A liquid chromatography detection method for Triton X-100 residue

Abstract

The present invention relates to the field of biological detection technology, and in particular to a liquid chromatography detection method for residual Triton X-100. The liquid chromatography detection method uses high performance liquid chromatography as a detection system, and the chromatographic conditions include: the chromatographic column is an octadecylsilane bonded silica gel column; the mobile phase includes mobile phase A and mobile phase B, mobile phase A is an isopropanol solution containing trifluoroacetic acid, and mobile phase B is water. A liquid chromatography detection method for residual Triton X-100 of the present invention uses an octadecylsilane bonded silica gel column as a chromatographic column, and a specific mobile phase can accurately determine the residual amount of Triton X-100 in a sample to be tested, with high sensitivity, accuracy and precision.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, and in particular to a liquid chromatography detection method for Triton X-100 residue. Background Art

[0002] Acellular matrix is ​​a dermal scaffold that removes epidermal cells and cellular components in the dermis of skin tissue by physical and chemical methods, and retains the normal three-dimensional structure of collagen and basement membrane. Acellular matrix has low antigenicity, good tissue compatibility and stable physical and chemical properties, and is an ideal biomaterial.

[0003] Triton X-100 is a nonionic surfactant that can combine with lipids such as phospholipids in biomembranes to form soluble complexes, thereby destroying the biomembrane. Therefore, it is often used in the preparation of decellularized matrix products. However, Triton X-100 is toxic to humans and may pose a safety hazard if it is not cleaned thoroughly. Therefore, its residual amount needs to be tested.

[0004] On the one hand, due to the characteristics of the molecular structure of Triton X-100 and its characteristics as a non-ionic surfactant, it has poor stability in conventional solvents, so the detection sensitivity is not high and the accuracy is average under conventional methods; on the other hand, the decellularized matrix is ​​a three-dimensional porous material with adsorption, which also makes it difficult to analyze the adsorbed Triton X-100 components in the matrix using conventional solvents and methods. The separation degree of Triton X-100 obtained by conventional methods is poor and the recovery rate is low, resulting in low test results. The above two reasons ultimately lead to the distortion of the detection results of Triton in the decellularized matrix, affecting the judgment of the safety of the decellularized matrix products.

[0005] In summary, the industry urgently needs a detection method that can accurately detect the residues of Triton X-100 in decellularized matrices. Summary of the invention

[0006] The invention provides a liquid chromatography detection method for Triton X-100 residues, which is used to solve the problems of low sensitivity, low accuracy and the like in the existing method for detecting Triton X-100 residues in acellular matrix.

[0007] According to a first aspect of the present invention, the present invention provides a liquid chromatography detection method for Triton X-100 residues, using high performance liquid chromatography as a detection system, and the chromatographic conditions include:

[0008] The chromatographic column is an octadecylsilane bonded silica gel column;

[0009] Mobile phase: includes mobile phase A and mobile phase B. Mobile phase A is an isopropanol solution containing trifluoroacetic acid, and mobile phase B is water.

[0010] In the above scheme, a liquid chromatography detection method for residual Triton X-100 of the present invention adopts an octadecylsilane bonded silica gel column as a chromatographic column, and simultaneously adopts a specific mobile phase to accurately determine the residual amount of Triton X-100 in the sample to be tested, and has high sensitivity, accuracy and precision.

[0011] In order to further improve the sensitivity, accuracy and precision of the liquid chromatography detection method, further, the mobile phase A is an isopropanol solution containing trifluoroacetic acid, and the volume percentage of the trifluoroacetic acid is 0.08% to 0.12%, preferably 0.1%; the volume ratio of the mobile phase A to the mobile phase B is 70:30-95:5, preferably 80:20.

[0012] Further, the following steps are included:

[0013] Step (1) Preparation of blank solution: Take isopropanol and trifluoroacetic acid, and dilute to volume with water to obtain a blank solution;

[0014] Step (2) Preparation of standard solution and test solution: taking a Triton X-100 standard, using the blank solution to make up the volume and diluting into standard solutions of different concentrations; placing the sample to be tested into the blank solution for extraction to obtain a test solution;

[0015] The setting of the chromatographic conditions in step (3) includes:

[0016] The chromatographic column is an octadecylsilane bonded silica gel column;

[0017] Mobile phase: including mobile phase A and mobile phase B, mobile phase A is an isopropanol solution containing trifluoroacetic acid, and mobile phase B is water;

[0018] Step (4) Detection method: Take the blank solution, the standard solution and the test solution and inject them into a liquid chromatograph respectively, record the peak area, and use the external standard method to calculate the residual amount of Triton X-100 in the sample to be tested.

