JP2559535B2 - Inorganic anion analysis method and analyzer - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and an analyzer for analyzing inorganic anions in a biological sample by liquid chromatography, and more particularly to an analyzer and a analyzer having a constitution capable of automating deproteinization. Regarding

[0002]

2. Description of the Related Art Conventionally, an analysis method utilizing ion chromatography has been known as a method for analyzing inorganic anions in a biological sample using liquid chromatography. However, there have been few reports on ion chromatography in which ions in biological samples are measured. "Ion Chromatography" (Instrument Analysis Practical Series, Japan Society for Analytical Chemistry, Kyoritsu Shuppan, pp. 204-205 (1988)) gives an example of anion measurement using serum as a sample. In this measuring method, a serum sample is deproteinized with acetonitrile, and thereafter, a treatment for removing excess chlorine ions contained in the sample is performed, and the sample is prepared as an injection sample. Then, an anion analysis column is used to detect anions by an electric conductivity meter. According to this measuring method, nitrite ion, nitrate ion, phosphate ion and sulfate ion can be measured in the order of elution.

As described above, when the sample is a biological sample such as serum, deproteinization must be performed in advance. Otherwise, a large amount of protein is contained in the biological sample, and if the protein is directly injected into the column, the protein is irreversibly adsorbed to the column and deteriorates the column. Therefore, in the analysis of the biological sample, it is necessary to perform the deproteinization treatment in advance as described above and to inject the sample subjected to the deproteinization treatment into the column. As the deproteinization treatment, an acid such as perchloric acid or trichloroacetic acid, or an organic solvent such as ethanol or acetonitrile is added to the sample to denature the protein, and the denatured protein is removed by centrifugation, and the supernatant is removed. The method used as an injection sample is general. However, these deproteinization treatments have problems that an interfering peak derived from the added acid is generated, or that phosphate ions, calcium ions, etc. are precipitated in the sample.

Therefore, in order to solve the above problems associated with the deproteinization treatment, a deproteinization method using an ultrafiltration membrane has been performed in recent years. Recently, disposable type ultrafilters are also on the market, and there are two types, syringe pressurized type and centrifugal type.
According to the deproteinization method using such an ultrafiltration membrane,
It is possible to improve the problem that has been caused by the deproteinization treatment using an acid or an organic solvent.

[0005]

However, when the deproteinization treatment is carried out using the ultrafiltration membrane, there are problems that the treatment requires a considerable amount of time and the cost is considerably high. In addition, in both the deproteinization treatment with an acid or an organic solvent and the deproteinization treatment with an ultrafiltration membrane, generally the analysis time becomes very long, it is difficult to automate the analysis, and the reproducibility of the measurement is not sufficient. There was also a problem.

Therefore, an object of the present invention is to allow a biological sample to be directly injected into liquid chromatography, and to automate the deproteinization process so that analysis can be performed in a short time, and further excellent reproducibility is achieved. Another object of the present invention is to provide a method and an analyzer for analyzing inorganic anions.

[0007]

The first invention of the present application is a method for analyzing inorganic anions in a biological sample by liquid chromatography, which comprises using a mobile phase for deproteinization to remove a hydrate.
The protein component in the biological sample is retained without retaining the inorganic anion in the sample in the protein retaining column packed with loxyapatite, and the inorganic anion in the sample is arranged on the downstream side of the protein retaining column. And a step of desorbing the inorganic anions from the anion holding column by an analytical mobile phase to guide them to the anion analysis column, and separating the inorganic anions with the anion analysis column. Equipped with.

A second invention of the present application is an analyzer for carrying out the above-mentioned first invention, which comprises a first mobile phase liquid feeding means,
The flow path switching means, the anion analysis column and the detection means are connected in series in this order, the first flow path, the second mobile phase liquid feeding means, the sample injection means and the hydroxyapatite.
The packed protein-retaining columns are connected in series in this order, and the second flow path is connected to the flow path switching means on the downstream side, and the shadow is connected to the flow path switching means on the inlet side and the outlet side. An ion retention column is provided so that the flow channel switching means can switch between a state in which the anion retention column is connected to the downstream end of the second flow channel and a state in which the anion retention column is connected to the first flow channel. It is configured.

