JPS62156559A - Preparation of reagent for liquid chromatography and liquid chromatography - Google Patents

Abstract

PURPOSE:To make microanalysis of PGs making use of the phenomenon in which a considerable difference in solubility exists between the ADAM itself and impurity when the temp. of an ADAM soln. is <=-10 deg.C. CONSTITUTION:The 9-anthryldiazomethane ADAM forms a deriv. by reacting with the carboxyl group of the PGs as shown in the formula and therefore the deriv. is subjected to a component sepn. by a liquid chromatograph and the fluorescent intensity thereof is measured. An example of PGE2 is shown here. The ADAM is dissolved in an org. solvent and after the soln. is cooled down to <=-10 deg.C, the insoluble component during cooling is removed in the absence of pressure. The refined 9-anthryldiazomethane soln. is then recovered. The soln. is subjected to the sepn. analysis by the liquid chromatography after the reaction to form the deriv., then the unreacted ADAM flows out of the sepn. column in precedence to the outflow of the PGs and therefore, the unreacted ADAM and the PGs can be fractionated. The impurities which are difficult to be separated from the PGs are removed prior to the formation of the deriv. and therefore, the trace detection of the PG deriv. is made possible.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for preparing a reagent used for analyzing prostaglandins by liquid chromatography and an analysis method using the reagent.

[Conventional technology]

Prostaglandins (PGs) are substances that have attracted clinical attention as they play an important physiological role. Examples of PGs include PGE.

P’GEz, PGEa, PGFti, PGFzm.

Approximately 20 types are known, including PGFg, <, 6-kedoPGFtv, thromboxane (TX)Bz, PG endoperoxide, and prostacyclin (PGIz).

Since it is difficult to measure such PGs in their original form with a fluorescence detector, attempts have been made to react them with a fluorescent pre-label reagent for carboxyl groups in advance and separate and analyze the reaction products using liquid chromatography. (For example, Japanese Patent Application Laid-Open No. 58-108457). A typical fluorescent prelabel reagent is 9-anthryldiazomethane (Ar
) AM) However, this method has not yet been generalized because of the practical mass points.

[Problem that the invention seeks to solve]

Since PGs are contained in extremely small amounts in biological samples (eg, human serum), highly sensitive detection is required.

On the other hand, the synthesized ADAM reagent contains many impurities, and even if powdered ADAM is dissolved in an organic solvent and reacted with PGs during use and separated and analyzed using liquid chroma 1~graph, PG'E1 It was difficult to separate ADAM derivatives from these impurities.

Therefore, it has been difficult to carry out practical and quick analysis, requiring a great deal of effort to extract and concentrate PGs before reacting them with ADAM.

An object of the present invention is to provide a reagent preparation method that reduces impurities in 9-anthryldiazomethane and enables the practical preparation of prostaglandins.
The object of the present invention is to provide an analytical method for performing liquid chromatography using the reagent thus prepared.

[Means for solving problems]

ADAM reacts with the carboxyl group of PGs as shown in the following formula to form a derivative, and the components of this derivative are separated using a liquid chromatograph and the fluorescence intensity is measured. Here, an example of PGE2 is shown.

When ADAM is processed at high temperatures or pressures, its activity is significantly reduced. Therefore, in the present invention, liquid chromatography reagents are prepared at low temperatures and high pressure is not applied.

In the reagent preparation method of the present invention, 9-anthryl diazomethane is dissolved in an organic solvent, cooled to -10°C or lower, and undissolved components upon cooling are removed under no pressure to obtain purified 9-anthryl diazomethane. It is characterized by recovering a diazomethane solution.

Moreover, in the method for analyzing PGs based on the present invention, 9-anthryldiazomethane is dissolved in an organic solvent.

