JPH0610675B2 - Packing agent for separating non-protein substances - Google Patents

Description

TECHNICAL FIELD The present invention relates to a deproteinizing column packing material used when measuring a non-protein substance in a sample containing protein such as serum.

(Prior art) Non-protein components such as steroid hormones such as aldosterone, aldosterone cortisol, pregnanediol, and estrogen contained in blood serum; amines such as catecholamines and polyamines; various drugs; etc. for pathological research and diagnosis. Analysis by liquid chromatography or the like is widely performed for use. Since a biological sample such as serum generally contains a protein, the analysis accuracy and the function of the analytical column are deteriorated or the life of the analytical column is shortened due to the influence of the protein.

Therefore, in the analysis of non-protein components, it is usual to use a sample from which proteins have been removed by a deproteinization operation in advance. However, the conventional method has a drawback in that a large amount of analytical components are lost or denatured because the deproteinization operation requires a lot of time and labor.

As a countermeasure, for example, a sample solution containing a protein and a non-protein substance is passed through a deproteinization column to adsorb only the non-protein component and remove the protein component, and then the adsorbed non-protein substance is eluted to obtain an analytical column. JP-A-58-2
It is disclosed in Japanese Patent No. 23061. As a packing material for the deproteinization column used in this method, fine particles such as an acrylic polymer are used. Examples of the acrylic polymer include bifunctional acrylic monomers such as triethylene glycol diacrylate and tetraethylene glycol diacrylate and / or trifunctional or higher functional acrylics such as tetramethylolmethane tetraacrylate and trimethylolpropane triacrylate. Examples thereof include polymers or copolymers containing a system monomer as a polymerization component. Porous fine particles are used because the higher the surface area of the fine particles, the higher the adsorption efficiency. Usually, a filler having a particle size of 10 to 20 μm and an average pore size of about 200Å is used.

According to the above method, the protein can be removed relatively effectively, which facilitates the measurement of the non-protein substance, but it has the following drawbacks: It takes a long time to separate the protein and the non-protein. For example, it takes 10 minutes or more at a flow rate of ion-exchanged water of 1.0 ml / minute to remove proteins in 10 μm of serum.

Since the proteins are not completely removed and a few% remain in the packing material, they elute with a time delay and interfere with the measurement.

Since the protein is not completely removed and a few% of the protein remains in the packing material and gradually accumulates, the pressure of the deproteinization column rises, and eventually measurement becomes impossible.

It is considered that the causes of the above phenomena are mainly caused by albumin, which is a typical protein in serum. Albumin is an ellipsoidal sphere having a short diameter of 40 Å and a long diameter of 140 Å. This is because the protein is not originally adsorbed by the filler having the above composition, but albumin enters the pores of the filler particles of 100 to 200 Å and is eluted with a time delay.

(Problems to be Solved by the Invention) The present invention is to solve the above-mentioned conventional drawbacks, and an object of the present invention is to provide a column packing material capable of accurately separating a protein from a non-protein in a short time. To provide. Another object of the present invention is to provide a packing material for non-protein separation, which can be repeatedly used over a long period of time and can inexpensively measure non-protein substances.

(Means and Actions for Solving Problems) The non-protein separating filler of the present invention does not adsorb proteins,
And fine particles capable of adsorbing a non-protein substance under a predetermined condition and desorbing the non-protein substance under another predetermined condition, wherein the fine particles are porous particles having pores. Has an average diameter of 40Å or less, thereby achieving the above object.

Any material can be used as the material of the filler of the present invention as long as it is hydrophilic and satisfies the conditions such as adsorption and desorption of the non-protein substance. Particularly, an acrylic (co) polymer developed by the inventors is preferably used. This acrylic (co) polymer is a monomer represented by the following formula (I) and / or x methylolalkane y (meth) acrylate [x, y is an integer, and x ≧ y ≧ 3] is a polymerization component. Is a polymer or copolymer: (R ₁ and R ₂ are each hydrogen or a methyl group, and n is an integer of 3 to 18).

