JP2003322655A - Apparatus for measuring concentration of endotoxin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an apparatus for measuring the concentration of endotoxin capable of incorporating part of a specimen liquid such as drugs from a storage tank, etc., for the specimen liquid by a pipe and continuously monitoring the concentration of endotoxin of a liquid of a low concentration of 50 EU/L or less in a short time. <P>SOLUTION: The apparatus for measuring the concentration of the endotoxin comprises a means for incorporating the specimen liquid containing the endotoxin in a measuring circuit and sealing it; a means for circulating the sealed specimen liquid in the measuring circuit; a means for injecting a limulus reagent in the circulating specimen liquid and mixing both liquids; a particulate counting means for continuously measuring the concentration of particulates with the passage of time which occur in the mixed liquids introduced to a measuring cell; and a means for discharging the mixed liquids from the measuring circuit and cleaning the measuring circuit. By measuring the time required for a count value of the particulate counting means to reach a predetermined amount of change or the rate of change of the amount of change with the passage of time, the concentration of the endotoxin is computed in the apparatus for measuring the concentration of the endotoxin. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a device for measuring endotoxin concentration, and more specifically, it is present in pharmaceutical products such as injection solutions, infusion solutions, dialysis solutions, intermediate products thereof, and purified water which is a part of these raw materials. , The concentration of endotoxin, which exhibits various biological activities and has harmful effects, is measured, and pharmaceuticals such as injections, infusions, dialysis fluids, and the like, which are in use during medical treatment or during manufacture or storage as pharmaceuticals, and their intermediate products, or these The present invention relates to an endotoxin concentration measuring device used for quality control and the like of purified water, which is a part of the raw material.

[0002]

2. Description of the Related Art Endotoxin is a thermostable toxin existing in the outer membrane of Gram-negative bacteria and is called endotoxin.
The body is said to be lipopolysaccharide. Lipopolysaccharide consists of lipid called lipid A and sugar chain, but the active center as endotoxin is lipid A, and almost all the activities are reproduced by separated lipid A.

Endotoxin promotes the reaction of blood coagulation lines,
Exhibits a variety of biological activities such as a decrease in platelets and white blood cells, a decrease in blood pressure, effects on the circulatory system such as shock, fever, induction of cytokines, effects on the immune system (edited by Shingo Takezawa, book on dialysate endotoxin, 15 -24 pages and 45-5
1 page (1995), Tokyo Medical Co., Ltd.). Gram-negative bacteria that have endotoxin as a component exist in the air, water, or foods, and the microbial cells are mechanically damaged,
Endotoxin is released into the solution when the dead cells are dissolved or the cells are divided, and the endotoxin is present in the solution.

[0004] Therefore, endotoxin is directly or indirectly associated with pharmaceutical products such as injectable solutions, infusion solutions and dialysates.
If mixed or present in the intermediate product or purified water that is a part of these raw materials, there is an adverse effect of exhibiting various biological activities as described above.Therefore, it is important to measure the concentration of endotoxin during medical use. Alternatively, it is necessary for quality control of pharmaceuticals such as injections, infusions, and dialysis fluids that are being manufactured or stored as pharmaceuticals, and intermediate products thereof, or purified water that is a part of these raw materials. Monitoring the endotoxin concentration is also effective as a means for early detection of the growth of viable bacteria. However, at present, there is no device that monitors the endotoxin concentration, and a sample solution is collected and the endotoxin concentration is measured off-line, which is useful for quality control.

The only method for quantitatively measuring the concentration of endotoxin is the Limulus test.
There is t). This method is based on the Limulus
Endotoxin activates the procoagulant enzyme (Proclotting Enzyme) present in the blood cells of
Enzyme) is used, and it is a component of Limulus amebocyte extract from Limulus amebocyte.
lysate) is used as a reagent, and there are three methods, a gelation method and a highly sensitive measurement method, a turbidimetric method and a colorimetric method.

According to the turbidimetric method, which is a highly sensitive assay method, the coagulant protein (Coaggulogue) present in the horseshoe crab blood cells is used.
The coagulation enzyme acts on n), and coagulation protein (Coagg
to form a gel (although if the endotoxin concentration is low, it does not lead to gelation of the whole)
This is a method of measuring the turbidity change occurring in the process by an optical analyzer, and there are an endpoint method and a kinetic method, which are quantitative methods. The endpoint method is
This is a method based on the fact that the turbidity (absorbance) that reaches within a certain period of time is proportional to the endotoxin concentration. The kinetic method is a turbidimetric reaction rate method based on the rate of change in turbidity (absorbance) with time being proportional to endotoxin concentration, and the logarithm of the time when the rate of change in turbidity reaches a preset threshold is the logarithm of endotoxin concentration. There is a turbidimetric reaction time method based on the inverse proportion to. The kinetic method is generally used (Takezawa Shingo ed., Book on dialysate endotoxin, page 36-39 (1995), Tokyo Medical Co., Ltd.).

With an existing endotoxin concentration measuring instrument by the nephelometric method, it is 90 to 1 to detect a concentration of 1 EU / L.
A reaction time of 50 minutes is required (supervised by Shingo Takezawa, Yoshi Ishizaki, edited by the Minami Tohoku Water Quality Study Group, dialysate endotoxin measurement collection, page 93 (1997), Medica Publishing Co., Ltd.). Low concentration liquid with endotoxin concentration of 50 EU / L or less, especially 10
It took too much time and was insufficient as a monitor of the endotoxin concentration of the HDF dialysate requiring a <1 EU / L level. In addition, the volume of the measurement cell and the like is limited due to the constraint that the amount of the sample to be measured is equal to the amount of Limulus reagent (0.1 to 0.05 cm ³ ), and it was difficult to circulate the mixed solution.

