CN114354817A - Method for measuring residual quantity of 4, 4' -diaminodiphenylmethane in hemodialyzer - Google Patents
Method for measuring residual quantity of 4, 4' -diaminodiphenylmethane in hemodialyzer Download PDFInfo
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Abstract
The invention discloses a method for measuring the residual quantity of 4, 4' -diaminodiphenylmethane in a hemodialyzer, which comprises the following steps: step 1: carrying out limit leaching on 4, 4' -diaminodiphenylmethane in a sample to be tested by adopting methanol to obtain a leaching solution or carrying out nitrogen-blown concentration on the obtained leaching solution, carrying out ultrasonic constant volume to 1-2mL by using methanol, and filtering by using a hydrophobic membrane to obtain a sample test solution of the sample to be tested; step 2: preparing 4, 4' -diaminodiphenylmethane methanol standard solution, combining a high performance liquid chromatography-PDA detector, testing the standard solution by adopting the chromatographic conditions of gradient elution of anhydrous methanol, phosphate buffer solution and deionized water, and establishing a standard curve equation of chromatographic peak area to concentration; and step 3: and (3) determining the leaching liquor or the test liquor of the sample to be determined by adopting the chromatographic conditions same as those in the step (2), substituting the peak area of the determined chromatographic peak into a standard curve equation to calculate the concentration, further obtaining the content of 4,4 '-diaminodiphenylmethane in the test liquor or the leaching liquor of the sample to be determined, and finally determining the residual amount of the 4, 4' -diaminodiphenylmethane in the sample to be determined.
Description
Technical Field
The invention relates to the technical field of chromatographic analysis, in particular to a method for measuring the residual quantity of 4, 4' -diaminodiphenylmethane in a hemodialyzer.
Background
The hemodialyzer mainly comprises a shell, hollow fibers, an end cover and sealing glue, wherein the sealing glue is mainly made of polyurethane glue. Diphenylmethane 4, 4' -diisocyanate reactive monomers are usually left in polyurethane adhesives, and these substances are toxic, can cause denaturation of human proteins, and have irritation to the skin, eyes and mucous membranes. In hemodialysis, residual monomers of diphenylmethane 4,4 '-diisocyanate can be rapidly degraded with water in blood to 4, 4' -diaminodiphenylmethane, which is classified as 2B by the international agency for research on cancer (IARC), i.e., has a possibility of carcinogenesis.
Currently, only the residual of diphenylmethane 4,4 ' -diisocyanate is considered when evaluating hemodialyzers, and some standards (YY/T1639) -2018), literatures, patents (CN 110887904A) and the like also only give a detection method of the residual monomer of diphenylmethane 4,4 ' -diisocyanate, and assume that the diphenylmethane 4,4 ' -diisocyanate is completely converted into 4,4 ' -diaminodiphenylmethane, so as to evaluate the safety of the residual diphenylmethane 4,4 ' -diaminodiphenylmethane to the use of instruments. The method is obviously incomplete because the hemodialysis machine needs to be hydraulically checked for leakage in the production process, the residual monomer of the diphenylmethane 4,4 '-diisocyanate in the sealing glue is inevitably degraded into the 4, 4' -diaminodiphenylmethane when being contacted with water, and the factors such as transportation, use environment and the like are inevitably influenced by water vapor. Therefore, it is necessary to consider the residual amount of 4, 4' -diaminodiphenylmethane in the safety evaluation of hemodialyzers.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a method for measuring the residual quantity of 4, 4' -diaminodiphenylmethane in a hemodialyzer, which has the following specific technical scheme:
a method for determining the residual amount of 4, 4' -diaminodiphenylmethane in a hemodialyzer comprises the following steps:
step 1: carrying out limit leaching on 4,4 ' -diaminodiphenylmethane in a sample to be tested by adopting methanol to obtain a leaching solution or carrying out nitrogen-blown concentration on the obtained leaching solution, carrying out ultrasonic constant volume to 1-2mL by using methanol, and filtering by using a hydrophobic membrane to obtain a sample test solution of the sample to be tested, wherein the residual quantity of the 4,4 ' -diaminodiphenylmethane in the sample to be tested is the sum of the content of the 4,4 ' -diaminodiphenylmethane in the test solution or the leaching solution of each limit leaching. The purpose of this patent is that the volume of remaining of 4,4 '-diaminodiphenylmethane in the sample that detects, so final foothold still adjusts the volume of remaining of 4, 4' -diaminodiphenylmethane in the survey sample by force. In detail, the hemodialysis machine is subjected to limit leaching by using methanol, and the total amount of 4, 4' -diaminodiphenylmethane content in each leaching is recorded as the residual amount of a target substance in the device; after the concentration is carried out by a nitrogen blowing method, methanol is subjected to constant volume of 1-2mL, and the main reason is to ensure that the concentration of a target substance in the liquid to be detected is within the concentration range of a standard working curve and ensure the accuracy of a detection result; the purpose of leaching in this step is to fully extract the 4, 4' -diaminodiphenylmethane residue in the hemodialyzer, and the data obtained can be used for related toxicological safety assessments, product supervision, and the like. Therefore, the limit extraction method is selected, namely, the content of the target substance is not higher than 10% of the content of the target substance extracted in the first time.
