JP3774888B2 - Highly sensitive detection method for steroid compounds by LC-MS - Google Patents

Description

【００２５】
質量分析計で測定したときのN-メチルピリジニウム誘導体のESIマススペクトルは、m/z382がインタクトモレキュラカチオンとして検出された（図１）。またm/z382をプレカーサーイオンとしたときのMS/MS測定を行うと、m/z255にプロダクトイオンが強い強度で検出される。MS/MS手法により測定することで一層特異性を増し、生体由来の夾雑物による妨害から逃れることが出来る。そこでm/z382をプレカーサーイオンとしたときのプロダクトイオンm/z255をモニターするselected reaction monitoring(SRM)法で測定したところ、5pgのジヒドロテストステロンが検出できた（図２）。内部標準物質には安定同位体で標識した17-18O,17-d1,19-d3ジヒドロテストステロンを用いた（N.Watanabeら: J.Mass Spectrom.Soc.Jpn.,45,367(1997)）。内部標準物質は5(10)-エストレン-3,17-ジオンを原料とし馬場らの方法に従い（S.Baba,ら:J.Labelled Com. Radiopharmaceuticals,14,783(1978)）、6工程でまず19位のメチル基に重水素標識した4-アンドロステン-3,17-ジオンを合成した。さらに17α位に重水素、17位に重酸素を標識したテストステロンに導き、接触還元でテストステロンからジヒドロテストステロンを合成した。このときに還元された5位の水素はαとβの異性体が生成した。生体中のジヒドロテストステロンはα体であるためHPLCで分離し、α体のピーク面積比を求めて、ジヒドロテストステロンの濃度を算出した。
【００２６】
この分析条件下で得られたジヒドロテストステロンの検量線を図３に示した。検量線はジヒドロテストステロン5〜100pgの濃度範囲で相関係数0.9997の良好な直線を得た。
試料中のN-メチルピリジニウム誘導体のピーク面積と内部標準物質とのピーク面積比を求めてジヒドロテストステロンの濃度を算出した。
患者Aの前立腺9.3mg中のジヒドロテストステロン量は36.4pgであった。患者Bの前立腺10.1mg中には50.8pg存在した。さらに血漿では、患者Cの血漿0.25ml中には80.7pg、患者Dの血漿0.25ml中には68.3pgのジヒドロテストステロンが検出された。健常人男性の0.1mlの血漿E、Fではそれぞれ42.7、41.3pgであった。これらの前立腺と血漿中ジヒドロテストステロンを定量した結果を表２に示した。
【００２７】
表2 前立腺及び血漿中ジヒドロテストステロン

【００２８】
誘導体化の反応は室温で１時間以内と簡便で迅速である。生成した誘導体は極めて安定である。この方法の定量限界は5pgであり、極少量の生体試料中の微量のジヒドロテストステロン量を定量できることから、数mgの生体試料があれば、ジヒドロテストステロン量を高感度に定量することが可能となった。
【００２９】
【発明の効果】
極少量の生体試料中ジヒドロテストステロンを高感度に測定するために、N-メチルピリジニウム誘導体化を行い、LC/MS法で測定する方法を発明した。本法を用いてヒト前立腺及び血漿中のジヒドロテストステロンを高感度に測定することができた。また、本発明は、極めて少量の生体試料を用いて、薬物による疾病の治療効果や病態の評価、更にはタンパク質同化ステロイドなどの不正使用の検査を行うために有効な手法である。本発明により、生体試料中の1個の水酸基を有するステロイド類化合物を検出するうえで高感度に測定できる有用な方法を提供できる。
【図面の簡単な説明】
【図１】ジヒドロテストステロンをN-メチルピリジニウム誘導体としたときのマススペクトルを図１に示す。
【図２】ジヒドロテストステロン5pgをN-メチルピリジニウム誘導体にしたときのSRMを図２に示す。
【図３】ジヒドロテストステロンの検量線を図３に示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a detection method for measuring by LC / MS (liquid chromatography-mass spectrometry) for detecting a steroid compound having one hydroxyl group contained in a trace amount in a biological sample. The present invention is an effective technique for evaluating the therapeutic effect and pathological condition of a disease caused by a drug using a very small amount of a biological sample, and further for examining illegal use of an anabolic steroid or the like.
