JP2000180428A - Method for analysis of estrogen - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a highly sensitive and highly accurate handy analysis method which enables microanalysis of estrogen such as estradiol, estriol and estrone. SOLUTION: In a method of analyzing estrogen in water, (i) weak acid buffer solution is added to a sample to perform a solid phase extraction, (ii) the sample is evaporated to dryness, (iii) acid is added to the obtained residue to be evaporated to dryness after being heated, (iv) the obtained residue is dissolved into a mixture of water and a water-soluble organic solvent, (v) the mixture is subjected to a liquid chromatography and (vi) detection is performed by a filter type electrode based electrochemical detector having a structure in which an activity test electrode with a through hole and a control electrode are arranged in lamination being electrically insulated therebetween.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for analyzing estrogen in water by liquid chromatography (LC).

[0002]

2. Description of the Related Art In recent years,
Certain compounds have attracted attention as an endocrine disruptor (sometimes called "environmental hormones") and their effects on the human body. Similarly, estradiol, S There are estrogens such as triol and estrone, and a technique for their trace analysis is required. In particular, when measuring the amount of estrogen in environmental water, the estrogen concentration in environmental water is usually very low, so that estrogen analysis methods that have been widely used in the past often fall below the detection limit. Therefore, there is a need for a technique for analyzing trace amounts of estrogen.

[0003]

According to the present invention, in the LC analysis of estrogens such as estradiol, estriol and estrone in water, (i) a weakly acidic buffer is added to a sample, and solid phase extraction is performed; The sample was evaporated to dryness and (ii)
i) Add acid to the obtained residue, heat it, evaporate to dryness,
(IV) dissolving the obtained residue in a mixture of water and a water-soluble organic solvent, (v) subjecting to liquid chromatography, and (vi)
Enables microanalysis of estrogen by detecting with a filter electrode type electrochemical detector that has a structure in which an active test electrode having a through hole and a reference electrode are electrically insulated from each other and arranged in a stacked configuration. Is what you do.

[0004]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, estrogen is a female hormone having a phenolic hydroxyl group such as estradiol, estriol and estrone.

In the analysis of estrogen contained in a water sample in an environment such as a river or a lake, first, (i) a weakly acidic buffer is added to the sample. Usually, the pH of the weakly acidic buffer is pH
4 to 6 are used, preferably a 1M acetate buffer at pH 5. The weakly acidic buffer is generally added in an amount of about 0.4 to 10 mL per liter of sample water. If the sample water contains insoluble matter, it is preferable to remove the insoluble matter by a filtration operation or the like in advance. that time,
In order to extract and analyze the estrogen contained in the insoluble matter, it is preferable to add the filter used for filtering the water of the sample to the sample while washing it with a water-soluble organic solvent such as methanol or acetonitrile. desirable. For example, the filter used to filter the water of the sample may be filtered using a mixture of methanol and 1 M acetate buffer at pH 5 (eg, 9/1 =
v / v mixture), 4 to 1 per liter of sample water
In the operation of adding to a sample while washing with 0 mL, it is convenient because a weakly acidic buffer can be added at the same time. The subsequent solid phase extraction is carried out according to a conventional method, for example, by adsorbing on a cartridge such as commercially available Seppak Plus C18 (manufactured by Waters), Seppak PS-2 (manufactured by Waters), and a solvent (for example, a mixed solvent of ethyl acetate / methanol). , Water / acetonitrile mixed solvent).

[0006] Next, (ii) the sample is evaporated to dryness, and this operation is performed, for example, by heating the sample to about 40 ° C as necessary under a nitrogen stream. Further, it can be carried out by heating the sample to about 40 ° C. under reduced pressure condition as needed. (Iii) Examples of the acid to be added to the obtained residue include hydrochloric acid and the like, and a methanol solution of hydrochloric acid of about 0.1 to 5 M is usually used. For example, 0.5 to 5 mL of a 1M methanolic hydrochloric acid solution is added to 1 liter of sample water. The subsequent evaporating and drying operation is performed by heating the sample to about 40 ° C. as necessary, for example, under a nitrogen stream in the same manner as described above. In addition, it can be carried out by heating the sample to about 40 ° C. under reduced pressure conditions as needed.

(IV) In dissolving the residue obtained above in a mixture of water and a water-soluble organic solvent, the water-soluble organic solvent used includes alcohols such as methanol and ethanol, acetonitrile, and mixtures thereof. Among them, it is preferable to dissolve the obtained residue in a mixed solvent of water / acetonitrile / methanol. In particular, the mixing ratio of water / acetonitrile / methanol is preferably 5: 3: 2 by volume. For example, 1 mL of the mixture may be used for 1 liter of sample water.

[0008] The solution obtained as described above is
Liquid chromatography followed by (vi) detection by a filter-type electrode-type electrochemical detector having a structure in which an active test electrode having a through-hole and a reference electrode are electrically insulated from each other and arranged in a layered manner. I do. As the filter type electrode type electrochemical detector of the step (vi),
JP-19093-A discloses an electrochemical detector (EC
D) For example, a commercially available ECD, Coulochem from ESA
An II 5200 detector can be used.

