Method for measuring the amount of fat-soluble vitamins immobilized

Abstract

To provide a method of accurately and efficiently measuring the amount of fat-soluble vitamins immobilized on an inner surface of a hollow fiber membrane of a blood purification module having the hollow fiber membrane therein.SOLUTION: Provided is a method of measuring an immobilized amount of fat-soluble vitamins for measuring the amount of fat-soluble vitamins immobilized on an inner surface of a hollow fiber membrane of a blood purification module including a hollow fiber membrane 14 containing fat-soluble vitamins and a container 18 having a blood inlet port 17 and a blood outlet port connected to the inside of the hollow fiber membrane, the method including: a step of feeding a solvent from the blood inlet port; a step of recovering the solvent discharged from the blood outlet port; and a step of measuring the amount of fat-soluble vitamins contained in the recovered solvent.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention primarily relates to a method for measuring the content of fat-soluble vitamins immobilized on the inner surface of a hollow fiber membrane contained in a blood purification module. [Background technology]

[0002] In recent years, blood purification therapy, which purifies bodily fluids such as blood and plasma, has become widespread and is being applied to the treatment of patients suffering from various diseases. Examples of blood purification devices using hollow fibers used in blood purification therapy include hemodialysis machines, hemodiafiltration machines, hemofilters, continuous hemofiltration (dialysis) machines, plasma separators, plasma component separators, ascites filters, ascites concentrators, and artificial lungs. These blood purification devices are being improved on a daily basis, with particular efforts being made to improve blood compatibility.

[0003] Taking hemodialysis as an example, long-term dialysis patients experience various complications such as dialysis amyloidosis and arteriosclerosis, and one contributing factor to their development is increased oxidative stress. Oxidative stress is a state in which oxidative capacity exceeds antioxidant capacity. In dialysis patients, oxidative stress is elevated due to increased free radical production by leukocytes activated by contact between the blood and the dialysis membrane, and a decrease in antioxidants in the blood, such as vitamin C and vitamin E. While the blood contains various antioxidants such as superoxide dismutase, uric acid, vitamin C, vitamin E, and carotenoids, a decrease in vitamin E concentration in the blood and red blood cell membranes has been reported in dialysis patients. It has been suggested that decreased vitamin E concentration leads to increased oxidation of lipids and a decrease in red blood cell lifespan, causing long-term complications in dialysis patients such as dialysis amyloidosis, arteriosclerotic diseases, increased malignant tumors, and anemia. Therefore, it has been pointed out that suppressing oxidative stress experienced by dialysis patients is likely to improve their quality of life (QOL) and prognosis.

[0004] Several methods can be considered to reduce oxidative stress experienced by dialysis patients. One such method involves using a hemodialysis membrane (hemodialyzer) with antioxidant properties, and numerous clinical cases have been reported. In recent years, hydrophobic synthetic polymers such as polysulfone polymers have become the main material for hemodialysis membranes due to their versatility as resins, heat and radiation resistance, and biocompatibility. However, if the hydrophobicity of the blood-contacting surface of the membrane is too strong, blood coagulation can occur. Therefore, membranes formed by polymer blends with hydrophilic polymers or surface modification with hydrophilic polymers are usually used (Patent Documents 1 and 2). Furthermore, it is known that hollow fiber membranes with immobilized lipid-soluble vitamins exhibit even better blood compatibility in hydrophobic polymer membranes with hydrophilic inner surfaces or the entire membrane.

[0005] It is known that as the fat-soluble vitamin content of hollow fiber membranes increases, cracks, regardless of size, are more likely to occur in the hollow fiber membrane during autoclave (AC) sterilization. Generally, a higher fat-soluble vitamin content leads to higher antioxidant capacity, but increasing the fat-soluble vitamin content to achieve sufficiently effective antioxidant capacity for practical use increases the likelihood of leakage in the hollow fiber membrane, indicating that there is a range of values ​​that are undesirable for practical purposes. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] HYPERLINK "javascript:void(0)" JP-A-6-238139 [Patent Document 2] HYPERLINK "javascript:void(0)" International Publication No. 98 / 52683 pamphlet [Patent Document 3] HYPERLINK "javascript:void(0)" JP-A-9-66225 [Patent Document 4] Japanese Patent Application Laid-Open No. 10-244000 [Patent Document 5] Japanese Patent Application Laid-Open No. 11-347117 [Summary of the Invention] [Problems to be Solved by the Invention]

[0007] As described above, since there is a practically suitable range for the content of fat-soluble vitamins, it is required to measure this accurately and efficiently. Conventional methods were useful for grasping the preferable amount of fat-soluble vitamins that does not cause leakage of the hollow fiber membrane because the content of fat-soluble vitamins is the extraction amount from the entire hollow fiber membrane. On the other hand, regarding the effects such as antioxidant ability expected for fat-soluble vitamins that come into contact with blood, if the amount present on the inner surface of the hollow fiber membrane can be measured more accurately than the amount of fat-soluble vitamins in the entire hollow fiber membrane, it can be grasped more precisely.

[0008] In view of the above circumstances, an object of the present invention is to provide a method for accurately and efficiently measuring the amount of fat-soluble vitamins immobilized on the inner surface of the hollow fiber membrane of a blood purification module having a hollow fiber membrane inside. [Means for Solving the Problems]

[0009] As a result of intensive studies to solve the above problems, the present inventors have found that in a blood purification module comprising a hollow fiber membrane containing fat-soluble vitamins and a container having a blood inlet port and a blood outlet port, a method having a step of feeding a solvent from the blood inlet port, a step of recovering the solvent discharged from the blood outlet port, and a step of measuring the fat-soluble vitamin content contained in the recovered solvent can accurately and efficiently measure the amount of fat-soluble vitamins immobilized on the inner surface of the hollow fiber membrane inside the blood purification module, and have completed the present invention.

