CN112611828B - Method for purifying, enriching and detecting steroid hormone in blood - Google Patents
Method for purifying, enriching and detecting steroid hormone in blood Download PDFInfo
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Abstract
The invention relates to a method for purifying, enriching and detecting steroid hormones in blood, belonging to the technical field of medical inspection and analysis. The sample containing steroid hormone is prepared by adopting a low-density three-point bonding C18 filler taking silica gel as a matrix, and the purification and enrichment of the steroid hormone in the sample are realized by optimizing the solvent dosage, the eluent ratio and other parameters in the preparation process. The prepared steroid hormone sample is analyzed by a liquid chromatography-tandem mass spectrometry method under certain conditions, so that the simultaneous detection of multiple steroid hormones at one time is realized. The method has the characteristics of good stability, high sensitivity, high recovery rate, simple and controllable operation, capability of realizing automatic operation and the like.
Description
Technical Field
The invention belongs to the technical field of medical inspection and analysis, and relates to a method for purifying, enriching and detecting steroid hormones in blood.
Technical Field
Steroid hormones are tetracyclic aliphatic hydrocarbon compounds having a cyclopentane-polyhydrophenanthrene nucleus, which are derived from cholesterol and regulate metabolic processes in the body through endocrine systems. Progestagens are precursor substances of all other steroid hormones. Corticosteroids are produced primarily by the adrenal gland and are primarily associated with the metabolism of glucose and minerals. Sex hormones are divided into two classes, male hormone and female hormone, which can be mutually transformed, and a balance exists in animals of different sexes. Androgens predominate in males, and estrogens predominate in females. When abnormal fluctuations in the levels of hormones occur in the body, significant diseases such as congenital adrenal cortical hyperplasia, adrenocortical insufficiency (Addison's disease), hyperandrogenism, Cushing's syndrome, polycystic ovary syndrome, congenital metabolic defects and malformations, and other metabolic disorders can be caused. Accurate assessment of hormone levels in the body is clinically significant.
At present, the steroid hormone is mainly determined by an immunoassay method, the defect of poor specificity exists, the result is often inaccurate due to the cross reaction of antibodies, and the result of multiple steroid hormones cannot be obtained in one detection. With the gradual application of high performance liquid-tandem mass spectrometry in steroid hormone detection in recent years, the high precision and accuracy of the method are more and more accepted by the industry. However, steroid hormone detection still has some problems, such as low content of the hormone in human body and detection sensitivity; the problem of interference of endogenous substances with hormone detection; variability of results due to matrix effects; the stability of the pretreatment technology of the biological sample and the like. Based on this, there is an urgent need to develop an effective purification and enrichment material for detecting steroid hormones and a detection method thereof.
Disclosure of Invention
The invention aims to provide a purification and enrichment method for detecting steroid hormones in blood and a detection method thereof.
In one embodiment, the present invention provides a method for detecting steroid hormones in a blood sample, the method comprising:
(1) enriching and purifying one or more steroid hormone components in the serum sample by using a solid phase extraction column;
(2) optionally, derivatizing the enriched purified steroid hormone component with a derivatizing agent;
(3) detecting the derivatized and/or underivatized one or more steroid hormone components using liquid chromatography-tandem mass spectrometry.
In one embodiment, the solid phase extraction column adopts bonded filler taking silica gel as a matrix, and the bonded density of the filler is 1.6-2.2 mu mol/m2And the carbon content is 10-12 percent, and the filler is C18.
Further, the silica gel particle size of the filler is 10-100 μm, and the pore diameter is
Specific surface area 100m2/g-600m2/g。
Further, the structural formula of the solid phase extraction material is as follows:
further, the solid phase extraction column is a solid phase extraction column with the specification of 30-100mg/1ml or 100-500mg/5 ml.
In some embodiments, the steroid hormone is selected from one or more of an estrogen, a progestin, an androgen or a corticosteroid.
Further, the estrogen is selected from one or more of estrone, estradiol and estriol; the progestogen is selected from one or more of progesterone, 17-hydroxyprogesterone, pregnenolone, and 17-hydroxyprogesterone; the androgen is selected from one or more of testosterone, androstenedione, dihydrotestosterone, dehydroepiandrosterone and dehydroepiandrosterone sulfate; the cortical hormone is one or more selected from cortisol, corticosterone, cortisone, 11-deoxycorticosterone, 11-deoxycorticosterol and 21-deoxycorticosterol.
In some embodiments, the enrichment purification process is performed by first using an organic solvent: activating the first mixed solution with water at 90:10 (v/v); and adding an organic solvent: water-5: 95(v/v) of the second mixed solution; then adding the blood sample to be detected into the extraction column according to the amount of 0.5-10% of the filler mass of the solid phase extraction column, and sequentially adding a first mixed solution and an organic solvent: water 30: leaching the third mixed solution of 70 (v/v); finally, eluting with an organic solvent, and collecting all eluted fractions.
Further, the organic solvent is selected from one or more of methanol, ethanol, isopropanol, acetonitrile, n-hexane, dichloromethane, chloroform and acetone.
