CN113552264A - High performance liquid chromatography tandem mass spectrometry detection method for 6 estrogens and metabolites thereof in human urine - Google Patents
High performance liquid chromatography tandem mass spectrometry detection method for 6 estrogens and metabolites thereof in human urine Download PDFInfo
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Abstract
The invention discloses a high performance liquid chromatography tandem mass spectrometry detection method for 6 estrogens and metabolites thereof in human urine, wherein the 6 estrogens and the metabolites thereof are estrone, 2-hydroxyestrone, 4-hydroxyestrone, 16 alpha-hydroxyestrone, 2-methoxyestrone and 4-methoxyestrone respectively, a pretreated human urine sample is detected by adopting a high performance liquid chromatography tandem mass spectrometry technology, the 6 estrogens and the metabolites thereof in the sample are separated by using high performance liquid chromatography, then a mass spectrometry isotope internal standard quantitation method is used, the concentration ratio of a standard substance and an internal standard substance is taken as an X axis, the peak area ratio of the standard substance and the internal standard substance is taken as a Y axis, a calibration curve is established, and the contents of the 6 estrogens and the metabolites thereof are calculated. The detection method of the invention takes human urine as a detection sample, and has the advantages of no wound, strong specificity, high sensitivity, high detection speed, high accuracy, good repeatability, simple pretreatment and no need of derivatization.
Description
Technical Field
The invention relates to the technical field of urine detection, in particular to a high performance liquid chromatography-tandem mass spectrometry detection method for 6 estrogens and metabolites thereof in human urine.
Background
The incidence of breast cancer is rising year by year, and accounts for the first place of female malignant tumors. Numerous studies have shown that the occurrence of breast cancer is closely related to the levels of estrogen and its metabolic abnormalities in the patient. The natural estrogen includes estrone (E1), estradiol (E2) and estriol (E3), and under the mediation of cytochrome enzyme P450, the C-2, C-4 and C-16 sites of estrogen are hydroxylated, and the main products are metabolites occurring in A-ring, namely 2-hydroxyestrone (2-OHE1) and 4-hydroxyestrone (4-OHE1), and a metabolite occurring in D-ring, namely 16 alpha-hydroxyestrone (16 alpha-OHE 1). 2-OHE1 and 4-OHE1 were methylated by catechol-o-methyltransferase to produce further 2-methoxyestrone (2-MeOE1) and 4-methoxyestrone (4-MeOE 1). Among them, the content of 2-OHE1 shows a balanced metabolic reaction, which can indicate whether the estrogen metabolism proceeds toward a more beneficial direction to the body. 2-MeOE1 has anti-cell proliferative effects, the production of 4-MeOE1 is considered to be an important detoxification pathway and therefore is a gastrointestinal beneficial metabolite, while 4-OHE1 and 16 α -OHE1 bind to estrogen receptors, have estrogenic effects and potential genotoxicity and are referred to as adverse metabolites. Thus, the levels of estrogen and its metabolites may be predictive of the risk of developing breast cancer.
However, the content of estrogen components in human bodies is very small and the structure is similar, so that the traditional analysis method is difficult to meet the detection requirement. Moreover, some of the detection methods use blood samples as the research objects, the sampling process will cause trauma to the patient, and the preliminary treatment is complicated. Therefore, there is a need to develop a detection method that can rapidly and accurately analyze the levels of estrogen and its metabolites in vivo, and is non-invasive to the patient.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a high performance liquid chromatography-tandem mass spectrometry detection method for 6 estrogens and metabolites thereof in human urine.
