阅读说明：本技术 血液透析器中二苯基甲烷-4,4′-二异氰酸酯残留的测定方法 (Method for determining diphenylmethane-4, 4' -diisocyanate residue in hemodialyzer ) 是由 郭利娟 沈永� 薄晓文 刘叶 赵长帅 秦洋 刘晓林 朱晓明 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二苯基甲烷-4,4′-二异氰酸酯残留的测定方法,为：将有机溶剂和衍生剂、内标物混合,对血液透析器密封层进行浸提,获得样品供试液；采用HPLC-荧光-内标法检测样品供试液中目标物含量,进而计算出残留量；激发波长250-260nm,发射波长410-415nm,乙腈、缓冲溶液和甲醇以30～100:0～60:0～15的体积比梯度洗脱,缓冲溶液pH为2.0～2.5,内标物为1,5-萘二异氰酸酯,衍生剂为9-N-甲氨甲基蒽。本发明能准确、灵敏、高效地测定血液透析器中的二苯基甲烷-4,4′-二异氰酸酯残留量,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 determining the residue of diphenylmethane-4, 4' -diisocyanate in a hemodialyzer, which comprises the following steps: mixing an organic solvent, a derivative and an internal standard substance, and leaching a sealing layer of a hemodialyzer to obtain a sample test solution; detecting the content of a target substance in a sample test solution by adopting an HPLC-fluorescence-internal standard method, and further calculating the residual quantity; the excitation wavelength is 250-260nm, the emission wavelength is 410-415nm, acetonitrile, a buffer solution and methanol are subjected to gradient elution according to the volume ratio of 30-100: 0-60: 0-15, the pH value of the buffer solution is 2.0-2.5, the internal standard substance is 1, 5-naphthalene diisocyanate, and the derivative agent is 9-N-methylaminomethylanthracene. The method can accurately, sensitively and efficiently determine the residual amount of the diphenylmethane-4, 4' -diisocyanate in the hemodialyzer, and has important significance for research and development, quality control and product supervision of the hemodialyzer.)

1. A method for measuring the residue of diphenylmethane-4, 4' -diisocyanate in a hemodialyzer, characterized by comprising the steps of:

mixing an organic solvent, a derivative and an internal standard substance, and leaching a sealing layer of a hemodialyzer to obtain a sample test solution; the internal standard substance is 1, 5-naphthalene diisocyanate, and the derivative agent is 9-N-methylaminomethylanthracene;

detecting the content of the diphenylmethane-4, 4 '-diisocyanate in the sample test solution by adopting a high performance liquid chromatography-fluorescence-internal standard method, and further calculating the residual amount of the diphenylmethane-4, 4' -diisocyanate 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: 1 to 2.5mL/min,

an excitation wavelength of 250-260nm, an emission wavelength of 410-415nm,

column temperature: 20 to 30 ℃,

sample introduction amount: 5 to 20 mu L of the composition,

mobile phase: the acetonitrile, the buffer solution and the methanol are eluted in a gradient mode according to the volume ratio of 30-100: 0-60: 0-15, preferably the volume ratio of 35-100: 0-55: 0-10, wherein the buffer solution is an inorganic salt buffer solution with the pH value of 2.0-2.5, preferably the pH value of 2-2.3;

and adding the derivatizing agent and the internal standard substance to the standard solution at the detection time.

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

mixing the organic solvent, the derivative and the internal standard substance, performing ultrasonic extraction on the sealing layer of the hemodialyzer for multiple times, and combining the extract to obtain the sample test solution.

3. The method according to claim 2, wherein after the combined leaching solutions are concentrated, the organic solvent is used for re-dissolving, and thus the sample test solution is obtained.

4. The assay of claim 1, wherein the derivatizing agent is added in an amount of 255 μ g or more, preferably 260 μ g or more, per gram of the sealing layer.

5. The assay according to claim 1, wherein the amount of the internal standard substance added per gram of the sealing layer is 0.9 μ g or more, preferably 0.93 μ g or more.

6. The method of claim 1, wherein the organic solvent is a halogenated alkane, preferably a C1-C3 halogenated alkane, preferably dichloromethane.

7. An assay method according to claim 1, wherein the sealing layer is chopped prior to the leaching.

8. The assay of claim 1, wherein the buffer solution consists of ammonium acetate, acetic acid and water.

9. 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.