Examples of the monomer represented by the formula (I) include triethylene glycol diacrylate, triethylene glycol dimethacrylate, tetraethylene glycol diacrylate, tetraethylene glycol dimethacrylate, nonaethylene glycol diacrylate, nonaethylene glycol dimethacrylate. , Tetradecaethylene glycol dimethacrylate, and tetradecaethylene glycol dimethacrylate. x Methylol alkane y
Examples of the (meth) acrylate include tetramethylolmethane tetraacrylate, tetramethylolmethane tetramethacrylate, tetramethylolmethane triacrylate, tetramethylolmethane trimethacrylate, trimethylolmethane triacrylate, trimethylolmethane trimethacrylate, and trimethylolpropane triacrylate. , Trimethylolpropane trimethacrylate. Particularly, tetramethylol methane tetraacrylate, tetramethylol methane triacrylate and tetramethylol methane trimethacrylate are suitable.

A polymer which does not adsorb the above-mentioned protein required for the filler of the present invention and has the ability to adsorb a non-protein substance under a predetermined condition and desorb the non-protein substance under another predetermined condition. The particles can be easily obtained by copolymerizing the monomer represented by the formula (I) and the x-methylolalkane y (meth) acrylate in a weight ratio of usually 1: 0.1-10.

Next, the preparation of the porous fine particles using the above-mentioned monomer can be usually carried out by a suspension polymerization method in an aqueous medium. It is recommended to adopt.

As a method for making polymer particles to be polymerized in an aqueous medium porous, it is usual to add an aromatic hydrocarbon which is a nonpolar solvent such as toluene or xylene to a polymerizable monomer as a solvent. As a solvent at the time of the polymerization, a higher alcohol (eg, isoamyl alcohol,
A polar solvent such as octyl alcohol) is used. The amount of the solvent is preferably 150 parts by weight or less per 100 parts by weight of the monomer.

When a copolymer containing the above monomer (I) and x-methylolalkane y (meth) acrylate is obtained, the content of x-methylolalkane y (meth) acrylate is increased to increase the crosslink density. For example, for monomer (I),
It is preferable to use 1 to 10 times the amount of x-methylolalkane y (meth) acrylate.

In addition to the above monomers, for example, divinylbenzene can be used as a copolymerization component of the monomer (I) to prepare (co) polymer fine particles having an average pore size of 40Å or less. It can be used as the filler of the present invention. In this case, 4 to 15 times the amount of divinylbenzene is used as a copolymerization component with respect to the monomer (I), because the fine polymer particles have pores of 40 Å or less, and It is preferable for satisfying the adsorption / desorption ability of.

The packing material thus obtained is added to a sample containing protein and non-protein substances (eg serum) at a predetermined pH, for example.
When contacted with, the non-protein substance is effectively adsorbed by the packing material and the protein is not adsorbed. Here, since the average pore size of the filler composed of the porous fine particles of the present invention is 40 liters or less, albumin, which occupies most of serum proteins, does not enter the pores. There are globulins and the like as serum proteins other than albumin, but since they are larger molecules than albnin, they do not penetrate into the pores of the packing material. Therefore, for example, when a serum sample containing a non-protein substance is flown through a deproteinization column packed with such a packing material, the protein component is promptly removed without being trapped in the column.

Next, a method for detecting non-protein using the packing material of the present invention will be described. FIG. 1 is an example of the analyzer, and a deproteinization column 5 is connected to a hexagonal valve 4 having connecting ends A to F via connecting ends B and E. The ion-exchanged water or the buffer solution in the water tank 1 is sent by the pump 2 to the deproteinization column 5 via the sample injector 3 and the connection ends A and B, and then discharged from the system through the connection ends E and F. The column 5 is packed with the packing material of the present invention. This filler can be used under certain conditions, such as water or a certain pH.
In the above buffer solution, the target substance for analysis, which is a non-protein substance, is adsorbed, and the protein is not adsorbed. Under other conditions, for example, in a buffer solution having a pH different from the above, it has a property of eluting the non-protein analysis target substance adsorbed on the column.

As a sample, for example, a serum sample that has not been subjected to deproteinization is used, and is injected into the system from the sample injector 3. The injected sample is transferred together with ion-exchanged water or a buffer solution by a pump and reaches the deproteinization column 5. Here, the non-protein analysis target substance in the serum sample is adsorbed by the packing material of the column. On the other hand, the protein in the sample passes through the column 5 without being adsorbed, and is discharged to the outside of the system through the connecting ends E and F of the hexagonal valve 4.