[0008]

SUMMARY OF THE INVENTION In view of the above-mentioned problems of the prior art, the present invention provides a storage tank or supply for medicines such as injections, infusions and dialysates, and intermediate products thereof, and specimen liquids such as purified water. A part of the sample solution can be taken in from the line with a pipe to continuously monitor the endotoxin concentration, and the endotoxin concentration is a low-concentration liquid of 50 EU / L or less, especially an extremely low level of 10 to <1 EU / L. An object of the present invention is to provide an endotoxin concentration measuring device capable of monitoring even a concentration liquid, and capable of monitoring the concentration of endotoxin in a short time.

[0009]

SUMMARY OF THE INVENTION In view of the above problems, the inventors of the present invention have proposed a storage tank or a supply line for a pharmaceutical product such as an injection solution, an infusion solution, a dialysate solution, an intermediate product thereof, and a sample solution such as purified water. By incorporating a part of the sample liquid, adopting a specific turbidimeter that can measure endotoxin even at low concentrations, and by combining each means to enable discharging the sample liquid whose concentration was measured, The inventors have found that good results can be obtained and completed the present invention.

That is, according to the first aspect of the present invention,
Means for capturing and sealing the sample liquid containing endotoxin in the measurement circuit including the measurement cell, means for circulating the captured and blocked sample liquid in the measurement circuit, and injecting a predetermined amount of Limulus reagent into the circulating sample liquid Means for mixing both solutions,
Fine particle counting means for continuously measuring the time-dependent change in the concentration of fine particles causing turbidity generated in the mixed solution introduced into the measurement cell, and the mixed solution is discharged from the measurement circuit after the measurement,
A device for measuring an endotoxin concentration, comprising: a means for washing a measurement circuit, wherein the fine particle counting means measures the time for the count value to reach a predetermined change amount or the rate of change over time of the change amount. An endotoxin concentration measuring device is provided, which is characterized by calculating the concentration of endotoxin contained therein.

According to the second aspect of the present invention, the first aspect
2. The endotoxin according to claim 1, wherein before the sample liquid containing endotoxin is taken into the measurement circuit, a filtering means capable of removing particles having a minimum diameter or more counted by the fine particle counting means is provided in advance. An apparatus for measuring concentration is provided.

According to the third aspect of the present invention, the first aspect
In the present invention, the fine particle counting means is a fine particle counter for setting the minimum diameter of fine particles to be 0.5 μm or less, and the time for the change amount to reach a predetermined amount from the count value or the change rate of the change amount with time. There is provided an endotoxin concentration measuring device, which comprises an electronic circuit for calculating and comparing the endotoxin concentration in a sample solution with a calibration curve prepared in advance.

Further, according to a fourth aspect of the present invention, in the first aspect, a plurality of measurement systems are switchably coupled in parallel to the flow path of the sample liquid, and are automatically switched in sequence.
There is provided an endotoxin concentration measuring device characterized in that measurement of fine particles and washing of a measuring circuit including a measuring cell are performed in parallel, or continuous measurement is performed at intervals shorter than the time required for one measurement. .

Further, according to a fifth aspect of the present invention, in the fourth aspect, the plurality of measurement systems are concentric circles having a rotation axis on which a single light source and a corresponding photodetector are fixed as a central axis. An endotoxin concentration measuring apparatus according to claim 4, comprising a measuring circuit including a plurality of measuring cells arranged on the circumference.

Further, according to the sixth invention of the present invention, in the fourth invention, the plurality of measurement systems correspond to a single light source that reciprocates in parallel with respect to the plurality of measurement cells arranged in a line. 5. A photodetector. 5.
An apparatus for measuring the endotoxin concentration is provided.

Further, according to the seventh invention of the present invention, in any one of the first to sixth inventions, when the endotoxin concentration in the sample liquid is at a low level, the sample liquid amount is relative to the Limulus reagent amount. An endotoxin concentration-measuring device is provided, which comprises measuring a mixed solution obtained by mixing the two solutions after mixing them in an amount of 10 times to 1000 times.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION The endotoxin concentration measuring apparatus of the present invention will be described in detail below for each item.

1. Means for capturing and sealing the sample liquid containing endotoxin in the measuring circuit including the measuring cell

1.1 Sample Liquid Containing Endotoxin In the present invention, the sample liquid containing endotoxin includes pharmaceutical products such as injectable solutions, infusion solutions and dialysates, and intermediate products thereof, or purified water as a part of these raw materials. The sample liquid is stored in a storage means such as a storage tank, a tank, a glass bottle, a plastic container, and a plastic bag, and when the sample liquid is used for its original purpose, a part of the storage means is used as a starting point. The sample liquid is sent to a place of use or a device to be used by a transportation means (1) such as a pipe, a pipe, or a hose. A part of the sample liquid stored in the storage means (not shown) or the sample liquid flowing in the transport means (1) is introduced into the endotoxin concentration measuring device of the present invention to measure the endotoxin concentration. For sampling, another transport means (2) (7 in FIG. 2) such as a pipe, pipe, hose or the like is provided in a part of the transport means (1), and a part of the sample liquid is used as a tributary of the present invention. Into the measurement circuit (15 in FIG. 3 (A)) of the endotoxin concentration measuring device of FIG.

1.2 Measurement Cell In the present invention, the measurement cell (16 in FIG. 3 (B), 1 in FIG. 4)
6) is a cell (room) for measuring the concentration of endotoxin in the sample liquid, which constitutes a part of the particle counter used in the present invention, and the endotoxin reacts with the Limulus reagent to coagulate protein (particles). The luminous flux from the light source is radiated to the mixed liquid in which the particle is generated, and the number of fine particles is measured. The measuring cell is part of the measuring circuit described in detail below.
As the material for the measuring cell, high-purity glass, quartz glass and the like are preferable because transparency to light is required.