In one embodiment, the present invention provides a method for detecting steroid hormones in a blood sample, the method comprising the steps of:
step (1):
a) filling a solid phase extraction column: the bonding density of the packing silica gel is 1.8 mu mol/m2The carbon content is 10%, the granularity is 30 mu m, and the pore diameter is
Specific surface area 300m2100mg of C18 filler with three-point bonding per gram is placed in a 1mL column tube;
b) preparing an internal standard working solution: accurately preparing 1mg/mL isotope labeled internal standard stock solution of steroid hormone components, and diluting with 50% methanol to obtain diluted mixed internal standard working solution for later use;
c) preparation of a sample: adding 100-500 mu L of serum sample into 10-50 mu L of the mixed internal standard working solution in the step (b), and uniformly mixing by vortex for later use;
d) solid phase extraction and separation: the solid phase extraction column first adopts 1mL of organic solvent: water 90:10(v/v) activation; and adding an organic solvent: water-5: 95(v/v) equilibrium; directly adding 500 mu L of the serum sample to be detected in the step c); add 500 μ L of organic solvent: water 30: 70(v/v) leaching; finally, eluting with 300-;
step (2): adding 30-200 mu L of derivatization reagent into the fraction obtained in the step d) for derivatization reaction to obtain a derivatization product; or directly adding the compound solution to obtain hormone prototype extract; and (3):
a) analysis of the samples: analyzing and detecting the derivative product or the hormone prototype extract, separating the components to be detected by adopting reverse chromatography, detecting by triple quadrupole tandem mass spectrometry to obtain a sample spectrogram, and detecting to obtain the positive ions and/or the negative ions of the steroid hormone;
wherein, the analysis conditions of the high performance liquid chromatography-tandem mass spectrometry are as follows:
A chromatographic column: waters CORTECS T32.7 μm, 2.1X 100mm
Mobile phase A: 0.2-5mM ammonium formate, 0.1% aqueous formic acid; mobile phase B: acetonitrile
The flow rate is 0.2-0.5 mL/min; the column temperature is 30-55 ℃; sample introduction amount: 1-10 μ L;
liquid chromatography Condition 2
A chromatographic column: luna C83 μm, 2 x 100mm
Mobile phase A: 0.2-5mM ammonium formate; mobile phase B: methanol
The flow rate is 0.2-0.5 mL/min; the column temperature is 30-55 ℃; sample introduction amount: 1-10 μ L;
mass spectrometric analysis Condition 1
Ionization mode: ESI positive ions or ESI positive and negative ions are scanned simultaneously; spraying voltage: 4.0 KV;
desolventizing gas temperature: 550 ℃; atomizing: 50 psi; assisting atomization gas: 50 psi; air curtain air: 30 psi; the scanning mode is as follows: multiple Reaction Monitoring (MRM);
mass spectrometric analysis Condition 2
Ionization mode: ESI positive ion; capillary voltage: 2.0 KV; taper hole voltage: 50V; desolventizing gas temperature: 450 ℃; desolventizing air flow rate: 800L/Hr; taper hole air flow rate: 150L/Hr; the scanning mode is as follows: multiple Reaction Monitoring (MRM);
b) and (3) analyzing a quantitative calculation result: and obtaining a linear regression equation according to the ratio of the peak area of the steroid hormone ion chromatographic peak in the sample chromatogram to the peak area of the corresponding isotope internal standard ion chromatographic peak and the corresponding concentration, and calculating the concentration of the steroid hormone in the serum sample to realize the detection of the steroid hormone in the serum.
In some embodiments, when the steroid hormone is estrone, estradiol and/or estriol, the dilution concentration of d 4-estrone and d 3-estradiol in the isotope mixed internal standard working solution is 2ng/ml, the dilution concentration of d 3-estriol is 20ng/ml, and the derivatization reagent is dansyl chloride;
in some embodiments, when the steroid hormone is testosterone, androstenedione, dihydrotestosterone, dehydroepiandrosterone, and/or dehydroepiandrosterone sulfate, the d3 testosterone, and the d-epiandrosterone sulfate are present in the isotopically mixed internal standard working solution,13The dilution concentration of C-androstenedione is 5ng/ml, the dilution concentration of d 3-dihydrotestosterone is 10ng/ml, the dilution concentration of d 6-dehydroepiandrosterone is 100ng/ml, the dilution concentration of d 6-dehydroepiandrosterone sulfate is 5 mu g/ml, and the derivatization reagent is hydroxylamine hydrochloride;
in some embodiments, when the steroid hormone is progesterone, 17 α -hydroxyprogesterone, pregnenolone and/or 17 α -hydroxyprogesterone, the dilution concentration of d 9-progesterone, d8-17 α -hydroxyprogesterone, d 4-pregnenolone and d3-17 α -hydroxyprogesterone in the isotope mixed internal standard working solution is 20ng/ml, and the double solution is an organic reagent;
in some embodiments, when the steroid hormone is cortisol, corticosterone, cortisone, 11-deoxycorticosterone, 11-deoxycorticosterol and/or 21-deoxycorticosterol, the dilution concentration of d 6-cortisol in the isotope mixing internal standard working solution is 200ng/mL, the dilution concentration of d 4-corticosterone is 10ng/mL, the dilution concentration of d 8-cortisone is 200ng/mL, the dilution concentration of d 7-11-deoxycorticosterone is 5ng/mL, the dilution concentration of d 5-11-deoxycorticosterol is 10ng/mL, and the dilution concentration of d 8-21-deoxycorticosterol is 5ng/mL, the compound solution is an organic reagent.
Has the advantages that:
(1) the enrichment purification filler adopted by the invention has a novel structure. The principle of purifying the enrichment material is to design a low-density three-point bonded C18 filler, wherein the low density can realize the tolerance of the filler to pure water substances and realize the direct sample loading of biological samples. Because the space structure of steroid hormone is bigger, according to the steric hindrance principle (as shown in figure 5), the low-density bonding phase is equivalent to increasing the item S (Steric), so that the volume of the hormone entering between the bonding phases is increased, and on one hand, the retention of the steroid hormone on the filler is favorably increased, and the enrichment effect is achieved; on the other hand, the steroid hormone is separated from interfering substances in the biological matrix, and the function of purification is achieved.
(2) The application range is wide. The solid phase extraction filler provided by the invention has purification and enrichment effects on steroid structures with cyclopentane-polyhydrophenanthrene parent nucleus.
(3) The method has the characteristics of good stability, high sensitivity, high recovery rate, simple and controllable operation, capability of realizing automatic operation and the like.
