CN111103383A - Method for simultaneously measuring concentrations of endogenous cortisol, corticosterone, androstenedione and testosterone in human plasma by liquid chromatography-mass spectrometry - Google Patents
Method for simultaneously measuring concentrations of endogenous cortisol, corticosterone, androstenedione and testosterone in human plasma by liquid chromatography-mass spectrometry Download PDFInfo
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Abstract
The invention aims to provide a method for simultaneously and accurately measuring the concentrations of endogenous cortisol, corticosterone, androstenedione and testosterone in human plasma. The method comprises the following steps: (1) selection of a surrogate substrate; (2) determining the concentrations of endogenous cortisol, corticosterone, androstenedione and testosterone in a control matrix (3) preparing and pretreating a working curve sample; (4) preparing and pretreating a quality control sample; (5) separating the analysis conditions; (6) by adopting an internal standard method, the concentration of series of cortisol, corticosterone, androstenedione and testosterone in the substitute matrix is taken as an abscissa, the peak area ratio of each of cortisol, corticosterone, androstenedione and testosterone to internal standard dexamethasone is taken as an ordinate, and the linear relation between theoretical concentration and response is obtained by linear regression by using a weight factor 1/x2 for quantification.
Description
Technical Field
The invention belongs to the technical field of medicine detection, and particularly relates to a method for measuring concentrations of endogenous cortisol, corticosterone, androstenedione and testosterone in human plasma.
Background
Sex hormones are main steroid hormones, which are lipid-soluble small molecule hormones, are obtained by cholesterol through a series of enzymes, are essential for body development, sexual maturation and metabolism, and play an important role in maintaining various vital activities, regulating body substance metabolism and the like (reference: 1.Andreu Fabregat, Aristotelis Kotromula, JoseppMarcos, et al. Detection, synthesis and characterization of metabolism of steric of complex treated with cysteine. Steroids,2013,78: 327) and the like. In clinic, gynecological endocrine and related reproductive diseases, abnormal uterine bleeding, amenorrhea, infertility, gynecological related tumors and the like are known through the level of sex hormones, so that the sex hormones can be used as a therapeutic drug for research and can also be used as a biomarker for the therapeutic effect of the anti-tumor drugs for research. However, it is difficult to determine whether or not sex hormones are endogenous substances and can be quantified accurately. The invention establishes a scientific, rapid and accurate method for measuring the concentrations of endogenous cortisol, corticosterone, androstenedione and testosterone in human plasma by liquid chromatography-mass spectrometry based on a substituted matrix method.
Disclosure of Invention
The invention aims to provide a method for simultaneously measuring the concentrations of endogenous cortisol, corticosterone, androstenedione and testosterone in human plasma by liquid chromatography-mass spectrometry.
In order to achieve the purpose, the invention adopts the following technical scheme:
(1) selection of alternative substrates
The present invention uses a "relative normalized recovery" method to screen for suitable surrogate matrices. Preparing 3% BSA solution with normal saline, and inspecting the standard recovery rate of the substitute matrix; adding 475. mu.L of diluent/W-GMQC/W-MQC/W-HQC working solution instead of the matrix/the contrast matrix, uniformly mixing, and subpackaging 4 parts of 100. mu.L each; adding 15.0 mu L of internal standard working solution into each part, adding 400 mu L of ethyl acetate for extraction, centrifuging, taking 200 mu L of supernatant, drying under nitrogen, adding 150 mu L of 50% methanol aqueous solution for redissolving, shaking and centrifuging, and determining by LC-MS/MS. Substituting the result into the relative standard addition recovery rate to calculate, wherein the relative standard addition recovery rate is subjected to the following standard: the relative standard recovery rate of the three concentrations is between 85 and 115 percent, and the percent CV does not exceed 15 percent. The calculation formula is as follows:
% relative normalized recovery ═ spiked SM-blank SM)/(spiked SM-blank SM)
spiked SM ═ spiked substituted peak area ratios of analyte to internal standard in the matrix
blank SM ═ blank substitution of peak area ratios of analyte to internal standard in the matrix
spiked CM-peak area ratio of analyte to internal standard in spiked control matrix
spiked CM-the peak area ratio of analyte to internal standard in the placebo matrix
The results show that the mass spectrum responses of the internal standards dexamethasone, cortisol, corticosterone, androstenedione and testosterone in the 3% BSA physiological saline solution and the human plasma sample basically tend to be consistent, the relative standard addition recovery rate of the three concentrations is between 90% and 115%, and the variation coefficient is not more than 15%. Therefore, 3% BSA physiological saline solution is selected as a substitute matrix to carry out a methodology verification test and a sample analysis test of cortisol, corticosterone, androstenedione and testosterone in human plasma.
(2) Determination of the concentration of endogenous Cortisol, Cortisone, androstenedione, Testosterone in control matrices (human plasma)
Preparing a standard curve sample: standard curve samples were prepared using 3% BSA saline as the surrogate matrix. Taking appropriate amount of cortisol, corticosterone, androstenedione and testosterone stock solution, diluting with 50% methanol water to obtain serial working solutions with cortisol concentration of 4.00, 8.00, 40.0, 200, 400, 1200, 1800 and 2000ng/mL and cortisol concentration of 160, 320, 800, 2400, 4800, 9600, 14400 and 16000 ng/mL; then mixing the two sets of working solutions in an equal volume according to corresponding concentration points to finally obtain a standard curve working solution required by preparing a standard curve sample, wherein the concentrations of corticosterone, androstenedione and testosterone are 2.00, 4.00, 20.0, 100, 200, 600, 900 and 1000 ng/mL; cortisol concentrations of 80.0, 160, 400, 1200, 2400, 4800, 7200, and 8000 ng/mL; adding 15.0 μ L of the above series of working solutions for standard curve preparation into 285 μ L of 3% BSA physiological saline (instead of the matrix) to prepare samples corresponding to corticosterone, androstenedione, testosterone concentrations of 0.100ng/mL, 0.200ng/mL, 1.00ng/mL, 5.00ng/mL, 10.0ng/mL, 30.0ng/mL, 45.0ng/mL, 50.0ng/mL, and cortisol concentrations of 4.00ng/mL, 8.00ng/mL, 20.0ng/mL, 60.0ng/mL, 120ng/mL, 240ng/mL, 360ng/mL, 400ng/mL, and dispensing 2 ng/100 μ L samples;
preparation of control matrix samples: directly taking 100 mu L of human plasma sample;
pretreatment of a biological sample: adding 15.0 mu L of internal standard working solution into each of the standard curve sample and the control matrix sample, adding 400 mu L of ethyl acetate for extraction, centrifuging, taking 200 mu L of supernatant, drying under nitrogen, adding 150 mu L of 50% methanol aqueous solution for redissolving, oscillating and centrifuging, and determining by LC-MS/MS.
