JPS63163276A - Method for measuring basic material in protein solution - Google Patents

Abstract

PURPOSE:To permit measurement of a material to be measured with high accuracy by supplying a sample soln. contg. the ion pairs formed by adding a negative charge material to a sample soln. contg. the basic material to be measured and protein component to a protein removing column and analyzing the ion pairs desorbed by supplying the eluate to the column in which the ion pairs are adsorbed. CONSTITUTION:The negative charge material is first added to the sample soln. contg. the basic material to be measured and protein component to form the ion pairs of the basic material to be measured and the negative charge material. The sample soln. contg. such ion pairs is supplied to the protein removing column so that the ion pairs are adsorbed to the column and the protein component is passed through the column without being adsorbed thereto and is thereby removed. The eluate is in succession supplied to the protein removing column to desorb the ion pairs adsorbed to the packing material in the column. The desorbed ion pairs are introduced into an analysis column and are thereby analyzed.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention is a method for measuring basic substances in solutions containing protein components, more specifically, a method for measuring basic substances contained in serum etc. using a reversed-phase liquid chromatography method. The present invention relates to a method for accurately measuring a basic substance to be measured without being interfered with by mixed protein components.

(Prior technology) Non-protein trace components such as various hormones, catecholamines, and various drugs contained in serum are measured by liquid chromatography, especially high-performance liquid chromatography, and used for pathological research and diagnosis. It is widely practiced. Biological samples such as serum generally contain protein components such as albumin, and the influence of these protein components can reduce analysis accuracy and analytical column functionality.

The life of the analytical column may be shortened. Therefore, when measuring non-protein components, it is common to use a sample from which protein components have been removed by a protein removal operation such as perchloric acid treatment. However, this method requires multiple steps and a long time for protein removal.
Loss or decomposition of the substance to be measured occurs. Furthermore, the above-mentioned protein removal operation requires a skilled technique and is subject to large variations between measurers or during measurement. As described above, conventional protein removal operations are complicated, have a low recovery rate of the analyte, and have poor reproducibility.

On the other hand, Japanese Patent Application Laid-Open No. 58-223061.

The sample solution containing protein components and non-protein analyte is passed through a protein removal column to adsorb only the non-protein components and remove the protein components.Then, the adsorbed non-protein components are eluted and transferred to the analytical column. A method is disclosed. According to this method, the substance to be measured in the sample is directly measured without the need for the above-mentioned protein removal operation, so that the measurement can be performed in a short time and with good reproducibility.

However, even if the method disclosed in the above publication is adopted, if the substance to be measured is a basic substance, that is, a substance such as a catecholamine that dissociates and has a positive charge, the Hfi substance cannot be packed in the above-mentioned reverse phase system. It is removed from the column along with protein components without any hydrophobic interaction with the agent (that is, without being retained by the packing material). Using a column packed with cation exchange packing to adsorb basic substances,
Although the recovery rate of basic substances is relatively good, protein components are adsorbed non-specifically, making the protein removal column useless.
Furthermore, it has the disadvantage of accelerating deterioration of the analytical column.

(Problem to be solved by the invention) The present invention solves the above-mentioned conventional drawbacks.

The purpose is to provide a method for directly measuring a basic analyte and a protein component in a sample containing the analyte without performing a complicated protein removal operation. Another object of the invention is.

The object of the present invention is to provide a method for accurately measuring the basic analyte by reverse-phase high-performance liquid chromatography equipped with a protein removal column.

(Means for solving problems) The method of measuring basic substances in a protein solution according to the present invention is as follows.

(a) a step of adding a negatively charged substance to a sample solution containing a basic analyte and a protein component to form an ion pair between the basic analyte and the negatively charged substance; (b)
a step of supplying the sample solution containing the ion pair to a protein removal column, allowing the column to adsorb the ion pair, and removing protein components by passing through the column without adsorbing protein components; (C) removing the protein components; A step of supplying an eluent to a column and desorbing the ion pair adsorbed on the packing material of the column.

and (d) introducing the desorbed ion pair to an analytical column and analyzing it, thereby achieving the above object.

