CN115963199A - Quantitative detection method and application of steroid hormone in human/animal body fluid - Google Patents
Quantitative detection method and application of steroid hormone in human/animal body fluid Download PDFInfo
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Abstract
The invention discloses a method for quantitatively detecting 24 steroid hormones in body fluids such as human/animal plasma, serum, urine and the like, which is based on ultra-high performance liquid chromatography-tandem mass spectrometry combined with online enzymolysis-solid phase extraction and can realize one-time detection of the total amount of free state and combined state of the 24 steroid hormones, wherein the detection limit of the method can reach pg/mL, and the methodological verification result meets 9101-analysis method verification guide principle. The detection method provided by the invention can simultaneously and accurately quantitatively detect the 24 steroid hormones in body fluids such as human/animal plasma, serum, urine and the like, has the advantages of high sensitivity, strong specificity, small required sample amount, short detection time, low cost, capability of improving the sensitivity, precision, detection efficiency and detection flux of the method, capability of avoiding resource waste and capability of providing efficient and powerful assistance for clinical diagnosis, monitoring, scientific research and the like.
Description
Technical Field
The invention belongs to the technical field of clinical examination, and relates to a quantitative detection method and application of 24 steroid hormones in body fluids such as human/animal plasma, serum, urine and the like.
Background
Steroid hormones, also known as steroid hormones, are structures consisting of four fused rings, the precursor of which is cholesterol. Cholesterol undergoes a series of metabolic reactions to produce five major steroid hormones: progestins, glucocorticoids, mineralocorticoids, estrogens, and androgens. The increase or decrease of steroid hormones in human bodies is closely related to some common clinical diseases, such as congenital adrenal hyperplasia, cushing's syndrome, polycystic ovary syndrome, adrenal cortex dysfunction and the like, and is one of the important means for clinical examination and diagnosis by monitoring the content change of steroid hormones in human bodies.
Due to the fact that steroid hormones and metabolic species thereof are various in types and low in common content, similar skeleton structures and physicochemical properties exist, and stereoisomers exist in the steroid hormones with the same structures, great difficulty and challenge are brought to efficient and accurate detection of the steroid hormones. Steroid hormones exist in free state or combined state in human body respectively, hormones in different states play different functions and roles, common combined steroid hormones mostly exist in sulfate or combined form with glucuronic acid, and the combined steroid hormone generated by different binding sites is increased in the number of isomers, so that a great technical problem exists in direct detection of the combined steroid hormone. In clinical steroid hormone detection, an immunoassay method is mostly adopted to detect one or more steroid hormones, and due to the factors of low sensitivity, poor specificity, low flux and the like of the detection method, false positive or false negative often exist, and the detection method is time-consuming and labor-consuming. In addition, due to the difference of reagents, methods, personnel and the like, the same sample has the problem of larger difference of results among different laboratories, and has larger influence on interpretation and treatment detection of related diseases. Currently, the conjugated steroid hormone is not considered and popularized yet, and not only is the conjugated steroid hormone greatly interfered by isomers in the aspect of direct detection, but also the complexity and complexity in operation exist, and more difficulties need to be overcome for further accurately quantifying the conjugated steroid hormone on the basis of ensuring the accurate quantification of the free steroid hormone. At present, no detection method capable of rapidly, accurately and efficiently quantifying various common steroid hormones in binding states and free states exists.
Disclosure of Invention
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology is one of the commonly used detection and analysis means, and due to the advantages of strong specificity, high sensitivity, high analysis speed and the like, the technology is more closely and deeply combined with clinical diagnosis, monitoring and the like in recent years, is favored by mass spectrometry students and medical researchers, and the clinical mass spectrometry is rapidly developed. The Solid Phase Extraction (SPE) is a sample pretreatment technology developed in recent years, is developed by combining a liquid-solid extraction column and a liquid chromatography technology, is mainly used for separating, purifying and concentrating samples, can improve the recovery rate of analytes compared with the traditional liquid-liquid extraction method, more effectively separates the analytes from interfering components, reduces the sample pretreatment process, is simple to operate, saves time and labor, and is widely applied to the fields of medicines, foods, environments, commercial inspection, chemical engineering and the like. In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a method and application for quantitative determination of 24 kinds of steroid hormones in human/animal body fluids such as plasma, serum and urine, which is based on an LC-MS, especially an UPLC-MS/MS analysis platform, and is used for simultaneously, rapidly and accurately quantitative determination of 24 kinds of steroid hormones in a bound state or a free state in human/animal body fluids such as plasma, serum and urine.
To achieve the above objects and other related objects, the present invention develops a high-throughput, high-coverage, high-sensitivity targeted quantitative method for steroid hormones, which can perform a precise quantitative analysis of 24 steroid hormones in 13 minutes. The 24 steroid hormones have wide coverage range, relate to estrogen, progestational hormone, cortical hormone, androgen, ecdysone and the like, and have more specificity and practicability compared with the prior clinical mass spectrometry detection technical method. Compared with the traditional enzyme-linked immunosorbent assay and most of the existing clinical mass spectrometric detection technologies, the invention simultaneously quantifies the levels of multiple steroid hormones through one-time analysis, effectively saves manpower and material resources, improves the flux and can also meet the detection requirements of most of steroid hormones in clinical laboratories. Compared with the method for directly detecting the combined steroid hormone, the method has the advantages that the technical problem is simplified through an online enzymolysis-solid phase extraction technology, the sum of different combined steroid hormones of any steroid hormone is directly detected, the possible influence caused by the isomer interference of the combined steroid hormones and a complex matrix is avoided, the detection of the sum of any combined steroid hormone is realized, and the detection efficiency, the flux and the convenience are greatly improved.
According to the invention, whether mixed enzyme solution is added for on-line enzymolysis or not can be used for detecting free steroid hormone and simultaneously making a difference value to obtain the content of the bound steroid hormone, and more portability, selectivity and comprehensiveness are provided aiming at the current application situation of the current clinical mass spectrometry technology in steroid hormone detection, so that the effects of steroid hormones in different states in the life activity process of human/animals can be better researched.
