JPS6071952A - Analyzing method of catecholamine or the like - Google Patents

Abstract

PURPOSE:To enable analysis of catecholamine with good sensitivity without breaking the peak of a chromatogram and disturbance in the base line by subjecting the catecholamic acid to solid phase extraction by using alumina, etc. then eluting the same with an acid or the like. CONSTITUTION:This analyzing method of catecholamine, etc. is characterized by passing preliminarily a high concn. of an acid buffer soln. consisting of higher alkane sulfonate or higher alkyl sulfate which is an ion pair reagent into an antiphase column 5 to condition the column then subjecting the sample to high speed liquid chromatography using a low concn. of an acidic buffer soln. 2 of the higher alkane sulfonate or higher alkyl sulfate which is an ion pair reagent as well as the salt of strong acid and strong base as a moving phase. The above- mentioned ion pair reagent is stable to acids and therefore the analysis is accomplished with good sensitivity by the chromatogram even if the acid of a high concn. is used in order to increase the recovering rate of the catecholamine in the stage of eluting the catecholamine extracted in the solid phase by the acid when the concn. of the catecholamine or the like in the sample is low.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Industrial application field This invention is directed to the analysis of catecholamines using high performance liquid chromatography using a chemically bonded reverse phase column with silica as a carrier and using the trihydroxyindole method as a post-column reaction. Concerning improvements in law. (b) Conventional technology The conventional method for analyzing catecholamines in biological samples such as plasma and urine is to extract the catecholamines in the sample in the same phase with alumina, etc., then elute with acid, and then apply this to high-performance liquid chromatography. A method is known in which the separated catecholamines are separated into trihydroxyindoles and subjected to fluorescence analysis.

In the above method, various methods are used as high performance liquid chromatography. Two types of columns are commonly used: a chemically bonded reverse phase column using silica as a carrier and an ion exchange column, and two types of reaction units are used: a flow reactor method and an air segment method. When using the above-mentioned reversed-phase column, an acidic buffer solution of lower alkanesulfonate (e.g., sodium pentanesulfonate) or lower alkyl sulfate (e.g., sodium pentyl sulfate), which is an ion pairing reagent, is conventionally used as the mobile phase. . However, if the concentration of catecholamines in the sample is low (e.g., a plasma sample), it is necessary to use a high concentration of acid to increase the recovery rate when eluting the in-phase extracted catecholamines with acid. It has a tendency to decompose, leading to problems such as splitting of peaks in chromatograms in fluorescence analysis and disturbance of the baseline, resulting in decreased sensitivity.

(c) Purpose of the Invention This invention was made to solve the above problems, and it is possible to avoid cracking of the peak of the chromatogram or disturbance of the baseline in the fluorescence analysis chromatogram even in samples with a low content of catecholamines. The purpose of this invention is to provide a method for analyzing catecholamines that can be analyzed with good sensitivity.

(d) Structure of the Invention This invention was made by focusing on the fact that higher alkanesulfonates or higher alkyl sulfates are stable against acids as ion pairing reagents. This acidic eluate is subjected to high-performance liquid chromatography using a chemically bonded reverse-phase column with silica as a carrier and eluted with an acidic buffer containing an ion pair reagent, and then separated.
A method for analyzing catecholamines in which the ion pair reagent is then converted into trihydroxyindole and then subjected to fluorescence analysis. After being conditioned by passing through it, it is subjected to high performance liquid chromatography using an acidic buffer solution of a low concentration higher alkanesulfonate or higher alkyl sulfate of an ion pair reagent and a salt of a strong acid and strong base as a mobile phase. The present invention provides a method for analyzing catecholamines, particularly a method for analyzing catecholamines in biological samples such as plasma and urine.

This invention has the above features, but as a conditioning liquid for the reverse phase column, a higher alkanesulfone Il! such as sodium dodecane sulfonate, which is an ion pair reagent, is used. Phosphate acidic buffer solutions, for example p)l 2.5, containing salts or higher alkyl sulfates such as sodium dodecyl sulfate are used. The ion pair reagent is used at a relatively high concentration of 3 to 1511M. After the above conditioning, the mobile phase used to analyze the above acidic catecholamine eluent includes a higher alkanesulfonate or higher alkyl sulfate 4- as an ion pairing reagent, and sodium sulfate or the like. For example, a phosphate edge solution containing a salt of a strong acid and a strong base at pH 2.5 is used. Ion pair reagents are used at relatively low concentrations, with higher alkanesulfonates or higher alkyl sulfates being 0.01-0.5mM, and salts of strong acids and bases being 0.01mM to 0.5mM.
05-0.3mM is used.

Also, known methods are used for solid phase extraction and elution of catecholamines in the sample. That is, a sample is treated with alumina or the like to form a complex with catecholamines and extracted, and then eluted with acetic acid, hydrochloric acid, or the like.