1.3 Measuring Circuit In the present invention, the measuring circuit (15 in FIG. 3A, 1 in FIG. 4)
5) is a circuit that recycles (circulates) the sample liquid, and may have any structure as long as it includes the above-described measurement cell in part and the rest can recycle the sample liquid, but it is a circular pipe. It is preferably composed of (tube). The material of the pipe (tube) is not particularly limited, and examples thereof include polytetrafluoroethylene (Teflon (R)), fluorine resin, lined metal, non-lined metal, ceramics, etc. From the viewpoint of, polytetrafluoroethylene (Teflon (R)) is particularly preferable. Although the mixed liquid amount is limited to a small amount, in the present invention,
By circulating in the measurement circuit, it is possible to continue the flow state in the measurement cell, which is a necessary condition for measurement by the particle counter, and to improve the measurement accuracy by uniformly mixing and reacting the sample liquid and Limulus reagent. And is the first proposed in the present invention. By providing a static mixer in a part of the measuring circuit, the mixing can be ensured quickly.

1.4 Incorporation Means Incorporation in the present invention means incorporation of a sample liquid containing endotoxin into a measurement circuit containing a measurement cell, which is carried out by the pipe described in 1.1 above. Kan,
It is a transportation means (2) such as a hose (7 in FIG. 2). The structure of the transportation means (2) may be any structure as long as it can transport the sample liquid, but it is preferably composed of a circular pipe. The material of the pipe (tube) is not particularly limited, and examples thereof include polytetrafluoroethylene (Teflon (R)), fluororesin, lined metal, non-lined metal, ceramics, etc.
Polytetrafluoroethylene (Teflon (R)) is particularly preferable from the viewpoint of detergency. The sample liquid intake pump 4 is an auxiliary pump, and can be omitted when the sample liquid is taken in from the line under pressure. Further, the sample solution filtration filter 5 can be omitted when the number of particles having a minimum diameter or more counted by the particle counter in the sample solution is small.

1.5 Means for Blocking In the present invention, the term “blocking” means blocking a sample liquid containing endotoxin in a measurement circuit containing a measurement cell, and blocking the inlet and outlet of the measurement circuit to form the measurement circuit. The sample liquid containing endotoxin can be circulated in the measurement circuit, and the sealing means may have any structure as long as it can block the inlet and the outlet of the measurement circuit. Valve), spherical valve (globe valve), cock (plug valve), ball valve and the like. The valve 9 for injecting the washing liquid and the valve 12 for injecting the Limulus reagent can be used as a partition having the ability to self-close a perforation by a check valve or a thin tube such as an injection needle by removing the thin tube. Specifically, in FIG. 3, valves such as V1, V3, and V4 or partitions that can be opened and closed may be closed. When the Lithmus reagent injector 10 is provided instead of V3, the Lithmus reagent injector may be closed.

2. Means for Circulating the Incorporated and Blocked Sample Liquid in the Measurement Circuit In the present invention, a means for circulating the captured and blocked sample liquid in the measurement circuit is a pump (17 in FIG. 3, FIG. 4).
17) is used.

3. Means for injecting a predetermined amount of Limulus reagent into the circulating sample liquid to mix both liquids In the present invention, as a means for injecting a predetermined amount of Limulus reagent into the circulating sample liquid and mixing both liquids to form a mixed liquid , A pump is used, but the above 2. The pump described in 1 may also be used. It is preferable to use a diaphragm pump that is easy to clean. The mixer 18 may be optionally installed in the measurement circuit in order to improve the mixing of the two liquids. As the mixer 18, there are various static mixers, fiber aggregates and the like. The means for injecting the Limulus reagent is a form in which the Limulus reagent can be injected,
Although it may be in any form, for example, a micropump (the check valve at the discharge port may also serve as the valve 12) or a microsyringe with an injection needle whose position can be controlled by a micromotor (referred to as a Limulus reagent injector 10) In some cases) etc. When the Limulus reagent injector 10 is used, the opening / closing valve 12 is replaced with an opening / closing valve to form a perforation by a thin tube such as an injection needle, and a partition having the ability to self-close when the thin tube is removed. In this case, the Limulus reagent injection pipe 11 may not be used.

4. Fine particle counting means In the present invention, the fine particle counting means is a continuous measurement of the time-dependent change in the count value of fine particles that causes turbidity generated in the mixed solution introduced into the measurement circuit, and the measured value is processed. Is a means for calculating the endotoxin concentration in the sample,
From the light source (13 in FIG. 4, 13 in FIG. 5), the photodetector (14 in FIG. 4, 14 in FIG. 5), the electronic circuit that calculates, displays, and records the output of the photodetector, and the measurement cell. Composed. An example of a particle counter that can be used for such particle counting means is the following means described in Japanese Patent No. 3151036. That is, the particle counting means is arranged near the coherent light source 13, an optical system that collects the light from the coherent light source, and the focal point of the light beam collected by this optical system, and the flow of the fluid that contains particles inside. Is passed through the measurement cell 16, a photodetector 14 arranged on the optical path of the light flux and on the side opposite to the light source of the light flux with respect to the measurement cell 16, and an electrical signal from the photodetector 14 determines that the And an electric circuit 23 for measuring the number of fine particles. A light beam from the coherent light source can be collected by a lens.