[0002]
[Prior art]
Steroid compounds are widely distributed in plants and animals, and are distributed in various organs in the body as important substances constituting living organisms. Most organisms have a function of biosynthesizing steroids, and therefore there are many intermediates and metabolites. In addition to its function as a component of biological membranes that are important for living organisms, steroids exhibit special physiological actions in small amounts, especially as hormones, and control in vivo, and their physiological functions are diverse (Shusuke Tsuda: Steroids, 1971, Asakura Shoten; Tamahiko Teruhiko: Understanding sex steroid hormones, 1999, Kinyoshido).
[0003]
For example, androgens have physiological effects such as genital development, vascularization, hair growth, and body volume increase by muscles and skeleton, and also have an inhibitory effect on the estrogen action, which is a female hormone. Androgen is converted into estradiol and the like which exerts estrogen action, and is also involved in estrogen action. A typical testosterone among androgens is converted into an active form of dihydrotestosterone and exhibits physiological action. Androgens, especially dihydrotestosterone (androstan-17β-ol-3-one), are said to be closely related to prostatic hypertrophy and prostate tumors, and their concentration measurements are used to evaluate the therapeutic effects and pathological conditions of diseases. In addition, dihydrotestosterone is listed as one of the prohibited substances in the inspection of illegal use of anabolic steroids in sports competitions.
[0004]
Hitherto, high separation and high sensitivity have been required for measuring steroid compounds having many similar structures in living bodies. For example, the following three methods were used for dihydrotestosterone measurement.
In the GC / MS (Gas Chromatography-Mass Spectrometry) method, measurement was performed as an oxime derivative in order to impart volatility to dihydrotestosterone, and the quantification limit at that time was 50 pg / ml. This derivatization reaction requires heating, and has the disadvantage that isomers are formed by derivatization.
In the LC / MS (liquid chromatography-mass spectrometry) method, carboxymethyl oxime derivatives are used for measurement in order to increase sensitivity. The limit of quantification at that time was 62.5 pg (Kotaro Gotanda, Kaoru Shinbo, Yoichi Nakano, Keiko Sasaki, Seijiro Honma, Katsuhiko Miyasaka: Clinical and new drugs, 36, 277 (1999)).
The radioimmunoassay method has a high sensitivity of 12.5 pg, but it requires time and labor to produce antibodies, and in order to avoid cross-reactivity with testosterone, pretreatment excluding testosterone by HPLC is necessary. (V. Puri et al: J. Steroid Biochem., 14, 877-881 (1981), M. Hill, R. Hampl, R. Petrik and L. Starka: Prostate, 28, 347 (1996)).
[0005]
[Problems to be solved by the invention]
Testing with a small amount of biological sample can reduce the burden on the subject. Particularly in the medical field, it is necessary to be able to detect with a small amount of biopsy sample and blood in order to be able to know the pathological condition of the target part and to be useful for the treatment method and policy.
For example, dihydrotestosterone, one of the androgens, is converted from testosterone to an active form by 5-α reductase. Dihydrotestosterone binds to the androgen receptor and develops an androgenic action. Knowing the abundance of dihydrotestosterone in the target tissue, which shows a stronger androgenic action in a minute amount, can consider a more accurate androgenic action. Dihydrotestosterone is only a few ng per gram of tissue in the prostate. The plasma concentration is 0.3 to 0.6 ng / ml in healthy adult men and 0.08 to 0.2 ng / ml in women (Hosaka Masahiko: Japanese Society of Infertility, 21, 135, (1976)). Further, in order to examine whether or not the anabolic steroid is used illegally, it is necessary to detect a trace amount remaining in blood or urine. Thus, a highly sensitive method is required to detect dihydrotestosterone that is present in a very small amount in the living body.
