CN110887903A - Method for measuring bisphenol A residue in hemodialyzer - Google Patents
Method for measuring bisphenol A residue in hemodialyzer Download PDFInfo
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- CN110887903A CN110887903A CN201811057196.2A CN201811057196A CN110887903A CN 110887903 A CN110887903 A CN 110887903A CN 201811057196 A CN201811057196 A CN 201811057196A CN 110887903 A CN110887903 A CN 110887903A
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- IISBACLAFKSPIT-UHFFFAOYSA-N bisphenol A Chemical group C=1C=C(O)C=CC=1C(C)(C)C1=CC=C(O)C=C1 IISBACLAFKSPIT-UHFFFAOYSA-N 0.000 title claims abstract description 107
- 238000000034 method Methods 0.000 title claims abstract description 43
- 239000012085 test solution Substances 0.000 claims abstract description 31
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 27
- 239000007853 buffer solution Substances 0.000 claims abstract description 19
- 239000003960 organic solvent Substances 0.000 claims abstract description 14
- 230000005284 excitation Effects 0.000 claims abstract description 10
- 238000002386 leaching Methods 0.000 claims abstract description 9
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 5
- 229910017053 inorganic salt Inorganic materials 0.000 claims abstract description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 39
- 239000007788 liquid Substances 0.000 claims description 20
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 15
- 238000010828 elution Methods 0.000 claims description 14
- 238000012360 testing method Methods 0.000 claims description 12
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 9
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 9
- 239000008280 blood Substances 0.000 claims description 9
- 210000004369 blood Anatomy 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 238000011010 flushing procedure Methods 0.000 claims description 8
- 238000001514 detection method Methods 0.000 claims description 7
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 5
- 239000005695 Ammonium acetate Substances 0.000 claims description 5
- 229940043376 ammonium acetate Drugs 0.000 claims description 5
- 235000019257 ammonium acetate Nutrition 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 4
- 229920001296 polysiloxane Polymers 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims 5
- 230000001351 cycling effect Effects 0.000 claims 1
- 238000002390 rotary evaporation Methods 0.000 claims 1
- 229920002379 silicone rubber Polymers 0.000 claims 1
- 238000002347 injection Methods 0.000 abstract description 2
- 239000007924 injection Substances 0.000 abstract description 2
- 238000003908 quality control method Methods 0.000 abstract description 2
- 238000012827 research and development Methods 0.000 abstract description 2
- 229940106691 bisphenol a Drugs 0.000 description 44
- 239000000243 solution Substances 0.000 description 13
- 238000011084 recovery Methods 0.000 description 10
- 239000012086 standard solution Substances 0.000 description 10
- 238000002360 preparation method Methods 0.000 description 7
- 239000000178 monomer Substances 0.000 description 5
- 238000005259 measurement Methods 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 238000010025 steaming Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000012417 linear regression Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 2
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000010812 external standard method Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 238000001631 haemodialysis Methods 0.000 description 2
- 230000000322 hemodialysis Effects 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000004451 qualitative analysis Methods 0.000 description 2
- 238000005096 rolling process Methods 0.000 description 2
- 229910052710 silicon Inorganic materials 0.000 description 2
- 239000010703 silicon Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 208000009304 Acute Kidney Injury Diseases 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 208000033626 Renal failure acute Diseases 0.000 description 1
- 206010074268 Reproductive toxicity Diseases 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 201000011040 acute kidney failure Diseases 0.000 description 1
- 208000012998 acute renal failure Diseases 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 208000020832 chronic kidney disease Diseases 0.000 description 1
- 208000022831 chronic renal failure syndrome Diseases 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
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- 230000007674 genetic toxicity Effects 0.000 description 1
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- 239000012510 hollow fiber Substances 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
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- 238000012544 monitoring process Methods 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
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- 230000007696 reproductive toxicity Effects 0.000 description 1
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- 238000011160 research Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—Temperature
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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Abstract
The invention provides a method for measuring bisphenol A residue in a hemodialyzer. The determination method comprises the following steps: leaching bisphenol A in a hemodialyzer by using an organic solvent to obtain a sample test solution; detecting the content of bisphenol A in a sample test solution by adopting a high performance liquid chromatography, and further calculating the residual quantity; a chromatographic column C18 with a flow rate of 0.8-1.7 mL/min, an excitation wavelength of 230-234nm, an emission wavelength of 310-320nm, a column temperature of 20-35 ℃, a sample injection amount of 5-15 muL, and acetonitrile and a buffer solution which are inorganic salt buffer solution with a pH value of 3.8-4.2, wherein the volume ratio of acetonitrile to buffer solution is 30-90: 20-70. The invention can accurately, sensitively and efficiently determine the bisphenol A residual quantity in the hemodialyzer, and has important significance for research and development, quality control and product supervision of the hemodialyzer.