The invention provides a quantitative detection method and application of 24 steroid hormones in human/animal body fluid, comprising the following steps:
step 1) preparation of a matrix mixed standard working solution: respectively adding multiple steroid hormone standards into a first solvent for ultrasonic dissolution to obtain steroid hormone single-standard stock solutions, simultaneously adding a mixed solution of methanol and water into the multiple steroid hormone single-standard stock solutions for ultrasonic dissolution to obtain steroid hormone mixed standard stock solutions, adding the steroid hormone mixed standard stock solutions into a second solvent for ultrasonic dissolution to obtain mixed standard solutions with different concentrations, and finally diluting the mixed standard solutions with different concentrations by using a BSA (bovine serum albumin) solution with a mass fraction of 1% to obtain matrix mixed standard working solutions with different concentrations;
wherein the steroid hormone is: pregnenolone, 17 alpha-hydroxypregnenolone, progesterone, 17 alpha-hydroxyprogesterone, corticosterone, 11-deoxycorticosterol, 21-deoxycorticosterol, 11-deoxycorticosterone, estrone, estradiol, estriol, dihydrotestosterone, dehydroepiandrosterone sulfate, testosterone, cortisone, melatonin, aldosterone, androstenedione, cortisol, androstenediol, androsterone, epitestosterone, 20-hydroxyecdysone.
Wherein, adding a second solvent for ultrasonic dissolution to obtain mixed standard solutions with different concentrations comprises the following steps: first, a high-concentration mixed standard solution (high-concentration standard koji) was prepared for further dilution to obtain a matrix mixed standard solution (high-concentration standard koji was diluted with 1% BSA to obtain low-concentration standard koji for quantification of unknown samples); specifically, the specific concentrations of dehydroepiandrosterone sulfate in the mixed standard solutions with different concentrations are as follows: 200000, 100000, 50000, 20000, 10000, 5000, 2000, 1000, 500, 200, 100, 50, 20, 10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05ng/mL, and the specific concentration of the rest 23 mixed standard solutions with different concentrations is 2000, 1000, 500, 200, 100, 50, 20, 10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05ng/mL.
The first solvent is methanol, acetonitrile, DMSO, etc.; preferably, the solvent is methanol or a mixed solvent of methanol and DMSO.
Wherein the volume ratio of methanol to DMSO is 1:1 to 1:9; preferably, 1:1.
the second solvent is methanol, acetonitrile, water and the like; preferably, it is a mixed solvent of methanol and water.
The volume ratio of the mixed solution of the methanol and the water is 1:1 to 2:1; preferably, 1:1.
2) Extracting a target object: extracting a sample to be detected and matrix mixed standard working solutions with different concentrations by an organic reagent protein precipitation method through an extracting agent 1 and an extracting agent 2 to respectively obtain supernate and protein precipitates;
3) Online enzymolysis-solid phase extraction: activating a solid phase extraction column (Cleanert PEP,2 mg) by 200 mu L of methanol, balancing by 200 mu L of water, loading a mixed solution of sulfatase and beta-glucuronidase to the solid phase extraction column, discarding an effluent liquid, bonding the sulfatase and the beta-glucuronidase to a filler of the solid phase extraction column, loading a supernatant obtained in the step 2) to the solid phase extraction column for the second time, closing an outflow valve, carrying out online enzymolysis for 60min at 37 ℃, slowly opening the outflow valve, discarding the effluent liquid, leaching the solid phase extraction column by using an eluent 1 and an eluent 2 respectively, discarding the eluent, eluting a target on the solid phase extraction column by using the eluent 1, collecting the target in a glass lining tube placed in a glass sample introduction vial, adding water to the initial proportion of a mobile phase, and mixing uniformly to obtain an on-machine sample to be tested and a standard test sample;
4) Analyzing by an instrument: and (3) measuring the to-be-measured on-machine sample and the standard test sample in the step 3) by adopting an ultra performance liquid chromatography-mass spectrometry combined method, identifying a target analyte according to retention time and parent-child ion pair information, and quantifying by an internal standard curve method according to the selected ion pair chromatographic peak area of the target analyte to determine the content of each steroid hormone in the to-be-measured sample.
The steps of loading the enzyme mixed solution and carrying out online enzymolysis at 37 ℃ for 60min are not carried out when only the content of the free 24 steroid hormones is detected, and the content of the 24 bound steroid hormones can be obtained by detecting the total content and the free content of the 24 free steroid hormones and the total content and the free content of the bound steroid hormones and carrying out difference.
a1 Conditions for ultrasonic dissolution are: temperature: carrying out ice water bath; ultrasonic time: 1-5 min;
a2 In the case of ultrasonic dissolution of the first solvent: estrone, 17 alpha-hydroxypregnenolone using methanol or acetonitrile to DMSO in a volume ratio of 1:1, dissolving the rest by using pure methanol or pure acetonitrile;
a3 The volume ratio of the mixed solution of methanol and water is 1:1 to 2:1;
a4 Specific concentrations of dehydroepiandrosterone sulfate in the mixed standard solutions of different concentrations are as follows: 200000, 100000, 50000, 20000, 10000, 5000, 2000, 1000, 500, 200, 100, 50, 20, 10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05ng/mL, the specific concentration of the rest 23 mixed standard solutions with different concentrations is 2000, 1000, 500, 200, 100, 50, 20, 10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05ng/mL;
a5 The dilution of each concentration of the mixed standard solution using a 1% BSA solution was specifically performed as follows: preparing an aqueous solution with the bovine serum albumin mass fraction of 1%, and diluting the mixed standard solution with each concentration by 5 times.
