CN113009036A - Kit for detecting sex hormone, sex hormone sample pretreatment method and method for simultaneously detecting multiple sex hormones - Google Patents
Kit for detecting sex hormone, sex hormone sample pretreatment method and method for simultaneously detecting multiple sex hormones Download PDFInfo
- Publication number
- CN113009036A CN113009036A CN202110243850.4A CN202110243850A CN113009036A CN 113009036 A CN113009036 A CN 113009036A CN 202110243850 A CN202110243850 A CN 202110243850A CN 113009036 A CN113009036 A CN 113009036A
- Authority
- CN
- China
- Prior art keywords
- sex hormone
- solution
- liquid
- concentration
- kit
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Library & Information Science (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention provides a kit for detecting sex hormone, a sex hormone sample pretreatment method and a method for simultaneously detecting multiple sex hormones, belonging to the technical field of hormone detection, wherein the kit comprises the following components: the device comprises a solid-phase supporting liquid extraction container, a dissociation agent, a stabilizing agent, a first extraction liquid, a second extraction liquid, an internal standard solution, a diluent, a quality control product and a chromatographic column; the medium filled in the solid-phase supporting liquid extraction container is modified diatomite; the modified diatomite is obtained by calcining diatomite at 400-800 ℃ for 2-6 h and purifying; the particle size of the modified diatomite is 30-600 mu m, and the pore diameter of the modified diatomite is
The internal standard solution is a methanol solution containing more than two sex hormone isotope internal standards. The kit provided by the invention can be used for rapidly and accurately detecting multiple sex hormones in serum at the same time, and has the advantages of high sensitivity, good specificity and low sample usage amount.
Description
Technical Field
The invention belongs to the technical field of hormone detection, and particularly relates to a kit for detecting sex hormones, a sex hormone sample pretreatment method and a method for simultaneously detecting multiple sex hormones.
Background
Sex hormones belong to the human endogenous sex steroid hormones including estrogens, progestagens and androgens. In human body, estrogen mainly comprises estrone, estradiol and estriol, and has wide and important physiological effects, not only has the physiological effects of promoting and maintaining female reproductive organs and second sexual characteristics, but also has obvious effects on endocrine, cardiovascular, metabolic systems, growth and maturity of bones and the like. More and more studies have confirmed that estrogen is closely related to the development of related diseases. The accurate measurement of progesterone in serum has important clinical significance for the confirmation of luteal function, intrauterine pregnancy, spontaneous abortion and the like and the diagnosis of other diseases. Androgens include mainly testosterone, androstenedione, dehydroepiandrosterone, and the like, and in males, androgens promote the development of the reproductive organs and maintain secondary sexual characteristics. In women, testosterone analysis can be used to assess hyperandrogenism and androgen deficiency. Sex hormone has important physiological functions in the aspects of reproduction, sexual differentiation, pregnancy, immune system regulation and the like, so that the monitoring of the content level of the sex hormone in a human body has important significance for accurately diagnosing and preventing related diseases such as infertility, hypogonadism, polycystic ovary syndrome and the like, and the diagnosis rate of the related diseases can be improved by simultaneously detecting a plurality of sex hormones.
The concentration of sex hormones in human serum is extremely low, the serum concentration of estradiol in men and postmenopausal women is as low as the level of dozens of picograms per milliliter, and the level of progesterone in different physiological stages of adult women shows periodic change and usually ranges from a few nanograms per milliliter to two hundred and more nanograms per milliliter; in women, the content of testosterone is usually lower than 0.5 nanogram per milliliter, so that the sex hormone is detected at low concentration, and the sensitivity and the specificity of a detection method are high.
Currently, immunoassay methods (radioimmunoassay RIA, enzyme-linked immunosorbent ELISA, chemiluminescence immunoassay CLLA and the like) and mass spectrometry methods (gas chromatography-mass spectrometry GC-MS, liquid chromatography-mass spectrometry LC-MS and the like) are mainly adopted for measuring endogenous hormones in clinical sex hormone samples. The immunoassay method has wide clinical application, but is gradually replaced by other new technologies due to the limitations of the method, such as low sensitivity, few types of measurement, low specificity, and poor accuracy and repeatability of the immunoassay result, especially in the detection of low-concentration hormone. The gas chromatography-mass spectrometry (GC-MS) can be used for detecting low-concentration sex hormones, but because the pretreatment process is complex, samples can be subjected to mass spectrometry after complicated derivatization treatment, so that the method has certain limitation in the detection of clinical sex hormones. The liquid chromatography-tandem mass spectrometry has become an important means for detecting endogenous hormones due to the characteristics of high selectivity, high accuracy, high sensitivity and the like. However, the performance of the currently developed mass spectrometry detection method is still insufficient to meet the requirements of clinical detection, and the precision and accuracy of the methods are different, so that a new sex hormone detection method based on mass spectrometry, which has good accuracy, high sensitivity and good reproducibility and can realize high-throughput detection, is urgently needed to be developed, and a corresponding detection kit is developed to provide convenience for clinical detection.
