JP6041125B2 - Ultrasensitive measurement method of estrogen - Google Patents

Description

  The present invention relates to a method for measuring estrogen contained in a sample with high sensitivity using a liquid chromatograph-mass spectrometer (LC-MS). The present invention is an effective technique for detecting and quantifying a very small amount of estrogen contained in a sample.

  Estrogens are also commonly referred to as follicular hormones or female hormones, and are used in various other diseases such as breast cancer, endometrial cancer, digestive organ cancer, cardiovascular disorders, congenital diseases of sex hormone metabolism, precocious puberty, osteoporosis It is believed that there is a close relationship. Monitoring the amount is useful for elucidating diseases and determining pharmacological effects. Estra-1,3,5 (10) -triene-3,17β-diol (estradiol: E2), which has the strongest action, is weakly affected by 17β-hydroxysteroid dehydrogenase type 2 (17βHSD2). Converted to 3,5 (10) -trien-3-ol-17-one (estrone: E1), E1 is reactivated to E2 by 17β-hydroxysteroid dehydrogenase type 1 (17βHSD1) . Circulating E2 concentration in adult premenopausal women who are not pregnant is about 1.5 to 4 times higher than E1, but E1 concentration is higher in men and postmenopausal women. Therefore, by monitoring not only E2 but also E1 at the same time, it becomes possible to grasp the state of metabolism and disease more accurately.

  In order to closely examine the relationship between the amount of E1 and E2 in the sample and the disease state, it is required to accurately measure a small amount of E1 and E2. However, in samples that are difficult to collect in large quantities, such as needle biopsy tissues, blood of newborns, and children, the amounts of E1 and E2 contained in the sample that can be used for measurement are naturally reduced. In the blood of rats and mice often used in animal experiments, male E1 and E2 are remarkably small. Moreover, in the case of humans, saliva has been increasingly used as a sample that can be collected non-invasively and easily, but the hormone contained in saliva is 1/20 to 1/100 of the hormone contained in serum, E1 and E2 contained in saliva of men and postmenopausal women are very small. The amount of E1 and E2 contained in these samples is expected to be less than 0.1 pg per analytical sample. Therefore, in order to accurately measure E1 and E2 in a sample, it is a problem to develop a measurement method having a lower limit of quantification lower than 0.1 pg / tube.

  Conventionally, immunoassays have been the mainstream for measuring estrogen. In the sample, structurally similar compounds such as E1 and E2 and sex hormone-binding globulin coexist, so that the measured value is likely to fluctuate between kits due to an increase in background due to cross-reaction. At present, a kit based on a highly sensitive enzyme immunoassay with a lower limit of quantification of 4.8 pg / mL is available on E1. In E2, in electrochemiluminescence measurement, the lower limit of quantification is generally 5 pg to 15 pg / mL, and a highly sensitive RIA measurement kit with a detection sensitivity of 1.4 pg / mL is also on the market. However, in the measurement by the immunization method, the variation becomes larger as the concentration is lower, so the reliability of the measurement is lowered at 10 pg / mL or less.

  A method of measuring with a gas chromatograph-mass spectrometer (GC-MS) has also been proposed. In GC-MS, a volatile reagent is generally reacted with estrogen in a sample, and a derivative thereof is measured by GC-MS (Non-patent Documents 1 and 2). However, since these derivatives are unstable, it is difficult to purify them as derivatives, and they are easily affected by impurities and reagents in the data during measurement. Further, in the measurement by GC-MS, the amount of sample injection is reduced to 5 to 10% or less of the total sample, and the loss increases. For this reason, sufficient sensitivity cannot be obtained, and a long time is required for analysis. Furthermore, GC-MS also has a drawback that its dynamic range is narrower than that of LC-MS. Therefore, it is difficult to measure E1 and E2 in a small amount of sample.