In order to realize the purpose, the invention adopts the technical scheme that:
a high performance liquid chromatography tandem mass spectrometry detection method for 6 estrogens and metabolites thereof in human urine, wherein the 6 estrogens and metabolites thereof are estrone, 2-hydroxyestrone, 4-hydroxyestrone, 16 alpha-hydroxyestrone, 2-methoxyestrone and 4-methoxyestrone respectively;
the detection method comprises the steps of detecting a pretreated human urine sample by adopting a high performance liquid chromatography tandem mass spectrometry technology, separating the 6 estrogens and metabolites thereof in the sample by utilizing the high performance liquid chromatography, then establishing a calibration curve by utilizing a mass spectrum isotope internal standard quantitative method, taking the concentration ratio of a standard substance to an internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and calculating the contents of the 6 estrogens and metabolites thereof, wherein the specific chromatographic conditions are as follows:
(1) high performance liquid chromatography conditions:
mobile phase A: 0.5mM ammonium fluoride in water;
mobile phase B: acetonitrile;
the type of the chromatographic column: CORTECS C182.1mm × 50mm, 1.6 μm;
the gradient elution mode was used, with the parameters given in the following table:
| Time | flow rate (mL/min) | ％A | ％B |
| 0.00 | 0.300 | 60 | 40 |
| 2.00 | 0.300 | 60 | 40 |
| 2.10 | 0.300 | 55 | 45 |
| 2.50 | 0.300 | 45 | 55 |
| 3.00 | 0.300 | 40 | 60 |
| 3.30 | 0.300 | 10 | 90 |
| 4.00 | 0.300 | 10 | 90 |
| 5.50 | 0.300 | 60 | 40 |
(2) Mass spectrum conditions:
adopting a mass spectrum scanning mode of MRM multi-reaction monitoring under an electrospray ionization negative ion mode;
capillary voltage: 3.00 kV;
taper hole voltage: 40V;
taper hole air flow rate: 150L/hr;
desolventizing gas temperature and flow rate: 630 deg.C, 1100L/hr;
atomizing: 7.0 bar;
collision gas: 4;
the mass spectrum parameters of the 6 estrogens and their metabolites are shown in the following table:
wherein the pretreated human urine sample is obtained according to the following method: centrifuging collected urine, putting 500 mu L of supernatant into a sample tube, adding 10 mu L of mixed internal standard working solution with the concentration of 100ng/mL, uniformly mixing by vortex, adding 0.5mL of enzymolysis solution, uniformly mixing by vortex, carrying out enzymatic hydrolysis at 37 ℃ for 16h, then carrying out reversed-phase medium solid-phase extraction and purification, and re-dissolving after drying by nitrogen.
The mixed internal standard working solution is obtained according to the following method: accurately weighing 1mg of 13C 3-estrone, 13C 6-2-hydroxyestrone and 3 isotope internal standard products of 13C, d 3-2-methoxyestrone respectively, placing the internal standard products into 1mL volumetric flasks respectively, adding methanol solutions containing 0.1% ascorbic acid respectively to prepare internal standard mother solutions with the concentrations of 1000 mu g/mL respectively, transferring appropriate solutions from the internal standard mother solutions respectively, and diluting the solutions with methanol to the concentration of 100ng/mL to obtain the mixed internal standard working solution.
The standard substance is obtained according to the following method: accurately weighing 1mg of each of estrone, 2-hydroxyestrone, 4-hydroxyestrone, 16 alpha-hydroxyestrone, 2-methoxyestrone and 4-methoxyestrone, adding a methanol solution containing 0.1% ascorbic acid to prepare standard mother solutions with the concentrations of 1000 mug/mL respectively, transferring a proper amount of solution from the standard mother solutions respectively, fixing the volume by using the methanol solution containing 0.1% ascorbic acid to obtain a mixed standard solution with the concentration of 10 mug/mL, and diluting the mixed standard solution with deionized water step by step into a mixed standard working solution with the concentrations of 100ng/mL, 10ng/mL and 1 ng/mL; diluting the mixed standard working solution with deionized water or PBS respectively to prepare standard curve calibration points of 0.002ng/mL, 0.005ng/mL, 0.02ng/mL, 0.05ng/mL, 0.2ng/mL, 0.5ng/mL, 2ng/mL, 5ng/mL and 20 ng/mL; respectively transferring 500 mu L of the standard curve calibration point solution, respectively adding 10 mu L of mixed internal standard working solution with the concentration of 100ng/mL, uniformly mixing in a vortex mode, then adding 0.5mL of enzymolysis solution, uniformly mixing in a vortex mode, carrying out enzymatic hydrolysis at 37 ℃ for 16h, then carrying out reversed phase medium solid phase extraction and purification, and re-dissolving after drying by nitrogen.
In the detection method, the adopted enzymolysis solution is obtained according to the following method: 250mg of ascorbic acid was weighed, 0.5mL of β -glucuronidase/arylsulfatase (β -glucuronidase/sulfatase) was added, and then 50mL of sodium acetate solution with a concentration of 0.15M, pH ═ 4.6 was added to the solution.