Basic substances that can be measured by the method of the present invention include:

Any of the common cationic substances that can dissociate and carry a positive charge are included. Representative examples include noradrenaline, epinephrine, nicotinamide, pyridoxine, thiamine, and the like. These are substances that are present in the blood or urine, for example, as biological components or externally administered.

The type of negatively charged substance added to measure the above-mentioned substance to be measured is not particularly limited, but is usually one.

Sulfonic acid having an alkyl group, aromatic group or alkyl aromatic group; Sulfonic acid salt having an alkyl group, aromatic group or alkyl aromatic group; Sulfuric acid having an alkyl group, aromatic group or alkyl aromatic group; and an alkyl group ,
At least one of the group of compounds consisting of sulfates having aromatic or alkyl aromatic groups is utilized. For example, sodium benzenesulfonate, sodium 1-dodecanesulfonate, sodium lauryl sulfate, sodium dodecylbenzenesulfonate, etc. are preferably used. The alkyl group of each of the above compounds preferably has 12 or less carbon atoms. Hydrophobicity increases when the number of carbon atoms in the alkyl group exceeds 13.

This substance does not dissolve in the sample solution to which it is added or in the protein removal solution (which consists mainly of buffers and contains almost no organic solvent).

The method of the present invention is embodied, for example, by the apparatus shown in FIGS. 1(A) and 1(B). This device connects terminals A-F.
The protein removal column 5 is connected to the hexagonal pulp 4 at the connecting end B-
It is connected via E. The protein removal solution 1 in the protein removal solution storage tank 11 is sent by the constant flow pump 2 to the protein removal column 5 via the sample introduction device 3 and the connection end A-B, and from there to the connection end E.

It passes through F and is discharged out of the system. The packing material packed in the protein removal column 5 adsorbs non-protein components and does not adsorb protein components under certain conditions 1, such as water or a buffer solution of a certain pH. and other conditions 2, such as a different pH from the above.
In a buffer solution containing an organic solvent or a buffer solution containing an organic solvent, it has the property of eluting the above-mentioned non-protein components adsorbed on the column. The adsorptivity of non-protein components is set lower than that of the packing material packed in the analysis column 8, which will be described later. As the packing material for the protein removal column 5, a conventional packing material for reversed phase chromatography, particularly a highly hydrophilic packing material, is used. For example, hydroxyl group,
A cylindrical gel having a hydrophilic group such as a carboxyl group or an ether group on its surface is used. Examples of the material include 2, for example, tetramethylolmethane triacrylate and n-ethylene glycol dimethacrylate (n is 2 to 4
) or copolymers of these and other monomers. In particular, it is preferable to use fine particles having a particle size of about 10 to 20 μm obtained by suspension polymerization.

The sample includes 2, for example, the above-mentioned basic substance to be measured.
A serum sample that has not been subjected to protein removal treatment is used. As the protein removal solution, water and various buffer solutions can be used, and low concentration buffer solutions containing no water or organic solvents are particularly suitable. If an organic solvent is contained, protein components in the sample may aggregate to form a precipitate.

The sample is introduced into the system from the sample introduction device 3. At this time, the above-mentioned negatively charged substance is mixed with the proof sample and/or
Or added to protein removal solution. The amount varies depending on the type and content of the substance to be measured.

It is necessary to be present in an excess amount relative to the substance to be measured, and for example, it is contained in a protein removal solution at a ratio of 10 to 800 μg/-. This negatively charged substance combines with a basic analyte (which dissociates and has a positive charge) in the sample to form an ion pair. Therefore, the polarity of the substance to be measured decreases.