Step 2: preparing 4, 4' -diaminodiphenylmethane methanol standard solution, combining a high performance liquid chromatography-PDA detector, testing the standard solution by adopting the chromatographic conditions of gradient elution of anhydrous methanol, phosphate buffer solution and deionized water, and establishing a standard curve equation of chromatographic peak area to concentration;
and step 3: and (3) determining the leaching liquor or the test liquor of the sample to be determined by adopting the chromatographic conditions same as those in the step (2), substituting the peak area of the determined chromatographic peak into a standard curve equation to calculate the concentration, further obtaining the content of 4,4 '-diaminodiphenylmethane in the test liquor or the leaching liquor of the sample to be determined, and finally determining the residual amount of the 4, 4' -diaminodiphenylmethane in the sample to be determined.
Preferably, a 4, 4' -diaminodiphenylmethane methanol standard is formulated at a concentration of 0.0 μ g/mL to 2.0 μ g/mL in step 2.
When methanol is adopted for carrying out limit extraction in the step 1, a hemodialyzer, a brown glass bottle and a peristaltic pump are connected in series by a Y-shaped glass tube and a silicone rubber tube, after the peristaltic pump is started, the Y-shaped glass tube, the silicone rubber tube and the hemodialyzer are ensured to be filled with the methanol, and the brown glass bottle is placed in a constant-temperature water bath at the temperature of 37 +/-1 ℃.
Preferably, the pore size of the hydrophobic membrane in step 1 is not more than 0.45 μm.
The phosphate buffer solution in the step 2 is prepared by dissolving ammonium dihydrogen phosphate and disodium hydrogen phosphate in water according to the mass ratio of 575:700, diluting, fixing the volume and adjusting the pH value to 6.9-7.0.
Further, the chromatographic conditions of the combination of the high performance liquid chromatography-PDA detector in step 2 are as follows: a chromatographic column SunFieC 18 or an equivalent chromatographic column, wherein the column temperature is 35-45 ℃; mobile phase: methanol, phosphate buffer solution and deionized water, wherein the flow rate is 0.8-1.0 mL/min; the sample injection volume is 10-20 μ L, and the detection wavelength range is 240-245 nm.
Furthermore, the chromatographic condition of gradient elution is selected when the high performance liquid chromatography-PDA detector combined technology is adopted for testing.
When the detection concentration of the 4, 4' -diaminodiphenylmethane in the primary leaching solution in the step 1 is higher than the 10 times of the quantitative limit of the method, nitrogen-blowing concentration operation on the leaching solution is not needed. This is because the concentration of the target substance in the sample leach liquor is sufficiently high that, when the limit leach is performed, if the target substance is not detected in the next leach liquor, the LOQ is used to calculate the target substance content to ensure that it is not more than 10% of the target substance content of the first leach, which meets the requirements of the limit leach.
And (3) refrigerating, shading and drying the standard solution in the step 2.
Preferably, a concentration interval with a standard working curve concentration range of about 0.0 mu g/mL-2.0 mu g/mL is configured.
