JPH10293129A - Method for measuring endotoxin in phospholipid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To determine endotoxin in phospholipid accurately by emulsifying or dispersing phospholipid into water containing an anionic polymer metal salt and an alkaline metal salt, removing phospholipid from the water and then determining endotoxin in the remaining water. SOLUTION: 0.01-0.5 pts.wt. of anionic polymer metal salt, e.g. sodium polyacrylate, having weight-average molecular weight of 500-30000, and 0.01-2 pts.wt. of alkaline metal salt, e.g. lithium fluoride, are added to 100 pts.wt. of sodium chloride brine. 0.1-5 pts.wt., preferably 0.5-2 pts.wt., of phospholipid derived from a natural product or synthesized phospholipid or liposome formed therefrom is then added to 100 pts.wt. of the aqueous solution and the phospholipid is emulsified or dispersed into the aqueous solution by means of a stirrer or an emulsifier. Subsequently, the phospholipid is separated and removed by filtering or centrifugal separation and the remaining aqueous solution is subjected to colorimetry thus determining endotoxin.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for measuring endotoxin in phospholipids.

[0002]

2. Description of the Related Art Endotoxin is known as a substance that exhibits various biological activities such as lethality, pyrogenicity, adjuvant action, etc., and also causes serious symptoms such as shock upon infection with Gram-negative bacteria and sepsis. . In addition, it is the most powerful substance as a pyrogen, and its incorporation into medicines, blood products, and vaccines causes serious side effects, and the degree of its incorporation requires strict control. In recent years, the application of phospholipids to liposome preparations as one of drug delivery systems has been vigorously pursued by pharmaceutical manufacturers. Currently, intravenous injection is the mainstream for the administration of liposome preparations. In the case of intravenous injection, the amount of endotoxin in the liposome preparation becomes a very serious problem. Therefore, the Japanese Pharmacopoeia sets up a test item for pyrogenic substances and regulates the amount of endotoxin in the preparation.
Therefore, there is a need for a method for measuring endotoxin in a phospholipid used as a base material of a liposome preparation.

[0003] Limulus amebocyte lysate
e, hereinafter abbreviated as LAL) As a method for measuring endotoxin using a component, two methods, a gelling method and a synthetic substrate method, have already been established. For example, gelling methods include Pregel (S) [Seikagaku Corporation] and Limulus (HS) Test Wako [Wako Pure Chemical Industries, Ltd.]. Synthetic substrate methods include Toxicolor System and Endotoxin Test. -D
Kits such as Seikagaku Kogyo Co., Ltd. are commercially available.

[0004] The gelation method is susceptible to vibration during reaction and measurement, and tends to be subjective in measurement. Furthermore, since it is a qualitative test method, in order to quantitatively determine the concentration of endotoxin, it is necessary to adjust the dilution series of the sample, and further, there is a problem that the quantitative accuracy is low. On the other hand, since the synthetic substrate method performs colorimetric quantification, it is an excellent method that enables objective measurement and can be quantified with a sensitivity several tens times or more that of the gelation method. However, since all of these measurement methods are performed in an aqueous system, it has been difficult to measure the amount of endotoxin in a phospholipid that is hardly soluble in water.

As a method for measuring endotoxin in phospholipids, there is a method disclosed in Japanese Patent Application Laid-Open No. 230083/1993, which extracts endotoxin in phospholipids with water and then extracts the phospholipids from water. And measuring the amount of endotoxin contained in the water.

However, Japanese Patent Application Laid-Open No. 5-320043 discloses that a liposome composed of phospholipid, cholesterol and saturated fatty acid or an aqueous solution containing the liposome can be an endotoxin-capturing agent.
Liposomes formed by phospholipids in water may trap endotoxin, and endotoxin may not be completely extracted into water, and accurate measurements may not be obtained.

[0007]

SUMMARY OF THE INVENTION The present invention solves the above-mentioned problems of the prior art and provides a method for accurately measuring endotoxin in a phospholipid by a synthetic substrate method using a LAL component. It is an object.