b1 The organic reagent protein precipitation method comprises the steps of putting 200 mu L of body fluid sample into a 1.5ml PP centrifuge tube, adding 200 mu L of extractant 1, uniformly mixing for 1min, adding 200 mu L of extractant 2, uniformly mixing for 1min, and centrifuging at 12000rpm at 3-5 ℃ for 10min;
b3 2M acetic acid-sodium acetate buffer solution of the extractant 2 has a pH of about 5.2;
c1 The specific concentration of the mixed solution of the sulfatase and the beta-glucuronidase is as follows: β -glucuronidase: 200-300U/mL, sulfatase: 600-800U/mL, the used solvent is water, and the use volume is 80-120 mu L; preferably, it is β -glucuronidase: 250U/mL, sulfatase: 700U/mL, the used solvent is water, and the used volume is 100 mu L;
c2 The volume of the supernatant of the secondarily loaded sample is 400 to 500 mu L; preferably, 500. Mu.L;
c3 The volume ratio of acetonitrile to water in the eluent 1 is 1:9, a volume of 150 to 250. Mu.L, preferably 200. Mu.L, is used;
c4 The volume of the eluent 2 is 150-250 mu L; preferably, 200. Mu.L;
c5 Eluent 1 acetonitrile to methanol in a volume ratio of 9:1, the using volume is 30-50 mu L; preferably, 35 μ L;
c6 Loading the enzyme mixture and carrying out on-line enzymolysis at 37 ℃ for 60min, wherein the step is not carried out when only the content of free 24 steroid hormones is detected.
In step 4 of the invention, the liquid chromatography-mass spectrometry combination method is an ultra-high liquid chromatography-tandem mass spectrometry combination method;
the measuring conditions of the ultra-high liquid chromatography are as follows:
a chromatographic column: a C8 liquid chromatography column; column temperature: 45 ℃; sample introduction amount: 5-20 mu L; flow rate: 0.3mL/min; the mobile phase A is 0.5mM ammonium fluoride aqueous solution; the mobile phase B is methanol; the analysis time is 13min; gradient elution;
the determination conditions of the mass spectrum are as follows:
an ionization mode: an electrospray ion source; the ion source temperature is 500 ℃; the air curtain air is 40psi; the parameters of collision induced ionization are medium; the voltage of ion spraying is-4500/5500V; ion source Gas1:60psi; gas2:55psi; the monitoring mode is that the multi-reaction monitoring MRM mode is used for carrying out primary/secondary mass spectrometry.
The specific procedure of the gradient elution of the invention is as follows: 0-0.5min, phase A: the volume ratio of the phase B is 55:45-55:45, a first step of; 0.5-6min, phase A: the volume ratio of the phase B is 55:45-45:55;6-10min, phase A: the volume ratio of the phase B is 45:55-5:95;10-12min, phase A: the volume ratio of the phase B is 5:95-5:95;12-12.2min, phase A: the volume ratio of the phase B is 5:95-55:45, a first step of; 12.2-13min, phase A: the volume ratio of the phase B is 55:45-55:45.
the steroid hormones provided by the invention are as follows: pregnenolone, 17 alpha-hydroxypregnenolone, progesterone, 17 alpha-hydroxyprogesterone, corticosterone, 11-deoxycorticosterol, 21-deoxycorticosterol, 11-deoxycorticosterone, estrone, estradiol, estriol, dihydrotestosterone, dehydroepiandrosterone sulfate, testosterone, cortisone, melatonin, aldosterone, androstenedione, cortisol, androstenediol, androsterone, epitestosterone, 20-hydroxyecdysone.
The concentration range of the steroid hormone standard substance after being redissolved by using a first solvent is 0.1-5 mg/mL; the first solvent adopted for redissolving the standard substance is one or more of methanol, acetonitrile and DMSO.
The extracting agent 1 is methanol containing 7 steroid hormone isotope internal standards;
the extractant 2 is 0.5-2M acetic acid-sodium acetate buffer solution; preferably, 2M;
the eluent 1 is a mixed solution of water and acetonitrile; wherein the volume ratio of water to acetonitrile is 9:1;
the eluent 2 is n-hexane;
the eluent 1 is a mixed solution of methanol and acetonitrile; wherein the volume ratio of methanol to acetonitrile is 1:9.
the solid phase extraction column is Cleanert PEP (2 mg).
The on-line enzymolysis of the invention uses a mixed solution of sulfatase and beta-glucuronidase.
As described above, the quantitative detection method and application of 24 steroid hormones in body fluids such as human/animal plasma, serum and urine provided by the invention can simultaneously, rapidly and accurately detect the 24 steroid hormones in the body fluids such as human/animal plasma, serum and urine on the basis of the UPLC-MS/MS analysis platform by combining online enzymolysis with a solid phase extraction technology, and has the advantages of high sensitivity and strong specificity. The detection method has the advantages of small sample amount, optimized pretreatment and detection methods, short detection time, improved sensitivity, precision, efficiency and detection flux, low cost, resource waste avoidance, and high-efficiency and strong assistance for clinical diagnosis, monitoring, scientific research and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly introduced below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is also possible for those skilled in the art to obtain other drawings based on the drawings without creative efforts.
FIG. 1 is a heat map showing the results of urine specimen testing of 41 volunteers in example 1 of the present invention.
FIG. 2 is a radar chart showing the results of urine sample testing of 41 volunteers in example 1 of the present invention
FIG. 3 is a graph showing a standard curve of steroid hormones classified in the middle of the standard test sample in example 1 of the present invention, FIG. 3A is a graph showing a standard curve of estrone, and FIG. 3B is a graph showing a standard curve of 17 α -hydroxypregnanolone.
FIG. 4 shows an LC-MS/MS extracted ion-diagram of steroid hormones from a urine sample of a volunteer according to example 1 of the present invention.
FIGS. 5 and 6 show the LC-MS/MS extracted ion-maps of the 24-steroid hormone standard test substance s14 in example 1 of the present invention.
FIGS. 7 and 8 show the LC-MS/MS extracted ion-graphs of steroid hormones from urine samples of 24 volunteers according to example 1 of the present invention.
Detailed Description
The invention is further described in detail with reference to the following specific examples and the accompanying drawings. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.
In a first aspect, the present invention provides a method for quantitatively detecting 24 steroid hormones in body fluids such as human/animal plasma, serum, urine, etc., wherein the method comprises the following steps of respectively using 24 steroid hormones and 7 steroid hormone isotope internal standards as shown in table 1 below.
