JP2011163944A - Method for quantifying estrogen - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To simply and accurately quantify estrogen in a sample originating from a living body. <P>SOLUTION: Estrogen is extracted from the sample originating from the living body and converted to pentahalogenated benzyl or pentahalogenated benzoyl derivative and, after the derivative is convertive to a 1-lower alkylpyridinium derivative, this derivative is measured by LC-MS to quantify estrogen in a skin horny layer. In this case, the pentahalogenated benzyl or pentahalogenated benzoyl derivative is purified by a sequential solid phase extraction method to be converted to 1-lower alkylpyridinium derivative. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for quantifying estrogen present in a biological sample.

  Estrogens are a type of sex hormone belonging to steroid hormones and are also called follicular hormones or female hormones. Estrogens are produced in the ovary, transported throughout the body, bind to receptors in the cytoplasm, and the conjugates move into the nucleus, exhibiting a wide variety of physiological effects in trace amounts. For example, for skin, (a) promote blood flow, (b) increase mucopolysaccharides such as hyaluronic acid, improve skin moisture retention, (c) increase elastin and collagen, It has important effects such as imparting elasticity to the.

  Therefore, attempts have been made to grasp the concentration of estrogen in the living body. For example, estrogen is extracted from a biological sample obtained from serum, saliva, urine, cultured cells, and organs, and then a pentahalogenated benzyl compound or pentane is added thereto. There is a method of reacting a halogenated benzoyl compound and then further reacting with 1-lower alkyl-2-halogenated pyridine, and measuring the resulting reaction mixture by LC-MS (Patent Document 1). Thereby, the detection sensitivity in LC-MS of estrogen can be improved. Further, a method has been proposed in which this method is applied to a skin stratum corneum collected by a tape stripping method so that the estrogen concentration in the skin can be easily measured (Patent Document 2).

JP 2006-138786 A JP 2009-85946

  However, since the estrogen content in biological samples such as the skin stratum corneum is a very small amount of pg order per 1 mg of protein, it has been a problem to improve the measurement accuracy of quantitative analysis.

  The present invention is intended to solve the above-described problems, and an object thereof is to improve the accuracy of quantification of estrogen in a biological sample.

  In order to improve the detection sensitivity of estrogen in LC-MS, the present inventors introduced a pentahalogenated benzyl group or a pentahalogenated benzoyl group into estrogen and further introduced a 1-lower alkylpyridinium group. A pentahalogenated benzyl derivative or a pentahalogenated benzoyl derivative of estrogen having a pentahalogenated benzyl group or a pentahalogenated benzoyl group introduced therein was purified by a normal phase solid phase extraction method, and 1-lower alkylpyridinium was added to the purified product. When the group was introduced, the background value at the time of LC-MS measurement was greatly reduced, and the detection accuracy of estrogen in the sample derived from the living body was dramatically improved, and the present invention was completed.

That is, the present invention is a method for quantifying estrogen in a biological sample,
(a) a step of extracting estrogen from a biological sample,
(b) introducing a pentahalogenated benzyl group or a pentahalogenated benzoyl group into the extracted estrogen to obtain a pentahalogenated benzyl derivative or a pentahalogenated benzoyl derivative of estrogen;
(c) Purified product of estrogen pentahalogenated benzyl derivative or pentahalogenated benzoyl derivative by purifying the pentahalogenated benzyl derivative or pentahalogenated benzoyl derivative obtained in step (b) by a normal phase solid phase extraction method Obtaining a step,
(d) introducing a 1-lower alkylpyridinium group into a purified product of a pentahalogenated benzyl derivative or a pentahalogenated benzoyl derivative of estrogen to obtain a 1-lower alkylpyridinium derivative of estrogen; and
(e) analyzing a 1-lower alkylpyridinium derivative of estrogen by LC-MS and quantifying estrogen;
A quantification method is provided.

  According to the present invention, in order to improve the estrogen detection sensitivity in LC-MS, a pentahalogenated benzyl group or a pentahalogenated benzoyl group is introduced into estrogen, and a 1-lower alkylpyridinium group is further introduced. -In the estrogen quantification method for measuring lower alkylpyridinium derivatives by LC-MS, the background value at the time of LC-MS measurement is greatly reduced, and the estrogen detection accuracy is dramatically improved. Therefore, the concentration of estrogen contained in the biological sample can be quantified with high accuracy even though the concentration of estrogen in the biological sample such as the stratum corneum is as small as pg order per 1 mg of protein.

