, with the optimal concentration of TAPS being 0.1 M.

[0063] (3) Changes in EDTA concentration Except for variations in EDTA concentration, all other detection conditions were fixed. Specific conditions: 10 mg of microsphere powder (15-30 μm) + 3 mL of TAPS buffer (pH 8.5-9.0) containing different concentrations of EDTA → vortex for 30 seconds → add 3 mL of 0.1% TNBS → gently shake to mix → incubate at 25°C in the dark, 120 rpm for 15 minutes → add 0.1 mL of 0.1M hydrochloric acid → incubate at 25°C in the dark, 120 rpm for 5 minutes → centrifuge / filter → measure A. 355 The results are shown in Table 7.

[0064] Table 7 Results of EDTA Concentration Optimization Note: Deviation from theoretical value (%) = (Theoretical value - Measured value) / Theoretical value 100%.

[0065] As shown in Table 7, when EDTA is not added to the reaction system or the EDTA concentration is <0.05 M, the blank concentration increases and the precision decreases; when the EDTA concentration exceeds 1 M, side reactions or turbidity occur, affecting the accuracy of the determination. Therefore, the EDTA concentration in the TNBS method needs to be controlled within the range of 0.1~1 M to ensure the accuracy of the detection results, with the optimal EDTA concentration being 0.1 M.

[0066] (4) Optimization of reaction time Except for the reaction time, all other detection conditions were fixed. Specific conditions: 10 mg of microsphere powder (15-30 μm) + 3 mL of TAPS buffer → vortex for 30 seconds → add 3.0 mL of 0.1% TNBS → gently shake to mix → 25 °C, protected from light, 120 rpm, shake for a certain time → add 0.1 mL of 0.1 M hydrochloric acid → 25 °C, protected from light, 120 rpm, 5 min → centrifuge / filter → measure A. 355 The results are shown in Table 8.

[0067] Table 8 Results of reaction time optimization Note: Deviation from theoretical value (%) = (Theoretical value - Measured value) / Theoretical value 100%.

[0068] As shown in Table 8, the reaction was only about 80% complete after 10 minutes, and it was not until 15 minutes that the reaction was complete and began to plateau. However, after 30 minutes, due to the partial degradation of TNBS, the measured residual TNBS amount decreased, resulting in an overestimation of the calculated amino content. Therefore, the reaction time needs to be controlled within 15-30 minutes.

[0069] Based on the optimization results of the TNBS method above, the detection steps under the optimal detection conditions can be determined as follows: 10 mg microsphere powder (15-30 μm) + 3 mL TAPS buffer (100 mL 0.1 M TAPS + 1 mL 0.1 M EDTA + 2 mL 1% SDS + 10 mL DMF, mix well, pH range 8.5) → Vortex for 30 s → Add 3 mL 0.1% TNBS → Gently shake to mix → React at 120 rpm for 15 min in the dark at 25 ℃ → Add 0.1 mL 0.1 M hydrochloric acid → React at 120 rpm for 5 min in the dark at 25 ℃ → Centrifuge / Filter → Measure A 355 .

[0070] (5) Standard curve and linear range A certain volume of 0.1% TNBS solution was taken and diluted with TAPS buffer to achieve TNBS concentrations of 0, 3.41, 6.82, 10.23, 13.64, 20.46, and 34.1 μmol. Reagents (except for the microspheres) were added according to the optimal detection conditions, and the reaction and standard curve were determined. The results are shown in Table 9. Figure 3 .

[0071] Table 9 Linear Range Results As shown in Table 9, TNBS exhibits good linearity within the range of 0–34.1 μmol. Since TNBS reacts with amino groups in a 1:1 stoichiometric ratio, the amino grafting rate can be calculated from the amount of TNBS consumed. Using the grafting rate of commercially available PS-NH2 microspheres (approximately 1.5 mmol / g) as a benchmark, the TNBS dosage corresponding to this linear range can cover the amino content detection requirements of 0.05–3 mmol / g, making it suitable for quantitative analysis of samples with different grafting rates.

[0072] (6) Limit of Detection (LOD) and Limit of Quantification (LOQ) Take a blank solution and perform 11 consecutive measurements (i.e., without grafted PS microspheres, and operate according to the established detection procedure under the optimal detection conditions), and calculate A. 355 The standard deviation (σ) is σ = 0.0023. The slope of the standard curve is k = 0.481 μmol⁻¹. According to the IUPAC definition: LOD = 3σ / k = 3 × 0.0023 / 0.481 = 0.014 μmol (equivalent to 0.0014 mmol / g for a 10 mg sample).