A solution-like reagent is obtained by removing insoluble components in a cooled state at -10°C or lower, and this solution-like reagent is mixed with a sample containing prostaglandins.
It is characterized by forming a reaction solution that has produced an anthryl diazomethane derivative, introducing this reaction solution into a separation column of a liquid chromatography device to separate the components, and measuring the eluate from the separation column by fluorescence photometry. .

One of the aspects of the above-mentioned removal of undissolved components under non-pressurized conditions is
The ADAM solution is allowed to flow down by gravity into a column packed with a coarse packing material, and the liquid is collected. In another embodiment, the centrifugation is performed by slowing down the speed changes during startup and shutdown, and the supernatant liquid is collected.

The PGs derivatized with the ADAM reagent are introduced into a liquid chromatography device, and a reverse phase distribution method is suitable for the liquid chromatography at this time.

[Effect]

In the present invention, since the ADAM solution is prepared by processing without applying high temperature and high pressure, decomposition of ADAM is prevented. In the present invention, the ADAM solution is minus 10
This method utilizes the fact that at temperatures below .degree. C., there is a significant difference in solubility between ADAM itself and impurities.

After the derivative formation reaction, when separated and analyzed by liquid chromatography, unreacted ADAM flows out from the separation column before PGs flow out, so unreacted ADAM and PG are separated.
It is possible to separate the types.

Since impurities that are difficult to separate from PGs are removed before derivatization, trace amounts of PG derivatives can be detected.

〔Example〕

First, the method for preparing the ADAM reagent will be explained.

5 mg of commercially available powdered ADAM is dissolved in 5 mQ of methyl alcohol. At this time, since ADAM is hardly soluble, it is dissolved while applying ultrasonic waves (operation 1).

Next, the solution was cooled to -20℃ for 24 hours.
Let stand for a while. This results in a gel-like or sherbet-like precipitate. This gel-like precipitate contains a large amount of impurities and only a small amount of ADAM. Then, gently collect the supernatant using a pipettor. The amount of liquid that can be collected is approximately 4 mQ (operation 2). Even in this state, a considerable amount of interfering coexisting substances are removed, but at -20℃
A somewhat suspended state remains.

Next, insert a syringe-shaped plastic tube (inner volume: 5 to 1
0 m Q) was filled with glass wool at the outlet end to form a filter, and biobeads S were placed on top of the glass wool layer.
An adsorption column is formed by filling 3 mQ of X2 (manufactured by Bio-Rad). This adsorption column is held on a stand, and the supernatant liquid drawn into the pipettor is gently dripped from above the adsorption column. The first 1 mQ of the liquid flowing out from the adsorption column is discarded, and the remaining liquid is collected in a container.

The amount of liquid recovered is approximately 2-2.5 mu (operation 3).
). By such operation, ADAM is hardly adsorbed, and insoluble components are filtered and adsorbed. Since the purification is now complete, this recovered solution, the AI)AM reagent, is stored at -20°C. This reagent can be used for about one week. If a precipitate occurs in this reagent solution,
It can be used by performing the above-mentioned operation 3 and purifying it.

When used in the fluorescent prelabeling reaction of PGs, methyl alcohol is added to the above-mentioned reagent solution to dilute it 5 times.

Methyl alcohol used in a series of operations is bubbled with nitrogen gas to remove dissolved oxygen in advance. Even during the treatment operation, it is better to use nitrogen gas for total oxygenation, which gives better results.

When trying to measure PGlff in human serum,
Preferably, the serum is pretreated. For pretreatment, first, 1 mQ of serum sample was transferred to Centriflow CF25
(manufactured by Amicon) at 4°C.

Ultrafilter at 10,000. Add 1 mfl of chloroform to this filtration and shake for 3 minutes to extract PGs into the chloroform layer. After leaving it to stand for about 10 minutes, collect the chloroform layer in a test tube. At this time, nitrogen gas is blown into the test tube. This test tube is immersed in a water bath at about 50° C. and heated to evaporate to dryness (operation 4).