[0009]

In the analysis method of the present invention, the biological sample is hydroxy.
The protein is introduced as it is to the protein holding column packed with apatite , and the protein is held in the protein holding column without holding the inorganic anion in the sample. The sample containing the inorganic anion that was not retained in the protein retention column is retained in the anion retention column. On the other hand, the inorganic anion is desorbed from the anion holding column by the mobile phase for analysis and guided to the anion analysis column to separate the inorganic anion in the anion analysis column. That is, in the present invention, the deproteinization treatment is performed in the protein retention column, and therefore, it is not necessary to deproteinize the biological sample in advance prior to the analysis. Also,
Unlike the conventional deproteinization method using an ultrafiltration membrane, the treatment does not take a long time and it is not necessary to use a relatively expensive disposable type product.

That is, the analysis method and apparatus of the present invention is a protein-retaining column packed with hydroxyapatite for deproteinization treatment which has caused various problems in the conventional method and apparatus for analyzing inorganic anions in biological samples. It has one feature in that it is solved by automating using. Further, in the analyzer of the present invention, when removing proteins from a biological sample, the proteins are held by a protein holding column filled with hydroxyapatite , and inorganic anions are held by an anion holding column, and then, , Desorbing the inorganic anions adsorbed on the anion holding column by the mobile phase for analysis by switching the flow path switching means,
It is possible to lead to an anion analysis column. Therefore, it is possible to regenerate the protein holding column connected to the second flow path while the anion holding column is connected to the first flow path by the flow path switching means to perform the separation and analysis of the inorganic anions. it can. Therefore, it is possible to significantly reduce the analysis time of inorganic anions in a biological sample, and
It is easy to automate the whole analysis system.

Hereinafter, the analysis method and apparatus of the present invention will be described more specifically with reference to FIG. FIG. 1 is a schematic configuration diagram for explaining the outline of the analysis method and apparatus of the present invention. The first flow path A is configured by connecting the first mobile phase liquid sending means 1, the flow path switching means 2, the anion analysis column 3 and the detection means 4 in series. Also, the second
The flow path B is configured by connecting the second mobile phase liquid sending means 5, the sample injection means 6 and the protein holding column 7 filled with hydroxyapatite in series, and the downstream end thereof is the flow path switching means 2 It is connected to the. further,
An anion holding column 8 is connected to the flow path switching means 2.

The flow path switching means 2 is in a state where the anion holding column 8 is connected to the second flow path B, that is, the flow path a- in FIG.
A state in which the anion holding column 8-the flow path c is connected,
It is configured so that the state where the anion holding column 8 is connected to the channel A, that is, the state where the anion holding column 8 is connected between the channels d and e can be switched. Next, the analysis method of the present invention will be described with reference to FIG.

The connection state of the sample injection flow path switching means 2 is a state in which the anion holding column 8 is connected to the second flow path B. That is, the flow path a-anion holding column 8-flow path c are connected. In this state, the mobile phase for deproteinization is sent from the second mobile phase sending means 5, and the sample is injected into the second channel B from the sample injection means 6.

[0014] The deproteinized second passage injected sample B, leading to a protein holding column 7, to retain the protein in the sample in the protein holding column 7. In this case, the inorganic anions in the sample are not retained by the protein retention column 7,
It is guided to the anion holding column 8 and held in the anion holding column 8.

Separation of Inorganic Anions Next, the connection state of the flow path switching means 2 is switched to connect the anion holding column 8 to the first flow path A. That is, the flow path d-anion holding column 8-flow path e is connected. Then, the mobile phase for analysis is sent by the first mobile phase sending means 1, the inorganic anions are desorbed from the anion holding column 8 and led to the anion analyzing column 3, and each of the anion analyzing columns 3 Separate into anions.

