JP2001328998A - Method and apparatus for separating and eluting estrogens - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method and apparatus for separating estrone and estradiol or 17α-ethynylestradiol whose structures are extremely similar. SOLUTION: A solution containing estradiol and estrone or a solution containing 17α-ethynylestradiol and estrone is brought into contact with a styrene-divinylbenzene copolymer and caught into the copolymer and then, estrone and estradiol or 17αethynylestradiol are separated and eluted.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION Regarding the separation of estrogens having the highest female hormone activity among endocrine disrupting chemicals, the structures are very similar after the estrogens are trapped in a styrene-divinylbenzene copolymer. Provided is a method and an apparatus capable of separating and obtaining estrone and estradiol or 17α-ethynylestradiol by separating them.

[0002]

2. Description of the Related Art In recent years, reports of spontaneous destruction have been rapidly increasing, and among them, a chemical substance having an estrogen action, such as binding to an estrogen receptor in a living body, has attracted a great deal of interest. However, the strongest female hormone activity in nature is naturally natural estrogens such as 17β-estradiol (abbreviated as E2) and synthetic estrogens such as 17α-ethynylestradiol and diethylstilbestrol which has been discontinued. (Adult Yamashita et al., Clinical Laboratory, Vol. 43, No. 11, P137)
5-1382, 1999). Natural estrogen is secreted in minute amounts from endocrine glands as needed, and signals are transmitted through estrogen receptors in cells in target tissues. Synthetic estrogens are mainly administered as pharmaceuticals to replace or supplement the action of natural estrogens. These substances have a strong binding activity to the estrogen receptor, so if they are inadvertently released into the environment and are present in high concentrations,
It may accumulate in humans or other animals that have taken it and may act as female hormones.
To avoid this danger, it is necessary to constantly monitor the amount of these substances in the environment and monitor changes in concentration.

[0003] Furthermore, in addition to the natural and synthetic estrogens described above, it has recently been reported that chemical substances such as bisphenol A, nonylphenol and polyvinyl chloride (PCB) have a weak female hormone action, and these chemicals in marine organisms have been reported. The causal relationship between substance concentrations and bioaccumulation and reproductive abnormalities and genital malformations was clarified, suggesting the risk of environmental pollution from these chemicals. On the other hand, the necessity of measuring these chemical substances has been recognized in order to grasp the pollution situation and take countermeasures and to monitor the natural environment.

[0004] As a method for measuring a chemical substance called an environmental hormone, conventionally, GC / MS, HPLC / MS, and bioassay (mammary tumor cells (MCF-7) showing estrogen-dependent growth) are cultured in an estrogen-free culture solution. Evaluation method for examining proliferation activity by exposure to a chemical substance having an estrogenic effect) and enzyme immunoassay have been reported and used. Among the various measurements, the one with the highest detection sensitivity is an enzyme immunoassay or a bioassay using breast cancer cells, rather than instrumental analysis. The bioassay requires skilled and complicated operations, and is not suitable for simple analysis because it requires special equipment and advanced technology.
It is excellent in sensitivity and has the advantage that endocrine disrupting effects can be directly detected as female hormone activity. On the other hand, the immunoassay is said to be 10 times more sensitive than the analysis using GC-MS. It is short and does not require special equipment or equipment. Therefore, it is the most effective method when you want to obtain many results quickly by environmental monitoring. In addition, by using an antigen-specific antibody, for example, an estradiol antibody can be used to convert estradiol without being affected by plant steroids, bisphenol A, and nonylphenol that have a similar structure and can bind to estrogen receptors. It can be specifically captured.

[0005]

However, when molecules having very similar structures, such as estradiol and estrone, which differ only in the structure at the 17-position, are mixed, the cross-reactivity of the antibody becomes a problem. This is a problem that occurs because the estradiol antigen molecule itself is small and the antigenic determinant in the molecule is a limited part in the molecule,
An antibody having a higher affinity for an antigenic determinant may not recognize a difference in a small molecular structure, for example, at position 17. By the way, in the environment, estrone which is a precursor and metabolite of estradiol is also present in addition to estradiol, and these are mutually converted by metabolic activity of microorganisms.