TABLE 1
In the detection method, the concentration ranges of the 24 steroid hormone standard products after ultrasonic redissolution are all 0.1-5 mg/mL, and the 24 steroid hormone standard products are 24 steroid hormone single-standard stock solutions; the standard substance is redissolved by adopting methanol or the volume ratio of methanol to DMSO (dimethyl sulfoxide) is 1:1, mixing the solution; the ultrasonic dissolving conditions are as follows: temperature: carrying out ice water bath; ultrasonic time: 1-5 min.
In the detection method, the mixed standard stock solution is obtained by mixing a 24-steroid hormone single-standard stock solution with a mixed solution of water and methanol; the volume ratio of the water to the methanol mixed solution adopted for preparing the mixed standard stock solution is 1:1.
in the detection method, the mixed standard solutions are obtained by diluting a mixed standard stock solution with a mixed solution of water and methanol, the number of the mixed standard solutions is 21, 21 mixed standard solutions are respectively represented by S1, S2, S3, S4, … …, S18, S19, S20 and S21, and the concentrations of the steroid hormones in S1 to S15 are sequentially ordered from small to large according to concentration gradients: 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000 and 2000ng/mL, wherein the dehydroepiandrosterone sulfate concentrations in S16-S21 are sequentially ordered from small to large according to concentration gradient: 5000. 10000, 20000, 50000, 100000 and 200000ng/mL; the volume ratio of water to methanol mixed solution adopted for diluting the mixed standard stock solution is 1:1.
in the detection method, the matrix mixed standard working solution is obtained by diluting a mixed standard solution with a Bovine Serum Albumin (BSA) solution with a mass fraction of 1%, the number of the matrix mixed standard working solutions is 21, 21 matrix mixed standard working solutions are respectively represented by s1, s2, s3, s4, … …, s18, s19, s20 and s21, and the steroid hormones in s1 to s15 have respective concentrations sequentially ordered from small to large according to a concentration gradient: 0.01, 0.02, 0.04, 0.1, 0.2, 0.4, 1, 2, 4, 10, 20, 40, 100, 200, 400ng/mL, the dehydroepiandrosterone sulfate concentrations in s 16-s 21 are sequentially ordered from small to large according to concentration gradient: 1000. 2000, 4000, 10000, 20000 and 40000ng/mL.
In the detection method, the reagent further comprises an extracting agent 1, an extracting agent 2, an eluting agent 1, an eluting agent 2 and an eluent 1, wherein the extracting agent 1 is methanol containing 7 steroid hormone isotope internal standards, the 7 steroid hormone isotope internal standards are shown in the table 1, the concentrations of the 7 steroid hormone isotope internal standards are all 50ng/mL, the extracting agent 2 is 2M acetic acid-sodium acetate buffer solution with the pH value of about 5.2, and the eluting agent 1 is an acetic acid-acetonitrile buffer solution with the volume ratio of water to acetonitrile of 9:1, wherein the eluent 1 is n-hexane, and the eluent 1 is a mixture of methanol and acetonitrile in a volume ratio of 1:9, and (c) a mixed solution.
The invention provides a quantitative detection method and application of 24 steroid hormones in body fluids such as human/animal plasma, serum, urine and the like, comprising the following steps:
1) Preparation of matrix mix standard working solution: adding methanol or a methanol and DMSO mixed solvent into a multiple steroid hormone standard substance for ultrasonic dissolution to obtain a steroid hormone single standard stock solution, adding a methanol and water mixed solution into the multiple steroid hormone single standard stock solution for ultrasonic dissolution to obtain a steroid hormone mixed standard stock solution, adding the steroid hormone standard stock solution into a water and methanol mixed solution for ultrasonic dissolution to obtain mixed standard solutions with different concentrations, and finally diluting the mixed standard solutions with different concentrations by using 1% BSA solution to obtain matrix mixed standard working solutions with different concentrations;
2) Extracting a target object: extracting a sample to be detected and matrix mixed standard working solutions with different concentrations by an organic reagent protein precipitation method through an extracting agent 1 and an extracting agent 2 to respectively obtain supernate and protein precipitates;
3) Online enzymolysis-solid phase extraction: activating a solid phase extraction column (Cleanert PEP,2 mg) with 200 muL of methanol, balancing with 200 muL of water, loading a mixed solution of sulfatase and beta-glucuronidase to the solid phase extraction column, discarding an effluent liquid, bonding the sulfatase and the beta-glucuronidase on a filler of the solid phase extraction column, taking 500 muL of supernatant obtained in the step 2), loading the supernatant to the solid phase extraction column for the second time, closing an outflow valve, placing the solid-phase extraction column in an environment at 37 ℃ for on-line enzymolysis for 60min, slowly opening an outflow valve, discarding effluent liquid, leaching the solid-phase extraction column with 200 mu L of leaching agent 1 and leaching agent 2 respectively, discarding the leaching agent, finally eluting a target on the solid-phase extraction column with 35 mu L of eluent 1, collecting the target in a glass lining tube placed in a glass sample injection vial, adding water to the initial proportion of a mobile phase, and uniformly mixing to obtain a computer sample to be tested and a standard test product;
4) Analyzing by an instrument: and (3) measuring the on-machine sample to be measured and the standard test sample in the step 3) by adopting an ultra performance liquid chromatography-mass spectrometry combined method, identifying a target analyte according to retention time and parent-child ion pair information, quantifying by adopting a standard curve method according to the selected ion pair chromatographic peak area of the target analyte, and determining the content of each steroid hormone in the sample to be measured.
The steps of loading the enzyme mixed solution and carrying out online enzymolysis at 37 ℃ for 60min are not carried out when only the content of the free 24 steroid hormones is detected, and the content of the 24 bound steroid hormones can be obtained by detecting the total content and the free content of the 24 free steroid hormones and the total content and the free content of the bound steroid hormones and carrying out difference.
In the step 1), BSA solutions with mass fractions of 0.5, 1 and 2 are tried, and the detection result of the step 4) shows that the BSA solution with the mass fraction of 1% can better simulate a blank serum matrix on the premise of not interfering the peak appearance and quantification of the target substance.
