JPS63163272A - Method for measuring acidic material in protein solution - Google Patents

Abstract

PURPOSE:To measure an acidic material to be measured with high accuracy by adding a positive charge material to a sample soln. contg. the acidic material to be measured and protein to form iron pairs and removing a protein component in a column. CONSTITUTION:The positive charge material is added to the sample soln. contg. the acidic material to be measured and the protein to form the ion pairs. The ion pairs are formed by adding alkylamine and tetraalkyl ammonium salt as the positive charge material to, for example, homovanillic acid, vanyllyl mandelic acid, etc., which are the acidic material to be measured. The sample soln. 1 contg. the ion pairs is passed through a selector valve 4 and the protein component is removed from an outlet F of the valve 4 without adsorbing the same in a protein removing column 5. An eluate 6 is then passed through the valve 4 and the adsorbed ion pairs are removed in the column 5 and are separated in a column 8, by which the acidic material to be measured is analyzed. Since the protein component is removed, the analysis of the acidic material to be measured with high accuracy without being affected by the protein component is permitted.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention is a method for measuring acidic substances in a solution containing protein components, more specifically, a method for measuring acidic substances contained in serum etc. using a reversed-phase liquid chromatography method. The acidic substance to be measured is
This invention relates to a method for accurately measuring 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 the performance of an analysis or the function of an analytical column.

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.

A sample solution containing protein components and non-protein substances to be measured is passed through a protein removal blade ram to adsorb only the non-fluorescent components and remove the protein components, and then the adsorbed non-protein components are eluted and analyzed. A method for introducing the column 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, the substance to be measured is an acidic substance, that is, a substance such as vanillylmandelic acid or homovanillic acid (both of which are metabolites of catecholamines) that dissociates and has a negative charge. In this case, the substance is removed from the column together with the protein component without hydrophobic interaction with the reversed-phase packing material (that is, without being adsorbed by the packing material). When a column packed with anion exchange type packing material is used to adsorb acidic substances, #J, the yield of acidic substances is relatively good.

Since protein components are non-specifically adsorbed, the protein removal blade ram is rendered useless, and furthermore, it has the disadvantage of accelerating the deterioration of the analytical column.

(Problem that the invention attempts to solve) The present invention solves the above-mentioned conventional drawbacks.

The purpose is to provide a method for directly measuring an acidic analyte in a sample containing an analyte and a fluorescent component without performing a complicated protein removal operation. Another object of the present invention is to provide a method for accurately measuring the above-mentioned acidic analyte by reverse-phase high-performance liquid chromatography equipped with a protein removal blade ram.

(Means for Solving the Problems) The method for measuring acidic substances in protein solutions of the present invention includes (a) adding a positively charged substance to a sample solution containing an acidic analyte substance and a protein component; A step of forming an ion pair between the substance to be measured and the positively charged substance.

(Step (C) Removal of protein components by supplying a sample solution containing bioion pairs to a protein removal blade ram, allowing the column to adsorb the ion pairs, and passing through the column without adsorbing protein components. The method includes a step of supplying an eluent to a protein removal column to desorb the ion pair adsorbed on the packing material of the column, and a step of guiding the tdl-desorbed ion pair to an analytical column and analyzing it. This achieves the above objective.

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

Any of the common anionic substances that can dissociate and have a negative charge are included. Representative examples include homovanillic acid, vanillylmandelic acid, aspirin, salicylic acid, and the like. these are.

For example, it is a substance that is present in blood or urine as a biological component or administered from the outside.

The type of positively charged substance added to measure the above-mentioned substance to be measured is not particularly limited, but is usually 9.

Alkylamines, tetraalkylammonium salts, and the like can be suitably used. The alkyl group of each of the above compounds preferably has 16 or less carbon atoms, and i and i1 preferably have 15 or less carbon atoms. When the number of carbon atoms in the alkyl group exceeds 20, it becomes highly hydrophobic, and therefore does not dissolve in the sample solution to which this substance is added or in the deproteinization solution (which contains a buffer as the main component and contains almost no organic solvent).