Washing and Regeneration of Protein Retaining Column In parallel with the above-mentioned step of separating inorganic anions, the mobile phase for washing is sent by the second mobile phase sending means 5, whereby it is held in the protein holding column 7. The protein that is present is eluted. After the washing is completed, in order to regenerate the protein holding column 7, the mobile phase fed by the second mobile phase liquid feeding means 5 is switched to the deproteinization mobile phase.

Preparation for the next measurement After the regeneration of the protein retention column 7 is completed, the flow path switching means 2
The connection state is switched so that the anion holding column 8 is connected to the second flow path B. As described above, in the analysis method of the present invention, by switching the flow path switching means 2, the state in which the anion holding column 8 is connected to the second flow path B,
The state of being connected to the first flow path A can be switched, whereby the sample injection step and the deproteinization treatment step of the next measurement can be simultaneously performed while performing the inorganic anion separation step. Therefore, when a large number of biological samples are sequentially measured, the analysis time per sample can be significantly shortened.

The packing material for the protein retention column 7 used in the analytical method and analytical apparatus of the present invention is:
It can hold proteins contained in the sample, and fillers der Ru hydroxy hardly retain inorganic anion
Limited to sheapatite. Hydroxyapatite
It has the same chemical composition as the inorganic components of teeth and bones, and is known as a material with excellent biocompatibility.It is used as a packing material for chromatography in the fields of medicine and biochemistry, such as proteins, nucleic acids, and enzymes. Used for separation and purification of molecular substances. Regarding the hydroxyapatite that can be used in the present invention, the specific surface area, the pore distribution, the ratio of calcium to phosphoric acid (Ca / P ratio) and the like are not particularly limited, but there is no irreversible adsorption of a biological sample and when it is columnized. It is necessary to use a material having a shape and particle size that allow liquid to pass through and a mechanical strength that does not collapse when passing through the liquid. As the packing material of the anion analysis column 3, any packing material that can separate inorganic anions can be used. For example, an ion exchanger having a quaternary ammonium group introduced as a functional group can be mentioned. Specifically, as the anion analysis column for ion chromatography, those commercially available from various manufacturers can be used.

As a packing material for the anion holding column 8,
The same packing material as the packing material of the anion analysis column 3 or a packing material having a weaker retention than the packing material used in the anion analysis column 3 may be used. As the detection means 4,
A substance that can detect various inorganic anions that are measurement target substances can be appropriately selected. In ion chromatography, it is common to use a conductivity detector. In the case of analyzing inorganic anions having ultraviolet absorption such as nitrite ion and nitrate ion, it is possible to use an ultraviolet absorbance detector.

The mobile phase for deproteinization is hydroxy
Apatite is held by the protein in the protein holding column 7 which is filled, will require an be those that do not retain the inorganic anion, if example embodiment, concentration is 1
0mM used following phosphate buffer or deionized water, as the cleaning mobile phase, will require an be those capable of eluting the protein retained in the protein holding column, for example, concentration 100mM Above 600
A phosphate buffer of mM or less is used.

As the mobile phase for analysis, those which can elute the inorganic anions held in the anion holding column 8 and can be separated into each anion in the anion analysis column 3 are used. For example, the concentration is 0.1 mM or more, 50 m
A buffer solution of M or less is used.