6 batches of human plasma were selected as a control matrix, and the concentrations of endogenous cortisol, corticosterone, androstenedione and testosterone in the control matrix were determined according to the above method. The average concentrations of endogenous cortisol, corticosterone, androstenedione and testosterone in 6 batches of human plasma were 79.6ng/mL, 1.94ng/mL, 1.06ng/mL and 1.70ng/mL, respectively.
(3) Preparation and pretreatment of standard curve sample
Preparing an internal standard working solution: taking a dexamethasone standard substance, correcting by a quality correction factor, and dissolving the dexamethasone standard substance in methanol to obtain a stock solution with the final concentration of 1.00 mg/mL; diluting the solution with 50% methanol water solution to obtain 200ng/mL internal standard working solution;
preparation of standard series of solutions: taking appropriate amount of cortisol, corticosterone, androstenedione and testosterone standard substance, respectively, and preparing stock solution with concentration of 1.00mg/mL with methanol; diluting with 50% methanol water to obtain serial working solutions with cortisone, androstenedione, and testosterone concentrations of 4.00, 8.00, 40.0, 200, 400, 1200, 1800, 2000ng/mL, and cortisol concentrations of 160, 320, 800, 2400, 4800, 9600, 14400, and 16000 ng/mL; then mixing the two sets of working solutions in an equal volume according to corresponding concentration points to finally obtain a standard curve working solution required by preparing a standard curve sample, wherein the concentrations of corticosterone, androstenedione and testosterone are 2.00, 4.00, 20.0, 100, 200, 600, 900 and 1000 ng/mL; cortisol concentrations of 80.0, 160, 400, 1200, 2400, 4800, 7200, and 8000 ng/mL;
pretreatment of a working curve sample: adding 15.0 μ L of the above series of working solutions for standard curve preparation into 285 μ L of 3% BSA physiological saline (instead of the matrix) to prepare samples corresponding to corticosterone, androstenedione, and testosterone concentrations of 0.100, 0.200, 1.00, 5.00, 10.0, 30.0, 45.0, 50.0ng/mL and cortisol concentrations of 4.00, 8.00, 20.0, 60.0, 120, 240, 360, and 400ng/mL, and subpackaging 2 parts of 100 μ L; adding 15.0 mu L of internal standard working solution into each part, adding 400 mu L of ethyl acetate for extraction, centrifuging, taking 200 mu L of supernatant, drying under nitrogen, adding 150 mu L of 50% methanol aqueous solution for redissolving, shaking and centrifuging, and determining by LC-MS/MS.
(4) Preparation and pretreatment of quality control sample
Preparation of standard series of solutions: taking appropriate amount of cortisol, corticosterone, androstenedione and testosterone standard substance, respectively, and preparing stock solution with concentration of 1.00mg/mL with methanol; diluting with 50% methanol water to obtain serial working solutions of corticosterone, androstenedione, testosterone with concentration of 1600ng/mL (W1-HQC), 800ng/mL (W1-MQC), 80.0ng/mL (W1-GMQC1), 40.0ng/mL (W1-GMQC2), 12.0ng/mL (W1-LQC) and cortisol with concentration of 10000ng/mL (W2-HQC), 6000ng/mL (W2-MQC), 2000ng/mL (W2-GMQC) and 480ng/mL (W2-LQC); and then mixing the two sets of working solutions in equal volume according to corresponding concentration points to finally obtain a quality control working solution required by preparing a standard curve sample: W-HQC (high concentration quality control working solution: W1-HQC is mixed with W2-HQC, the concentrations of corticosterone, androstenedione and testosterone are 800ng/mL, and the concentration of cortisol is 5000 ng/mL); W-MQC (medium concentration quality control working solution: W1-MQC and W2-MQC are mixed, the concentrations of corticosterone, androstenedione and testosterone are 400ng/mL, and the concentration of cortisol is 3000 ng/mL); W-GMQC (quality control working solution with middle concentration in arithmetic: W1-GMQC1 and W2-GMQC are mixed, the concentrations of corticosterone, androstenedione and testosterone are 40.0ng/mL, and the concentration of cortisol is 1000 ng/mL); W-LQC (low-concentration quality control working solution: W1-LQC and W2-LQC are mixed, the concentrations of corticosterone, androstenedione and testosterone are 6.00ng/mL, and the concentration of cortisol is 240 ng/mL);
pretreatment of a quality control sample: 35.0 mu LW1-GMQC2 working solution, W-MQC working solution and W-HQC working solution are respectively added into 665 mu L of human blank plasma to prepare corresponding GMQC (corticosterone, androstenedione, testosterone: 2.00ng/mL, cortisol: 0.00ng/mL), MQC (corticosterone, androstenedione, testosterone: 20.0ng/mL, cortisol: 150ng/mL) and HQC (corticosterone, androstenedione, testosterone: 40.0ng/mL, cortisol: 250ng/mL) quality control samples*Taking human plasma with a certain volume, then respectively diluting the human plasma with a substitute matrix (3% BSA physiological saline solution) to the concentration close to the low quality control concentration and the quantitative lower limit concentration, preparing LQC and LLOQ quality control samples, and subpackaging 6 parts of each quality control sample, wherein each part is 100 mu L; adding 15.0 mu L of internal standard working solution into each part, adding 400 mu L of ethyl acetate for extraction, centrifuging, taking 200 mu L of supernatant, drying under nitrogen, adding 150 mu L of 50% methanol aqueous solution for redissolving, shaking and centrifuging, and determining by LC-MS/MS.
The concentrations were not calculated for the content of Androstenedione, Testosterone, Corticosterone, Hydrocortisone in the control matrices, the actual sample concentration being (working fluid concentration + working fluid volume + control matrix concentration) volume of control matrix/total volume.
(5) Conditions for separation analysis
Liquid chromatography conditions:
a chromatographic column: ACQUITY UPLC BEH C181.7 μm (50X 2.1 mm);
mobile phase: a 0.1% aqueous formic acid (containing 2% methanol); b-0.1% formic acid in methanol (5% water) with the following gradient:
0-0.60min, 70.0% of mobile phase A and 30.0% of mobile phase B; 0.60-1.50min, the mobile phase A is 70.0-40.0%, and the mobile phase B is 30.0-60.0%; 1.50-3.80min, the mobile phase A is from 40.0% to 30.0%, and the mobile phase B is from 60.0% to 70.0%; 3.80-4.00min, the mobile phase A is from 30.0% to 0%, and the mobile phase B is from 70.0% to 100%; 4.00-4.30min, keeping the mobile phase A at 0% and the mobile phase B at 100%; 4.30-4.40min, the mobile phase A is from 0% to 70.0%, and the mobile phase B is from 100% to 30.0%; 4.40-5.00min, the mobile phase A is kept at 70.0%, and the mobile phase B is kept at 30.0%;
flow rate: 0.500 mL/min;
column temperature: 40 ℃;
sample introduction amount: 10.0. mu.L.