The measuring cell can be made of any material such as glass, but the part through which the light flux passes must be transparent. A semiconductor laser can be used as the coherent light source, and the photodetector can be a photodiode. The coherent light source can be composed of a laser oscillator and a collimator lens. Any laser oscillator can be used, but the shorter the oscillation wavelength, the higher the detection sensitivity. A feature of the detection method is that a laser oscillator having a small oscillation output, for example, a semiconductor laser can be used as a light source. Output is 1m
It is possible to measure even with a semiconductor laser of W or less, specifically 0.2 mW.

The focal lengths of the focusing system and the optical lens are selected according to the size of the fine particles to be detected. For example, when detecting fine particles having a particle diameter of 0.2 μm, a lens having a focal length of f = 10 mm can be used. The measuring cell is
It is necessary to make at least the light receiving surface and the transmitting surface of the focused light transparent. Since this measuring cell does not need to worry about stray light, it can have a simple structure, but means for preventing turbulence from occurring in the flow of the test fluid containing fine particles, for example, a laminar flow plate as an optical cell. It is preferable to provide it inside. In an actual device, it is preferable to provide position adjusting means for arranging the optical lens so that the focused light emitted from the laser is focused at a predetermined position of the measurement cell, for example, the center of the measurement cell.

Since the function of the photodetector 14 is to detect the diffraction image hidden in the transmitted light, the sensitivity is not so required. A single photodiode can be used in principle, but it is preferable to use a photodiode array. This photodiode array is preferably arranged perpendicular to the direction of the flow containing the particles to be detected and also perpendicular to the optical axis. Using the above-mentioned particle counting means, the light flux from the coherent light source is condensed, the flow of the fluid containing the fine particles is passed near the focus of the focused light flux, and the diffracted light diffracted by the fine particles in the fluid is converted into fine particles. A photodetector placed on the optical path of the light flux on the side opposite to the light source of the light flux with respect to the fluid flow containing Thus, it is possible to detect (measure) fine particles having a minimum diameter of 0.5 μm or less.

5. Means for discharging In the present invention, discharging means that a sample liquid containing endotoxin is taken into a measuring circuit including a measuring cell, and the blocked sample liquid is circulated in the measuring circuit, and a predetermined amount of limulus reagent is circulated in the circulating sample liquid. Is injected to mix both solutions, and after continuously measuring the change with time of the fine particles of the mixed solution, the mixed solution is discharged from the measurement circuit.
A transport means such as a pipe, a pipe and a hose (20 in FIG. 3) and a circulation pump as a transport power source. The open / close valve of the discharge pipe is in a closed state when the mixed liquid is circulated in the measurement circuit, and is in an open state when the mixed liquid is discharged.

The structure of the transportation means may be any structure as long as it can transport the sample liquid, but it is preferable that it is constituted by a circular pipe. The material of the pipe (pipe) is not particularly limited. It is also possible to use a soft tube (silicone rubber, soft PVC).

6. Filtration Means In the present invention, the filtration means is means for removing particles having a minimum diameter or more counted by the fine particle counting means in advance before the sample liquid containing endotoxin is taken into the measurement circuit (5 in FIG. 2, FIG. 4). 5), and a ceramic microfilter, a fluororesin microfilter, or the like, which does not easily adsorb the active ingredient in the sample liquid, is particularly preferable. In addition, it is preferable to provide a deaeration means (not shown) for removing bubbles after the microfilter. The description of the means in this section corresponds to claim 2.

7. Automatic switching continuous measuring means for a plurality of measurement systems In the present invention, the automatic switching continuous measuring means for a plurality of measurement systems is defined in claim 4 as "a plurality of measurement systems are switchably coupled in parallel to a sample liquid flow path, There are two cases of "means for automatically switching sequentially and continuously measuring", and the "plurality of measurement systems has a single light source and a corresponding rotation axis on which a photodetector is fixed as a central axis. And a plurality of measuring cells arranged on a concentric circle, and a plurality of measuring systems include a single light source that reciprocates in parallel with respect to the plurality of measuring cells and a photodetector corresponding thereto. There is a case that consists of. The case of claim 5 will be described in more detail in 7.1 below, and the case of claim 6 will be described in more detail in 7.2 below.

Here, a plurality of measuring systems means a system in which two or more single measuring systems are arranged in parallel. A singular measurement system is as shown in the conceptual diagram of FIG.
The measuring means 2 in one unit of the endotoxin concentration measuring device comprising a means 1 for taking in and sealing the sample liquid, a measuring means 2 for the sample liquid, and a means 3 for discharging and sealing the sample liquid. As shown in FIG. 4B, one (single) measurement system has two series, which are arranged in parallel. FIG. 4 shows a more specific configuration of this. As shown in FIG. 1C, the three measurement systems are one (single) measurement system in three series, which are arranged in parallel.

The means 1 for taking in and sealing the sample liquid is the connecting structure of the above-mentioned means, that is, the connecting structure shown in FIG. 2A, and the sample liquid taking-in pipe 7 and the filtration filter 5, It is composed of a detergent liquid injection pipe 8, a sample liquid opening / closing valve 6, and a detergent liquid opening / closing valve 9. A deaerator (not shown) may be provided after the filtration filter 5. The measuring means 2 for the sample liquid is as shown in FIG.
It is the coupling structure shown in (B), and has a measuring circuit 15, a measuring cell 16, a light source 13, a detector 14, and a circulation pump 1.
7, Limulus reagent injection pipe 11, open / close valve 12
And a mixer 18 (optional). The means 3 for discharging and sealing the sample liquid is shown in FIG.
This is a connection structure of the discharge pipe 20 and the opening / closing valve 19 of the discharge pipe.

In the case of a plurality of measurement systems, a part of the sample liquid intake means can also be used, and therefore (B) and (C) in FIG.
As shown in, the upstream of the part including the filtration filter 5 is
It is composed of one sample liquid intake pipe 7 and one filtration filter 5, and since it cannot be used downstream from these,
The pipe is also divided into two or three. The use of a plurality of measurement systems has the effect of making it possible to shorten the interval for measuring the endotoxin concentration as shown in FIG.