  In addition, there are some reports of detection by GC-MS by reacting a phenolic hydroxyl group of a compound with perfluoroaryl ether (Patent Documents 3 to 6), but these documents contain detailed descriptions of E1 and E2. Not. There is also a report in which pentafluoropyridine is reacted with the phenolic hydroxyl group of E2 in human male urine on a solid phase column and the tetrafluoropyridine derivative of the reaction product is measured by GC-MS (Non-patent Document 7). However, the measurement range is 1 to 40 ng / mL, and the sensitivity is insufficient to measure E2 in a low concentration sample.

  Attempts to derivatize and measure estrogen have also been made in 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 electrospray ionization (ESI) method. (Non-patent document 8). However, this method has a drawback that a product ion derived from a compound cannot be obtained and lacks specificity.

  A method for measuring the carbonyl group of estrone by derivatization with p-toluenesulfonyl hydrazide has also been reported (Non-patent Document 9). In this method, estrone can be measured from 10 pg / on column, but the sensitivity is insufficient to measure blood E1 in children and postmenopausal women.

  In E2 measurement, a method is also reported in which a phenolic hydroxyl group is converted to a pentafluorobenzyl derivative and then the 17-position hydroxyl group is converted to 1-lower alkyl-2-pyridinium and measured by LC-MS (Patent Document 1). In addition, in E1 measurement, a method has been reported in which a phenolic hydroxyl group is converted to a pentafluorobenzyl derivative, and then a 17-position carbonyl group is converted to hydrazino-1 lower alkylpyridine and measured by LC-MS (Patent Document 2). The measurement sensitivity of Patent Document 1 is 0.1 pg / tube, and the measurement sensitivity of Patent Document 2 is 0.25 pg / tube, both of which are highly sensitive. Insufficient sensitivity to measure E1 and E2 in male serum.

Japanese Patent No. 4667832 JP 2011-174710 A

Steroids, 57, 319-324 (1992) Endocr. J. et al. , 50, 783-792 (2003) J. et al. Org. Chem. , 32, 3163-3168 (1967) J. et al. Chem. Soc. Chem. Commun. 125-127 (1984) J. et al. Chromatogr. A, 984, 237-243 (2003) J. et al. Chromatogr. A, 1042, 1-7 (2004) J. et al. Chromatogr. A, 1218, 9135-9141 (2011) J. et al. Pharm. Biomed. Anal. , 54, 830-837 (2011) Anal. Chem. , 76, 5829-5836 (2004)

  An object of the present invention is to provide a method for detecting and quantifying an extremely small amount of estrogen present in a sample with high sensitivity using LC-MS.

  As a result of intensive studies, the present inventors have developed a method capable of highly sensitively measuring estrogen in a very small amount of a sample that is smaller than 0.1 pg / tube by reviewing the reagents and purification methods used for derivatization, Overcame the problem. In order to measure a small amount of estrogen, the present inventors first selectively pentafluoropyridine a phenolic hydroxyl group under alkaline conditions, and then derivatize various carbonyl groups and alcoholic hydroxyl groups of the reaction product. The measurement sensitivity of the derivatives was investigated. As a result, it was found that the 1-lower alkylpyridinium hydrazone formation of the carbonyl group and the alcoholic hydroxyl group can be measured with good sensitivity in LC-MS by derivatizing with a pyridinecarboxylic acid ester, thereby completing the present invention.

  In the present invention, pentafluoropyridine is introduced into the phenolic hydroxyl group of estrogen in a sample, and then a 1-lower alkylhydrazinopyridinium salt (hereinafter referred to as hydrazino-1 lower alkylpyridine) is added to the carbonyl group of the reaction product. And / or reacting pyridinecarboxylic acid with the alcoholic hydroxyl group of the reactant and measuring by LC-MS.