Mass detector and conditions: the mass spectrum detector is Triple Quad 6500+ AB Sciex; the ion source is an electrospray ion source; the spraying voltage is 5500V; ion source temperature: 550; curtain gas (CUR) at ° c: 20psi, collision gas (CAD): 9psi, GS 1: 50psi, GS 2: 20 psi; detecting in a positive ion mode; the scanning mode is as follows: MRM; the quantitative ion pair of androstenedione is 287.2 → 97.1, and the collision voltage is 28.0 eV; the testosterone quantitative ion pair is 289.2 → 109.1, and the collision voltage is 31.0 eV; the corticosterone quantitative ion pair is 347.2 → 293.1, and the collision voltage is 25.0 eV; the cortisol quantitative ion pair is 363.2 → 327.3, and the collision voltage is 23.0 eV; the internal standard dexamethasone quantitative ion pair is 393.1 → 373.3, and the collision voltage is 13.0 eV.
(6) Adopting an internal standard method, taking the concentrations of serial cortisol, corticosterone, androstenedione and testosterone in the substitute matrix as horizontal coordinates, taking the peak area ratios of cortisol, corticosterone, androstenedione and testosterone to internal standard dexamethasone as vertical coordinates, and performing linear regression operation with the weight factor of 1/x2And obtaining a linear regression equation, namely a working curve, and respectively measuring the concentrations of endogenous cortisol, corticosterone, androstenedione and testosterone in human plasma by using the working curve.
Has the advantages that:
the method for simultaneously and accurately measuring the concentrations of endogenous cortisol, corticosterone, androstenedione and testosterone in human plasma measures the concentrations of endogenous cortisol, corticosterone, androstenedione and testosterone in a plasma sample by a substrate replacing method and liquid chromatography-mass spectrometry.
Drawings
FIG. 1 is a blank surrogate matrix chromatogram; FIG. 1a is an analyte androstenedione ion channel chromatogram; FIG. 1b is a chromatogram of the ion channel of analyte testosterone; FIG. 1c is a chromatogram of an analyte corticosterone ion channel; FIG. 1d is a chromatogram of an analyte cortisol ion channel; FIG. 1e is an internal standard dexamethasone ion channel chromatogram;
FIG. 2 is a chromatogram of the lower limit of quantitation of a standard curve; FIG. 2a is a chromatogram of the analyte androstenedione (0.100ng/mL) in an alternative matrix; FIG. 2b is a chromatogram of the analyte testosterone (0.100ng/mL) in a surrogate matrix; FIG. 2c is a chromatogram of the analyte corticosterone (0.100ng/mL) in an alternative matrix; FIG. 2d is a chromatogram of the analyte cortisol (4.00ng/mL) in an alternative matrix; FIG. 2e is a chromatogram of internal standard dexamethasone (200ng/mL) in surrogate matrix;
FIG. 3 is a quantitative upper limit chromatogram of a standard curve; FIG. 3a is a chromatogram of the analyte androstenedione (50.0ng/mL) in an alternative matrix; FIG. 3b is a chromatogram of the analyte testosterone (50.0ng/mL) in a surrogate matrix; FIG. 3c is a chromatogram of the analyte corticosterone (50.0ng/mL) in an alternative matrix; FIG. 3d is a chromatogram of the analyte cortisol (400ng/mL) in an alternative matrix; FIG. 3e is a chromatogram of internal standard dexamethasone (200ng/mL) in surrogate matrix;
FIG. 4 is a chromatogram of the quality-controlled LLOQ concentration in a control matrix; FIG. 4a is a chromatogram of the analyte androstenedione (0.133ng/mL, obtained after dilution of a control matrix with an alternative matrix) in a control matrix; FIG. 4b is a chromatogram of the analyte testosterone (0.213ng/mL, obtained after dilution of a control matrix with a surrogate matrix) in a control matrix; FIG. 4c is a chromatogram of the analyte corticosterone (0.243ng/mL, obtained after dilution of the control matrix with a surrogate matrix) in a control matrix; FIG. 4d is a chromatogram of analyte cortisol (9.95ng/mL, obtained from a control matrix diluted with an alternative matrix) in a control matrix; FIG. 4e is a chromatogram of internal standard dexamethasone (200ng/mL) in a control matrix;
FIG. 5 is a chromatogram of the quality-controlled LQC concentration in the control matrix; FIG. 5a is a chromatogram of the analyte androstenedione (0.353ng/mL, obtained after dilution of a control matrix with a surrogate matrix) in a control matrix; FIG. 5b is a chromatogram of the analyte testosterone (0.567ng/mL, obtained after dilution of a control matrix with a surrogate matrix) in a control matrix; FIG. 5c is a chromatogram of the analyte corticosterone (0.647ng/mL, obtained after dilution of a control matrix with an alternative matrix) in a control matrix; FIG. 5d is a chromatogram of analyte cortisol (26.5ng/mL, obtained from a control matrix diluted with surrogate matrix) in a control matrix; FIG. 5e is a chromatogram of internal standard dexamethasone (200ng/mL) in a control matrix;
FIG. 6 is a chromatogram of quality control GMQC concentration in control matrix; FIG. 6a is a chromatogram of the analyte androstenedione (3.01ng/mL, control matrix endogenous concentration + added concentration) in a control matrix; FIG. 6b is a chromatogram of the analyte testosterone (3.62ng/mL, control matrix endogenous concentration + added concentration) in a control matrix; FIG. 6c is a chromatogram of the analyte corticosterone (3.84ng/mL, control matrix endogenous + added concentration) in a control matrix; FIG. 6d is a chromatogram of analyte cortisol (75.6ng/mL, obtained from a control matrix diluted with surrogate matrix) in a control matrix; FIG. 6e is a chromatogram of internal standard dexamethasone (200ng/mL) in a control matrix;
FIG. 7 is a chromatogram of the quality-controlled MQC concentration in the control matrix; FIG. 7a is a chromatogram of the analyte androstenedione (21.0ng/mL, control matrix endogenous concentration + added concentration) in a control matrix; FIG. 7b is a chromatogram of the analyte testosterone (21.6ng/mL, control matrix endogenous concentration + added concentration) in a control matrix; FIG. 7c is a chromatogram of the analyte corticosterone (21.8ng/mL, control matrix endogenous + added concentration) in a control matrix; FIG. 7d is a chromatogram of the analyte cortisol (226ng/mL, control matrix endogenous + added concentration) in a control matrix; FIG. 7e is a chromatogram of internal standard dexamethasone (200ng/mL) in a control matrix;
FIG. 8 is a chromatogram of the HQC concentration in control matrix; FIG. 8a is a chromatogram of the analyte androstenedione (41.0ng/mL, control matrix endogenous concentration + added concentration) in a control matrix; FIG. 8b is a chromatogram of the analyte testosterone (41.6ng/mL, control matrix endogenous concentration + added concentration) in a control matrix; FIG. 8c is a chromatogram of the analyte corticosterone (41.8ng/mL, control matrix endogenous + added concentration) in a control matrix; FIG. 8d is a chromatogram of analyte cortisol (326ng/mL, control matrix endogenous + added concentration) in a control matrix; FIG. 8e is a chromatogram of internal standard dexamethasone (200ng/mL) in a control matrix;
Detailed Description
The invention will be further elucidated with reference to specific embodiments and with reference to the drawing, without being limited thereto.
