阅读说明：本技术 血液透析器中双酚a残留的测定方法 (Method for measuring bisphenol A residue in hemodialyzer ) 是由 郭利娟 孙兴霞 许凯 刘爱娟 陈方 刘栋 胡建宁 于 2018-09-11 设计创作，主要内容包括：本发明提供了血液透析器中双酚A残留的测定方法。测定方法为：用有机溶剂浸提血液透析器中的双酚A,得到样品供试液；采用高效液相色谱法检测样品供试液中的双酚A的含量,进而计算出残留量；色谱柱C18柱,流速0.8～1.7mL/min,激发波长230-234nm,发射波长310-320nm,柱温20～35℃,进样量5～15μL,乙腈与缓冲溶液以30～90:20～70的体积比梯度洗脱,所述缓冲溶液为pH为3.8～4.2的无机盐缓冲液。本发明能准确、灵敏、高效地测定血液透析器中的双酚A残留量,这对于血液透析器的研究开发,质量控制及产品监管都具有重要的意义。(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.)

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

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.

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.