The method uses methanol to leach the hemodialyzer, and adopts a high performance liquid chromatography-PDA detector combined technology to further determine the residual amount of the 4, 4' -diaminodiphenylmethane in the hemodialyzer. The invention provides reasonable reference for the research and development work of the polyurethane adhesive for the products of the hemodialysis machine of the sealing adhesive, and provides important basis for the detection and safety evaluation of the instruments.
The core of the detection method is the design of the limit extraction experiment and the setting of chromatographic detection conditions. Firstly, methanol is used as a conventional reagent, has the advantages of low price, low toxicity, easy operation and the like, and 4, 4' -diaminodiphenylmethane is very easy to dissolve in methanol. Therefore, the invention selects methanol to effectively extract the 4, 4' -diaminodiphenylmethane in the hemodialyzer. The method uses cheap, common and low-toxicity methanol as an extraction solvent to determine the residual amount of 4, 4' -diaminodiphenylmethane in a hemodialyzer, and has simple and convenient operation method, high accuracy and high sensitivity; meanwhile, in order to effectively separate the chromatographic peaks of the target substance and the interfering impurities, the invention uses the chromatographic conditions of gradient elution and is matched with a PDA detector (namely a photodiode matrix detector), so that the 4, 4' -diaminodiphenylmethane in the leaching solution can be accurately and efficiently measured. The verification proves that the method can be used for measuring the residual quantity of the 4, 4' -diaminodiphenylmethane in the hemodialyzer, and provides important basis for the research and development, supervision, quality control and related safety evaluation of the appliance products.
The invention aims to determine the residual quantity of 4,4 '-diaminodiphenylmethane in a dialyzer, and provides a method for detecting the residual quantity of 4, 4' -diaminodiphenylmethane in a hemodialyzer.
In summary, compared with the prior art, the invention has the following beneficial technical effects:
the method for detecting the residual quantity of the 4,4 '-diaminodiphenylmethane in the hemodialyzer is provided for the first time, the cheap, common and low-toxicity methanol is used as an extraction solvent, the residual quantity of the 4, 4' -diaminodiphenylmethane in the hemodialyzer can be effectively extracted, and the method is innovative.
The method can be used for measuring the residue of 4,4 '-diaminodiphenylmethane in the hemodialyzer, fills in the blank of the method for measuring the residue of 4, 4' -diaminodiphenylmethane in the hemodialyzer, provides important basis for safety evaluation and supervision of toxic and harmful substances in the instruments, and has practicability.
The method uses a high performance liquid chromatography-PDA detector combined technology, adopts the chromatographic condition of gradient elution, can effectively separate impurities and target substances in the sample, and has the advantages of high accuracy and sensitivity, simple operation and low cost.
Drawings
FIG. 1 is a schematic view of a hemodialyzer sample leaching test apparatus in accordance with embodiment 1 of the present invention;
FIG. 2 is an HPLC chromatogram of a standard solution of 4, 4' -diaminodiphenylmethane in example 1 of the present invention;
FIG. 3 is an HPLC chromatogram of a sample blank control solution and a sample leaching solution in example 1 of the present invention;
FIG. 4 is a standard curve of peak area-concentration of chromatographic peak of 4, 4' -diaminodiphenylmethane standard solution measured in example 1 of the present invention.
Wherein 1, a constant temperature water bath kettle; 2-a peristaltic pump; 3-Y type glass tube and silicon rubber tube.
Detailed Description
The following detailed description and specific examples are given for clarity in understanding the principles and technical solutions of the present invention. The following examples are described as only a part of the examples of the present invention, not all of the examples, and are only illustrative of the present invention and should not be construed as limiting the scope of the claims of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention. The examples were carried out under the conventional conditions or conditions recommended by the manufacturer, unless otherwise specified. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1
The materials, reagents and instrumentation used in this example were as follows:
materials: disposable hemodialyzer for adults
Reagent: 4, 4' -diaminodiphenylmethane standard, methanol, secondary water (self-made) in an analytical laboratory, ammonium dihydrogen phosphate, disodium hydrogen phosphate and ultra-pure nitrogen.
