JP2011174710A - Method for measuring estrones with high sensitivity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a compound for measuring estrones contained in a sample with high sensitivity by liquid chromatography-mass analysis (LC-MS), and a measuring method using the same. <P>SOLUTION: A pentahalogenated benzyl or pentahalogenated benzoyl group is introduced into the phenolic hydroxyl group of estrones in the sample and hydrazino-1-lower alkylpyridine is subsequently introduced into the 17-carbonyl group thereof to obtain an estrone derivative. An LC-MS measuring method uses the compound. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

In the present invention, liquid chromatography mass spectrometry (LC-MS) of 3-hydroxyestradi-1,3,5 (10) -trien-17-one (estrone) contained in a sample is used. The present invention relates to a compound for measuring with high sensitivity and a measuring method using the compound. More specifically, after introducing a pentahalogenated benzyl group or a pentahalogenated benzoyl group into the phenolic hydroxyl group of estrone in the sample, a 1-lower alkylhydrazinopyridinium salt (hereinafter referred to as hydrazino-1- Lower alkylpyridine), and a derivative thereof is measured by LC-MS.

Estrogens are produced from androgens by aromatase. Estrogens are closely related to various diseases such as breast cancer, digestive organ cancer, cardiovascular disorders, congenital diseases of sex hormone metabolism, precocious puberty, osteoporosis, etc. It is useful for elucidating diseases and determining pharmacological effects. The most effective estra-1,3,5 (10) -triene-3,17β-diol (estradiol) is converted to less effective estrone by 17β-hydroxysteroid dehydrogenase type 2 (17β-HSD2) And estrone is reactivated to estradiol by 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1). Circulating estradiol levels in premenopausal women who are not pregnant and adults are 1.5 to 4 times higher than estrone. In contrast, in men and postmenopausal women, estradiol is reduced to about 1/10 and the estradiol / estrone ratio is reversed. Therefore, for pre-pubertal girls, postmenopausal women, and men, the state of metabolism and disease can be grasped more accurately by monitoring not only strong estradiol but also estrone, which is a major estrogen.

Polycystic ovary syndrome (PCOS) is a disease in which follicles become cysts and ovulation is difficult to occur, but is thought to be caused by various endocrine inconsistencies. As one of the diagnostic methods, the ratio of estrone and estradiol in blood is useful, and is listed as a test method for PCOS.
It is known that estrogen is also greatly involved in the growth and suppression of breast cancer, and even if the concentration of estrogen in the blood is low, as in postmenopausal women, adrenal androgens and sulfate-conjugated estrone in organs other than the ovary It is suggested that estradiol and estrone are locally produced from a relatively large amount of steroid hormones by aromatase and 17β-HSD in tissues, and that these may be greatly involved in the development and growth of breast cancer. . (Non-Patent Document 1)
In endometrial cancer, it has been reported that the amount of estrone increases in cancer tissues and the ratio of estrone and estradiol is different compared to other tissues. (Non-Patent Document 2)

In any case, it is important to analyze the causal relationship between aromatase and the pathophysiology in order to diagnose and elucidate the etiology, but in order to grasp the whole picture of aromatase, only estradiol with strong physiological action is used. It is important to consider the amount of estrone. However, samples such as blood obtained from small animals and children, tissues collected by needle biopsy, and saliva contain only a small amount of estrogen, and high sensitivity is required for these measurements.

In normal clinical tests, estrone in a sample is quantified by an immunoassay such as radioimmunoassay or enzyme immune reaction using ³ H-estrone as a ligand, but the general quantification limit of immunity is 10 pg / mL. In addition, the structure-similar compounds of estrone and sex hormone-binding globulin coexist in the blood, so the quantification value is likely to fluctuate greatly depending on the quantification method used and differences between kits, and the results are There may be a gap. Recently, a high-sensitivity estrone EIA measurement kit with a detection sensitivity of 4.8 pg / mL has been put on the market, but there is a large variation in measurement values at low concentrations.

On the other hand, a method for measuring by gas chromatography-mass spectrometry (GC-MS) has also been proposed. In GC-MS, in general, estrogen in a sample is converted into a volatile reagent such as trifluoroacetic anhydride or heptafluorobutyric anhydride, or a silylating agent such as N, O-bis (trimethylsilyl) trifluoroacetamide. And the derivative is measured by GC-MS. (Non-Patent Documents 3 and 4) However, since these derivatives are all unstable, it is difficult to purify them as derivatives, and they are easily affected by impurities and reagents in the sample during measurement. In addition, in the measurement by GC-MS, the injection amount of the sample is reduced to 5 to 10% or less of the total sample and the loss increases, so that sufficient sensitivity cannot be obtained and the analysis takes a long time. This is a major drawback. Furthermore, GC-MS has a drawback that its dynamic range is narrower than that of LC-MS. Thus, there are many limitations on the measurement using GC-MS, and therefore it is difficult to measure a biological sample obtained from postmenopausal women and small animals by GC-MS.