Examples of the positively charged substance include cetyltrimethylammonium bromide and tetraheptylammonium bromide.

The method of the invention is embodied, for example, by the apparatus shown in FIGS. 1(^) and 1(B). This device connects terminals A-F.
A protein removing blade ram 5 is connected to a six-way valve 4 having a 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 blade ram 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 filler filled in the protein removal blade ram 5 adsorbs non-protein components and does not adsorb protein components under certain conditions 9, such as water or a buffer solution of a certain pH. and other conditions 9, e.g. a different pH than 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 typical packing material for reversed phase chromatography, particularly a highly hydrophilic packing material, is used. For example, hydroxyl group,
A polymer gel having hydrophilic groups such as carboxyl groups and ether groups on its surface is used. Examples of materials include 3, 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 with a particle size of about 10 to 20 μm obtained by suspension polymerization.

As the sample, for example, a serum sample containing the above-mentioned acidic substance to be measured and which 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 positively charged substance is added to the sample and/or the protein removal solution in advance. 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 positively charged substance combines with the acidic analyte (which dissociates and has a negative charge) in the sample to form an ion pair. Therefore, the polarity of the substance to be measured decreases. Furthermore, since the positively 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 blade ram 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 positively charged substance molecule, the more
For example, the longer the alkyl chain, the higher the degree of adsorption of the ion pair to the protein removal column 5. The fluorescent component in the sample is not adsorbed and passes through the column 5, and is discharged to the outside of the system via the connection ends E and F of the six-way valve 4.

On the other hand, the eluent 6 in the eluent tank 61 is sent to the analytical column 8 of the liquid chromatography via the connection end 5 and C by the constant flow pump 7. Next, as shown in FIG. 1(B), when the hexagonal pulp 4 is switched and the connecting end is connected to the other side, the eluent 6 in the eluent tank 61 is removed by the constant flow pump 7 through the connecting end and E. It is supplied to the protein blade ram 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 second analytical column 8. The analytical column 8 is filled with a packing material having higher adsorption properties than the packing material used in the protein removal blade ram 5. By using such a highly adsorbent packing material, the ion pairs adsorbed on the protein removal blade ram are completely desorbed by the eluent, which is then directly passed through the analytical column where the analyte is accurately separated (separated). and can be analyzed.

(Function) In the method of the present invention, ion pairs are formed by adding a positively charged substance, which is an oppositely charged substance, to an acidic analyte. Therefore, the polarity of the substance to be measured becomes low. Furthermore, because of the alkyl group or aromatic group that the positively charged substance has, the ion pair has a high degree of hydrophobicity. Therefore,
It becomes possible to adsorb protein to a protein removal blade ram filled with gel for reversed phase chromatography. In this way, the acidic analyte, which is a negatively charged substance, has properties similar to those of ordinary nonionic non-protein components, so that it is effectively separated from the protein components in the protein removal blade ram. The analyte from which protein components have been removed.

It is separated and measured using the analytical column 8 in the same manner as in ordinary high performance liquid chromatography.

In such a method, the acidic substance is easily measured in a short period of time with high accuracy without loss or deterioration during the measurement. Therefore, it can be used in many ways as a general method for measuring acidic substances in samples containing protein components.

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

(8) Preparation of protein removal blade ram filler gel: 400ml of 4% by weight polyvinyl alcohol aqueous solution, 40g of tetraethylene glycol dimethacrylate, log of tetramethylolmethane triacrylate, 50g of methacrylic acid,
40 g of t-toluene and 1.5 g of -hnzoyl peroxide were added. 80° while stirring at 400 rpm
After reacting with C for 10 hours, the mixture was washed with hot water and acetone to obtain 9 spherical porous polymer particles each having a particle size of 20 to 30 μm.