Description
Technical Field
The invention relates to the field of analytical chemistry, in particular to a method for determining bisphenol A in a hemodialyzer.
Background
The hemodialyzer is one kind of medical equipment for treating acute and chronic renal failure and consists of hollow fiber, casing, sealing layer and end caps. The casing and the end cover of the hemodialyzer are made of polycarbonate, and the main synthetic raw material of the hemodialyzer is bisphenol A. Bisphenol A is easily soluble in organic solvents such as methanol, ethanol, acetone, toluene and the like, and is hardly soluble in water. When the hemodialyzer is clinically used, the end cover and the shell of the hemodialyzer are in direct and indirect contact with blood, and residual bisphenol A monomer in the product can be dissolved out or migrated to the blood. According to the toxicological research of bisphenol A at home and abroad, the bisphenol A has certain reproductive toxicity and genetic toxicity to mice and lowers the immune defense function of the mice. The residual bisphenol A monomer in the hemodialyzer has obvious safety risk, so that it is very necessary to control the residual amount of bisphenol A monomer.
In the prior art, many methods for detecting bisphenol A exist, but no relevant report is provided for detecting bisphenol A residue in a hemodialyzer.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method for measuring bisphenol A residue in a hemodialyzer, which can accurately, sensitively and efficiently measure the bisphenol A residue in the hemodialyzer.
In order to achieve the above purpose, the invention provides the following technical scheme:
a method for determining bisphenol A residual in a hemodialyzer, comprising the steps of:
leaching bisphenol A in a hemodialyzer by using an organic solvent to obtain a sample test solution;
detecting the content of bisphenol A in the sample test solution by using a high performance liquid chromatography-fluorescence detector, and further calculating the residual amount of bisphenol A in a hemodialyzer;
the chromatographic conditions for detection are as follows:
a chromatographic column: a column of C18 is arranged on the column,
flow rate: 0.8 to 1.7mL/min,
the excitation wavelength 230-234nm, the emission wavelength 310-320nm,
column temperature: 20 to 35 ℃,
sample introduction amount: 5 to 15 mu L of the composition,
mobile phase: the acetonitrile and the buffer solution are eluted in a gradient mode according to the volume ratio of 30-90: 20-70, preferably 31-80: 20-69, wherein the buffer solution is an inorganic salt buffer solution with the pH value of 3.8-4.2, preferably the pH value of 4-4.1.
The core of the determination method is chromatographic conditions, a high performance liquid chromatography-fluorescence detector is adopted, and a specific mobile phase and a specific stationary phase are adopted, so that the bisphenol A can be effectively separated from the leaching liquor, and the separation degree of a target object and an adjacent chromatographic peak is ensured to be more than 1.5, so that the accuracy, precision and efficiency of the detection method are improved, the requirements on specificity, linearity, accuracy, precision and the like in the verification process of the method are met, and the method has important significance on research and development, quality control and product supervision of a hemodialyzer.
In the present invention, the flow rate can be arbitrarily selected from the range of 0.8 to 1.7mL/min, for example, 0.8mL/min, 1.0mL/min, 1.5mL/min, 1.6mL/min, 1.7mL/min, etc.