The method for the solid phase extraction and purification of the reversed phase medium comprises the following steps: the hydrolyzed treated sample was added to 3mL methanol and 3mL aqueous activated SPE cartridges, and 2 column volumes of methanol eluate were collected in a new 96-well collection plate.
The temperature for blowing the nitrogen is 60 ℃.
The redissolved solvent is a mixed solution of methanol, acetonitrile and water, and the volume ratio of the methanol to the acetonitrile to the water is 4:36: 60.
The invention has the beneficial effects that: the detection method provided by the invention takes human urine as a detection sample, is noninvasive, can complete separation and detection of 6 trace estrogens, namely 2-hydroxyestrone, 4-hydroxyestrone, 16 alpha-hydroxyestrone, 2-methoxyestrone and 4-methoxyestrone, and metabolites thereof within 5.5min, and has the advantages of strong specificity, high sensitivity, high detection speed, high accuracy, good repeatability, high flux, simple pretreatment and capability of meeting the requirements of batch quantitative analysis of endogenous estrogens and metabolites thereof in human urine without derivatization.
Drawings
FIG. 1 is a line graph of the chromatogram, signal-to-noise ratio and calibration curve at calibration point 0.005ng/mL of the standard curve for Compound E1;
FIG. 2 is a linear plot of the chromatogram, signal-to-noise ratio, and calibration curve at the calibration point 0.005ng/mL of the standard curve for Compound 2-OHE 1;
FIG. 3 is a linear plot of the chromatogram, signal-to-noise ratio, and calibration curve at the calibration point 0.005ng/mL of the standard curve for Compound 4-OHE 1;
FIG. 4 is a linear plot of the chromatogram, signal-to-noise ratio, and calibration curve at calibration point 0.005ng/mL of the standard curve for Compound 16 α -OHE 1;
FIG. 5 is a line graph of a chromatogram, signal-to-noise ratio, and calibration curve at the calibration point 0.005ng/mL of a standard curve for Compound 2-MeOE 1;
FIG. 6 is a line graph of a chromatogram, signal-to-noise ratio, and calibration curve at the calibration point 0.005ng/mL of a standard curve for Compound 4-MeOE 1;
FIG. 7 shows typical chromatograms of 2-hydroxyestrone, 4-hydroxyestrone, 16 α -hydroxyestrone, 2-methoxyestrone and 4-methoxyestrone.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention is further illustrated by the following examples. It should be understood that the embodiments of the present invention are only for illustrating the technical effects of the present invention, and are not intended to limit the scope of the present invention. In the examples, the methods used were all conventional methods unless otherwise specified. Meanwhile, in the examples, materials, reagents and the like used therein are commercially available unless otherwise specified.
Example 1
1. Material
Urine samples used for testing were from members of the Bo-ao's first-year life maintenance center.
2. The instrument equipment comprises:
LC-MS (liquid chromatography-mass spectrometer): WatersACQUITYLC I-Class ultra high Performance liquid phase System and Xevo TQ-S Mass spectrometer, Milli-Q Direct 8 ultrapure water treatment System, high speed refrigerated centrifuge (Thermo Fisher Scientific Freesco 21), high speed centrifuge (Thermo Fisher Scientific ST 16), Vortex mixer (Scientific Industries Vortex-Genie2), Adjustable pipettor (Germany Brand), analytical balance (Shanghai Mei Teller-Tolydo).
3. Reagent consumables:
ultrapure water, methanol (lcmsm grade), ammonium fluoride (lcmsm grade), β -glucuronidase >100000units/mL, arylsulfatase <20000units/mL, sodium acetate (chromatographic grade), L-ascorbic acid (chromatographic grade), solid phase extraction cartridge: Sep-Pak C1896-well Plate,40mg Sorbent perWell96 well Plate, 96-well collection Plate, different volume EP tube, different size pipette tips, chromatography column CORTECS C1890A,1.6 μm,2.1mm × 150 mm.