For LC-MS, a direct quantification method by atmospheric pressure photoionization (APPI) has been reported. However, APPI has many drawbacks, such as that ionization tends to be inhibited when acetonitrile is used as an eluent, and the baseline is likely to be disturbed by the pulsating flow of the dopant solution. Compared with electrospray ionization (ESI). It is not universal as a detector for LC-MS. (Non-Patent Documents 5 and 6)

Attempts have also been made to derivatize estrogens and measure them by LC-MS. For example, a method has been reported in which the phenolic hydroxyl group of estrogen is dansylated (5-dimethylaminonaphthalen-1-ylsulfonate) and measured by two-dimensional chromatography using positive ion detection ESI. (Non-patent document 7) However, this method has a defect that a product ion derived from a compound cannot be obtained and lacks specificity.

Further, since the Sanger reagent easily reacts with a phenolic hydroxyl group in the presence of an alkali, a method of measuring the LC-MS by reacting the piperazine derivative of the Sanger reagent with estrogen has been reported. (Non-Patent Document 8) Although this method can measure estrogen in pregnant women at a relatively high concentration, it cannot detect blood estrogen in men.

A method for measuring the carbonyl group of estrone by derivatizing with p-toluenesulfonyl hydrazide has also been reported. (Non-patent document 9) In this method, estrone can be measured from 10 pg (oncolumn), but the sensitivity is insufficient for quantification of blood estrone in children and menopause.

The inventors have so far converted the phenolic hydroxyl group of in vivo estradiols to a pentahalogenated benzyl or pentahalogenated benzoyl group, and then converted the 17-position hydroxyl group to a halogeno-1-lower alkylpyridinium salt (hereinafter referred to as halogeno-1- A method of derivatization with lower alkylpyridine) was developed. However, this method cannot be applied to the measurement of estrogens that do not have an alcoholic hydroxyl group such as estrone.

JP 2006-138786 A

Japanese clinical 65th volume extra number 6 (Vol.917) 106-110 Clin. Cancer. Res. 15 (2009) 6038-6034 Steroids 57 (1992) 319-324 Endocr. J. et al. 50 (2003) 783-792 Clin. Chim. Acta 409 (2009) 78-84 Bunseki (2009) 278-284 Am. J. et al. Clin. Pathol. 129 (2009) 530-539 J. et al. Pharm. Biomed. Anal. 44 (2007) 786-795 Anal. Chem. 76 (2004) 5829-5836

An object of the present invention is to provide a compound for measuring estrones contained in a sample with high sensitivity using LC-MS and a measurement method using the compound.

In order to measure a trace amount of an estrogen having a carbonyl group such as estrone, the present inventors first made a phenolic hydroxyl group selectively pentahalogenated benzyl ether or pentahalogenated benzoyl ester under alkaline conditions, The 17-position carbonyl group was derivatized in various ways, and the measurement sensitivity of the derivatives was investigated. As a result, it was found that estrogen having a carbonyl group can be measured with good sensitivity by LC-MS by converting the 17-position carbonyl group to a 1-lower alkylpyridinium hydrazone, thereby completing the present invention.

（式中、Ｒは低級アルキル基を表し、Ｘはハロゲン原子を表し、ＹはＣＨ_２又はＣＯを表す）
で示される化合物を提供するものである。 That is, the present invention provides the following formula (1) as a compound for measuring estrone in a sample by LC-MS.

(Wherein R represents a lower alkyl group, X represents a halogen atom, and Y represents CH ₂ or CO)
The compound shown by these is provided.

（式中、Ｘはハロゲン原子を表し、ＹはＣＨ_２又はＣＯを表し、Ｌは脱離基を表す）
で示されるペンタハロゲン化ベンジル化合物又はペンタハロゲン化ベンゾイル化合物をアルカリ条件化で反応させる工程、
３） 上記２）で得られた反応物に含まれるエストロン類のペンタハロゲン化ベンジル誘導体又はペンタハロゲン化ベンゾイル誘導体にヒドラジノ−１−低級アルキルピリジンを酸性下で反応させる工程、及び
４） 上記３）で得られた反応物に含まれるエストロン類のペンタハロゲン化ベンジル−又はペンタハロゲン化ベンゾイル−１−低級アルキルピリジニウムヒドラゾンをＬＣ−ＭＳで測定する工程、
を含むことを特徴とする、試料中エストロン類の測定方法を提案するものである。 Furthermore, the present invention is a method for measuring estrones in a sample by LC-MS,
1) a process of extracting steroids from a sample,
2) The following formula (2) for the extract obtained in 1) above