The instrument equipment comprises: high performance liquid chromatography (Waters), photodiode matrix detector (PDA) (Waters), SunFireC18(5 μm, 4.6mm × 250mm) chromatography column (Waters), peristaltic pump, nitrogen blower, ultrasonic cleaner, electric thermostat water bath, glass test tube with plug scale, pipette, volumetric flask (brown), pH meter.
The detection method of the embodiment comprises the following steps:
1. firstly, solution preparation is carried out: preparing a standard solution, namely accurately weighing 0.1016g of 4,4 ' -diaminodiphenylmethane standard substance into a 100mL brown volumetric flask, dissolving and diluting the 4,4 ' -diaminodiphenylmethane standard substance by using methanol, diluting the solution to a constant volume to obtain 1016 mu g/mL of standard stock solution, diluting the standard stock solution step by step to obtain 4,4 ' -diaminodiphenylmethane methanol standard solutions with the concentrations of 0.00, 0.10, 0.20, 0.51, 1.02 and 2.04 mu g/mL, refrigerating, keeping out of the sun, and drying for storage;
2. then preparing a sample leaching solution: taking a hemodialyzer sample, connecting the hemodialyzer, a brown glass bottle and a peristaltic pump in series by using a shortest silicone rubber tube according to the connection mode shown in figure 1, adding a certain volume of methanol into the brown glass bottle, setting the temperature of a constant-temperature water bath to be 37 +/-1 ℃, placing the brown glass bottle filled with the methanol into the constant-temperature water bath, starting the peristaltic pump, ensuring that the pipelines and the devices are full of the methanol, and performing circulating extraction. According to the clinical use condition of the hemodialyzer, the leaching time is 4h each time, and a sample leaching solution is obtained. No sample was leached during the process as a blank test. And repeating the leaching steps for multiple times by adopting a limit leaching method until the leaching is finished when the content of the target substance in the subsequent leaching is not higher than 10% of the content of the target substance in the first leaching.
3. And preparing a sample test solution, accurately measuring 10mL of the sample leaching solution and the sample blank control solution obtained in the previous step by using a 10mL pipette, concentrating by using a nitrogen blowing instrument at 40 ℃, ultrasonically measuring the volume to 2mL by using methanol, filtering by using a 0.45-micrometer hydrophobic filter membrane, and measuring.
4. Setting chromatographic conditions:
column SunFieC 18(5 μm, 4.6 mm. times.250 mm) or equivalent, column temperature 40 deg.C; mobile phase: methanol, phosphate buffer solution and deionized water, the elution gradient is shown in table 1, and the flow rate is 1.0 mL/min; the sample volume is 10 μ L, and the detection wavelength is 240 nm.
Table 1 mobile phase elution gradient in example 1
| Time/min | Methanol/% of | Phosphate buffer solution/%) | De-ionized water/%) |
| 0 | 25 | 75 | 0 |
| 22 | 55 | 45 | 0 |
| 23 | 0 | 0 | 100 |
| 28 | 0 | 0 | 100 |
| 29 | 25 | 75 | 0 |
| 37 | 25 | 75 | 0 |
Wherein, the phosphate buffer solution is prepared as follows: 0.575g of ammonium dihydrogen phosphate and 0.700g of disodium hydrogen phosphate are weighed, dissolved by water, diluted to a constant volume of 1L, and the pH is adjusted to 6.9-7.0.
And (3) testing the 4,4 '-diaminodiphenylmethane methanol standard solution which is prepared in the first step and has the concentration of 0.00 mu g/mL-2.04 mu g/mL under the chromatographic conditions, establishing a standard curve equation of chromatographic peak area to standard solution concentration, testing the sample test solution and the leaching solution, substituting the determined chromatographic peak area into the standard curve equation to obtain the concentration, and further calculating the content of 4, 4' -diaminodiphenylmethane.