As described above, the immunoassay is suitable for estradiol analysis of river water, sediment, soil, and the like. However, as described above, the measured value is reported to be higher than the true value of estradiol due to cross-reactivity with estrone. It has been pointed out that there is a danger of doing so. Further, estrone is not as strong as estradiol but has female hormone activity, and is easily converted to estradiol in nature as described above. Therefore, the amount of estrone itself is also a chemical substance to be monitored in the environment. Methods for measuring estrone include immunoassay and bioassay, in addition to instrumental analysis, which is insensitive. In the immunoassay, an estrone measurement system was constructed in the same manner as the estradiol measurement system,
It is necessary to selectively measure the amount of estrone in the concentrated solution of estradiol and estrone obtained by solid-phase extraction, but the cross-reactivity of the estrone immunoassay system to estradiol again becomes a problem. , There is a risk of false highs due to an increase in the measured values due to the mixture of estradiol. Also in the bioassay, it is difficult to simultaneously and independently obtain the measurement results of the amount of estradiol and the amount of estrone, which are mixed in the extract of the conventional method, since detection is performed by the action of female hormones.

[0007]

Means for Solving the Problems The present inventors have made intensive studies on the above problems, and as a result, have reached the present invention. That is, the present invention is to contact a solution containing estradiol and estrone with a styrene-divinylbenzene copolymer,
A method for separating and eluting estradiol and / or estrone, comprising capturing estradiol and / or estrone in a solution with the copolymer, and then separating and eluting estradiol and / or estrone.

The present invention also relates to a method of contacting a solution containing 17α ethinyl estradiol and estrone with a styrene-divinylbenzene copolymer, capturing 17α ethinyl estradiol and estrone in the solution, and then capturing 17α ethinyl estradiol. A method for separating and eluting 17α ethinylestradiol and / or estrone, comprising separating and eluting estrone.

Further, the present invention is an apparatus for separating and eluting estradiol and / or estrone, wherein a styrene-divinylbenzene copolymer is used as a separating means.

Further, the present invention is an apparatus for separating and eluting 17α-ethynylestradiol and / or estrone, wherein a styrene-divinylbenzene copolymer is used as a separating means. Hereinafter, the present invention will be described in more detail.

First, a method for separating and eluting estradiol and / or estrone will be described.

It is preferable that the solution containing estradiol and estrone has previously been subjected to filtration to remove insoluble matter, but the origin is not particularly limited, and there is no limitation on environmentally-derived samples and biological samples. When an organic solvent or the like is mixed therein, the amount thereof is preferably less than 50% (v / v) in the case of methanol, and more preferably 20%.
Less than (v / v) is preferred.

The solution is brought into contact with a styrene-divinylbenzene copolymer, but the styrene-divinylbenzene copolymer used is not particularly limited. The degree of polymerization of the copolymer is not specified, but if the degree of crosslinking is low, the gel becomes soft and weak to pressure.
About 0-55% is desirable. The excluded molecular weight is not specified, but if the excluded molecular weight is increased, the copolymer surface area is reduced.
The porosity of the gel is not specified, but if it is too large, the gel may be crushed by pressure,
About 0.4-0.7 is preferable. The particle size of the copolymer is not specified, but considering the possibility of sending a large volume of liquid, one having a very small particle size is not preferred, and 10-300 μm is preferred.

Examples of the method include using the polymer by filling it in a column in advance, or dispersing it in a solution containing estradiol and estrone, and then filling it in a column bed. Is not done.
In particular, a method of pumping a solution containing estradiol and estrone into a column packed with the polymer by a pump is preferable. The liquid sending speed at this time is not particularly limited.
Flow rates of 5 mL or less are preferred. Further, the reaction can be carried out under an open column system or under high pressure conditions such as using high performance liquid chromatography.

Thus, estradiol and estrone in the solution are captured by the styrene-divinylbenzene copolymer.

Next, estradiol and / or estrone captured by the copolymer are separated and eluted. It is preferable to use a mixed solution of an organic solvent miscible with water and an aqueous buffer as the solution used for separation and elution. At this time, the organic solvent is preferably methanol, and the eluate of estradiol preferably has a methanol concentration of 70 to 80%, and the elution of estrone has a methanol concentration of 80 to 90%. The pH of the buffer is preferably 9 or more, and particularly preferably 9.5 to 10.5 in consideration of the durability of the styrene gel. There is no particular limitation on the type of buffer, but Tris buffer is preferable, and the concentration is preferably 10 to 100 mM.

The flow rate at the time of separation and elution is not particularly limited, but is preferably about 0.3 to 1.5 ml / min.

In this manner, estradiol and / or
Alternatively, estrone can be separated and eluted.