(Wherein X represents a halogen atom, Y represents CH ₂ or CO, and L represents a leaving group)
A step of reacting the pentahalogenated benzyl compound or pentahalogenated benzoyl compound represented by
3) a step of reacting a pentahalogenated benzyl derivative or a pentahalogenated benzoyl derivative of estrone contained in the reaction product obtained in 2) above with hydrazino-1-lower alkylpyridine, and 4) above 3) A step of measuring by LC-MS the pentahalogenated benzyl- or pentahalogenated benzoyl-1-lower alkylpyridinium hydrazone of the estrone contained in the reaction product obtained in step 1.
The present invention proposes a method for measuring estrone in a sample, characterized in that

The present invention also includes the following steps:
1) After reacting a pentahalogenated benzyl compound or a pentahalogenated benzoyl compound under alkaline conditions, using a normal phase column, the pentahalogenated benzyl or pentahalogenated benzoyl derivative of an estradiol compound and the pentahalogenated benzyl of an estrone compound Or separating the pentahalogenated benzoyl derivative,
2) a step of reacting a pentahalogenated benzyl or pentahalogenated benzoyl derivative of the estradiol obtained in 1) above with a halogeno-1-lower alkylpyridine, and 3) a pentahalogenated benzyl or pentahalogenated estrone. When benzoyl-1-lower alkylpyridinium hydrazone is measured by LC-MS, the pentahalogenated benzyl- or pentahalogenated benzoyl-1-lower alkylpyridinium derivative of the estradiol obtained in 2) above is measured simultaneously. The process of
A method for measuring estrone in a sample as described above is further proposed.

The present invention also proposes a method for measuring estrone in a sample as described above, further comprising a step of removing neutral steroid from the sample using a solid phase extraction column.

By using the compound and measurement method of the present invention, estrones present in a trace amount in a sample can be measured at a level of 0.25 pg / tube (0.05 pg / on column).

In the present invention, “estrone” includes, for example, estrone, 2-hydroxyestrone, 4-hydroxyestrone, 2-hydroxyestrone 3-methyl ether, 4-hydroxyestrone 3-methyl ether, 2-methoxyestrone, 4- Mention may be made of estrogens present in vivo or in nature, such as methoxyestrone, 16α-hydroxyestrone, 16oxoestradiol.

In the present specification, the “solid phase extraction column” represents a general cartridge column, and in the present invention, a commercially available one can be used. The separation mode of the filler includes adsorption, distribution, ion exchange, molecular sieving, affinity, etc. Among them, adsorption and distribution are preferable. Moreover, although it divides roughly into a reverse phase and a normal phase from the relationship of the polarity of a mobile phase and a stationary phase, a reverse phase, a normal phase, or ion exchange is suitable among these. Further, the “normal phase column” in the present specification represents the “solid phase extraction column” in which the separation mode of the filler is adsorption.

In this specification, “LC-MS” represents an analysis method performed using an apparatus combining a liquid chromatograph and a mass spectrometer. Among them, a tandem type mass analysis in which a plurality of mass analysis units are combined. (Liquid Chromatography-Tandem Mass Spectrometry: LC-MS / MS) is preferably used. Examples of ionization in LC-MS include atmospheric pressure chemical ionization (ESP), ESI, and APPI. Among these, positive ion detection ESI is preferably used.

Examples of the mass spectrometer include a magnetic field type, a quadrupole type, and a time-of-flight type.In the present invention, a quadrupole type having a good quantitative property, a wide dynamic range, and a good linearity is used. It is preferable to use it.

Furthermore, as ion detection in quantification, selective ion monitoring (Selected IonMonitoring) that selectively detects only the target ion or one of the ion species purified by the first mass spectrometer is a precursor ion. And a reaction reaction monitoring (Selected Reaction Monitoring: SRM) in which a product ion generated by the cleavage of the precursor ion is detected in the second mass spectrometer. In the present invention, measurement by SRM, in which the signal / noise ratio is improved by increasing the selectivity and reducing the noise, is preferable.

Further, in the present specification, the “sample” is not particularly limited, and may be derived from any organism, environment, or industrial product. Examples of biological samples include blood, saliva, tears, sweat, urine, feces, bile, tissue, living cells, tissues or cell cultures or organs obtained from animals including humans, or plants. The extract of etc. can be mentioned. Examples of the environment-derived sample include soil, sewage, wastewater, river water, seawater and the like. Furthermore, food products, pharmaceuticals, etc. are mentioned as samples derived from industrial products. Among them, blood, saliva, urine, tissue, living cells, and the like of animals including humans are preferable as biological samples, waste water, river water, and the like are preferable as environment-derived samples, and pharmaceutical products are preferable as samples derived from industrial products.