1: determination of detection method
1.1 laboratory instruments and reagents
The instrument comprises the following steps:
the liquid chromatograph is Shimadzu Nexera UHPLC liquid phase system (Shimadzu corporation), and the chromatographic column is ACQUITY UPLC BEH C181.7 μm (50 x 2.1 mm);
the mass spectrometer is Triple Quad 6500+, AB Sciex, and is provided with Analyst 1.6.3 analysis software;
information management software: watson LIMS 7.5 SP1(Thermo Scientific);
the main instruments, see table 1 below:
TABLE 1 Main Instrument
Reagent:
original reagents:
ultrapure water (made by Milli-Q purification systems);
formic acid, LC/MS, Fisher Scientific, batch number: 190284, respectively;
methanol, chromatographic grade, Fisher Chemical, lot number: 190894, 191614;
acetonitrile, chromatographic grade, Fisher Chemical, lot number: 186341, respectively;
ethyl acetate, chromatographic grade, Fisher Chemical, lot number: 180735, respectively;
sodium chloride injection, medical, ann hui double crane pharmaceutical industry, batch number: 1701087V;
bovine serum albumin, cell culture grade, Sigma-aldrich, lot number: WXBD 0889V.
And (3) standard substance:
androstenedione (Androstenedione), lot number: 40701, purity: 99.5 percent; the manufacturer: dr ehrenstorfer gmbh; and (3) expiration date: year 2018, month 06, day 25; correction factor: 0.994, free molecular weight: 286.41, and storing at-20 + -4 deg.C.
Testosterone (Testosterone), batch number: 50213, purity: 99.0 percent; the manufacturer: dr Ehrenstorfer GmbH; and (3) expiration date: year 2019, month 08, day 10; correction factor: 0.984, free molecular weight: 288.42, and storing at-20 + -4 deg.C.
Corticosterone (Corticosterone), lot number: 50729, purity: 99.8%, moisture: 0.1 percent; the manufacturer: DrEhrenstorfer GmbH; and (3) expiration date: 11/13/2019; correction factor: 0.997, free molecular weight: 346.51, and storing at-20 + -4 deg.C.
Cortisol (Hydrocortisone), batch No.: 50320, purity: 99.8 percent; the manufacturer: dr ehrenstorfer gmbh; and (3) expiration date: 19 months 03 and 2019; correction factor: 0.998, free molecular weight: 362.5, and storing at-20 + -4 deg.C.
Dexamethasone (Dexamethasone), batch No.: 100129-; the content is as follows: 99.8 percent; the manufacturer: china institute for testing and testing food and drug; and (3) expiration date: releasing in the next batch; correction factor: 0.998, free molecular weight: 392.47, storing at room temperature under sealed condition and protected from light.
Preparing a reagent:
preparing an internal standard stock solution: taking a dexamethasone standard substance, correcting by a quality correction factor, and dissolving the dexamethasone standard substance in methanol to obtain a stock solution with the final concentration of 1.00 mg/mL;
standard stock solutions: taking appropriate amount of cortisol, corticosterone, androstenedione and testosterone standard substances, and preparing stock solutions with concentration of 1.00mg/mL with methanol;
preparing an internal standard working solution: taking a proper amount of internal standard stock solution mother liquor, and diluting the mother liquor with 50% methanol aqueous solution to obtain 200ng/mL internal standard working solution;
preparing a standard curve working solution: taking appropriate amount of cortisol, corticosterone, androstenedione and testosterone standard substance, respectively, and preparing stock solution with concentration of 1.00mg/mL with methanol; diluting with 50% methanol water to obtain serial working solutions with cortisone, androstenedione, and testosterone concentrations of 4.00, 8.00, 40.0, 200, 400, 1200, 1800, 2000ng/mL, and cortisol concentrations of 160, 320, 800, 2400, 4800, 9600, 14400, and 16000 ng/mL; then mixing the two sets of working solutions in an equal volume according to corresponding concentration points to finally obtain a standard curve working solution required by preparing a standard curve sample, wherein the concentrations of corticosterone, androstenedione and testosterone are 2.00, 4.00, 20.0, 100, 200, 600, 900 and 1000 ng/mL; cortisol concentrations of 80.0, 160, 400, 1200, 2400, 4800, 7200, and 8000 ng/mL;
preparing a quality control working solution: taking appropriate amount of cortisol, corticosterone, androstenedione and testosterone standard substance, respectively, and preparing stock solution with concentration of 1.00mg/mL with methanol; diluting with 50% methanol water to obtain serial working solutions of corticosterone, androstenedione, testosterone with concentration of 1600ng/mL (W1-HQC), 800ng/mL (W1-MQC), 80.0ng/mL (W1-GMQC1), 80.0ng/mL (W1-GMQC2), 12.0ng/mL (W1-LQC) and cortisol with concentration of 10000ng/mL (W2-HQC), 6000ng/mL (W2-MQC), 2000ng/mL (W2-GMQC) and 480ng/mL (W2-LQC); and then mixing the two sets of working solutions in equal volume according to corresponding concentration points to finally obtain a quality control working solution required by preparing a standard curve sample: W-HQC (high concentration quality control working solution: W1-HQC is mixed with W2-HQC, the concentrations of corticosterone, androstenedione and testosterone are 800ng/mL, and the concentration of cortisol is 5000 ng/mL); W-MQC (medium concentration quality control working solution: W1-MQC and W2-MQC are mixed, the concentrations of corticosterone, androstenedione and testosterone are 400ng/mL, and the concentration of cortisol is 3000 ng/mL); W-GMQC (quality control working solution with middle concentration in arithmetic: W1-GMQC1 and W2-GMQC are mixed, the concentrations of corticosterone, androstenedione and testosterone are 40.0ng/mL, and the concentration of cortisol is 1000 ng/mL); W-LQC (low-concentration quality control working solution: W1-LQC and W2-LQC are mixed, the concentrations of corticosterone, androstenedione and testosterone are 6.00ng/mL, and the concentration of cortisol is 240 ng/mL).