Next, a method for separating and eluting 17α ethinyl estradiol and / or estrone will be described. 1
Separation and elution of 7α ethinyl estradiol and / or estrone can be carried out in the same manner as the above-mentioned separation and elution of estradiol and / or estrone, and the solution to be separated and eluted, the copolymer to be used, the separation and elution method, etc. The same is true.

Further, an apparatus for separating and eluting estradiol and / or estrone or an apparatus for separating and eluting 17α ethinylestradiol and / or estrone can be constituted by using a styrene-divinylbenzene copolymer as a separating means.

[0021]

According to the present invention, by using a styrene-divinylbenzene copolymer, the estradiol fraction and the estrone fraction having a high purity or the 17α ethinyl estradiol fraction and the estrone fraction can be obtained by a single operation. Can be obtained separately.

Therefore, estradiol or 17α containing less estrone than conventional solid-phase extraction.
Ethinylestradiol can be obtained. Therefore, when measuring estradiol or 17α-ethynyl estradiol in an immunoassay or a bioassay, it is possible to prevent an increase in the measured value due to the mixture of estrone, and to measure estradiol or 17α-ethynyl estradiol closer to the true value by using these measurement systems. The value can be determined.

Conversely, if an immunoassay or bioassay capable of detecting estrone is used, the estrone concentration in the sample can be determined more accurately.

When a separation eluate according to the present invention which comprises methanol and a buffer solution and does not contain other organic solvents is used, the influence on the enzyme immunoassay or bioassay, which is a measurement system composed of proteins, is not obtained. Since it is small, it can be brought into such a measurement system only by diluting it several times by a dilution operation, and the solvent replacement operation can be omitted. As a result, loss of estradiol due to solvent replacement can be prevented, and the reliability of the measured value is further increased.

[0025]

EXAMPLES The present invention will be described below with reference to examples. However, the present invention is not limited to only these examples.

Example 1 Elution patterns of estradiol (abbreviated as E2) and estrone (abbreviated as E1) by various gels 0.2 mL of various commercially available gels (G3000S, G5000)
PW, Butyl-TP, Hexyl-TP, Phen
yl-TP, OD-2PW, both manufactured by Tosoh Corporation)
Was packed in a column bed, washed with 3 mL of methanol, equilibrated with 8 mL of 50 mM phosphate buffer (pH 7), and a solution containing 500 pg of E2 was passed through the column to capture estradiol (E2) on the column. . The gels used are as follows. G3000S Styrene-divinylbenzene copolymer G5000PW Hydrophilic vinyl polymer Butyl-TP Gel with butyl groups introduced into hydrophilic vinyl polymer base Hexyl-TP Gel with hexyl groups introduced into hydrophilic vinyl polymer base Phenyl-TP OD-2PW A gel in which octadecyl groups are introduced into a hydrophilic vinyl polymer substrate.

A methanol / 50 mM phosphate buffer (pH 7) mixture in which the methanol content was changed from 20% to 100% in 10% steps was prepared, and 0.75 mL of the mixture was added to the column in descending order of methanol content. Apply and
The methanol content was varied stepwise, and at which methanol content E2 eluted was examined. Finally, put the column in 100 mL of methanol
After washing with, 8m of 50 mM phosphate buffer (pH 7)
After equilibration with L, then 1000 pg of El was passed through the column and eluted as in E2. All liquid sending rates for elution were 0.6 mL / min.

The eluted E2 was measured by a one-step enzyme immunoassay based on a competition method using an anti-estradiol antibody. That is, 12 beads with goat anti-rabbit IgG immobilized in a cup, and 0.2 mA of alkaline phosphatase-labeled 17β estradiol are further contained (1 mA means absorbance at 280 nm of 1.
0) Add 50 μL of the conjugate diluent (the conjugate diluent mentioned here is 50 mM Tris-HCl buffer (pH 7.5), 2.5% gelisate (manufactured by Becton Dickinson), 2% sucrose (Wako Pure Chemical Industries, Ltd.) Medicine), 5
mM magnesium chloride, 0.1 mM zinc chloride, 0.1%
50 μL of a solution obtained by diluting rabbit anti-17β estradiol antiserum 60000-fold with a conjugate diluent was added thereto, followed by freeze-drying to prepare a prepared measurement cup. To this cup, 75 μL of a column eluate diluted and prepared with 50 mM phosphate buffer (pH 7) at a final methanol concentration of 5% was added, and the solution was added to a commercially available fully automatic immunodiagnostic apparatus (AIA-21, manufactured by Tosoh Corporation). After incubation for 40 minutes, the binding reaction between E2 in the column eluate and enzyme-labeled E2 to the anti-estradiol antibody was performed. B / F separation was performed, and the amount of alkaline phosphatase bound to the beads was measured using 4-methylumbelliferyl phosphate as a substrate. At this time, the amount of the enzyme bound was represented by the activity of decomposing 4-methylumbelliferyl phosphate per second.