For accurate quantitative detection of serum neutral hormones, a pretreatment step is crucial, and due to the low content of the sex hormones in serum, the pretreatment process must be capable of efficiently extracting a target substance from a serum matrix and removing as many impurities as possible on the premise of retaining and concentrating the target substance. In LC-MS/MS analysis, the existence of components such as esters in a serum matrix can generate ion inhibition, and further reduce the signal response value of target ions. Most of the existing detection methods need to use a sample amount of not less than 500 μ L to satisfy the signal response of the target in the serum sample, which is very disadvantageous for clinical samples with precious sources. Therefore, it is required to develop a pretreatment method suitable for clinical low sample size and capable of extracting a target substance with high efficiency and maximally removing unnecessary impurities.
Disclosure of Invention
In view of the above, the present invention aims to provide a sex hormone sample pretreatment method, a kit and a method for simultaneously detecting multiple sex hormones, which have high extraction efficiency, high impurity removal rate, low sample usage amount and simple operation.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a kit for simultaneously detecting multiple sex hormones, which comprises the following components: the device comprises a solid-phase supporting liquid extraction container, a dissociation agent, a stabilizing agent, a first extraction liquid, a second extraction liquid, an internal standard solution, a diluent, a quality control product and a chromatographic column;
the medium filled in the solid-phase supporting liquid extraction container is modified diatomite;
the modified diatomite is obtained by calcining the diatomite at 400-800 ℃ for 2-6 h and purifying, preferably 3-4 h, and more preferably 3.5 h.
The particle size of the modified diatomite is 30-600 mu m, and the pore diameter of the modified diatomite is
The internal standard solution is a methanol solution containing more than two sex hormone isotope internal standards;
the dissociating agent is 0.4-0.6 mol/L ammonium acetate water solution;
the stabilizer is 0.1-0.3 mol/L sodium carbonate aqueous solution;
the first extraction liquid is a mixed liquid of ethyl acetate and n-hexane; the second extraction liquid is n-hexane.
Preferably, the solid-phase supporting liquid-liquid extraction container is a solid-phase supporting liquid-liquid extraction perforated plate, the particle size of the modified diatomite is 50-200 mu m, and the pore diameter of the modified diatomite is
Preferably, the standard substance is a mixed solution containing more than two sex hormones, and the standard substance comprises a first standard substance and a second standard substance; the concentration of the first standard substance is 80-120 ng/mL by a concentration meter of a single sex hormone; and the concentration of the second standard substance is 8-12 ng/mL by using a concentration meter of a single sex hormone.
Preferably, the concentration of each sex hormone isotope internal standard in the internal standard solution is 8000-12000 pg/mL independently.
Preferably, the sex hormone isotope internal standard comprises testosterone-D3, androstenedione-D3, progesterone-D9, estrone-D4, or estradiol-D5.
Preferably, the quality control product is a blank serum matrix solution comprising a standard product; the quality control products comprise a first quality control product, a second quality control product and a third quality control product; the concentration of the standard substance in the first quality control substance is 80-120 pg/mL by a single sex hormone concentration meter, the concentration of the standard substance in the second quality control substance is 1800-2200 pg/mL by the single sex hormone concentration meter, and the concentration of the standard substance in the third quality control substance is 7500-8500 pg/mL by the single sex hormone concentration meter.
Preferably, the diluent comprises a first diluent and a second diluent; the first diluent is a blank serum matrix solution, and the second diluent is methanol.
The invention also provides a sex hormone sample pretreatment method, which comprises the following steps:
1) mixing and dissociating the internal standard solution, the sex hormone sample to be detected and the dissociating agent to obtain a dissociation solution;
2) transferring the dissociation solution to the solid-phase supporting solution extraction container, performing first elution by using a first extraction solution, and then stabilizing by using a stabilizing agent to obtain a stabilized sample;
3) and carrying out second elution on the stabilized sample by using a second extraction liquid to obtain an eluent, drying the eluent, and then re-dissolving the eluent by using methanol.
