JP2792038B2 - Analysis method and pretreatment method for sample in which water-soluble polymer substance and low-molecular component coexist, and filler for chromatography - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to the analysis of a sample in which a water-soluble polymer substance and a low-molecular component coexist using a modified silica gel obtained by introducing a specific group into a silica gel surface. The present invention relates to a method, a pretreatment method and a packing material for chromatography.

[Conventional technology]

In the fields of medicine, biochemistry, food chemistry, polymer chemistry, etc.,
Analysis of trace components coexisting with proteins, such as drugs present in serum, has been performed by reversed-phase high-performance liquid chromatography. What has introduced an alkyl group, and as a bath separation liquid, those mainly composed of a polar solvent such as water, alcohol and acetonitrile have been conventionally used.

[Problems to be solved by the invention]

However, when the components coexisting with the protein are analyzed by liquid chromatography, if the sample is directly analyzed without pretreatment using the above-mentioned hydrophobic alkyl group-introduced silica gel as a filler, the protein adsorbs on the silica gel surface. In addition, the peak of the protein and the peak of the target component overlap to make accurate analysis difficult, or the protein is deposited on the silica gel surface, deteriorating the packing material or clogging the column. For this reason, it is indispensable to subject the sample containing the protein to a deproteinization treatment such as a solvent extraction operation or a protein precipitation removal operation as a pretreatment, so that the analysis operation becomes extremely complicated.

As a packing material for liquid chromatography that does not adsorb or deposit proteins, a hydrophobic group is introduced into the inner surface of the pores of silica gel, and a hydrophilic organic group is introduced into the outer surface. Silica gel is known (see JP-A-62-158113). The partially hydrophilized silica gel has unique properties having both hydrophilic and hydrophobic properties. However, in the production of the partially hydrophilized silica gel, the silica gel introduced with a hydrophobic group such as an octadecyl group is subjected to a plasma treatment to reveal silanol groups on the outer surface of the silica gel. An operation of introducing an organic group is required, and the production process is complicated.

The present invention has been made in view of the above circumstances, and does not require the protein to be adsorbed or deposited.Therefore, even when analyzing or separating and separating components coexisting with the protein, it is necessary to subject the sample to a deproteinization pretreatment. Therefore, the sample was directly injected into the chromatography to allow the target component to be analyzed or separated and collected quickly, easily and accurately, and a modified silica gel which was not deteriorated by the deposition of protein was used. An object of the present invention is to provide a method for analyzing a sample in which a water-soluble polymer substance and a low-molecular component coexist, a pretreatment method, and a packing material for chromatography.

[Means and actions for solving the problem]

In order to achieve the above object, the present invention provides the following formula (1) -AB (1) wherein a part or all of the hydrogen atoms of the surface silanol group is bonded through a silicon atom. A hydrophobic column of Formulas 2 to 24, and B represents a hydrophilic group.) A chromatography column packed with a modified silica gel substituted with a group shown in After eluting the high molecular substance in the sample containing, the low molecular component is separated and eluted, and the low molecular component separated from the high molecular substance is detected. Provided is a method for analyzing a sample in which a molecular component coexists.

In addition, the present invention provides the following formula (1) -AB (1) wherein a part or all of the hydrogen atoms of the surface silanol group is linked through a silicon atom (where A is a hydrophobic group having 2 to 24 carbon atoms). Group, B represents a hydrophilic group.) A modified silica gel substituted with a group represented by the following formula is packed in a pretreatment column of a chromatograph, and a sample containing a water-soluble polymer substance and a low-molecular component is charged to the pretreatment column. Pretreatment of a sample in which a water-soluble polymer material and a low-molecular component coexist, wherein the low-molecular component in the sample is separated and retained in the column, and the high-molecular material is eluted and removed. Provide a way.

Further, the present invention provides a packing material for chromatography comprising a modified silica gel in which part or all of the hydrogen atoms of the surface silanol group is replaced with only a group represented by the following formula (2) via a silicon atom.