[0010] That is, the present invention is as follows. [1] A method for measuring the amount of fat-soluble vitamins immobilized on the inner surface of a hollow fiber membrane in a blood purification module, comprising a hollow fiber membrane containing fat-soluble vitamins and a container having a blood inlet port and a blood outlet port connected to the inside of the hollow fiber membrane, The process of supplying a solvent through the aforementioned blood inlet port, A step of recovering the solvent discharged from the blood outlet port, The process includes a step of measuring the fat-soluble vitamin content contained in the recovered solvent. Method for measuring the amount of fat-soluble vitamins immobilized. [2] The method for measuring the amount of fat-soluble vitamins immobilized according to [1] above, wherein the solvent is an alcohol. [3] The method for measuring the amount of fat-soluble vitamins immobilized according to [2] above, wherein the alcohols are an aqueous solution of alcohol with an alcohol concentration of 75% (by volume) or higher. [4] A method for measuring the amount of fat-soluble vitamins immobilized according to any one of the above [1] to [3], wherein the amount of extract Y in the solvent recovery step satisfies the following formula (1). 0.1 × solvent flow rate (mL / min) + 5.2 ≤ extract volume Y (mL) (1) [5] A method for measuring the amount of fat-soluble vitamins immobilized according to any one of [1] to [4] above, wherein the amount of extract Y in the solvent recovery step satisfies the following formula (2). Extract volume Y (mL) ≤ {(k × membrane area)} 2 (m 2 ) / solvent flow rate (mL / min)) + 61} × 7.5 (2) (In equation (2), k = 800.) [6] A method for measuring the amount of fat-soluble vitamins immobilized according to any one of the above [1] to [4], wherein the amount of extract Y in the solvent recovery step satisfies the following formula (3). Extract volume Y (mL) ≤ {(k × membrane area)} 2 (m 2 ) / solvent flow rate (mL / min)) + 61} × 3.5 (3) (In equation (3), k = 800.) [7] A method for measuring the amount of fat-soluble vitamins immobilized according to any one of [1] to [4] above, wherein the amount of extract Y in the solvent recovery step satisfies the following formula (4). Extract volume Y (mL) ≤ {(k × membrane area)} 2 (m 2 ) / solvent flow rate (mL / min)) + 61} × 2.5 (4) (In equation (4), k = 800.) [8] A method for measuring the amount of fat-soluble vitamins immobilized according to any one of [1] to [4] above, wherein the amount of extract Y in the solvent recovery step satisfies the following formula (5). Extract volume Y (mL) ≤ {(k × membrane area)} 2 (m 2 ) / solvent flow rate (mL / min) + 61} × 2 (5) (In equation (5), k = 800.) [9] A method for measuring the amount of fat-soluble vitamins immobilized according to any one of [1] to [4] above, wherein the amount of extract Y in the solvent recovery step satisfies the following formula (6). Extract volume Y (mL) ≤ Solvent flow rate (mL / min) × 5 (6) [Effects of the Invention]

[0011] The method of the present invention makes it possible to accurately and efficiently measure the amount of lipid-soluble vitamins immobilized on the inner surface of the hollow fiber membrane in a blood purification module having a hollow fiber membrane inside. [Brief explanation of the drawing]

[0012] [Figure 1] This figure shows one embodiment of the blood purification module of the present invention. [Modes for carrying out the invention]

[0013] The following describes in detail an embodiment of the present invention (hereinafter referred to as "this embodiment"), but the present invention is not limited thereto, and various modifications are possible without departing from its essence.

[0014] The method for measuring the amount of fat-soluble vitamins immobilized in this embodiment is: A method for measuring the amount of fat-soluble vitamins immobilized on the inner surface of a hollow fiber membrane in a blood purification module, comprising a hollow fiber membrane containing fat-soluble vitamins and a container having a blood inlet port and a blood outlet port connected to the inside of the hollow fiber membrane, The process of supplying a solvent through the aforementioned blood inlet port, A step of recovering the solvent discharged from the blood outlet port, The process includes a step of measuring the fat-soluble vitamin content contained in the recovered solvent. This is a method for measuring the amount of fat-soluble vitamins immobilized.

[0015] [Blood purification module]

[0016] The blood purification module of this embodiment is not particularly limited, but comprises at least a hollow fiber membrane containing fat-soluble vitamins, and a container having a blood inlet port and a blood outlet port connected to the inside of the hollow fiber membrane.

[0017] For example, as shown in Figure 1, the blood purification module 10 consists of a container 13 having a dialysate flow channel inlet 11 and a dialysate flow channel outlet 12 near both ends, into which a bundle of hollow fiber membranes 14 is inserted, and then both ends of the container are sealed liquid-tightly with sealing parts 15 and 16. Headers 19 and 20, each equipped with a blood inlet port 17 and a blood outlet port 18, are attached to both ends of the container 13, respectively, and the headers 19 and 20 are fixed to the container 13. When processing bodily fluids, tubes 23 and 24 for connection to the human body are connected to the blood inlet port 17 and the blood outlet port 18. The materials for the outer cylinder and headers include polycarbonate and polypropylene. The material for the sealing parts includes polyurethane.