Matrix:
alternative matrix: 3% BSA normal saline solution is used as a substitute matrix for preparing a standard curve sample in method verification;
control matrix: detecting the concentrations of cortisol, corticosterone, androstenedione and testosterone in the blood plasma of different individuals, selecting individual blood plasma with lower contents of cortisol, corticosterone, androstenedione and testosterone to mix, using the mixed blood plasma as a reference matrix, detecting the concentrations of cortisol, corticosterone, androstenedione and testosterone in the mixed blood plasma, and using the concentrations to verify the preparation of quality control samples;
plasma sample: plasma from 6 healthy volunteers using EDTA-K2 as an anticoagulant was provided by the third national hospital, Zhenjiang, and packed in polypropylene centrifuge tubes and frozen in a freezer at-75 + -15 deg.C.
1.2 plasma sample analysis method
Plasma sample pretreatment: taking 100 mu L of plasma of healthy volunteers, adding 15.0 mu L of internal standard working solution into each part, adding 400 mu L of ethyl acetate for extraction, centrifuging, taking 200 mu L of supernatant, drying under nitrogen, adding 150 mu L of 50% methanol aqueous solution for redissolving, shaking and centrifuging, and determining by LC-MS/MS.
1.2.1 liquid phase conditions
A chromatographic column: ACQUITY UPLC BEH C181.7 μm (50X 2.1 mm); mobile phase: a 0.1% aqueous formic acid (containing 2% methanol); b ═ 0.1% methanoic acid solution (5% water) in methanol, gradient elution is given in table 2; column temperature: 40 ℃; autosampler temperature: 8 ℃; sample introduction amount: 10.0. mu.L.
TABLE 2 liquid phase gradient elution procedure
1.2.2 Mass Spectrometry conditions
The ion source is an electrospray ion source; the spraying voltage is 5500V; ion source temperature: 550 ℃; curtain gas (CUR): 20psi, collision gas (CAD): 9psi, GS 1: 50psi, GS 2: 20 psi; detecting in a positive ion mode; the scanning mode is as follows: and (4) MRM. Specific parameters are shown in table 3:
TABLE 3 mode acquisition with MRM, main Mass Spectrometry parameters
2: methodology validation experiment
Linearity of the method: taking a proper amount of stock solution mother liquor of a standard product, and preparing the stock solution with the concentration of 1.00mg/mL by using methanol; diluting with 50% methanol water to obtain serial working solutions with cortisone, androstenedione, and testosterone concentrations of 4.00, 8.00, 40.0, 200, 400, 1200, 1800, 2000ng/mL, and cortisol concentrations of 160, 320, 800, 2400, 4800, 9600, 14400, and 16000 ng/mL; then mixing the two sets of working solutions in an equal volume according to corresponding concentration points to finally obtain a standard curve working solution required by preparing a standard curve sample, wherein the concentrations of corticosterone, androstenedione and testosterone are 2.00, 4.00, 20.0, 100, 200, 600, 900 and 1000 ng/mL; cortisol concentrations of 80.0, 160, 400, 1200, 2400, 4800, 7200, and 8000 ng/mL; adding 15.0 μ L of the above series of working solutions for standard curve preparation into 285 μ L of 3% BSA physiological saline (instead of the matrix) to prepare samples corresponding to corticosterone, androstenedione, and testosterone concentrations of 0.100, 0.200, 1.00, 5.00, 10.0, 30.0, 45.0, 50.0ng/mL and cortisol concentrations of 4.00, 8.00, 20.0, 60.0, 120, 240, 360, and 400ng/mL, and subpackaging 2 parts of 100 μ L; adding 15.0 mu L of internal standard working solution into each part, adding 400 mu L of ethyl acetate for extraction, centrifuging, taking 200 mu L of supernatant, drying under nitrogen, adding 150 mu L of 50% methanol aqueous solution for redissolving, shaking and centrifuging, and determining by LC-MS/MS.
Taking 10.0 mu L of the mixture to perform LC-MS/MS analysis, and recording a chromatogram; taking the concentration of the serial to-be-measured substances in the substitute matrix as a horizontal coordinate, taking the peak area ratio of the to-be-measured substances to the internal standard as a vertical coordinate, performing linear regression operation, and taking the weight factor of 1/x2And obtaining a linear regression equation, namely the working curve.
The linear range of the work curve of cortisol in the substituted matrix is 4.00-400ng/mL, the typical work curve is that y is 0.010197x +0.007497, the correlation coefficients are all larger than 0.9831, and the quantitative detection requirement is met; the linear range of the work curve of corticosterone in the substituted matrix is 0.100-50.0ng/mL, the typical work curve is that y is 0.008110x +0.000033, the correlation coefficients are all larger than 0.9920, and the quantitative detection requirement is met; the linear range of the working curve of androstenedione in the substituted matrix is 0.100-50.0ng/mL, the typical working curve is that y is 0.049359x-0.000653, the correlation coefficients are all larger than 0.9921, and the requirement of quantitative detection is met; (ii) a The linear range of the working curve of testosterone in the substitute matrix is 0.100-50.0ng/mL, the typical working curve is that y is 0.046444x +0.000311, the correlation coefficients are all larger than 0.9951, and the requirement of quantitative detection is met.