[0008]

That is, the present invention provides a method for emulsifying or dispersing a phospholipid in water containing an anionic polymer metal salt and an alkali metal salt and then removing endotoxin from the remaining water from which the phospholipid has been removed. A method for measuring endotoxin in phospholipids, characterized by quantification

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The phospholipid for measuring endotoxin is not particularly limited, and for example, soybean lecithin,
Phospholipids derived from natural products or synthesized by natural substances such as egg yolk lecithin, dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, and liposomes formed by these are widely used as measurement targets. The ratio of the phospholipid to water is 0.1 to 5 parts by weight, preferably 0.5 to 2 parts by weight, based on 100 parts by weight of water. If the amount is less than 0.1 part by weight, the measurement accuracy of endotoxin deteriorates, and if it exceeds 5 parts by weight, separation of phospholipid and water deteriorates, and measurement accuracy of endotoxin also deteriorates.

The anionic polymer metal salt used in the present invention may be any one which does not affect the reaction between the LAL component and endotoxin. Examples thereof include sodium polyacrylate, sodium polyvinylsulfonate and polystyrenesulfonic acid. Sodium salt, styrene / maleic acid copolymer sodium salt, vinyl acetate / maleic acid copolymer sodium salt and the like can be mentioned. Further, the molecular weight is from 500 to 30,000, preferably from 1,000 to 10,000, and more than 500 or more than 30,000 results in poor measurement accuracy. The amount used is 0.01 to 0.5 part by weight, preferably 0.02 to 0.04 part by weight, based on 100 parts by weight of water,
If the amount is less than 0.01 parts by weight or more than 0.5 parts by weight, the measurement accuracy is deteriorated.

The alkali metal salt used in the present invention includes:
Lithium fluoride, sodium fluoride, potassium fluoride, lithium chloride, sodium chloride, potassium chloride, lithium bromide, sodium bromide, potassium bromide, lithium iodide,
Examples thereof include sodium iodide and potassium iodide, and preferred examples include lithium chloride, sodium chloride, and potassium chloride. The amount used is preferably in the range of 0.01 to 2 parts by weight with respect to 100 parts by weight of water. If less than 0.01 or more than 2 parts by weight, the measurement accuracy tends to decrease.

The water used in the present invention preferably has a low endotoxin content, and includes, for example, distilled water, distilled water for injection, and physiological saline, and is preferably a physiological saline containing sodium chloride. is there. In the present invention, as a method for emulsifying or dispersing the phospholipid and water, a commonly used stirrer, emulsifier, or the like can be used. Removal of phospholipids is performed by filtration or centrifugation. For example, in the case of centrifugation, the higher the rotation speed, the better the separation, and the better the separation efficiency and measurement accuracy.

[0013]

According to the method for measuring endotoxin in phospholipid of the present invention, endotoxin can be quantitatively determined with high accuracy.

[0014]

Next, the present invention will be described specifically with reference to examples. The instruments used in the examples were all 250
Heat treated at 5 ° C. for 5 hours. “EU” used in the measurement results is an endotoxin unit, which is a unit indicating the gelation activity of LAL by endotoxin.

Example 1 A 100 ml screw tube was filled with an aqueous solution of sodium polyacrylate (Aldrich, weight average molecular weight 12).
(00, 45% by weight), and physiological saline (containing 0.9% by weight of sodium chloride, manufactured by Otsuka Pharmaceutical Co., Ltd.) was added to make a total volume of 60 ml. This test solution 1
After adding 100 mg of dipalmitoylphosphatidylcholine (hereinafter abbreviated as DPPC) to 10 ml of the mixture, the mixture was stirred with a vortex mixer, and then ultrasonically treated (UT-105, manufactured by Sharp Corporation) for 30 minutes to emulsify. This emulsion is centrifuged (H-2000C manufactured by Domestic Centrifuge Co., Ltd.).
And centrifuged at 5000 rpm for 30 minutes.
Water from which C was removed was obtained. This is designated as test liquid 2. Also,
Separately, 20 μl of an endotoxin aqueous solution (100 EU / ml) was placed in each of three 50 ml screw tubes,
Take 50 μl and 100 μl, add test solution 1 to make the total volume 10 ml each, further add 100 mg of DPPC, and prepare water from which DPPC was removed by the same operation as above. Each of the obtained test liquids was used as test liquid 3 (endotoxin content 0.02 EU / ml), test liquid 4 (endotoxin content 0.05 EU / ml), and test liquid 5 (endotoxin content 0.1 EU / ml). Using 100 μl of each of Test Solutions 1 to 5, colorimetric quantification was performed at 545 nm according to the operation method of Toxicolor System (manufactured by Seikagaku Corporation). Table 1 shows the results.