(4) The method of the invention is accurate in qualitative and quantitative determination. The method adopts high performance liquid chromatography-tandem mass spectrometry for detection, the biological sample is corrected by adding an isotope internal standard, the standard curve is quantitative, the result accuracy is high and stable, and the method can be used for qualitative and quantitative determination of steroid hormone in clinical blood samples.
Drawings
FIG. 1 is an MRM chromatogram of estrogen in the serum of example 1;
FIG. 2 is an MRM chromatogram of androgen in the serum of example 2;
FIG. 3 is an MRM chromatogram of progestin in serum of example 3;
FIG. 4 is an MRM chromatogram of cortical hormone in serum of example 4;
FIG. 5 is a schematic diagram of the steric hindrance of a three point bonded C18 filler employed in the present invention.
Detailed Description
The invention will now be further described with reference to the following examples, which are intended to be illustrative of the invention and are not to be construed as limiting the invention.
Example 1
Preparation of three-point bonded C18 filler
Using dry silica gel, adding into toluene for stirring, then dropwise adding silane (mainly dimethyl octadecyl chlorosilane), heating to 120 ℃, and stirring for reacting for 6 hours. Cooling to room temperature, filtering, and washing with toluene, methanol, water and methanol sequentially. Drying at 80 deg.C for 12 hr.
The bonding density of the C18 solid-phase extraction filler is 1.6-2.2 mu mol/m through the identification of a solid nuclear magnetic resonance method2And the carbon content is 10-12 percent, and the filler is C18. The particle size of the silica gel is 10-100 μm, and the pore diameter is
The specific surface area is 100m2/g-600m 2/g. The structure is as follows:
example 2
Detection of estrogens in serum
1. Filling a solid phase extraction column: the filler is filled in 100mg and placed in a 1mL column tube (the bonding density of the filled silica gel is 1.8 mu mol/m)2The carbon content is 10%, the granularity is 30 mu m, and the pore diameter is
Specific surface area 300m2A three-point bonded C18 filler,/g);
2. sample pretreatment process
(1) Preparing a standard working solution: accurately preparing stock solutions (1mg/mL) of estrone, estradiol and estriol standard products, diluting the stock solutions with 50% methanol step by step to obtain standard-mixed working solutions, wherein the concentrations of estrone and estradiol are 20ng/mL, 10ng/mL, 5ng/mL, 1ng/mL, 0.2ng/mL, 0.1ng/mL and 0.05ng/mL, and the concentrations of estriol are 200ng/mL, 100ng/mL, 50ng/mL, 10ng/mL, 2ng/mL, 1ng/mL and 0.5ng/mL for later use;
(2) preparing an internal standard working solution: accurately preparing an isotope internal standard stock solution (1mg/mL) of estrone, estradiol and estriol, diluting with 50% methanol to obtain a mixed internal standard working solution, wherein the concentrations of d 4-estrone and d 3-estradiol are 2ng/mL, and the concentration of d 3-estriol is 20ng/mL for later use;
(3) preparation of a standard curve: and (3) respectively taking 30 mu L of the serial concentration standard-mixed working solution in the step (1), adding 270 mu L of the blank matrix solution without hormone, adding 30 mu L of the mixed standard working solution in the step (2), and uniformly mixing by vortex for later use.
(4) Preparation of a sample: and (3) adding 300 mu L of serum into 30 mu L of the mixed internal standard working solution in the step (2), and uniformly mixing by vortex for later use.
(5) Solid phase extraction and separation: the solid phase extraction column is activated by 1mL of 90% methanol; then adding 5% methanol water for balancing; adding 300 mu L of serum sample to be detected directly; adding 500 mu L of 5% methanol water and 500 mu L of 30% methanol water in sequence for leaching; finally, the mixture is eluted by 400 mu L of methanol, and all elution fractions are collected and dried by nitrogen.
(6) And (3) adding 100 mu L of 1mg/mL dansyl chloride into the fraction obtained in the step (5) for derivatization to obtain a derivatization product.
3. Analysis of the samples: analyzing and detecting the sample to be detected in the step 2 and the step 6, separating the component to be detected by adopting a hydrophilic reverse chromatogram, and detecting by a triple quadrupole tandem mass spectrometry to obtain a sample spectrogram.
4. Analysis conditions of high performance liquid chromatography-tandem mass spectrometry
(1) Conditions of liquid chromatography
A chromatographic column: waters CORTECS T32.7 μm, 2.1X 100mm
Mobile phase A: 2mM ammonium formate, 0.1% aqueous formic acid; mobile phase B: acetonitrile
The flow rate is 0.4 mL/min; the column temperature is 35 ℃; sample introduction amount: 2 mu L of the solution;
TABLE 1 Estrogen gradient elution conditions
(2) Conditions for mass spectrometry
Ionization mode: ESI positive ion; spraying voltage: 4.0 KV; desolventizing gas temperature: 550 ℃; atomizing: 50 psi; assisting atomization gas: 50 psi; air curtain air: 30 psi; the scanning mode is as follows: multiple Reaction Monitoring (MRM).
TABLE 2 Estrogen MRM Mass Spectrometry parameters
5. And (3) analyzing a quantitative calculation result: and (3) obtaining a linear regression equation according to the ratio of the peak area of the estrone, estradiol and estriol ion chromatographic peak to the peak area of the corresponding isotope internal standard ion chromatographic peak in the sample chromatogram and the corresponding concentration, and calculating the concentrations of the estrone, estradiol and estriol in the serum sample to realize the detection of the estrogen in the serum (the result is shown in figure 1 and table 3).