Accuracy and precision of the method: taking appropriate amount of cortisol, corticosterone, androstenedione and testosterone standard substance, respectively, and preparing stock solution with concentration of 1.00mg/mL with methanol; diluting with 50% methanol water to obtain serial working solutions of corticosterone, androstenedione, testosterone with concentration of 1600ng/mL (W1-HQC), 800ng/mL (W1-MQC), 80.0ng/mL (W1-GMQC1), 80.0ng/mL (W1-GMQC2), 12.0ng/mL (W1-LQC) and cortisol with concentration of 10000ng/mL (W2-HQC), 6000ng/mL (W2-MQC), 2000ng/mL (W2-GMQC) and 480ng/mL (W2-LQC); and then mixing the two sets of working solutions in equal volume according to corresponding concentration points to finally obtain a quality control working solution required by preparing a standard curve sample: W-HQC (high concentration quality control working solution: W1-HQC is mixed with W2-HQC, the concentrations of corticosterone, androstenedione and testosterone are 800ng/mL, and the concentration of cortisol is 5000 ng/mL); W-MQC (medium concentration quality control working solution: W1-MQC and W2-MQC are mixed, the concentrations of corticosterone, androstenedione and testosterone are 400ng/mL, and the concentration of cortisol is 3000 ng/mL); W-GMQC (quality control working solution with middle concentration in arithmetic: W1-GMQC1 and W2-GMQC are mixed, the concentrations of corticosterone, androstenedione and testosterone are 40.0ng/mL, and the concentration of cortisol is 1000 ng/mL); W-LQC (low-concentration quality control working solution: W1-LQC and W2-LQC are mixed, the concentrations of corticosterone, androstenedione and testosterone are 6.00ng/mL, and the concentration of cortisol is 240 ng/mL). Adding 35.0 μ L of geometric middle concentration quality control working solution (W1-GMQC2) and middle concentration quality control working solution (W-MQC 2) into 665 μ L of human blank plasma) And high-concentration quality control working solution (W-HQC) to prepare corresponding GMQC (corticosterone, androstenedione, testosterone: 2.00ng/mL, cortisol: 0.00ng/mL), MQC (corticosterone, androstenedione, testosterone: 20.0ng/mL, cortisol: 150ng/mL) and HQC (corticosterone, androstenedione, testosterone: 40.0ng/mL, cortisol: 250ng/mL) quality control sample*Taking human plasma with a certain volume, then respectively diluting the human plasma with a substitute matrix (3% BSA physiological saline solution) to the concentration close to the low quality control concentration and the quantitative lower limit concentration, preparing LQC and LLOQ quality control samples, and subpackaging 6 parts of each quality control sample, wherein each part is 100 mu L; adding 15.0 mu L of internal standard working solution into each part, adding 400 mu L of ethyl acetate for extraction, centrifuging, taking 200 mu L of supernatant, drying under nitrogen, adding 150 mu L of 50% methanol aqueous solution for redissolving, shaking and centrifuging, and determining by LC-MS/MS. (concentration: the content of androstenone, Testosterone, Corticosterone, Hydrocortisone in the control matrices was not calculated, and the actual sample concentration (working fluid concentration + working fluid volume + control matrix concentration)/total volume)
Batch precision and accuracy studies were determined by analyzing 5 concentration levels of quality control samples (LLOQ, LQC, GMQC, MQC and HQC, n ═ 6) within a batch. Batch precision and accuracy studies were determined by 5 concentration levels (LLOQ, LQC, GMQC, MQC and HQC, n ═ 6) of at least 3 batches over at least two days. The measurement results are shown in table 4, and the results in table 4 show that the method for determining the concentrations of endogenous cortisol, corticosterone, androstenedione and testosterone in plasma by LC-MS/MS meets the requirements of analytical measurement on accuracy in batch and precision. The method has good accuracy and precision, and can fully verify the operability of the established method.
TABLE 4 results of Intra-and Inter-batch precision and accuracy
Cortisol
Corticosterone
Androstenedione
Testosterone
Recovery and matrix effects of the process: the extraction recovery and matrix effect of the control matrix was demonstrated by comparing the similarity of the analyte/internal standard extraction recovery/matrix effect in the control matrix and the surrogate matrix. Control matrices (actual plasma matrices) were determined for 6 individual subjects, and the mean value was determined as the theoretical endogenous concentration for the subject.
The extraction recovery and matrix effect of the control matrix was demonstrated by comparing the similarity of the analyte/internal standard extraction recovery/matrix effect in the control matrix and the surrogate matrix. Determining normal plasma of at least 6 different individuals, and determining the average value as the theoretical endogenous concentration of the individuals; adding LQC, GMQC and HQC quality control working solutions into an actual matrix, preparing at least 3 parts of each concentration sample, and performing quantification by using a standard curve replacing the matrix. This approach further investigated parallelism among different individuals. Acceptance criteria: the precision of the endogenous concentration of 6 different individuals is not more than 20%, the accuracy deviation of the LQC concentration after dilution of each individual is not more than +/-20%, and the precision is not more than 20%. The results are shown in Table 5:
TABLE 5 recovery and substrate Effect results
Cortisol
Corticosterone
Androstenedione
Testosterone
As can be seen from Table 5, the matrix effect/recovery rate of endogenous cortisol, corticosterone, androstenedione and testosterone meets the experimental requirements, which shows that the mass spectrum and chromatographic conditions effectively avoid the matrix effect, the requirements of human plasma endogenous cortisol, corticosterone, androstenedione and testosterone analysis and determination are met, and the operability of the established method is fully verified.
3: determination of actual samples
The plasma of 6 healthy volunteers was selected for the determination of the concentrations of endogenous cortisol, corticosterone, androstenedione and testosterone. Plasma samples were processed as "pretreatment of plasma samples under item 1.2" and liquid quality analysis was performed according to the liquid phase and mass spectrometry conditions as described in example 1, and the analysis batches contained two standard curves, requiring at least 4 concentration level quality control samples (HQC, MQC, GMQC and LQC), at least 2 replicates per concentration, and in amounts not less than 5% of the total amount of samples in each analysis batch. The concentrations of the quality control sample and the unknown sample were calculated from the standard curve. The accuracy deviation of the standard samples of the standard curve samples is not more than +/-15%, the accuracy deviation of the quantitative lower limit of the standard samples is not more than +/-20%, the standard curve samples which do not accord with the acceptance standard do not participate in the regression of the standard curve, the elimination of the standard curve samples starts from the maximum deviation, then the regression is carried out again, the standard curve samples which do not accord with the acceptance standard and have the maximum deviation are eliminated, the standard samples which do not accord with the acceptance standard are eliminated in sequence until all the samples which participate in the regression of the standard curve meet the acceptance standard of the accuracy deviation, the samples which participate in the regression of the standard curve are not less than 75% of non-zero standard samples, and each standard sample with the concentration of at least 50% meets the acceptance standard or the standard; the coefficient of determination of the linear regression (R2) should not be less than 0.9801. At least 67% of the quality control samples in the quality control samples and at least 50% of the quality control samples in each concentration level have accuracy deviation of not more than +/-20%; the average accuracy deviation of each concentration quality control sample of all received analysis batches is not more than +/-20%, and the precision is within 20%. The results are shown in Table 6.