[0016]

[Table 1]

As is clear from Table 1, the added endotoxin was well measured, and the endotoxin in the sample could be easily measured by the method for measuring endotoxin in the phospholipid of the present invention.

Example 2 20 μl, 50 μl of an endotoxin aqueous solution (100 EU / ml) were placed in three 50 ml screw tubes, respectively.
100 μl was taken, and the total volume was adjusted to 10 ml with physiological saline (containing 0.9% by weight of sodium chloride, manufactured by Otsuka Pharmaceutical Co., Ltd.). Further, 100 mg of each DPPC was added, and the mixture was stirred with a vortex mixer, and then sonicated (UT-105, manufactured by Sharp Corporation) for 30 minutes to emulsify, and 0.02 EU / ml, 0.05 EU / ml, and 0. 1
DPPC containing endotoxin at a concentration of EU / ml
Three liposome solutions were obtained. Each of these three DPPC liposome solutions was added to a polyacrylic acid sodium salt aqueous solution (Aldrich, weight average molecular weight 1200, 45).
(% By weight), added, mixed, and sonicated (UT-105, manufactured by Sharp Corporation) for 20 minutes after mixing. By the same operation as above, water from which DPPC had been removed was prepared. Each of the obtained test solutions was used as a test solution 6 (endotoxin content 0.02 EU / ml), a test solution 7 (endotoxin content 0.05 EU / ml), and a test solution 8 (endotoxin content 0.1 EU / ml). , Each 100μ
l, Toxicolor system (Seikagaku Corporation)
Colorimetric determination at 545 nm according to the operating method of Table 2 shows the results.

[0019]

[Table 2]

As is clear from Table 2, the added endotoxin was well measured, and the endotoxin contained in the liposome could be easily measured by the method for measuring endotoxin in the phospholipid of the present invention. .

Comparative Example 1 10 ml of distilled water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was placed in a 50 ml screw tube, 100 mg of DPPC was added thereto, and the mixture was stirred with a vortex mixer, followed by sonication for 30 minutes (Sharp Corporation). UT-105) and emulsified. This emulsified product is centrifuged by a centrifuge (Hokuto Centrifuge Co., Ltd.)
-2000C) for 30 minutes at 5000 rpm to obtain water from which DPPC had been removed. Test solution 1
Set to 0. Separately, 20 μl, 50 μl, and 100 μl of an endotoxin aqueous solution (100 EU / ml) were respectively taken in three 50 ml screw tubes, and the total amount was made up to 10 ml with distilled water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.).
DPPC-free water was prepared by adding 100 mg of DPPC and performing the same operation as above. Each of the obtained test solutions was used as a test solution 11 (endotoxin content 0.02 EU / ml), a test solution 12 (endotoxin content 0.05 EU / ml), and a test solution 13 (endotoxin content 0.1 EU / ml). 545 nm according to the operation method of Toxicolor System (manufactured by Seikagaku Corporation) using 100 μl of each of test solutions 10 to 13
Was used for colorimetric determination. Table 3 shows the results.

[0022]

[Table 3]

As is apparent from Table 3, in the absence of the anionic polymer metal salt and the alkali metal salt, the detection rate of the added endotoxin was as low as 10 to 15%, and an accurate measurement value could not be obtained. I understand.