Specifically, the determination of sample leaching solution and test solution
1) Determination of sample leach liquor
According to the set chromatographic conditions, adopting a high performance liquid chromatography-PDA detector combined technology to measure the blank reference solution and the sample leaching solution to obtain the peak area and the peak emergence time of the chromatographic peak of a target substance in the leaching solution, and carrying out qualitative analysis by combining with special attribute verification in methodology verification; bringing the peak area of a chromatographic peak of a target substance in the leaching solution into a corresponding standard curve equation, calculating the concentration of the target substance, and calculating the content according to the detection concentration of the target substance;
the calculation formula of the content of the target substance is as follows: qn is cn × Vn, wherein Q is the content of the target substance, n is the number of times of leaching, c is the measured concentration of the target substance, and V is the volume of the obtained leaching solution;
2) measurement of sample test solution
According to the set chromatographic conditions, adopting a high performance liquid chromatography-PDA detector combined technology to measure the sample test solution to obtain the peak area of the chromatographic peak of the target substance in the test solution; bringing the peak area of the chromatographic peak of the target substance in the test solution into a corresponding standard curve equation, calculating the concentration of the target substance, and calculating the content of the target substance according to the detection concentration of the target substance in the test solution;
the calculation formula of the content of the target substance is as follows:
wherein Q is the content of the target substance, n is the leaching frequency, c is the measured concentration of the target substance, V1 is the volume of the sample test solution, V2 is the volume of the leaching solution, and V3 is the volume measured by the liquid pipe before nitrogen-blowing concentration;
taking 3 hemodialyzer samples of the same type as the hemodialyzer samples to carry out parallel tests, wherein the detection concentration of 4, 4' -diaminodiphenylmethane in the first leaching liquor of the samples is lower than the method quantitative limit (0.055 mu g/mL), and the sample leaching liquor is prepared into sample test solution for determination. According to the steps, the total amount of the 4,4 '-diaminodiphenylmethane content detected each time is recorded as the residual amount of the target substance in the device, and finally the residual amount of the 4, 4' -diaminodiphenylmethane in the hemodialyzer of the model is measured to be 60.2-66.5 mu g/device.
The method is verified in the following aspects:
(a) the specificity is as follows: measuring the 4,4 '-diaminodiphenylmethane standard solution according to chromatographic conditions to obtain an HPLC chromatogram of the 4, 4' -diaminodiphenylmethane standard solution, which is shown in figure 2; sampling blank reference solution and leaching solution, and determining according to chromatographic conditions to obtain HPLC chromatogram of the blank reference solution and the leaching solution, as shown in FIG. 3. Determining the peak appearance of 4, 4' -diaminodiphenylmethane in the sample leaching solution according to the peak appearance time, finding that the peak appearance of the target substance is intact and the separation degree of the adjacent chromatographic peak is more than 1.5, and the blank reference solution has no interference to the chromatographic peak of the target substance, thereby indicating that the method meets the detection requirements.
(b) The linear relationship is: and (3) according to the set chromatographic conditions, measuring the 4, 4' -diaminodiphenylmethane standard solution with each concentration by adopting a high performance liquid chromatography-PDA detector combined technology. Drawing a standard curve of peak area-concentration of 4, 4' -diaminodiphenylmethane chromatographic peak in the concentration range of 0.00-2.04 mu g/mL, and establishing a linear regression equation: 74485X-226.31, R2See fig. 4 for 0.9997, indicating that the process of the invention is linear.
(c) Precision: taking 4,4 ' -diaminodiphenylmethane standard solutions with high, medium and low 3 different levels of concentration, and parallelly measuring the peak area of 4,4 ' -diaminodiphenylmethane chromatographic peak for 6 times according to the set chromatographic conditions, wherein the RSD% of the peak area of the 4,4 ' -diaminodiphenylmethane chromatographic peak is 0.73% -5.96% (see table 2). The recovery rate test is used for measuring the recovery rate of 4, 4' -diaminodiphenylmethane at different levels of high, medium and low concentrations with high precision, each standard addition level concentration is measured in parallel for 3 times, and the relative standard deviation RSD is 0.76-1.21% (see table 3), which shows that the method of the invention has good precision.
Table 2 verification data on precision of the measurement method of example 1
(d) Sensitivity: taking 4, 4' -diaminodiphenylmethane standard solution with minimum concentration of 0.10 mu g/mL, carrying out HPLC test according to chromatographic conditions, and determining the concentration for 7 times. The detection limit is according to the formula: LOD ═ t (n-1,1- α ═ 0.99) × S calculated, where S is the standard deviation of the samples, t (n-1,1- α ═ 0.99) is the Students t value with 99% confidence, with 3.3 times LOD as the limit of quantitation (LOQ). The detection limit of the method for determining 4, 4' -diaminodiphenylmethane is 0.017 mu g/mL, and the quantitative limit is 0.055 mu g/mL.
