KR20120059257A - Quantification of active androgens in urine and blood by isotope dilution-mass spectrometry - Google Patents

Abstract

PURPOSE: A method for quantitatively analyzing androgen in a biological specimen is provided to obtain data for evaluating alopecia medicine based on the concentration distribution of the androgen. CONSTITUTION: Androgen substituted with isotope as an internal standard material is added into a biological specimen. The androgen is extracted from the biological specimen using a copolymer absorbent with hydrophilic property and lipophilic property. The extracts is re-extracted using an organic solvent under pH 8 to 12. The re-extract is derivatized into flophemesyl-trimethylsilyl. The androgen is selectively detected by a gas chromatography-mass spectrometry. The biological specimen is urine or blood.

Description

Quantification of active androgen hormones in biological samples using isotope dilution-mass spectrometry {quantification of active androgens in urine and blood by isotope dilution-mass spectrometry}

The present invention relates to a method for rapid and accurate quantification of active male hormones in a biological sample.

Recently, as a metabolite study, researches on metabolic changes in various diseases and the relationship between diseases have been extensively conducted based on metabolic profile studies on metabolite changes in specific physiological and pathological states. If a small amount of urine and plasma are used to determine the concentration of steroid hormones, immunoassays that quantify using selective antigenic antibody responses are commonly used, but cross-response, inaccuracy, Problems such as limitations on linearity range, low reproducibility, etc. appear. Therefore, isotope dilution mass spectrometry is applied as a primary analysis method, and analytical methods for quantitative analysis along with gas chromatography with excellent separation are widely used. Clin. Biochem., 2009, 42: 484-90].

In general, the analysis of steroid hormones has limited analysis of steroid hormones related to a specific disease rather than simultaneously analyzing various types of steroid hormones in one sample pretreatment, and testosterone in the urine or blood of a patient with androgenetic alopecia compared to a normal person The increase in 5α-reductase activity, an enzyme that converts to dihydroxytestosterone in rats, has been experimentally demonstrated [CNS Drug Rev., 2006, 12: 53-76; J. Clin. Endocrinol. Metab., 1994, 79: 703 ?? 706. It has also been predicted that epitestosterone, a structural isomer of testosterone, can inhibit hair loss by acting as an anti-male hormone [J. Steroid Biochem., 1989, 33: 1019-1021; J. Invest. Dermatol., 2001, 116: 57-61. Therefore, it is very important to accurately measure the active male hormone including testosterone in urine and blood, and a technique for quantitative analysis at a low detection limit is required as a quick and reproducible selective method.

 Accordingly, the present inventors have conducted research and efforts to more quickly and accurately measure active male hormones. As a result, enzymatic hydrolysis of a biological sample in which isotopically substituted male hormones are added as an internal standard of a specific molar ratio, solid phase extraction process, After extracting the active male hormones through liquid-liquid extraction, derivatizing it with flophemesyl-trimethylsilyl and using gas chromatography-mass spectrometry, quantitative analysis of male hormones is possible. The present invention has been completed by discovering that it can.

Accordingly, an object of the present invention is to provide a method for directly and quantitatively evaluating a concentration of an active male hormone and an internal standard in a biological sample.

The present invention,

Adding an isotope-substituted male hormone as an internal standard to the biological sample;

Extracting androic hormone from the biological sample with a copolymer adsorbent having lipophilic and hydrophilicity;

Re-extracting the extract with an organic solvent at a pH of 8 to 12; And

Derivatizing the reextract with flophemesyl-trimethylsilyl and selectively detecting male hormone using a gas chromatography-mass spectrometer

Characterized in that the method for quantitative analysis of male hormones in a biological sample comprising a.

The present invention can effectively extract and quantify testosterone-containing active male hormones from biological samples such as urine, blood, etc. by adding an isotopically substituted internal standard, thereby determining the concentration and distribution of male hormones in biological samples. I can figure it out. Therefore, the present invention can be applied to provide data for the evaluation of hair loss patients and hair loss-related therapeutic agents through the distribution of male hormones.

1 is a chromatogram of a urine sample in which the molar ratio of testosterone and its internal standard quantitative ion is 1 or less as an example of an analyte.
2 is a chromatogram of a urine sample in which the molar ratio of testosterone and its internal standard quantitative ion is 1 as an example of an analyte.
3 is a chromatogram of a urine sample in which the molar ratio of testosterone and its internal standard quantitative ion is confirmed as one or more as an example of an analyte.
Figure 4 is a graph confirming the ratio of 5α-reductase activity in the sample of hair loss patients before and after hair loss treatment for one month.