Also, known chemically bonded reverse phase columns with silica as a carrier can be used, such as Solpax ODS (
(manufactured by DuPont). A known method is used to convert catecholamines into trihydroxyindoles. Potassium ferricyanide, ascorbic acid, and sodium hydroxide are used as the first to third reagents, respectively, and the fluorescence intensity of the trihydroxyindoles obtained is determined by Catecholamines are detected and analyzed using known methods.

(e) Examples Next, the method of the present invention will be explained by way of examples.

As a sample, 9100 μg of 0.4 N vinegar containing 1 oopo each of noradrenaline and adrenaline was used, and this was analyzed using an analyzer whose system diagram is shown in FIG. 1, and the method of the present invention and the conventional method were compared.

(a) An embodiment of the analytical method of the present invention Before injecting the above sample into the sample injection part (4), (1)
20mM phosphate buffer (pH 2,5>) containing 5mM sodium dodecyl sulfate at 112/mtn.
It was conditioned by passing it through a Solpax ODS reverse phase column (5) using a minute pump (3). Next, 20IIIM phosphate buffer (pH 2,5) containing 0.1111M sodium dodecyl sulfate and 0.2M sodium sulfate (pH 2,5) from (2) was added to the reversed phase column (2) using a pump (3).
5) After that, send the first reagent of (6), potassium ferricyanide 0.04w/v% gold-containing 0.2M phosphate buffer (pH 6,8) to 0.31! Ascorbic acid 0.04 of the second reagent of ('7) at a flow rate of f/l1in.
0.3ν of an aqueous solution containing w/v% and mercaptoethanol 0.2v/v%! /min, and 0.3y of 10N sodium hydroxide solution as the third reagent in (8).
After sending the sample at a flow rate of f/sin, the sample was injected from the sample injection part (4) and analyzed with a Shimadzu RF500LCA fluorescence detector (121) to obtain the chromatogram shown in Figure 2 (a). Middle (1) and (2) are the peaks of noradrenaline and adrenaline, respectively].

(b) Comparative example of conventional analytical method Without conditioning the reversed phase column in Example (a) above, a 20mM phosphate buffer (E) containing 51M sodium pentanesulfonate was used as the mobile phase.
Catecholamines were analyzed in the same manner as in Example (a) above, except that I H2,5) was used at a flow rate of 0.6 t7/min. The obtained chromatogram is shown in Figure 2 [in the figure, (1) and (2 are the peaks of noradrenaline and adrenaline, respectively]).

As is clear from (a) and (b) in Figure 2, -/
- In contrast to the peaks in the chromatogram in (b), the peaks in the chromatogram in 11) are sharp, and even extremely low concentrations of catecholamines, such as 1001) 0/100 μΩ, can be analyzed with good sensitivity. .

(f) Effect of the invention According to the analysis method of this invention, 1100p / 100μ
Even extremely low concentrations of catecholamines, such as 1.5 g, can be analyzed with good sensitivity without cracking the chromatogram peaks or disrupting the baseline.

[Brief explanation of the drawing]

Figure 1 is a system diagram of an example of an analytical device used in the method of this invention, and Figures 2 (a) and (b) are a chromatogram of an example of the analytical method of this invention and a chromatogram of a conventional analytical method, respectively. This is a chromatogram of a comparative example. (1)...Reverse phase column conditioning solution, (2)...Mobile phase, (3105]...
・Pump section, (4)...Sample injection section, (5)・
...Reversed phase column, 8- (6) ...First reagent, (Katana...Second reagent, (8) ...Third reagent, (9)・・・・・・
・First reagent reaction tube, (g)...Second reagent reaction tube, (11)...Third reagent reaction tube, Oz...
. . . Fluorescence detector, 03) . . . Analysis data processor, and (14) . . . Analysis liquid discharge pipe.

Claims (1)

[Claims] 1. Catecholamines in a sample are extracted in solid phase using alumina or the like, then eluted with acid, and this acidic eluent is used in a chemically bonded reverse phase column with silica as a carrier containing an ion pair reagent. A method for analyzing catecholamines, which is separated by high-performance liquid chromatography eluting with an acidic buffer, and then converted to trihydroxyindole for fluorescence analysis; After conditioning by passing through an acidic buffer of higher alkanesulfonate or higher alkyl sulfate, use a low concentration of higher alkanesulfonate or higher alkyl sulfate M salt of ion pair reagent as a mobile phase and a salt of strong acid and strong base. A method for analyzing catecholamines, which comprises subjecting them to high performance liquid chromatography using an acidic buffer solution. 2. The analytical method according to claim 1, wherein the higher alkanesulfonate, higher alkyl sulfate, and strong acid/strong base salt of the ion pair reagent are sodium dodecane sulfonate, sodium dodecyl sulfate, and sodium sulfate, respectively.