JPS58108457A - Analizying method for prostaglandin - Google Patents

Abstract

PURPOSE:To remove a fatty acid which is abundantly present in a living body sample, by a method wherein a reverse-phase partition chromatography by a octadecylran fixed phase is conducted as a pretreatment of a living body sample. CONSTITUTION:A living sample such as homo serum, urine, sperm is diluted with a 1 in 2 physiological salt solution, it is held in a cartridge for preheating used for filling an octadecylran treating carrier, it is cleansed, in order, by 4lm distilled water and 4lm 50% methanol, and it is eluated by 55% methanol. After the eluate is pressure-reduced, dried, and hardened, 10mul an internal standard dipropyl acetic acid-ethanol solution (20ng/mul) is added, and 90mul a 0.2% ADAM-ethyl acetate solution is added to react with each other. 5-10mul is applied to a high-speed liquid chromatography, and a separated component is measured and quantitively determined by a fluorescent detector.

Description

[Detailed description of the invention] This invention relates to a method for analyzing prostaglandins.

Glotaglandin (PG) is an important substance that is used clinically and is a substance that neutralizes animals, including humans, and exhibits a variety of strong physiological activities. PG is a derivative of chemical [U brostanoic acid, and due to the difference in functional groups AK↓ in its cyclopenkune 11, E, F, A,
It is classified into types B, C, and D. At present, the naturally occurring substances are E*, EH, and Es.
, FHa, F, a, and Fss are known.

Such PGDIII assays are important in analyzing the significance and mechanism of their physiological effects, but the amount of PGDIII in each sample is extremely small, and high sensitivity is required, as well as specificity and other factors. Separation from biological components is necessary. Generally G
O,GC-MS is widely used, but some problems remain in terms of complicated pretreatment and derivatization for purification.In this sense, high-performance liquid talomatography (HPLC) Although the measurement of PG is expected to be a new means of separation and analysis, PG itself has a characteristic UV
It does not absorb or emit light, making it difficult to detect at the biological level in its original form. This is why HPLC is often used as a means of purifying PGO.%t)
(1) Carr, K., Swee-tm
an, B, J, *Fr1l iek, J,
C.: Promtaglandins.

H, L mouth 11g) 2) Tuss@1. J.
lL e Ge1pt, E, :J, Chroma
togr, , 1111.11L (1980))
@ Therefore, using the esterification reaction with the carboxyl group of PG, the P-N loph@meyl enter
CII) Morxo-wieh, W., Dougla
s, 8. Meeting: Prostaglandins, *1G
, (114G) 4) M@rritt, M, V
, ,Brouson, G.

:Anal, Bl@eh@z, u! IH 1 mouth 1 fan?
)), p-N1trob@nzyl @st@r
6) Fizpatriek, F.A.

W7nalda, M, A, e Kais@r,
D, G, ;Anal, Ch@m.

49, 1110 (111??)) and L? Currently, UV detection is being performed. These methods are not sufficient in terms of t1 reaction conditions and detection sensitivity. Therefore, a specific derivatization method using mild reaction conditions and simple operations is desired. The inventors of this invention completed this invention as a result of research aimed at establishing a highly sensitive analysis method in HPLC by applying fluorescent labeling reagents such as fatty acids to PGK.

Thus, according to this publication W14, the biological sample was subjected to reversed-phase partition chromatography using an octadecylsilane stationary phase, and then the prostaglandin-containing fraction was reacted with a fluorescent labeling agent for carboxyl groups, and the reaction product was This invention provides a prostaglandin analysis method characterized by high performance liquid chromatography (KNL), in which separated components are measured and quantified using a fluorescence detector.

One of the features of the method of this invention is that the biological sample is pretreated by reversed-phase partition chromatography using an octadecylsilane (ODS) stationary phase. The purpose is to Traditionally this fat #! Although it was considered difficult to completely remove O, the invention by Jin O allows this removal to be done easily. On the other hand, in the treatment step with the optical pre-labeling agent, which is another feature of the method of the present invention, this fatty acid reacts more easily than PG, so there is no adverse effect on the accuracy of PG quantification and other aspects. However, this can be avoided by the preamble 11.

Now, this invention includes biological samples such as human serum, urine, and semen. These biological sample companies,
Before applying the pre-treatment to reverse phase distribution 0-v togelafy, it is used after being appropriately diluted with physiological saline.

As the stationary phase for reversed-phase partition talomadography, a surface-treated octadecylsilane tubular porous carrier (IJ force) is used. The mobile phase is preferably a methanol-water system, such as 60-thimethanol or si-thimethanol. Through this process, PG flows out as an eluent and is separated from fatty acids. This place
? dL, using a Kashito Azalea packed with such a stationary phase, a high-performance liquid lipograph was attached to a reversed-phase partition chromatograph, and even one animal was detected.