7.1 Plural Measuring Cells Arranged on Concentric Circles with a Single Light Source and a Photodetector Corresponding to the Single Light Source Fixed as a Center of Rotation Axis In the present invention, one measuring system (measuring cell ), A set of light source and photodetector is used. However, since the light source and photodetector are expensive, in case of multiple measurement systems (measurement cells) Attaching a pair of light source and photodetector increases cost, so fix a single light source and its corresponding photodetector to a specific part of the measuring device, and rotate that specific part to multiple measuring cells. Then, the number of fine particles in the measurement cell is measured by sequentially corresponding to each measurement system. It has the effects of reducing the cost and volume and weight of the concentration measuring device, making it compact, saving space and making it easy to carry, and reducing the number of maintenances.

The description of the means in this section corresponds to claim 5, and will be described in more detail with reference to FIGS. 5 (A) and 5 (B). In FIG. 5A, one set of light source 13 and detector 14 is used for one measurement system (measurement cell 16), and this set of 13 + 14 is represented by being enclosed by a rectangle. In FIG. 5B, four measurement systems (four measurement cells 16 are arranged) are shown in the upper stage,
There is a set of rotatably movable light source 13 and photodetector 14 fixed in the middle stage, and 13 + 14 is shown surrounded by a rectangle. The multiple measurement systems consist of a single light source and a measurement circuit that includes multiple measurement cells arranged concentrically around a rotation axis that has a fixed photodetector and is fixed to the upper left end. One measuring system (measurement cell 16)
Then, a pair of light sources 13 and photodetectors 14 in the middle stage, which are rotatable and movable, are moved and united to be in the state of the lower stage, and the number of fine particles is measured. Then, a pair of light sources 13 and photodetectors 14 in the middle stage, which are rotatably movable, are moved and united with the second fixed measurement system (measurement cell 16) from the left in the upper stage. Then, the number of fine particles is measured. Sequentially, and the number of fine particles is measured. The rotation axis may be fixed and the measurement circuit including a plurality of measurement cells arranged on the concentric circumference may be rotated.

7.2 A single light source that reciprocates in parallel to a plurality of measurement cells and a photodetector corresponding thereto In the present invention, in principle, one set of light sources is provided for one measurement system (measurement cell). However, in the case of multiple measurement systems (measurement cells), one set of light source and photodetector is used for each measurement system (measurement cell). Attaching a single light source that moves back and forth in parallel to multiple measurement cells and a photodetector corresponding to it will reduce the cost and reduce the volume and weight of the concentration measurement device, downsize it, and save space. It has the effect of making it easy to carry and reducing the number of maintenance.

The description of the means in this section corresponds to claim 6, and will be described in more detail with reference to FIGS. 6A and 6B. In FIG. 6 (A), one set of light source 13 and photodetector 14 is used for one measurement system (measurement cell 16), and this set of 13 + 14 is surrounded by a rectangle. In FIG. 6B, a pair of light sources 13 and photodetectors 14 that can move to the upper stage are shown, and 13 + 14 is surrounded by a rectangle. The movement is indicated by an arrow, and the measurement system is combined with four measurement systems (four measurement cells 16 are arranged side by side) which are fixed to the lower stage. That is,
One set of movable light source 13 and one set of photodetector 14 (13
+14) is the measurement system (measurement cell 1
6) and the number of fine particles is measured. Then, a pair of movable light source 13 and photodetector 14 (13 + 1
4) is the measurement system in the lower state (measurement cell 16)
And the number of fine particles is measured. Furthermore, the number of fine particles is measured by incorporating the measurement system (measurement cell 16) in the states of and.

8. Endotoxin Concentration Measuring Device The endotoxin concentration measuring device of the present invention is a structure in which the above-mentioned means are organically combined. As a superordinate concept, in the case of a single measurement system, as shown in the conceptual diagram of FIG. 1A, a means 1 for taking in and blocking a sample solution, a measuring means 2 for a sample solution, and discharging and blocking the sample solution It is a device for measuring the endotoxin concentration of 1 unit, which is Means 3. In the case of two measurement systems, as shown in FIG. 1 (B), there is one (single) measurement system in two series, and two units are in parallel to measure the endotoxin concentration. A more specific configuration of this is shown in FIG. In the case of three measurement systems, as shown in (C) of FIG. 1, there is one (single) measurement system in three series, which is an apparatus for measuring endotoxin concentration of three units in parallel.

9. Method of Measuring Endotoxin Concentration In the present invention, the endotoxin concentration is measured by the following method using the endotoxin concentration measuring device of the present invention. First, a sample liquid containing endotoxin (injection liquid,
Medicines such as infusions and dialysates, and intermediate products or purified water that is a part of these raw materials) are stored in storage tanks, tanks, glass bottles, plastic containers, storage means such as plastic bags, or A sample liquid is pumped from a supply line through which these are flowing to a measuring circuit including a measuring cell by a means such as a pipe. The pump can be omitted when the pressure is fed from the supply line. In this case, before being taken into the measuring circuit, a filter provided in a part of the pipe removes particles having a minimum diameter or more counted by the particle counting means. Then, after the sample liquid is put into the measurement circuit, the inlet of the measurement circuit and the valve at the inlet are closed to close the measurement circuit.