[0018] The blood purification module is divided into two compartments by a hollow fiber membrane and a sealing section. A blood flow path is formed by the space created by the lumen of the hollow fiber membrane, the sealing section, and the header, and a second flow path is formed by the outer surface of the hollow fiber membrane and the container. When the blood purification module is used, blood flows through the blood flow path and dialysate flows through the second flow path.

[0019] The method for measuring the amount of fat-soluble vitamins immobilized in this embodiment is a method for measuring the amount of fat-soluble vitamins immobilized on the inner surface of the hollow fiber membrane of a blood purification module. From the viewpoint of enhancing biocompatibility and antioxidant capacity, it is necessary to have a higher fat-soluble vitamin content on the blood flow path side of the hollow fiber than on the filtrate flow path side, in other words, near the inner surface of the hollow fiber. On the other hand, if the amount of fat-soluble vitamins immobilized near the inner surface is excessively high, the hydrophobicity of the inner surface may become too high, potentially stimulating the blood coagulation system. Therefore, it is important to accurately and efficiently measure whether the amount of fat-soluble vitamins immobilized on the inner surface of the hollow fiber membrane of a blood purification module is within an appropriate range. Here, the inner surface of the hollow fiber membrane of the blood purification module is not particularly limited as long as the functions such as antioxidant capacity of the solidified lipid-soluble vitamins are fully expressed, and refers to a region preferably from 1 to 1000 nm, more preferably from 1 to 500 nm, and even more preferably from 1 to 5 nm, from the membrane surface of the hollow fiber membrane lumen (blood flow channel side).

[0020] [Fat-soluble vitamins] In this embodiment, fat-soluble vitamins are not particularly limited, but generally refer to vitamins that are poorly soluble in water and soluble in alcohol or oils and fats. Examples include vitamin A, vitamin D, vitamin E, vitamin K, and ubiquinone, among which vitamin E is preferred. Examples of vitamin E include α-tocopherol, α-tocopherol acetate, α-tocopherol nicotinate, β-tocopherol, γ-tocopherol, and δ-tocopherol. Among these, α-tocopherol is particularly preferred because it has various physiological effects such as antioxidant activity, biomembrane stabilization activity, and platelet aggregation inhibition activity.

[0021] [Hollow fiber membrane] The hollow fiber membrane contained within the blood purification module in this embodiment is not particularly limited and is, for example, a hollow fiber membrane used in hemodialysis machines, hemodiafiltration machines, hemofilters, continuous hemofiltration (dialysis) machines, plasma separators, plasma component separators, ascites filters, ascites concentrators, artificial lungs, etc., and is a hollow fiber membrane made of hydrophobic polymers and hydrophilic polymers containing lipid-soluble vitamins, having pore sizes suitable for various applications. Considering that oxidative stress experienced by patients accumulates through repeated and prolonged extracorporeal circulation treatments, it is preferable to use the blood purification module of this embodiment as a hemodialysis machine or hemodiafiltration machine in hemodialysis or hemodiafiltration performed frequently and over a long period of time.

[0022] The hydrophobic polymers that constitute hollow fiber membranes are synthetic or natural polymers that do not dissolve in water or show no affinity for water. Examples include polysulfone, polyethersulfone, polyamide, polyarylate, polyethersulfone-polyarylate polymer alloys, polymethyl methacrylate, polycarbonate, polyetheretherketone, polyallyletherketone, cellulose triacetate, and cellulose diacetate. Among these, synthetic polymers are preferred due to the uniformity of their composition as polymers, and polysulfone is particularly preferred because it has a large number of suitable clinical results in blood purification applications and is excellent in terms of stable supply as a raw material. In this invention, polysulfone includes not only those in which part of the aromatic ring has been chemically modified, but also so-called related compounds such as polyphenylsulfone and polyallylethersulfone.

[0023] Hydrophilic polymers that constitute hollow fiber membranes are substances that are soluble in water and can be crosslinked by physical and / or chemical treatment, thereby becoming insoluble in water. Examples include polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), polypropylene glycol (PPG), hydroxypropyl cellulose (HPC), starch, and hydroxyethyl starch (HES). Among these, polyvinylpyrrolidone and polyethylene glycol are preferred because they have good pore-forming properties for hollow fiber membranes, with polyvinylpyrrolidone being particularly preferred. The hollow fiber membrane in this invention includes the hydrophobic polymer and hydrophilic polymer described above, but their form of existence is not particularly limited. For example, it may be a polymer blend of highly compatible components, or a hydrophilic polymer grafted onto a hydrophobic polymer membrane substrate. Alternatively, a copolymer polymer consisting of a hydrophobic component and a hydrophilic component can be used. Examples include block copolymers of PVP and PSf, and block copolymers of PEG and PSf.

[0024] In this embodiment, the blood-side flow path in a blood purification module filled with hollow fiber membranes refers to the space inside the hollow fiber membrane separated by the membrane, or the space formed between the outside of the hollow fiber membrane and the inside of the container, and is the space through which blood flows. In cases where blood flows through the lumen of the hollow fiber membrane, such as in a hemodialysis machine, it refers to the space on the inner side of the hollow fiber membrane. However, in cases where gas flows through the lumen of the hollow fiber membrane, such as in an artificial lung, it refers to the space formed between the outside of the hollow fiber membrane and the inside of the container.

[0025] In this embodiment, the filtrate-side flow path in a blood purification module filled with hollow fiber membranes refers to the space separating the blood-side flow path from the hollow fiber membrane, specifically the space on the opposite side of the blood-side flow path, and is the space through which the filtrate or dialysate flows. In cases where blood flows through the lumen of the hollow fiber membrane, such as in a hemodialyzer, it refers to the space formed between the outside of the hollow fiber membrane and the inside of the container. However, in cases where gas flows through the lumen of the hollow fiber membrane, such as in an artificial lung, it refers to the space inside the hollow fiber membrane.