[0073] LOQ = 10σ / k = 10 × 0.0023 / 0.481 = 0.048 μmol (equivalent to 0.0048 mmol / g for a 10 mg sample).

[0074] The results above show that the detection limit under optimal detection conditions is as low as 0.014 μmol (approximately 0.0014 mmol / g), which is far lower than the typical amino grafting rate of blood perfusion adsorbent intermediates (>0.05 mmol / g), and can meet the requirements for accurate determination of samples with low grafting rates.

[0075] (7) Repeatability The same batch of microsphere powder (particle size 15-30 μm) was independently measured 6 times according to the detection procedure under the determined optimal detection conditions. The results are as follows: Table 10 Repeatability Results As can be seen from Table 10, the precision is excellent (RSD≈0.6%) under the optimal detection conditions, making it suitable for batch quality control.

[0076] (8) Spike recovery rate Add a known amount of L-leucine standard solution to 10 mg of microsphere powder with known amino content (15-30 μm range, TNBS predictive value of 0.568 mmol / g), mix well, and determine the total amino content according to the detection procedure under the determined optimal detection conditions, and calculate the recovery rate.

[0077] Table 11 Spike Recovery Results As shown in Table 11, the average recovery rate under optimal detection conditions was 99.03%. Within the range of 95–105%, the method accuracy was good, and there were no obvious matrix effects or systematic biases.

[0078] (9) Summary of method validation Table 12 Summary of Method Validation The above methodological validation results show that the TNBS method has the characteristics of good linearity (R²=0.9995), high sensitivity (LOD=0.0014 mmol / g), excellent precision (RSD=0.56%), and reliable accuracy (recovery rate 99.03%) under optimal conditions, which fully meets the quality control requirements for the amino grafting rate on the surface of blood perfusion adsorbent intermediate microspheres.

[0079] V. Comparison of different types of buffer solutions used as reaction media for detecting amino grafting rate of PS microspheres In the TNBS method, the core role of the buffer is to activate the nucleophilic reaction of the primary amine by controlling the pH of the reaction system and to maintain the overall stability of the colorimetric process. To determine the role of the tertiary amine-type Good's buffer in determining the amino grafting rate of PS microspheres, a commonly used buffer in the TNBS method was used as a control instead of the tertiary amine-type Good's buffer. This allowed for an independent evaluation of the effect of the tertiary amine-type Good's buffer on the colorimetric reaction, excluding interference from other variables.

[0080] Tertiary amine-type Good's buffers (TAPS, AMPSO), borate buffer, Bicine buffer, and Tris buffer were used as buffers for the TNBS method. Each 100 mL of these buffers contained 1 mL of 0.1 M EDTA, 2 mL of 1% SDS, and 10 mL of DMF; the pH ranged from 8.5 to 9.0. Other solution preparation, detection procedures, and calculation methods were consistent with the optimal detection conditions determined under "IV. Method Validation of the TNBS Method" for the detection of PS-NH2-1 microspheres. After six independent measurements for each group, the mean, standard deviation, and RSD of the measured values ​​were calculated. The results are shown in Table 13.

[0081] Table 13 Detection results for different buffer types As shown in Table 13, the AMPSO system (pH 8.8) and the TAPS system (pH 8.5) exhibited highly consistent performance in detecting the amino grafting rate of pure PS-NH2 microspheres: the measured –NH2 value deviation was <0.5%, and the RSD was approximately 0.5%. AMPSO has a pKa of 9.0, and although its buffering capacity at pH 8.8 is slightly higher than that of TAPS at pH 8.5, it still meets the pH 8.5-9.0 stability requirements for the TNBS reaction. Therefore, AMPSO can be an effective alternative to TAPS, especially suitable for scenarios requiring a slightly higher reaction pH (e.g., compatibility with temperature-sensitive ligands). However, the borate system does not meet the requirements of high precision and low blank for hemoperfusion adsorbent intermediates; Bicine and Tris buffers lead to inflated TNBS consumption and degraded precision, making them unsuitable for determining the amino grafting rate on solid matrix surfaces using the TNBS method. The above results indicate that tertiary amine buffers (TAPS / TAPSO / TABS / AMPSO) can ensure the accuracy, precision, and repeatability of the determination of amino grafting rate of solid PS microspheres, and are a necessary condition for the determination by the TNBS method.