The amount of eluted E1 was measured using the E1 solution having a known concentration in the same measurement system as E2 described above.
Using the cross-reactivity of the measurement system to E1, a standard curve for E1 was prepared, and the amount of E1 in the eluate was measured. The concentration of methanol in the measurement sample was set to 5% as in the measurement of E2.

In all gels, no E2 or E1 was detected in the flow-through fraction, and no E2 and E1
And E1 were confirmed to be completely captured. Next, the results of elution by flowing 0.75 ml each from the lower methanol content are shown in FIGS. 1 to 6. G5000P
W (FIG. 2), Butyl-TP (FIG. 3), Hexyl-
TP (FIG. 4), Phenyl-TP (FIG. 5), OD-2
In various gels of PW (FIG. 6), captured E2 and E1 were eluted at almost the same methanol concentration, and E2 and E1
Separation was unsuccessful, while G3000S (Fig. 1)
In Table 2, the elution positions of E2 and E1 were different, and it was confirmed that separation and elution of E1 and E2 could be performed by changing the methanol concentration.

Example 2 Separation of styrene-divinylbenzene copolymer Effect of buffer type and pH in eluate Styrene-divinylbenzene copolymer G3000
Using S, the pH of the buffer solution in the separation eluate was changed to examine the change in separation and elution. As the buffer in the separation eluate, the 50 mM phosphate buffer (pH
7.0), 1% acetic acid (pH 5.0), 50 mM
Tris-HCl buffer (pH 9.0), same (pH 10.0)
Was used. The washing and equilibration of the column and the separation and elution using a mixed solution of methanol / each buffer were carried out according to Example 2 according to E2 and E2.
The measurement of the amount was in accordance with the method described in Example 1.

The results are shown in FIGS. FIG. 7: 0.1% acetic acid (pH 5.0), FIG.
mM Tris buffer (pH 9.0), Fig. 9: 50 mM Tris buffer (pH 10.0) was used. Separation of E2 and E1 was possible using any of the aqueous solutions, but the highest separation and recovery of E2 and E1 occurred in the 50 mM Tris-HCl buffer (pH 10.
0) was used as the buffer.

Example 3 Investigation of Methanol Concentration in Separation of E2 and E1 As in Example 1, 500 pg of E2 or 1000
pg of E1 was applied to a column packed with 0.2 ml of G3000S. Then, methanol 60%, 70%
Elution was performed using a mixture of 50%, 75%, 80%, 85%, 90%, and 100% methanol / 50 mM Tris-HCl buffer (pH 10.0).

FIG. 10 shows the results. E2 is methanol 7
Elution up to 5% recovered 78% of the total volume, while E1 was up to 75% methanol, 33% of the total volume, methanol 80%.
%, 85% of the total elution, 67.2% of the total amount,
From these results, it was found that 75% of methanol was used for elution of E2, and 80% or more of methanol was used for elution of E1.
It was shown that separation and elution of 2 and E1 were possible.

Example 4 Fractionation from a mixed solution of E2 and E1 From the results of Example 3, it was possible to elute E2 with 75% methanol and E1 with 85% methanol. 1 mL of 50 mM phosphate buffer (pH 7) containing 5 pg and 363 pg of E1 was added to G300
Apply to a 0S packed column (gel volume 0.2 mL)
Separation and fractionation of E2 and E1 were attempted using a mixture of 50%, 75%, 85%, and 100% methanol / 50 mM Tris-HCl buffer (pH 10.0). Note that 50
Use 0.75 mL for the elution of the 75% methanol mixture and the 85% methanol mixture.
1.5 mL of a methanol mixture was used for elution.

