阅读说明：本技术 血液透析器中n-甲基吡咯烷酮、聚维酮k30残留的测定方法 (Method for measuring residues of N-methyl pyrrolidone and povidone K30 in hemodialyzer ) 是由 沈永� 郭利娟 吴长岩 杜福映 孟凯 刘斌 高晋权 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N-甲基吡咯烷酮、聚维酮K30残留的测定方法。血液透析器中N-甲基吡咯烷酮、聚维酮K30残留的测定方法为：用水浸提血液透析器中的目标物,得到样品供试液；采用高效液相色谱法检测所述样品供试液中的目标物的含量,进而计算出血液透析器中目标物的残留量；所述高效液相色谱法的色谱条件为：色谱柱C18柱,流速0.3～0.7mL/min,检测波长190～210nm,柱温20～30℃,进样量50～100μL。本发明能准确、灵敏、高效地测定血液透析器中的N-甲基吡咯烷酮、聚维酮K30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 residues of N-methylpyrrolidone and povidone K30 in a hemodialyzer. The method for measuring the residues of N-methylpyrrolidone and povidone K30 in the hemodialyzer comprises the following steps: leaching a target object in a hemodialyzer by using water to obtain a sample test solution; detecting the content of the target in the sample test solution by adopting a high performance liquid chromatography, and further calculating the residual quantity of the target in a hemodialyzer; the chromatographic conditions of the high performance liquid chromatography are as follows: a chromatographic column C18, the flow rate is 0.3-0.7 mL/min, the detection wavelength is 190-210 nm, the column temperature is 20-30 ℃, and the sample injection amount is 50-100 mu L. The method can accurately, sensitively and efficiently determine the residual quantity of the N-methylpyrrolidone and the povidone K30 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 residues of N-methylpyrrolidone and povidone K30 in the hemodialyzer is characterized by comprising the following steps of:

leaching N-methyl pyrrolidone and povidone K30 in a hemodialyzer by using water to obtain a sample test solution;

detecting the content of N-methylpyrrolidone and povidone K30 in the sample test solution by adopting a high performance liquid chromatography, and further calculating the residual quantity of N-methylpyrrolidone and povidone K30 in a hemodialyzer;

the chromatographic conditions of the high performance liquid chromatography are as follows:

a chromatographic column: a column of C18 is arranged on the column,

flow rate: 0.3 to 0.7mL/min,

detection wavelength: the particle size of the nano-particles is 190-210 nm,

column temperature: 20 to 30 ℃,

sample introduction amount: 50 to 100 mu L of the composition,

mobile phase: the acetonitrile and the buffer solution are in a volume ratio of 1-5: 95-99, and the buffer solution is a phosphate buffer solution with the pH value of 2.5 +/-0.1.

2. An assay as claimed in claim 1, wherein the method of leaching is:

and circulating the blood chamber and the dialysate chamber of the hemodialyzer by using water, and taking out circulating liquid after circulating for preset time, wherein the circulating liquid is sample test liquid.

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 method according to claim 2, wherein the water used in the circulation is water distilled three or more times.

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 N-methylpyrrolidone, povidone K30 in the hemodialyzer, a blank test solution is also subtracted; 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 water, wherein the flow rate and the temperature of flushing are the same as the circulating conditions for obtaining the sample test solution.

9. The assay according to any one of claims 1 to 8, wherein the buffer solution consists of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, phosphoric 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 0.5-0.7 mL/min,

preferably, the detection wavelength is: the particle size of the particles is 200-210 nm,

preferably, the column temperature is: at the temperature of 20-25 ℃,

preferably, the sample volume: 80-100 μ L of the composition is added,

preferably, the mobile phase: the acetonitrile and the buffer solution are mixed according to a volume ratio of 3-5: 95-97.

Technical Field

The invention relates to the field of analytical chemistry, in particular to a method for determining residues of N-methylpyrrolidone and povidone K30 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 hemodialysis machine uses a processing aid N-methyl pyrrolidone and an additive povidone K30 at a dialysis membrane part in the production process. Both N-methylpyrrolidone and povidone K30 are readily soluble in water. In clinical use, there is a risk of residual N-methylpyrrolidone and povidone K30 in the hemodialyzer leaching into the blood. The chronic action of N-methylpyrrolidone is reported to cause the functional damage of the central nervous system, so that the pathological changes of respiratory organs, kidneys and vascular systems are caused. Residual N-methylpyrrolidone in the hemodialyzer carries an obvious safety risk.

Disclosure of Invention

The invention aims to provide a method for measuring residues of N-methylpyrrolidone and povidone K30 in a hemodialyzer, which can accurately, sensitively and efficiently measure the residues of N-methylpyrrolidone and povidone K30 in the hemodialyzer.

In order to achieve the above purpose, the invention provides the following technical scheme:

the method for measuring the residue of N-methylpyrrolidone and povidone K30 in the hemodialyzer comprises the following steps:

leaching N-methyl pyrrolidone and povidone K30 in a hemodialyzer by using water to obtain a sample test solution;

detecting the content of N-methylpyrrolidone and povidone K30 in the sample test solution by adopting a high performance liquid chromatography, and further calculating the residual quantity of N-methylpyrrolidone and povidone K30 in a hemodialyzer;

the chromatographic conditions of the high performance liquid chromatography are as follows:

a chromatographic column: a column of C18 is arranged on the column,

flow rate: 0.3 to 0.7mL/min,

detection wavelength: the particle size of the nano-particles is 190-210 nm,

column temperature: 20 to 30 ℃,

sample introduction amount: 50 to 100 mu L of the composition,

mobile phase: the acetonitrile and the buffer solution are in a volume ratio of 1-5: 95-99, and the buffer solution is a phosphate buffer solution with the pH value of 2.5 +/-0.1.