In the step 2), the extractant 2 is used for providing a suitable pH environment for enzymolysis in the step 3), and plays a role in diluting the supernatant generated in the step 2), so as to avoid excessive loss of the target substance during secondary loading in the step 3).
In the step 2), the extractant 1 is tried to be replaced by MTBE containing 7 isotope internal standards and acetonitrile containing 7 isotope internal standards, and the detection result in the step 4) shows that the extractant 1 has higher response intensity and signal-to-noise ratio for most target detection objects when methanol containing 7 isotope internal standards is used.
In the step 2), the organic reagent protein precipitation method comprises the steps of putting 200 mu L of body fluid sample into a 1.5ml PP centrifugal tube, adding 200 mu L of extractant 1, uniformly mixing for 1min, adding 200 mu L of extractant 2, uniformly mixing for 1min, and centrifuging at 12000rpm at 3-5 ℃ for 10min.
In the above step 3), an attempt was made to replace eluent 1 with water, water and acetonitrile at a volume ratio of 8:2, and the detection result of the step 4) shows that the volume ratio of water to acetonitrile used in the extractant 1 is 9:1, the mixed solution has higher response intensity and signal-to-noise ratio to most target detection objects.
In the above step 3), the eluent agent 1 was tried to be changed to methanol and acetonitrile in a volume ratio of 1:1, acetonitrile, and the detection result of the step 4) shows that the volume ratio of methanol to acetonitrile used in the extractant 1 is 1: and 9, the kit has higher response intensity and signal-to-noise ratio to most target detection objects.
In the step 3), the specific concentration of the mixed solution of the sulfatase and the beta-glucuronidase is as follows: β -glucuronidase: 250U/mL, sulfatase: 700U/mL, the used solvent is water, and the used volume is 100 mu L;
in the step 3), the steps of loading the enzyme mixed solution and carrying out on-line enzymolysis at 37 ℃ for 60min are not carried out when only the content of free 24 steroid hormones is detected.
In the step 4), in the ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), the measurement conditions of the Ultra Performance Liquid Chromatography (UPLC) are as follows:
and (3) chromatographic column: a C8 liquid chromatography column; column temperature: 45 ℃; sample introduction amount: 5-20 mu L; flow rate: 0.3mL/min; the mobile phase A is 0.5mM ammonium fluoride aqueous solution; the mobile phase B is methanol; the analysis time is 13min; gradient elution;
in a further preferred embodiment, the measurement conditions for ultra high liquid chromatography (UPLC) are:
and (3) chromatographic column: an ACQUITYUPLC BEH C8 chromatographic column (column length 100 mm. Times.inner diameter 2.1mm, particle size 1.7 um); column temperature: 45 ℃; sample introduction amount: 20 mu L of the solution; flow rate: 0.3mL/min; the mobile phase A is 0.5mM ammonium fluoride aqueous solution; the mobile phase B is methanol; the analysis time is 13min; gradient elution.
In a further preferred embodiment, the specific procedure for the gradient elution is shown in table 2 as:
0-0.5min, phase A: the volume ratio of the phase B is 55:45-55:45, a first step of;
0.5-6min, phase A: the volume ratio of the phase B is 55:45-45:55;
6-10min, phase A: the volume ratio of the phase B is 45:55-5:95;
10-12min, phase A: the volume ratio of the phase B is 5:95-5:95;
12-12.2min, phase A: the volume ratio of the phase B is 5:95-55:45, a first step of;
12.2-13min, phase A: the volume ratio of the phase B is 55:45-55:45.
TABLE 2 gradient elution parameters
| Retention time (min) | Flow rate (mL/min) | Mobile phase A | Mobile phase B |
| 0 | 0.3 | 55 | 45 |
| 0.5 | 0.3 | 55 | 45 |
| 6 | 0.3 | 45 | 55 |
| 10 | 0.3 | 5 | 95 |
| 12 | 0.3 | 5 | 95 |
| 12.2 | 0.3 | 55 | 45 |
| 13 | 0.3 | 55 | 45 |
In the step 4), the measurement conditions of the ultra high liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) are as follows:
an ionization mode: an electrospray ion source; the ion source temperature is 500 ℃; the air curtain air is 40psi; the parameters of collision induced ionization are medium; the voltage of ion spraying is-4500/5500V; ion source Gas1:60psi; gas2:55psi; the monitoring mode is a multi-reaction monitoring MRM mode to carry out primary/secondary mass spectrometry.
The mass spectrum (MS/MS) includes a secondary mass spectrum, and the secondary mass spectrum can take fragment ions of the primary mass spectrum as parent ions and then carry out fragmentation analysis.
In one embodiment, the ultra high liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) results in declustering voltages, collision voltages, parent ions, daughter ions, retention times for the 24 steroid hormone and 7 steroid hormone isotope internal standards are shown in table 3.
TABLE 3
In the step 4), the standard curve method includes the steps of:
a1 Carrying out liquid chromatography-mass spectrometry combined analysis after processing a series of 24 steroid hormone standard mixed working solutions with different concentrations through the steps 2) and 3), obtaining linear relations between the chromatographic peak areas of the 24 steroid hormones and the peak areas of 7 steroid hormone isotope internal standard substances and corresponding concentrations, drawing corresponding standard curves, and carrying out regression operation through a weighted least square method to obtain a linear regression equation of the substance to be detected;
a2 Processing a sample to be detected in the steps 2) and 3) and then carrying out UPLC-MS/MS analysis, substituting the obtained chromatographic peak area of 24 steroid hormones and the peak area of 7 steroid hormone isotope internal standard substance into a regression equation of the standard working curve of the corresponding component in the step A1), and calculating to obtain the concentration of the corresponding analyte in the sample to be detected.
The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and not to limit the scope of the invention.
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
The reagents and instruments used in the following examples are conventional products which are commercially available, although manufacturers thereof are not indicated. For example, a vortex shaker for sample homogenization, a bench top high speed refrigerated centrifuge for centrifugation. The ultra-high performance liquid chromatograph (UPLC) for sample analysis and detection is produced by Shimadzu corporation, and the mass spectrometer for sample analysis and detection is a 6500 (QQQ/Qtrap) triple quadrupole mass spectrometry system produced by Sciex corporation.