The present invention will be described in detail as follows.

The present invention extracts active male hormones from a biological sample to which isotope-substituted male hormones as internal standards are extracted through a solid phase extraction process and a liquid-liquid extraction method, followed by flophemesyl-trimethylsilyl derivatives. The present invention relates to a method for quantitative analysis of male hormones using gas chromatography-mass spectrometry.

The male hormone that is the subject of quantitative analysis in the present invention may be selected from testosterone, dihydrotestosterone, and epitestosterone, but is not limited thereto.

As the biological sample, urine or blood may be used. First, an isotope-substituted male hormone is added to the biological sample in a specific molarity as an internal standard.

In this case, in the case of the urine sample, after the addition of an isotope-substituted male hormone as an internal standard, a hydrolyzate is obtained by adding an enzyme at pH 5-8 and 40-70 ° C. In this case, it is preferable to use β-glucuronidase in order to accurately evaluate steroid hormones having a specific form (3β-hydroxy-5-ene). Also, the amount of urine used is preferably 0.1 to 5 mL, more preferably 0.2 to 2 mL. If the amount of urine is less than 0.2 mL, there is a problem in the detection limit for analysis, and if it is larger than 5 mL, there is a problem in that a large amount of sampling is required.

In addition, in the case of blood samples, under a slightly acidic condition of pH 4-7, heat cultured to 40 ~ 70 ℃ to obtain a decomposition product, the amount of blood used is preferably 0.4 ~ 2 mL. If the amount of blood is less than 0.4 mL, there is a problem in the detection limit for the analysis of steroid hormones, and if it is larger than 2 mL, the level of interference by fatty compounds in the sample is high, and a large amount of blood must be collected. there is a problem.

Thereafter, the active male hormones are extracted using the solid phase extraction method using the copolymer as an adsorbent, respectively. Since liquid at pH 8 ~ 12 conditions with an organic solvent - by transplanting the liquid extraction, and selective re-extract the active androgen, at this time, the methyl-tertiary-butyl ether (methyl- tert -butyl ether, MTBE), ethyl acetate ( It is preferable to use one or two or more mixed solvents selected from ethylacetate) and hexane (n-hexane) in terms of extraction efficiency, and more preferably to use methyl tert-butyl ether at pH 9-11. good.

Flows that do not react with enol-ethers and sterically hindered hydroxyl groups to facilitate detection via the gas chromatography-mass spectrometer and for effective separation from steroid compounds other than targets Derivatization with flophemesyl chloride. Thereafter, the mixture is evaporated to dryness and trimethylsilyl derivatization is performed to form a flophemesyl-trimethylsilyl derivative. The selectivity on the chromatogram is excellent compared to the case where the flopemethyl-trimethylsilyl derivatization is carried out as compared with a derivative such as trimethylsilyl, alkyl halide or alkyl anhydride.

Thereafter, the derivative is analyzed using a gas chromatography-mass spectrometer as an analyte, and the concentration of the analyte can be simply quantified by comparing the area ratio of the analyte and the internal standard in the chromatogram.

In particular, the present invention does not use a quantitative curve that requires samples of various concentrations, and adds an isotopically-substituted substance of the compound to a specific molarity as an internal standard, and then obtains an analyte and an internal standard. By comparing the area ratio of the chromatograms and confirming the molar ratio of the two substances, it is possible to quickly and accurately measure the concentration of testosterone, an analyte.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited to the following examples.

Example: Analysis of male hormones in a biological sample

1) Addition of Isotope Standards

In order to quantify male hormones in urine samples, the internal standard of three male hormones isotopically substituted with three active male hormones including testosterone (d ₃ -testosterone, 1 μg / mL; d _3- 40 μl of a mixed solution of epitestosterone, 1 μg / mL; d ₃ -dihydrotestosterone, 0.5 μg / mL) was added (Test Tube 1).

On the other hand, for the quantification of male hormones in blood samples, the internal standard of three male hormones in which three active male hormones including isosterone are substituted in vitro (d ₃ -testosterone, 100 ng / mL; d ₃ 40 μl of a mixed solution of -epitestosterone, 20 ng / mL; d ₃ -dihydrotestosterone, 50 ng / mL) was added (Test Tube 2).

2) Hydrolysis of Urine Samples

2 mL of the urine sample was added to the test tube 1, 1 mL of 0.2 M phosphate buffer (pH 7.2) was added to adjust the pH to 6.4 to 6.6, and 50 μl of glucuronidase (β-glucuronidase). ) Was further added and hydrolyzed at 55 ° C. for 1 hour.