The pre-labeling reaction of PGs is performed between the 5-fold dilution of the ADAM reagent obtained in Step 3 and the dried residue obtained in Step 4. That is, dilute AI)A to the dry residue of step 4.
Add 50 μQ of M reagent to dissolve the residue and add 1
React for a period of time to generate ADAM derivatives of PGs (
Operation 5). This reaction solution is introduced into a liquid chromatograph to separate the components.

The separation column for liquid chromatography has an inner diameter of 4+
mm, and the length was 150 mm. Octadecylsilane (OI') with a particle size of 3 μm as a column packing material
The eluent to be passed through the 0 separation column using water 2
A mixture of 5% and 75% methyl alcohol by volume was added to 1
It was flowed at a flow rate of mm/lll1n. Using a fluorometer as a detector and setting the excitation wavelength to 365 nm,
The fluorescence wavelength was set at 412 nm.

An example of the chromatograph thus obtained is shown in FIG. The number on the shoulder of the peak represents the elution time (retention time) in minutes. The measurement example shown in FIG. 1 is a chromatogram obtained when human serum was processed according to the procedure described above and 5 μQ of the reaction solution was injected. 1st
In the figure, A is the peak of T X B z, and B is the peak of P G
It is a mixed peak of D2 and PGEz, and C is P G
It is the peak of E 1, and D is 6-kedo PG F x
This is the peak of <. These PGs are contained in only a few picograms (pg) of each in 100 μΩ of serum equivalent to injection, but according to Honzuke, even such a small amount can be detected, and the detection rate is more than 10 times that of conventional methods. Can be measured with high sensitivity.

FIGS. 2 and 3 are reagent blank chromatograms in which only the ADAM reagent was measured using a liquid chromatograph under similar conditions. Figure 2 is a chromatogram obtained when 5 μQ of the unpurified ADAM solution obtained in step 1 above diluted with metal alcohol was injected into a liquid chromatograph, and many peaks appeared even after 20 minutes. ing. FIG. 3 is a chromatogram obtained when 5 μQ of the purified ADAM reagent solution obtained in Step 3 above was diluted 5 times and injected. Fewer peaks due to impurity components, 2
Almost no peaks appear after 6 minutes. In this way, impurities derived from the ADAM reagent are significantly reduced by this soaking.

In the above example, the suspended matter after Step 2 was removed using an adsorption column at low temperature and normal pressure, but the removal of the S turbidity after Step 2 was performed by applying the above-mentioned centrifugation method and removing the supernatant. It can also be removed by collecting it.

In addition to being used for the analysis of PCs, the purified ADAM reagent can also be applied to the analysis of organic acids with carbon numbers of 16 or less, which has been considered difficult in the past.

〔Effect of the invention〕

According to the present invention, since impurities in the ADAM reagent can be significantly reduced, it is possible to perform trace analysis of PGs.

[Brief explanation of drawings]

Figure 1 is a diagram showing an example of a chromatogram obtained by measuring an actual sample containing 11) lI amount of PGs based on the present invention, Figure 2 is an example of a chromatogram showing a reagent blank of ADAM before purification, and Figure 3 The figure is an example chromatogram showing a reagent blank of ADAM after purification.

Claims (1)

[Claims] 1,9-anthryldiazomethane is dissolved in an organic solvent,
A solution-like reagent is obtained by removing insoluble components in a cooled state of -10°C or less, and this solution-like reagent is mixed with a sample containing prostaglandins, and by this mixing, the above-mentioned 9-
It is characterized by forming a reaction solution that has produced an anthryl diazomethane derivative, introducing this reaction solution into a separation slum of a liquid chromatography device to separate the components, and measuring the eluate from the separation column by fluorescence photometry. liquid chromatography. Dissolving 2,9-anthryldiazomethane in an organic solvent,
1. A method for preparing a reagent for liquid chromatography, which comprises cooling to -10° C. or below, removing insoluble components during cooling without pressure, and recovering a purified 9-anthryldiazomethane solution.