10. The method according to any one of claims 1 to 8, wherein the flow rate under the chromatographic conditions is 1.5 to 2.0mL/min,

preferably, the excitation wavelength is 250-254nm, the emission wavelength is 410-412nm,

preferably, the column temperature is: 25 to 30 ℃,

preferably, the sample volume: 5 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 diphenylmethane-4, 4' -diisocyanate 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 two ends of the hemodialyzer are sealed by medical polyurethane adhesive and fixed with the shell. Diphenylmethane-4, 4-diisocyanate (MDI) or polymeric MDI is one of the main materials of medical polyurethane adhesives, and is commonly used in hemodialyzers. MDI has 2 hetero-accumulated double bonds and electron-withdrawing effect of aromatic ring, so that the MDI has very active chemical property and is very easy to react with other compounds containing active hydrogen atoms to generate a carbamate structure. When the hemodialyzer is used, the sealing layer is directly contacted with human blood, and free MDI in the sealing layer of the hemodialyzer reacts with protein containing amino, hydroxyl and sulfhydryl groups and peptide to generate different hapten-protein complexes or antigens, thereby inducing body sensitization reaction, damaging immune system and possibly generating genetic toxicity and reproductive toxicity. In addition, MDI is rapidly hydrolyzed to 4, 4-diaminodiphenylmethane (MDA) after contacting blood, and MDA has potential carcinogenicity. The MDI monomer of the seal layer of the hemodialyzer has a significant safety risk, so it is very necessary to control the MDI monomer residue. At present, no report on the determination of MDI monomer residue of a hemodialyzer exists.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a method for measuring the residual amount of diphenylmethane-4, 4 '-diisocyanate in a hemodialyzer, which can accurately, sensitively and efficiently measure the residual amount of the diphenylmethane-4, 4' -diisocyanate in the hemodialyzer.

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

a method for determining the residue of diphenylmethane-4, 4' -diisocyanate in a hemodialyzer, comprising the steps of:

mixing an organic solvent, a derivative and an internal standard substance, and leaching a sealing layer of a hemodialyzer to obtain a sample test solution; the internal standard substance is 1, 5-naphthalene diisocyanate, and the derivative agent is 9-N-methylaminomethylanthracene.

Detecting the content of the diphenylmethane-4, 4 '-diisocyanate in the sample test solution by adopting a high performance liquid chromatography-fluorescence-internal standard method, and further calculating the residual amount of the diphenylmethane-4, 4' -diisocyanate 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: 1 to 2.5mL/min,

an excitation wavelength of 250-260nm, an emission wavelength of 410-415nm,

column temperature: 20 to 30 ℃,

sample introduction amount: 3 to 10 mu L of the composition,

mobile phase: the acetonitrile, the buffer solution and the methanol are eluted in a gradient mode according to the volume ratio of 30-100: 0-60: 0-15, preferably the volume ratio of 35-100: 0-55: 0-10, wherein the buffer solution is an inorganic salt buffer solution with the pH value of 2.0-2.5, preferably the pH value of 2-2.3;

and adding the derivatizing agent to the standard solution at the time of detection.

The core of the assay method of the invention lies in the internal standard, derivatizing agent and chromatographic conditions. Compared with the traditional detection method, the sensitivity and the accuracy are higher when the internal standard substance and the derivative are used for detecting the diphenylmethane-4, 4' -diisocyanate.

According to the invention, 9-N-methylaminomethylanthracene is used as a derivative to protect a target object, and 1, 5-naphthalene diisocyanate is used as an internal standard substance to reduce the influence of errors on results and improve the recovery rate; the high performance liquid chromatography-fluorescence detector is adopted, and a specific mobile phase and a specific fixed phase are adopted to effectively separate the diphenylmethane-4, 4' -diisocyanate from the leaching liquor, and the separation degree of the derivative of the target object and the adjacent chromatographic peak is ensured to be more than 1.5, so that the accuracy, precision and high 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 for research and development, quality control and product supervision of a hemodialyzer.

In the present invention, the flow rate can be arbitrarily selected within the range of 1-2.5 mL/min, for example, 1mL/min, 1.2mL/min, 1.5mL/min, 2.0mL/min, 2.2mL/min, etc.

In the present invention, the excitation wavelength can be arbitrarily selected within the range of 250-260nm, such as 250nm, 251nm, 252nm, 255nm, 254nm, 255nm, 256nm, 257nm, 258nm, 259nm, etc., and the emission wavelength can be arbitrarily selected within the range of 410-415nm, such as 410nm, 412nm, 414nm, 415nm, etc.