（式中、Ｒは水素原子、ハロゲン原子、Ｃ１−４アルキル基、Ｃ１−４アルコキシ基、アリルＣ１−４アルコキシ基、Ｃ１−４アルキルアミノ基、ジ（Ｃ１−４アルキル）アミノ基、ニトロ基又はシアノ基を表し、ＣＯＸはカルボン酸又はその反応性官能基を表す。）
を含む測定方法に関する。That is, the present invention is a method for measuring estrogen by LC-MS,
1. 1. reacting the compound present in the sample with pentafluoropyridine under alkaline conditions; 2. a step of reacting hydrazino-1-lower alkylpyridine with a carbonyl group of the tetrafluoropyridyl ether derivative, which is the reaction product of 1 above, under acidic conditions; A step of reacting the alcoholic hydroxyl group of the tetrafluoropyridyl ether derivative obtained in 1 with a compound represented by the following formula (I):

(In the formula, R is a hydrogen atom, a halogen atom, a C1-4 alkyl group, a C1-4 alkoxy group, an allyl C1-4 alkoxy group, a C1-4 alkylamino group, a di (C1-4 alkyl) amino group, a nitro group. Or represents a cyano group, and COX represents a carboxylic acid or a reactive functional group thereof.)
It is related with the measuring method containing.

  The estrogen measurement method of the present invention is a safe and simple operation, and has an advantage that estrogen in a sample, for example, E1 can be detected from 0.05 pg / tube and E2 can be detected from 0.01 pg / tube. In the present invention, since both estrogen having a carbonyl group and estrogen having a hydroxyl group can be processed and measured by a series of operations, E1 and E2 can be highly sensitive even with a small amount of sample. Can be measured.

FIG. 1 is a mass spectrum of an E1-3-tetrafluoropyridyl ether-17- (1′-methylpyridinium-2 ′)-hydrazone derivative. FIG. 2 is a mass spectrum of an E2-3-tetrafluoropyridyl ether-17-fusaric acid derivative. FIG. 3 is a calibration curve of 0.05 to 100 pg / tube of E1-3-tetrafluoropyridyl ether-17- (1′-methylpyridinium-2 ′)-hydrazone derivative. FIG. 4 is a calibration curve of 0.01 to 100 pg / tube of the E2-3-tetrafluoropyridyl ether-17-fusaric acid derivative.

  In the present invention, “estrogen” means a steroid compound having a phenolic hydroxyl group. For example, estrone, estradiol, 2-hydroxyestrone, 4-hydroxyestrone, 2-hydroxyestrone 3-methyl ether, 4-hydroxyestrone 3-methyl ether, 2-methoxyestrone, 4-methoxyestrone, 16α-hydroxyestrone, 16 Examples thereof include estrogens existing in vivo or in nature such as oxoestradiol, 2-hydroxyestradiol, and 4-hydroxyestradiol.

  In the present specification, the “solid phase extraction column” represents a general cartridge column, and a commercially available one can be used in the present invention. Examples of the separation mode of the filler include normal phase, reverse phase, distribution, ion exchange, molecular sieving, affinity, etc. Among them, reverse phase, normal phase and ion exchange are preferable.

  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. It is preferable to use (LC-MS / MS). Examples of ionization in LC-MS include atmospheric pressure chemical ionization, ESI, and atmospheric pressure photoionization. Among these, positive ion detection ESI is preferably used.

  Examples of the mass spectrometer include a magnetic field type, a quadrupole type, a time-of-flight type, etc., but it is preferable to use a quadrupole type that has good quantitativeness, a wide dynamic range, and good linearity. .

  Furthermore, the detection of ions in the quantification is performed by selecting one of the ion species purified by the selected ion monitoring or the first mass analysis unit, which selectively detects only the target ion, as a precursor ion. For example, selective reaction monitoring (SRM), which detects product ions generated by cleavage of the precursor ions in the first mass spectrometer, can be mentioned. 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, etc. are preferable as environmental samples, and pharmaceutical products are preferable as samples derived from industrial products.

In the present invention, “pentafluoropyridine” represents a compound represented by the following formula (II) having CAS registration number 700-16-3.

  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, n-butyl, 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 them, the reactivity is excellent. Derivatization using 2-hydrazino-1-methylpyridine with good sensitivity is preferred. This hydrazino-1-lower alkylpyridine is also a known compound or can be synthesized from a known compound by a known method.