This eluent is concentrated as necessary, dissolved in a suitable reaction solvent, and then treated with a fluorescent prelabel agent for carboxyl groups, such as 9.
-Anthryldiazomethane, 9-bromomethyl-7-medoxycoumarin, 9.10-cyanophenanthrene or L1-bromomethylpyrene. It can be used as a reaction solvent as long as it has sufficient dissolving ability for the reagents, prosmeglandin, and the reaction solvent, and is inert. Particular preference is given to ethyl acetate. However, methanol, ethanol and sometimes ethyl ether have been found to be less preferred. Reaction temperature [Generally, room temperature or slightly elevated temperature (40 to 60°C for iFl) is preferred.Reaction time is mainly affected by temperature; for example, for 40 cycles,
It is a minute.

In the above O process, PG is made into a fluorescent ester, and this is added with Hr i, cyc, and various P
Separate the derivative of G. As a column for HPLC, an octadecylsilane stationary phase similar to that used in reversed phase partition chromatography can be used. The mobile phase is preferably a methanol-water system, for example a mixture of methanol (S) and water (1).

If the fluorescence intensity of the components separated by ζ is adjusted using a fluorescence detector according to a conventional method and compared with the standard, the quantitative determination of each PG will be clouded.

Next, this invention will be explained using examples.

Example 1) Reagents - PGs to be analyzed are PG El, PGE
ffi, P G F S sw 1P G F! m
4 types (all manufactured by Wako Tsumugi) and methanol for HPLC were used as the mobile phase. ADAM (Funakoshi Pharmaceutical Co., Ltd.), ethanol, ethyl acetate, etc. used for other pretreatments were all Wako Tsumugi Special Grade.

2) Equipment; High performance liquid chromatograph (LC-II)
A), fluorescent detector (RF-690LC).

-kk capacity t! Pj), ultraviolet at absorbance needle (UVD-1
, 264 mm fixed wavelength), and an ocmedecylsilane-treated column (Zorbax ODS (do
(manufactured by Pont) (461@i, d, X2601
1)] was used.

8) Conditions: Mobile phase Kti methanol-water (8:l), flow rate 1.2 l/-, column temperature 40°C, detection u
EX・8g5nm Ern41! One light was detected at a wavelength of n.

4) Pretreatment: Dilute the biological sample with 2x physiological saline,
Cartridge for bending processing filled with octadecylsilane-treated carrier, Bond] Jute TM CH
iehem Int@rnationaj Inc.) K was retained, distilled water 4 ml, 60-methanol 4-
and eluted with 55Ls methanol. Next, after drying the eluate under reduced pressure, the internal standard dipropylacetic acid-ethanol solution (Snow Onf/PI) was prepared using 10711
and add 90 μl of og% ADAM-ethyl acetate solution.
r was added and reacted. HPLC the 6 to 10 liters
injected into. ADAM reacted quickly with PG to yield a relatively stable ester.

The reaction solution was mixed with ammonia, and the structure of the product was confirmed using a mass spectrum determined by copper ionization detection method.
Saturation was reached in 5 minutes. Furthermore, as the reaction temperature increased, the time until saturation was shortened, and at 40'C, a constant light intensity was exhibited over a period of approximately 20 minutes.

FIG. 1 shows the excitation and fluorescence x-vectors (in ethanol) of the company PG Ex-ADA M ester.

Figure 2 Strains, PG mixture (1: PG Ex, 2:
PGF*a, 11: PGEt, 4: PGFIII,
6:18 chromatogram of Kagome diisopropyl acetate).

FIG. 8 shows the calibration curve of each PG. This calibration curve shows good linearity in a wide range (10@pfN100nf).

Figure 4 shows the analysis results of human semen OPG.

[Brief explanation of the drawing]

Figure 1 shows the optical spectra of PG)i:x-mul)AM esters; Figure 8 shows P G Et , P G L , P G F
*a, PGF- calibration curve; FIG. 4 is a human semen O chromatogram.

Claims (1)

[Claims] 1. A biological sample is subjected to "reversed phase partition chromatography using an octadecylsilane stationary phase, and then subjected to 7 o x
Glosstaglan, characterized by Yr, is produced by reacting a carboxyl group-containing fluorescent prelabeling agent with p/Randin-containing fraction, subjecting the reaction product to high-performance liquid chromatography, and quantifying the separated components using a strange theta photodetector. Gin analysis method. 112. The method according to claim 111, wherein the reverse phase partition chroma dog 9 fee uses a methanol aqueous system as a transfer phase. 8. The method according to claim 1, wherein the Ikkou pre-labeling agent for carboxyl groups is 9-anthowardiazomethane.