4. And (3) standard substance:
estrone (E1), 2-hydroxyestrone (2-OHE1), 4-hydroxyestrone (4-OHE1), 16 α -hydroxyestrone (16 α -OHE1), 2-methoxyestrone (2-MeOE1) and 4-methoxyestrone (4-MeOE1) were purchased from iso sciences.
Isotopic internal standards 13C 3-estrone, 13C 6-2-hydroxyestrone and 13C, d 3-2-methoxyestrone were purchased from Cambridge Isotope.
5. Preparing a reagent:
(1) sodium acetate: sodium acetate is weighed, purified water is added for dilution, the pH value is adjusted, and finally, the sodium acetate solution with the concentration of 0.15M, pH-4.6 is obtained.
(2) Enzymolysis liquid: 250mg of ascorbic acid was weighed out, 0.5mL of β -glucuronidase/sulfate was added, and then 50mL of sodium acetate solution was added thereto at 0.15M, pH ═ 4.6 to make a volume of 50 mL.
(3) Preparing a standard substance: accurately weighing 1mg of each of the estrone, 2-hydroxyestrone, 4-hydroxyestrone, 16 alpha-hydroxyestrone, 2-methoxyestrone and 4-methoxyestrone standard substances, respectively, placing the weighed standard substances into a 1mL volumetric flask, adding a methanol solution containing 0.1% ascorbic acid, and preparing standard substance mother liquor with the concentration of 1000 mu g/mL; respectively transferring appropriate amount of solution from the above mother solution of standard product, and diluting to constant volume with 0.1% ascorbic acid-containing methanol solution to obtain mixed standard solution with concentration of 10 μ g/mL; then, the 10 mu g/mL mixed standard solution is diluted by deionized water to 100ng/mL, 10ng/mL and 1ng/mL mixed standard working solution in a stepwise manner.
(4) Preparing a mixed internal standard working solution: accurately weighing 1mg of each internal standard (namely 3C 3-estrone, 13C 6-2-hydroxyestrone and 13C, d 3-2-methoxyestrone) respectively, placing the internal standard in a 1mL volumetric flask respectively, adding a methanol solution containing 0.1% ascorbic acid, and preparing internal standard mother liquor with the concentration of 1000 mug/mL respectively; and respectively transferring a proper amount of solution from the internal standard mother liquor, and diluting the solution with methanol to the concentration of 100ng/mL to obtain a mixed internal standard working solution.
(5) Preparation of standard curve calibration point solution: the mixed standard working solution is diluted by deionized water or PBS respectively to prepare standard curve calibration points of 0.002ng/mL, 0.005ng/mL, 0.02ng/mL, 0.05ng/mL, 0.2ng/mL, 0.5ng/mL, 2ng/mL, 5ng/mL and 20 ng/mL.
6. Pretreatment:
(1) transferring 500 mu L of collected centrifuged urine supernatant sample or 500 mu L of standard curve calibration point solution into a 1.5mL EP tube, adding 10 mu L of mixed internal standard working solution with the concentration of 100ng/mL, and vortexing for 30 seconds;
(2) continuously adding 0.5mL of enzymolysis solution into an EP tube, uniformly shaking for 3min by vortex, and hydrolyzing for 16h in an incubator at 37 ℃;
(3) 1mL of the above-hydrolyzed treated sample was added to an SPE cartridge (Sep-Pak C1896-well Plate,40mg Sorbentperwell) activated with 3mL of methanol and 3mL of an aqueous solution, 2 column volumes of methanol eluates were collected in a new 96-well collection Plate and dried under nitrogen at 60 ℃ and finally dissolved in 100. mu.L of a methanol/acetonitrile aqueous solution (4:36:60) for LC-MS-MS analysis.