  On the other hand, since estrogen shows a strong physiological action in a very small amount in the living body, accurate quantification of estrogen in the sample derived from the living body is important for diagnosis and analysis of physiological functions and diseases related to estrogen. It is. Therefore, the present invention is useful in clinical diagnosis, pathological analysis, beauty diagnosis and the like.

  Above all, the skin stratum corneum is in the outermost layer of the skin and remarkably reflects the properties inside the skin. Therefore, the present invention is particularly useful for analyzing the properties of the entire skin including the inside of the skin in cosmetic diagnosis and the like.

  The present invention is a method for quantitatively analyzing estrogen in a sample derived from a living body by LC-MS, and includes steps (a) to (e) described below. Here, estrogen refers to female hormone estradiol and derivatives of estradiol.

  Moreover, the biological sample in which the present invention quantifies estrogen refers to a sample derived from a living body, and a sample collected from a living body or a sample collected after being discharged from a living body. Specific examples include skin stratum corneum, blood, saliva, urine, feces, living cells or cultured cells thereof, preparations obtained from living cells, tissues or organs, and the like. These biological samples are selected optimally according to the purpose of diagnosis and analysis for the subject. For example, the skin stratum corneum is preferable for analysis of skin properties. In addition, blood is preferable for diagnosing systemic physiological functions and the like.

  In addition, the collection method in particular of a biological sample is not restrict | limited, The general method in the field | area of medical treatment or beauty is used.

  For example, as a method for collecting the skin stratum corneum, preferably the tape stripping method, a method for collecting the scraped material of the skin stratum corneum, and removing the dermis and epidermis by treating the collected skin with a protease such as dispase or trypsin. Examples thereof include a method of exfoliating only the stratum corneum, and exfoliation of the stratum corneum from a cultured skin model. Among them, the tape stripping method is a method of applying a tape having an adhesive layer on the surface to the test site, peeling it off, attaching the surface layer of the test site to the adhesive surface of the tape, and collecting it. It is more preferable because the burden of

  The tape for performing the tape stripping method is not particularly limited as long as it can be peeled off after being in close contact with the skin, and a commercially available adhesive tape can be used as such a tape. More specifically, the pressure-sensitive adhesive layer can be a rubber-based, acrylic-based, or silicon-based pressure-sensitive adhesive, and the tape body that supports the pressure-sensitive adhesive layer is a paper made of natural fibers or synthetic fibers. Alternatively, it may be a cloth, or a plastic sheet made of polyester, polyimide, polyphenylene sulfide, polypropylene, or the like. Among these, polyphenylene sulfide (PPS) is preferable from the viewpoints of solvent resistance and background (amount of estrogen-like compound detected from the tape).

  On the other hand, the part of the living body-derived sample on the body is not particularly limited, and for example, a skin stratum corneum collected from the face, neck, arm or the like where cosmetic skin properties are important can be used.

In the present invention, step (a) is a step of extracting estrogen from a biological sample.
As a method for extracting estrogen from a biological sample, general pretreatment methods in the field of biochemistry such as centrifugation, liquid-liquid extraction using an organic solvent, column chromatography, etc. can be appropriately selected and combined.

  For example, as a method of extracting estrogen from blood, blood is centrifuged to obtain a serum component, and this serum component is liquid-liquid extracted using an organic solvent to separate an organic solvent layer. The organic solvent is removed from the solution. It should be noted that the preparation method for extracting estrogen from samples such as saliva, urine, feces, living cells and the like can be performed using a preparation method substantially similar to that for blood.

  When estrogen is extracted from the skin stratum corneum sampled by the tape stripping method, as shown in the examples described later, the tape to which the skin stratum corneum is attached is cut or left as it is, with ethyl alcohol. It is preferable to extract the estrogen by immersing in a solvent such as hexane, acetone or ethyl acetate.

  In the present invention, when the extracted estrogen is quantified by LC-MS, in order to improve the detection sensitivity, a pentahalogenated benzyl group or a pentahalogenated benzoyl group is introduced into the estrogen in advance in step (b). A pentahalogenated benzyl derivative or a pentahalogenated benzoyl derivative is obtained, purified in step (c) by a solid phase extraction method of normal phase, and in step (d), a 1-lower alkylpyridinium group is introduced into the purified product. The 1-lower alkylpyridinium derivative of estrogen thus obtained is quantified by LC-MS.