Then, the sample liquid in the measuring circuit is circulated. Open the valve of the Limulus reagent injection pipe, inject a predetermined amount of Limulus reagent into the circulating sample liquid in the measurement circuit, circulate the sample liquid and Limulus reagent to make a mixed solution of both, and in the process of gelation reaction The concentration of endotoxin is calculated by measuring with a particle counting means for continuously measuring the time-dependent change of the generated particles. After the measurement is completed, the mixed solution is discharged from the measuring circuit. For that purpose, the intake valve 6 and the valve 19 of the discharge pipe are opened, and the sample solution to be measured in the measuring circuit is discarded from the discharge pipe with the sample liquid to be newly introduced. In this case, in order to clean the inside of the measurement circuit, the valve 9 of the cleaning liquid pipe is opened and the cleaning liquid is introduced into the measurement circuit,
After cleaning the inside, or circulating and retaining for a certain time,
The mixed solution in the measurement circuit may be discharged together with the cleaning solution and discarded.

10. Measuring method when the endotoxin concentration in the sample solution is low level Usually, the sample solution amount and the Limulus reagent amount are 0.1: 1 of a mixed solution of 0.1 to 0.2 ml obtained by mixing both solutions at a ratio of 1: 1. Endotoxin concentration is measured by measuring the turbidity at rest. However, in order to measure the number of fine particles (concentration calculated from them) that is the cause of turbidity with a fine particle counter, it is a necessary condition to flow the mixed solution across the light flux. However, with a normal mixed liquid of 0.1 to 0.2 ml,
Even in the case of circulation, it is very difficult to make it flow because it requires an extra volume for it. Even if you try to sprinkle the mixed solution, the Limulus reagent is very expensive, so increasing the amount used to the amount of the sample liquid will result in a significant economic burden, and the particle counting It becomes a big obstacle when measuring with a meter. It is considered that most of the Limulus reagent mixed with the normal amount of the sample solution remains without reacting with the endotoxin when the endotoxin concentration of the sample solution is low. Therefore, 10 times to 100 times the amount of reagent
It is considered that even if 0 times the amount of the sample solution is mixed, the amount of the endotoxin in the sample solution that reacts with the Limulus reagent is almost the same as in the case of mixing the same amount. That is, in the present invention, since the measurement is performed on a sample liquid having a low endotoxin concentration,
The amount of sample liquid is 10 times the amount of Limulus reagent to 1000
For the first time, it was possible to measure fine particles generated during the gelation reaction by using a mixed solution obtained by mixing both solutions after doubling the amount. The description of the method in this section corresponds to claim 7.

11. Automatic switching diagram when parallel measurement cells are 3 series As shown in FIG. 1C, when the measurement cells are 3 series, the first series, the second series, and the third series have constant time intervals. Fine particles are sequentially measured at intervals, and then cleaning with each series of cleaning solutions is sequentially performed at regular intervals. When the washing is completed, the fine particles are measured again. FIG. 9 shows an automatic switching diagram in the case where three parallel measurement cells are used. In the present invention, the washing solution dissolves glycolipids and proteins, and even if a trace amount remains, it does not adversely affect the sample solution, endotoxin, limulus reagent, etc., and promotes the reaction between endotoxin and limulus reagent. Any type may be used as long as it does not inhibit or inhibit, and it can be selected from an aqueous hydrochloric acid solution and a surfactant.

12. Particularly preferred embodiment In the turbidimetric method for measuring the turbidity generated by the reaction between endotoxin and Limulus reagent, the turbidimetric method for measuring the time to reach a preset turbidity is used as a method for measuring a sample solution having a low endotoxin concentration. The reaction time method or the turbidimetric reaction rate method for measuring the rate of change of turbidity with time is often used (Takezawa Shingo, ed.
Page 8 (1995), Tokyo Medical Co., Ltd.).

When the concentration of endotoxin is low, the logarithm of the time to reach the preset turbidity increases in inverse proportion to the logarithm of the endotoxin concentration or the logarithm of twice, so the set turbidity is set to the lowest measurable value. Therefore, the monitor is required to minimize the measurement time. Therefore, it is important to measure at lower turbidity. Therefore, it is conceivable to use a fine particle counter instead of the turbidimeter as a highly sensitive measuring method.

However, since the light flux of the fine particle counter is thin and trapping (= counting) fine particles in it is a stochastic phenomenon, the target liquid can be counted up to a statistically reliable count (several tens or more). Is scanned (the light flux is fixed and the liquid is made to flow transversely), and the concentration is obtained by dividing the light flux by the volume (transverse flow rate × time) across the light flux. That is, when the concentration of the fine particles becomes low, it is necessary to increase the volume to be measured in inverse proportion, and the measurement time becomes longer. Furthermore, the measurement target of the turbidimeter is held in a limited space, whereas the measurement target of the particle counter is required to flow across the light flux, so the sample to be measured and the Limulus reagent are Since the volume of the mixed reaction solution is as small as 0.2 to 0.1 cm ³ , it is not easy to circulate the reaction solution in the measurement cell in a closed system.

[Embodiment 1] As a basic example of a system in which a plurality of devices are switchably connected in parallel to a flow path of a sample and automatically switched in sequence to perform continuous measurement, two sets of devices (one set of FIG. 5 shows an embodiment in which a light source and a light receiver can be shared) is connected to the flow path of the target liquid in parallel in a switchable manner, and is automatically switched in sequence to perform continuous measurement. FIG. 9 shows an example of an automatic switching operation diagram when the parallel devices are three series.