[0019] In the above scheme, the present invention uses a blank solution containing isopropanol and trifluoroacetic acid to extract the sample to be tested, which can change the ionic environment on the surface of the sample to be tested, so that the Triton X-100 adsorbed on the surface of the sample to be tested can be fully dissolved to the maximum extent, and then further combined with the chromatographic column and specific chromatographic conditions provided by the present invention, the residual amount of Triton in the sample to be tested can be more accurately determined, and the sensitivity, accuracy and precision of the detection can be further improved.

[0020] Furthermore, in step (1), in the blank solution, the volume ratio of isopropanol, trifluoroacetic acid and water is 6:2:2-8:1:1, preferably 7:1:2.

[0021] Furthermore, in step (2), the concentration of the standard solution is 20-100 ppm.

[0022] Furthermore, the weight ratio of the sample to be tested to the blank solution is 1: (4-6), preferably 1: 5. By selecting a reasonable range of the weight ratio of the sample to be tested to the blank solution, it is beneficial to fully extract Triton X-100 in the sample to be tested.

[0023] Furthermore, the extraction time is 1-4 hours, preferably 4 hours. The extraction temperature is room temperature. It should be noted that room temperature generally refers to 10-30° C. Through the reasonable design of the extraction conditions, the Triton X-100 in the sample to be tested can be fully and effectively extracted, which is conducive to improving the accuracy of the detection.

[0024] Furthermore, the setting of the chromatographic conditions in step (3) also includes: the detector is an ultraviolet detector, and the detection wavelength is 200-235nm. The ultraviolet absorption detector not only has high sensitivity, low noise, wide linear range, and good selectivity, but also is not very sensitive to ambient temperature, mobile phase composition changes, and flow rate fluctuations. By selecting a detection wavelength within a reasonable range, the sensitivity of the detection can be improved.

[0025] Furthermore, the setting of the chromatographic conditions in step (3) also includes: a flow rate of 0.8-1.2 mL / min; a column temperature of 25-40° C., preferably, a flow rate of 1.0 mL / min; and a column temperature of 30° C. By setting the flow rate and column temperature within a reasonable range, it is beneficial to separate Triton X-100 from other impurities in the sample to be tested.

[0026] Furthermore, the specifications of the chromatographic column are 4.6 mm×250 mm, 5 μm. By selecting a suitable specification of the chromatographic column, Triton X-100 in the sample to be tested can be effectively separated.

[0027] Further, the formula for calculating the residual amount of Triton X-100 in the sample to be tested is as follows:

[0028]

[0029] Where: A 供 —Peak area of ​​the test solution; A 标 —Peak area of ​​standard solution; C 标 —Concentration of standard solution, ppm; W 供 —mass of the test sample, g; V—volume of blank solution, mL.

[0030] Furthermore, the sample to be tested is a decellularized matrix.

[0031] Beneficial effects of the present invention:

[0032] The liquid chromatography detection method of residual Triton X-100 of the present invention adopts blank solution extraction under a specific proportion composition, which can change the ion environment of the surface of the decellularized matrix, so that the Triton X-100 adsorbed on the surface of the decellularized matrix can be dissolved to the maximum extent. With the joint support of the chromatographic column and specific chromatographic conditions provided by the present invention, the residual amount of Triton X-100 in the decellularized matrix can be accurately determined, with high sensitivity, accuracy and precision. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the 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 creative work.

[0034] Figure 1 is a chromatogram of a 20 ppm Triton X-100 standard solution in Example 1 of the present invention;

[0035] Figure 2 Chromatogram of 20 ppm Triton X-100 standard solution in Example 2 of the present invention;

[0036] Figure 3 Chromatogram of 20 ppm Triton X-100 standard solution in Example 3 of the present invention;

[0037] Figure 4 Chromatogram of 20 ppm Triton X-100 standard solution in Example 4 of the present invention;

[0038] Figure 5 Chromatogram of 20 ppm Triton X-100 standard solution in Example 5 of the present invention;

[0039] Figure 6 Chromatogram of 20 ppm Triton X-100 standard solution in Example 6 of the present invention;

[0040] Figure 7 Chromatogram of 20 ppm Triton X-100 standard solution in Example 7 of the present invention.

[0041] Figure 8 Chromatogram of 20 ppm Triton X-100 standard solution in Comparative Example 1 of the present invention.

[0042] Fig. 9 Chromatogram of 20 ppm Triton X-100 standard solution in Comparative Example 2 of the present invention.

[0043] Fig.10Chromatogram of 20 ppm Triton X-100 standard solution in Comparative Example 3 of the present invention.

[0044] Fig.11 Chromatogram of 20 ppm Triton X-100 standard solution in Comparative Example 4 of the present invention. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] Example 1

[0047] This embodiment provides a liquid chromatography detection method for residual Triton X-100 in a decellularized matrix, comprising the following steps:

[0048] Preparation of blank solution: Accurately weigh appropriate amounts of isopropanol and trifluoroacetic acid, and make up to volume with purified water. The volume ratio of isopropanol, trifluoroacetic acid and water is 7:1:2.