-X-Y ··· (2) (where X is a hydrophobic group having 4 to 24 carbon atoms, Y is -CHOH-CH ₂ OH
Shows a group containing a group. In the present invention, a part or all of the hydrogen atoms of the surface silanol group may be converted to the above formula (1) or (2) through a silicon atom.
In the modified silica gel substituted with the group of the formula, since the groups of the formulas (1) and (2) are composed of a hydrophilic part and a hydrophobic part, the hydrophobic part is located near the surface of the silica gel and the hydrophilic part is hydrophilic. The structure has a structure in which many molecules are arranged on the surface of the silica gel, with the active part moving away from the surface of the silica gel.As a group of molecules, the part near the silica gel surface is lipophilic, and the part far away is hydrophilic. The layer structure is composed of many molecules. Therefore, this modified silica gel has a unique property having both hydrophilic and hydrophobic properties, and does not particularly adsorb or deposit water-soluble polymer substances such as proteins, and therefore has high water-soluble properties such as proteins. When this modified silica gel is used as a filler for the analysis, preparative, and pretreatment columns of components coexisting with molecular substances, only the target components are retained by this filler, and water-soluble polymer substances are eluted immediately without retention I do. Therefore, when the modified silica gel is used as a filler for an analytical column, there is no need to subject the sample to a polymer substance removal treatment such as a protein removal pretreatment, and a sample containing a protein or the like can be directly injected into a chromatograph to quickly and quickly. The target component can be easily and accurately separated and analyzed, and does not deteriorate due to deposition of a high molecular substance such as a protein. When used as a filler for a pretreatment column, proteins and the like can be removed and only the target component can be easily concentrated. Further, the filler of the present invention can be easily produced by reacting silica gel with a specific compound as described later. Among the modified silica gels in which the hydrogen atom of the surface silanol group is substituted by the group of the above formula (1), those in which A is a hydrophobic group having 3 carbon atoms have been conventionally used as a filler for gel permeation chromatography. The use of this modified silica gel as a filler for reversed-phase high-performance liquid chromatography, such as for analyzing low-molecular components coexisting with a water-soluble polymer substance, is a new finding of the present inventors. In the modified silica gel substituted with the group of the formula (1), A is a hydrophobic group having 4 to 24 carbon atoms;
There shall a group containing a -CHOH-CH ₂ OH group, namely claim 3
The filler is a novel substance found by the present inventor, and is effectively used for thin-layer chromatography in addition to liquid chromatography because of the above-mentioned unique properties.

 Hereinafter, the present invention will be described in more detail.

As described above, the modified silica gel used in the present invention is obtained by substituting a part or all of the surface silanol group with the group of the above formula (1) or (2) via a silicon atom. Fig. 1 shows this model schematically, 1 is silica gel,
Reference numeral 2 denotes pores, in which hydrophilic groups B and Y are arranged in the surface layer 3 and hydrophobic groups A and X are arranged in the inner layer.

The modified silica gel has the following formula (1a)
Examples shown in (1d) can be given.

In the above formulas (1a) to (1d), A and B are the same as above, and R ¹ and R ² each represent an alkyl group having 1 to 5 carbon atoms or a hydroxyl group. Further, as shown in the formula (1d), there is also a case where OH groups are ether-bonded between molecules.

Here, in the modified silica gel, examples of the hydrophobic groups A and X include an alkyl group, an alkylene group, and a halogenated alkyl group. When a sample in which a protein and a target component coexist is directly injected into a chromatograph, the protein may be denatured and precipitated by an organic solvent in a mobile phase. It may be necessary to limit the solvent content to below a certain concentration, in which case the sample will not elute. However, in the filler of the present invention, by selecting the types of the hydrophobic groups A and X and appropriately setting the hydrophobicity of the hydrophobic groups, the target component having high lipophilicity can be eluted without increasing the content of the organic solvent. Thus, a wide range of objectives can be analyzed.

The base as the hydrophilic group B including, for example, -CH ₂ OH group,
—CHOH—CH ₂ OH group-containing groups, amide groups, ketone groups, ether groups, cyano groups, amino groups, quaternary ammonium groups, carboxyl groups, and the like.