TABLE 3 Estrogen Linear regression equation and limits of detection
Example 3
Detection of androgens in serum
1. Filling a solid phase extraction column: the filling specifications and procedure were the same as in example 1.
2. Sample pretreatment process
(1) Preparing a standard working solution: accurately preparing standard stock solution (1mg/mL) of testosterone, androstenedione, dihydrotestosterone, dehydroepiandrosterone and dehydroepiandrosterone sulfate, diluting with 50% methanol step by step to obtain mixed standard working solution, wherein the concentrations of testosterone and androstenedione are 150ng/mL, 75ng/mL, 15ng/mL, 6ng/mL, 1.5ng/mL, 0.6ng/mL and 0.3ng/mL, the concentrations of dihydrotestosterone are 250ng/mL, 125ng/mL, 25ng/mL, 10ng/mL, 2.5ng/mL, 1ng/mL and 0.5ng/mL, the concentrations of dehydroepiandrosterone are 2500ng/mL, 1250ng/mL, 250ng/mL, 100ng/mL, 25ng/mL, 10ng/mL and 5ng/mL, and the concentrations of dehydroepiandrosterone sulfate are 100 μ g/mL, 50 μ g/mL, 5ng/mL, 10. mu.g/mL, 4. mu.g/mL, 1. mu.g/mL, 0.4. mu.g/mL and 0.2. mu.g/mL for use;
(2) preparing an internal standard working solution: accurately preparing testosterone, androstenedione, dihydrotestosterone, dehydroepiandrosterone and dehydroepiandrosterone sulfate isotope internal standard stock solution (1mg/mL), diluting with 50% methanol to obtain mixed internal standard working solution, d 3-testosterone,13The concentration of C-androstenedione is 5ng/mL, the concentration of d 3-dihydrotestosterone is 10ng/mL, the concentration of d 6-dehydroepiandrosterone is 100ng/mL, and the concentration of d 6-dehydroepiandrosterone sulfate is 5 mu g/mL for later use;
(3) preparation of a standard curve: and (3) respectively taking 30 mu L of the serial concentration standard-mixed working solution in the step (1), adding 270 mu L of the blank matrix solution without hormone, adding 30 mu L of the mixed standard working solution in the step (2), and uniformly mixing by vortex for later use.
(4) Preparation of a sample: and (3) adding 300 mu L of serum into 30 mu L of the mixed internal standard working solution in the step (2), and uniformly mixing by vortex for later use.
(5) Solid phase extraction and separation: the solid phase extraction column is activated by 1mL of 90% methanol; then adding 5% methanol water for balancing; adding 300 mu L of serum sample to be detected directly; adding 500 mu L of 5% methanol water and 500 mu L of 30% methanol water in sequence for leaching; finally, the mixture is eluted by 400 mu L of methanol, and all elution fractions are collected and dried by nitrogen.
(6) And (3) adding 100 mu L of 100mM hydroxylamine hydrochloride into the fraction obtained in the step (5) for derivatization to obtain a derivatization product.
3. Analysis of the samples: analyzing and detecting the sample to be detected in the step 2 and the step 6, separating the component to be detected by adopting a hydrophilic reverse chromatogram, and detecting by a triple quadrupole tandem mass spectrometry to obtain a sample spectrogram.
4. Analysis conditions of high performance liquid chromatography-tandem mass spectrometry
(1) Conditions of liquid chromatography
A chromatographic column: waters CORTECS T32.7 μm, 2.1X 100mm
Mobile phase A: 2mM ammonium formate, 0.1% aqueous formic acid; mobile phase B: acetonitrile
The flow rate is 0.4 mL/min; the column temperature is 35 ℃; sample introduction amount: 2 mu L of the solution;
TABLE 4 androgen gradient elution conditions
(2) Conditions for mass spectrometry
Capillary voltage: 2.0 KV; taper hole voltage: 50V; desolventizing gas temperature: 450 ℃; desolventizing air flow rate: 800L/Hr; taper hole air flow rate: 150L/Hr; the scanning mode is as follows: multiple Reaction Monitoring (MRM).
TABLE 5 androgen MRM Mass Spectrometry parameters
5. And (3) analyzing a quantitative calculation result: and obtaining a linear regression equation according to the ratio of the peak area of the chromatographic peak of testosterone, androstenedione, dihydrotestosterone, dehydroepiandrosterone and dehydroepiandrosterone sulfate ion in the sample chromatogram to the peak area of the corresponding isotope internal standard ion chromatographic peak and the corresponding concentration, and calculating the concentrations of testosterone, androstenedione, dihydrotestosterone, dehydroepiandrosterone and dehydroepiandrosterone sulfate in the serum sample to realize the detection of the androgen in the serum (the result is shown in figure 2 and table 6).
TABLE 6 androgen linear regression equation and detection limits
Example 4
Detection of progestagens in serum
1. Filling a solid phase extraction column: the filling specifications and procedure were the same as in example 1.
2. Sample pretreatment process
(1) Preparing a standard working solution: accurately preparing standard stock solutions (1mg/mL) of progesterone, 17 alpha-hydroxyprogesterone, pregnenolone and 17 alpha-hydroxyprogesterone, diluting with 50% methanol step by step to obtain mixed standard working solutions with the concentrations of 400ng/mL, 200ng/mL, 100ng/mL, 20ng/mL, 4ng/mL, 2ng/mL and 1ng/mL for later use;
(2) preparing an internal standard working solution: accurately preparing progesterone, 17 alpha-hydroxyprogesterone, pregnenolone and 17 alpha-hydroxyprogesterone isotope internal standard stock solution (1mg/mL), diluting with 50% methanol to obtain mixed internal standard working solution, wherein the concentrations of d 9-progesterone, d8-17 alpha-hydroxyprogesterone, d 4-pregnenolone and d3-17 alpha-hydroxyprogesterone are 20ng/mL for later use;
(3) preparation of a standard curve: and (3) respectively taking 20 mu L of the serial concentration standard-mixed working solution in the step (1), adding 180 mu L of the blank matrix solution without hormone, adding 20 mu L of the mixed standard working solution in the step (2), and uniformly mixing by vortex for later use.
(4) Preparation of a sample: and (3) adding 200 mu L of serum into 20 mu L of the mixed internal standard working solution in the step (2), and uniformly mixing by vortex for later use.