[0049] Preparation of standard solution: Accurately weigh an appropriate amount of Triton X-100 standard, dilute with blank solution to make standard solutions with concentrations of 20ppm, 40ppm, 60ppm, 80ppm, and 100ppm. The chromatogram of the standard solution with a concentration of 20ppm is as follows: Figure 1 shown.

[0050] Preparation of test solution: Accurately weigh an appropriate amount of decellularized matrix and put it into the blank solution. The mass ratio of decellularized matrix to blank solution is 1:5, and extract for 4 hours.

[0051] Setting of HPLC detection conditions:

[0052] The chromatographic column is an octadecylsilane bonded silica gel column (C18 column), 4.6 mm×250 mm, 5 μm; the mobile phase includes mobile phase A and mobile phase B, mobile phase A is an isopropanol solution containing 0.1% (V / V) trifluoroacetic acid, mobile phase B is purified water, and the volume ratio of A to B is 80:20; the detector is an ultraviolet detector with a detection wavelength of 210 nm; the flow rate is 1.0 mL / min; and the column temperature is 30°C.

[0053] Detection: Accurately measure 10 μL of blank solution, standard solution and test solution, inject into liquid chromatograph, record the peak area, use external standard method, and calculate according to the following formula:

[0054]

[0055] Where: A 供 —Peak area of ​​the test solution; A 标 —Peak area of ​​standard solution; C 标 —Concentration of standard solution, ppm; W 供 —mass of the test sample, g; V—volume of blank solution, mL.

[0056] Combination Figure 1 The test results are shown in Tables 1 and 2 below. The linear fitting R 2 =0.9998, indicating that the test results have a high degree of linear fitting, and the recovery rate is between 99.87% and 100.24%, and the test results have high accuracy and precision.

[0057] Table 1 Peak area and linear relationship table of Example 1

[0058]

[0059] Table 2 Accuracy results of Example 1

[0060]

[0061] Example 2

[0062] This embodiment provides a liquid chromatography detection method for residual Triton X-100 in a decellularized matrix, comprising the following steps:

[0063] Preparation of blank solution: Accurately weigh appropriate amounts of isopropanol and trifluoroacetic acid, and make up to volume with purified water. The volume ratio of isopropanol, trifluoroacetic acid and water is 7:1:2.

[0064] Preparation of standard solution: Accurately weigh an appropriate amount of Triton X-100 standard, dilute with blank solution to make standard solutions with concentrations of 20ppm, 40ppm, 60ppm, 80ppm, and 100ppm. The chromatogram of the standard solution with a concentration of 20ppm is as follows: Figure 2 shown.

[0065] Preparation of test solution: Accurately weigh an appropriate amount of decellularized matrix and put it into the blank solution. The mass ratio of decellularized matrix to blank solution is 1:5, and extract for 4 hours.

[0066] Setting of HPLC detection conditions:

[0067] The chromatographic column is an octadecylsilane bonded silica gel column (C18 column), 4.6 mm×250 mm, 5 μm; the mobile phase includes mobile phase A and mobile phase B, mobile phase A is an isopropanol solution containing 0.1% (V / V) trifluoroacetic acid, and mobile phase B is purified water, and the volume ratio of A to B is 80:20; the detector is an ultraviolet detector with a detection wavelength of 200 nm; the flow rate is 1.2 mL / min; and the column temperature is 25°C.

[0068] Detection: Accurately measure 10 μL of blank solution, standard solution and test solution, inject into liquid chromatograph, record the peak area, use external standard method, and calculate according to the following formula:

[0069]

[0070] Where: A 供 —Peak area of ​​the test solution; A 标 —Peak area of ​​standard solution; C 标 —Concentration of standard solution, ppm; W 供 —mass of the test sample, g; V—volume of blank solution, mL.

[0071] Combination Figure 2 The test results are shown in Tables 3 and 4 below. The linear fitting R 2 =0.9996, indicating that the test results have a high degree of linear fitting, and the recovery rate is between 99.65% and 99.79%, and the test results have high accuracy and precision.

[0072] Table 3 Peak area and linear relationship table of Example 2

[0073]

[0074] Table 4 Accuracy results of Example 2

[0075]

[0076] Example 3

[0077] This embodiment provides a liquid chromatography detection method for residual Triton X-100 in a decellularized matrix, comprising the following steps:

[0078] Preparation of blank solution: Accurately weigh appropriate amounts of isopropanol and trifluoroacetic acid, and make up to volume with purified water. The volume ratio of isopropanol, trifluoroacetic acid and water is 7:1:2.