で示されるペンタハロゲン化ベンジル化合物において、Ｘは、具体的には、フッ素原子、臭素原子、塩素原子、ヨウ素原子を表す。また、脱離基Ｌとしては、ハロゲン原子： トシルオキシ、メシルオキシ、又はトリフルオロメタンスルホニルオキシ基等の脱離基を挙げることができ、好適には、ハロゲン原子を挙げることができる。上記ペンタハロゲン化ベンジル化合物の具体例としては、例えば、ペンタフルオロベンジルクロライド、ペンタフルオロベンジルブロマイド、ペンタブロモベンジルクロライド、ペンタブロモベンジルブロマイド等を挙げることができ、中でも、ペンタフルオロベンジルブロマイドが好適である。 In the present invention, the following formula (2) that can be used:

In the pentahalogenated benzyl compound represented by X, specifically, X represents a fluorine atom, a bromine atom, a chlorine atom, or an iodine atom. Further, examples of the leaving group L include a leaving group such as a halogen atom: tosyloxy, mesyloxy, or trifluoromethanesulfonyloxy group, and preferably a halogen atom. Specific examples of the pentahalogenated benzyl compound include pentafluorobenzyl chloride, pentafluorobenzyl bromide, pentabromobenzyl chloride, pentabromobenzyl bromide, etc. Among them, pentafluorobenzyl bromide is preferable. .

These pentahalogenated benzyl compounds and pentahalogenated benzoyl compounds are known compounds or can be synthesized from known compounds by known methods.

In the present specification, “lower alkyl” means a linear or branched alkyl group having 1 to 6 carbon atoms, preferably 1 to 3 carbon atoms. Examples of such lower alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, tert-butyl, n-pentyl, isopentyl, tert-pentyl and the like. Preferable examples include methyl, ethyl and n-propyl. Examples of the “hydrazino-1-lower alkylpyridine” that can be used in the present invention include Girard reagent P and 2-hydrazino-1-methylpyridine, among which the reactivity is good and the sensitivity is high. The excellent derivatization with 2-hydrazino-1-methylpyridine is preferred. This hydrazino-1-lower alkylpyridine is also a known compound or can be synthesized from a known compound by a known method.

The present invention will be specifically described below.

1. Hydrazino-1 For preparation methods for preparing <br/> hydrazino-1-lower alkyl pyridine lower alkyl pyridine, although described in detail in the Examples below, hydrazine and 2-halogeno-1-lower alkyl pyridinium p- toluene It can be obtained by dissolving a general commercial synthetic raw material such as a sulfonate in an inert solvent such as acetonitrile and reacting it. This reaction can be performed in the range of −10 ° C. to 50 ° C., but it is preferable to perform the reaction at a temperature within the range of −5 ° C. to 5 ° C. for 5 to 15 minutes from the start of the reaction. Thereafter, the range of 10 ° C. to 40 ° C. is suitable. Moreover, as a reaction solvent, a general inert organic solvent, for example, chloroform, dichloromethane, acetonitrile, ethyl acetate, tetrahydrofuran, etc. can be used.

2. For preparation method of estrogens from Preparation <br/> serum sample in the sample, will be described in detail in Examples below, a general preparation method of separation and purification by a simple column chromatography on extraction and solid phase extraction with an organic solvent as appropriate It can be selected and used. In addition, as a method for preparing a sample such as a preparation obtained from saliva, urine, feces, cultured cells, organs, and sewage, a technique almost similar to that for serum can be used. Moreover, only a phenolic steroid is selectively used using a commercially available solid phase extraction column, for example, Oasis (registered trademark) MAX manufactured by Waters, or InertSep (registered trademark) MA-1 manufactured by GL Sciences. The separation can be performed as needed.

3. Preparation of pentahalogenated benzyl derivative or pentahalogenated benzoyl derivative The sample prepared above is dissolved in an inert solvent such as acetonitrile and reacted with pentahalogenated benzyl compound or pentahalogenated benzoyl compound under alkaline conditions. The estrogen prepared above is converted into a pentahalogenated benzyl derivative or a pentahalogenated benzoyl derivative. This reaction can be carried out at a temperature within the range of -10 ° C to 60 ° C, preferably a temperature within the range of 5 ° C to 60 ° C. Further, the use ratio of the pentahalogenated benzyl compound or the pentahalogenated benzoyl compound is not particularly limited, but in general, it can be 1 mg to 100 mg, preferably 2 mg to 5 mg per 1 ng of the sample. mg is suitable. Furthermore, in the above reaction, the pH of the reaction solution can be in the range of 8 to 14, preferably 11 to 13, and the reaction can be carried out from bases such as potassium hydroxide, sodium hydroxide and potassium carbonate. A base and an amount of a base sufficient to bring the liquid into the above pH range can be appropriately selected and used. Here, by performing this reaction under alkaline conditions, among the hydroxyl groups in estrogen, the phenolic hydroxyl group is selectively converted into a pentahalogenated benzyl ether or a pentahalogenated benzoyl ester.