(B) Measurement of acidic substances: Homovanillic acid and vanillylmandelic acid in serum were measured using the measuring device shown in FIG. As the protein removal blade ram 5, a column made of stainless steel 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; manufactured by Yamamura Chemical Co., Ltd.; column size, inner diameter: 6 dragons) was used.

150 mm in length) was used. 0.5m as protein removal solution
mol/l- of tetraheptyl ammonium bromide aqueous solution (using ion-exchanged water), and as the eluent 6,
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 (8), the protein removal solution 1 is pumped into the hexagonal valve 4 at a rate of 0.6rn1/min using the constant flow pump 2.
20 μl of sample serum was injected from the sample introduction device 3 while flowing into the protein removal blade ram 5 through the connecting end A-B of the sample. This sample contains 1.0% of standard homovanillic acid in normal male serum.
μg and 0.7 μg of vanillylmandelic acid were added. Eight minutes after sample injection, the six-way valve 4 was switched and connected as shown in FIG. 1(B). The pump 7 pumps the eluent 6 from the eluent tank 61 at a rate of 1.0 d/min through the six-way valve connecting end D-E, the protein removal blade ram 5, and the six-way valve connecting end B-C for liquid chromatograph analysis. Passed through column 8. Ion pairs containing the analyte are eluted by the flow of the eluent and separated by two analytical columns.
Detected by a detector (not shown).

As a detector, an absorbance meter (UVIDEC100-Vl
; manufactured by JASCO Corporation), the absorption intensity of 28011111 was measured. The obtained chromatogram is shown in Figure 2 (8). In FIG. 2(A), peak a of homovanillic acid and peak b of vanillylmandelic acid are confirmed. The recovery rates of homovanillic acid and vanillylmandelic acid were each 98% or higher.

Comparison 4 with Example 1 Same as Example 1 except that ion-exchanged water was used as the protein removal solution. The obtained chromatogram is shown in Figure 2 (B
). In FIG. 2(B), the peaks of homovanillic acid and vanillylmandelic acid are not confirmed.

Charcoal 1 Samples include 1.0 μg of aspirin and 1.0 μg of salicylic acid.
20 μl of normal male serum containing 0 μg was used, 0.1 M phosphate buffer (pl(2,5)-methanol mixture (volume ratio 50:50) was used as the eluent, and the measurement wavelength was set to 2.
The same as in Example 1 except that the thickness was 45 nm. The obtained chromatogram is shown in FIG. 3(A).

In FIG. 3(A), the peaks of aspirin C and salicylic acid d are confirmed. The recoveries of aspirin and salicylic acid were each over 95%.

This example was the same as in Example 2 except that ion exchange water was used as the protein removal solution. The obtained chromatogram is shown in Figure 3 (B
). In FIG. 3(B), the peaks of aspiric acid and salicylic acid are not confirmed.

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

4. Figures 1 (A) and (B) are connection diagrams showing an example of a measuring device used in carrying out the method of the present invention; Figures 2 (A) and 3 ( A) is a chromatogram obtained in an example of the present invention; FIG. 2(B) and FIG. 3(B) are.

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

DESCRIPTION OF SYMBOLS 1... Protein removal solution, 3... Sample introduction device, 4... Hexagonal valve, 5... Protein removal blade ram, 6... Eluent, 8
...Analytical column.

Figure 1 (A) Figure 1 (B) Figure 2 (A) Figure 2 (B) Figure 3 (A) Figure 3 (B)

Claims (1)

[Claims] 1. (a) A step of adding a positively charged substance to a sample solution containing an acidic analyte and a protein component to form an ion pair between the acidic analyte and the positively 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) ) a step of supplying an eluent to the protein removal column to desorb the ion pair adsorbed on the packing material of the column; and (d) a step of guiding the desorbed ion pair to an analytical column and analyzing it. A method for measuring acidic substances in protein solutions containing. 2. The measuring method according to claim 1, wherein the positively charged substance is a tetraalkylammonium salt and/or an alkylamine.