[0079] Preparation of standard solution: Accurately weigh an appropriate amount of Triton X-100 standard, dilute with blank solution to make standard solutions with concentrations of 20ppm, 40ppm, 60ppm, 80ppm, and 100ppm. The chromatogram of the standard solution with a concentration of 20ppm is as follows: Figure 3 shown.

[0080] Preparation of test solution: Accurately weigh an appropriate amount of decellularized matrix and put it into the blank solution. The mass ratio of decellularized matrix to blank solution is 1:5, and extract for 1 hour.

[0081] Setting of HPLC detection conditions:

[0082] The chromatographic column is an octadecylsilane bonded silica gel column (C18 column), 4.6 mm×250 mm, 5 μm; the mobile phase includes mobile phase A and mobile phase B, mobile phase A is an isopropanol solution containing 0.1% (V / V) trifluoroacetic acid, and mobile phase B is purified water, and the volume ratio of A to B is 80:20; the detector is an ultraviolet detector with a detection wavelength of 235 nm; the flow rate is 1.2 mL / min; and the column temperature is 40°C.

[0083] Detection: Accurately measure 10 μL of blank solution, standard solution and test solution, inject into liquid chromatograph, record the peak area, use external standard method, and calculate according to the following formula:

[0084]

[0085] Where: A is the peak area of ​​the test solution; A 标 —Peak area of ​​standard solution; C 标 —Concentration of standard solution, ppm; W 供 —mass of the test sample, g; V—volume of blank solution, mL.

[0086] Combination Figure 3 The test results are shown in Tables 5 and 6 below. The linear fitting R 2 =0.9991, indicating that the test results have a high degree of linear fitting, and the recovery rate is between 99.94% and 100.18%, indicating that the test results have high accuracy and precision.

[0087] Table 5 Peak area and linear relationship table of Example 3

[0088]

[0089]

[0090] Table 6 Accuracy results of Example 3

[0091]

[0092] Example 4

[0093] This embodiment provides a liquid chromatography detection method for residual Triton X-100 in a decellularized matrix, comprising the following steps:

[0094] Preparation of blank solution: Accurately weigh appropriate amounts of isopropanol and trifluoroacetic acid, and make up to volume with purified water. The volume ratio of isopropanol, trifluoroacetic acid and water is 7:1:2.

[0095] Preparation of standard solution: Accurately weigh an appropriate amount of Triton X-100 standard, dilute with blank solution to make standard solutions with concentrations of 20ppm, 40ppm, 60ppm, 80ppm, and 100ppm. The chromatogram of the standard solution with a concentration of 20ppm is as follows: Figure 4 shown.

[0096] Preparation of test solution: Accurately weigh an appropriate amount of decellularized matrix and put it into the blank solution. The mass ratio of decellularized matrix to blank solution is 1:5, and extract for 2 hours.

[0097] Setting of HPLC detection conditions:

[0098] The chromatographic column is an octadecylsilane bonded silica gel column (C18 column), 4.6 mm×250 mm, 5 μm; the mobile phase includes mobile phase A and mobile phase B, mobile phase A is an isopropanol solution containing 0.1% (V / V) trifluoroacetic acid, mobile phase B is purified water, and the volume ratio of A to B is 70:30; the detector is an ultraviolet detector with a detection wavelength of 210 nm; the flow rate is 0.8 mL / min; and the column temperature is 30°C.

[0099] Detection: Accurately measure 10 μL of blank solution, standard solution and test solution, inject into liquid chromatograph, record the peak area, use external standard method, and calculate according to the following formula:

[0100]

[0101] Where: A 供 —Peak area of ​​the test solution; A 标 —Peak area of ​​standard solution; C 标 —Concentration of standard solution, ppm; W 供 —mass of the test sample, g; V—volume of blank solution, mL.

[0102] Combination Figure 4 The test results are shown in Tables 7 and 8 below. The linear fitting R 2 =0.9992, indicating that the test results have a high degree of linear fitting, and the recovery rate is between 99.71% and 99.88%, and the test results have high accuracy and precision.

[0103] Table 7 Peak area and linear relationship table of Example 4

[0104]

[0105] Table 8 Accuracy results of Example 4

[0106]

[0107] Example 5

[0108] This embodiment provides a liquid chromatography detection method for residual Triton X-100 in a decellularized matrix, comprising the following steps:

[0109] Preparation of blank solution: Accurately weigh appropriate amounts of isopropanol and trifluoroacetic acid, and make up to volume with purified water. The volume ratio of isopropanol, trifluoroacetic acid and water is 7:1:2.

[0110] Preparation of standard solution: Accurately weigh an appropriate amount of Triton X-100 standard, dilute with blank solution to make standard solutions with concentrations of 20ppm, 40ppm, 60ppm, 80ppm, and 100ppm. The chromatogram of the standard solution with a concentration of 20ppm is as follows: Figure 5 shown.