[0009] It is preferable to use an ODS-based filler or the like for the liquid chromatography column. As the filler, for example, SU manufactured by Sumika Chemical Analysis Service, Ltd.
MIPAX ODS A-212 or the like can be used.

In the analysis method of the present invention, qualitative analysis can be performed using a standard estrogen, and quantitative analysis can be easily performed by a calibration curve method or the like.

[0011]

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples. First, an example of a general method will be described from Example 1- (i) to Example 1- (vi). Example 1- (i) One liter of sample water is filtered through a glass filter. The glass filter is washed with 10 mL of a 1 M acetate buffer (9: 1, v / v) of methanol / pH 5.0, and the washing solution is added to the filtrate. The mixed solution of the filtrate and the washing solution is passed through a solid phase extraction cartridge (Seppack Plus C18 cartridge manufactured by Waters) sequentially washed with 10 mL of methanol and 20 mL of water, and the cartridge is sequentially washed with 5 mL of pure water and 5 mL of hexane. I do. Next, the sample components are eluted from the cartridge with 5 mL of ethyl acetate / methanol (5: 1, v / v).

Example 1- (ii) The eluate obtained in Example 1- (i) is placed in an eggplant-shaped flask, heated to 40 ° C. under reduced pressure, and evaporated to dryness.

Example 1- (iii) 1M was added to the evaporated and dried residue obtained in Example 1- (ii).
Add 1 mL of methanolic hydrochloric acid solution, seal and add
Heat to 0 ° C. After cooling, it is evaporated to dryness at 40 ° C. under a stream of nitrogen.

Example 1- (iv) A water / acetonitrile / methanol mixture (5: 3: 2, v) was added to the evaporated residue obtained in Example 1- (iii).
(V / v) Add 1 mL to dissolve.

Example 1- (v) 100 μL of the solution obtained in Example 1- (iv) is subjected to liquid chromatography under the following conditions. High-performance liquid chromatograph: Hitachi L-7000 (manufactured by Hitachi, Ltd.) Analysis column: SUMIPAX ODS-A-211 (manufactured by Sumika Chemical Analysis Center) 5 μm, 4.6 mmi.d. × 25 mmL Mobile phase: 50 mM sodium acetate (pH 4.8) / Methanol / acetonitrile = 5/2/3 Flow rate: 1.1mL / min Column temperature: room temperature

Example 1- (vi) The high performance liquid chromatograph of Example 1- (v) was used as a filter type electrode type ECD as a Coulochem II 5200 manufactured by ESA.
Detector is connected, set potential is 500 mV, guard cell 55
The measurement is performed at 0 mV.

Next, an example of preparing a calibration curve for β-estradiol will be described. Example (Preparation of calibration curve) 10 mg of β-estradiol was dissolved in methanol and 100
mL. This 1 mL is diluted 100 times with methanol to prepare a concentration of 1 μg / mL. 1 mL of this diluted solution is further diluted 100 times with methanol to obtain a reference solution adjusted to a concentration of 10 ng / mL. 0.1m of reference liquid obtained
L, 0.2 mL, 0.4 mL, 0.8 mL, and 1.6 mL were each placed in a stoppered pressure-resistant Spitz tube, evaporated to dryness at 40 ° C. under a nitrogen stream, and a water / acetonitrile / methanol mixture (5: 3:
(2, v / v / v) dissolved in 1 mL and subjected to LC / ECD measurement under the same conditions as in the analysis of the above sample to prepare a calibration curve.

Example (Analysis of β-estradiol in Environmental Water) According to the above operation, β-estradiol in 1 liter of environmental water at a certain place was analyzed by a calibration curve method, and the result of measurement was 7 μg / L (7 ppt). Obtained.

[0019]

As described above, the analysis method of the present invention provides a method for estrogen L
C enables trace analysis and is a highly sensitive, accurate, and simple analysis method.

Claims (3)

[Claims] (1) In a method for analyzing estrogen in water, (i) solid phase extraction is performed by adding a weakly acidic buffer to a sample, (ii) the sample is evaporated to dryness, and (iii) a residue obtained is obtained. Add an acid, heat and evaporate to dryness. (IV) Dissolve the resulting residue in a mixture of water and water-soluble organic solvent, (v) subject to liquid chromatography, (vi) An estrogen analysis method, wherein a test electrode and a reference electrode are detected by a filter-type electrode-type electrochemical detector having a structure in which they are electrically insulated from each other and are arranged in a layered manner. 2. A method for analyzing estrogen in water, comprising the steps of (i) adding a buffer having a pH of 4 to 6 to a sample, performing solid phase extraction, (ii) evaporating the sample to dryness, and (iii) obtaining the sample. An acid was added to the residue, and the mixture was heated and evaporated to dryness. (IV) The obtained residue was dissolved in a mixture of water and a water-soluble organic solvent, and (v)
Liquid chromatography, (vi) detection by a filter-type electrode-type electrochemical detector having a structure in which an active test electrode having a through-hole and a reference electrode are electrically insulated from each other and arranged in a layered manner; An estrogen analysis method, characterized in that: 3. The method according to claim 1, wherein the estrogen is estradiol.