Example 1
1. Treating a sample to be detected:
obtaining 41 tubes of urine from 41 volunteers as samples to be detected, taking 200 mu L of each sample to be detected in a 1.5ml PP centrifugal tube, adding 200 mu L of extractant 1, then mixing uniformly for 1min, adding 200 mu L of extractant 2, then mixing uniformly for 1min, and centrifuging at 12000rpm at 3-5 ℃ for 10min to obtain supernatant and protein precipitate. Activating a solid phase extraction column (Cleanert PEP,2 mg) by 200 mu L of methanol, balancing by 200 mu L of water, loading 100 mu L of mixed solution of sulfatase and beta-glucuronidase to the solid phase extraction column, discarding effluent liquid, bonding the sulfatase and the beta-glucuronidase on a filler of the solid phase extraction column, loading 500 mu L of supernatant liquid obtained after centrifugation to the solid phase extraction column for the second time, closing an outflow valve, placing the solid phase extraction column in an environment of 37 ℃ for on-line enzymolysis for 60 minutes, slowly opening the outflow valve, discarding the effluent liquid, respectively eluting the solid phase extraction column by 200 mu L of eluent 1 and eluent 2, discarding the eluent, eluting and collecting a target object on the solid phase extraction column by 35 mu L of eluent 1 into a glass lining tube placed in a glass sample injection vial, supplementing 43 mu L of water and uniformly mixing to obtain an on-machine sample to be detected.
The various solvent compositions involved are shown in table 4 below.
TABLE 4
* The 7 isotopic internal standards were: estrone-d 4, estradiol-d 4, pregnenolone- d 4, 17 alpha-hydroxypregnenolone-d 3, progesterone-d 9, testosterone-d 3 and cortisol-d 4, wherein the concentration is 50ng/mL.
The sample to be tested is stored in an environment of 20 ℃ below zero before being tested and analyzed on the computer, and the whole sample preparation process except the on-line enzymolysis process ensures low temperature.
2. Preparation and treatment of matrix mixed standard working solution
Preparing 21 matrix mixed standard working solutions with different concentrations according to the step 1), wherein the 21 matrix mixed standard working solutions are respectively represented by s1, s2, s3, s4, … …, s18, s19, s20 and s21, and the concentrations of the steroid hormones in the s 1-s 15 are sequentially ordered from small to large according to concentration gradients: 0.01, 0.02, 0.04, 0.1, 0.2, 0.4, 1, 2, 4, 10, 20, 40, 100, 200, 400ng/mL, the dehydroepiandrosterone sulfate concentrations in s 16-s 21 are sequentially ordered from small to large according to the concentration gradient: 1000. 2000, 4000, 10000, 20000 and 40000ng/mL.
And (3) mixing 200 mu L of matrix with the standard working solution in a 1.5ml PP material centrifuge tube, and performing subsequent operation according with the processing procedure of the sample to be tested to obtain the standard test product.
The standard test sample is stored in an environment of 20 ℃ below zero before being put on a computer for analysis, and the low temperature is ensured in the whole sample preparation process except the on-line enzymolysis process.
3. Instrumental analysis
Respectively measuring an on-machine sample to be measured and a standard test product by using UPLC-MS/MS, identifying characteristic peaks of an object to be measured and an internal standard substance according to relative retention time and the parent-character ion pair information, and quantifying according to a standard curve to determine the content of each object to be measured in the sample to be measured.
In the UPLC-MS/MS analysis method, the measurement conditions of UPLC are as follows:
a chromatographic column: an ACQUITYUPLC BEH C8 column (column length 100 mm. Times. Inner diameter 2.1mm, particle size 1.7 um); column temperature: 45 ℃; sample injection amount: 20 mu L of the solution; flow rate: 0.3mL/min; the mobile phase A is 0.5mM ammonium fluoride aqueous solution; the mobile phase B is methanol; the analysis time is 13min; gradient elution.
The specific procedure for gradient elution was:
0-0.5min, phase A: the volume ratio of the phase B is 55:45-55:45, a first step of;
0.5-6min, phase A: the volume ratio of the phase B is 55:45-45:55;
6-10min, phase A: the volume ratio of the phase B is 45:55-5:95;
10-12min, phase A: the volume ratio of the phase B is 5:95-5:95;
12-12.2min, phase A: the volume ratio of the phase B is 5:95-55:45, a first step of;
12.2-13min, phase A: the volume ratio of the phase B is 55:45-55:45.
in the UPLC-MS/MS analysis method, the measurement conditions of MS/MS are as follows:
an ionization mode: an electrospray ion source; the ion source temperature is 500 ℃; the air curtain air is 40psi; the parameters of collision induced ionization are medium; the voltage of ion spraying is-4500/5500V; ion source Gas1:60psi; gas2:55psi; the monitoring mode is a multi-reaction monitoring MRM mode to carry out primary/secondary mass spectrometry.
In the UPLC-MS/MS analysis method, the declustering voltage, collision voltage, parent ion, child ion, retention time of the target analyte are shown in table 3.
The test results of 41 volunteer urine samples are shown in FIG. 1 and FIG. 2.
Example 2
In order to ensure the stability, sensitivity, accuracy and reliability of the detection method, the invention carries out methodology investigation on the aspects of sensitivity, linear range, recovery rate, precision and the like.
1. Linear and sensitivity investigation
The standard test samples s1 to s21 prepared in step 2 of example 1 of the present invention were measured by UPLC-MS/MS method under the detection conditions of step 3 of example 1 of the present invention, and the linear range and detection limit of the detection method were examined, and the detection results are shown in table 5 below. As can be seen from Table 5, the detection method has high sensitivity as low as pg/mL level, has wide linear method as wide as 3-5 orders of magnitude, and can meet the quantitative detection requirement of 24 steroid hormones in body fluids such as human/animal plasma, serum, urine and the like.