[0111] Preparation of test solution: Accurately weigh an appropriate amount of decellularized matrix and put it into the blank solution. The mass ratio of decellularized matrix to blank solution is 1:5, and extract for 3 hours.

[0112] Setting of HPLC detection conditions:

[0113] The chromatographic column is an octadecylsilane bonded silica gel column (C18 column), 4.6 mm×250 mm, 5 μm; the mobile phase includes mobile phase A and mobile phase B, mobile phase A is an isopropanol solution containing 0.1% (V / V) trifluoroacetic acid, and mobile phase B is purified water, and the volume ratio of A to B is 95:5; the detector is an ultraviolet detector with a detection wavelength of 210 nm; the flow rate is 0.8 mL / min; and the column temperature is 30°C.

[0114] Detection: Accurately measure 10 μL of blank solution, standard solution and test solution, inject into liquid chromatograph, record the peak area, use external standard method, and calculate according to the following formula:

[0115]

[0116] Where: A 供 —Peak area of ​​the test solution; A 标 —Peak area of ​​standard solution; C 标 —Concentration of standard solution, ppm; W 供 —mass of the test sample, g; V—volume of blank solution, mL.

[0117] Combination Figure 5 The test results are shown in Tables 9 and 10 below. The linear fitting R 2=0.9998, indicating that the test results have a high degree of linear fitting, and the recovery rate is between 99.96% and 100.07%, and the test results have high accuracy and precision.

[0118] Table 9 Peak area and linear relationship table of Example 5

[0119]

[0120] Table 10 Accuracy results of Example 5

[0121]

[0122] Example 6

[0123] This embodiment provides a liquid chromatography detection method for residual Triton X-100 in a decellularized matrix, comprising the following steps:

[0124] Preparation of blank solution: Accurately weigh appropriate amounts of isopropanol and trifluoroacetic acid, and make up to volume with purified water. The volume ratio of isopropanol, trifluoroacetic acid and water is 6:2:2.

[0125] Preparation of standard solution: Accurately weigh an appropriate amount of Triton X-100 standard, dilute with blank solution to make standard solutions with concentrations of 20ppm, 40ppm, 60ppm, 80ppm, and 100ppm. The chromatogram of the standard solution with a concentration of 20ppm is as follows: Figure 6 shown.

[0126] Preparation of test solution: Accurately weigh an appropriate amount of decellularized matrix and put it into the blank solution. The mass ratio of decellularized matrix to blank solution is 1:5, and extract for 4 hours.

[0127] Setting of HPLC detection conditions:

[0128] The chromatographic column is an octadecylsilane bonded silica gel column (C18 column), 4.6 mm×250 mm, 5 μm; the mobile phase includes mobile phase A and mobile phase B, mobile phase A is an isopropanol solution containing 0.1% (V / V) trifluoroacetic acid, and mobile phase B is purified water, and the volume ratio of A to B is 80:20; the detector is an ultraviolet detector with a detection wavelength of 210 nm; the flow rate is 0.8 mL / min; and the column temperature is 30°C.

[0129] Detection: Accurately measure 10 μL of blank solution, standard solution and test solution, inject into liquid chromatograph, record the peak area, use external standard method, and calculate according to the following formula:

[0130]

[0131] Where: A 供 —Peak area of ​​the test solution; A标 —Peak area of ​​standard solution; C 标 —Concentration of standard solution, ppm; W 供 —mass of the test sample, g; V—volume of blank solution, mL.

[0132] Combination Figure 6 As shown in the following table, the test results are shown in the following table, and the linear fitting R 2 =0.9999, indicating that the test results have a high degree of linear fitting, and the recovery rate is between 99.87% and 99.90%, indicating that the test results have high accuracy and precision.

[0133] Table 11 Peak area and linear relationship table of Example 6

[0134]

[0135] Table 12 Accuracy results of Example 6

[0136]

[0137] Example 7

[0138] This embodiment provides a liquid chromatography detection method for residual Triton X-100 in a decellularized matrix, comprising the following steps:

[0139] Preparation of blank solution: Accurately weigh appropriate amounts of isopropanol and trifluoroacetic acid, and make up to volume with purified water. The volume ratio of isopropanol, trifluoroacetic acid and water is 8:1:1.

[0140] Preparation of standard solution: Accurately weigh an appropriate amount of Triton X-100 standard, dilute with blank solution to make standard solutions with concentrations of 20ppm, 40ppm, 60ppm, 80ppm, and 100ppm. The chromatogram of the standard solution with a concentration of 20ppm is as follows: Figure 7 shown.

[0141] Preparation of test solution: Accurately weigh an appropriate amount of decellularized matrix and put it into the blank solution. The mass ratio of decellularized matrix to blank solution is 1:5, and extract for 4 hours.