The substitution rate of the surface silanol group by the group of the above formula (1) or (2) is preferably 10 to 100%.

There is no limitation on the particle shape of the modified silica gel used in the present invention, and it can be formed into an appropriate shape such as a spherical shape or a crushed shape. In addition, the particle type, the size of the pores, the surface area, and the like are also appropriately selected.In the present invention, the pore diameter is desirably set to 60 to 120 °, whereby the protein enters the pores,
In addition to preventing the target component from being retained in the column, the target component is a low-molecular-weight component, so that it can freely enter and exit the pores and be retained in the column.

Although there is no limitation on the method for producing the modified silica gel used in the present invention, silica gel and the following formula (3): (However, in the formula (3), A and B are the same as above, R ⁴ ,
R ⁵ and R ⁶ each represent a halogen atom, an alkyl group having 1 to 5 carbon atoms or an —OR group (R is an alkyl group having 1 to 5 carbon atoms), and at least one of R ⁴ , R ⁵ and R ⁶ One is a halogen atom or an -OR group. ) Can be easily obtained by reacting the compound with

Specific examples of the compound of the above formula (3) include the following. However, Me is a methyl group,
Et is an ethyl group, and n is an integer of 2 to 24.

[Effects of the Invention] As described above, in the present invention, since a water-soluble polymer substance such as a protein is not adsorbed or deposited on a filler,
Quick, simple and accurate analysis of target components coexisting with water-soluble polymer substances such as proteins is possible, and concentration can be performed easily, and the filler does not deteriorate due to deposition of proteins and the like. Things. That is, as an example of a protein as a water-soluble polymer substance, the modified silica gel used as a filler in the present invention does not interact with protein at all, and therefore does not adsorb or deposit proteins on the filler. When the target component is simultaneously introduced into the column filled with the packing material, the target component is retained by the hydrophobic groups of the packing material, while the protein is discharged out of the column as it is. Therefore, when analyzing the trace components coexisting with the protein, the complicated separation of protein is not required as in the past, and the protein is separated and eluted at first by simply passing the sample through the column packed with the modified silica gel of the present invention. The trace components are retained and can be easily analyzed. Also,
The modified silica gel of the present invention is suitably used when only the target component is concentrated in the pretreatment column by utilizing such properties, and also for fractionation.

Hereinafter, the present invention will be described in detail with reference to Examples,
The present invention is not limited to the following examples.

〔Example〕

 The fillers of the present invention Nos. 1 to 8 were produced by the following methods.

Filler No.1 (filler silylated with 3-glycidoxypropyltrimethoxysilane) 10 g of spherical silica gel with a pore size of about 60 mm and an average particle size of 5 μm
Was dried at 120 ° C. under reduced pressure for 2 hours, and water was added thereto by adding 0.6 ml of water. This silica gel was put into a three-necked flask, and 35 ml of toluene, 10% p-toluenesulfonic acid, 200 μm of acetonitrile solution and 5 μm of 3-glycidoxypropyltrimethoxysilane were added, and heated at 110 ° C. for 16 hours. After cooling, the mixture was suction-filtered using a crow filter, further washed with 200 ml of toluene and 200 ml of acetone in this order, and suction-filtered using a glass filter.

This silica gel was placed in a 200 ml eggplant-shaped flask, 50 ml of a 10 ^-2 N sulfuric acid aqueous solution was added, and the mixture was heated under reflux for 1 hour. After cooling, suction filtration was performed using a glass filter (G4), 200 ml of water was washed, and suction filtration was performed. Next, the silica gel was placed again in a 200 ml eggplant flask, 50 ml of a 10 mM phosphate buffer (pH 8.0) was added, and the mixture was heated under reflux for 1 hour. After cooling, the solution was suction-filtered using a glass filter (G4).
l, 100 ml of diethyl ether in that order, suction filtration, and drying in a vacuum drier at 60 ° C. for 2 hours to obtain a filler of the present invention.