TABLE 5 detection limit and Linear Range examination of targets
Note: S/N: signal/noise.
2. Investigation of recovery and precision
The method comprises the following steps of setting and inspecting LQC, MQC and HQC in a sample labeling mode, wherein the concentrations of 24 steroid hormones corresponding to the LQC, the MQC and the HQC are respectively 2, 20 and 200ng/mL, the method is prepared by using a commercially available blank urine matrix labeling mode, and the subsequent processing and detection processes are consistent with the steps 2 and 3 in the embodiment 1; 3 different persons are arranged to carry out the experiment content 3 times respectively to obtain 3 batches of different data so as to calculate the precision among batches, one batch is selected to carry out 3 times of detection in one day so as to calculate the precision in one day, and the detection is carried out again on the 2 nd day and the 3 rd day so as to calculate the precision in one day; the detection samples are placed in an environment of-20 ℃ when not detected, and are placed in an instrument sample plate in the detection process, and the set temperature is 4 ℃.
And calculating the recovery rate and the precision of the target analyte, wherein the obtained recovery rate and the method precision are shown in the following table 6, the method recovery rate is 80-120%, the precision is within 20%, and the method is stable and reliable.
TABLE 6 methods recovery and precision
Note: taking the average value of a plurality of batches of data for the result with a plurality of batches of data; n =3 for intra-day, inter-day, and batch precision.
In conclusion, the quantitative detection method and application of the 24 steroid hormones in the body fluids such as the human/animal plasma, the serum and the urine provided by the invention can simultaneously, rapidly and accurately quantitatively detect the content of the 24 steroid hormones in the body fluids such as the human/animal plasma, the serum and the urine, and has the advantages of high sensitivity, strong specificity, small required sample amount, short detection time, improvement of the sensitivity, the precision, the efficiency and the detection flux of the method, low cost and resource waste avoidance. Therefore, the invention effectively overcomes various defects in the prior art and has high industrial utilization value.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Those skilled in the art can modify or change the above-described embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which may be made by those skilled in the art without departing from the spirit and scope of the present invention as defined in the appended claims.
Claims (11)
1. A method for the quantitative determination of steroid hormones in human/animal body fluids, comprising the steps of:
step 1, preparing a matrix mixed standard working solution: respectively adding multiple steroid hormone standards into a first solvent for ultrasonic dissolution to obtain steroid hormone single-standard stock solutions, simultaneously adding a mixed solution of methanol and water into the multiple steroid hormone single-standard stock solutions for ultrasonic dissolution to obtain steroid hormone mixed standard stock solutions, adding the steroid hormone mixed standard stock solutions into a second solvent for ultrasonic dissolution to obtain mixed standard solutions with different concentrations, and finally diluting the mixed standard solutions with different concentrations by using a BSA (bovine serum albumin) solution with a mass fraction of 1% to obtain matrix mixed standard working solutions with different concentrations;
step 2, extracting a target: extracting a sample to be detected and matrix mixed standard working solutions with different concentrations by an organic reagent protein precipitation method through an extracting agent 1 and an extracting agent 2 to respectively obtain supernate and protein precipitates;
step 3, online enzymolysis-solid phase extraction: activating a solid phase extraction column by using 200 mu L of methanol, balancing by using 200 mu L of water, loading a mixed solution of sulfatase and beta-glucuronidase to the solid phase extraction column, discarding an effluent liquid, bonding the sulfatase and the beta-glucuronidase on a filler of the solid phase extraction column, secondarily loading a supernatant obtained in the step 2 to the solid phase extraction column, closing an outflow valve, carrying out online enzymolysis for 60 minutes at 37 ℃, slowly opening the outflow valve, discarding the effluent liquid, respectively eluting the solid phase extraction column by using an eluent 1 and an eluent 2, discarding the eluent, finally eluting a target on the solid phase extraction column by using the eluent 1, collecting the target into a glass lining tube placed in a glass sample introduction vial, supplementing water to the initial proportion of a mobile phase, and uniformly mixing to obtain an on-machine sample to be detected and a standard test product;
step 4, analyzing by an instrument: and (4) determining the on-machine sample to be detected and the standard test product in the step (3) by adopting an ultra-high performance liquid chromatography-mass spectrometry combined method, identifying a target analyte according to retention time and parent-child ion pair information, and quantifying by an internal standard curve method according to the chromatographic peak area of a selected ion pair of the target analyte to determine the content of each steroid hormone in the sample to be detected.
2. A method of detecting steroid hormones in human/animal body fluids according to claim 1, characterised in that step 1 includes any one or more of the following conditions:
a1 Conditions for ultrasonic dissolution are: temperature: carrying out ice water bath; ultrasonic time: 1-5 min;
a2 In the case of ultrasonic dissolution of the first solvent: estrone and 17 alpha-hydroxypregnenolone are prepared by using methanol or acetonitrile and DMSO in a volume ratio of 1:1, dissolving the rest by using pure methanol or pure acetonitrile; the first solvent is one or more of methanol, acetonitrile and DMSO; the second solvent is one or more of methanol, acetonitrile and water;
a3 The volume ratio of the mixed solution of methanol and water is 1:1 to 2:1;
a4 Specific concentrations of dehydroepiandrosterone sulfate in the mixed standard solutions of different concentrations are as follows: 200000, 100000, 50000, 20000, 10000, 5000, 2000, 1000, 500, 200, 100, 50, 20, 10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05ng/mL, the specific concentration of the rest 23 mixed standard solutions with different concentrations is 2000, 1000, 500, 200, 100, 50, 20, 10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05ng/mL;
a5 The dilution of each concentration of the mixed standard solution using a 1-percent BSA solution was specifically carried out by: preparing an aqueous solution with the bovine serum albumin mass fraction of 1%, and diluting the mixed standard solution with each concentration by 5 times.