The invention provides a method for simultaneously detecting multiple sex hormones by using the kit, which comprises the following steps:
1) pretreating a serum sample by using the sex hormone sample pretreatment method to obtain a pretreated serum sample;
2) and detecting the pretreated serum sample and the standard substance by adopting an ultra performance liquid chromatography tandem mass spectrometry method, establishing a calibration curve by taking the concentration ratio of the standard substance to the internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and calculating the content of the neutral hormone in the serum sample according to the calibration curve.
Preferably, the sex hormones include any two or more of testosterone, androstenedione, progesterone, estrone, and estradiol.
The kit for detecting sex hormone provided by the invention can be used for quickly and accurately detecting multiple sex hormones in serum at the same time, and has the advantages of high sensitivity, good specificity and low sample consumption. The use amount of samples in the existing hormone detection method based on mass spectrum is more than 500 mu L, but the kit of the invention can achieve the aim of low-concentration detection by only using 100 mu L serum samples, and is more suitable for the detection of clinical samples.
The sex hormone sample pretreatment method provided by the invention adopts a solid-phase support liquid-liquid extraction method, takes the modified diatomite as a liquid-liquid distribution carrier, and the modified diatomite has the largest specific surface area and the lowest surface activity, so that an ideal liquid-liquid distribution support surface can be provided, and the emulsification phenomenon can be avoided. After the sex hormone sample is loaded, the aqueous phase solution forms a thin liquid film on the surface of the modified diatomite through capillary action. The elution is carried out by using an organic solvent which is not mutually soluble with water, and the distribution of the target substance between the water phase and the organic phase is very efficient, so that the results with high recovery rate and high repeatability can be obtained.
Furthermore, the solid-phase support liquid-liquid extraction container is made into a porous plate, so that the extraction time is shortened by more than half, the operation is simple, the flux is high, the automation is easy to realize, the detection of a large batch of samples is facilitated, and the method is suitable for screening related diseases in crowds.
According to the invention, ammonium acetate is used as a dissociation agent, the sex hormone in a human body is mainly combined with serum protein, the dissociation agent can completely dissociate the sex hormone from the binding protein, and meanwhile, incomplete balance of an internal standard and the binding protein is avoided, so that the recovery rate of an analyte tends to be consistent, and the detection precision of the kit is improved; according to the invention, sodium carbonate is used as a stabilizer, and n-hexane is used for extraction, so that a large amount of acidic impurities such as fatty acid and phospholipid can be removed, polar lipid is prevented from being accumulated on a chromatographic column, the separation effect is improved, and ion inhibition is reduced; the kit provided by the invention is subjected to two-time extraction in the using process, so that the precision and the chromatographic separation consistency are improved. The kit is used for secondary extraction, but the extraction efficiency reaches 80 percent, which is enough for quantitatively measuring the sex hormone with low concentration. Further, the ethyl acetate n-hexane is used as an extracting agent, and the safety of the ethyl acetate n-hexane is higher than that of ethers.
In conclusion, the kit and the method for detecting the sex hormone in the serum are adopted, the sample pretreatment is more convenient, compared with the existing detection means, the detection method of the kit improves the detection sensitivity, increases the recovery rate of the method, requires shorter analysis time, and provides a feasible method and a detection kit for detecting the sex hormone in the clinical serum sample.
Drawings
FIG. 1 is a MRM plot of testosterone and its isotope testosterone-D3 in serum;
FIG. 2 is a MRM map of androstenedione and its isotope androstenedione-D3 in serum;
FIG. 3 is a MRM graph of progesterone in serum and its isotope progesterone-D9;
FIG. 4 is a MRM map of estrone and its isotope estrone-D4 in serum;
FIG. 5 is a MRM map of estradiol and its isotope estradiol-D5 in serum;
FIG. 6 is a MRM chart of blank serum, blank serum plus standard, human serum.