3) Hydrolysis of Blood Samples

After putting 0.4 mL of the blood sample in Test Tube 2, 2.6 mL of 0.2 M sodium acetate buffer (pH 5.2) was added to adjust the pH to 5.7 to 6.0, and hydrolyzed at 50 ° C. for 15 minutes.

4) Solid phase extraction

The solid phase extraction method uses an Oasis HLB cartridge [Oasis HLB ^™ , 60 mg, Waters, Co., Milford, MA, USA]. Methanol and 2 mL of distilled water were flowed into the cartridge, respectively, and the decomposition products of 2) and 3) were flowed into each cartridge. To remove impurities, 2 mL of distilled water was poured, followed by 4 mL of methanol to elute the decomposition products of 2) and 3) adsorbed to the cartridge, and the eluate was received in a clean test tube. The eluate received in the test tube was evaporated through a rotary evaporator at 40 ℃.

5) Liquid-Liquid Extraction

Add 1 mL of 0.2 M phosphate buffer and 0.5 mL of 5% potassium carbonate (K ₂ CO ₃ ) solution to the evaporated test tube of 4) to adjust the pH to 10.0, and use MTBE (methyl-tert-butyl ether). Extracted by. The eluate was evaporated using a Turbo Vap LV nitrogen evaporator at 40 ° C. and then dried again in a vacuum desiccator for 30 minutes or more using P ₂ O ₅ / KOH.

6) Gas Chromatography-Mass Spectrometry

Increase the volatility of the sample for easy detection through gas chromatography-mass spectrometry, selectively derivatize only certain substituents for separation from other steroid compounds, and reduce noise compared to other derivatization methods In order to proceed with flophemesyl-trimethylsilyl derivatization, derivatization was performed as follows.

First, 50 μl of flofemesyl chloride solution was added to the dried product of 5) and reacted at room temperature for 15 minutes. Then, to increase the volatility of the solution, 1 mL of n -hexane was added to evaporate at 70 ° C., and P ₂ It was sufficiently dried in a vacuum desiccator for 30 minutes using O ₅ / KOH. 40 μl of a mixed solution of MSTFA (N-methyl-N-trifluorotrimethylsilyl acetamide) / NH ₄ I (ammonium iodide) / dithioerythritol (500: 4: 2, v / w / w) was added to the dried test tube. The analytes were derivatized with flophemesyl-trimethylsilyl.

For the analysis of male hormones, Agilent 6890 series Gas Chromatograph connected to a mass spectrometer (Agilent's 5975 Mass Selective Detector) was used. ) And scan mode.

The male hormones were separated using Agilent Ultra-1 as a fused-silica capillary column. The length of the column was 25 m, the inner diameter was 0.2 mm, and the film thickness was 0.33 μm. The flow rate of the carrier gas helium was 1.0 mL / min. Sample injection was performed using a split mode of 8: 1, and the temperature conditions for analysis were as follows. The temperature of the inlet was 280 ° C. and the temperature was raised to 320 ° C. at a rate of 8 ° C./min and maintained for 6 minutes. The temperature of the detector was 280 ° C and the temperature of the ion source was 230 ° C. The ionization method was applied to the electron impact method (electron impact ionization, EI), the electron energy used was 70 eV.

7) Determination of active male hormone

The retention time in the quantitative ion and column in the selective ion detection method used for the quantitative analysis of three active male hormones is shown in Table 1 below, and the area ratio of the analyte and the isotopically substituted internal standard is shown. In comparison, the male hormone in the sample was quantified.

Steroid hormones sign Fixed ion Retention time
(minute) Male Hormone Testosterone T 584 5.49 Dihydrotestosterone DHT 586 5.34 Epitestosterone ET 584 4.93 Standard material d ₃ -testosterone d ₃ -T 587 5.48 d ₃ -dihydrotestosterone d ₃ -DHT 589 5.33 d ₃ -epitestosterone d ₃ -ET 587 4.92

The chromatogram of a sample whose molar ratio of testosterone and its internal standard in the urine sample was found to be 1 or less is shown in FIG. 1, and the chromatogram of the sample whose molar ratio is 1 is shown in FIG. The chromatogram of the sample confirmed above is shown in FIG.

Through the above procedure, the concentrations of the active male hormones of testosterone, dihydrotestosterone and epitestosterone were measured using an internal standard, and the molar ratio of quantitative ions to the corresponding peaks of the analyte was measured without using a separate quantitative correction curve. The concentration could be measured quickly.