For the measurement of the eluted E2, Examples 1 to 3
And a one-step enzyme-linked immunosorbent assay based on a competition method using an anti-estradiol antibody different from that described in the above section. Since the cross-reactivity with E1 of the E2 measurement system used in this example is 5%, it can be said that the E2 measurement system is an E2-specific measurement system. Twelve beads on which goat anti-rabbit IgG is immobilized are placed in a cup, and 50 μL of a conjugate diluent containing 0.08 mA of alkaline phosphatase-labeled 17β estradiol (1 mA is absorbance at 280 nm of 1.0) is added (here, referred to as “1”). The conjugate diluent is a 50 mM Tris-HCl buffer (pH 7.5),
2.5% Gerizate (manufactured by Becton Dickinson),
2% sucrose (manufactured by Wako Pure Chemical Industries, Ltd.), a solution comprising 5 mM magnesium chloride, 0.1 mM zinc chloride, and 0.1% sodium azide), and rabbit anti-17β estradiol antiserum in a conjugate dilution of 70,000. 50 μL of the diluted solution was added and freeze-dried to prepare an E2 measurement cup.

On the other hand, the eluted E1 was measured by a one-step enzyme immunoassay using an anti-estrone antibody and based on a competition method. The cross-reactivity of the E1 measurement system used in this example with E2 was as small as 6%, and is considered to be an E1-specific measurement system. The preparation of the measuring cup was performed according to the above E2
According to the measurement, alkaline phosphatase-labeled estrone was diluted to 0.05 mA, and rabbit anti-estrone antiserum was diluted 80,000-fold with a conjugate diluent.

To each of the E2 measurement cup and the E1 measurement cup, 75 μL of a column eluate diluted to a final methanol concentration of 25% with 50 mM Tris-HCl buffer (pH 10.0) was added in an amount of 75 μL each, and commercially available fully automatic immunization was performed. Incubation was performed for 40 minutes on a diagnostic device (AIA-21, manufactured by Tosoh Corporation) to carry out an antigen-antibody reaction. After washing, the amount of bound labeled antigen was measured. E2 prepared with a methanol / 50 mM Tris-HCl buffer (pH 10.0) solution
A standard curve was prepared in both measurement systems using the standard product and the E1 standard product, and E2 and E1 in each fraction were prepared based on the standard curves.
The amount was determined.

FIG. 11 shows the results. E2 is methanol 7
87% of the total amount was recovered in the 5% fraction, while E1 was eluted only in the 25% fraction in the methanol 75% fraction. In the 85% methanol fraction, the remaining 13% of E2 was eluted, and 75% of E1 was recovered. As a result, an E2 solution containing less E1 was obtained by eluting 75% of methanol, and an E1 solution containing less E2 was obtained by eluting 85% of methanol. The mixed solution was obtained by using a column packed with a styrene-divinylbenzene copolymer. 2 from
It was shown that separate fractionation was possible.

Example 5 Fractionation from a mixed solution of 17α ethinyl estradiol and E1 Separation and fractionation of both from a mixed solution of 17α ethinyl estradiol and E1 was attempted using a column packed with a styrene-divinylbenzene copolymer. 1 mL of 50 mM phosphate buffer (pH 7) containing 1020 pg of 17α ethinyl estradiol and 660 pg of E1 was added to G300
Apply to a 0S packed column (gel volume 0.2 mL)
As in Example 4, methanol 50%, 75%, 85%
Fractionation was carried out using a mixture of 100% and 100% methanol / 50 mM Tris-HCl buffer (pH 10.0).

The eluted 17α ethinyl estradiol was measured by a one-step enzyme immunoassay using an anti-17α ethinyl estradiol antibody and based on the competition method. The cross-reactivity to E1 of the 17α ethinyl estradiol measurement system used in this example was 0.1%.
Therefore, it can be said that the measurement system is specific to 17α ethinyl estradiol. Goat anti-rabbit I in cup
Twelve beads to which gG was immobilized were added, and 50 μL of a conjugate diluent containing 0.15 mA of alkaline phosphatase-labeled 17α ethinyl estradiol (1 mA: absorbance at 280 nm: 1.0) was added (here, the conjugate diluent and the conjugate diluent). Are 50 mM Tris-HCl buffer (pH 7.5), 2.5% gelizate (manufactured by Becton Dickinson), 2% sucrose (manufactured by Wako Pure Chemical Industries), 5 mM magnesium chloride, 0.1 mM zinc chloride,
A solution consisting of 0.1% sodium azide),
A solution obtained by diluting rabbit anti-17α ethinyl estradiol antiserum 15000-fold with a conjugate diluent was added to 50 μl.
L was added and freeze-dried to prepare a prepared measurement cup.