[0026] The hollow fiber membrane filled in the blood purification module of the present embodiment contains fat-soluble vitamins in the range of 10 mg / m or more and 300 mg / m or less in terms of membrane area. Here, the membrane area refers to the effective total inner surface area of the hollow fiber membrane involved in filtration or dialysis, and is represented by the product of the average inner diameter, the circumference ratio, the number, and the effective length of the hollow fiber membrane. If the fat-soluble vitamin is less than 10 mg / m in terms of membrane area, uneven coating of the fat-soluble vitamin is likely to occur, and thus the antioxidant ability tends to be inferior. On the other hand, if it is more than 300 mg / m, a large amount of fat-soluble vitamins will adhere to the entire surface of the membrane, for example, the inside of the hollow fiber where blood other than the inner surface of the hollow fiber does not come into contact, and the outer surface of the hollow fiber. As a result, not only does the permeation ability of blood components, filtrate, or dialysate decrease, but the antithrombotic property also decreases and residual blood occurs. Furthermore, due to the heat history during sterilization or contact with hot water, the fat-soluble vitamin may cause local aggregation and cracks may occur in the hollow fiber. Therefore, a more preferable content range is 50 mg / m or more and 270 mg / m or less, more preferably 80 mg / m or more and 250 mg / m or less, and particularly preferably 100 mg / m or more and 200 mg / m or less. 2 or less in terms of membrane area. 2 Here, the membrane area refers to the effective total inner surface area of the hollow fiber membrane involved in filtration or dialysis, and is represented by the product of the average inner diameter, the circumference ratio, the number, and the effective length of the hollow fiber membrane. If the fat-soluble vitamin is less than 10 mg / m in terms of membrane area, uneven coating of the fat-soluble vitamin is likely to occur, and thus the antioxidant ability tends to be inferior. 2 On the other hand, if it is more than 300 mg / m, a large amount of fat-soluble vitamins will adhere to the entire surface of the membrane, for example, the inside of the hollow fiber where blood other than the inner surface of the hollow fiber does not come into contact, and the outer surface of the hollow fiber. As a result, not only does the permeation ability of blood components, filtrate, or dialysate decrease, but the antithrombotic property also decreases and residual blood occurs. 2 Furthermore, due to the heat history during sterilization or contact with hot water, the fat-soluble vitamin may cause local aggregation and cracks may occur in the hollow fiber. Therefore, a more preferable content range is 50 mg / m or more and 270 mg / m or less, more preferably 80 mg / m or more and 250 mg / m or less, and particularly preferably 100 mg / m or more and 200 mg / m or less. 2 or less, more preferably 80 mg / m or more and 250 mg / m or less, and particularly preferably 100 mg / m or more and 200 mg / m or less. 2 Here, the membrane area refers to the effective total inner surface area of the hollow fiber membrane involved in filtration or dialysis, and is represented by the product of the average inner diameter, the circumference ratio, the number, and the effective length of the hollow fiber membrane. If the fat-soluble vitamin is less than 10 mg / m in terms of membrane area, uneven coating of the fat-soluble vitamin is likely to occur, and thus the antioxidant ability tends to be inferior. 2 On the other hand, if it is more than 300 mg / m, a large amount of fat-soluble vitamins will adhere to the entire surface of the membrane, for example, the inside of the hollow fiber where blood other than the inner surface of the hollow fiber does not come into contact, and the outer surface of the hollow fiber. As a result, not only does the permeation ability of blood components, filtrate, or dialysate decrease, but the antithrombotic property also decreases and residual blood occurs. 2 Furthermore, due to the heat history during sterilization or contact with hot water, the fat-soluble vitamin may cause local aggregation and cracks may occur in the hollow fiber. Therefore, a more preferable content range is 50 mg / m or more and 270 mg / m or less, more preferably 80 mg / m or more and 250 mg / m or less, and particularly preferably 100 mg / m or more and 200 mg / m or less. 2 or less, more preferably 80 mg / m or more and 250 mg / m or less, and particularly preferably 100 mg / m or more and 200 mg / m or less. 2

[0027] The membrane area of the hollow fiber membrane filled in the blood purification module of the present embodiment is preferably 0.5 to 3.0 m. When the membrane area is 0.5 m or more, a sufficient blood volume for treatment can be ensured, and thus a treatment effect tends to be achieved within the treatment time. When it is 3.0 m or less, problems such as a decrease in blood pressure due to an excessive increase in the blood undergoing extracorporeal circulation and a decrease in the patient's internal blood can be reduced. 2 When the membrane area is 0.5 m or more, a sufficient blood volume for treatment can be ensured, and thus a treatment effect tends to be achieved within the treatment time. 2 When it is 3.0 m or less, problems such as a decrease in blood pressure due to an excessive increase in the blood undergoing extracorporeal circulation and a decrease in the patient's internal blood can be reduced. 2

[0028] ​​The method for manufacturing the blood purification module in this embodiment is not particularly limited, but for example, it can be manufactured by filling a container with a bundle of hollow fiber membranes for blood purification made of hydrophobic polymers and hydrophilic polymers using a known method, assembling and molding it into the shape of a general hollow fiber membrane type blood purification device, and then sequentially going through a lipid-soluble vitamin coating step and a sterilization step. In this case, a hollow fiber membrane wetting step and a sterilization protective agent addition step can also be added between the coating step and the sterilization step.

