JPS63149561A - Analysis of prostaglandin - Google Patents

Abstract

PURPOSE:To enhance the measuring accuracy of prostaglandin (PG), by analyzing the recovered liquid from a gel permeation column by a liquid chromatograph equipped with a reversed-phase distribution column. CONSTITUTION:9-anthryldiazomethane (ADAM) is reacted with PG to form a PG derivative and ethyl acetate is used as the solvent of an ADAM solution at the time of this reaction to extract PG. Next, the residue after the removal of the solvent is dissolved in methanol and the resulting solution is cooled to precipitate impurities which are, in turn, removed by a centrifugal separation method to remove high-molecular impurities. Subsequently, the solvent is again removed and the liquid of the residue dissolved by ethyl acetate is passed through a gel permeation column and a solution eluted at an early stage is discharged while the residual eluate is recovered. Low-molecular impurities are removed from this recovered liquid. The liquid containing the PG derivative thus obtained is analyzed by a liquid chromatograph equipped with a reversed- phase distribution column. Since ethyl acetate is used as the solvent, the modifi cation of ADAM is reduced and the separation from a water-soluble component becomes good.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for analyzing prostaglandins, and particularly to a method for analyzing prostaglandins in a biological sample using a liquid chromatograph.

[Conventional technology]

Prostaglandins are substances that have important hormone-like functions that control biological functions, and it is known that biological tissues or fluids containing prostaglandins can be processed and analyzed using liquid chromatography.

JP-A No. 61-202164 discloses that 9-anthryldiazomethane C or less ADAM, which has been pre-prepared to mm, is added to biological samples.
A method is disclosed in which prostaglandins (hereinafter abbreviated as PG) are derivatized by the action of PG (hereinafter abbreviated as PG), subjected to various pretreatments, and then analyzed with a liquid chromatograph.

[Problem that the invention seeks to solve]

Conventionally, ADAM was purified before reacting with a sample containing PG. This is because commercially available ADAM contains many coexisting substances and cannot be used as an analytical reagent as it is. On the other hand, there are problems in that ADAM itself is unstable and easily deteriorates, and PG in the sample also tends to change rapidly over time. However, PG is ADAM
The derivative produced by the reaction with is stable over time and has fluorescence, so the fluorescence can be detected and quantified with a chromatographic minute hand, so it is used practically.

When purified before the reaction with ADAMt-PG, there was a problem in that the quality deteriorated during the purification operation.

Furthermore, conventional methods have the problem of not being able to sufficiently remove impurities.

An object of the present invention is to provide a method for analyzing PG that has high ADAM utilization efficiency and can reduce PG denaturation.

[Means for solving problems]

The present invention derivatizes the prostaglandin by mixing a sample containing prostaglandin with a solution of 9-anthryldiazomethane using ethyl acetate as a solvent, and removes the solvent by volatilizing the solution, and then removes the residue. Dissolve in methanol or acetonitrile, cool this solution to precipitate insoluble matter, remove the precipitated insoluble matter by evaporating the solvent of the liquid, and dissolve the residue in ethyl acetate,
The method is characterized in that the dissolved solution is passed through a gel permeation column to separate and remove low-molecular substances, and the recovered solution from the gel permeation column is analyzed using a liquid chromatograph equipped with a reverse phase distribution column.

[Effect]

In the present invention, instead of first purifying ADAM that has been stored in cold storage, it is first reacted with PG to form a PG derivative before purification. During this reaction, ethyl acetate was used as a solvent for the ADAM solution.

Let PG be extracted.

Next, a solution obtained by dissolving the solvent-removed residue in methanol or acetonitrile is cooled to a low temperature (-20'C) to precipitate impurities, and the precipitate is removed by centrifugation or filtration. This removes polymeric impurities.

Next, the solvent is removed again, and the residue dissolved in ethyl acetate is passed through a gel permeation column (hereinafter referred to as GPC column), the initially eluted solution is discharged, and the remaining eluate is collected. Low molecular impurities have been removed from this recovered liquid. The liquid containing the PG derivative thus obtained is analyzed using a liquid chromatograph equipped with a reverse phase distribution column.