[0142] Setting of HPLC detection conditions:

[0143] The chromatographic column is an octadecylsilane bonded silica gel column (C18 column), 4.6 mm×250 mm, 5 μm; the mobile phase includes mobile phase A and mobile phase B, mobile phase A is an isopropanol solution containing 0.1% (V / V) trifluoroacetic acid, mobile phase B is purified water, and the volume ratio of A to B is 90:10; the detector is an ultraviolet detector with a detection wavelength of 210 nm; the flow rate is 0.8 mL / min; and the column temperature is 30°C.

[0144] Detection: Accurately measure 10 μL of blank solution, standard solution and test solution, inject into liquid chromatograph, record the peak area, use external standard method, and calculate according to the following formula:

[0145]

[0146] Where: A 供 —Peak area of ​​the test solution; A 标 —Peak area of ​​standard solution; C 标 —Concentration of standard solution, ppm; W 供 —mass of the test sample, g; V—volume of blank solution, mL.

[0147] Combination Figure 7 The test results are shown in Tables 13 and 14 below. The linear fitting R 2 =0.9999, indicating that the test results have a high degree of linear fitting, and the recovery rate is between 99.88% and 99.96%, indicating that the test results have high accuracy and precision.

[0148] Table 13 Peak area and linear relationship table of Example 7

[0149]

[0150] Table 14 Accuracy results of Example 7

[0151]

[0152] Comparative Example 1

[0153] This comparative example provides a liquid chromatography detection method for residual Triton X-100 in a decellularized matrix, comprising the following steps:

[0154] Preparation of blank solution: Accurately weigh an appropriate amount of methanol and make up to volume with purified water. The volume ratio of methanol to water is 90:10.

[0155] Preparation of standard solution: Accurately weigh an appropriate amount of Triton X-100 standard, dilute with blank solution to make standard solutions with concentrations of 20ppm, 40ppm, 60ppm, 80ppm, and 100ppm. The chromatogram of the standard solution with a concentration of 20ppm is as follows: Figure 8 shown.

[0156] Preparation of test solution: Accurately weigh an appropriate amount of decellularized matrix and put it into the blank solution. The mass ratio of decellularized matrix to blank solution is 1:5, and extract for 4 hours.

[0157] Setting of HPLC detection conditions:

[0158] The chromatographic column is an octadecylsilane bonded silica column (C18 column), 4.6mm×250mm, 5μm; the mobile phase includes mobile phase A and mobile phase B, mobile phase A is methanol, mobile phase B is purified water, and the volume ratio of A to B is 90:10; the detector is an ELSD detector with a detection wavelength of 210nm; the flow rate is 1.0mL / min; and the column temperature is 35°C.

[0159] Detection: Accurately measure 20 μL of blank solution, standard solution and test solution, inject into liquid chromatograph, record the peak area, use external standard method, and calculate according to the following formula:

[0160]

[0161] Where: A 供 —Peak area of ​​the test solution; A 标 —Peak area of ​​standard solution; C 标 —Concentration of standard solution, ppm; W 供 —mass of the test sample, g; V—volume of blank solution, mL.

[0162] Combination Figure 8 The test results are shown in Tables 15 and 16. From the results in the table, it can be seen that the linear fitting result is R 2 =0.9589, the result is poor, the recovery rate of the test sample is 88.10%~93.39%, the result difference is large, and the accuracy and precision of the method are insufficient.

[0163] Table 15 Peak area and linear relationship table of comparative example 1

[0164]

[0165]

[0166] Table 16 Comparative Example 1 Accuracy Results

[0167]

[0168] Comparative Example 2

[0169] In order to better illustrate the accuracy and precision of the detection method of the present invention, a common solution and a common treatment method are used, purified water is selected as an extraction medium, and acetonitrile and purified water are selected as mobile phases. This comparative example provides a liquid chromatography detection method for residual Triton X-100 in a decellularized matrix, comprising the following steps:

[0170] Preparation of blank solution: purified water.

[0171] Preparation of standard solution: Accurately weigh an appropriate amount of Triton X-100 standard, dilute with blank solution to make standard solutions with concentrations of 20ppm, 40ppm, 60ppm, 80ppm, and 100ppm. The chromatogram of the standard solution with a concentration of 20ppm is as follows: Fig. 9 shown.

[0172] Preparation of test solution: Accurately weigh an appropriate amount of decellularized matrix and put it into the blank solution. The mass ratio of decellularized matrix to blank solution is 1:5, and extract for 4 hours.

[0173] Setting of HPLC detection conditions:

[0174] The chromatographic column is an octadecylsilane bonded silica gel column (C18 column), 4.6mm×250mm, 5μm; the mobile phase includes mobile phase A and mobile phase B, mobile phase A is acetonitrile, mobile phase B is purified water, and the volume ratio of A to B is 90:10; the detector is an ultraviolet detector with a detection wavelength of 210nm; the flow rate is 1.0mL / min; and the column temperature is 35°C.