[0022]

DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 2 is a block diagram showing an inorganic anion analyzer according to an embodiment of the present invention. In FIG. 2, the first
In the flow path A, a first mobile phase liquid sending means including a mobile phase container 11a storing a mobile phase 11 for analysis and a liquid sending pump 12, a high pressure flow path switching valve 13 as a flow path switching means, anion analysis. The column 14 and the detector 15 are connected in series in this order. A low-pressure flow passage switching valve 16 is connected to the base end of the second flow passage B. The low-pressure flow path switching valve 16 is connected to mobile phase containers 17a and 18a so that the deproteinization mobile phase 17 or the protein desorption (washing) mobile phase 18 is supplied. A liquid feed pump 19 is connected to the downstream side of the low pressure flow path switching valve 16. The mobile phase 17 for deproteinization, the mobile phase container 17a, the mobile phase 18 for protein desorption, the mobile phase container 18a, the low-pressure flow path switching valve 16 and the liquid feed pump 19 constitute a second mobile phase liquid feed means. An injector 20 as sample injection means is connected to the downstream side of the liquid feed pump 19. A hydroxyapatite packed column 21 as a protein holding column is connected to the downstream side of the injector 20, and a downstream side of the hydroxyapatite packed column 21 is connected to a high-pressure flow path switching valve 13.

Further, the anion holding column 22 is connected between the ports h and k of the high pressure flow path switching valve 13. The high-pressure flow path switching valve 13 is composed of a hexagonal valve, and the outlet side of the hydroxyapatite packed column 21 is connected to the port g of the hexagonal valve. Also,
The downstream side of the liquid feed pump 12 in the first flow path A is a port i,
The inlet side of the anion analysis column 14 is connected to the port j. Each port g to l of the high-pressure flow path switching valve 13
In the meantime, the connection state I shown by the solid line in the figure and the connection state II shown by the broken line are configured to be switched.

Reference numeral 23 denotes a data processor, which is provided to calculate a measurement value from the detection amount obtained by the detector 15. Also, with a controller not shown,
On / off of the liquid feed pumps 12 and 19, switching of the low pressure flow passage switching valve 16, operation of the injector 20, switching of the high pressure flow passage switching valve 13, operation of the detector 15 and the like are controlled.

Next, a description will be given of a concrete experimental example performed by using the analyzer of the embodiment shown in FIG. Each device used is as follows. Liquid-sending pumps 12, 19 ... Shimadzu LC-9A injector 20 ... Shimadzu SIL-6B detector 15 ... Shimadzu ultraviolet absorption detector (SPD-6AV) Low pressure flow path switching valve 16. Sekisui Chemical Co., Ltd. SGR-720 high pressure flow path switching valve 13 ... Shimadzu FCV-2AH data processor 23 ... Shimadzu C-R3A controller ... Shimadzu SCL-6B hydroxyapatite packed column 21 ... Mitsui Toatsu Chemicals hydroxyapatite Packed column HCA-Column A-4007, (column diameter 4.0 mm x length 75 mm) Anion analysis column 14 ... Japan Millipore's IC-Pak Anion anion holding column 22 ... Tosoh's IC-Conc-A (ion Exchange group is quaternary ammonium group) Also, transfer used The phases are as follows:

Mobile phase for deproteinization 17: Deionized water (specific resistance 16 MΩ-cm) Mobile phase for protein desorption (washing) 18: 300 mM potassium phosphate buffer (pH 6.8) Mobile phase for analysis 11: 2 mM phosphoric acid Potassium buffer solution (pH 7.8) The liquid feed rate of each mobile phase was as follows.
The analytical mobile phase 11 was 1.2 ml / min, the deproteinization mobile phase 17 was 1.0 ml / min, and the protein desorption mobile phase 18 was 1.0 ml / min.

For the measurement, the high pressure flow path switching valve 1
The connection state of No. 3 and the type of the second mobile phase to be fed by the liquid feed pump 19 were changed over time as shown in Table 1 below.