3. A method of detecting steroid hormones in human/animal body fluids according to claim 1, characterised in that step 2 includes any one or more of the following conditions:
b1 The organic reagent protein precipitation method comprises the steps of putting 200 mu L of a body fluid sample into a 1.5ml PP centrifugal tube, adding 200 mu L of an extracting agent 1, uniformly mixing for 1min, adding 200 mu L of an extracting agent 2, uniformly mixing for 1min, and centrifuging at 12000rpm at 3-5 ℃ for 10min;
b2 The 7 internal isotope standards contained in the extractant 1) were: estrone-d 4, estradiol-d 4, pregnenolone-d 4, 17 alpha-hydroxypregnenolone-d 3, progesterone-d 9, testosterone-d 3 and cortisol-d 4, wherein the concentrations are all 50ng/mL;
b3 2M acetic acid-sodium acetate buffer solution of the extractant 2 had a pH of 5.2.
4. A method of detecting steroid hormones in human/animal body fluids according to claim 1, characterised in that step 3 includes any one or more of the following conditions:
c1 The specific concentration of the mixed solution of sulfatase and beta-glucuronidase is as follows: β -glucuronidase: 200-300U/mL, sulfatase: 600-800U/mL, the used solvent is water, and the use volume is 80-120 mu L;
c2 The volume of the sample supernatant of the second loading is 400-500 μ L;
c3 The eluent 1 has a volume ratio of acetonitrile to water of 1:9, the using volume is 150-250 mu L;
c4 Eluent 2 is used in a volume of 150-250 μ L;
c5 Eluent 1 acetonitrile to methanol in a volume ratio of 9:1, the using volume is 30-50 mu L;
c6 Loading the enzyme mixture and carrying out on-line enzymolysis at 37 ℃ for 60min, wherein the step is not carried out when only the content of free 24 steroid hormones is detected.
5. The method for detecting steroid hormones in human/animal body fluids according to claim 1, wherein in step 4, the liquid chromatography-mass spectrometry is ultra high liquid chromatography-tandem mass spectrometry;
the measuring conditions of the ultra-high liquid chromatography are as follows:
and (3) chromatographic column: a C8 liquid chromatography column; column temperature: 45 ℃; sample introduction amount: 5-20 mu L; flow rate: 0.3mL/min; the mobile phase A is 0.5mM ammonium fluoride aqueous solution; the mobile phase B is methanol; the analysis time is 13min; gradient elution;
the mass spectrum measurement conditions are as follows:
an ionization mode: an electrospray ion source; the ion source temperature is 500 ℃; the air curtain air is 40psi; the parameters of collision induced ionization are medium; the voltage of ion spraying is-4500/5500V; ion source Gas1:60psi; gas2:55psi; the monitoring mode is a multi-reaction monitoring MRM mode to carry out primary/secondary mass spectrometry.
6. Method for the detection of steroid hormones in human/animal body fluids according to claim 1, characterised in that the specific procedure of the gradient elution is: 0-0.5min, phase A: the volume ratio of the phase B is 55:45-55:45, a first step of; 0.5-6min, phase A: the volume ratio of the phase B is 55:45-45:55;6-10min, phase A: the volume ratio of the phase B is 45:55-5:95;10-12min, phase A: the volume ratio of the phase B is 5:95-5:95;12-12.2min, phase A: the volume ratio of the phase B is 5:95-55:45, a first step of; 12.2-13min, phase A: the volume ratio of the phase B is 55:45-55:45.
7. the method for detecting steroid hormones in human/animal body fluids according to claim 1, characterized in that the steroid hormones are: pregnenolone, 17 alpha-hydroxypregnenolone, progesterone, 17 alpha-hydroxyprogesterone, corticosterone, 11-deoxycorticosterol, 21-deoxycorticosterol, 11-deoxycorticosterone, estrone, estradiol, estriol, dihydrotestosterone, dehydroepiandrosterone sulfate, testosterone, cortisone, melatonin, aldosterone, androstenedione, cortisol, androstenediol, androsterone, epitestosterone, 20-hydroxyecdysone.
8. The method for detecting steroid hormones in human/animal body fluids according to claim 1, wherein the concentration ranges of the steroid hormone standard substance after being reconstituted with the first solvent are all 0.1-5 mg/mL; the first solvent adopted for redissolving the standard substance is one or more of methanol, acetonitrile and DMSO.
9. The method of claim 1, wherein the extraction reagent 1 is methanol containing an internal standard of 7 steroid hormones isotopes; the extractant 2 is 0.5-2M acetic acid-sodium acetate buffer solution; the eluent 1 is a mixed solution of water and acetonitrile; the eluent 2 is n-hexane, and the eluent 1 is a mixed solution of methanol and acetonitrile.
10. The method for detecting steroid hormones in human/animal body fluids according to claim 1, wherein the solid phase extraction column is Cleanertpp (2 mg).
11. The method of claim 1, wherein the on-line enzymatic hydrolysis is performed using a mixture of sulfatase and β -glucuronidase.
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| CN116519850A (en) * | 2023-06-29 | 2023-08-01 | 四川大学华西医院 | Method for rapidly detecting 16 hormone concentrations in serum sample |
| CN117571882A (en) * | 2024-01-09 | 2024-02-20 | 北京豪思生物科技股份有限公司 | Liquid chromatography-tandem mass spectrometry detection method for steroid hormone in serum |
| CN117571882B (en) * | 2024-01-09 | 2024-04-30 | 北京豪思生物科技股份有限公司 | Liquid chromatography-tandem mass spectrometry detection method for steroid hormone in serum |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116519850A (en) * | 2023-06-29 | 2023-08-01 | 四川大学华西医院 | Method for rapidly detecting 16 hormone concentrations in serum sample |
| CN116519850B (en) * | 2023-06-29 | 2023-09-19 | 四川大学华西医院 | Method for rapidly detecting 16 hormone concentrations in serum sample |
| CN117571882A (en) * | 2024-01-09 | 2024-02-20 | 北京豪思生物科技股份有限公司 | Liquid chromatography-tandem mass spectrometry detection method for steroid hormone in serum |
| CN117571882B (en) * | 2024-01-09 | 2024-04-30 | 北京豪思生物科技股份有限公司 | Liquid chromatography-tandem mass spectrometry detection method for steroid hormone in serum |
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