TABLE 66 measurement of endogenous Cortisol, Cortisone, androstenedione, Testosterone in the plasma of healthy volunteers
The linear range of endogenous corticosterone, androstenedione and testosterone in human plasma is 0.100-50.0ng/mL by LC-MS (liquid chromatography-mass spectrometry), the lower limit of the quantification is 0.100ng/mL, and the linear relation is good in the range of 0.100-50.0 ng/mL; the linear range of endogenous cortisol is 4.00-400ng/mL, the lower limit of quantitation is 4.00ng/mL, and the linear relation is good in the range of 4.00-400 ng/mL. 3% BSA normal saline is selected as a substitute matrix, so that the interference of endogenesis on accurate quantification can be avoided; the accuracy, precision, recovery rate, matrix effect and other methodological verification and investigation meet the requirements, and the requirements of detecting the concentrations of endogenous cortisol, corticosterone, androstenedione and testosterone in human plasma are met.
In conclusion, the method for quantitatively detecting the concentrations of endogenous cortisol, corticosterone, androstenedione and testosterone in human plasma by using liquid chromatography-mass spectrometry can accurately, quickly and reliably detect the concentrations of cortisol, corticosterone, androstenedione and testosterone in blood samples.
The foregoing is merely an example of the embodiments of the present invention, and it should be noted that, for the technical field, several improvements and modifications can be made without departing from the technical principle of the present invention, and these improvements and modifications should be construed as the protection scope of the present invention.
Claims (2)
1.A method for simultaneously measuring the concentrations of endogenous cortisol, corticosterone, androstenedione and testosterone in human plasma by liquid chromatography-mass spectrometry, which is characterized by comprising the following steps:
(1) selection of alternative matrices: selecting 3% BSA normal saline solution as a substitute matrix to carry out a methodological verification test and a sample analysis test of cortisol, corticosterone, androstenedione and testosterone in human plasma;
(2) determination of the endogenous cortisol, corticosterone, androstenedione, testosterone concentrations in human plasma: preparing a standard curve sample by using the substitute matrix determined in the step (1), determining the concentrations of background cortisol, corticosterone, androstenedione and testosterone in human plasma of each used batch, and determining the average concentrations of cortisol, corticosterone, androstenedione and testosterone in the mixed control matrix to be 79.6ng/mL, 1.94ng/mL, 1.06ng/mL and 1.70ng/mL respectively so as to calculate the theoretical concentration of an actual quality control sample;
(3) preparation and pretreatment of a standard curve sample: comprises the preparation of content solution, the preparation of standard series solution and the pretreatment of a working curve sample, wherein,
preparing an internal standard solution by taking a dexamethasone standard substance, correcting by a mass correction factor, and dissolving the dexamethasone standard substance in methanol to obtain a stock solution with the final concentration of 1.00 mg/mL; diluting the solution with 50% methanol water solution to obtain 200ng/mL internal standard working solution;
preparing a standard series of solutions by taking a proper amount of cortisol, corticosterone, androstenedione and testosterone standard products respectively and preparing stock solutions with the concentration of 1.00mg/mL by using methanol; diluting with 50% methanol water to obtain serial working solutions with cortisone, androstenedione, and testosterone concentrations of 4.00, 8.00, 40.0, 200, 400, 1200, 1800, 2000ng/mL, and cortisol concentrations of 160, 320, 800, 2400, 4800, 9600, 14400, and 16000 ng/mL; then mixing the two sets of working solutions in an equal volume according to corresponding concentration points to finally obtain a standard curve working solution required by preparing a standard curve sample, wherein the concentrations of corticosterone, androstenedione and testosterone are 2.00, 4.00, 20.0, 100, 200, 600, 900 and 1000 ng/mL; cortisol concentrations of 80.0, 160, 400, 1200, 2400, 4800, 7200, and 8000 ng/mL;
the pretreatment of the working curve sample comprises the steps of respectively adding 15.0 mu L of the series working solutions used for standard curve preparation in the step into 285 mu L of alternative matrixes to prepare samples corresponding to corticosterone, androstenedione and testosterone with the concentrations of 0.100ng/mL, 0.200ng/mL, 1.00ng/mL, 5.00ng/mL, 10.0ng/mL, 30.0ng/mL, 45.0ng/mL, 50.0ng/mL and cortisol with the concentrations of 4.00ng/mL, 8.00ng/mL, 20.0ng/mL, 60.0ng/mL, 120ng/mL, 240ng/mL, 360ng/mL and 400ng/mL, and subpackaging 2 samples with each 100 mu L; adding 15.0 mu L of internal standard working solution into each part, adding 400 mu L of ethyl acetate for extraction, centrifuging, taking 200 mu L of supernatant, drying under nitrogen, adding 150 mu L of 50% methanol aqueous solution for redissolving, shaking and centrifuging, and determining by LC-MS/MS;
(4) preparation and pretreatment of a quality control sample: comprises the preparation of standard series solution and the pretreatment of quality control samples, wherein,
preparing a standard series of solutions by respectively taking appropriate amounts of cortisol, corticosterone, androstenedione and testosterone standard substances, and respectively preparing stock solutions with the concentration of 1.00mg/mL by using methanol; diluting with 50% methanol water to obtain a series of working solutions containing corticosterone, androstenedione, W1-HQC with testosterone concentration of 1600ng/mL, W1-MQC with 800ng/mL, W1-GMQC1 with 80.0ng/mL, W1-GMQC2 with 40.0ng/mL, W1-LQC with 12.0ng/mL, W2-HQC with cortisol concentration of 10000ng/mL, W2-MQC with 6000ng/mL, W2-GMQC with 2000ng/mL and W2-LQC with 480 ng/mL; and then mixing the two sets of working solutions in equal volume according to corresponding concentration points to finally obtain a quality control working solution required by preparing a standard curve sample: the high-concentration quality control working solution W-HQC is W1-HQC and W2-HQC are mixed, the concentrations of corticosterone, androstenedione and testosterone are 800ng/mL, and the concentration of cortisol is 5000 ng/mL; the medium-concentration quality control working solution W-MQC is a mixture of W1-MQC and W2-MQC, the concentrations of corticosterone, androstenedione and testosterone are 400ng/mL, and the concentration of cortisol is 3000 ng/mL; the geometric concentration quality control working solution W-GMQC is W1-GMQC1 and W2-GMQC are mixed, the concentrations of corticosterone, androstenedione and testosterone are 40.0ng/mL, and the concentration of cortisol is 1000 ng/mL; the low-concentration quality control working solution W-LQC is W1-LQC mixed with W2-LQC, the concentrations of corticosterone, androstenedione and testosterone are 6.00ng/mL, and the concentration of cortisol is 240 ng/mL;
pretreatment of a quality control sample: respectively adding 35.0 μ L of the above W1-GMQC2 working solution, W-MQC working solution and W-HQC working solution into 665 μ L of human blank plasma, and preparing corresponding GMQC quality control samples, MQC quality control samples and HQC quality control samples; taking human plasma with a certain volume, diluting the human plasma with a substitute matrix to be close to low quality control concentration and quantitative lower limit concentration respectively, preparing LQC and LLOQ quality control samples, and subpackaging 6 parts of each quality control sample, wherein each part is 100 mu L; adding 15.0 mu L of internal standard working solution into each part, adding 400 mu L of ethyl acetate for extraction, centrifuging, taking 200 mu L of supernatant, drying under nitrogen, adding 150 mu L of 50% methanol aqueous solution for redissolving, shaking and centrifuging, and determining by LC-MS/MS;
(5) by adopting an internal standard method, taking the concentrations of series of cortisol, corticosterone, androstenedione and testosterone in a substitute matrix as a horizontal coordinate, taking the peak area ratios of the cortisol, corticosterone, androstenedione and testosterone to internal standard dexamethasone as vertical coordinates, performing linear regression operation, wherein the weight factor is 1/x2 to obtain a linear regression equation, namely a working curve, and determining the concentrations of endogenous cortisol, corticosterone, androstenedione and testosterone in human plasma by using the working curve.