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 can be arbitrarily selected from the range of 5 to 20. mu.L, for example, 5. mu.L, 6. mu.L, 8. mu.L, 10. mu.L, 15. mu.L, 18. mu.L, 20. mu.L, and the like.

The invention adopts gradient elution, and the initial volume ratio of acetonitrile/buffer solution can be 30:60:10, or 35:55:10, or 35:60:5, etc.; the end point volume ratio may be 30:60:10, or 35:55:10, etc.; the initial volume ratio is the same as the end volume and the flow rates at different gradients can 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 1.5-2.0 mL/min,

preferably, the excitation wavelength is 250-254nm, the emission wavelength is 410-412nm,

preferably, the column temperature is: 25 to 30 ℃,

preferably, the sample volume: 5 to 10 μ L.

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 into a 1L volumetric flask, dissolved, diluted and subjected to constant volume by water, and the pH is adjusted to 2.3 by acetic acid.

In the invention, the purpose of leaching is to fully extract the residual diphenylmethane-4, 4' -diisocyanate in the sealing layer of the hemodialyzer so as to detect the residual quantity and provide accurate data for the safety monitoring of the hemodialyzer. Therefore, leaching needs to be complete, and a limit leaching test needs to be carried out to determine conditions required for complete leaching, including feed-liquor ratio, time, repetition times and the like.

Preferably, the leaching method comprises the following steps:

mixing the organic solvent, the derivative and the internal standard substance, performing ultrasonic extraction on the sealing layer of the hemodialyzer for multiple times, and combining the extract to obtain the sample test solution.

Taking a 1g sample of the sealant layer as an example, the preferred leaching procedure is: precisely weighing 1g (accurate to 0.01g) of sample to be tested in a brown glass bottle, adding 10mL of dichloromethane and 1.0mL of derivative reagent, sealing, performing ultrasonic treatment at room temperature for 30min, placing the leaching solution in another bottle, washing the rest sample with dichloromethane twice, and combining the washing solution and the leaching solution. The remaining sample residue was derivatized by repeating the leaching as above. And (3) concentrating the nitrogen of the three-time leaching liquor to be dry, precisely adding 5.0mL of derivative solvent, carrying out ultrasonic dissolution, filtering with a 0.45-micron filter membrane, and measuring by adopting a high-performance liquid phase-fluorescence method.

Preferably, after said combining of the leach liquors, concentration is carried out, followed by re-dissolution with said organic solvent.

The above operation can enrich the target substance on the one hand and remove part of impurities on the other hand.

Preferably, the derivatizing agent is added in an amount of 255 μ g or more, preferably 260 μ g or more, per gram of the sealant layer.

Usually, the amount of the derivatizing agent is excessive, but too much is not good for accurate detection, and it is sufficient to add more than 255 μ g of derivatizing agent per gram of sealing layer, preferably 260 μ g, for a conventional hemodialyzer.

Also, preferably, the amount of the internal standard substance added per gram of the sealant layer is 0.9 μ g or more, preferably 0.93 μ g or more.

In order to improve the detection accuracy, the organic solvent is preferably halogenated alkane, preferably halogenated alkane of C1-C3, preferably dichloromethane.

Preferably, the seal layer is chopped prior to the leaching.

In addition, in order to reduce the interference of impurities with the measurement, it is recommended that, after the sample test solution is obtained, a portion of the solution is blown dry with liquid nitrogen and then redissolved with the same organic solvent, if necessary, by filtration through a filter (usually 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, a diameter of 3.5 μm or equivalent;

flow rate: 1.5-2.0 mL/min;

excitation wavelength: 254nm, emission wavelength: 412 nm;

column temperature: 25 ℃;

sample introduction amount: 5 mu L of the solution;

mobile phase: gradient elution was performed as per table 1.

TABLE 1 gradient elution

When the method of the invention is used for detecting the residual amount of the diphenylmethane-4, 4' -diisocyanate in the hemodialyzer, the concentration range has certain influence on the accuracy when a standard curve is drawn.

Preferably, a standard sample with the concentration of about 20 ng/mL-2000 ng/mL is selected to draw a curve.

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

(1) the method can efficiently, accurately and sensitively determine the residue of the diphenylmethane-4, 4' -diisocyanate 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 comparison of HPLC of the test sample and the standard sample provided in example 1;

FIG. 2 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.