Examples of the pentahalogenated benzyl derivative or pentahalogenated benzoyl derivative of estrogen prepared by the above method 3 include the following.
Estrone 3-pentafluorobenzyl ether,
Estrone 3-pentafluorobenzoyl ester,
2-methoxyestrone 3-pentafluorobenzyl ether,
4-methoxyestrone 3-pentafluorobenzyl ether,
2-hydroxyestrone 2,3-dipentafluorobenzyl ether,
4-hydroxyestrone 3,4-dipentafluorobenzyl ether 16α-hydroxyestrone 3-pentafluorobenzyl ether,
Estradiol 3-pentafluorobenzyl ether,
Estradiol 3-pentafluorobenzoyl ester,
2-methoxyestradiol 3-pentafluorobenzyl ether,
4-methoxyestradiol 3-pentafluorobenzyl ether and the like.

As described above, after estrogen is converted into a pentahalogenated benzyl derivative or pentahalogenated benzoyl derivative, a general normal phase column such as InertSepSI (registered trademark) manufactured by GL Sciences, Sep-Pak (registered by Waters) is registered. Trademark) Using a silica cartridge or the like, the derivative of estradiol and the derivative of estrone can be separated as necessary.

4.1 Preparation of 1-lower alkylpyridinium hydrazone The pentahalogenated benzyl derivative or pentahalogenated benzoyl derivative of estrone prepared by the above method was dissolved in an inert solvent such as acetonitrile, and then trifluoroacetic acid. Can be converted to 1-lower alkylpyridinium hydrazone under acidic conditions such as.

This reaction can be carried out at a temperature within the range of -10 ° C to 60 ° C, preferably a temperature within the range of 15 ° C to 40 ° C. Moreover, as a reaction solvent, a general inert organic solvent, for example, chloroform, dichloromethane, acetonitrile, ethyl acetate, tetrahydrofuran, etc. can be used.

Further, the use ratio of hydrazino-1-lower alkylpyridine to the pentahalogenated benzyl derivative or pentahalogenated benzoyl derivative of estrone is not particularly limited, but generally 0.1 μg to 1 ng of the sample It can be 500 μg, preferably 1 μg to 50 μg is suitable.

4. above. Examples of the 1-lower alkylpyridinium hydrazone prepared by the above method include the following.
3-pentafluorobenzyloxyestradi-1,3,5 (10) -trien-17-one 17- (1′-methylpyridinium-2 ′)-hydrazone,
3-pentafluorobenzoyloxyestradi-1,3,5 (10) -trien-17-one 17- (1′-methylpyridinium-2 ′)-hydrazone,
16α-hydroxy-3-pentafluorobenzyloxyestradi-1,3,5 (10) -trien-17-one 17- (1′-methylpyridinium-2 ′)-hydrazone,
16α-hydroxy-3-pentafluoroenzoyloxyestradi-1,3,5 (10) -trien-17-one 17- (1′-methylpyridinium-2 ′)-hydrazone,
2-methoxy-3-pentafluorobenzyloxyestradi-1,3,5 (10) -trien-17-one 17- (1′-methylpyridinium-2 ′)-hydrazone,
4-methoxy-3-pentafluorobenzyloxyestradi-1,3,5 (10) -trien-17-one 17- (1′-methylpyridinium-2 ′)-hydrazone,
2,3-dipentafluorobenzyloxyestradi-1,3,5 (10) -trien-17-one 17- (1′-methylpyridinium-2 ′)-hydrazone,
3,4-dipentafluorobenzyloxyestradi-1,3,5 (10) -trien-17-one 17- (1′-methylpyridinium-2 ′)-hydrazone and the like.

For separation and purification from the above reaction and other contaminants and reagents after the reaction in the present invention, a general solid phase extraction column such as InertSep (registered trademark) Pharma, Bond Elut (registered trademark) is used. C18 (Varian) or the like can be used.

The LC-MS measurement in the measurement method of the present invention can be performed by a method common to those skilled in the art.

The following examples illustrate the invention in more detail, but the invention is not limited thereto.

Synthesis of 3-pentafluorobenzyloxyestradi-1,3,5 (10) -trien-17-one 17- (1′-methylpyridinium-2 ′)-hydrazone
Preparation of 1-1.2-hydrazino-1-methylpyridine 30 mL of acetonitrile added with 132 μL of 80% hydrazine hydrate solution and acetonitrile added with 300 mg of 2-fluoro-1-methylpyridinium p-toluenesulfonate 6 mL was mixed at 0 ° C. and stirred for 10 minutes, then placed under nitrogen for 20 minutes at room temperature. The reaction was concentrated and the residue was redissolved with 2 mL of acetonitrile and filtered. The crude product obtained from the filtrate was recrystallized twice with acetonitrile-ethyl acetate (5: 1) to obtain 2-hydrazino-1-methylpyridine.