2. The method for simultaneous LC-MS/MS measurement of the concentrations of endogenous cortisol, corticosterone, androstenedione and testosterone in human plasma according to claim 1, wherein said LC-MS/MS measurement conditions comprise:
liquid chromatography conditions:
a chromatographic column: ACQUITY UPLC BEH C181.7 μm, 50X 2.1 mm;
mobile phase: a 0.1% aqueous formic acid solution containing 2% methanol; 0.1% formic acid in methanol, 5% water, gradient as follows:
0-0.60min, 70.0% of mobile phase A and 30.0% of mobile phase B; 0.60-1.50min, the mobile phase A is 70.0-40.0%, and the mobile phase B is 30.0-60.0%; 1.50-3.80min, the mobile phase A is from 40.0% to 30.0%, and the mobile phase B is from 60.0% to 70.0%; 3.80-4.00min, the mobile phase A is from 30.0% to 0%, and the mobile phase B is from 70.0% to 100%; 4.00-4.30min, keeping the mobile phase A at 0% and the mobile phase B at 100%; 4.30-4.40min, the mobile phase A is from 0% to 70.0%, and the mobile phase B is from 100% to 30.0%; 4.40-5.00min, the mobile phase A is kept at 70.0%, and the mobile phase B is kept at 30.0%;
flow rate: 0.500 mL/min;
column temperature: 40; c
Sample introduction amount: 10.0 μ L;
mass detector and conditions: the mass spectrum detector is Triple Quad 6500+ AB Sciex; the ion source is an electrospray ion source; the spraying voltage is 5500V; ion source temperature: 550; curtain gas CUR at DEG C: 20psi, collision gas CAD: 9psi, GS 1: 50psi, GS 2: 20 psi; detecting in a positive ion mode; the scanning mode is as follows: MRM; the quantitative ion pair of androstenedione is 287.2 → 97.1, and the collision voltage is 28.0 eV; the testosterone quantitative ion pair is 289.2 → 109.1, and the collision voltage is 31.0 eV; the corticosterone quantitative ion pair is 347.2 → 293.1, and the collision voltage is 25.0 eV; the cortisol quantitative ion pair is 363.2 → 327.3, and the collision voltage is 23.0 eV; the internal standard dexamethasone quantitative ion pair is 393.1 → 373.3, and the collision voltage is 13.0 eV.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114487229A (en) * | 2022-02-09 | 2022-05-13 | 杭州佰辰医疗器械有限公司 | Preparation method of endogenous micromolecular compound quality control product for blood plasma and blood serum |
| CN115236246A (en) * | 2022-09-26 | 2022-10-25 | 北京金域医学检验实验室有限公司 | Method for extracting multiple steroid hormones from organism fluid sample |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140349885A1 (en) * | 2012-01-05 | 2014-11-27 | Dh Technologies Development Pte. Ltd. | Enhancement of sensitivity and specificity of ketosteroids and keto or aldehyde containing analytes |
| CN107064400A (en) * | 2017-04-20 | 2017-08-18 | 博厚健康科技股份有限公司 | The method for detecting five steroids hormones in serum simultaneously |
| CN109001335A (en) * | 2018-09-13 | 2018-12-14 | 山东师范大学 | Method for measuring 17 steroids hormones in krill |
-
2019
- 2019-12-25 CN CN201911361003.7A patent/CN111103383A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140349885A1 (en) * | 2012-01-05 | 2014-11-27 | Dh Technologies Development Pte. Ltd. | Enhancement of sensitivity and specificity of ketosteroids and keto or aldehyde containing analytes |
| CN107064400A (en) * | 2017-04-20 | 2017-08-18 | 博厚健康科技股份有限公司 | The method for detecting five steroids hormones in serum simultaneously |
| CN109001335A (en) * | 2018-09-13 | 2018-12-14 | 山东师范大学 | Method for measuring 17 steroids hormones in krill |
Non-Patent Citations (5)
| Title |
|---|
| FATIH YESILDAL ET AL.: "A practical ID-LC-MS/MS method for the most commonly analyzed steroid hormones in clinical laboratories", 《TURK J BIOCHEM》 * |
| 任进民 等: "液相色谱-质谱法同时测定大鼠血浆中游离型神经甾体", 《解放军药学学报》 * |
| 童鸿斌 等: "液相色谱-串联质谱法检测血清中15种甾体激素的动态变化", 《色谱》 * |
| 米晓霞: "禽蛋及猪血浆中类固醇激素含量的检测——液相色谱串联质谱法", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
| 闫映飞: "基于LC-MS/MS技术的心脑血管疾病脂质类标志物研究", 《万方数据 华中科技大学硕士学位论文》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114487229A (en) * | 2022-02-09 | 2022-05-13 | 杭州佰辰医疗器械有限公司 | Preparation method of endogenous micromolecular compound quality control product for blood plasma and blood serum |
| CN115236246A (en) * | 2022-09-26 | 2022-10-25 | 北京金域医学检验实验室有限公司 | Method for extracting multiple steroid hormones from organism fluid sample |
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