Filler No.2 (3-glycoxyalkylsilylated filler with monofunctional silylating agent) Manufactured in the same manner as No.1 above, using dimethylethoxy-3-glycidoxypropylsilane as the silylating agent did.

Filler No.3 (filler 3-glycoxyalkylsilylated with silylating agent having a different alkyl group) Using 4-glycidoxybutyltrimethoxysilane as a silylating agent, manufactured in the same manner as No.1 above did. However, the amount of the silylating agent was 2.6 ml, and the hydrolysis time was 1 hour each.

Filler No. 4 (same as above) Manufactured in the same manner as in No. 1 above, using 4-glycidoxy-1-methylbutyltrimethoxysilane as a silylating agent. However, the amount of the silylating agent was 2.8 ml, and the hydrolysis time was 1 hour each.

Filler No. 5 (same as above) It was produced in the same manner as in No. 1 above, using 6-glycidoxyhexyltrimethoxysilane as a silylating agent. However, the amount of the silylating agent was 2.9 ml, and the hydrolysis time was 1 hour each.

Filler No.6 (filler dihydroxyalkylsilylated with silylating agent containing no ether bond) Same as No.1 above using 2- (3,4-epoxycyclohexylethyl) trimethoxysilane as silylating agent Manufactured by the method. However, the amount of the silylating agent was 2.6 ml, and the hydrolysis time was 1 hour each.

Filler No. 7 (same as above) A filler was produced in the same manner as in No. 1 above, using 1,2-epoxyhexyltrimethoxysilane as a silylating agent. However, the amount of the silylating agent was 2.4 ml, and the hydrolysis time was 1 hour each.

Filler No. 8 (same as above) Using 1,2-epoxyoctyltrimethoxysilane as a silylating agent, it was produced in the same manner as in No. 1 above. However, the amount of the silylating agent was 2.6 ml, and the hydrolysis time was 3 hours each.

Next, each of the fillers No. 1 to 8 was 4.6 mm in inner diameter,
A stainless steel column with a length of 150 mm was packed by the equilibrium slurry method to prepare packed columns.Using these packed columns, the following bovine serum albumin recovery tests, benzene and naphthalene analysis tests, and serum sample injection tests were performed. Done.

Bovine serum albumin recovery test Pass bovine serum albumin through each column under the following analytical conditions,
The recovery was examined. The results are shown in Table 1.

Analysis conditions; sample 1% serum albumin / 100 mM phosphate buffer (pH
6.8) Mobile phase Acetonitrile: 100 mM phosphate buffer (20:80) Flow rate 1 ml / min Detection wavelength 295 nm Analysis test of benzene and naphthalene Using each of the above columns, a mixed solution of uracil, benzene and naphthalene was analyzed under the following analysis conditions, and the retention time of each component was examined. The results are shown in Table 2.

Analysis conditions; mobile phase acetonitrile: water (10:90); however, in the case of columns using No. 4 and No. 8 packing materials, acetonitrile: water (30:70) flow rate 1 ml / min detection wavelength 254 nm Serum Sample Injection Test 1 Serum containing 10 μg / ml of carbamazepine was directly injected under the following analysis conditions into columns packed with the above No. 1, No. 5, and No. 8 packing materials, and carbamazepine was analyzed. The obtained chromatograms are shown in FIGS.

Analysis conditions; mobile phase Acetonitrile: 100 mM phosphate buffer (pH 6.
8) (10:90 for columns packed with No. 1 packing material, N
20:80 for column packed with o.8 packing material) Flow rate 1ml / min Detection wavelength 220nm Sample injection volume 20μ Serum sample injection test 2 Phenobarbital 20μg / ml, phenytoin 20μg / ml
And sera containing 10 μg / ml of carbamazepine were directly injected into the column packed with the No. 2 packing material under the following analysis conditions, and analyzed. The obtained chromatogram is shown in FIG.