Detailed Description
The invention provides a kit for simultaneously detecting multiple sex hormones, which comprises the following components: the device comprises a solid-phase supporting liquid extraction container, a dissociation agent, a stabilizing agent, a first extraction liquid, a second extraction liquid, an internal standard solution, a diluent, a quality control product and a chromatographic column; the medium filled in the solid-phase supporting liquid extraction container is modified diatomite; the modified diatomite is obtained by calcining diatomite at 400-800 ℃ for 2-6 h and purifying; the particle size of the modified diatomite is 30-600 mu mThe pore diameter of the diatomite is
The internal standard solution is a methanol solution containing more than two sex hormone isotope internal standards; the dissociating agent is 0.4-0.6 mol/L ammonium acetate water solution; the stabilizer is 0.1-0.3 mol/L sodium carbonate aqueous solution; the first extraction liquid is a mixed liquid of ethyl acetate and n-hexane; the second extraction liquid is n-hexane.
In the invention, the solid-phase supporting liquid-liquid extraction container is preferably a solid-phase supporting liquid-liquid extraction perforated plate, and the invention has no specific requirement on the number of holes of the solid-phase supporting liquid-liquid extraction perforated plate, and any number of holes can be selected; in the specific implementation process, the solid-phase support liquid-liquid extraction multi-well plate is a 96-well plate or a 384-well plate. In the invention, the medium filled in the solid-phase support liquid-liquid extraction porous plate is modified diatomite; the modified diatomite is obtained by calcining diatomite at 400-800 ℃ for 2-6 h and purifying; the calcining and purifying temperature is preferably 450-600 ℃, and more preferably 500 ℃; the calcining and purifying time is preferably 3-4 h, and more preferably 3.5 h; in the invention, the modified diatomite is calcined and purified, surface water and other organic matters are removed, the modified diatomite has large specific surface area and low surface activity, the pore structure is obviously improved, and the adsorbability of the diatomite is improved. In the invention, the particle size of the modified diatomite is preferably 50-200 μm, and the pore diameter of the modified diatomite is preferably 50-200 μm
In the invention, the kit comprises a dissociation agent, wherein the dissociation agent is 0.4-0.6 mol/L ammonium acetate aqueous solution, and preferably 0.5 mol/L; in the invention, the dissociation agent is used for separating lipid and polar components in serum, testosterone is mainly combined with serum protein, and the dissociation agent can completely dissociate testosterone from binding protein and simultaneously avoid incomplete balance of an internal standard solution and the binding protein, so that the recovery rate of an analyte tends to be consistent, and the precision of the method is improved.
In the invention, the kit comprises a stabilizer which is 0.1-0.3 mol/L sodium carbonate aqueous solution, preferably 0.2 mol/L. In the present invention, the stabilizer functions to remove acidic impurities such as phospholipids and fatty acids; such polar lipids generally accumulate on the chromatography column, worsen the separation effect, and increase ion suppression.
In the invention, the kit comprises a first extraction liquid and a second extraction liquid; the first extraction liquid is a mixed liquid of ethyl acetate and n-hexane, and the volume ratio of ethyl acetate to n-hexane in the first extraction liquid is preferably (2.5-3.5): 2, and more preferably 3: 2; the second extraction liquid is n-hexane. The invention can reach ideal extraction rate of sex hormone after two liquid-liquid extractions; in the present invention, the first extraction liquid separates the sex hormones from the matrix to the maximum extent, and the second extraction liquid removes the polar lipids from the sample extract to a large extent, thereby improving the accuracy and consistency of the chromatographic separation.
In the invention, the kit comprises an internal standard solution, wherein the internal standard solution is a methanol solution comprising more than two sex hormone isotope internal standards; the concentration of each sex hormone isotope internal standard in the internal standard solution is preferably 8000-12000 pg/mL independently, and more preferably 9000-11000 pg/mL; in the present invention, the sex hormone isotope internal standard comprises testosterone-D3, androstenedione-D3, progesterone-D9, estrone-D4, or estradiol-D5.
In the invention, the kit comprises a standard substance, wherein the standard substance is a mixed solution containing more than two sex hormones, and the standard substance comprises a first standard substance and a second standard substance; the concentration of the first standard substance is preferably 80-120 ng/mL, more preferably 90-110 ng/mL, and most preferably 100ng/mL by a concentration meter of a single sex hormone; the concentration of the second standard substance is preferably 8-12 ng/mL, more preferably 9-11 ng/mL, and most preferably 10ng/mL based on the concentration of the single sex hormone. In the present invention, the solvent of the first standard and the second standard is preferably methanol, and the methanol is preferably chromatographically pure. In the present invention, the standard solution is preferably prepared by the following method: preparing different sex hormone standard substances into mixed standard substance mother liquor by using methanol as a solvent; and then diluting the mixture step by using methanol to obtain a first standard substance and a second standard substance. In the invention, the first standard substance is used for diluting to obtain a part of series of concentration calibration points, and the second standard substance is used for serving as a first high-value concentration calibration point and preparing a first quality control substance, a second quality control substance and a third quality control substance with low, medium and high concentrations.