[0028]

[Table 1]

In the measurement, first, a standard sample having a nitrite ion concentration of 5 ppm and a nitrate ion concentration of 5 ppm was prepared, and the wavelength was 220 nm and the injection amount was 10 μl, and the measurement was carried out under the above conditions. The calibration curve was prepared by the one-check curve method (calculated by the peak area value). The results are shown in FIG. Next, an example using a biological sample will be described. Example Blood of a healthy person was collected using a vacuum blood collection tube containing a separating agent (Insepack SI-0706S manufactured by Sekisui Chemical Co., Ltd.), coagulated and then centrifuged to obtain serum. 200 μl of the obtained serum was sampled in a sample bottle, and the injector 20
Set to. Next, the measurement was performed under the same measurement conditions as when the standard sample was measured. The injection volume of the serum sample was 10 μl. The results are shown in FIG. Comparative Example 1 The serum used in Example 1 and deionized water were mixed at a volume ratio of 1: 1 and 1 ml of a mixed sample was subjected to an ultrafiltration membrane (Millipore, Molcut II, LGC molecular weight cutoff 1). 10,000). 1 to get 0.5 ml of filtrate
It took 7 minutes. Next, using this filtrate as a sample, the injection amount was set to 20 μl, and the measurement was performed in the same manner as in Example 1.
Results are shown in FIG.

In Example 1 and Comparative Example 1, substantially the same amount of serum was injected, but as is clear from the comparison of the results in FIGS. 4 and 5, nitrite ion and nitrate ion It can be seen that the two peaks of 1 are separated in the same manner. Therefore, it can be seen from Example 1 that nitrate ion and nitrite ion in a serum sample can be accurately measured without performing deproteinization treatment in a separate step in advance.

[0031]

As described above, according to the inorganic anion analysis method and apparatus of the present invention, hydroxyapatite is filled.
Since the protein is removed in the packed protein retention column, it is not necessary to perform deproteinization treatment on the biological sample in advance in a separate step. That is, the deproteinization process can be automated.

Therefore, the protein can be removed in an extremely short time as compared with the conventional method using the deproteinization treatment by the ultrafiltration method, and the time required for the whole analysis method can be greatly shortened. . Further, in the analyzer of the present invention, by switching the connection state of the anion holding column by the flow path switching means, it is possible to regenerate the protein holding column in parallel with the detection of inorganic anions in the anion holding column. As a result, the measurement time during continuous measurement can be significantly shortened and the analysis method can be easily automated.

[Brief description of drawings]

FIG. 1 is a schematic configuration diagram for explaining an outline of an inorganic anion analysis method and apparatus of the present invention.

FIG. 2 is a configuration diagram of an inorganic anion analyzer of an example.

FIG. 3 is a diagram showing a chromatogram of a standard sample.

FIG. 4 is a diagram showing a chromatogram when nitrite ion and nitrate ion are analyzed in Example 1.

5 is a diagram showing a chromatogram when nitrite ion and nitrate ion are analyzed in Comparative Example 1. FIG.

[Explanation of Codes] 1 ... First mobile phase liquid sending means 2 ... Flow path switching means 3 ... Anion analysis column 4 ... Detection means 5 ... Second mobile phase liquid sending means 6 ... Sample injection means 7 ... Protein holding column 8 ... Anion retention column A ... 1st flow path B ... 2nd flow path

Claims (2)

(57) [Claims] 1. A method for analyzing inorganic anions in a biological sample by liquid chromatography, which comprises removing a protein component in a sample from a hydroxy component without retaining the inorganic anions in the sample by a deproteinizing mobile phase.
It is retained on a protein retention column packed with Patite , and the inorganic anion in the sample is retained on an anion retention column located downstream of the protein retention column. And a step of desorbing the inorganic anions, introducing the inorganic anions to an anion analysis column, and separating the inorganic anions by the anion analysis column. 2. A first flow path in which a first mobile phase liquid sending means, a flow path switching means, an anion analysis column and a detection means are connected in series in this order, a second mobile phase liquid sending means, and sample injection. Means and hydroxya
A protein holding column filled with pattetes is connected in series, and a downstream end of the protein holding column is connected to the flow passage switching means, and a flow passage switching means has an inlet side and an outlet side. A connected anion retention column, wherein the flow path switching means connects the anion retention column to the downstream end of the second flow path, and connects the anion retention column to the first flow path. An inorganic anion analyzer characterized by being configured to switch between states.