[0029] The method for measuring the amount of fat-soluble vitamins immobilized in this embodiment is a method for measuring the amount of fat-soluble vitamins immobilized on the inner surface of the hollow fiber membrane of the blood purification module described above. The process of supplying a solvent through the aforementioned blood inlet port, A step of recovering the solvent discharged from the blood outlet port, The method comprises the step of measuring the fat-soluble vitamin content contained in the recovered solvent.

[0030] If the blood purification module contains a filling solution, the filling solution can be removed from the module before delivering the solvent through the blood inlet port. The module can then be rinsed with water to remove any remaining filling solution, and then dried for several hours before use.

[0031] [solvent] The solvent used in the measurement method of this embodiment is not particularly limited as long as it dissolves fat-soluble vitamins and does not dissolve the hollow fiber membrane. Examples include alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, sec-butanol, and 2-ethylhexanol; ethers such as diethyl ether; glycol ethers such as methyl cellsolve (ethylene glycol monomethyl ether) and ethyl cellsolve (ethylene glycol monoethyl ether); chlorofluorinated hydrocarbons such as 1,2,2-trichloro-1,2,2-trifluoroethane, trichlorofluoromethane, and 1,1,2,2-tetrachloro-1,2-difluoroethane; perfluorocycloalkanes such as methyl fluoride, carbon tetrafluoride, tetrafluoroethane, tetrafluoroethylene, perfluoromethylpropylcyclohexane, and perfluorobutylcyclohexane; fluorinated hydrocarbons such as perfluorodecane, perfluoromethyldecalin, and perfluoroalkyltetrahydropyran; or hydrocarbons such as hexane, heptane, and decane. The solvent can be appropriately selected from among these depending on the type of hollow fiber membrane used. In particular, for industrial production, a solvent that is inexpensive, has a relatively high boiling point for safety reasons, and has high dissolving power in order to contain a large amount of fat-soluble vitamins is preferable. For example, when polysulfone is used as the base material for the hollow fiber membrane and vitamin E is used as the fat-soluble vitamin, it is preferable to use alcohols as the solvent, with isopropanol and ethanol being more preferable, and ethanol being particularly preferable.

[0032] Furthermore, when using alcohols as a solvent, it is preferable to use an aqueous alcohol solution with an alcohol concentration of 75% (by volume) or higher. When the alcohol concentration of the aqueous alcohol solution is 75% or higher, there is a tendency for sufficient elution of lipid-soluble vitamins on the inner surface of the hollow fiber membrane. There is no particular upper limit to the alcohol concentration, but if the concentration is too high, the substrate used as the hollow fiber membrane may elute, which may adversely affect the measurement of lipid-soluble vitamins, so it is preferable that it be 93% or lower. The alcohol concentration of the aqueous alcohol solution is more preferably 78-93%, and even more preferably 83-91%.

[0033] [Amount of extract] The method for measuring the amount of fat-soluble vitamins immobilized according to this embodiment includes the steps of supplying a solvent from a blood inlet port and recovering the solvent discharged from a blood outlet port, and the amount of solvent discharged from the blood outlet port at this time is defined as the "extract volume".

[0034] In this embodiment, it is preferable that the amount of extract Y in the solvent recovery step satisfies the following formula (1). 0.1 × solvent flow rate + 5.2 ≤ extract volume Y (mL) (1) When the extract volume Y satisfies the above equation (1), the variation in the measured amount of fat-soluble vitamin immobilized for each module tends to decrease, enabling more stable measurements.

[0035] Furthermore, in this embodiment, it is preferable that the amount of extract Y in the solvent recovery step satisfies the following formula (2). Extract volume Y (mL) ≤ {(k × membrane area)} 2 (m 2 ) / solvent flow rate (mL / min)) + 61} × 7.5 (2) (In equation (2), k = 800.) When the amount of extract Y in the solvent recovery step satisfies equation (2), there is a tendency for sufficient elution of lipid-soluble vitamins immobilized on the inner surface of the hollow fiber membrane. The basis for the above calculation formula is as follows: Reason 1: A larger membrane surface area means a greater amount of immobilized vitamins, thus requiring a larger volume of extract. Therefore, the volume of extract is proportional to the membrane surface area. Reason 2: The volume of extract is inversely proportional to the linear velocity.

[0036] Furthermore, in this embodiment, it is preferable that the amount of extract Y in the solvent recovery step satisfies the following formula (3). Extract volume Y (mL) ≤ {(k × membrane area)} 2 (m 2 ) / solvent flow rate (mL / min)) + 61} × 3.5 (3) (In equation (3), k = 800.) When the amount of extract Y in the solvent recovery process satisfies equation (3), the elution of fat-soluble vitamins immobilized inside the membrane other than on the inner surface can be reduced, and therefore, the amount of fat-soluble vitamins immobilized on the inner surface of the hollow fiber membrane tends to be measured more accurately. In this embodiment of the measurement method, it was determined that when the difference Δ of the change in the fat-soluble vitamin concentration per unit volume of the extraction solvent reached 1 μg / ml, the fat-soluble vitamins on the inner surface were extracted, and thereafter only the fat-soluble vitamins inside the membrane were extracted.