で示される化合物におけるＣＯＸは、カルボン酸又はカルボン酸の反応性官能基を表す場合が好ましい。「カルボン酸」としては、ピコリン酸、ニコチン酸、イソニコチン酸、６−メチルピコリン酸、フザリン酸、キナルジン酸等が挙げられるが、この中でも、特にピコリン酸、フザリン酸が好ましい。「反応性官能基」としては、例えば、酸ハライド、酸無水物、混合酸無水物、活性アミド、活性エステル等を挙げることができる。この中、ＣＯＸが酸ハライドを表す場合が好ましく、特にＣＯＸが酸クロライドを表す場合が好適である。Furthermore, the following formula (I) that can be used in the present invention:

COX in the compound represented by is preferably a carboxylic acid or a reactive functional group of a carboxylic acid. Examples of the “carboxylic acid” include picolinic acid, nicotinic acid, isonicotinic acid, 6-methylpicolinic acid, fusaric acid, quinaldic acid, etc. Among them, picolinic acid and fusaric acid are particularly preferable. Examples of the “reactive functional group” include acid halides, acid anhydrides, mixed acid anhydrides, active amides, and active esters. Among these, the case where COX represents an acid halide is preferable, and the case where COX represents an acid chloride is particularly preferable.

In the compound represented by the above formula (I), R represents a halogen atom, a C _1-4 alkyl group, a C _1-4 alkoxy group, an allyl C _1-4 alkoxy group or a di (C _1-4 alkyl) amino group. The case where it represents is preferable, and especially the case where R represents a _C1-4 alkyl group is preferable.

  Furthermore, in the measurement method of the present invention, the substitution position of —COX in the compound represented by the formula (I) is not particularly limited, but is preferably the 2-position of the pyridine ring.

  Most of the compounds of formula (I) are known, and even if they are new, they can be easily synthesized from known compounds by known methods.

  The measurement method of the present invention will be specifically described below.

1. Preparation of hydrazino-1-lower alkylpyridine A method for preparing hydrazino-1-lower alkylpyridine is disclosed in JP 2010-36810. Briefly, hydrazine and 2-halogeno-1-lower alkylpyridine are disclosed. It can be obtained by dissolving a general commercial synthetic raw material such as pyridinium p-toluenesulfonate in an inert solvent such as acetonitrile. This reaction can be carried out in the range of −10 ° C. to 50 ° C., but it is desirable to carry out 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. Preparation of samples Preparation of estrogens from samples obtained from serum, plasma, waste water, saliva, urine, cultured cells and tissue homogenates of animals including humans is performed by extraction with organic solvents or simple column chromatography using solid phase extraction. A general preparation method for separation and purification can be appropriately selected and used. As the organic solvent, commercially available organic solvents such as ethyl acetate, acetonitrile, diethyl ether and the like can be used. In addition, it is also necessary to selectively separate only estrogen using a commonly used solid-phase extraction column, for example, Oasis (registered trademark) MAX manufactured by Waters, or adane: lPAX (registered trademark) manufactured by Shiseido. Can be done according to. It should be noted that the conditions for adjustment and extraction may be appropriately changed based on the properties of each sample, the amount collected, and the like.

3. Preparation of pentafluoropyridine derivative The sample prepared above is dissolved in an inert solvent such as acetonitrile and reacted with pentafluoropyridine under alkaline conditions to convert the estrogen in the sample prepared above into a pentafluoropyridine derivative. . This reaction can be carried out at a temperature within the range of -10 ° C to 80 ° C, preferably a temperature within the range of 5 ° C to 40 ° C. The proportion of pentafluoropyridine used is not particularly limited, but can generally be 0.01 to 0.5 mL per test tube, and preferably 0.025 to 0.075 mL. Furthermore, the alkaline condition means that the pH of the reaction solution is in the range of 10 to 14, preferably 12 to 14, such as potassium hydroxide, sodium hydroxide, potassium carbonate, triethylamine, triethanolamine, diethylamine, etc. From these bases, the kind and amount sufficient to bring the reaction solution into the above pH range can be appropriately selected and used.