Analysis conditions; mobile phase Tetrahydrofuran: acetonitrile: 100
mM phosphate buffer (pH 6.8) (5: 5: 90) Flow rate 1ml / min Detection wavelength 220nm Sample injection volume 10μ Next, as a pretreatment column filled with 6-glycidoxyhexylsilylated silica gel Using the apparatus shown in FIG. 6, serum carbamazepine was analyzed by a column switching method. In this case, the N
The packing material of o.5 was packed into a stainless steel column having an inner diameter of 4.6 mm and a length of 30 mm by the equilibrium slurry method to prepare a packed column for pretreatment. Also, first, the sample is concentrated in the pretreatment column by flowing the pretreatment mobile phase through the flow path indicated by the dotted line of the valve, and then the valve is switched, and the analysis mobile phase is caused to flow through the flow path indicated by the solid line of the valve. The sample in the pretreatment column was introduced into the analysis column. Serum containing 10 μg / ml of carbamazepine was analyzed by the high performance liquid chromatograph equipped with this processor under the following conditions. The obtained chromatogram is shown in FIG.

This method is effective for the analysis of trace metabolites in serum, etc., because trace components can be easily concentrated in a pretreatment column.

Pretreatment conditions; mobile phase 100 mM phosphate buffer (pH 6.8) Flow rate 1 ml / min Pretreatment time 10 min Sample injection volume 20 μ Analysis conditions; analytical column ODS (4.6 mmφ × 150 mmL.) Mobile phase acetonitrile: 100 mM Phosphate buffer (pH 6.
8) (30:70) Flow rate 1 ml / min Based on the measurement wavelength of 220 nm or more, the modified silica gel of the present invention does not adsorb water-soluble polymer substances such as proteins and has excellent properties as a packing material for liquid chromatography. Is recognized.

[Brief description of the drawings]

FIG. 1 is an explanatory view schematically illustrating a denatured silica gel used in the present invention, FIGS. 2 to 5 are chromatograms obtained by analyzing carbamazepine and the like in serum by the analysis method of the present invention, and FIG. 6 is a pretreatment. FIG. 7 is a schematic diagram showing an analyzer using a column, and FIG. 7 is a chromatogram obtained by concentrating carbamazepine in serum on a pretreated column by the pretreatment method of the present invention and analyzing the carbamazepine by an ODS column.

Continuation of the front page (72) Inventor Yasushi Takahata 4-1-1 Higashi-Mukojima, Sumida-ku, Tokyo Inside the Chemical Inspection Association (56) References JP-A-57-3043 (JP, A) (58) Field surveyed (Int.Cl. ⁶ , DB name) G01N 30/48 G01N 30/88 G01N 30/14

Claims (3)

(57) [Claims] (1) A compound represented by the following formula (1) -AB (1) wherein a part or all of the hydrogen atoms of the surface silanol group is linked via a silicon atom, wherein A is a hydrophobic group having 2 to 24 carbon atoms; B represents a hydrophilic group.) A chromatography column packed with denatured silica gel substituted with a group shown in the above is used in a reversed-phase system, and the above-mentioned polymer in a sample containing a water-soluble polymer substance and a low-molecular component is used. After the substance is eluted, the low-molecular component is separated and eluted, and the low-molecular component separated from the high-molecular substance is detected. Analysis method. 2. A method according to claim 1, wherein a part or all of the hydrogen atoms of the surface silanol group are bonded through a silicon atom to the following formula (1) -AB (1) (where A is a hydrophobic group having 2 to 24 carbon atoms, B represents a hydrophilic group.) A modified silica gel substituted with a group shown in the following is packed in a pretreatment column of a chromatograph, and a sample containing a water-soluble polymer substance and a low-molecular component is passed through the pretreatment column. A pretreatment method for a sample in which a water-soluble polymer substance and a low-molecular component coexist, wherein the low-molecular component in the sample is separated and held in the column, and the high-molecular substance is eluted and removed. 3. A packing material for chromatography comprising a modified silica gel in which part or all of the hydrogen atoms of the surface silanol group is replaced with only a group represented by the following formula (2) via a silicon atom. -X-Y ... (2) (where, X is a hydrophobic group having 4 to 24 carbon atoms, Y is -CHOH-CH ₂ O
Shows a group containing an H group. )