  Here, the introduction of the pentahalogenated benzyl group or the pentahalogenated benzoyl group in the step (b) is performed, for example, as described in JP-A-2006-138786 (Patent Document 1). Can be selectively reacted with a pentahalogenated benzyl compound or a pentahalogenated benzoyl compound. By extracting the reaction solution with an organic solvent such as ether, a pentahalogenated benzyl derivative or a pentahalogenated compound can be obtained. A crude product of the benzoyl derivative can be obtained.

  The purification of the benzyl derivative or benzoyl derivative of estrogen in step (c) by normal phase solid phase extraction is a characteristic step of the present invention. Rather than extracting the benzyl derivative or benzoyl derivative of estrogen with ether or the like from the reaction solution obtained in step (b), or extracting the solvent extract more precisely by HPLC, it is a normal phase solid phase extraction method. As a result, the background when the 1-lower alkylpyridinium derivative of estrogen is finally measured by LS-MS can be greatly reduced, and the estrogen detection accuracy can be improved.

  Here, the solid phase extraction method is a method in which a sample is held in a mini-column having a solid phase of a filler such as chemically bonded silica, porous polymer, alumina, activated carbon, etc., and a target component in the sample is selectively selected using a solvent. Extraction, separation and purification means a normal phase in the solid phase extraction method means a method using a high polarity filler for the solid phase and a low polarity solvent for the solvent.

  As the normal phase solid phase used in the step (c) of the invention, it is preferable to use a silica gel-based filler having a stronger polarity than the silanol group. More specifically, examples include silica gel having a particle size of 40 to 90 μm and an average pore size of 50 to 70 angstrom.

  The mini-column used in the solid phase extraction method is large and includes a cartridge type, a Luer device type, and a disk type. In the present invention, it is preferable to use a cartridge type because it is simple and has good operability.

  In the step (c), specific product names of mini-columns that can be used for the solid phase extraction method include product name InertSep SI manufactured by GL Sciences Inc., product name Bond Elut CN manufactured by Varian, etc. .

  On the other hand, as a low-polarity solvent used for conditioning, cleaning, and elution in the normal phase solid-phase extraction method, for example, ethyl acetate, hexane, benzene, heptane, etc. may be used by changing their mixing ratio as appropriate. it can.

  In the present invention, the minicolumn used in the solid-phase extraction method is preferably used as a disposable column in consideration of prevention of contamination due to the previous operation, workability of the washing operation, and the like, and is preferably replaced for each measurement sample. .

  Introduction of 1-lower alkylpyridinium in the step (d) is described in a purified product of a pentahalogenated benzyl derivative or a pentahalogenated benzoyl derivative of estrogen, for example, in JP-A-2006-138786 (Patent Document 1). As shown, it can be carried out by reacting 1-lower alkyl-2-halogenated pyridine.

  In the step (e), after the estrogen is converted to a 1-lower alkylpyridinium derivative as described above, the 1-lower alkylpyridinium derivative is detected by LC-MS (liquid chromatograph-mass spectrometer) to quantify the estrogen. To do.

  Prior to this quantification, the 1-lower alkylpyridinium derivative is preferably purified by a reverse phase solid phase extraction method. In this case, an ODS (C18) column or the like is used as the solid phase used in the solid phase extraction method, and methanol, formic acid, water, or the like is used as a solvent for conditioning, cleaning, or elution, and the mixing ratio thereof is set. It can be used as appropriate.

  Moreover, as LC-MS, LC-MS / MS (liquid chromatograph-tandem type mass spectrometer), LC-ESI-MS / MS, LC-APCI-MS / MS, etc. can be used. The measurement itself can be performed by a general method.

  According to the present invention, since the amount of estrogen in a sample derived from a living body can be accurately quantified, the present invention is useful for diagnosis and analysis of physiological functions and diseases related to estrogen. In particular, the amount of estrogen in the skin can be estimated by quantifying estrogen in the stratum corneum of the skin, research on the relationship between the skin properties and the amount of estrogen, and the effect of cosmetics or cosmetic methods on the amount of estrogen. It becomes useful for etc.

Hereinafter, based on an Example and a comparative example, this invention is demonstrated concretely.
[Example 1]
(1) Collection of biological samples As the tape for stripping, PPS tape (Nichiban Co., Ltd.) Upper arm of 9 subjects (male 20-50 years old, subjects number 1-9) each with 5 pieces of 5cm x 2.5cm tape It peeled after sticking to an inner side part, and the skin stratum corneum of each test subject was extract | collected.