On the other hand, the eluted E1 was measured by a one-step enzyme immunoassay based on the competition method using the anti-estrone antibody described in Example 4.

A final methanol concentration of 25% was added to the 17α ethinyl estradiol measurement cup and the E1 measurement cup with 50 mM Tris-HCl buffer (pH 10.0).
The column eluate prepared by dilution was added in an amount of 75 μL each, and the mixture was incubated for 40 minutes on a commercially available automatic immunodiagnostic apparatus (AIA-21, manufactured by Tosoh Corporation) to carry out an antigen-antibody reaction. The amount of labeled antigen was measured. Methanol / 50 mM Tris-HCl buffer (pH 1
0.0) A standard curve was prepared in both measurement systems using the 17α ethinyl estradiol standard product and the E1 standard product prepared in the solution, and the amounts of 17α ethinyl estradiol and E1 in each fraction were determined based on the standard curves. .

FIG. 12 shows the results. The 17α ethinyl estradiol recovered 67% of the total amount in the 75% methanol fraction, while E1 eluted only 16% of the total amount in the 75% methanol fraction. In the methanol 85% fraction, the remaining 33% of 17α-ethynylestradiol was eluted, and 62% of E1 was recovered. Further, 16% of the total amount of E1 was recovered in the 100% methanol fraction.
As a result, a 17α-ethynyl estradiol solution with a small amount of E1 was obtained by eluting 75% of methanol, while an E1 solution with a small amount of 17α-ethynyl estradiol was obtained by elution of 85% or more of methanol.
It was shown that by using a column packed with a divinylbenzene copolymer, separation and fractionation of the two from the mixed solution was possible.

[Brief description of the drawings]

FIG. 1 is a view showing an elution pattern when G3000S in Example 1 is used.

FIG. 2 is a view showing an elution pattern when G5000PW is used in Example 1.

FIG. 3 is a view showing an elution pattern when Butyl-TP is used in Example 1.

FIG. 4 is a view showing an elution pattern when Hexyl-TP is used in Example 1.

FIG. 5 is a diagram showing an elution pattern when Phenyl-TP is used in Example 1.

FIG. 6 is a diagram showing an elution pattern when OD-2PW is used in Example 1.

FIG. 7 is a diagram showing an elution pattern when 0.1% acetic acid (pH 5.0) is used as a buffer in Example 2.

FIG. 8 is a diagram showing an elution pattern when a 50 mM Tris buffer (pH 9.0) is used as a buffer in Example 2.

FIG. 9 is a diagram showing an elution pattern when a 50 mM Tris buffer (pH 10.0) is used as a buffer in Example 2.

FIG. 10 is a graph showing the relationship between the methanol concentration and E1,
It is a figure which shows the relationship with E2 collection rate.

FIG. 11 is a graph showing the relationship between the methanol concentration and E1,
It is a figure which shows the relationship with E2 collection rate.

FIG. 12 is a graph showing the relationship between the methanol concentration and E1,
It is a figure which shows the relationship with 17 (alpha) -ethynyl estradiol recovery.

Claims (9)

[Claims] 1. A solution containing estradiol and estrone is brought into contact with a styrene-divinylbenzene copolymer, estradiol and estrone in the solution are captured by the copolymer, and then estradiol and / or estrone are separated and eluted. A method for separating and eluting estradiol and / or estrone, characterized in that: 2. A solution containing 17α ethinyl estradiol and estrone is brought into contact with a styrene-divinylbenzene copolymer, and 17α ethinyl estradiol and estrone in the solution are trapped in the copolymer.
A method for separating and eluting 17α ethinyl estradiol and / or estrone, comprising separating and eluting α ethinyl estradiol and / or estrone. 3. The method according to claim 1, wherein
A method characterized in that the solution used for separation and elution is a mixed solution of an organic solvent and an aqueous buffer that are miscible with water. 4. The method according to claim 3, wherein the water-miscible organic solvent is methanol. 5. The method according to claim 4, wherein the concentration of methanol is 70% or more. 6. The method according to claim 4, wherein the methanol concentration is 80% or more. 7. The method according to claim 3, wherein the pH of the aqueous buffer is 9 or more. 8. An apparatus for separating and eluting estradiol and / or estrone, wherein a styrene-divinylbenzene copolymer is used as a separating means. 9. An apparatus for separating and eluting 17α ethinyl estradiol and / or estrone, wherein a styrene-divinylbenzene copolymer is used as a separating means.