[0082] In summary, this invention employs the TNBS method, using a tertiary amine-type Good's buffer solution with specific concentrations of EDTA, SDS, and DMF as the reaction medium, combined with a pretreatment step of pulverizing the solid matrix to a specific particle size. This effectively eliminates interference from components other than primary amines in the solid matrix, achieving highly specific quantitative detection and possessing practical application value.

Claims

1. A method for determining the amino grafting rate on the surface of a solid matrix, characterized in that: This includes using the TNBS method to detect the amino grafting rate on the surface of the solid matrix; The buffer solution used in the TNBS method is a tertiary amine type Good's buffer solution containing EDTA, SDS and DMF.

2. The determination method according to claim 1, characterized in that: The tertiary amine type Good's buffer contains 1 mL of 0.1-1 M EDTA solution, 2 mL of 1% SDS solution, and 10 mL of DMF per 100 mL; the concentration of the tertiary amine compound in the tertiary amine type Good's buffer is 0.05-0.2 M, and the pH value is 8.5-9.

0.

3. The determination method according to claim 2, characterized in that: The tertiary amine type Good's buffer includes TAPS buffer, AMPSO buffer, TAPSO buffer, or TABS buffer.

4. The determination method according to claim 1, characterized in that: When the specific surface area of ​​the solid matrix does not exceed 500 m² / g, it is freeze-pulverized to a particle size of <60 μm during testing; when the specific surface area is above 500 m² / g, it is freeze-pulverized to a particle size of <30 μm during testing.

5. The determination method according to claim 4, characterized in that: The solid matrix comprises amino-modified polystyrene microspheres; the amino-modified polystyrene microspheres include those used in hemoperfusion adsorbents.

6. The determination method according to any one of claims 1 to 5, characterized in that: Includes the following steps: 1) Draw the standard curve Take TNBS solution and dilute it with tertiary amine Good's buffer to obtain a series of concentrations for the standard curve working solution; after reacting the standard curve working solution in the dark, add HCl solution to terminate the reaction, and measure A in the solution after the reaction. 355 Values, with the concentration of the standard curve working solution as the x-axis and A as the y-axis. 355 Use the value as the ordinate to plot a standard curve; 2) Detection a. Take amino-modified polystyrene microspheres and unmodified polystyrene microspheres from the same batch, freeze-crush and dry them to obtain the test sample and blank control. b. Take the test sample and blank control separately, disperse them in tertiary amine-type Good's buffer, then add TNBS solution and mix well. After the reaction is complete in the dark, add HCl solution to terminate the reaction. Finally, take the reaction solution to measure A. 355 value; 3) Calculation of amino grafting rate A of the blank control and the test sample 355 Substitute the values ​​into the standard curve to calculate the initial and remaining TNBS amounts, then calculate the grafting rate using the following formula: Amino grafting rate on solid surface (mmol / g) = (initial amount of TNBS - remaining amount of TNBS) / mass of microspheres.

7. The determination method according to claim 6, characterized in that: Step 1) The volume ratio of the working solution of the series concentration standard curve to the HCl solution is 6 mL: 100 μL; the concentration of the working solution of the series concentration standard curve is 0~10 μmol / mL.

8. The determination method according to claim 6, characterized in that: The cryogenic pulverization described in step a is performed by grinding or homogenizing in a liquid nitrogen environment; the grinding time is 2-5 min; the homogenization is performed twice, 30 s each time; the drying is performed under vacuum at a temperature of 60 ℃ for 6 h.

9. The determination method according to claim 6, characterized in that: The mass-to-volume ratio of the test sample or blank control in step b to the tertiary amine-type Good's buffer, TNBS solution, and HCl solution is 10 mg: 3 mL: 3 mL: 100 μL; the dispersion is performed by ultrasonic dispersion or vortex dispersion for 30 s.

10. The determination method according to claim 6, characterized in that: The concentration of the TNBS solution is 0.1~0.2%; the concentration of the HCl solution is 0.1 M; the reaction conditions are: protection from light, temperature 25 ℃, reciprocating shaking at 120 rpm, and reaction time 15~30 min.

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