(5) Solid phase extraction and separation: the solid phase extraction column is activated by 1mL of 90% methanol; then adding 5% methanol water for balancing; directly adding 200 mu L of serum sample to be detected; adding 500 mu L of 5 percent methanol water for balancing and 500 mu L of 30 percent methanol water for leaching in sequence; finally, eluting with 500 μ L of methanol, collecting all the eluted fractions, and drying with nitrogen.
(6) And (5) adding 100 mu L of methanol into the fraction obtained in the step (5) for redissolving to obtain the substance to be detected.
3. Analysis of the samples: analyzing and detecting the sample to be detected in the step 2 and the step 6, separating the components to be detected by adopting reverse chromatography, and detecting by triple quadrupole tandem mass spectrometry to obtain a sample spectrogram.
4. Analysis conditions of high performance liquid chromatography-tandem mass spectrometry
(1) Conditions of liquid chromatography
A chromatographic column: luna C83 μm, 2 x 100mm
Mobile phase A: 2mM ammonium formate; mobile phase B: methanol
The flow rate is 0.4 mL/min; the column temperature is 35 ℃; sample introduction amount: 10 mu L of the solution;
TABLE 7 gradient elution conditions for progestin
(2) Conditions for mass spectrometry
Ionization mode: simultaneously scanning ESI positive ions and ESI negative ions; spraying voltage: 4.0 KV; desolventizing gas temperature: 550 ℃; atomizing: 50 psi; assisting atomization gas: 50 psi; air curtain air: 30 psi; the scanning mode is as follows: multiple Reaction Monitoring (MRM).
TABLE 8 progestogen MRM mass spectral parameters
5. And (3) analyzing a quantitative calculation result: and obtaining a linear regression equation according to the ratio of the peak area of the ion chromatographic peak of the progesterone, 17 alpha-hydroxyprogesterone, pregnenolone and 17 alpha-hydroxyprogesterone to the peak area of the corresponding isotope internal standard ion chromatographic peak in the sample chromatogram and the corresponding concentration, and calculating the concentrations of the progesterone, 17 alpha-hydroxyprogesterone, pregnenolone and 17 alpha-hydroxyprogesterone in the serum sample to realize the detection of the progestogen in the serum (the result is shown in fig. 3 and table 9).
TABLE 9 progestogen linear regression equation and limit of detection
Example 5
Detection of corticosteroids in serum
1. Filling a solid phase extraction column: the filling specifications and procedure were the same as in example 1.
2. Sample pretreatment process
(1) Preparing a standard working solution: accurately preparing standard stock solutions (1mg/mL) of cortisol, cortisone, 11-deoxycorticosterone and 21-deoxycorticosterol, diluting with 50% methanol step by step to obtain mixed standard working solution, wherein the cortisol concentration is 5000ng/mL, 2000ng/mL, 500ng/mL, 200ng/mL, 50ng/mL, 20ng/mL and 10ng/mL, the cortisone concentration is 500ng/mL, 200ng/mL, 50ng/mL, 20ng/mL, 5ng/mL, 2ng/mL and 1ng/mL, the cortisone concentration is 2000ng/mL, 800ng/mL, 200ng/mL, 80ng/mL, 20ng/mL, 8ng/mL and 4ng/mL, the 11-deoxycorticosterol concentration is 200ng/mL, 1ng/mL, and the cortisone concentration is 1ng/mL, 80ng/mL, 20ng/mL, 8ng/mL, 2ng/mL, 0.8ng/mL and 0.4ng/mL, 11-deoxycorticosterone and 21-deoxycorticosterol concentrations of 100ng/mL, 40ng/mL, 10ng/mL, 4ng/mL, 1ng/mL, 0.4ng/mL and 0.2ng/mL, for use;
(2) preparing an internal standard working solution: accurately preparing isotope internal standard stock solutions (1mg/mL) of corticosterone, cortisol, cortisone, 11-deoxycorticosterone, 11-deoxycorticosterol and 21-deoxycorticosterol, diluting with 50% methanol to obtain a mixed internal standard working solution, wherein the dilution concentration of d 6-cortisol is 200ng/mL, the dilution concentration of d 4-corticosterone is 10ng/mL, the dilution concentration of d 8-cortisone is 200ng/mL, the dilution concentration of d 7-11-deoxycorticosterone is 5ng/mL, and the dilution concentration of d 5-11-deoxycorticosterol is 10ng/mL
The dilution concentration of the alcohol and the d 8-21-deoxycorticosterol is 5ng/mL, and the dilution concentration is 10 ng/mL;
(3) preparation of a standard curve: and (3) respectively taking 40 mu L of the serial concentration standard-mixed working solution in the step (1), adding 360 mu L of the blank matrix solution without hormone, adding 40 mu L of the mixed standard working solution in the step (2), and uniformly mixing by vortex for later use.
(4) Preparation of a sample: and (3) adding 400 mu L of serum into 40 mu L of the mixed internal standard working solution in the step (2), and uniformly mixing by vortex for later use.
(5) Solid phase extraction and separation: the solid phase extraction column is activated by 1mL of 90% methanol; then adding 5% methanol water for balancing; directly adding 400 mu L of serum sample to be detected; adding 500 mu L of 5% methanol water and 500 mu L of 30% methanol water in sequence for leaching; finally, eluting with 500 μ L of methanol, collecting all the eluted fractions, and drying with nitrogen.
(6) And (5) adding 100 mu L of methanol into the fraction obtained in the step (5) for redissolving to obtain a sample to be detected.
3. Analysis of the samples: and (3) carrying out sample loading detection on the sample to be detected in the step (2) and (5), separating the component to be detected by adopting reverse chromatography, and carrying out triple quadrupole tandem mass spectrometry detection to obtain a sample spectrogram.