Comparative Example 2 Into a 100 ml screw tube, 60 ml of distilled water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was taken, and an aqueous solution of sodium salt of polyacrylate (manufactured by Aldrich, weight average molecular weight: 120)
0, 45% by weight) and mixed with 60 μl of test solution 14.
I got 100 ml of DPPC is added to 10 ml of this test solution 14.
g and vortex mixer, then
Ultrasonic treatment for 30 minutes (UT-105, manufactured by Sharp Corporation)
And emulsified. This emulsion was centrifuged at 5,000 rpm using a centrifuge (H-2000C manufactured by Domestic Centrifuge Co., Ltd.).
Centrifugation was performed for 0 minutes to obtain water from which DPPC had been removed. This is designated as test liquid 15. Separately, an endotoxin aqueous solution (100 EU /
20 μl, 50 μl, and 100 μl, respectively, and the total volume was adjusted to 10 ml with test solution 14, and DPPC 10
0 mg was added, and DPPC was performed in the same manner as above.
From which water was removed. Each of the obtained test liquids was sequentially added to test liquid 16 (endotoxin content 0.02 EU /
ml), test solution 17 (endotoxin content 0.05E)
U / ml), test solution 18 (endotoxin content 0.1
EU / ml).
Using 0 μl, colorimetric quantification was performed at 545 nm according to the operation method of Toxicolor System (manufactured by Seikagaku Corporation). Table 4 shows the results.

[0025]

[Table 4]

As apparent from Table 4, the detection rate of added endotoxin was 25% in the absence of an alkali metal salt.
%, Which indicates that accurate measurement values cannot be obtained.

Comparative Example 3 10 ml of physiological saline (containing 0.9% by weight of sodium chloride, manufactured by Otsuka Pharmaceutical Co., Ltd.) was placed in a 50 ml screw tube, 100 mg of DPPC was added, and the mixture was stirred with a vortex mixer. The mixture was sonicated (UT-105, manufactured by Sharp Corporation) for 10 minutes to emulsify. This emulsion was centrifuged at 5,000 rpm for 30 minutes using a centrifuge (H-2000C manufactured by Kokusan Centrifuge Co., Ltd.) to obtain water from which DPPC had been removed. This is designated as test solution 19. Separately, 20 μl each of an endotoxin aqueous solution (100 EU / ml) was placed in three 50 ml screw tubes.
μl and 100 μl, and the total amount was adjusted to 10 with physiological saline (containing 0.9% by weight of sodium chloride, manufactured by Otsuka Pharmaceutical Co., Ltd.).
ml of water, and 100 mg of DPPC was added, and the same operation as above was performed to prepare water from which DPPC had been removed. Each of the obtained test liquids was used as a test liquid 20 (endotoxin content 0.02 EU / ml), a test liquid 21 (endotoxin content 0.05 EU / ml), and a test liquid 22 (endotoxin content 0.1 EU / ml). , Test liquid 19
And 100 μl of the test solution 22 was used to prepare the solution according to the operation method of Toxicolor System (manufactured by Seikagaku Corporation).
Colorimetry was performed at 5 nm. . Table 5 shows the results.

[0028]

[Table 5]

As is clear from Table 5, in the absence of the anionic high-molecular metal salt, the detection rate of the added endotoxin was as low as 20 to 30%, indicating that accurate measurement values could not be obtained.

Example 3 An aqueous solution of sodium polyacrylate (Aldrich, weight average molecular weight 12) was placed in a 100 ml screw tube.
(00, 45% by weight), 60 ml of physiological saline (containing 0.9% by weight of sodium chloride, manufactured by Otsuka Pharmaceutical Co., Ltd.) was added to make a total volume of 60 ml, and a test solution 23 was obtained. To 10 ml of this test solution 23, 100 mg of distearoyl phosphatidylcholine (hereinafter abbreviated as DSPC) was added, and the mixture was stirred with a vortex mixer, followed by sonication (UT-105, manufactured by Sharp Corporation) for 30 minutes to emulsify. This emulsion is centrifuged (H-2000 manufactured by Domestic Centrifuge Co., Ltd.).
C), and centrifuged at 5000 rpm for 30 minutes.
Water from which SPC was removed was obtained. This is designated as test liquid 24.
Separately, an endotoxin aqueous solution (100 EU / ml) was added to each of three 50 ml screw tubes.
Take 50 μl, 50 μl, and 100 μl, add test solution 23 to make the total volume 10 ml each, and further add DSPC100
mgPC was added, and water from which DSPC was removed was prepared in the same manner as above. Each of the obtained test liquids was sequentially applied to test liquid 25 (endotoxin content 0.02 EU / m
l), test solution 26 (endotoxin content 0.05 EU)
/ Ml), test solution 27 (endotoxin content 0.1E)
U / ml).
Use Toxicolor System (Seikagaku Corporation)
Colorimetric determination at 545 nm according to the operating method of . Table 6 shows the results.