By using ethyl acetate as a solvent, denaturation of ADAM can be reduced and separation from water-soluble components can be improved, compared to the case where a normal solvent is used.

〔Example〕

FIG. 1 shows an analytical operation according to an embodiment of the present invention.

Biological samples are pretreated to remove proteins.

1 ml of serum sample or biological tissue is treated by ultrafiltration and the filtrate is collected. As this ultrafilter, Sentry Flow CF 25 manufactured by Amicon was used.

Next, 1 m + 2 of ADAM reagent solution (0.1%) dissolved in ethyl acetate was added to 1 m Q of the filtrate containing PG, shaken for 3 minutes, and then placed in a centrifuge at 1000
Centrifuge at G gravity. The PG derivative produced by reaction with ADAM is dissolved in the ethyl acetate liquid layer. This liquid layer is taken out into a test tube, and the solvent is volatilized and removed while blowing nitrogen gas over a hot bath.

The dried residue was dissolved in 1 mfl of methanol or acetonitrile, and then cooled at -20°C for 12 hours.
Precipitates polymeric impurities. Next, this suspension is centrifuged to separate the precipitate and the liquid layer is taken out. In this case, filtration may be performed instead of centrifugation.

The taken out liquid is taken out into a test tube, and the solvent is volatilized and removed while nitrogen gas is passed through the hot bath again, and the test tube is dried. The residue is dissolved in 100 μΩ ethyl acetate, the solution is introduced into a GPC column and separated using chloroform as eluent, the initial eluate from the GPC column is drained and the remainder is collected. As a GPC column, GELPADK-R420 manufactured by Hitachi Chemical Co., Ltd. was used. This removes low molecular weight impurities.

The liquid obtained as a preparative liquid in this way.

Impurities are significantly reduced compared to conventional products.

This fractionated liquid is separated and analyzed using a liquid chromatograph equipped with a reverse phase distribution column to obtain a chromatogram of derivatized PG. A fluorometer is used as a detector.

Figure 2 shows a chromatogram obtained based on the conventional method, and Figure 3 shows a chromatogram obtained for the same sample based on the present invention. Peak 1 is thromboxane Bz (TXBz). .

Peak 2 is P G E z and peak 3 is P G
Fzα, and peak 4 is 6-keto PGFzα.

Peak 5 is P G E s. Based on the present invention,
It is understood that the peaks due to impurities are reduced.

According to the above embodiments, denaturation of prostaglandin during sample pretreatment can be reduced, and the labeling agent can be purified after one reaction, so stable derivatization is possible. Especially prostaglandins and A
Both DAM and DAM are unstable in themselves, but the derivatized products are stable, so water droplets are effective. Chromatogram is also A
The quantitative accuracy was improved by removing the high molecular region of the DAM reagent with methanol and the low molecular region with GPC method.

〔Effect of the invention〕

According to the invention, ADAM can be used for reaction before deterioration progresses, and the accuracy of PG measurement can be improved.

[Brief explanation of the drawing]

Fig. 1 is a diagram showing the analytical operation of one embodiment of the present invention;
is a diagram showing a chromatogram obtained by a conventional method. FIG. 3 is a diagram showing a chromatogram based on the present invention.
1, fl. ,
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Claims (1)

[Claims] 1. In a method of analyzing prostaglandin in biological tissues or fluids using liquid chromatography, the above-mentioned prostaglandin is extracted by mixing a 9-anthryldiazomethane solution using ethyl acetate as a solvent with a sample containing prostaglandin. After derivatization and removing the solvent of this liquid by volatilization, the residue is dissolved in methanol or acetonitrile, this solution is cooled to precipitate insoluble materials, and the solvent of the liquid from which the precipitated insoluble materials are separated is removed. The residue was dissolved in ethyl acetate, and the solution was passed through a gel permeation column to separate and remove low-molecular substances.
A method for analyzing prostaglandin, comprising analyzing the liquid recovered from the gel permeation column using a liquid chromatograph equipped with a reversed phase distribution column.