The eluent in the eluent tank 6 is passed through the connection ends D and C by the pump 7 to the analytical column 8 for liquid chromatography.
Sent to. Next, as shown in FIG. 2, when the hexagonal valve 4 is switched to connect the connection end D to E, the eluent having the elution condition of the adsorbent in the eluent tank 6 is connected to the connection end D and E by the pump 7. It is supplied to the deproteinization column 5 via E. As the eluent passes through the column 5, the adsorbed non-protein substance is eluted and reaches the analytical column 8 together with the eluent through the connection ends B and C. The substance to be analyzed is separated by the analysis column 8 and detected by an appropriate detector (not shown) provided after the analysis column 8.

Adopting such an analyzer using the packing material of the present invention provides the following advantages.

Deproteinization is promptly performed.

Since proteins are reliably removed, non-protein substances can be detected accurately.

Since the protein component is not trapped in the column, the column can be used for a long time, and as a result, the non-protein substance can be detected inexpensively.

(Example) Hereinafter, the present invention will be described with reference to Examples.

Example 1 5 equipped with a condenser, stirrer, thermometer and dropping funnel
The separable flask of was prepared. 400 ml of 4% by weight polyvinyl alcohol aqueous solution; 40 g of tetraethylene glycol dimethacrylate as a polymerizable monomer and 10 g of tetramethylolmethane triacrylate; 40 g of isoamyl alcohol as an organic solvent; and 1.5 g of benzoyl peroxide as a polymerization initiator. And supplied to the flask. Nitrogen gas is supplied to this at a flow rate of 150 ml / min, while stirring at a stirring speed of 150 rpm.
The temperature was raised to 0 and the polymerization reaction was carried out for 10 hours. After cooling, the polymerization product was separated from the mother liquor and washed successively with hot water and acetone to obtain a porous polymer having a particle size of 10 to 50 μm. When the pore distribution of the obtained porous polymer was examined by the BET method, the average pore diameter was 10Å.

200 mg of a filler having a particle size of 25 to 35 μm obtained by removing fine particles and coarse particles from this porous polymer is dispersed in 40 ml of distilled water, and is applied to a stainless steel column having an inner diameter of 4 mm and a length of 20 mm by a high pressure constant flow pump. Distilled water was pressure-fed at a flow rate of 2 ml / min to fill the column, and a deproteinization column was obtained.

The high-pressure hexagonal valve 4 was connected to the deproteinization column 5 and the analytical column 8 incorporated in liquid chromatography (Waters, Model 6000A) as shown in FIG. As an analytical sample, normal male serum without deproteinization (prednisolone, a corticosteroid, at 10 pp
(added at a ratio of m) was prepared. While the ion-exchanged water in the water tank 1 was caused to flow by the pump 2 at the flow rate of 1.0 ml / min into the deproteinization column 5 via the connection ends A and B of the hexagonal valve 4, 10 μm of the sample serum was injected from the sample injector 3. 5
After a minute, the hexagonal valve 4 was switched and connected as shown in FIG. By means of the pump 7, the eluent containing 70:30 of methanol and water from the eluent tank 6 at a flow rate of 0.5 ml / min was used to connect the hexagonal valve connection ends D and E, the deproteinization column 5, and the hexagonal valve connection ends B and C. Through liquid chromatography analytical column 8
I went to The non-protein substance (prednisolone) eluted by the circulation of the eluent was analyzed. The analytical column used was HYPERSIL-ODS (trade name: column size inner diameter 6 mm; length 10 cm) manufactured by SHANDON, and detection was performed by absorption intensity at 240 nm. The obtained chromatogram is shown in FIG. In FIG. 3 (A), 11 is the peak of prednisolone.

The above measurement was repeated 300 times without replacing the column. The chromatogram of the 300th specimen is shown in FIG. 3 (B). 3 (A) and 3 (B) have almost the same pattern, no interference due to protein contamination at the time of detection was observed, and it can be seen that the performance of the deproteinization column is not deteriorated.

The following table collectively shows the charging composition at the time of polymerization of the present Example, Examples 2 to 3 described later, and Comparative Examples 1 to 3 and the average pore diameter of the obtained polymer particles.