[0175] Detection: Accurately measure 20 μL of blank solution, standard solution and test solution, inject into liquid chromatograph, record the peak area, use external standard method, and calculate according to the following formula:

[0176]

[0177] Where: A 供 —Peak area of ​​the test solution; A 标 —Peak area of ​​standard solution; C 标 —Concentration of standard solution, ppm; W 供 —mass of the test sample, g; V—volume of blank solution, mL.

[0178] Combination Fig. 9 The test results are shown in Tables 17 and 18. From the results in the table, it can be seen that the linear fitting result is R 2 =0.7166, the result is poor, the recovery rate of the test sample is 69.54%~89.57%, the result difference is large, and the accuracy and precision of the method are insufficient.

[0179] Table 17 Comparative Example 2 Peak Area and Linear Relationship Table

[0180]

[0181] Table 18 Comparative Example 2 Accuracy Results

[0182]

[0183] Comparative Example 3

[0184] Comparative Example 3 provides a liquid chromatography detection method for residual Triton X-100 in a decellularized matrix, comprising at least the following steps:

[0185] Preparation of blank solution: Accurately weigh appropriate amounts of isopropanol and acetic acid, and make up to volume with purified water. The volume ratio of isopropanol, acetic acid and water is 7:1:2.

[0186] Preparation of standard solution: Accurately weigh an appropriate amount of Triton X-100 standard, dilute with blank solution to make standard solutions with concentrations of 20ppm, 40ppm, 60ppm, 80ppm, and 100ppm. The chromatogram of the standard solution with a concentration of 20ppm is as follows: Fig.10 shown.

[0187] Preparation of test solution: Accurately weigh an appropriate amount of decellularized matrix and put it into the blank solution. The mass ratio of decellularized matrix to blank solution is 1:5, and extract for 4 hours.

[0188] Setting of HPLC detection conditions:

[0189] The chromatographic column is an octadecylsilane bonded silica gel column (C18 column), 4.6 mm×250 mm, 5 μm; the mobile phase includes mobile phase A and mobile phase B, mobile phase A is an isopropanol solution containing 0.1% (V / V) acetic acid, mobile phase B is purified water, and the volume ratio of A to B is 80:20; the detector is an ultraviolet detector with a detection wavelength of 210 nm; the flow rate is 1.0 mL / min; and the column temperature is 30°C.

[0190] Detection: Accurately measure 10 μL of blank solution, standard solution and test solution, inject into liquid chromatograph, record the peak area, use external standard method, and calculate according to the following formula:

[0191]

[0192] Where: A 供 —Peak area of ​​the test solution; A 标 —Peak area of ​​standard solution; C 标 —Concentration of standard solution, ppm; W 供 —mass of the test sample, g; V—volume of blank solution, mL.

[0193] Combination Fig.10 The test results are shown in Tables 19 and 20 below. The linear fitting R 2 =0.9544, indicating that the linear fitting degree of the detection result is not high, and the recovery rate is 97.49-98.13%. The accuracy and precision of the detection result are lower than the detection result of the liquid chromatography detection method of the present invention.

[0194] Table 19 Comparative Example 3 Peak Area and Linear Relationship Table

[0195]

[0196]

[0197] Table 20 Comparative Example 3 Accuracy Results

[0198]

[0199] Comparative Example 4

[0200] This comparative example provides a liquid chromatography detection method for residual Triton X-100 in a decellularized matrix, which at least comprises the following steps:

[0201] Preparation of blank solution: Accurately weigh appropriate amounts of ethanol and trifluoroacetic acid, and make up to volume with purified water. The volume ratio of ethanol, trifluoroacetic acid and water is 7:1:2.

[0202] Preparation of standard solution: Accurately weigh an appropriate amount of Triton X-100 standard, dilute with blank solution to make standard solutions with concentrations of 20ppm, 40ppm, 60ppm, 80ppm, and 100ppm. The chromatogram of the standard solution with a concentration of 20ppm is as follows: Fig.11 shown.

[0203] Preparation of test solution: Accurately weigh an appropriate amount of decellularized matrix and put it into the blank solution. The mass ratio of decellularized matrix to blank solution is 1:5, and extract for 4 hours.

[0204] Setting of HPLC detection conditions:

[0205] The chromatographic column is an octadecylsilane bonded silica gel column (C18 column), 4.6 mm×250 mm, 5 μm; the mobile phase includes mobile phase A and mobile phase B, mobile phase A is an ethanol solution containing 0.1% (V / V) trifluoroacetic acid, mobile phase B is purified water, and the volume ratio of A to B is 80:20; the detector is an ultraviolet detector with a detection wavelength of 210 nm; the flow rate is 1.0 mL / min; and the column temperature is 30°C.