4. Analysis conditions of high performance liquid chromatography-tandem mass spectrometry
(1) Conditions of liquid chromatography
(2) A chromatographic column: luna C83 μm, 2 x 100mm
(3) Mobile phase A: 2mM ammonium formate; mobile phase B: methanol
The flow rate is 0.4 mL/min; the column temperature is 35 ℃; sample introduction amount: 10 mu L of the solution;
TABLE 10 gradient elution conditions for corticosteroids
(4) Conditions for mass spectrometry
Ionization mode: ESI positive ion; spraying voltage: 4.0 KV; desolventizing gas temperature: 550 ℃; atomizing: 50 psi; assisting atomization gas: 50 psi; air curtain air: 30 psi; the scanning mode is as follows: multiple Reaction Monitoring (MRM).
TABLE 11 cortical hormone MRM Mass Spectrometry parameters
5. And (3) analyzing a quantitative calculation result: according to the cortisol, corticosterone, cortisone in a sample chromatogram map,
The ratio of the peak area of the 11-deoxycorticosterone, 11-deoxycorticosterol and 21-deoxycorticosterol ion chromatographic peak to the peak area of the corresponding isotope internal standard ion chromatographic peak and the corresponding concentration obtain a linear regression equation, and the concentrations of cortisol, corticosterone, cortisone, 11-deoxycorticosterone, 11-deoxycorticosterol and 21-deoxycorticosterol in the serum sample are calculated to realize the detection of the corticoid in the serum (the results are shown in fig. 4 and table 12).
TABLE 12 corticoid Linear regression equation and limits of detection
Example 6
The representative compounds of each steroid hormone (estradiol, testosterone, 17 alpha-hydroxyprogesterone, corticosterone) are taken as examples to illustrate the comparison of the pretreatment processes of the solid phase extraction column C8, Phenyl and the specific solid phase extraction column of the invention:
1. the three pretreatment processes of the solid phase extraction column C8, the Phenyl and the specific solid phase extraction column of the invention comprise the following steps:
(1) preparing a standard working solution: accurately preparing estradiol, testosterone, 17 alpha-hydroxyprogesterone and corticosterone stock solutions respectively at a concentration of 1mg/mL, and diluting with 50% methanol water to obtain estradiol, testosterone, 17 alpha-hydroxyprogesterone and corticosterone working solutions at a concentration of 10 ng/mL;
(2) preparation of a sample: adding 300 mu L of blank serum substrate (without target compound) into 30 mu L of the standard substance working solution in the step (1), and mixing for 5min in a vortex manner for later use;
(3) pretreatment process of a solid phase extraction column C8: activating by using 1mL of methanol; balancing 1mL of pure water; loading 300uL of the standard serum sample added in the step (2); adding 500 mu L of pure water and 500 mu L of 20% methanol water in sequence for leaching; finally, eluting with 1mL of methanol, collecting all elution fractions, and drying by nitrogen. The samples were processed by reconstitution and derivatization procedures and analyzed for testing in examples 1, 2, 3, and 4, respectively.
(4) Solid phase extraction column Phenyl pretreatment process: activating by using 1mL of methanol; balancing 1mL of pure water; loading 300uL of the standard serum sample added in the step (2); adding 500 mu L of pure water and 500 mu L of 20% methanol water in sequence for leaching; finally, eluting with 1mL of methanol, collecting all elution fractions, and drying by nitrogen. The samples were processed by reconstitution and derivatization procedures and analyzed for testing in examples 1, 2, 3, and 4, respectively.
(5) Pretreatment of a specific C18 solid-phase extraction column: activating by using 1mL of 90% methanol; then adding 5% methanol water for balancing; adding 300 mu L of serum sample to be detected directly; adding 500 mu L of 5% methanol water and 500 mu L of 30% methanol water in sequence for leaching; finally, eluting with 500 μ L of methanol, collecting all the eluted fractions, and drying with nitrogen. The samples were processed by reconstitution and derivatization procedures and analyzed for testing in examples 1, 2, 3, and 4, respectively.
2. And (3) sample analysis: the samples to be tested in steps 1(3), 1(4) and 1(5) are analyzed and tested in the same way as in examples 1, 2, 3 and 4.
3. And (3) analyzing a detection result:
TABLE 13 results of extraction of analytes in three different solid-phase extraction columns
The comparison of the results of three different types of solid phase extraction columns can show that the signal response and the RSD% of the same serum sample processed by the specific C18 filler solid phase extraction column are obviously superior to those of C8 and Phenyl solid phase extraction columns.
Example 7
The representative compounds (estradiol, testosterone, 17 alpha-hydroxyprogesterone and corticosterone) of each steroid hormone are taken as examples to show that the pretreatment processes of different bonding sites, different bonding densities and different carbon contents of the solid phase extraction column and the specific solid phase extraction column of the invention are compared:
1. the pretreatment process of the solid phase extraction column bonding site single-point bonding C18 filler, the double-point bonding C18 filler and the specific solid phase extraction column three-point bonding C18 filler comprises the following steps:
(1) preparing a standard working solution: accurately preparing estradiol, testosterone, 17 alpha-hydroxyprogesterone and corticosterone stock solutions respectively at a concentration of 1mg/mL, and diluting with 50% methanol water to obtain estradiol, testosterone, 17 alpha-hydroxyprogesterone and corticosterone working solutions at a concentration of 10 ng/mL;
(2) preparation of a sample: adding 300 mu L of blank serum substrate (without target compound) into 30 mu L of the standard substance working solution in the step (1), and mixing for 5min in a vortex manner for later use;
(3) three solid phase extraction column pretreatment processes: activating by using 1mL of 90% methanol; then adding 5% methanol water for balancing; adding 300 mu L of serum sample to be detected directly; adding 500 mu L of 5% methanol water and 500 mu L of 30% methanol water in sequence for leaching; finally, eluting with 500 μ L of methanol, collecting all the eluted fractions, and drying with nitrogen. The samples were processed for reconstitution and derivatization, and analyzed for testing, according to examples 2, 3, 4, and 5, respectively.