1-2. Synthesis of estrone 3-pentafluorobenzyl ether 276 mg of estrone was dissolved in 15 mL of acetonitrile, 361 mg of pentafluorobenzyl bromide and 0.6 g of anhydrous potassium carbonate were added, and the mixture was stirred at 53 ° C for 1 hour. After the reaction, the mixture was filtered through filter paper, the filtrate was concentrated, and the residue was poured into chloroform (60 mL) and washed with water. The organic layer was separated and the solvent was distilled off, and then recrystallized from methanol to obtain 185 mg of estrone 3-pentafluorobenzyl ether.

表１． Ｅ_１−ＰＦＢＺＨＭＰのＨＲ−ＭＳ、融点及びＮＭＲデータ 1-3.3-Pentafluorobenzyloxyestradi-1,3,5 (10) -trien-17-one 17- (1'-methylpyridinium-2 ')-hydrazone synthesis 1-2 above After adding 50 mg of estrone 3-pentafluorobenzyl ether and 30 mg of 2-hydrazino-1-methylpyridine obtained in 1 to 1.5 mL of toluene, 13 μL of 1% trifluoroacetic acid was added, and the mixture was stirred at 60 ° C. for 2 hours. . After the reaction, the solvent was distilled off under reduced pressure, and the oil was separated by preparative thin-layer chromatography with chloroform-methanol-triethylamine (20: 1: 0.05) and mixed with ethyl acetate-methanol (2: 1). Eluted with. After removal of the solvent, recrystallization from methanol gave 3-pentafluorobenzyloxyestradi-1,3,5 (10) -trien-17-one 17- (1′-methylpyridinium-2 ′)-hydrazone (E ₁ -PFBZHMP) was obtained in an amount of 27 mg.
High resolution mass spectrometry (High-resolution Mass Spectrometry: HR-MS), melting point, and nuclear magnetic resonance (NMR) data of the obtained E ₁ -PFBZHMP are shown in Table 1 below.

Table 1. HR-MS, melting point and NMR data of the E ₁ -PFBZHMP

Reproducibility of simultaneous determination of estrone and estradiol
2-1. Preparation of calibration curve and preparation of QC sample As calibration curve samples, methanol solutions of estrone and estradiol (0.25, 0.5, 1, 10, 100, 500 pg / 50 μL, 0.1, 0, respectively) .25, 1, 10, 100, 500 pg / 50 μL) was added to 1 mL with purified water. As a QC (Quality Control) sample, 0.25 mL of activated carbon-treated serum and methanol solution of estrone and estradiol (0.25, 0.5, 10, 500 pg / 50 μL, 0.1, 0.25, 10, (500 pg / 50 μL) was added and made up to 1 mL with purified water. Then, internal standard (estrone ^- 13 _{C 4} and estradiol ^- 13 _{C 4} each 100 pg / 50 [mu] L), diethyl ether 5mL added, after shaking for 10 minutes, the aqueous layer was freeze-separated, followed by distilling off the resulting solvent .

2-2. Preparation of estrone 3-pentafluorobenzyl ether and estradiol 3-pentafluorobenzyl ether To the residue obtained in the previous section 2-1, 100 μL and 0.8% of 2.5% pentafluorobenzyl bromide in acetonitrile was added. 50 μL of a potassium hydroxide / ethanol solution was added, and the mixture was heated at 53 ° C. for 1 hour. After the reaction, the reaction reagent was distilled off with nitrogen gas while heating, 0.75 mL of purified water was added, and the mixture was extracted with 4 mL of diethyl ether, and the solvent was distilled off.

2-3. Separation of estrone 3-pentafluorobenzyl ether and estradiol 3-pentafluorobenzyl ether The residue obtained in the preceding section 2-2 was dissolved by adding 0.1 mL of ethyl acetate and 1 mL of hexane, Loaded to an InertSepSI cartridge conditioned with 3 mL and 3 mL of hexane. The column was sequentially washed with 1 mL of hexane and 1 mL of ethyl acetate-hexane (3:17), and then estrone 3-pentafluorobenzyl ether was eluted with 3.5 mL of ethyl acetate-hexane (3:17). Subsequently, estradiol 3-pentafluorobenzyl ether was eluted with 3 mL of ethyl acetate-hexane (1: 1). Each solvent was distilled off under reduced pressure.