[0037] Furthermore, in this embodiment, it is preferable that the amount of extract Y in the solvent recovery step satisfies the following formula (4). Extract volume Y (mL) ≤ {(k × membrane area)} 2 (m 2 ) / solvent flow rate (mL / min)) + 61} × 2.5 (4) (In equation (4), k = 800.) When the amount of extract Y in the solvent recovery process satisfies equation (4), approximately 95% of the lipid-soluble vitamins immobilized on the inner surface of the hollow fiber membrane can be extracted, and solvent costs and extraction time tend to be reduced.

[0038] Furthermore, in this embodiment, it is preferable that the amount of extract Y in the solvent recovery step satisfies the following formula (5). Extract volume Y (mL) ≤ {(k × membrane area)} 2 (m2 ) / solvent flow rate (mL / min) + 61} × 2 (5) (In equation (5), k = 800.) When the amount of extract Y in the solvent recovery step satisfies equation (5), nearly 100% of the fat-soluble vitamins present near the inner surface of the hollow fiber membrane (preferably 1-5 nm from the inner surface), which reliably reflects the functional expression of fat-soluble vitamins, can be extracted. Furthermore, the amount of fat-soluble vitamins extracted from inside the membrane is kept below 5%, which tends to allow for more accurate measurement of fat-soluble vitamins immobilized near the inner surface.

[0039] Furthermore, in this embodiment, it is preferable that the amount of extract Y in the solvent recovery step satisfies the following formula (6). Extract volume Y (mL) ≤ Solvent flow rate (mL / min) × 5 (6)

[0040] When the amount of extract Y in the solvent recovery process satisfies equation (6), the cost of the solvent and the extraction time can be reduced, and the amount of fat-soluble vitamins tends to be measured more efficiently.

[0041] The method for measuring the amount of fat-soluble vitamins immobilized in this embodiment includes a step of measuring the fat-soluble vitamin content contained in the recovered solvent. The method for measuring the fat-soluble vitamin content contained in the recovered solvent is not particularly limited and can be measured, for example, by high-performance liquid chromatography (HPLC) or UV meter that measures the absorption wavelength of fat-soluble vitamins, or by a microbiological quantitative method. More specifically, it can be measured according to the method described in the examples below. [Examples]

[0042] The present invention will be described in more detail below with reference to examples, but the present invention is not limited in any way by these examples. The measurement method in these examples is as follows.

[0043] <Method for measuring the amount of lipid-soluble vitamins immobilized on the inner surface of hollow fiber membranes> After rinsing the hollow fiber membrane type blood purification device with water, the rinsing water was removed and the device was dried. Next, an ethanol aqueous solution of a desired concentration was passed through the blood inlet port at a desired flow rate, and the liquid coming out of the blood outlet port was collected in a desired quantity to be used as the extract. The quantitative analysis was performed using liquid chromatography with the following apparatus, and the lipid-soluble vitamin content of the extract was determined using a calibration curve obtained from the peak area of ​​the lipid-soluble substance standard solution. High-performance liquid chromatography (H-PLC) system (pump: JASCO PU-1580, detector: Shimadzu RID-6A, auto-injector: Shimadzu SIL-6B, data processing: Tosoh GPC-8020, column oven: GL Sciences 556) was fitted with a column (Shodex Asahipak ODP-506E packed column for H-PLC). At a column temperature of 40°C, methanol for high-performance liquid chromatography was passed through the column at a flow rate of 1 mL / min, and the concentration of fat-soluble vitamins was determined from the area of ​​the absorption peak in the ultraviolet region. From the determined concentration of fat-soluble substances, the fat-soluble vitamin content (mg / m³) was calculated assuming an extraction efficiency of 100%. 2 ) was sought. When the fluid exiting the blood outlet port is collected in 25 mL fractions, it is defined that all fat-soluble vitamins present on the inner surface of the hollow fiber are extracted when the difference in fat-soluble vitamin concentration between the fractions is Δ = -1 μg / ml. After that point, only fat-soluble vitamins present inside the hollow fiber are extracted. The approximate straight line created from the fat-soluble vitamin content of the five fractions where |Δ|<1 was defined as the amount of fat-soluble vitamins inside the hollow fiber (a). The amount of fat-soluble vitamins on the inner surface of the hollow fiber (b) was calculated using the following equation (X). b = Fat-soluble vitamin content in fraction - a (X) The sum of b from the start of extraction until Δ=-1 was defined as the total amount of fat-soluble vitamins present on the inner surface of the hollow fiber.

[0044] <Method for measuring the fat-soluble vitamin content present throughout the entire hollow fiber membrane (inner surface and inside the membrane)> After disassembling a blood purification device and collecting the hollow fiber membranes used for blood purification, they were washed with water, dried, and their weight was measured. The entire volume was then finely chopped, 400 mL of 100% ethanol was added, and fat-soluble vitamins were extracted at room temperature for 60 minutes while applying ultrasonic vibrations. The quantitative analysis was performed using liquid chromatography with the following apparatus, and the lipid-soluble vitamin content of the extract was determined using a calibration curve obtained from the peak area of ​​the lipid-soluble substance standard solution. High-performance liquid chromatography (H-PLC) system (pump: JASCO PU-1580, detector: Shimadzu RID-6A, auto-injector: Shimadzu SIL-6B, data processing: Tosoh GPC-8020, column oven: GL Sciences 556) was fitted with a column (Shodex Asahipak ODP-506E packed column for H-PLC). At a column temperature of 40°C, methanol for high-performance liquid chromatography was passed through the column at a flow rate of 1 mL / min, and the concentration of fat-soluble vitamins was determined from the area of ​​the absorption peak in the ultraviolet region. From the determined concentration of fat-soluble substances, the fat-soluble vitamin content (mg / m³) was calculated assuming an extraction efficiency of 100%. 2 ) was sought.