4.1 Preparation of 1-lower alkylpyridinium hydrazone Among the elastogen pentafluoropyridine derivatives prepared by the above method 3, the estrogen carbonyl group having an estrone or other carbonyl group is dissolved in an inert solvent such as acetonitrile. It can be converted to 1-lower alkylpyridinium hydrazone under acidic conditions such as trifluoroacetic acid.

  This reaction can be carried out at a temperature in the range of −10 ° C. to 60 ° C., preferably a temperature in the range of 15 ° C. to 40 ° C. is suitable. As the reaction solvent, a general inert solvent such as chloroform, dichloromethane, acetonitrile, ethyl acetate, tetrahydrofuran and the like can be used. Further, the use ratio of hydrazino-1 lower alkylpyridine to the pentafluoropyridine derivative of estrogen having a carbonyl group is not particularly limited, but can generally be 0.1 μg to 500 μg per test tube, preferably 1 μg to 50 μg is suitable.

（式中、Ｒは水素原子、ハロゲン原子、Ｃ１−４アルキル基、Ｃ１−４アルコキシ基、アリルＣ１−４アルコキシ基、Ｃ１−４アルキルアミノ基、ジ（Ｃ１−４アルキル）アミノ基、ニトロ基又はシアノ基を表し、Ｘはヒドロキシ基又は脱離基を表す。）
で示される化合物を反応させ、ピリジンカルボン酸エステル誘導体にすることで検出感度が上昇する。 5. Preparation of Pyridine Carboxylate Derivative Of the estrogen pentafluoropyridine derivatives prepared by the method 3 above, the alcoholic hydroxyl group of estrogen having other alcoholic hydroxyl groups is represented by the following formula (I):

(In the formula, R is a hydrogen atom, a halogen atom, a C1-4 alkyl group, a C1-4 alkoxy group, an allyl C1-4 alkoxy group, a C1-4 alkylamino group, a di (C1-4 alkyl) amino group, a nitro group. Or a cyano group, and X represents a hydroxy group or a leaving group.)
The detection sensitivity is increased by reacting the compound represented by the formula (1) with a pyridinecarboxylic acid ester derivative.

  This reaction is carried out by using an inert solvent such as acetonitrile, for example, ethers such as dioxane, tetrahydrofuran and dimethoxyethane, aromatic hydrocarbons such as benzene, toluene and xylene, amides such as dimethylformamide and dimethylacetamide, acetonitrile, dimethyl The reaction can be carried out in the presence of a suitable base such as triethylamine or pyridine in a state dissolved or suspended in an inert organic solvent such as sulfoxide. The reaction temperature can be usually within the range of −5 ° C. to 80 ° C., and preferably within the range of 10 ° C. to 60 ° C. The reaction time can usually be a time within a range of 5 to 240 minutes, and preferably a time within a range of 10 to 90 minutes is suitable.

  For separation and purification from the above reaction and other reagents and the like after the reaction in the present invention, a general solid-phase extraction column such as Oasis WCX (registered trademark, Waters), InertSep Pharma (registered trademark, GL Sciences), Bond Elut C18 (registered trademark, 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.

Reproducibility of simultaneous determination of estrone and estradiol
1-1. Preparation of calibration curve and QC sample As calibration curve samples, methanol solutions of E1 and E2 (0.05, 0.1, 0.25, 1, 10, 100 pg / 50 μL, 0.01,. (05, 0.1, 1, 10, 100 pg / 50 μL) was added and made up to 1 mL with purified water. As a QC (Quality Control) sample, 50 μL of a methanol solution of estrone and estradiol (0.05, 0.075, 0.1 pg / 50 μL, 0.01, 0.02, 0.03 pg / 50 μL, respectively) was added to a test tube. Add to 1 mL with purified water.
Subsequently, the internal standard (E1- ¹³ C ₄ and E2- ¹³ C ₄ 10 pg / 50 μL each) and 3 mL of ethyl acetate were added, and after shaking for 5 minutes, the aqueous layer was freeze-separated and the solvent obtained was distilled off. After the residue was dissolved in 0.5 mL of methanol, 1 mL of purified water was added and loaded onto an Oasis (registered trademark) MAX cartridge (Waters) previously conditioned with 3 mL of methanol and 3 mL of purified water. 1 mL of 1% acetic acid, 1 mL of 30% acetonitrile, 1 mL of 1M sodium hydroxide, 3 mL of methanol, 1 mL of 1% acetic acid, 1 mL of pyridine / 60% methanol (1: 100), and then 1 mL of pyridine / 90% methanol (1: 100) E1 and E2 were eluted with, and the eluate was distilled off with a centrifugal evaporator.