The method solves the problem that the residues of N-methylpyrrolidone and povidone K30 in the hemodialyzer cannot be detected simultaneously.

The core of the determination method lies in chromatographic conditions, N-methylpyrrolidone and povidone K30 can be effectively separated by adopting a specific mobile phase and a specific stationary phase under a certain wavelength, and the separation degree of the N-methylpyrrolidone and povidone K30 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 0.3 to 0.7mL/min, for example, 0.3mL/min, 0.4mL/min, 0.5mL/min, 0.6mL/min, 0.7mL/min, etc.

In the invention, the detection wavelength can be arbitrarily selected within the range of 190-210 nm, such as 190nm, 195nm, 200nm, 205nm, 210nm and the like.

In the present invention, the column temperature can be arbitrarily selected from the range of 20 to 30 ℃ such as 20 ℃, 22 ℃, 25 ℃, 27 ℃, 29 ℃, 30 ℃ and the like.

In the present invention, the amount of sample may be arbitrarily selected from the range of 50 to 100. mu.L, for example, 50. mu.L, 60. mu.L, 70. mu.L, 80. mu.L, 90. mu.L, 100. mu.L, etc.

In the mobile phase, acetonitrile and a buffer solution are prepared in any volume ratio within the range of 1-5: 95-99, such as 1:99, 3:97, 4:96, 5:95 and the like.

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 0.5-0.7 mL/min,

preferably, the detection wavelength is: the particle size of the particles is 200-210 nm,

preferably, the column temperature is: at the temperature of 20-25 ℃,

preferably, the sample volume: 80-100 μ L of the composition is added,

preferably, the mobile phase: the acetonitrile and the buffer solution are mixed according to a volume ratio of 3-5: 95-97.

Preferably, the buffer solution is composed of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, phosphoric acid and water, and taking a buffer solution with a constant volume of 1L as an example, the preparation process is as follows:

2.085g of monopotassium phosphate and 0.2175g of anhydrous dipotassium phosphate are precisely weighed into a 1L volumetric flask, dissolved and diluted by water, 1.0mL of phosphoric acid is added, and the volume is determined to the scale by water.

In the invention, the purpose of leaching is to fully extract the residual N-methyl pyrrolidone and povidone K30 at the dialysis membrane part so as to detect the residual quantity and provide accurate data for safety monitoring of a hemodialyzer. Therefore, it is preferred to simulate the working conditions of a hemodialyzer to leach N-methylpyrrolidone, povidone K30, as detailed below.

And circulating the blood chamber and the dialysate chamber of the hemodialyzer by using water, 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 N-methyl pyrrolidone and the povidone K30 residue to human bodies.

Wherein, the circulation time is not suitable to be too long or too short, the two compounds cannot be fully extracted by too short, the time and energy are wasted by too long, and the circulation is preferably carried out for more than 5 hours after screening.

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 and reduce the influence of impurities on the measurement, the circulating water is preferably tertiary water.

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 N-methylpyrrolidone and the povidone K30 in the hemodialyzer is calculated, blank test solutions are 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 water, wherein the flushing conditions (including temperature, flow rate and the like) are the same as the leaching conditions for obtaining the sample test solution.

Preferably, the amount of the circulated water 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.

Considering the sample acquisition method comprehensively, more preferred chromatographic conditions are as follows:

a chromatographic column: a C18 column with a length of 150mm, an inner diameter of 4.6mm, a diameter of 3.5 μm or equivalent;

flow rate: 0.5 mL/min;

detection wavelength: 200 nm;

column temperature: 25 ℃;

sample introduction amount: 100 uL;

mobile phase: acetonitrile: preparing a buffer solution, namely 3: 97: 2.085g of monopotassium phosphate and 0.2175g of anhydrous dipotassium phosphate are precisely weighed into a 1L volumetric flask, dissolved and diluted by water, 1.0mL of phosphoric acid is added, and the volume is determined to the scale by water.

When the method provided by the invention is used for detecting the residual quantity of N-methylpyrrolidone and povidone K30 in the hemodialyzer, the concentration range has certain influence on the accuracy when a standard curve is drawn.

It is preferred to draw a curve for a standard sample at a concentration in the interval 0.5. mu.g/mL to 30. mu.g/mL, for example 0.5. mu.g/mL to 20. mu.g/mL, for N-methylpyrrolidone (NMP).

For povidone K30(PVP), a standard sample with a concentration in the interval of 0.5 to 100. mu.g/mL is preferably chosen to plot, for example, 0.5 to 80. mu.g/mL.

In summary, compared with the prior art, the invention achieves the following technical effects:

(1) the method can efficiently, accurately and sensitively determine the residues of the N-methylpyrrolidone and the povidone K30 in the hemodialyzer, and solves the problems of sampling and simultaneous detection of two compounds.

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 water flow diagram of a pre-flush hemodialyzer as provided in example 1 of the present invention;

FIG. 2 is a water flow diagram for obtaining a sample test solution according to example 1 of the present invention;

FIG. 3 is a chromatogram of a sample test solution provided in example 1;

FIG. 4 is a high performance liquid chromatogram of a standard solution of N-methylpyrrolidone as provided in example 1;

FIG. 5 is a high performance liquid chromatogram of a standard solution of povidone K30 provided in example 1;

FIG. 6 is a chromatogram of a blank test solution 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.