Comparative Example 1 The procedure of Example 1 was repeated except that 40 g of toluene was used instead of isoamyl alcohol, and the particle size was 10 to 50 μm.
A porous polymer of When the pore distribution of the obtained porous polymer was examined by the BET method, the average pore diameter was 140Å.

In the same manner as in Example 1, a packing material having a particle size of 25 to 35 μm obtained by removing fine particles and coarse particles from the porous polymer was packed to obtain a deproteinized column. Using this,
Serum was analyzed in the same manner as in Example 1. In the first measurement, the same chromatogram as in FIG. 3 (A) was obtained, but as the measurement was repeated, the pressure in the deproteinization column gradually increased and peak 11 became broad. For the 15th sample, the column pressure was 50 kg / cm ² , and the chromatogram shown in FIG. 3 (C) was obtained, making measurement difficult.

Example 2 Polymerization was carried out in the same manner as in Example 1 using 70 g of diethylene glycol dimethacrylate and 200 g of tetramethylolmethane triacrylate as the polymerizable monomer; 200 g of toluene as the organic solvent; and 5.0 g of benzoyl peroxide as the polymerization initiator. A porous polymer having a particle size of 10 to 50 μm was obtained. When the pore distribution of the obtained porous polymer was examined by the BET method, the average pore diameter was 35Å.

In the same manner as in Example 1, a packing material having a particle diameter of 25 to 35 μm obtained by removing fine particles and coarse particles from the porous polymer was packed to obtain a deproteinized column.

An apparatus similar to that in Example 1 was assembled using the above deproteinization column. Deproteinization was not performed as an analytical sample. Normal male serum (cholic acid, a kind of bile acid, was added at a rate of 200 ng / ml) was prepared.

While the ion-exchanged water in the water tank 1 was caused to flow by the pump 2 at the flow rate of 1.0 ml / min into the deproteinization column 5 via the connection ends A and B of the hexagonal valve 4, 10 μm of the sample serum was injected from the sample injector 3. After 10 minutes, the hexagonal valve 4 was switched and connected as shown in FIG. By means of the pump 7, the eluent containing 0.3: 4 ammonium carbonate aqueous solution and acetonitrile at 14: 4 from the eluent tank 6 at a flow rate of 1.0 ml / min, the hexagonal valve connection ends D and E, the deproteinization column 5, and the hexagonal valve connection end B. , C to the analysis column 8 for liquid chromatography. The non-protein substance (cholic acid) eluted by the circulation of the above eluent was analyzed. The analytical column used was μ-Bandapak Phenyl (trade name: column size inner diameter 3.9 mm; length 30 cm) manufactured by Waters, and detection was performed by absorption intensity at 254 nm. The obtained chromatogram is the fourth
It is shown in FIG. In FIG. 4 (A), the peak 12 of cholic acid is clearly confirmed.

Comparative Example 2 300 g of diethylene glycol dimethacrylate and 40 g of tetramethylolmethane triacrylate as polymerizable monomers; 150 g of toluene as an organic solvent; and 5.0 g of benzoyl peroxide as a polymerization initiator,
Polymerization reaction is performed according to Example 1, and the particle size is 10 to 50 μm.
A porous polymer of When the pore distribution of the obtained porous polymer was examined by the BET method, the average pore diameter was 155Å.

In the same manner as in Example 1, a packing material having a particle diameter of 25 to 35 μm obtained by removing fine particles and coarse particles from the porous polymer was packed to obtain a deproteinized column.

Using this deproteinized column, cholic acid was analyzed in the same manner as in Example 2. The obtained chromatogram is shown in FIG. 4 (B). From FIG. 4 (B), it can be seen that in this comparative example, it is difficult to accurately analyze cholic acid due to contamination with serum proteins.

Example 3 Using 30 g of nonaethylene glycol dimethacrylate as a polymerizable monomer and 250 g of divinylbenzene; 300 g of toluene as an organic solvent; and 5.0 g of benzoyl peroxide as a polymerization initiator, a polymerization reaction was carried out according to Example 1. A porous polymer having a particle size of 10 to 50 μm was obtained.
When the pore distribution of the obtained porous polymer was examined by the BET method, the average pore diameter was 20Å. In the same manner as in Example 1, a packing material having a particle diameter of 25 to 35 μm obtained by removing fine particles and coarse particles from the porous polymer was packed to obtain a deproteinized column.