(2) Extraction of estrogen from biological samples
¹³ C ₄ (100 pg) / 50 μL was added as an internal standard estradiol to a 30 mL centrifuge tube. On the other hand, 27 ml of ethanol was put into a beaker, and each tape from which the skin stratum corneum was collected was cut and immersed, transferred to the above centrifuge tube, and shaken at 50 ° C. for 1 hour.

  The above ethanol was collected in a new 30 mL centrifuge tube. Add 8 mL of ethanol to the remaining tape from which ethanol was collected, shake vigorously for another 10 minutes with an extractor, extract the ethanol again from the tape, combine it with the ethanol collected earlier, and mix it with a 40 ° C centrifugal evaporator. Concentrate and make it up to 4 mL with ethanol to obtain a sample concentrate.

Meanwhile, in order to prepare a sample for calibration curve, estradiol - ¹³ C ₄ (100pg) and estradiol a standard and weighed prescribed amount of ethanol was added 3.75 mL, was shaken in a standard sample.

  For each of the sample concentrate and the standard sample, add 0.5 mL of purified water, stir it, leave it at 5-15 ° C for 1 hour, centrifuge (3000 rpm, 3 minutes), and remove the supernatant. Collected. On the other hand, 1 mL of 85% ethanol was added to the precipitate, shaken, and centrifuged to collect a supernatant, which was combined with the previously collected supernatant.

  To the combined supernatant (ethanol layer), 1.5 mL of hexane was added and shaken. After centrifugation, the hexane layer was discarded, the supernatant was washed with hexane, and the washed ethanol layer was distilled off with a centrifugal evaporator ( 40-45 ° C., 2.5 hours), this was dissolved in 250 μL of methanol, 1 m of purified water was added, and shaken to obtain a sample for estradiol determination and a sample for calibration curve.

  In addition, extraction operation was also performed on the tape from which the stratum corneum had not been collected by the same method to obtain a sample for zero test.

(3) Determination of estradiol
(3-1) Preparation of estradiol-3-pentafluorobenzyl ether The sample for estradiol determination obtained in (2) was loaded on crushed acidic silica (Shimadzu GLC, Bond Elut C18) conditioned with 6 ml of methanol and 6 ml of purified water. After washing sequentially with 1 ml of purified water and 4 mL of 30% acetonitrile aqueous solution, the estradiol fraction was eluted with 2.5 mL of 70% acetonitrile aqueous solution, and the eluate was distilled off with a centrifugal evaporator (53 ° C, 2.5 hours). And dried for 1 hour.

  Next, 50 μL of 0.8% potassium hydroxide / ethanol solution and 100 μl of 2.5% pentafluorobenzyl bromide / acetonitrile solution were added to the estradiol determination sample, respectively, and left in a reaction incubator at 53 ° C. for 1 hour. Reaction with fluorobenzyl bromide.

  After distilling off the solvent of this reaction solution with nitrogen gas, 0.75 ml of purified water and 4 ml of ether solution were added, and the mixture was shaken for 10 minutes to extract the reaction product of estradiol and pentafluorobenzyl bromide into ether. After centrifugation (3000 rpm, 3 minutes), the ether solution was separated from the supernatant by freezing, and the ether was distilled off with nitrogen gas to obtain a crude purified product of estradiol-3-pentafluorobenzyl ether. . To this, 100 μl of ethyl acetate and 1 ml of hexane were added and shaken to obtain a crude product solution.

(3-2) Purification of estradiol-3-pentafluorobenzyl ether Next, the crude product solution of estradiol-3-pentafluorobenzyl ether prepared in (3-1) was conditioned with 3 mL of ethyl acetate and 3 mL of hexane. Load onto a silica gel-based disposable column (GL Science, InertSep SI) for normal phase solid phase extraction, wash sequentially with 1 mL of hexane, 4.5 mL of 15% ethyl acetate / hexane solution, and then 50% ethyl acetate / hexane solution Estradiol was eluted with 3 mL. This eluate was distilled off with a centrifugal evaporator (40 ° C., 1 hour), and further dried at 40 ° C. for 1 hour to obtain a purified product of estradiol-3-pentafluorobenzyl ether.

