JPH05296989A - Method for separating optical isomer in sample - Google Patents

Abstract

PURPOSE:To accurately separate an optical isomer from a sample by feeding the sample to a column for liquid chromatograph/ filled with a filler in which a protein having an optically splitting ability is immobilized. CONSTITUTION:A separation column (a) filled with a filler in which ovomucoid is immobilized is connected to an analysis column (b) filled with the same filler. After about 20mul of a sample solution prepared by dissolving about 100g of a mixture of propranolol and ceramics in a human blood serum is fed to the column (a) at a rate of about 1ml/min and a cleaning solution is fed to the column (a) for about three minutes, an eluent is fed to the column (a). These operations are repeated, for example, five times. Then, by switching the eluent from the column (a) to the column (b), propranolol is fed to the column (b). The optical isomer eluted from the column (b) is measured with a fluorescence detector (e) Therefore, even when a sample contains a protein, an optical isomer contained in the sample can be separated and analyzed. Therefore, no pre- processing is required for removing the protein and the occurrence of a measurement error associated with the pre-processing can be eliminated.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention can detect a specific amount of a protein-containing sample, for example, a mixture of photoisomers in a biological fluid represented by blood, serum, urine, etc., without being affected by the protein. The present invention relates to a method for separating optical isomers. Furthermore, the present invention relates to a method for separating a protein-containing sample without performing pretreatment such as deproteinization operation prior to separation of the optical isomer.

[0002]

2. Description of the Related Art As a method for separating optical isomers in a sample,
A method by liquid chromatography is known, which uses a column packed with a packing material having a variety of functional groups and having an optical resolution.

[0003]

When the above method is applied to a sample containing a protein such as blood, serum or urine,
There is a problem that the protein is adsorbed on the packing material and the resolution is lowered.

As a solution to this problem, when a sample contains a protein, the organic solvent or acidic solution is added to the sample to remove the protein prior to the separation operation of the optical isomers. It has been proposed to perform deproteinization treatment of

However, such pretreatment operation not only reduces the advantage of liquid chromatography that the operation is simple and can be carried out rapidly, but also in the case of separation of optical isomers for a large number of samples, There is a possibility that an error may occur in the preprocessing stage.

[0006]

[Means for Solving the Problems] The inventors of the present invention have conducted earnest studies for the purpose of providing a separation method which enables accurate separation of optical isomers without reducing the advantages of liquid chromatography. As a result, they have found that this object can be achieved by using a packing material in which a protein having an optical resolution is immobilized, and completed the present invention.

That is, the present invention provides a method for separating an optical isomer from a mixture of optical isomers in a sample containing a protein, in which a sample is packed with a packing material on which a protein having an optical resolution is immobilized. -The column, and then the eluate is used. The present invention will be described below.

The present invention can be suitably applied to an optical isomer solution containing a protein as a contaminant. As such a sample, a biological sample such as blood, serum or urine can be specifically exemplified.

The protein having an optical resolution is not particularly limited, and examples thereof include bovine serum albumin, ovalbumin, and α-acid glycoprotein. Considering the properties of the optical isomers to be separated, etc. It is advisable to appropriately select and use it by conducting preliminary experiments. As a substrate for immobilizing these proteins, silica gel, synthetic polymer carrier, etc. can be used without particular limitation.

Although the present invention separates optical isomers by a liquid chromatography method, the liquid chromatography can be carried out not only by ordinary liquid chromatography but also by conventional liquid chromatography.
High performance liquid chromatography may be used. When the present invention is carried out by high performance liquid chromatography,
It is preferable to use a material having a high pressure resistance as a base material for immobilizing proteins. In addition, there is no particular limitation on the flow rate or the like when performing liquid chromatography, and it may be appropriately determined by conducting a preliminary experiment or the like.

Contaminant proteins in the sample supplied to the column are
It elutes outside the column without being adsorbed by the packing material, but the optical isomers in the sample are adsorbed by the protein with optical resolution,
To be captured. Therefore, although the adsorbed optical isomers can be separated and eluted by applying the eluate described below to the column after the operation of providing the sample to the column, in order to complete the separation from contaminating proteins, etc. For this purpose, it is preferable to wash the inside of the column by applying a suitable buffer solution or the like that does not separate or elute the adsorbed optical isomers.

Separation and elution of the adsorbed optical isomers may be carried out by changing the salt concentration or pH of the solution in the column, or by changing the methanol concentration.
It can be carried out by adding an organic solvent such as alcohol, ethanol, isopropanol or acetonitrile. Therefore, as the above-mentioned washing buffer, etc., the same salt concentration and pH as the sample are used.
Or when the sample is diluted with an appropriate buffer or the like, such as the buffer used for the dilution does not change the conditions under which the optical isomers in the sample are adsorbed to the column good. On the other hand, as the above-mentioned eluent, it is preferable to use a buffer solution in which at least one of pH and salt concentration is changed, or an organic solvent. In this case, the amount of the organic solvent added is preferably up to about 30% by volume in order to prevent alteration of the protein having an optical resolution ability which is immobilized on the substrate.