2-4.17 Preparation of [beta]-(1'-methylpyridinium-2 ')-oxy-3-pentafluorobenzyloxyestradi-1,3,5 (10) -triene Obtained in item 2-3 above To the residue containing estradiol 3-pentafluorobenzyl ether was added 200 μL of a 2% 2-fluoro-1-methylpyridinium p-toluenesulfonate / dichloromethane solution, followed by 30 μL of triethylamine-dichloromethane (1: 9) at room temperature. For 1.5 hours. After the reaction, the solvent was distilled off under nitrogen, the residue was dissolved with 0.5 mL of methanol, diluted with 0.5 mL of purified water, and then the Bond Elut C18 cartridge conditioned in advance with 6 mL of methanol and 6 mL of purified water. Loaded. The column was washed with 1 mL of purified water, 4 mL of 0.3% ammonia, 3 mL of methanol, 3 mL of 0.1% formic acid-methanol (1: 1), and then 17β with 3 mL of 10% formic acid-acetonitrile (1: 9). - (1'-methyl-pyridinium-2 ') - oxy-3-pentafluoro-benzyloxy-1,3,5 (10) - was eluted triene _(E 2 -PFBZPY). The solvent was then distilled off under reduced pressure.

2-5. E obtained above 1-1 residue containing the resulting estrone 3-pentafluorobenzyl ether ₁ -PFBZHMP Preparation <br/> preceding 2-3, 1% 2-hydrazino-1-methylpyridine 1mg 140 μL of a solution dissolved in trifluoroacetic acid-acetonitrile (1: 1400) was added and left at room temperature for 1 hour. After the reaction, 50 μL of acetone was added to the reaction solution, and the solvent was distilled off under reduced pressure. The residue was dissolved in 0.5 mL of methanol, diluted with 0.5 mL of purified water, and loaded onto InertSepPharmaca previously conditioned with 3 mL of ethyl acetate, 6 mL of methanol, and 6 mL of purified water. Subsequently, after washing with 1 mL of purified water, 2 mL of 0.3% ammonia, 3 mL of acetonitrile-purified water (2: 3), E ₁ -PFBZHMP was dissolved in ethyl acetate-acetonitrile-0.05% formic acid (40: 9: 1) Elute with 3 mL. After distilling off the solvent under reduced pressure, 0.5 mL of 0.3% ammonia was added to the residue, followed by extraction with 3 mL of ethyl acetate, and the solvent was distilled off under reduced pressure. E ₂ -PFBZPY obtained in the preceding item 1-4 was dissolved in 115 μL of acetonitrile-methanol-0.1% formic acid (7: 7: 6), and E ₁ -PFBZHMP was further dissolved in the solution.

エストロンの検量線は、０．２５〜 ５００ｐｇの範囲において、エストラジオールの検量線は、０．１ 〜 ５００ ｐｇの範囲において、各々相関係数０．９９９以上と良好な直線性を示した。また、再現性試験結果を下記表３及び表４に示す。

エストロン及びエストラジオールのいずれも、同時並びに日差再現性ともに真度が８０〜 １２０％並びに精度が２０％以内と良好な再現性が得られた。 2-6. Measurement by LC-MS / MS Of the solution obtained in the previous section 2-5, 20 μL was used for the measurement. LC-MS / MS was measured by positive ion detection ESI using a material analyzer (ABSciex, API 5000) connected to a liquid chromatograph (Shimadzu Corporation, Prominence). The measurement conditions are shown in Table 2 below.

The calibration curve for estrone was in the range of 0.25 to 500 pg, and the calibration curve for estradiol was in the range of 0.1 to 500 pg, showing a good linearity with a correlation coefficient of 0.999 or more. The reproducibility test results are shown in Tables 3 and 4 below.

For both estrone and estradiol, good reproducibility was obtained with a precision of 80 to 120% and accuracy within 20% for both simultaneous and daily difference reproducibility.

Simultaneous determination of estrone and estradiol in human serum
3-1. Internal standard serum sample 0.25 mL obtained from human serum samples prepared <br/> human blood ^{(- -} 13 _{C 4} and estradiol estrone 13 _{C 4} each 100 pg / 50 [mu] L) was added and the 1mL with purified water Later, 5 mL of diethyl ether was added, and after shaking for 10 minutes, the aqueous layer was frozen and separated, and the resulting solvent was distilled off. Hereinafter, the calibration curve was prepared in the same manner as in the above 2-1, and the others were operated in the same manner as in the above 2-2 to 2-6 to quantify E ₁ -PFBZHMP and E ₂ -PFBZPY. The results are shown in Table 5 below.