Furthermore, since the negatively charged substance has an alkyl group or an aromatic group, the formed ion pair has a high degree of hydrophobicity. The sample solution containing such ion pairs is transported together with the protein removal solution by the constant flow pump 2 and reaches the protein removal column 5 . The substance to be measured that has formed an ion pair has high hydrophobicity as described above, and therefore is adsorbed to the packing material of the protein removal column 5. The larger the hydrophobic part of the negatively charged substance molecule is, for example, the longer the alkyl chain and/or the more it contains an aromatic ring, the higher the degree of adsorption of the ion pair to the protein removal column 5 will be. The protein component in the sample is not adsorbed and passes through the column 5, and is discharged from the system via the connecting end EF of the hexagonal pulp 4.

On the other hand, the eluent 6 in the eluent tank 61 is sent by the constant flow pump 7 to the analytical column 8 of liquid chromatography via the connecting end and C. Next, as shown in FIG. 1(B), when the hexagonal pulp 4 is switched and the connecting end is connected to E, the eluent 6 in the eluent tank 61 is removed by the constant flow pump 7 through the connecting end and E. Supplied to protein column 5. As the eluent passes through the column 5, the adsorbed ion pairs are eluted (desorbed) and reach the analytical column 8 through the connecting ends B and C together with the eluent. The ion pair containing the substance to be measured is separated by the analytical column 8 and detected by an appropriate detector (not shown) provided after the first analytical column 8. The packing material packed in the analytical column 8 is one that has higher adsorption properties than the packing material used in the protein removal column 5. By using such a highly adsorbent packing material, the ion pairs adsorbed on the protein removal column are completely desorbed by the eluent, and the eluent is passed directly to the analytical column, where the analyte is accurately separated and analyzed. can do.

(Function) In the method of the present invention, an ion pair is formed by adding a negatively charged substance, which is an oppositely charged substance, to a basic analyte. Therefore, the polarity of the substance to be measured becomes low. Furthermore, because of the alkyl group or aromatic group that the negatively charged substance has, the ion pair has a high degree of hydrophobicity. Therefore, it becomes possible to adsorb onto a protein removal column packed with a gel for reverse phase chromatography. In this way, the basic analyte, which is a positively charged substance, has properties similar to ordinary nonionic non-protein components, and is therefore effectively separated from protein components in the protein removal column. The substance to be measured from which protein components have been removed is separated in an analytical column 8 and measured in the same manner as in ordinary high performance liquid chromatography. In such a method, basic substances are not lost or altered during the measurement (they can be easily measured in a short time with high accuracy). It can be used in many ways as a general method for measuring sexual substances.

(Example) The invention will be explained below with reference to examples.

On the practical plate (8) Preparation of protein removal column packing gel = 400g of 4% by weight aqueous polyvinyl alcohol solution, 40g of tetraethylene glycol dimethacrylate, 10g of tetramethylolmethane triacrylate, 50g of methacrylic acid, 40g of toluene and benzoyl peroxide. 1.5g was added. After reacting at 80°C for 10 hours while stirring at 400 rpm, the particles were washed with hot water and acetone to reduce the particle size to 2.
A spherical porous polymer body with a diameter of 0 to 30 μm was obtained.

(B) Measurement of basic substances: Noradrenaline and epinephrine in serum were measured using the measuring device shown in FIG. As the protein removal column 5, a stainless steel column with an inner diameter of 4 mm and a length of 150 mm was used, which was filled with the packing gel obtained in section (A).

As the analytical column 8, a commercially available column for ODS-based reverse phase chromatography (backed column AM-312) was used.
; Manufactured by Yamamura Chemical Co., Ltd.; Column size: inner diameter 6, length 150 m
m) was used. The protein removal solution was a 10 mM phosphate buffer (pH 3,0) containing sodium lauryl sulfate at a ratio of 2 mmol/1, and the eluent 6 was a 10 mM phosphate buffer (pH 3,0).

A mixed solution of 0.1M phosphate buffer (pH 3.0) and acetonitrile (volume ratio 90:10) was used.