2. Solid phase extraction column packing silica gel bonding density 1 mu mol/m2、1.8μmol/m2And 3. mu. mol/m2The pretreatment process of the C18 filler comprises the following steps:
(1) preparing a standard working solution: accurately preparing estradiol, testosterone, 17 alpha-hydroxyprogesterone and corticosterone stock solutions respectively at a concentration of 1mg/mL, and diluting with 50% methanol water to obtain estradiol, testosterone, 17 alpha-hydroxyprogesterone and corticosterone working solutions at a concentration of 10 ng/mL;
(2) preparation of a sample: adding 300 mu L of blank serum substrate (without target compound) into 30 mu L of the standard substance working solution in the step (1), and mixing for 5min in a vortex manner for later use;
(3) three solid phase extraction column pretreatment processes: activating by using 1mL of 90% methanol; then adding 5% methanol water for balancing; adding 300 mu L of serum sample to be detected directly; adding 500 mu L of 5% methanol water and 500 mu L of 30% methanol water in sequence for leaching; finally, eluting with 500 μ L of methanol, collecting all the eluted fractions, and drying with nitrogen. The samples were processed for reconstitution and derivatization, and analyzed for testing, according to examples 2, 3, 4, and 5, respectively.
3. The pretreatment process of filling C18 filler with the carbon content of 8%, 12% and 16% of silica gel in a solid phase extraction column comprises the following steps:
(1) preparing a standard working solution: accurately preparing estradiol, testosterone, 17 alpha-hydroxyprogesterone and corticosterone stock solutions respectively at a concentration of 1mg/mL, and diluting with 50% methanol water to obtain estradiol, testosterone, 17 alpha-hydroxyprogesterone and corticosterone working solutions at a concentration of 10 ng/mL;
(2) preparation of a sample: adding 300 mu L of blank serum substrate (without target compound) into 30 mu L of the standard substance working solution in the step (1), and mixing for 5min in a vortex manner for later use;
(3) three solid phase extraction column pretreatment processes: activating by using 1mL of 90% methanol; then adding 5% methanol water for balancing; adding 300 mu L of serum sample to be detected directly; adding 500 mu L of 5% methanol water and 500 mu L of 30% methanol water in sequence for leaching; finally, eluting with 500 μ L of methanol, collecting all the eluted fractions, and drying with nitrogen. The samples were processed for reconstitution and derivatization, and analyzed for testing, according to examples 2, 3, 4, and 5, respectively.
4. And (3) sample analysis: the samples to be tested in steps 1(3), 2(3), 3(3) are analyzed and tested in the same way as in examples 2, 3, 4 and 5.
5. And (3) analyzing a detection result:
TABLE 14 extraction results of the substances to be tested in the solid-phase extraction column with fillers of different bonding sites
TABLE 15 extraction results of substances to be tested in solid-phase extraction columns with fillers of different bonding densities
TABLE 16 extraction results of the samples with different carbon contents in solid-phase extraction column
The comparison of the results of the solid phase extraction columns filled with different bonding sites, different bonding densities and different carbon contents can show that the signal response and RSD% of the same serum sample processed by the specific filler with the bonding sites, the bonding densities and the carbon contents of the solid phase extraction column are obviously superior to those of the solid phase extraction columns with other bonding sites, bonding densities and carbon contents.
The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations of the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.
Claims (9)
1. A method for detecting a steroid hormone in a blood sample, the method comprising:
(1) enriching and purifying one or more steroid hormone components in the serum sample by using a solid phase extraction column;
(2) optionally, derivatizing the enriched purified steroid hormone component with a derivatizing agent;
(3) detecting the derivatized and/or underivatized one or more steroid hormone components using liquid chromatography-tandem mass spectrometry;
wherein the solid phase extraction column adopts bonding filler with silica gel as a matrix, and the filler is bonding density of 1.6-2.2 mu mol/m2The carbon content is 10 to 12 percent, and the low-density three-point bonding C18 filler;
the structural formula of the solid phase extraction material is as follows:
2. the detection method according to claim 1, characterized in that the filler has a silica gel particle size of 10 μm to 100 μm, a pore diameter of 60A to 200A, and a specific surface area of 100m2/g-600m2/g。
3. The method as claimed in claim 1, wherein the solid phase extraction column is a 30-100mg/1ml or 100-500mg/5ml solid phase extraction column.
4. The assay of claim 1, wherein the steroid hormone is selected from one or more of an estrogen, a progestin, an androgen or a corticosteroid.
5. The assay of claim 4, wherein the estrogen is selected from one or more of estrone, estradiol, estriol; the progestogen is selected from one or more of progesterone, 17-hydroxyprogesterone, pregnenolone, and 17-hydroxyprogesterone; the androgen is selected from one or more of testosterone, androstenedione, dihydrotestosterone, dehydroepiandrosterone and dehydroepiandrosterone sulfate; the corticoids are selected from one or more of corticosterone, cortisol, cortisone, 11-deoxycorticosterone, 11-deoxycorticosterol and 21-deoxycorticosterol.
6. The detection method according to claim 1, wherein the enrichment and purification method comprises the following steps of firstly adopting an organic solvent: activating a first mixed solution with the water volume ratio of 90: 10; and adding an organic solvent: balancing a second mixed solution with the volume ratio of water being 5: 95; then adding the blood sample to be detected into the extraction column according to the amount of 0.5-10% of the filler mass of the solid phase extraction column, and sequentially adding a first mixed solution and an organic solvent: the volume ratio of water is 30: 70, leaching the third mixed solution; finally, eluting with an organic solvent, and collecting all eluted fractions.