In the present invention, the excitation wavelength can be selected arbitrarily within the range of 230-234nm, such as 230nm, 231nm, 232nm, 233nm, 234nm, etc., and the emission wavelength can be selected arbitrarily within the range of 310-320nm, such as 310nm, 312nm, 314nm, 316nm, 318nm, 320nm, etc.
In the present invention, the column temperature can be arbitrarily selected from the range of 20 to 35 ℃ such as 20 ℃, 22 ℃, 25 ℃, 27 ℃, 29 ℃, 30 ℃, 32 ℃, 35 ℃ and the like.
In the present invention, the amount of sample can be arbitrarily selected from the range of 5 to 15. mu.L, for example, 5. mu.L, 7. mu.L, 8. mu.L, 10. mu.L, 15. mu.L, and the like.
The invention adopts gradient elution, the initial volume ratio of acetonitrile/buffer solution can be 30:70, or 31:69, or 40:60, etc.; the end-point volume ratio may be 80:20, or 85:15, or 70:30, or 90:10, etc. The flow rates at different gradients may be the same or different.
Within the above range, preferable ranges of the respective parameters are as follows.
Preferably, the high performance liquid chromatography column is a 150mm x 4.6mm x 3.5 μm C18 column or equivalent;
preferably, the flow rate is 1.0-1.5 mL/min,
preferably, the excitation wavelength is 232-,
preferably, the column temperature is: 25 to 30 ℃,
preferably, the sample volume: 8 to 10 mu L of the composition,
preferably, the gradient elution is performed according to table 1:
TABLE 1 gradient elution
| Time/min | Acetonitrile/%) | Buffer solution/%) |
| 0 | 31 | 69 |
| 25 | 31 | 69 |
| 27 | 80 | 20 |
| 32 | 80 | 20 |
| 34 | 31 | 69 |
| 42 | 31 | 69 |
Preferably, the buffer solution is composed of ammonium acetate, acetic acid and water, taking a buffer solution with a constant volume of 1L as an example, the preparation process is as follows:
1.54g of ammonium acetate is precisely weighed in a 1L volumetric flask, dissolved, diluted and subjected to constant volume by water, and the pH value is adjusted to a preset value by acetic acid.
In the invention, the purpose of leaching is to fully extract the bisphenol A remained on the end cover and the shell part, further detect the residual quantity and provide accurate data for safety monitoring of the hemodialyzer, therefore, the working condition of the hemodialyzer is preferably simulated to leach the bisphenol A, which is as follows.
And circulating the blood chamber and the dialysate chamber of the hemodialyzer by using an organic solvent, and taking out circulating liquid after circulating for preset time, wherein the circulating liquid is sample test liquid.
The method simulates the clinical hemodialysis process, and can fully examine the harm of the bisphenol A residue to human bodies.
Wherein, the circulation time is not suitable to be too long or too short, the bisphenol A can not be fully leached out, and the circulation time is preferably more than 5 hours after screening, because of too long time, time and energy are wasted.
The circulating temperature and flow rate preferably simulate the clinical hemodialysis conditions, namely the circulating temperature is 35-38 ℃, and preferably 37-38 ℃;
preferably, the flow rate of the circulation is 150-250 mL/min, preferably 150-200 mL/min.
In order to improve the detection accuracy, the organic solvent used for the circulation is preferably methanol, ethanol, acetone or toluene, preferably methanol.
At the same time, the hemodialyzer is preferably pre-flushed before being recirculated;
the pre-flushing method comprises the following steps: the silicone tube is respectively connected with the blood chamber and the dialysate chamber of the hemodialyzer to form two independent circulating systems, and the two independent circulating systems are respectively flushed by water.