By the above operation, the optical isomers in the protein-containing sample can be separated, which means that the optical isomers can be identified at the same time. That is, by comparing the elution pattern of various optical isomers with the elution pattern of an unknown optical isomer under the same eluate conditions, the optical isomers present in the sample can be determined. You can know (analyze) whether it was of a kind.

Regarding the analysis of the optical isomers in the sample, an operation for analysis can be added separately from the operation of the present invention. For example, it is possible to exemplify the above operation as a pretreatment for analysis, and subsequently introducing the separated optical isomers into an analytical column for analysis by a liquid chromatography method. In this case, it is required to introduce the adsorbed optical isomers into the analytical column promptly, but it is particularly preferable to use an organic solvent as an eluent to achieve this purpose. Further, it is preferable to use an organic solvent in order to improve reproducibility.

In the case of adding the operation for analysis as described above, a column packed with a packing material for reversed phase liquid chromatography having an alkyl group such as octadecyl group bonded thereto,
A column packed with an ion exchange resin can be used as an example, but a column packed with a packing material in which the above-mentioned protein having optical resolution is immobilized can also be used. In this case, it becomes a tandem type in which two similar columns are connected.

[0016]

EXAMPLES Examples will be described below to explain the present invention in more detail, but these examples do not limit the present invention.

[0017]

Example 1 A liquid flow path was constructed as shown in FIG. Separation column a (Tosoh Corporation, TSK gel Enantio) packed with a packing material in which ovomucoid is immobilized.
-OVM, inner diameter 4.6 mm, length 5 mm) and analytical column b packed with the same packing material (manufactured by Toso Corporation, TSK g)
el Enantio-OVM, inner diameter 4.6mm, length 15c
m) was linked.

20 μl of a sample solution prepared by dissolving 100 ng of a racemic mixture of propranolol in 20 μl of human serum was applied to a separation column at a flow rate of 1 ml / min, and a washing solution (20 mM phosphate buffer (p
H 6.8)) for 3 minutes, and then the eluate (20 mM phosphate buffer (pH 6.8) / acetonitrile = 70/30 (volume ratio)) for 5 times in total. Column switching was performed on the eluate from the separation column, and propranolol was applied to the analytical column. The optical isomers eluted from the analytical column were measured using a fluorescence detector e at an excitation wavelength of 305 nm and a measurement wavelength of 350 nm.

As a result, an elution pattern as shown in FIG. 2 was obtained. The two peaks (peaks 1 and 2) shown in FIG. 2 are the propranolol-containing ones from the results of the same operation individually performed on the racemic mixture of propranolol or human serum.
It was possible to identify that it originated from Le.

From the above results, it was shown that the optical isomers can be separated and further analyzed according to the present invention even when a contaminant protein is present in the sample. Note that the first sample
The heights (mv) of the peaks 2 obtained in the fifth injection are 10.1, 10.2, 10.0, 10.1, 10.0, respectively. Considering that the samples used for each operation are the same amount, The fact that the peak height is constant indicates that the deterioration of the separation ability due to the adsorption of the contaminant protein on the gel is not observed.

[0021]

INDUSTRIAL APPLICABILITY According to the present invention, the separation of optical isomers in a sample containing a protein can be carried out by a liquid chromatography method without performing a pretreatment such as removing the protein from the sample. Therefore, according to the present invention, since there is no possibility of causing a measurement error due to the pretreatment operation, a method for separating optical isomers which is a simple operation and has high reproducibility is provided.

The present invention also provides a simple method for analyzing optical isomers. This method provides a highly reproducible analysis of optical isomers by a simple operation even if a sample contains a contaminant protein.

[Brief description of drawings]

FIG. 1 shows a configuration of a liquid flow path according to a first embodiment. In the figure, a is a separation column, b is an analysis column, c is an inlet of a sample or the like to a separation column, d is an inlet of an eluate or the like to an analysis column, and e is a fluorescence detector. F is a 6-way valve for high pressure, g is an outlet from the separation column, and a pump for feeding a sample or the like is omitted. In addition, d and g are connected.

FIG. 2 shows a chromatogram obtained by a fluorescence detector in Example 1, in which 1 and 2 respectively show two peaks derived from propranolol.

Claims (1)

[Claims] 1. A method for separating optical isomers from a mixture of optical isomers in a protein-containing sample, wherein the sample is packed with a packing material on which a protein having an optical resolution is immobilized. And then the eluate.