(3-3) Preparation of estradiol-3-pentafluorobenzyl ether 17-O-methylpyridinium
Add 200 μL of 2% 2-fluoro-1-methylpyridinium p-toluenesulfonate / dichloromethane solution and 30 μL of 10% triethylamine / dichloromethane solution to the purified estradiol-3-pentafluorobenzyl ether prepared in (3-2) For 1.5 hours at room temperature. The solvent was distilled off from this reaction solution with nitrogen gas, dissolved in 500 μL of methanol, diluted with 500 μL of purified water and shaken to obtain an estradiol-3-pentafluorobenzyl ether 17-O-methylpyridinium solution.

(3-4) Determination of estradiol-3-pentafluorobenzyl ether 17-O-methylpyridinium
Load the estradiol-3-pentafluorobenzyl ether 17-O-methylpyridinium solution prepared in (3-3) onto a mini-column of crushed acidic silica (Shimadzu GLC, Bond Elut C18) conditioned with 6 mL of methanol and 6 mL of purified water. After washing sequentially with 1 mL of purified water, 4 mL of 0.3% aqueous ammonia, 3 mL of methanol, 3 mL of 0.1% formic acid aqueous solution / methanol (1: 1), the estradiol fraction was mixed with 3 mL of acetonitrile / 10% formic acid aqueous solution (9: 1). The eluate was distilled off with a centrifugal evaporator (53 ° C., 2.5 hours). Furthermore, the reaction sample was dissolved in 100 μL of a mixed solution of acetonitrile / 0.1% aqueous formic acid / methanol (1: 2: 1) to obtain a sample for LC-MS / MS measurement of Example 1.

[Comparative Example 1]
To the crude product of estradiol-3-pentafluorobenzyl ether obtained by the same procedure as in (3-1) of Example 1 above, 110 μL of 50% ethanol / hexane was added and shaken to obtain the crude product. It was set as the solution.

This crude product solution was purified by HPLC under the following conditions to elute and fractionate the estradiol fraction. The eluate was distilled off with a centrifugal evaporator to obtain a purified product of estradiol-3-pentafluorobenzyl ether.
Precolumn: Shiseido CapcelPak NH2 UG80, particle size 5μm, size 4.6mm × 35mm
This column: Shiseido CapcelPak NH2 UG80, particle size 5μm, size 4.6mm x 150mm
Mobile phase: 5% ethanol / hexane, flow rate 1 mL / min
Elution site: 4-8 minutes

  The purified product of estradiol-3-pentafluorobenzyl ether obtained was derivatized with pyridinium by the same procedure as in (3-3) and (3-4) of Example 1 and the estradiol fraction was fractionated. A sample for LC-MS / MS measurement of Comparative Example 1 was prepared.

About the LC-MS / MS measurement sample of the said Example 1 and the comparative example 1, LC-MS / MS measurement was performed on the following conditions.
1) LC part Column: Xterra 3μm 2.1 × 100mm
Column temperature: 40 ° C
Mobile phase, flow rate: acetonitrile / 0.05% formic acid aqueous solution (3: 1) mixture, 0.2 ml / min
Injection volume: 20ml
2) MS part
MS: API-5000 (Applied Biosystems)
Ionization method: Positive ion ESI
Capillary voltage: 3.5kv
Cone voltage: 35, 40v
Collision energy: 18ev
Ion source temperature: 120 ° C
Measurement ions: 544.4 → 339, 110.1 (Estradiol), 547.4 → 339 (IS)

  When MS measurement is further performed on the peak of m / z = 544.4 detected by this MS measurement, peaks of m / z = 339 and 110.1 are obtained, and this is measured by SRM (selected reaction monitoring) chromatogram, thereby estradiol. Was detected.

  For the determination of estradiol, a calibration curve was used using the aforementioned calibration curve sample.

  The quantitative results of LC-MS / MS of estradiol purified by the methods of Example 1 and Comparative Example 1 are shown in Table 1 below. In addition, the numerical values of the measurement samples No. 1 to 9 in the table are correction values obtained by subtracting the average value (background value) of the zero test from the numerical values quantified using the calibration curve, and are collected with 5 sheets of PPS tape. It shows the amount of estradiol in the stratum corneum.

  From the results in Table 1, the benzyl derivative of estrogen was purified by a normal phase solid-phase extraction method, then converted into a 1-lower alkylpyridinium derivative, and quantified by LS-MS. It can be seen that the ground value is very small. On the other hand, in the quantitative method for purifying the benzyl derivative of estrogen of Comparative Example 1 by HPLC, it can be seen that the measured value of the zero test is 50 times or more compared to Example 1.