In addition, when the residual quantity of the bisphenol A in the hemodialyzer is calculated, blank test solution is preferably deducted so as to improve the accuracy of the experiment; the blank test solution is obtained by the following method: and connecting the silicon tubes into a circulating system, and flushing the circulating system by using an organic solvent, wherein the flushing condition is the same as the leaching condition for obtaining the sample test solution. The organic solvent used here is the same as that used for obtaining the sample test solution.
Preferably, the amount of the recycled organic solvent is 400-600 mL, preferably 500mL, so as to ensure that the concentration of the target object is large enough to be accurately detected. The volume is usually up to 600mL before final injection.
In addition, in order to improve the sensitivity of the test solution, after obtaining the sample test solution and the blank test solution, it is recommended to take part of the solution to be rotary evaporated to dryness, then re-dissolved with the same organic solvent, and filtered with a filter (usually with a 0.45 μm filter).
Considering the sample acquisition method, the preferred chromatographic conditions are as follows:
a chromatographic column: a C18 column with a length of 150mm, an inner diameter of 4.6mm, 3.5um or equivalent;
flow rate: 1.0-1.5 mL/min;
excitation wavelength: emission wavelength at 232 nm: 314 nm;
column temperature: 30 ℃;
sample introduction amount: 10 mu L of the solution;
mobile phase: gradient elution was performed as per table 2.
TABLE 2 gradient elution
| Time/min | Acetonitrile/%) | Buffer solution/%) |
| 0 | 31 | 69 |
| 25 | 31 | 69 |
| 27 | 80 | 20 |
| 32 | 80 | 20 |
| 34 | 31 | 69 |
| 42 | 31 | 69 |
When the method of the invention is used for detecting the residual quantity of bisphenol A in the hemodialyzer, the concentration range has certain influence on the accuracy when a standard curve is drawn.
Preferably, a standard sample with a concentration ranging from about 50ng/mL to 10000ng/mL is selected to draw a curve, for example, from 50ng/mL to 1020 ng/mL.
In summary, compared with the prior art, the invention achieves the following technical effects:
(1) the method of the invention can efficiently, accurately and sensitively determine the residue of bisphenol A in the hemodialyzer, and solves the problems of sampling and high-efficiency separation.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a circuit diagram of a pre-flush hemodialyzer according to example 1 of the present invention;
FIG. 2 is a schematic diagram of a cycle for obtaining a sample test solution according to example 1 of the present invention;
FIG. 3 is a comparison of HPLC of bisphenol A standard and test sample provided in example 1;
FIG. 4 is a blank solvent high performance liquid chromatogram provided in example 1.
Detailed Description
The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and the detailed description, but those skilled in the art will understand that the following described embodiments are some, not all, of the embodiments of the present invention, and are only used for illustrating the present invention, and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1
Materials and reagents
Materials: hemodialyzer (Xinhua medical devices, Inc.).
Reagent: methanol, acetic acid, ammonium acetate, acetonitrile, bisphenol-a, tertiary water, hplc (ex agilent), XBridge RP C18 column (ex Waters).
Secondly, the detection method steps
(1) Solution preparation: the following solutions were required in the preparation of the experimental procedure
a. Standard solution preparation
Precisely weighing 5.12mg of bisphenol A standard substance in a 50mL brown volumetric flask, dissolving with methanol, diluting, and fixing volume to obtain a standard stock solution with a concentration of 102.4 mug/mL, and preparing standard solutions with concentrations of 51.2, 102.4, 204.8, 409.6 and 1024ng/mL respectively by a stepwise dilution method.
b. Preparation of buffer solution
1.54g of ammonium acetate is precisely weighed into a 1L volumetric flask, dissolved, diluted and subjected to constant volume by using water, and the pH value is adjusted to 4.0 by using acetic acid.
(2) Chromatographic conditions are as follows: determining the following chromatographic conditions according to the requirements of the test solution
c. A chromatographic column: a C18 column with a length of 150mm, an inner diameter of 4.6mm, 3.5um or equivalent;
d. flow rate: see table 3;
e. excitation wavelength: 232nm, emission wavelength: 314 nm;
f. column temperature: 30 ℃;
g. sample introduction amount: 10 μ L
h. Mobile phase: a, acetonitrile; b buffer solution, elution gradient is shown in Table 3.