[0006]
[Means for Solving the Problems]
As a result of intensive studies, the present inventors have introduced a derivative of LC / ESI-MS (liquid) by introducing an N-lower alkylpyridinium group into a hydroxyl group in order to detect a steroid compound having one hydroxyl group in a biological sample. It was found that high-sensitivity detection is expected by measuring by a chromatography / electrospray ionization-mass spectrometry method. Furthermore, a method for extracting and purifying trace amounts of steroidal compounds from a small amount of biological sample has been devised. That is, a solid phase extraction method was employed to remove excess derivative reagents and biological contaminants. Specifically, after loading the N-lower alkylpyridinium derivative onto the solid phase, impurities were completely removed using methanol, and then the N-methylpyridinium derivative was eluted from the solid phase. After pretreatment with solid phase, steroid compounds are detected by LC / ESI-MS method. In addition, by using the MS / MS (MS / MS) method in particular, selectivity has been improved and background noise from the HPLC mobile phase and MS equipment has been improved to improve the signal-to-noise ratio (signal-to-noise ratio). High sensitivity detection is possible.
[0007]
That is, the present invention relates to the following (1) to (8).
(1) A detection method in which a derivative in which an N-lower alkylpyridinium group is introduced into a hydroxyl group of the steroid compound is measured by LC / MS in detecting a steroid compound having one hydroxyl group in a biological sample.
(2) The detection method according to item 1, wherein the N-lower alkylpyridinium group is an N-methylpyridinium group.
(3) The detection method according to 1 or 2 above, wherein the steroid compound having one hydroxyl group is a compound selected from any of an androgen compound, an estrogen compound, a progesterone compound, and a sterol compound.
(4) The detection method according to 1 or 2 above, wherein the steroid compound having one hydroxyl group is an androgen compound.
(5) The detection method according to item 4, wherein the androgenic compound is dihydrotestosterone.
(6) After generating a derivative at the hydroxyl group of the steroid compound, pretreating with methanol washing by solid phase extraction method, and then eluting with methanol containing acid to obtain a sample for LC / MS measurement The detection method of crab.
(7) The detection method according to any one of 1 to 6, wherein in the LC / MS measurement, ionization of mass spectrometry is performed by electrospray ionization.
(8) The detection method according to any one of 1 to 7 above, wherein in the LC / MS measurement, ions are performed by an MS / MS method.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
The present invention will be described in more detail.
In the present invention, the target biological sample includes tissue, blood, urine, bile and the like obtained from a human, and these can be collected from a healthy person or a patient.
In the present invention, a steroid compound having one hydroxyl group refers to a compound in which one hydroxyl group is substituted for a compound having a steroid skeleton. In the present invention, a substance derived from a living body is particularly preferable, and examples thereof include sterol compounds, progesterone compounds, androgen compounds, and estrogen compounds.
When the present invention is applied to steroids having two or more hydroxyl groups, the resulting derivative is a multivalent ion having a plurality of quaternary ammonium ions. The molecular ion appearing on the mass spectrum (m / z value) is a value shifted to the low mass side, where a large amount of impurities are present, which is disadvantageous for high sensitivity analysis. Therefore, the present invention is preferably applied to steroids having one hydroxyl group.
[0009]
Examples of the sterol compound include lanosterol, dihydrolanosterol, timosterol, 4α-methyl cholesterol, desmosterol, 7-dehydrocholesterol, latosterol, metostenol, cholesterol and the like.
Examples of the progesterone-based compound include pregnenolone, 11-deoxycorticosterone, 17α-hydroxypregnenedione, 17α-hydroxyprogesterone, 20α-hydroxyprogesterone, and the like.
[0010]
Examples of androgenic compounds include 11-ketoandrosterone, 11-ketoetiocholanolone, testosterone, androsterone, epiandrosterone, ethiocolanolone, dehydroepiandrosterone, 11-hydroxyandrostenedione, 7-ketodehydroepiandrosterone. Androstenen-3α-ol, dihydrotestosterone and the like.
Examples of the estrogen compound include estrone.
In the present invention, the steroid compound having one hydroxyl group is preferably an androgenic compound, particularly preferably dihydrotestosterone.
These steroidal compounds having one hydroxyl group used in the present invention are well-known substances and can be appropriately synthesized by known methods or available as reagents.
[0011]
In the present invention, the N-lower alkylpyridinium group refers to a group in which C1-C4 alkyl is substituted at the N-position of pyridine. Examples of the C1-C4 alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, t-butyl and the like. In the present invention, an N-methylpyridinium group substituted with methyl is preferred. The introduction of the N-lower alkylpyridinium group into the hydroxyl group is prepared, for example, with a 2-fluoro-1-methylpyridinium reagent (manufactured by Aldrich).