7. The detection method according to claim 6, wherein the organic solvent is selected from methanol.
8. The detection method according to claim 1,
the step (1) comprises the following steps:
a) filling a solid phase extraction column: the bonding density of the filled silica gel is 1.6-2.2 mu mol/m2The carbon content is 10-12%, the granularity is 10-100 μm, the pore diameter is 60-200A, and the specific surface area is 100m2/g-600m230-100mg of low-density three-point bonded C18 filler is placed in a 1mL column tube or 100-500mg is placed in a 5mL column tube;
b) preparing an internal standard working solution: accurately preparing 1mg/mL of isotope labeling mixed internal standard stock solution of steroid hormone components, and diluting with 50% methanol to obtain diluted mixed internal standard working solution for later use;
c) preparation of a sample: adding 100-fold 500 mu L serum sample into 10-50 mu L mixed internal standard working solution in the step (b), and uniformly mixing by vortex for later use;
d) solid phase extraction and separation: the solid phase extraction column first adopts 1mL of organic solvent: activating the water at a volume ratio of 90: 10; and adding an organic solvent: the volume ratio of water is 5: 95; directly adding 100-500 mu L of the to-be-detected serum sample in the step c); add 500 μ L of organic solvent in sequence: the volume ratio of water is 5:95 and 500. mu.L of organic solvent: the volume ratio of water is 30: 70, leaching; finally, eluting with 300-;
the step (2) comprises the following steps: adding 30-200 mu L of derivatization reagent into the fraction obtained in the step d) for derivatization reaction to obtain a derivatization product; or directly adding the compound solution to obtain hormone prototype extract;
the step (3) comprises the following steps:
a) analysis of the samples: analyzing and detecting the derivative product or the hormone prototype extract, separating the components to be detected by adopting reverse phase chromatography, detecting by triple quadrupole tandem mass spectrometry to obtain a sample spectrogram, and detecting to obtain the positive ions and/or the negative ions of the steroid hormone;
wherein, the analysis conditions of the high performance liquid chromatography-tandem mass spectrometry are as follows:
liquid chromatography Condition 1
A chromatographic column: waters CORTECS T32.7 μm, 2.1 x 100mm
Mobile phase A: 0.2-5mM ammonium formate, 0.1% aqueous formic acid; mobile phase B: acetonitrile
The flow rate is 0.2-0.5 mL/min; the column temperature is 30-55 ℃; sample introduction amount: 1-10 μ L;
liquid chromatography Condition 2
A chromatographic column: luna C83 mu m, 2 x 100mm
Mobile phase A: 0.2-5mM ammonium formate; mobile phase B: methanol
The flow rate is 0.2-0.5 mL/min; the column temperature is 30-55 ℃; sample introduction amount: 1-10 μ L;
mass spectrometric analysis Condition 1
Ionization mode: ESI positive ions or ESI positive and negative ions are scanned simultaneously; spraying voltage: 4 KV; desolventizing gas temperature: 550 ℃; atomizing: 50 psi; assisting atomization gas: 50 psi; air curtain air: 30 psi; the scanning mode is as follows: multiple Reaction Monitoring (MRM);
mass spectrometric analysis Condition 2
Ionization mode: ESI positive ion; capillary voltage: 2.0 KV; taper hole voltage: 50V; desolventizing gas temperature: 450 ℃; desolventizing air flow rate: 800L/Hr; taper hole air flow rate: 150L/Hr; the scanning mode is as follows: multiple Reaction Monitoring (MRM);
b) and (3) analyzing a quantitative calculation result: and obtaining a linear regression equation according to the ratio of the peak area of the steroid hormone ion chromatographic peak in the sample chromatogram to the peak area of the corresponding isotope internal standard ion chromatographic peak and the corresponding concentration, and calculating the concentration of the steroid hormone in the serum sample to realize the detection of the steroid hormone in the serum.
9. The detection method according to claim 8,
when the steroid hormone is estrone, estradiol and/or estriol, the dilution concentration of d 4-estrone and d 3-estradiol in the isotope mixed internal standard working solution is 2ng/ml, the dilution concentration of d 3-estriol is 20ng/ml, and the derivatization reagent is dansyl chloride;
when the steroid hormone is testosterone, androstenedione, dihydrotestosterone, dehydroepiandrosterone and/or dehydroepiandrosterone sulfate, d 3-testosterone in the internal standard working solution,13The dilution concentration of C-androstenedione is 5ng/ml, the dilution concentration of d 3-dihydrotestosterone is 10ng/ml, the dilution concentration of d 6-dehydroepiandrosterone is 100ng/ml, the dilution concentration of d 6-dehydroepiandrosterone sulfate is 5 mu g/ml, and the derivatization reagent is hydroxylamine hydrochloride;
when the steroid hormone is progesterone, 17 alpha-hydroxyprogesterone, pregnenolone and/or 17 alpha-hydroxyprogesterone, the dilution concentration of d 9-progesterone, d8-17 alpha-hydroxyprogesterone, d 4-pregnenolone and d3-17 alpha-hydroxyprogesterone in the isotope mixed internal standard working solution is 20ng/ml, and the complex solution is an organic reagent;
when the steroid hormone is cortisol, corticosterone, cortisone, 11-deoxycorticosterol and/or 21-deoxycorticosterol, the dilution concentration of d 6-cortisol in the isotope mixed internal standard working solution is 200ng/mL, the dilution concentration of d 4-corticosterone is 10ng/mL, the dilution concentration of d 8-cortisone is 200ng/mL, the dilution concentration of d 7-11-deoxycorticosterone is 5ng/mL, the dilution concentration of d 5-11-deoxycorticosterol is 10ng/mL, the dilution concentration of d 8-21-deoxycorticosterol is 5ng/mL, and the compound solution is an organic reagent.
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| CN114487210B (en) * | 2022-03-11 | 2024-05-10 | 天津国科医疗科技发展有限公司 | Liquid chromatography tandem mass spectrometry detection method of steroid hormone based on magnetic solid phase extraction |
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