Simultaneous determination of estrone and estradiol concentrations in human tissues
4-1. Preparation of human tissue sample The weighed tissue was pulverized in liquid nitrogen, and then 1 mL of purified water was added and homogenized with Ultra Turrax. Internal standard (estrone ^- 13 _{C 4} and estradiol ^- 13 _{C 4} each 100 pg / 50 [mu] L) was added to and 3 volumes of ethanol were separate homogenates tissue and shaken for 2 hours at 50 ° C.. Thereafter, the mixture was allowed to stand in a cold place for 2 hours, and then centrifuged at 4 ° C., 3000 rpm, and 10 minutes. The solvent of the obtained supernatant was distilled off under reduced pressure. The residue was dissolved in 0.25 mL of methanol, diluted with 1 mL of purified water, and loaded onto a Bond Elut C ₁₈ cartridge column previously conditioned with 6 mL of methanol and 6 mL of purified water. After sequentially washing with 1 mL of purified water and acetonitrile-purified water (3: 7), estrone and estradiol were eluted with acetonitrile-purified water (4: 1), and the solvent was distilled off under reduced pressure. The obtained residue was dissolved in 1 mL of methanol-purified water (1: 4), and preliminarily conditioned with 6 mL of methanol, 3 mL of purified water, 2 mL of 0.1 M sodium hydroxide solution, and 4 mL of purified water. Loaded on the column. After washing with 3 mL of methanol-purified water (1: 1) and 3 mL of methanol, estrogen was eluted with acetonitrile-purified water (4: 1), and the solvent was distilled off under reduced pressure. Thereafter, a calibration curve was prepared in the same manner as in 2-1, and the other operations were performed in the same manner as in 2-2 to 2-6 to quantify E ₁ -PFBZHMP and E ₂ -PFBZPY. The results are shown in Table 6 below.

Simultaneous determination of estrone and estradiol in human saliva
5-1. Internal standard in saliva samples 2 mL taken from Preparation <br/> human human saliva sample ^{(- -} 13 _{C 4} and estradiol estrone 13 _{C 4} each 100 pg / 50 [mu] L) was added, diethyl ether 5mL addition, vibration 10 minutes After that, it was centrifuged at 4 ° C., 3000 rpm, and 5 minutes. The aqueous layer was frozen and separated, and the resulting solvent was distilled off. Thereafter, a calibration curve was prepared in the same manner as in 2-1, and the other operations were performed in the same manner as in 2-2 to 2-6 to quantify E ₁ -PFBZHMP and E ₂ -PFBZPY. The results are shown in Table 7 below.

According to the measurement method of the present invention, it is possible to detect the presence or absence of estrogens, particularly estrones, and to measure the amount of estrogens in a sample. The present invention can be used in the fields of medicine, pharmacy, biochemistry, public health, food inspection, and the like.

Claims (7)

The following formula (1) (wherein R represents a lower alkyl group, X represents a halogen atom, and Y represents CH ₂ or CO).
A compound represented by

A method for measuring estrones in a sample by LC-MS,
1) a process of extracting steroids from a sample,
2) The following formula (2) for the extract obtained in 1) above

(Wherein X represents a halogen atom, Y represents CH ₂ or CO, and L represents a leaving group.)
A step of reacting a pentahalogenated benzyl compound or pentahalogenated benzoyl compound represented by
3) a step of reacting a pentahalogenated benzyl derivative or a pentahalogenated benzoyl derivative of estrone contained in the reaction product obtained in the above 2) with hydrazino-1-lower alkylpyridine under acidic conditions, and 4) the above 3 A step of measuring by LC-MS the pentahalogenated benzyl- or pentahalogenated benzoyl-1-lower alkylpyridinium hydrazone of estrone contained in the reaction product obtained in (1),
A measurement method comprising: The following steps:
1) After reacting a pentahalogenated benzyl compound or a pentahalogenated benzoyl compound under alkaline conditions, using a normal phase column, the pentahalogenated benzyl or pentahalogenated benzoyl derivative of an estradiol compound and the pentahalogenated benzyl of an estrone compound Or separating the pentahalogenated benzoyl derivative,
2) a step of reacting a pentahalogenated benzyl or pentahalogenated benzoyl derivative of the estradiol obtained in 1) above with a halogeno-1-lower alkylpyridine, and 3) a pentahalogenated benzyl or pentahalogenated estrone. When benzoyl-1-lower alkylpyridinium hydrazone is measured by LC-MS, the pentahalogenated benzyl- or pentahalogenated benzoyl-1-lower alkylpyridinium derivative of the estradiol obtained in 2) above is measured simultaneously. The process of
The measurement method according to claim 2, further comprising: The measurement method according to claim 2, further comprising a step of removing neutral steroid from the sample using a solid phase extraction column. The measurement method according to claim 2, wherein the hydrazino-1-lower alkylpyridine is 2-hydrazino-1-lower alkylpyridine. In the LC-MS measurement, the ionization is performed by positive ion detection electrospray ionization. The measurement method according to claim 2, wherein the sample is selected from the group consisting of blood, saliva, urine, tissue and living cells or waste water, river water, or pharmaceuticals of animals including humans.