TABLE 3 elution conditions
(3) Pre-washing a test sample: pre-flushing a hemodialyzer according to a diagram of figure 1, connecting a blood side and a dialysate side of a product of the hemodialyzer with 1 glass beaker by using a silicon hose to form 2 respectively independent circulating systems, adding 1000mL of three times of water into each of the 2 glass beakers, filling the whole closed-circuit system by using a rolling pump, flushing the blood side and the dialysate side of the hemodialyzer for 10 minutes at a flow rate of 200mL/min, and blowing air to remove the liquid in the hemodialyzer as much as possible.
(4) Preparing a sample test solution and a blank test solution: and (3) circulating the blood chamber and the dialysate chamber of the pre-flushed dialyzer at 37 +/-1 ℃ at the flow rate of 200mL/min by using 500mL of methanol through a rolling pump in a mode shown in figure 2, taking out circulating liquid after circulating for 5 hours, and metering the volume to 600mL by using the methanol to obtain sample test solution for later use. And (3) forming a circulating system by using 500mL of methanol through a silica gel tube, circulating at 37 +/-1 ℃ at the flow rate of 200mL/min, taking out circulating liquid after circulating for 5 hours, and metering the volume to 600mL by using the methanol to obtain blank test solution.
(5) And (3) measuring a sample test solution and a blank test solution: measuring 100mL of sample circulating liquid, rotary-steaming to dryness, precisely transferring 2.00mL of methanol dissolved rotary-steaming residue, filtering with 0.45 μm filter membrane to obtain sample liquid, and measuring according to the chromatographic condition (2);
(6) standard solution determination: taking a series of standard solutions with the concentrations of 51.2, 102.4, 204.8, 409.6 and 1024ng/mL, and determining according to the chromatographic condition (2);
(7) and (4) analyzing results: and (3) carrying out qualitative analysis by adopting retention time, and carrying out quantitative analysis by adopting an external standard method.
And (3) measuring the bisphenol A standard solution and the sample test solution according to the chromatographic condition (2), comparing the bisphenol A standard product with the high performance liquid chromatography of the test product (see figure 3), and knowing that the retention time of the bisphenol A and the bisphenol A standard product in the test product is basically consistent.
Quantitatively analyzing bisphenol A of a test sample of a hemodialyzer, taking 3 parts of the test sample, operating according to the steps (3), (4) and (5), calculating by a linear regression equation according to an external standard method, and measuring that the total residual quantity of bisphenol A in the test sample is 808.75 ng/set-863.96 ng/set.
Method verification
(1) The specificity is as follows: weighing 100mL of blank circulating liquid, rotary-steaming to dry, precisely transferring 2.00mL of methanol-dissolved rotary-steaming residue, filtering with 0.45 μm filter membrane to obtain blank test solution, measuring according to the chromatographic condition (2), and obtaining a blank high performance liquid chromatogram shown in figure 4. The qualitative analysis of the test sample is combined, the peak shape of the bisphenol A chromatographic peak is good, the separation degree of the bisphenol A chromatographic peak and the separation degree of the adjacent chromatographic peak are both greater than 1.5, and the blank solvent has no interference to the measurement of the bisphenol A, which indicates that the technical method meets the analysis requirement.
(2) Standard working solution linearity
Taking a series of standard solutions with the concentrations of 51.2, 102.4, 204.8, 409.6 and 1024ng/mL, determining according to the chromatographic condition (2), and drawing a standard working curve by taking the concentration of bisphenol A (ng/mL) as a horizontal coordinate and the peak area of a chromatographic peak of the bisphenol A as a vertical coordinate to obtain a linear regression equation (see Table 4).