(e) Accuracy: the accuracy is verified by adopting a leaching liquor labeling recovery test, and the direct labeling recovery verification of the leaching liquor and the labeling recovery verification of the leaching liquid nitrogen blowing and concentration to be tested liquid can be divided into the direct labeling recovery verification of the leaching liquor and the labeling recovery verification of the leaching liquid nitrogen blowing and concentration according to whether the content of the target substance in the first leaching liquor is higher than the method quantitative limit of 10 times: direct labeling, recycling and verifying of the leaching liquor: taking 4 parts of leaching solution, adding methanol into one part of leaching solution, respectively adding 4,4 '-diaminodiphenylmethane standard solution with known content into the other 3 parts of leaching solution, preparing 4, 4' -diaminodiphenylmethane standard-adding recovery solution with high, medium and low concentrations according to the method, and measuring according to chromatographic conditions; and (3) standard addition recovery verification of the test solution obtained by blowing and concentrating leaching liquid nitrogen into test solution: taking 4 parts of leaching solution, adding 3 parts of standard solution of 4,4 '-diaminodiphenylmethane with known content of methanol into the leaching solution, preparing high, medium and low concentration 4, 4' -diaminodiphenylmethane standard-adding recovery solution according to the preparation method of the sample test solution, and determining according to chromatographic conditions. Through determination, the method detection limit that the detection concentration of the 4, 4' -diaminodiphenylmethane in the sample primary leaching solution is lower than 10 times is determined, the recovery rate of the obtained method is 90.8-96.6% according to the standard addition recovery verification method that the leaching liquid nitrogen is blown and concentrated into the test solution, and the verification data is shown in table 3.
Table 3 verification data on recovery rate of the measurement method of example 1
Proved by methodology, the method has high accuracy and sensitivity, can efficiently determine the 4, 4' -diaminodiphenylmethane residue in the hemodialyzer, and has great method advantages.
Example 2
The same method for obtaining the sample leaching solution, the test solution and the blank control solution as in example 1 was used, and the same method for preparing the standard solution of the target substance was used. The difference is the detection chromatographic conditions. Chromatographic conditions in this example: column sunfirmec 18(5 μm, 4.6mm × 250mm) or equivalent column, column temperature: 40 ℃; mobile phase: methanol, phosphate buffer and deionized water (elution gradient see table 4), flow rate 1.0 mL/min; the sample introduction volume is 10 mu L, and the detection wavelength range is 242 nm.
Table 4 mobile phase elution gradient in example 2
Proved by methodology, the method has excellent specificity, high accuracy and precision, good linear relation and sensitivity meeting the detection requirement, and related methodology parameters are shown in table 5:
table 5 verification parameters for the determination of example 2
Example 3
The same method for obtaining the sample leaching solution, the test solution and the blank control solution as in example 1 was used, and the same method for preparing the standard solution of the target substance was used. The difference lies in the different detection chromatographic conditions: column sunfirmec 18(5 μm, 4.6mm × 250mm) or equivalent column, column temperature: 35 ℃; mobile phase: methanol, phosphate buffer and deionized water (elution gradient see table 6), flow rate 0.8 mL/min; sample introduction volume is 15 muL, and detection wavelength range is 245 nm.
Table 6 mobile phase elution gradient in example 3
Proved by methodology, the method has excellent specificity, high accuracy and precision, good linear relation and sensitivity meeting the detection requirement, and the related methodology parameters are shown in Table 7:
table 7 verification parameters for the determination of example 3
Example 4
The same procedure for obtaining the sample leaching solution and the blank control solution of example 1 was followed, the same procedure for preparing the standard solution of the target substance was followed, and the same conditions for testing all chromatographic conditions were followed. The difference lies in that the sample leaching liquor has the first detection concentration higher than the method quantitative limit of 10 times, and the test and the methodological inspection are directly carried out according to the detection method and the methodological verification of the leaching liquor without preparing a sample test solution.