[Embodiment 2] The amount of the reaction solution to be measured is as small as 0.1 to 0.2 cm ³ because it is prepared by mixing the same amount of the sample as the amount of the reagent usually used. Therefore, it is necessary to set the internal volume of the measuring cell to 0.1 cm ³ or less. However, the Limulus reagent should contain an active ingredient in an amount of 0.1 to 0.05 cm ³ which is usually used, in an amount capable of coping with endotoxin having a concentration of 10 ³ EU / L or more. Therefore, in the case of the 10 ¹ EU / L digit level sample, it is considered that most of the Limulus reagent mixed with the normal amount of the sample solution remains without reacting with endotoxin. Even if 10 times to 1000 times the amount of the sample liquid is mixed with respect to the amount of the reagent, the amount of the endotoxin in the sample liquid that reacts with the Limulus reagent is considered to be almost the same as in the case of mixing the same amount. In the present invention, since a sample liquid having a low endotoxin concentration is measured, the amount of the sample liquid is adjusted to 10 times to 1000 times the amount of Limulus reagent, and then a mixed liquid obtained by mixing both liquids is used. For the first time, it has become possible to measure the fine particles generated during the gelation reaction. That is, the amount of the mixed liquid is 1 to 20 cm ³ , and it is much easier to circulate the mixed liquid in the measurement cell as compared with the case where the amount of the mixed liquid is 0.1 to 0.2 cm ³ .

[Embodiment 3] It is expected that the sensitivity is increased by reducing the size of fine particles measured by a turbidimeter. The number of microparticles decreases in inverse proportion to the cube of the size during the process where endotoxin reacts with Limulus reagent to generate minute coagulation bodies (microparticles) that cause turbidity and associate with each other to finally gelate. . Therefore, if the size of the fine particles to be measured is set small, the volume of the mixed solution to be measured is reduced by narrowing the light beam, but the frequency of the fine particles crossing the light beam becomes higher, and the measurement time is Shortening is almost inversely proportional to the size of the number. Also, the fact that the detection size can be set small enables measurement at an early stage of the gelation reaction of endotoxin and Limulus reagent, which contributes to reduction in measurement time.

The weight concentration of endotoxin was 0.2 [ng / L] when the concentration of coagulant fine particles (counting concentration) in the sample liquid having an extremely low endotoxin concentration of 1 [EU / L] was estimated.
(If it is derived from E. coli 0113: H10, it will be the already-explained “Dialysate ET well-known book” page 29), and endotoxin in the dialysate has a molecular weight of about 1 in an associated state.
Since it is about 0 ⁶ [g / mol], 1.2 × 10 ⁸ [pieces /
The number density is about L]. On the other hand, the diameter of the amphipathic aggregate having a molecular weight of about 10 ⁶ is about (1-2) × 10 ⁻² [μ
m], the solidified bodies generated by the reaction with the Limulus reagent are gradually aggregated, and the lower limit of the particle counter,
For example, when the diameter grows from 0.1 to 0.5 to 0.1 [μm], the concentration of the first unreacted ET as fine particles is about 1.2.
× 10 ⁸ [pieces / L] decreases in inverse proportion to the cube of the diameter ratio, and the lower limit of detection of the particle counter, for example, 0.1 [μm]
At 10 ² to 10 ³ cells / mL, at 0.5 [μm] 10 ⁰ to
10 ¹ / mL, 10 ⁻² to 10 ⁻¹ / [μm]
Becomes mL.

When it is attempted to measure the concentration of the coagulated substance with a fine particle counter, fine particles such as air bubbles (preliminarily degassed and removed) and dust existing in the dialysate (preliminarily filtered and removed).
Those that cannot be completely removed interfere with noise, so that measurement with the count of microparticles derived from endotoxin lowered to the level of the noise number loses the reliability of the measured value. Therefore, the lower the endotoxin concentration of the sample,
It is necessary to measure at the stage where the diameter of the coagulated body particles is small. 0.5 when the endotoxin concentration is 1 EU / L
It is estimated that it will be 10 ¹ / mL or less at the stage of growth up to μm, and it may be buried in noise depending on the sample, so it is at least about 0.5 μm or less, preferably 0.2 μm or less.
Counting at about 0.1 μm is an essential condition for measuring the endotoxin concentration with a fine particle counting turbidimeter. This condition also matches the purpose of shortening the measurement time by measuring at an early stage of the reaction of endotoxin and Limulus reagent.

[0054]

The present invention will be described in more detail based on the following examples, but the invention is not intended to be limited by these examples.

[Example 1] Sample liquid intake pipe 7, opening / closing valve 6, detergent liquid injection pipe 8 (not shown), opening / closing valve 9 (not shown), filtration filter 5 (not shown), deaeration device (not shown) ), Limulus reagent injection pipe 11, opening / closing valve 12, measuring circuit 15, measuring cell 16, circulation pump 17,
Static mixer 18 (not shown), open / close valve 1
9, a series of endotoxin concentration measuring devices consisting of a combination of a discharge pipe 20, a thermostat 21 (not shown), a movable light source 13 and a photodetector 14, and 3 series of endotoxin concentration measuring devices arranged in parallel. Created the device. This is shown in FIG.

[Embodiment 2] In Embodiment 1, the limulus reagent injection pipe 11 and the on-off valve 12 were replaced with a microsyringe (limulus reagent injector 10) with an injection needle whose position can be controlled by a micromotor. An endotoxin concentration measuring device having the same structure as the device of Example 1 was prepared.

[0057]

According to the device of the present invention, an injection solution, an infusion solution,
From the storage tank or supply line of the drug such as dialysate and its intermediate product, the sample liquid such as purified water, a part of the sample liquid can be taken in with a pipe and the concentration of endotoxin can be continuously monitored. Concentration is 50E
Even a low-concentration liquid of U / L or less, particularly an extremely low-concentration liquid of 10 to <1 EU / L level can be monitored, and the endotoxin concentration can be monitored in a short time.

[Brief description of drawings]

FIG. 1 is a conceptual diagram showing a configuration of a device. Figure (A) shows
Means for capturing and sealing the sample liquid, means for measuring the sample liquid,
The means for discharging and blocking the sample liquid is one series, FIG. (B) is two series, and FIG. (C) is three series.