[0206] Detection: Accurately measure 10 μL of blank solution, standard solution and test solution, inject into liquid chromatograph, record the peak area, use external standard method, and calculate according to the following formula:

[0207]

[0208] Where: A 供 —Peak area of ​​the test solution; A 标 —Peak area of ​​standard solution; C 标—Concentration of standard solution, ppm; W 供 —mass of the test sample, g; V—volume of blank solution, mL.

[0209] Combination Fig.11 The test results are shown in Table 21 and Table 22. The linear fitting R 2 =0.9873, indicating that the linear fitting degree of the detection result is not high, and the recovery rate is between 97.54% and 98.84%. The accuracy and precision of the detection result are lower than those of the detection result using the liquid chromatography detection method of the present invention.

[0210] Table 21 Comparative Example 4 Peak Area and Linear Relationship Table

[0211]

[0212] Table 22 Comparative Example 4 Accuracy Results

[0213]

[0214] 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 aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A liquid chromatography detection method for Triton X-100 residue, characterized in that: High performance liquid chromatography was used as the detection system, and the chromatographic conditions included: The steps include: Step (1) Preparation of blank solution: Take isopropanol and trifluoroacetic acid, and dilute to volume with water to obtain a blank solution; Step (2) Preparation of standard solution and test solution: taking a Triton X-100 standard, using the blank solution to make up the volume and diluting into standard solutions of different concentrations; placing the sample to be tested into the blank solution for extraction to obtain a test solution; The setting of the chromatographic conditions in step (3) includes: The chromatographic column is an octadecylsilane bonded silica gel column; Mobile phase: comprising mobile phase A and mobile phase B, wherein mobile phase A is an isopropanol solution containing trifluoroacetic acid, and mobile phase B is water; the volume percentage of the trifluoroacetic acid is 0.08% to 0.12%; the volume ratio of the mobile phase A to the mobile phase B is 70:30-95:5; The chromatographic conditions of step (3) are also set as follows: the detector is an ultraviolet detector, the detection wavelength is 200-235nm; the flow rate is 0.8-1.2mL / min; the column temperature is 25-40°C; Step (4) detection method: take the blank solution, the standard solution and the test solution and inject them into a liquid chromatograph respectively to detect the residual amount of Triton X-100 in the sample to be tested; the sample to be tested is a decellularized matrix.

2. The liquid chromatography detection method according to claim 1, characterized in that: The volume percentage of the trifluoroacetic acid is 0.1%.

3. The liquid chromatography detection method according to claim 1, characterized in that: The volume ratio of the mobile phase A to the mobile phase B is 80:20 or 90:

10.

4. The liquid chromatography detection method according to any one of claims 1 to 3, characterized in that: The steps include: The detection method of step (4) is specifically as follows: inject the blank solution, the standard solution and the test solution into a liquid chromatograph respectively, record the peak area, and calculate the residual amount of Triton X-100 in the sample to be tested using an external standard method.

5. The liquid chromatography detection method according to claim 1, characterized in that: In step (1), in the blank solution, the volume ratio of isopropanol, trifluoroacetic acid and water is (6-8): (1-2): (1-2).

6. The liquid chromatography detection method according to claim 5, characterized in that: In step (1), in the blank solution, the volume ratio of isopropanol, trifluoroacetic acid and water is 7:1:

2.

7. The liquid chromatography detection method according to claim 1, characterized in that: In step (2), the concentration of the standard solution is 20-100 ppm; and / or the weight ratio of the sample to be tested to the blank solution is 1:(4-6); and / or the extraction time is 1-4 h.

8. The liquid chromatography detection method according to claim 7, characterized in that: The weight ratio of the sample to be tested to the blank solution is 1:5; and / or the extraction time is 4 hours.

9. The liquid chromatography detection method according to claim 1, characterized in that: The setting of the chromatographic conditions in step (3) also includes: the detector is an ultraviolet detector, and the detection wavelength is 200nm, 210nm, 230nm or 235nm.

10. The liquid chromatography detection method according to claim 1, characterized in that: The flow rate was 1.0 mL / min; the column temperature was 30°C.

11. The liquid chromatography detection method according to claim 1, characterized in that: The specifications of the chromatographic column are 4.6 mm×250 mm, 5 μm.

12. The liquid chromatography detection method according to claim 4, characterized in that: The formula for calculating the residual amount of Triton X-100 in the sample to be tested is as follows: Where: A 供 —Peak area of ​​the test solution; A 标 —Peak area of ​​standard solution; C 标 —Concentration of standard solution, ppm; W 供 —mass of the test sample, g; V—volume of blank solution, mL.

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