Proved by methodology, the method has excellent specificity, high precision, good linear relation and higher recovery rate, the sensitivity meets the detection requirement, and the related methodology parameters are shown in Table 7:
table 7 verification parameters for the determination of the method of example 4
In each of the above embodiments, the emphasis is placed on the differences from the other embodiments, and the same or similar parts in the respective embodiments may be mutually referred to.
Finally, the above embodiments are only for illustrating the technical principles of the present invention, and not for limiting the same, so that those skilled in the art can understand or realize the present invention. It is obvious that those skilled in the art can make various modifications to the technical solutions in the above embodiments, and can make equivalent substitutions for part or all of the technical features, and these modifications or substitutions do not depart from the spirit or scope of the present invention, and are within the protection scope of the technical solutions in the embodiments of the present invention.
Claims (8)
1. A method for determining the residual amount of 4, 4' -diaminodiphenylmethane in a hemodialyzer is characterized by comprising the following steps:
step 1: carrying out limit leaching on 4, 4' -diaminodiphenylmethane in a sample to be tested by adopting methanol to obtain a leaching solution or carrying out nitrogen-blown concentration on the obtained leaching solution, carrying out ultrasonic constant volume to 1-2mL by using methanol, and filtering by using a hydrophobic membrane to obtain a sample test solution of the sample to be tested;
step 2: preparing 4, 4' -diaminodiphenylmethane methanol standard solution, combining a high performance liquid chromatography-PDA detector, testing the standard solution by adopting the chromatographic conditions of gradient elution of anhydrous methanol, phosphate buffer solution and deionized water, and establishing a standard curve equation of chromatographic peak area to concentration;
and step 3: and (3) determining the leaching liquor or the test liquor of the sample to be determined by adopting the chromatographic conditions same as those in the step (2), substituting the peak area of the determined chromatographic peak into a standard curve equation to calculate the concentration, further obtaining the content of 4,4 '-diaminodiphenylmethane in the test liquor or the leaching liquor of the sample to be determined, and finally determining the residual amount of the 4, 4' -diaminodiphenylmethane in the sample to be determined.
2. The method according to claim 1, wherein the critical extraction with methanol in step 1 is carried out by connecting a hemodialyzer, a brown glass bottle and a peristaltic pump in series with a Y-shaped glass tube and a silicone rubber tube, wherein after the peristaltic pump is started, the Y-shaped glass tube, the silicone rubber tube and the hemodialyzer are filled with methanol, and the brown glass bottle is placed in a thermostatic water bath at 37 ± 1 ℃.
3. The method for determining the residual amount of 4, 4' -diaminodiphenylmethane in a hemodialyzer according to claim 1, characterized in that the pore size of the hydrophobic membrane in step 1 is not more than 0.45 μm.
4. The method according to claim 1, wherein the phosphate buffer in step 2 is prepared from ammonium dihydrogen phosphate and disodium hydrogen phosphate in a mass ratio of 575:700 by dissolving in water, diluting and fixing the volume, and adjusting the pH to 6.9-7.0.
5. The method for determining the residual amount of 4, 4' -diaminodiphenylmethane in a hemodialyzer according to claim 1 or 4, characterized in that the chromatographic conditions for the HPLC-PDA detector combination in step 2 are: a chromatographic column SunFieC 18 or an equivalent chromatographic column, wherein the column temperature is 35-45 ℃; mobile phase: methanol, phosphate buffer solution and deionized water, wherein the flow rate is 0.8-1.0 mL/min; the sample injection volume is 10-20 μ L, and the detection wavelength range is 240-245 nm.
6. The method of claim 5, wherein the HPLC-PDA detector combination uses gradient elution chromatography.
7. The method of claim 1, wherein the concentration of 4, 4' -diaminodiphenylmethane in the first leach solution is higher than 10 times the limit of quantitation in the method in step 1, and the nitrogen-blowing concentration operation is not required.
8. The method for determining the residual amount of 4,4 '-diaminodiphenylmethane in a hemodialyzer according to claim 1, characterized in that the 4, 4' -diaminodiphenylmethane standard solution in step 2 should be refrigerated, protected from light, and stored dry.
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