  Since the estrogen content in biological samples such as skin stratum corneum is extremely small at the pg level per mg of protein, the background value should be kept as low as possible in quantitative analysis by LC-MS (for example, the lower limit of quantification) It is desirable that the background value be kept very low as in Example 1, which is extremely useful in the determination of trace amounts of estrogen.

[Example 2]
(1) Collection of biological samples 3 mL of blood was collected from 12 postmenopausal women, and serum was obtained by centrifugation (4-10 ° C, 1,500 xg, 10 minutes). 5 mL of ether was added, shaken for 10 minutes, and further centrifuged (5 ° C., 1500 × g, 5 minutes). This was frozen and the supernatant was separated, the solvent was distilled off, and this was used as a sample for elastdiol determination. To this estradiol quantification sample, add 50 μL of 0.8% potassium hydroxide / ethanol solution and 100 μl of 2.5% pentafluorobenzyl bromide / acetonitrile solution, respectively, and then leave it in a reaction incubator at 53 ° C. for 1 hour. Estradiol and pentafluorobenzyl bromide And reacted. The crude product solution was then loaded onto a silica gel-based disposable column (GL Science, InertSep SI) for normal phase solid phase extraction conditioned with 3 mL of ethyl acetate and 3 mL of hexane, and 1 mL of hexane, 15% ethyl acetate. After sequentially washing with 4.5 mL / hexane solution, estradiol was eluted with 3 mL of 50% ethyl acetate / hexane solution. This eluate was distilled off with a centrifugal evaporator (40 ° C., 1 hour), and further dried at 40 ° C. for 1 hour to obtain a purified product of estradiol-3-pentafluorobenzyl ether. To the purified product of estradiol-3-pentafluorobenzyl ether, 200 μL of 2% 2-fluoro-1-methylpyridinium p-toluenesulfonate / dichloromethane solution and 30 μL of 10% triethylamine / dichloromethane solution were added and left at room temperature for 1.5 hours. The solvent was distilled off from this reaction solution with nitrogen gas, dissolved in 500 μL of methanol, diluted with 500 μL of purified water and shaken to obtain an estradiol-3-pentafluorobenzyl ether 17-O-methylpyridinium solution.

(2) Quantification of elast diol In the same manner as in Example 1, a sample for LC-MS / MS measurement was prepared from a sample for quantification of elast diol, and elast diol was quantified by LC-MS / MS. The results are shown in Table 2.

ゼロ試験：活性炭で処理してエストラジオールを除去したゼロ血清

Zero test: Zero serum treated with activated charcoal to remove estradiol

  From the results in Table 2, it can be seen that the background value is also very low in this example.

  The estrogen quantification method of the present invention is useful in fields such as clinical diagnosis, pathological analysis, and cosmetic diagnosis because estrogen in a sample derived from a living body can be accurately quantified. In particular, it is useful in fields such as research on the relationship between skin properties and the amount of estrogen and other hormones contained in the skin, and research and development of cosmetics and beauty methods based on the research.

Claims (5)

A method for quantifying estrogen in a biological sample,
(a) a step of extracting estrogen from a biological sample,
(b) introducing a pentahalogenated benzyl group or a pentahalogenated benzoyl group into the extracted estrogen to obtain a pentahalogenated benzyl derivative or a pentahalogenated benzoyl derivative of estrogen;
(c) Purified product of estrogen pentahalogenated benzyl derivative or pentahalogenated benzoyl derivative by purifying the pentahalogenated benzyl derivative or pentahalogenated benzoyl derivative obtained in step (b) by a normal phase solid phase extraction method Obtaining a step,
(d) introducing a 1-lower alkylpyridinium group into a purified product of a pentahalogenated benzyl derivative or a pentahalogenated benzoyl derivative of estrogen to obtain a 1-lower alkylpyridinium derivative of estrogen; and
(e) analyzing a 1-lower alkylpyridinium derivative of estrogen by LC-MS and quantifying estrogen;
A quantitative method comprising:   The method according to claim 1, wherein the biological sample is a skin stratum corneum, blood, saliva, urine, feces, or biological cells.   The method according to claim 2, wherein the biological sample is a skin stratum corneum collected by a tape stripping method, and estrogen is extracted from the skin stratum corneum in the step (a).   The quantification method according to any one of claims 1 to 3, wherein in the solid phase extraction method of step (c), a cartridge type column using a highly polar silica gel with silanol groups as a solid phase is used.   The method according to any one of claims 1 to 4, wherein estrogen is quantified by LC-MS / MS in step (e).