TABLE 4 bisphenol A Standard solution-Peak area data sheet
| Concentration of Standard solution (ng/mL) | 51.2 | 102.4 | 204.8 | 409.6 | 1024 |
| Peak area | 2.7 | 6.1 | 12.3 | 25.7 | 66.1 |
The linear regression equation y is 0.0652x-0.7942, the correlation coefficient r is 0.9999, and bisphenol A has good linearity in the range of 51.2-1024 ng/mL.
(3) Precision (reproducibility): bisphenol A standard solutions with concentrations of 51.2, 204.8 and 1024ng/mL were taken, and the peak area was measured by performing the chromatographic conditions (2) 6 times (see Table 5). The results of 6 times of parallel measurement of the standard substance solution show that the relative standard deviation is 0.64-2.99 percent, which indicates that the method has good measurement precision of the standard substance. Meanwhile, the low, medium and high 3-level recovery rate precision measurement of bisphenol A is carried out by a 6.9 recovery rate test, each standard addition level is measured in parallel for 3 times, and the relative standard deviation is 2.15% -3.37%, which shows that the precision of the sample measured by the method is good.
TABLE 5 bisphenol A precision test results (n ═ 6)
| Number of times | 1 | 2 | 3 | 4 | 5 | 6 | Average peak area | RSD％ |
| Peak area (51.2ng/mL) | 2.5 | 2.5 | 2.5 | 2.6 | 2.6 | 2.4 | 2.52 | 2.99 |
| Peak area (204.8ng/mL) | 11.8 | 11.7 | 11.7 | 11.8 | 11.9 | 11.8 | 11.78 | 0.64 |
| Peak area (1024ng/mL) | 63.4 | 63.9 | 63.2 | 61.0 | 61.9 | 61.4 | 62.47 | 1.90 |
(4) And (3) recovery rate: the test solution is used as a substrate, the low, medium and high addition recovery rates and precision of 3 levels of bisphenol A are respectively measured, each addition standard level is measured in parallel for 3 times, and the method recovery rate and the Relative Standard Deviation (RSD) are shown in Table 6. The result shows that the average recovery rate of the bisphenol A is 96.94-113.44%, the relative standard deviation is 2.15-3.37%, and the accuracy of the method meets the analysis requirement.
Table 6 results of bisphenol a recovery and precision (n ═ 3)
| Standard concentration (ng/set) | Average recovery (%) and n-3 | RSD(％),n＝3 |
| 648 | 113.44 | 2.50 |
| 1228.8 | 107.42 | 2.15 |
| 2457.6 | 96.94 | 3.37 |
The data can confirm that after methanol tightening simulated leaching of the bisphenol A residual monomer in the sample to be tested of the hemodialyzer, the bisphenol A is separated by adopting a C18 liquid chromatographic column, and the high-efficiency liquid phase-fluorescence detection method is used for measuring, so that the bisphenol A residual monomer of the hemodialyzer can be accurately, sensitively and efficiently measured, and the method has great advantages.
Example 2
The method is the same as the method for obtaining the sample test solution and the blank test solution and the preparation of the standard solution in example 1, and the difference is that the chromatographic detection conditions are different:
a chromatographic column: c18 column with length 150mm, inner diameter 4.6mm, 3.5 um;
excitation wavelength 230nm, emission wavelength 310 nm;
column temperature: 20 ℃;
sample introduction amount: 5 mu L of the solution;
mobile phase: gradient elution is shown in table 7, and the buffer solution is the same as in example 1.
TABLE 7 gradient elution Table
The results show that the method also exhibits: the specificity is good, the specificity is strong, the accuracy is high, the precision and the linearity are good, the correlation coefficient r of the standard working curve is more than 0.999, the relative standard deviation RSD of the precision of the method is 0.84-3.96%, and the average recovery rate of low, medium and high concentrations is 94.92-115.51%.