[0045] <Method for measuring normalized peak intensity of lipid-soluble vitamins on the membrane surface> The inner surface of a dried hollow fiber, exposed by making a longitudinal cut and opening it, was measured using a TOF-SIMS instrument (TRIFTIII, Physical Electronics). The measurement conditions were: primary ion Ga+, acceleration voltage 15kV, current 600pA (as DC), analysis area 200μm×200μm, and integration time 5min. Negative ions (as Mass, 163 for fat-soluble vitamins) were detected as the detection ion by the detector. Due to the characteristics of this measuring instrument, the measurement depth corresponds to a depth of 5nm from the surface. The ionic intensity (IV) of the obtained fat-soluble vitamin peak was used to calculate the normalized peak intensity of the fat-soluble vitamin using the following formula (Y), with the ionic intensity IH of protons and the total ionic intensity IT. Normalized peak intensity = IV / (IT-IH) (Y)

[0046] [Example 1] Dried film area: 2.1 m² 2 An 85 v / v% ethanol aqueous solution was passed through the blood inlet port of a hollow fiber membrane blood purification device at a flow rate of 100 mL / min. 100 mL of the extract was collected, and the amount of lipid-soluble vitamins immobilized on the inner surface of the hollow fiber membrane was measured to be 48 mg / m². 2 That was the case.

[0047] [Example 2] The same procedure as in Example 1 was followed, except that a 90 v / v% ethanol aqueous solution was used. The amount of lipid-soluble vitamins immobilized on the inner surface of the hollow fiber membrane was measured to be 52 mg / m². 2 That was the case.

[0048] [Example 3] The same procedure as in Example 1 was followed, except that a 100 v / v% ethanol aqueous solution was used. The amount of lipid-soluble vitamins immobilized on the inner surface of the hollow fiber membrane was measured to be 38 mg / m². 2 That was the case.

[0049] [Example 4] The same procedure as in Example 1 was followed, except that a 70 v / v% ethanol aqueous solution was used. The amount of lipid-soluble vitamins immobilized on the inner surface of the hollow fiber membrane was measured to be 32 mg / m². 2 That was the case. [Example 5] The same procedure as in Example 1 was followed, except that a 65 v / v% ethanol aqueous solution was used. The amount of lipid-soluble vitamins immobilized on the inner surface of the hollow fiber membrane was measured to be 16 mg / m². 2 That was the case.

[0050] [Example 6] (Method that satisfies formulas (1), (2), and (6)) Dried film area: 2.1 m² 2 When an 85 v / v% ethanol aqueous solution was passed through the blood inlet port of a hollow fiber membrane blood purification device at a flow rate of 100 mL / min and collected in 25 mL fractions, the change in fat-soluble vitamin concentration in the fractions when the total volume of extracted liquid reached 400 mL was Δ = -4.56 μg / mL, and the fat-soluble vitamin content in the fractions was 0.53 mg / mL.2 That was the case. Of the total amount of fat-soluble vitamins in the extract, 11% were extracted from inside the hollow fibers, while 89% were extracted from the inner surface of the hollow fiber membrane. The extracted fat-soluble vitamins from the inner surface of the hollow fiber membrane accounted for 99% of the total amount of fat-soluble vitamins present on the inner surface. The extraction time for 400 mL was 4 minutes.

[0051] [Example 7] (Method that satisfies formula (1)) The same procedure as in Example 6 was followed. When the total volume of extract reached 1000 mL, the change in the concentration of fat-soluble vitamins in the fraction was Δ = -0.43 μg / mL, and the fat-soluble vitamin content in the fraction was 0.13 mg / m³. 2 That was the case. Of the total amount of fat-soluble vitamins in the extract, 18% were extracted from inside the hollow fibers, while 82% were extracted from the inner surface of the hollow fiber membrane. The amount of fat-soluble vitamins extracted from the inner surface of the hollow fiber membrane was 100% of the total amount of fat-soluble vitamins present on the inner surface of the membrane. The extraction time for 1000 mL was 10 minutes.

[0052] [Example 8] (Method that satisfies formulas (1), (3), and (6)) The same procedure as in Example 6 was followed. When the total volume of extract reached 300 mL, the change in fat-soluble vitamin concentration in the fraction was Δ = -9.91 μg / mL, and the fat-soluble vitamin content in the fraction was 0.82 mg / m³. 2 That was the case. Of the total amount of fat-soluble vitamins in the extract, 9% were extracted from inside the hollow fibers, while 81% were extracted from the inner surface of the hollow fiber membrane. The extracted fat-soluble vitamins from the inner surface of the hollow fiber membrane accounted for 97% of the total amount of fat-soluble vitamins present on the inner surface.

[0053] [Example 9] (Method that satisfies formulas (1), (4), and (6)) The same procedure as in Example 6 was followed. When the total volume of extract reached 200 mL, the change in the fat-soluble vitamin concentration in the fraction was Δ = -28.6 μg / mL, and the fat-soluble vitamin content in the fraction was 1.42 mg / m³. 2 That was the case. Of the total amount of fat-soluble vitamins in the extract, 7% were extracted from inside the hollow fibers, while 93% were extracted from the inner surface of the hollow fiber membrane. The extracted fat-soluble vitamins from the inner surface of the hollow fiber membrane accounted for 93% of the total amount of fat-soluble vitamins present on the inner surface of the membrane.