7. LC-MS-MS analysis:
firstly, separating 6 estrogens and metabolites thereof by using a high performance liquid chromatography, then establishing a calibration curve by using a mass spectrum isotope internal standard quantitative method and taking the concentration ratio of a standard substance to an internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and calculating the contents of the 6 estrogens and the metabolites thereof, wherein the specific chromatographic conditions are as follows:
(1) high performance liquid chromatography conditions:
mobile phase A: 0.5mM ammonium fluoride in water;
mobile phase B: acetonitrile;
the type of the chromatographic column: CORTECS C182.1mm × 50mm, 1.6 μm;
the gradient elution mode was used, with the parameters given in the following table:
| Time | flow rate (mL/min) | ％A | ％B |
| 0.00 | 0.300 | 60 | 40 |
| 2.00 | 0.300 | 60 | 40 |
| 2.10 | 0.300 | 55 | 45 |
| 2.50 | 0.300 | 45 | 55 |
| 3.00 | 0.300 | 40 | 60 |
| 3.30 | 0.300 | 10 | 90 |
| 4.00 | 0.300 | 10 | 90 |
| 5.50 | 0.300 | 60 | 40 |
(2) Mass spectrum conditions:
adopting a mass spectrum scanning mode of MRM multi-reaction monitoring under an electrospray ionization negative ion mode;
capillary voltage: 3.00 kV;
taper hole voltage: 40V;
taper hole air flow rate: 150L/hr;
desolventizing gas temperature and flow rate: 630 deg.C, 1100L/hr;
atomizing: 7.0 bar;
collision gas: 4;
the mass spectrometric parameters of the 6 estrogens and their metabolites are given in the following table:
(3) results
Linearity and detection limit
As shown in the table below, each compound has good linearity, the correlation coefficient is greater than 0.998, the detection Limit (LOD) of each compound corresponding to 3 times of signal-to-noise ratio is between 0.002 and 0.005ng/mL, and the quantification Limit (LOQ) of each compound corresponding to 10 times of signal-to-noise ratio is between 0.005 and 0.015 ng/mL. The method linearity and sensitivity are shown in the following table and in FIGS. 1-6.
From the detection results, two groups of trace isomers of 6 estrogens and metabolites thereof in human urine tested by the invention are 2-hydroxyestrone (2-OHE1), 4-hydroxyestrone (4-OHE1), 16 alpha-hydroxyestrone (16 alpha-OHE 1), 2-methoxyestrone (2-MeOE1) and 4-methoxyestrone (4-MeOE1), and the detection method can detect the 6 pg/mL urine estrogens and metabolites thereof under the condition of baseline separation of the two groups of isomers within 5.5min without derivatization, and has the advantages of high sensitivity, strong specificity, high detection speed, simple pretreatment and capability of meeting the requirement of simultaneous batch quantitative analysis of clinical endogenous estrogens and metabolites thereof without derivatization.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (8)
1. The high performance liquid chromatography tandem mass spectrometry detection method of 6 estrogens and their metabolites in human urine is characterized in that the 6 estrogens and their metabolites are estrone, 2-hydroxyestrone, 4-hydroxyestrone, 16 alpha-hydroxyestrone, 2-methoxyestrone and 4-methoxyestrone, respectively;
the detection method comprises the steps of detecting a pretreated human urine sample by adopting a high performance liquid chromatography tandem mass spectrometry technology, separating the 6 estrogens and metabolites thereof in the sample by utilizing the high performance liquid chromatography, then establishing a calibration curve by utilizing a mass spectrum isotope internal standard quantitative method, taking the concentration ratio of a standard substance to an internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and calculating the contents of the 6 estrogens and metabolites thereof, wherein the specific chromatographic conditions are as follows:
(1) high performance liquid chromatography conditions:
mobile phase A: 0.5mM ammonium fluoride in water;
mobile phase B: acetonitrile;
the type of the chromatographic column: CORTECS C182.1mm × 50mm, 1.6 μm;
the gradient elution mode was used, with the parameters given in the following table:
(2) Mass spectrum conditions:
adopting a mass spectrum scanning mode of MRM multi-reaction monitoring under an electrospray ionization negative ion mode;
capillary voltage: 3.00 kV;
taper hole voltage: 40V;
taper hole air flow rate: 150L/hr;
desolventizing gas temperature and flow rate: 630 deg.C, 1100L/hr;
atomizing: 7.0 bar;
collision gas: 4;
the mass spectrum parameters of the 6 estrogens and their metabolites are shown in the following table:
2. the HPLC-MS detection method for 6 estrogens and their metabolites in human urine as claimed in claim 1, wherein said pre-treated human urine sample is obtained as follows: centrifuging collected urine, putting 500 mu L of supernatant into a sample tube, adding 10 mu L of mixed internal standard working solution with the concentration of 100ng/mL, uniformly mixing by vortex, adding 0.5mL of enzymolysis solution, uniformly mixing by vortex, carrying out enzymatic hydrolysis at 37 ℃ for 16h, then carrying out reversed-phase medium solid-phase extraction and purification, and re-dissolving after drying by nitrogen.