[0012]
The solid phase extraction method used as a pretreatment method is, for example, by adsorbing to an ODS type solid phase such as Bond Elut C18 (manufactured by Varian), washing with methanol and eluting with methanol containing acid. Biological impurities can be effectively removed.
Furthermore, in LC / MS measurements, mass spectrometry ionization is performed by selecting atmospheric pressure chemical ionization (APCI), fast atom bombardment (FAB), thermospray ionization (TSP), and electrospray ionization (ESI) as appropriate. Is called. ESI improves the ionization efficiency by using polar derivatives, and does not require high heat compared to other ionizations such as APCI and TSP. In addition, FAB has poor ionization efficiency and high sensitivity cannot be obtained. Therefore, in the present invention, it is preferable to use electrospray ionization (ESI).
In the present invention, the MS / MS technique is a method of detecting precursor ions obtained by MS1 (here, intact molecular cations), decomposing the precursor ions with argon gas in MS2, and detecting the resulting product ions. Selectivity increases the signal-to-noise ratio and increases sensitivity.
[0013]
Next, a method for detecting a steroid compound in an actual biological sample will be described. The conditions for preparation, extraction, and measurement may be appropriately changed depending on the properties / collection amount of each biological sample and the physicochemical properties of each steroid compound.
A. Biological samples to be collected and stored are used by collecting tissues, blood, urine, bile and the like from healthy persons or patients. Blood is subjected to heparin treatment immediately after blood collection and separated into blood cells and plasma fractions by high-speed centrifugation to obtain plasma. Store plasma immediately at around -80 ° C. Other specimens should be stored at about -80 ° C immediately after biopsy at the time of surgery or directly after collection.
[0014]
B. Extraction of steroid compounds from living tissue To extract steroid compounds in a very small amount of living tissue of about 5 to 10 mg efficiently in an alkaline solution containing methanol at 70-80 ° C for 1 hour. Prepare. After diluting the lysate with water, Bond Elut C18 and Abselut Nexus (Varian), Oasis (Waters), Superclin (Spelco), Empore Disc (3M), Isoroute (International Solvent) Technology Corp.), etc., wash with water, and elute steroid compounds with ethyl acetate. The solvent is distilled off to obtain a derivative sample.
[0015]
C. Extraction of steroid compounds from plasma Prepare 0.05-0.5 ml of plasma by adding about 10 times the amount of water and diluting, and adding a small amount of methanol and alkali to mix. This is loaded onto Bond Elut C18, etc., washed with water, and then the steroid compound is eluted with ethyl acetate. Evaporate the solvent, dissolve in hexane / ethyl acetate (10: 1), and apply to Bond Elut Silica. After washing with about hexane / ethyl acetate (10: 1), the steroid compound is appropriately eluted with a hexane / ethyl acetate mixture. After distilling off the solvent, a derivative sample is obtained.
[0016]
D. Derivative production and purification method Samples for derivatives extracted and purified from tissues and plasma were dissolved in a small amount of dichloromethane, chloroform, tetrahydrofuran, acetonitrile, etc., and then derivatized reagents (2-fluoro-1-methylpyridinium reagent (Aldrich) dissolved in acetonitrile. Etc.), triethylamine is further added, and the mixture is stirred at room temperature for 1 hour. Then, after distilling off the solvent, water is added to dissolve the reaction product. After loading on Bond Elut C18, wash with water and methanol sequentially. N-methylpyridinylated steroids are eluted with a methanol solution containing 2-10% acid such as formic acid, acetic acid, TFA (trifluoroacetic acid).
[0017]
E. Analytical detection by LC / ESI-MS / MS method uses electrospray ionization (ESI) that can measure N-lower alkylpyridinium derivatives with high sensitivity, or mass spectrometer by ionization of FAB, APCI, TSP. preferable. An on-line concentrated semi-micro HPLC system is used for HPLC (High Performance Liquid Chromatography). After injecting almost the entire amount of the sample, the sample may be concentrated using a concentration column and separated from impurities using an analytical column by column switching.