Example 3
The method is the same as the method for obtaining the sample test solution and the blank test solution and the preparation of the standard solution in example 1, and the difference is that the chromatographic detection conditions are different:
a chromatographic column: c18 column with length 150mm, inner diameter 4.6mm, 3.5 um;
excitation wavelength 234nm and emission wavelength 320 nm;
column temperature: 25 ℃;
sample introduction amount: 15 mu L of the solution;
mobile phase: gradient elution is shown in table 8, and the buffer solution is the same as in example 1.
TABLE 8 gradient elution Table
The results show that the method also exhibits: the specificity is good, the specificity is strong, the accuracy is high, the precision and the linearity are good, the correlation systems r of the standard working curve are all larger than 0.999, the relative standard deviation RSD of the precision of the method is 0.84-4.02%, and the average recovery rate of low, medium and high concentrations is 98.71-118.22%.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. The method for measuring the bisphenol A residue in the hemodialyzer is characterized by comprising the following steps:
leaching bisphenol A in a hemodialyzer by using an organic solvent to obtain a sample test solution;
detecting the content of bisphenol A in the sample test solution by using a high performance liquid chromatography-fluorescence detector, and further calculating the residual amount of bisphenol A in a hemodialyzer;
the chromatographic conditions for detection are as follows:
a chromatographic column: a column of C18 is arranged on the column,
flow rate: 0.8 to 1.7mL/min,
the excitation wavelength 230-234nm, the emission wavelength 310-320nm,
column temperature: 20 to 35 ℃,
sample introduction amount: 5 to 15 mu L of the composition,
mobile phase: mixing acetonitrile and a buffer solution in a proportion of 30-90: gradient elution with a volume ratio of 20-70, preferably 31-80: 20-69, wherein the buffer solution is an inorganic salt buffer solution with a pH of 3.8-4.2, preferably a pH of 4-4.1.
2. An assay as claimed in claim 1, wherein the method of leaching is:
circulating between a blood chamber and a dialysate chamber of the hemodialyzer by using an organic solvent, and taking out circulating liquid after circulating for preset time, wherein the circulating liquid is sample test liquid;
preferably, after the withdrawal of the circulating liquid, rotary evaporation is carried out, followed by redissolution with the organic solvent.
3. The method of claim 2, wherein the cycle time is 5 hours or more.
4. The method of claim 2, wherein the temperature of the cycle is 35 to 38 ℃, preferably 37 to 38 ℃.
5. The method according to claim 2, wherein the flow rate of the circulation is 150 to 250mL/min, preferably 150 to 200 mL/min.
6. The assay according to claim 2, wherein the organic solvent is methanol, ethanol, acetone or toluene, preferably methanol.
7. The assay method according to claim 2, wherein prior to the cycling, the hemodialyzer is also pre-flushed;
the pre-flushing method comprises the following steps: the silicone tube is respectively connected with the blood chamber and the dialysate chamber of the hemodialyzer to form two independent circulating systems, and the two independent circulating systems are respectively flushed by water.
8. The assay method according to claim 7, wherein in calculating the residual amount of bisphenol A in the hemodialyzer, a blank test solution is also subtracted; the blank test solution is obtained by the following method: and connecting the silicon rubber tubes into a circulating system, and flushing the circulating system by using the organic solvent under the same flushing condition as the leaching condition for obtaining the sample test solution.
9. The assay according to any one of claims 1 to 8, wherein the buffer solution consists of ammonium acetate, acetic acid and water.
10. The method according to any one of claims 1 to 8, wherein the high performance liquid chromatography column is a 150mm x 4.6mm x 3.5 μm C18 column or an equivalent column;
preferably, the flow rate is 1.0-1.5 mL/min,
preferably, the excitation wavelength is 232-,
preferably, the column temperature is: 25 to 30 ℃,
preferably, the sample volume: 8 to 10 mu L of the composition,
preferably, the gradient elution is performed according to table 1:
TABLE 1 gradient elution
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| CN114354817A (en) * | 2022-01-24 | 2022-04-15 | 苏州苏大卫生与环境技术研究所有限公司 | Method for measuring residual quantity of 4, 4' -diaminodiphenylmethane in hemodialyzer |
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