First, as shown in FIG. 1(A), while flowing the protein removal solution 1 through the connection end A-B of the six-way valve 4 to the protein removal column 5 at a rate of 0.6 mj/min using the constant flow pump 2, 20 μl of sample serum was injected from the sample introduction device 3. This sample contains standard noradrenaline 1.5 in normal male serum.
µg and 1.5 µg adrenaline were added. 8 minutes after sample injection, switch the six-way valve 4 to the first
The connections were made as shown in Figure (11). The pump 7 pumps the eluent 6 from the eluent tank 61 into 1. It was passed through the six-way valve connecting end D-E, the protein removal column 5, and the six-way valve connecting end B-C to the analysis column 8 of the liquid chromatograph at a rate of 0 mg/min. Ion pairs containing the substance to be measured are eluted by the flow of the eluent, separated by 9 analytical columns, and detected by a detector (not shown). The absorbance intensity at 280 nm was measured using detectors L and 7C and an absorbance meter (UVIDIEC100-Vl; manufactured by JASCO Corporation). The obtained chromatogram is shown in FIG. 2(A).

In FIG. 2(A), noradrenaline peak a and adrenaline peak b are confirmed. The recoveries of noradrenaline and epinephrine were each 98% or higher.

The same procedure as in Example 1 was used except that a protein removal solution containing no sodium laurauryl sulfate was used. The obtained chromatogram is shown in FIG. 2(B). In FIG. 2(B), the peaks of noradrenaline and adrenaline are not observed.

0.1 gg of standard nicotinic acid amide as a sample of charcoal
- 0.1 gg of pyridoxine and 0.1 gg of thiamine
The procedure was the same as in Example 1, except that 20 μl of normal male serum containing 20 μl of normal male serum was used and the detection wavelength of the absorbance meter was set to 210 nm. The obtained chromatogram is shown in Figure 3 (8). Third
In figure (A), nicotinamide C.

Pyridoxine d and thiamine e peaks are confirmed. The recovery rate of each compound was 95% or more.

Comparison sample 1 Same as Example 2 except that a protein removal solution not containing sodium lauryl sulfate was used. The obtained chromatogram is shown in FIG. 3(B). In FIG. 3(B), the peaks of nicotinamide, pyridoxine, and thiamine are not observed.

(Effects of the Invention) According to the method of the present invention, in a sample (serum, urine, etc.) in which protein components coexist, the basic analyte can be measured with high precision without being affected by the protein components. be measured. Since it has become possible to accurately measure basic substances that could not be measured directly using high-performance liquid chromatography, various diagnoses and pathological studies can be carried out effectively using the two-pronged method.

4. Figures 1 (A) and (B) are connection diagrams showing an example of the measuring device used to carry out the method of the present invention; Figures 2 (8) and 3 (A) are Chromatograms obtained in Examples of the invention; FIG. 2(B) and FIG. 3(B).

The second example in which a protein removal solution different from the method of the present invention was used.
FIG. 3A is a chroma 1-gram corresponding to FIG. 3(A) and FIG. 3(A).

1...Protein removal solution, 3...4 sample device, 4...Hexagonal pulp, 5...Protein removal column, 6...Eluent, 8
...Analytical column.

that's all

Claims (1)

[Claims] 1. (a) Adding a negatively charged substance to a sample solution containing a basic analyte and a protein component to form an ion pair between the basic analyte and the negatively charged substance. (b) a step of supplying a sample solution containing the ion pair to a protein removal column, allowing the column to adsorb the ion pair, and removing protein components by passing through the column without adsorbing protein components; (c) supplying an eluent to the protein removal column to desorb the ion pair adsorbed on the column packing material; and (d) guiding the desorbed ion pair to an analytical column and analyzing it. A method for measuring a basic substance in a protein solution, comprising the steps of: 2. A sulfonic acid in which the negatively charged substance has an alkyl group, an aromatic group, or an alkyl aromatic group; a sulfonic acid salt in which the negatively charged substance has an alkyl group, an aromatic group, or an alkyl aromatic group;
Claim 1, which is at least one compound consisting of sulfuric acid having an alkyl group, an aromatic group, or an alkyl aromatic group; and a sulfate having an alkyl group, an aromatic group, or an alkyl aromatic group. measurement method.