FIG. 2 is a diagram showing an example of means for taking in and sealing a sample liquid. FIG. (A) shows a case where the mixed liquid circulates in the measurement circuit, and FIG. (B) shows that a part of the upper portion of the measurement circuit is a measurement cell.

FIG. 3 is a diagram showing an example of means for measuring a sample liquid. The figure (A) shows the case of one series, the figure (B) shows the case of two series, and the figure (C) shows the case of three series.

FIG. 4 is a diagram showing an example of a device in which a plurality of devices are installed in parallel and are automatically switched to successively measure in sequence.

FIG. 5 shows a plurality of measuring cells rotating around a single light source and its corresponding photodetector.

FIG. 6 is a diagram showing a single light source that moves back and forth in parallel with respect to a plurality of measurement cells and a corresponding detector.

FIG. 7 is a device diagram showing a plurality of measurement systems, each of which is composed of a single light source that reciprocates in parallel with respect to a plurality of measurement cells juxtaposed in a row and a photodetector corresponding thereto, and FIG. Figure (A)
Is a bird's-eye view, FIG. (B) is a side view, and (C) is a plan view.

FIG. 8 is a view showing a plurality of measurement systems each including a single light source and a measurement circuit including a plurality of measurement cells arranged concentrically around a rotation axis having a fixed photodetector as a central axis; FIG. Figure (A) is a bird's eye view, Figure (B) is a side view, and (C) is a plan view.

FIG. 9 is a diagram showing an automatic switching diagram in the case where three parallel measurement cells are provided.

[Explanation of symbols]

1 Means for Capturing and Sealing Sample Liquid 2 Means for Measuring Sample Liquid 3 Means for Discharging and Sealing Sample Liquid 4 Pump for Capturing Sample Liquid 5 Filtration Filter 6 for Sample Liquid 6 Opening / closing valve for sample liquid intake pipe 7 Sample liquid intake pipe 8 Detergent Liquid injection pipe 9 Open / close valve for detergent liquid injection pipe 10 Limulus reagent injector 11 Limulus reagent injection pipe 12 Open / close valve or partition for Limulus reagent injection pipe 13 Light source 14 Photodetector 15 Measuring circuit 15-1 Circulating mixture inlet 15- 2 Circulating mixed liquid outlet 16 Measuring cell 17 Circulating pump (also serves as intake pump, cleaning / discharging pump) 18 Static mixer 19 Discharge pipe opening / closing valve 20 Discharge pipe 21 Constant temperature bath 22 Luminous flux 23 Electric circuit (calculation unit)

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Tokuzo Harada             3-13-2 Ojimachi, Abeno-ku, Osaka-shi, Osaka (72) Inventor Kaoru Miura             2-8-1-401 Todaishima, Urayasu City, Chiba Prefecture F term (reference) 2G052 AA06 AB18 AB22 AD06 AD26                       AD49 BA03 BA14 CA03 CA04                       CA12 CA20 CA35 EA03 FB03                       FB06 FC04 FC11 FC15 GA09                       GA11 HC07 HC10 HC39 JA07

Claims (7)

[Claims] 1. A means for capturing and sealing a sample liquid containing endotoxin in a measuring circuit including a measuring cell, a means for circulating the captured and blocked sample liquid in the measuring circuit, and a predetermined amount of limulus in the circulating sample liquid. A means for injecting a reagent to mix both solutions, a particle counting means for continuously measuring the time-dependent change in the concentration of particles causing turbidity generated in the mixed solution introduced into the measurement cell, and after measurement A device for measuring the endotoxin concentration, which comprises a means for discharging the mixed solution from the inside of the measurement circuit and washing the measurement circuit, wherein the time for the count value to reach a predetermined change amount in the fine particle counting means or the change amount An endotoxin concentration measuring device, wherein the concentration of endotoxin contained in a sample liquid is calculated by measuring the rate of change over time. 2. The filtering means for removing particles having a minimum diameter or more counted by the particle counting means in advance before the sample liquid containing endotoxin is taken into the measuring circuit. Measuring device for endotoxin concentration. 3. The fine particle counting means is a fine particle counter for setting the minimum diameter of fine particles to be counted to 0.5 μm or less, and the time for the amount of change to reach a predetermined amount from the count value, or the rate of change over time. The endotoxin concentration measuring device according to claim 1, further comprising an electronic circuit for calculating the endotoxin concentration in the sample liquid by comparing with the calibration curve prepared in advance. 4. A plurality of measurement systems are switchably coupled in parallel to a flow path of a sample liquid and are automatically switched in sequence to measure fine particles and wash a measurement circuit including a measurement cell in parallel. The endotoxin concentration measuring apparatus according to claim 1, wherein the endotoxin concentration measuring method is performed or continuously measured at intervals shorter than a time required for one measurement. 5. A plurality of measuring systems comprises a single light source and a measuring circuit including a plurality of measuring cells arranged concentrically around a rotation axis having a fixed photodetector as a central axis. The endotoxin concentration measuring device according to claim 4. 6. The plurality of measurement systems comprises a single light source that reciprocates in parallel with respect to a plurality of measurement cells juxtaposed in a row, and a photodetector corresponding to the single light source. Measuring device for endotoxin concentration. 7. When the endotoxin concentration in the sample liquid is at a low level, the amount of the sample liquid is adjusted to 10 times to 1000 times the amount of Limulus reagent, and then a mixed liquid obtained by mixing both liquids is used. It measures, The measuring apparatus of the endotoxin density | concentration in any one of Claims 1-6 characterized by the above-mentioned.