[0018]
For measurement with a mass spectrometer, the intact molecular cation can be detected from the mass spectrum of each derivative. In addition, for example, when MS / MS measurement is performed using intact molecular cations as precursor ions, product ions are detected. If this ion is measured, it becomes a more specific measurement method, and interference by contaminants derived from living organisms. You can escape.
In addition, it is necessary to add an internal standard substance for quantification. In this case, a compound similar to the steroid compound to be measured may be used, but preferably a stable isotope such as a heavy water label of the steroid compound to be measured. It is preferable to use a labeling compound. These internal standard substances are added when preparing a biological sample.
[0019]
【Example】
Next, as a working example, the method for detecting dihydrotestosterone (androstan-17β-ol-3-one) in the biological tissue of the present invention will be described in detail. Note that the present invention is not limited to these examples.
(1) Collection and storage of biological samples Prostate samples were immediately placed in sample tubes after biopsy and stored at -80 ° C until measurement. The blood was treated with heparin immediately after blood collection and separated into blood cells and plasma fractions by high speed centrifugation to obtain plasma. Plasma was immediately stored at -80 ° C.
[0020]
(2) Extraction of dihydrotestosterone from living tissue Weigh approximately 10 mg of living tissue, add a fixed amount of 50 μl of internal standard substance dissolved in acetonitrile, and heat in an alkaline solution containing methanol for 1 hour at 70-80 ° C. Dissolve. The lysate is diluted with water, loaded onto Bond Elut C18, washed with water, and dihydrotestosterone is eluted with ethyl acetate. The solvent is distilled off to obtain a derivative sample.
[0021]
(3) Extraction of dihydrotestosterone from plasma To 0.1 to 0.25 ml of plasma, add a certain amount of internal standard substance, add 10 times the amount of water, add a small amount of methanol and alkali, and mix. This is loaded onto Bond Elut C18, washed with water, and dihydrotestosterone is eluted with ethyl acetate. Evaporate the solvent, dissolve in hexane / ethyl acetate (10: 1) and load onto Bond Elut Silica. After washing with hexane / ethyl acetate (10: 1), dihydrotestosterone is eluted with hexane / ethyl acetate (6: 1). After distilling off the solvent, a derivative sample is obtained.
[0022]
(4) Derivative production and purification method The derivative sample extracted and purified from tissues and plasma was dissolved in a small amount of dichloromethane, and then 2-fluoro-1-methylpyridinium reagent (manufactured by Aldrich) dissolved in acetonitrile was added, and further triethylamine was added. And a derivative is formed by stirring for 1 hour at room temperature. Then, after distilling off the solvent, water is added to dissolve the reaction product. After loading on Bond Elut C18, wash sequentially with 50% methanol solution containing 0.3% aqueous ammonia, water, methanol, and 0.1% formic acid. N-methylpyridinylated dihydrotestosterone is eluted with a methanol solution containing 10% formic acid.
[0023]
(5) The sample obtained by evaporating the analytical solvent by the LC / ESI-MS / MS method was redissolved in 0.1 ml of the mobile phase of HPLC for concentration, and almost the entire amount was injected. The sample was allowed to flow through the concentration column for 4 minutes, and then a column switch was performed to switch the flow path to the analysis column and introduce it into the mass spectrometer. For ionization, an electrospray ionization (ESI) method was used. The analysis conditions are shown in Table 1.
[0024]
[Table 1]

[0025]
In the ESI mass spectrum of the N-methylpyridinium derivative as measured with a mass spectrometer, m / z382 was detected as an intact molecular cation (FIG. 1). In addition, when MS / MS measurement is performed using m / z 382 as a precursor ion, a product ion is detected at a strong intensity at m / z 255. By measuring by MS / MS method, the specificity can be further increased, and it is possible to escape from interference by biological contaminants. Therefore, when measured by the selected reaction monitoring (SRM) method that monitors the product ion m / z255 when m / z382 is used as a precursor ion, 5 pg of dihydrotestosterone was detected (FIG. 2). As an internal standard, 17-18O, 17-d1,19-d3 dihydrotestosterone labeled with a stable isotope was used (N. Watanabe et al .: J. Mass Spectrom. Soc. Jpn., 45, 367 (1997)). The internal standard is 5 (10) -estrene-3,17-dione as a raw material according to the method of Baba et al. (S. Baba, et al .: J. Labelled Com. Radiopharmaceuticals, 14,783 (1978)), first 19th in 6 steps 4-androstene-3,17-dione was synthesized with deuterium on the methyl group. Furthermore, dihydrotestosterone was synthesized from testosterone by catalytic reduction by deuterium labeled at 17α position and deuterium labeled at 17 position. The reduced 5-position hydrogen produced α and β isomers. Since dihydrotestosterone in the living body is α-form, it was separated by HPLC, and the peak area ratio of α-form was determined to calculate the concentration of dihydrotestosterone.