Test Example 1

The concentrations of three male hormones in urine and blood were measured from 130 patients with male-pattern baldness (MPB) and 14 patients with female-patten baldness (FPB) through the analysis method of the above example. The results are shown in Tables 2 and 3 below.

Analyte Male blood
(ng / mL, mean ± SD) Male urine
(ng / mL, mean ± SD) Testosterone 4.94 ± 1.47 15.89 ± 21.92 Epitestosterone 0.37 ± 0.39 29.57 ± 21.73 Dihydrotestosterone 0.48 ± 0.39 5.03 ± 5.55 5α-reductase activity ^a 0.10 ± 0.11 1.01 ± 2.12  a: metabolite / precursor (testosterone / dihydrotestosterone, mean ± SD)

Analyte Female blood
(ng / mL, mean ± SD) Women's urine
(ng / mL, mean ± SD) Testosterone 0.64 ± 0.33 1.31 ± 1.93 Epitestosterone 0.45 ± 0.60 9.77 ± 10.54 Dihydrotestosterone 0.26 ± 0.16 1.27 ± 0.87 5α-reductase activity ^a 0.41 ± 0.17 1.73 ± 1.97  a: metabolite / precursor (testosterone / dihydrotestosterone, mean ± SD)

The concentrations detected in the samples of male baldness patients were testosterone 4.94 ± 1.47 ng / mL, epitestosterone 0.37 ± 0.39 ng / mL and dihydrotestosterone 0.48 ± 0.39 ng / mL for blood samples, and testosterone for urine samples. 15.89 ± 21.92 ng / mL, epitestosterone 29.57 ± 21.73 ng / mL, dihydrotestosterone 5.03 ± 5.55 ng / mL. In addition, the 5α-reductase activity of the precursor (testosterone) to the product (dihydrotestosterone) (5α-reductase activity) was 0.10 ± 0.11, 1.01 ± 2.12 in blood and urine samples, respectively.

Blood samples showed testosterone 0.64 ± 0.33 ng / mL, epitestosterone 0.37 ± 0.39 ng / mL, and dihydrotestosterone 0.48 ± 0.39 ng / mL in the sample of female alopecia, and testosterone 1.31 ± 1.93 ng in the urine sample. / mL, epitestosterone 9.77 ± 10.54 ng / mL, dihydrotestosterone 1.27 ± 0.87 ng / mL. In addition, 5α-reductase activity was found to be 0.41 ± 0.17 and 1.73 ± 1.97 in blood and urine samples, respectively.

Test Example  2

Blood and urine samples collected on the first visit to a clinic for 23 hair loss patients using the analytical method of the above example, and 5α-reductase activity through blood and urine samples collected after 1 month of hair growth treatment It was possible to evaluate the effect of hair growth treatment by comparing the results are shown in FIG.

Claims (7)

Adding an isotope-substituted male hormone as an internal standard to the biological sample;
Extracting androic hormone from the biological sample with a copolymer adsorbent having lipophilic and hydrophilicity;
Re-extracting the extract with an organic solvent at a pH of 8 to 12; And
Derivatizing the reextract with flophemesyl-trimethylsilyl and selectively detecting male hormone using a gas chromatography-mass spectrometer
Method for quantitative analysis of male hormones in a biological sample, characterized in that consisting of.
The method of claim 1, wherein the biological sample is urine or blood.
The method of claim 2, wherein after the addition of the isotope-substituted male hormone as an internal standard to the urine sample, the quantification of the male hormone in the biological sample, characterized in that the hydrolysis at pH 5 ~ 8 and 40 ~ 70 ℃ Analytical Method.
[Claim 3] The quantitative analysis of male hormone in a biological sample according to claim 2, wherein the blood sample is added with an isotopically substituted male hormone as an internal standard, and then cultured at pH 4-7 and 40-70 ° C. Way.
According to claim 1, wherein the organic solvent is a male in a biological sample, characterized in that selected from methyl tert -butyl ether (MTBE), ethyl acetate (ethylacetate) and hexane (n-hexane) Method of quantitative analysis of hormones.
The method of claim 1, wherein the male hormone is testosterone, dihydrotestosterone or epitestosterone, characterized in that the quantitative analysis of male hormone in a biological sample.
The method according to any one of claims 1 to 6, characterized in that to compare the area ratio of the analyte and the internal standard in the chromatogram obtained using a gas chromatography-mass spectrometer. Quantitative Analysis Method.

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