2-2. Preparation of E1-3-tetrafluoropyridyl ether and E2-3-tetrafluoropyridyl ether To the residue containing E1 and E2 obtained in the preceding section 1-1, pentafluoropyridine 0.05 mL, acetonitrile 0.1 mL, 1 M water Sodium oxide 0.04 mL and ethanol 0.015 mL were added, shaken well, and allowed to stand at room temperature for 20 minutes. After the reaction, the reaction reagent was distilled off under reduced pressure, 0.5 mL of purified water and 2 mL of hexane were added, and after shaking for 5 minutes, the aqueous layer was freeze-separated and the solvent obtained was distilled off under reduced pressure.

2-3. Preparation of E1-3-tetrafluoropyridyl ether-17- (1′-methylpyridinium- 2 ′) -hydrazone derivative E1-3-tetrafluoropyridyl ether and E2-3-tetrafluoropyridyl obtained in 2-2 above 0.14 mL of 1% 2-hydrazino-1-methylpyridine dissolved in 1% trifluoroacetic acid-acetonitrile (1: 1400) was added to the residue containing ether and allowed to stand at room temperature for 1 hour. A method for producing 2-hydrazino-1-methylpyridine is described in JP 2010-36810 A, and is therefore omitted. After the reaction, the solvent was distilled off under reduced pressure. The residue was dissolved in 0.5 mL of methanol, 0.5 mL of purified water was added, and loaded onto an Oasis WCX cartridge that had been conditioned in advance with 3 mL of methanol and 3 mL of purified water. The cartridge column was washed with 1 mL of 0.1 M potassium dihydrogen phosphate and 1 mL of 40% acetonitrile, and then E2-3-tetrafluoropyridyl ether was eluted with 1 mL of methanol, and the solvent was distilled off under reduced pressure. Next, the cartridge column was washed with 2 mL of acetonitrile, 0.5 mL of purified water, and 1 mL of formic acid / 65% methanol (1:50), and then E1-3-tetrafluoropyridyl ether-17- with 1 mL of formic acid / methanol (1:50). The (1′-methylpyridinium-2 ′)-hydrazone derivative (E1-TfpyHMP) was eluted. The solvent was distilled off from the eluate under reduced pressure. The residue was dissolved in 0.1 mL of 70% acetonitrile to prepare an E1-TfpyHMP solution.

2-4. Preparation of E2-3-tetrafluoropyridyl ether-17-fusaric acid derivative The residue containing E2-3-tetrafluoropyridyl ether obtained in 2-3 above was dried under reduced pressure for 30 minutes or more and then derivatized. Reagents (40 mg of fusaric acid, 40 mg of 2-methyl-6nitrobenzoic anhydride, 20 mg of 4-dimethylaminopyridine / 1 mL of acetonitrile) and 0.01 mL of triethylamine were added and left at room temperature for 30 minutes. After the reaction, 0.5 mL of hexane / acetic acid (50: 1) was added to the reaction solution, shaken well, and then loaded onto an InertSep (registered trademark) SI cartridge previously conditioned with 3 mL of ethyl acetate and 3 mL of hexane. The cartridge column was washed with 1 mL of hexane and 2 mL of ethyl acetate / hexane (15:85), and then E2-3-tetrafluoropyridyl ether-17-fusaric acid derivative (E2-TfpyFU) with ethyl acetate / hexane (45:55). ) And the eluate was distilled off under reduced pressure. The residue was dissolved in 0.1 mL of 80% acetonitrile to prepare an E2-TfpyFU solution.