[0054] [Example 10] (Method that satisfies formulas (1), (5), and (6)) The same procedure as in Example 6 was followed. When the total volume of extract reached 75 mL, the change in fat-soluble vitamin concentration in the fraction was Δ = -481.0 μg / mL, and the fat-soluble vitamin content in the fraction was 8.10 mg / m³. 2 That was the case. Of the total amount of fat-soluble vitamins in the extract, 4% were extracted from inside the hollow fibers, while 96% were extracted from the inner surface of the hollow fiber membrane. The extracted fat-soluble vitamins from the inner surface of the hollow fiber membrane accounted for 68% of the total amount of fat-soluble vitamins present on the inner surface of the membrane. When collecting the extract, an error of 2 seconds, or 3.3 mL, occurs. When collecting 75 mL of extract, the amount of fat-soluble vitamins lost due to the 3.3 mL error accounted for 0.1% of the total fat-soluble vitamin content in the 75 mL of extract, according to calculations.

[0055] [Example 11] (Method that satisfies formula (6)) When 10 mL of the extract was collected using the same method as in Example 6, the proportion of the fat-soluble vitamin content in the 10 mL of extract that was generated due to an error of 3.3 mL was calculated to be 28.3%.

[0056] [Table 1]

[0057] [Example 12] Dry film area: 1.3 m² 2 Using a hollow fiber membrane blood purification device, an 85 v / v% ethanol aqueous solution was passed through at a flow rate of 100 mL / min. After collecting 75 mL of extract, the amount of lipid-soluble vitamins present at approximately 1-5 nm of the inner surface of the hollow fiber membrane was measured using TOF-SIMS. The results showed a 95% decrease compared to before the ethanol aqueous solution was passed through, and the values ​​were equivalent to those of a membrane without lipid-soluble vitamin coating.

[0058] [Example 13] Membrane area 1.5m 2 The same procedure as in Example 12 was followed, except that 80 mL of extract was collected using a hollow fiber membrane type blood purification device. The amount of lipid-soluble vitamins present at approximately 1-5 nm on the inner surface of the hollow fiber membrane decreased by 95% compared to before the passage of the ethanol aqueous solution, showing a value equivalent to that of a membrane not coated with lipid-soluble vitamins.

[0059] [Comparative Example 1] After disassembling a blood purification device and collecting the hollow fiber membranes used for blood purification, they were washed with water, dried, and their weight was measured. The entire volume was then finely chopped, 400 mL of 100% ethanol was added, and fat-soluble vitamins were extracted at room temperature for 60 minutes while applying ultrasonic vibrations. Quantitative analysis was performed using liquid chromatography, and the lipid-soluble vitamin content of the extract was determined using a calibration curve obtained from the peak area of ​​standard solutions of lipid-soluble substances. While the above method makes it possible to measure the fat-soluble vitamins contained throughout the entire hollow fiber membrane, it was difficult to measure only the fat-soluble vitamins immobilized on the surface of the hollow fiber membrane.

Claims

1. A method for measuring the amount of fat-soluble vitamins immobilized on the inner surface of a hollow fiber membrane in a blood purification module, comprising a hollow fiber membrane containing fat-soluble vitamins and a container having a blood inlet port and a blood outlet port connected to the inside of the hollow fiber membrane, The process of supplying a solvent through the aforementioned blood inlet port, A step of recovering the solvent discharged from the blood outlet port, The process includes a step of measuring the fat-soluble vitamin content contained in the recovered solvent, A method for measuring the amount of fat-soluble vitamins immobilized, wherein the amount of extract Y in the solvent recovery step satisfies the following formula (1). 0.1 × solvent flow rate (mL / min) + 5.2 ≤ extract volume Y (mL) (1)

2. The method for measuring the amount of fat-soluble vitamins immobilized according to claim 1, wherein the solvent is an alcohol.

3. The method for measuring the amount of fat-soluble vitamins immobilized according to claim 2, wherein the alcohols are an aqueous alcohol solution with an alcohol concentration of 75% (by volume) or more.

4. The method for measuring the amount of fat-soluble vitamins immobilized according to any one of claims 1 to 3, wherein the amount of extract Y in the solvent recovery step satisfies the following formula (2). Extract volume Y (mL) ≤ {(k × membrane area)} 2 (m 2 (2) (In equation (2), k = 800.)

5. The method for measuring the amount of fat-soluble vitamin immobilized according to any one of claims 1 to 3, wherein the amount of extract Y in the solvent recovery step satisfies the following formula (3). Extract volume Y (mL) ≤ {(k × membrane area)} 2 (m 2 ) / solvent flow rate (mL / min)) + 61} × 3.5 (3) (In equation (3), k = 800.)

6. The method for measuring the amount of fat-soluble vitamins immobilized according to any one of claims 1 to 3, wherein the amount of extract Y in the solvent recovery step satisfies the following formula (4). Extract volume Y (mL) ≤ {(k × membrane area)} 2 (m 2 ) / solvent flow rate (mL / min)) + 61} × 2.5 (4) (In equation (4), k = 800.)

7. The method for measuring the amount of fat-soluble vitamin immobilized according to any one of claims 1 to 3, wherein the amount of extract Y in the solvent recovery step satisfies the following formula (5). Extract volume Y (mL) ≤ {(k × membrane area)} 2 (m 2 ) / solvent flow rate (mL / min)) + 61} × 2 (5) (In equation (5), k = 800.)

8. The method for measuring the amount of fat-soluble vitamin immobilized according to any one of claims 1 to 3, wherein the amount of extract Y in the solvent recovery step satisfies the following formula (6). Extract volume Y (mL) ≤ Solvent flow rate (mL / min) × 5 (6)

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