The above deproteinization column was used and assembled in the same apparatus as in Example 1. As a sample for analysis, a normal male serum that had not been deproteinized (theophylline as an asthma drug was added at a rate of 100 ng / ml) was prepared.

While the ion-exchanged water in the water tank 1 was caused to flow by the pump 2 at the flow rate of 1.0 ml / min into the deproteinization column 5 via the connection ends A and B of the hexagonal valve 4, 10 μm of the sample serum was injected from the sample injector 3. After 6 minutes, the hexagonal valve 4 was switched and connected as shown in FIG. By the pump 7, the eluent of acetonitrile: purified water 8:92 from the eluent tank 6 was passed through the hexagonal valve connections D and E, the deproteinization column 5 and the hexagonal valve connection ends B and C at a flow rate of 1.0 ml / min. It was passed through an analytical column 8 for liquid chromatography. The non-protein substance (theophylline) eluted by the flow of the eluent was analyzed. The analytical column used was NUCLEOSIL 5C manufactured by Chemco.
18 (trade name: column size inner diameter 4 mm; length 150 mm), and detection was performed by absorption intensity at 254 nm. The obtained chromatogram is shown in FIG. In FIG. 5 (A), peak 13 of theophylline is clearly confirmed.

Comparative Example 3 Using 300 g of divinylbenzene, 80 g of methacrylic acid as a polymerizable monomer, 350 g of toluene as an organic solvent, and 5.0 g of benzoyl peroxide as a polymerization initiator,
Polymerization reaction is performed according to Example 1, and the particle size is 10 to 50 μm.
A porous polymer of When the pore distribution of the obtained porous polymer was examined by the BET method, the average pore diameter was 100Å. In the same manner as in Example 1, a packing material having a particle diameter of 25 to 35 μm obtained by removing fine particles and coarse particles from the porous polymer was packed to obtain a deproteinized column.

Using this deproteinized column, theophylline was analyzed in the same manner as in Example 3. The obtained chromatogram is shown in FIG. 5 (B). From FIG. 5 (B), it can be seen that in this comparative example, accurate analysis of theophylline is difficult due to contamination with serum proteins.

Next, using a new device, the time until the hexagonal valve 4 was switched after the sample was injected was extended so that the ion-exchanged water from the water tank 1 could pass through the deproteinization column 5 for a long time. When the time was extended to 21 minutes, a chromatogram with the same pattern as in FIG. 5 (A) was obtained. This is 3 times or more compared to 6 minutes in Example 3, and it can be seen that deproteinization requires a long time.

(Effect of the Invention) According to the present invention, a column packing material capable of accurately separating a protein and a non-protein substance in a short time can be obtained. Such a packing material is suitably used, for example, for detecting or quantifying a non-protein substance in serum. Since the filler can be used for a long period of time, it is possible to inexpensively detect non-protein substances.

[Brief description of drawings]

1 and 2 are connection diagrams showing an example of an analyzer using the packing material of the present invention; FIGS. 3 (A) and (B), 4
Fig. (A) and Fig. 5 (A) are chromatograms obtained by measuring non-protein substances using the packing material of the present invention;
FIG. 3 (C), FIG. 4 (B), and FIG. 5 (B) are chromatograms obtained by measuring nonprotein using a conventional packing material. 1 ... Water tank, 2, 7 ... Pump, 3 ... Sample injector, 4 ... Hexagon valve, 5 ... Deproteinization column, 6 ...
Eluent tank, 8 ... Analytical column.

Claims (2)

[Claims] 1. A fine particle which does not adsorb protein and has an ability to adsorb a non-protein substance under a predetermined condition and desorb the non-protein substance under another predetermined condition, wherein the fine particle is a pore. A packing material for separating non-protein substances, wherein the packing material is a porous particle having an average diameter of 40 Å or less. 2. The fine particles are a monomer represented by the following formula (I) and / or x methylolalkane y (meth) acrylate [x, y is an integer, and x ≧ y ≧ 3] is a polymerization component. The filler for separating non-protein substances according to claim 1, which comprises a polymer or a copolymer: (R ₁ and R ₂ are each hydrogen or a methyl group, and n is an integer of 3 to 18).