3. The HPLC-MS detection method for 6 estrogens and their metabolites in human urine as claimed in claim 2, wherein said mixed internal standard working solution is obtained by the following method: accurately weighing 1mg of 13C 3-estrone, 13C 6-2-hydroxyestrone and 3 isotope internal standard products of 13C, d 3-2-methoxyestrone respectively, placing the internal standard products into 1mL volumetric flasks respectively, adding methanol solutions containing 0.1% ascorbic acid respectively to prepare internal standard mother solutions with the concentrations of 1000 mu g/mL respectively, transferring appropriate solutions from the internal standard mother solutions respectively, and diluting the solutions with methanol to the concentration of 100ng/mL to obtain the mixed internal standard working solution.
4. The method for detecting 6 estrogens and their metabolites in human urine according to claim 1, wherein the standard is obtained by the following method: accurately weighing 1mg of each of estrone, 2-hydroxyestrone, 4-hydroxyestrone, 16 alpha-hydroxyestrone, 2-methoxyestrone and 4-methoxyestrone, adding a methanol solution containing 0.1% ascorbic acid to prepare standard mother solutions with the concentrations of 1000 mug/mL respectively, transferring a proper amount of solution from the standard mother solutions respectively, fixing the volume by using the methanol solution containing 0.1% ascorbic acid to obtain a mixed standard solution with the concentration of 10 mug/mL, and diluting the mixed standard solution with deionized water step by step into a mixed standard working solution with the concentrations of 100ng/mL, 10ng/mL and 1 ng/mL; diluting the mixed standard working solution with deionized water or PBS respectively to prepare standard curve calibration points of 0.002ng/mL, 0.005ng/mL, 0.02ng/mL, 0.05ng/mL, 0.2ng/mL, 0.5ng/mL, 2ng/mL, 5ng/mL and 20 ng/mL; respectively transferring 500 mu L of the standard curve calibration point solution, respectively adding 10 mu L of mixed internal standard working solution with the concentration of 100ng/mL, uniformly mixing in a vortex mode, then adding 0.5mL of enzymolysis solution, uniformly mixing in a vortex mode, carrying out enzymatic hydrolysis at 37 ℃ for 16h, then carrying out reversed phase medium solid phase extraction and purification, and re-dissolving after drying by nitrogen.
5. The HPLC-MS/MS detection method for 6 estrogens and their metabolites in human urine as claimed in claim 2 or 4, wherein the said enzymatic hydrolysate is obtained by the following method: 250mg of ascorbic acid was weighed out, 0.5mL of β -glucuronidase/arylsulfatase was added, and then 50mL of sodium acetate solution with a concentration of 0.15M, pH ═ 4.6 was added to the solution.
6. The HPLC-MS/MS detection method for 6 estrogens and their metabolites in human urine as claimed in claim 2 or 4, wherein the reversed phase medium solid phase extraction and purification method comprises: the hydrolyzed treated sample was added to 3mL methanol and 3mL aqueous activated SPE cartridges, and 2 column volumes of methanol eluate were collected in a new 96-well collection plate.
7. The HPLC-MS/MS detection method for 6 estrogens and their metabolites in human urine as claimed in claim 2 or 4, wherein the temperature for drying with nitrogen is 60 ℃.
8. The method for detecting 6 estrogens and their metabolites in human urine as claimed in claim 2 or 4, wherein the redissolved solvent is a mixed solution of methanol, acetonitrile and water, and the volume ratio of methanol, acetonitrile and water is 4:36: 60.
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| Title |
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| JING-FANG HSU等: "Evaluation of electrospray ionization and atmospheric pressure chemical ionization for simultaneous detection of estrone and its metabolites using high-performance liquid chromatography/tandem mass spectrometry", 《JOURNAL OF CHROMATOGRAPHY B》 * |
| NILESH W. GAIKWAD等: "Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Profiling of Steroid Metabolome in Human Tissue", 《ANAL. CHEM》 * |
| 方玲等: "***代谢与乳腺癌关系的研究进展", 《中国普外基础与临床杂志》 * |
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