[0031]

[Table 6]

As is clear from Table 6, the added endotoxin was well measured, and the endotoxin in the sample could be easily measured by the method for measuring endotoxin in the phospholipid of the present invention.

Example 4 20 μl and 50 μl of an endotoxin aqueous solution (100 EU / ml) were placed in three 50 ml screw tubes, respectively.
Take 100 μl of physiological saline (0.9% sodium chloride).
% By weight, manufactured by Otsuka Pharmaceutical Co., Ltd.). Further, 100 mg of DSPC was added to each,
After stirring with a vortex mixer, the mixture was sonicated (UT-105, manufactured by Sharp Corporation) for 30 minutes to emulsify, and 0.02 EU / ml, 0.05 EU / ml, and 0.
DSP containing endotoxin at a concentration of 1 EU / ml
Three types of C liposome solutions were obtained. An aqueous solution of sodium polyacrylate (Aldrich, weight average molecular weight 1200, 45) was added to each of these three DSPC liposome solutions.
(% By weight), added, mixed, and sonicated (UT-105, manufactured by Sharp Corporation) for 20 minutes after mixing. By the same operation as above, water from which DSPC was removed was prepared. Each of the obtained test liquids was used as a test liquid 28 (endotoxin content 0.02 EU / ml), a test liquid 29 (endotoxin content 0.05 EU / ml), and a test liquid 30 (endotoxin content 0.1 EU / ml). , Each one
Using 00 μl, colorimetric quantification was performed at 545 nm according to the operation method of Toxicolor System (manufactured by Seikagaku Corporation). Table 7 shows the results.

[0034]

[Table 7]

As is clear from Table 7, the added endotoxin was well measured, and the endotoxin contained in the liposome can be easily measured by the method for measuring endotoxin in the phospholipid of the present invention. Was.

Example 5 A 100 ml screw tube was charged with an aqueous solution of sodium polyacrylate (Aldrich, weight average molecular weight 12).
(00, 45% by weight), and a 0.45% aqueous solution of sodium chloride [physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.)] and distilled water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) at a weight ratio of 1: 1. [Mixing] to make the total amount 60 ml, thereby obtaining a test solution 31. 100 mg of dimyristoyl phosphatidylcholine (hereinafter abbreviated as DMPC) was added to 10 ml of this test solution 31, and the mixture was stirred with a vortex mixer, followed by sonication (UT-105, manufactured by Sharp Corporation) for 30 minutes to emulsify. This emulsion is centrifuged (H-manufactured by Domestic Centrifuge Co., Ltd.).
(2000C) for 30 minutes at 5000 rpm to obtain water from which DMPC had been removed. This was added to test solution 32
And Separately, a 50 ml screw tube 3
Take 20 μl, 50 μl, and 100 μl of an endotoxin aqueous solution (100 EU / ml) into the book, and add test solution 3
1 to make the total volume 10 ml each, and then DMP
C was added, and the same operation as above was carried out.
Water from which MPC was removed was prepared. Each of the obtained test liquids was sequentially applied to test liquid 33 (endotoxin content 0.02
EU / ml), test solution 34 (endotoxin content 0.
05 EU / ml) and test solution 35 (endotoxin content 0.1 EU / ml), and using 100 μl of each of test solution 31 to test solution 35, according to the operation method of Toxicolor System (manufactured by Seikagaku Corporation). Colorimetry was performed at 545 nm. Table 8 shows the results.

[0037]

[Table 8]

As is clear from Table 8, the added endotoxin was well measured, and the endotoxin in the sample could be easily measured by the method for measuring endotoxin in the phospholipid of the present invention.

Claims (1)

[Claims] 1. A phospholipid characterized by emulsifying or dispersing a phospholipid in water containing an anionic polymer metal salt and an alkali metal salt, and then quantifying endotoxin from the remaining water from which the phospholipid has been removed. For measuring endotoxin in blood.