2-5. LC-MS measurement of E1-TfpyHMP 0.02 mL of the E1-TfpyHMP solution obtained in 2-3 above was injected into the LC-MS for measurement. In the ESI mass spectrum when E1-TfpyHMP was measured with a mass spectrometer, an intact proton-added ion was detected at m / z 525 (FIG. 1). Further, as a result of performing MS / MS measurement using m / z 525 as a precursor ion, m / z 308 was the product ion with the highest specificity. Therefore, SRM measurement was performed using these ions. The measurement conditions are shown in Table 1 below.

2-6. LC-MS measurement of E2-TfpyFU 0.02-mL of the E2-TfpyFU solution obtained in 2-4 above was injected into LC-MS for measurement. In the ESI mass spectrum when E2-TfpyFU was measured with a mass spectrometer, an intact protonated ion was detected at m / z 583 (FIG. 2). Further, as a result of performing MS / MS measurement using m / z 583 as a precursor ion, m / z 308 was the product ion having the highest specificity. Therefore, SRM measurement was performed using these ions. The measurement conditions are shown in Table 2 below.

  The results of the reproducibility test of E1 obtained in the above 2-5 and 2-6 are shown in Table 3 below, and the results of the reproducibility test of E2 are shown in Table 4.

  From the results of Tables 3 and 4 above, it was shown that E1 can be determined from a concentration of 0.05 pg / tube and E2 can be determined from a concentration of 0.01 pg / tube using the method of the present invention.

  In the same manner as in Example 1, quantification of E1 and E2 in the same serum was performed 5 times to confirm reproducibility. The results are shown in Tables 5 and 6 below.

  From the results of Tables 5 and 6, it was shown that E1 and E2 in serum can be quantified with good reproducibility.

  The present invention can separate a very small amount of estrogen contained in a sample and quantify it with very high sensitivity. The reactions are both simple and rapid, with a reaction time of 10 to 60 minutes at room temperature. The resulting derivative is extremely stable. By using the present invention, it is possible to detect estrogen even from a very small amount of sample or a sample containing very little estrogen contained in postmenopausal women and children. The present invention is a very effective means for the evaluation of disease treatment effects and pathological conditions caused by drugs.

Claims (6)

A method for measuring estrogen by LC-MS, comprising:
1. 1. reacting the compound present in the sample with pentafluoropyridine under alkaline conditions; A step of reacting the carbonyl group of the tetrafluoropyridyl ether derivative, which is the reactant of 1 above, with hydrazino-1-lower alkylpyridine under acidic conditions, and / or
3. In the alcoholic hydroxyl group of the tetrafluoropyridyl ether derivative which is the reactant of the above 1,
Formula (I)

(In the formula, R is a hydrogen atom, a halogen atom, a _C1-4 alkyl group, a _C1-4 alkoxy group, an allyl _C1-4 alkoxy group, a _C1-4 alkylamino group, di ( _C1-4 alkyl). (Amino group, nitro group or cyano group is represented, and COX represents a carboxylic acid or a reactive functional group thereof.)
A step of reacting a compound represented by:
4). A step of measuring the compound obtained in the steps 2 and / or 3 using LC-MS,
Measuring method including The measuring method according to claim 1, wherein the estrogen is estrone and estradiol. The measuring method of any one of Claim 1 or 2 whose hydrazino-1 lower alkyl pyridine is 2-hydrazino 1-lower alkyl pyridine. The measuring method according to claim 1, wherein R represents a hydrogen atom or a C _1-4 alkyl group. The measuring method according to any one of claims 1 to 4, wherein the substitution position of COX is the 2-position of the pyridine ring. The measurement method according to claim 1, wherein the LC-MS is LC-MS / MS.

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