[0026]
A calibration curve of dihydrotestosterone obtained under these analytical conditions is shown in FIG. The calibration curve was a good straight line with a correlation coefficient of 0.9997 in the concentration range of dihydrotestosterone from 5 to 100 pg.
The concentration of dihydrotestosterone was calculated by determining the peak area ratio between the peak area of the N-methylpyridinium derivative in the sample and the internal standard substance.
The amount of dihydrotestosterone in patient A's prostate 9.3 mg was 36.4 pg. There was 50.8 pg in the 10.1 mg prostate of patient B. In plasma, 80.7 pg of dihydrotestosterone was detected in 0.25 ml of patient C's plasma and 68.3 pg of dihydrotestosterone in 0.25 ml of patient D's plasma. It was 42.7 and 41.3 pg in 0.1 ml of plasma E and F in healthy men, respectively. The results of quantifying dihydrotestosterone in the prostate and plasma are shown in Table 2.
[0027]
Table 2 Dihydrotestosterone in prostate and plasma

[0028]
The derivatization reaction is simple and rapid, within 1 hour at room temperature. The resulting derivative is extremely stable. The limit of quantification of this method is 5 pg, and the amount of dihydrotestosterone in a very small amount of biological sample can be quantified. Therefore, if there is a biological sample of several mg, the amount of dihydrotestosterone can be quantified with high sensitivity. It was.
[0029]
【The invention's effect】
In order to measure dihydrotestosterone in a very small amount of biological sample with high sensitivity, N-methylpyridinium derivatization was performed and a method for measuring by LC / MS was invented. Using this method, dihydrotestosterone in human prostate and plasma could be measured with high sensitivity. In addition, the present invention is an effective technique for evaluating the therapeutic effect and pathological condition of a disease caused by a drug using a very small amount of a biological sample, and further for examining illegal use of an anabolic steroid or the like. The present invention can provide a useful method that can be measured with high sensitivity in detecting a steroid compound having one hydroxyl group in a biological sample.
[Brief description of the drawings]
FIG. 1 shows a mass spectrum when dihydrotestosterone is an N-methylpyridinium derivative.
FIG. 2 shows the SRM when dihydrotestosterone 5 pg is converted to an N-methylpyridinium derivative.
FIG. 3 shows a calibration curve of dihydrotestosterone.

Claims (8)

A detection method in which a derivative obtained by introducing an N-lower alkylpyridinium group into a hydroxyl group of the steroid compound is measured by LC / MS in detecting a steroid compound having one hydroxyl group in a biological sample. The detection method according to claim 1, wherein the N-lower alkylpyridinium group is an N-methylpyridinium group. The detection method according to claim 1 or 2, wherein the steroid compound having one hydroxyl group is a compound selected from any of an androgen compound, an estrogen compound, a progesterone compound, and a sterol compound. The detection method according to claim 1 or 2, wherein the steroid compound having one hydroxyl group is an androgen compound. The detection method according to claim 4, wherein the androgenic compound is dihydrotestosterone. A derivative is generated at a hydroxyl group of a steroid compound, pretreated by washing with methanol by a solid phase extraction method, and then eluted with methanol containing an acid to obtain a sample for LC / MS measurement. The described detection method. The detection method according to claim 1, wherein in the LC / MS measurement, ionization of mass spectrometry is performed by electrospray ionization (ESI). In the LC / MS measurement, the detection method according to any one of claims 1 to 7, wherein ions are performed by an MS / MS technique.

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