In the invention, the kit comprises a quality control product, wherein the quality control product is a blank serum matrix solution comprising a standard product; the quality control products comprise a first quality control product, a second quality control product and a third quality control product; the concentration of the standard substance in the first quality control product is preferably 80-120 pg/mL, more preferably 90-110 pg/mL, and most preferably 100pg/mL by a concentration meter of a single sex hormone; the concentration of the standard substance in the second quality control product is preferably 1800-2200 pg/mL, more preferably 1900-2100 pg/mL and most preferably 2000pg/mL by a concentration meter of a single sex hormone; the concentration of the standard substance in the third quality control product is preferably 7500-8500 pg/mL, more preferably 7800-8200 pg/mL, and most preferably 8000pg/mL based on the concentration of a single sex hormone. In the invention, the blank serum matrix solution is a blank serum sample which is purified by activated carbon adsorption and does not contain a target substance to be detected. In the invention, the first quality control product, the second quality control product and the third quality control product are used for evaluating the recovery rate and the precision of the method.
In the present invention, the kit comprises a diluent; in the present invention, the diluent preferably includes a first diluent and a second diluent; the first diluent is a blank serum matrix solution, and the second diluent is methanol. In the invention, the first diluent is used for preparing a series of standard solutions and quality control products, and the second diluent is used for diluting a mother solution of the standard products and re-dissolving the samples.
In the present invention, the kit comprises a chromatography column, preferably a UPLCC18 chromatography column; the inner diameter of the chromatographic column is preferably 1.0-3.0 mm, and more preferably 2.1 mm; the length of the chromatographic column is preferably 30-150 mm, and more preferably 100 mm; the particle size of the filler of the chromatographic column is 1.4-2.0 μm, and more preferably 1.7 μm.
The invention also provides a sex hormone sample pretreatment method, which comprises the following steps: 1) mixing and dissociating the internal standard solution, the sex hormone sample to be detected and the dissociating agent to obtain a dissociation solution; 2) transferring the dissociation solution to the solid-phase supporting solution extraction container, performing first elution by using a first extraction solution, and then stabilizing by using a stabilizing agent to obtain a stabilized sample; 3) and carrying out second elution on the stabilized sample by using a second extraction liquid to obtain an eluent, drying the eluent, and then re-dissolving the eluent by using methanol.
In the invention, the internal standard solution, the sex hormone sample to be detected and the dissociation agent are mixed and dissociated to obtain dissociation liquid. In the present invention, the internal standard solution is preferably placed in a 96-well sample collection plate; according to the invention, the internal standard solution is preferably dried by nitrogen and then mixed with the sex hormone sample to be detected and the dissociation agent for dissociation. In the invention, the dissociation temperature is preferably 20-30 ℃; more preferably 25 ℃; the dissociation time is preferably 25-35 min, and more preferably 30 min. In the present invention, the volume ratio of the internal standard solution, the sex hormone sample to be detected and the dissociation agent is preferably 1: (8-12): (8-12), more preferably 1:10: 10; in the specific implementation process of the invention, the volume of the internal standard solution is preferably 8-12 μ L, and more preferably 10 μ L; the volume of the sex hormone sample to be detected and the dissociation agent is preferably 80-120 mu L, and more preferably 100 mu L.
According to the invention, after obtaining the dissociation solution, the dissociation solution is transferred to the solid-phase supporting solution extraction container, first elution is carried out by using a first extraction liquid, and then a stabilized sample is obtained by using a stabilizing agent for stabilization. In the invention, the solid-phase supporting liquid-liquid extraction container is a solid-phase supporting liquid-liquid extraction 96-well plate filled with modified diatomite. After the dissociation solution is transferred to a solid-phase support liquid-liquid extraction 96-well plate, preferably standing for 4-6 min, and more preferably standing for 5 min. The invention uses the first extract liquid to carry out the first elution, and adds the stabilizing agent into the eluent after the eluent of the first elution completely flows out. In the invention, the volume of the first extraction liquid is preferably 5-10 times, more preferably 6-8 times, and most preferably 7 times of the volume of the sex hormone sample to be detected; in the invention, the stabilizing time is preferably 4-6 min, and more preferably 5 min. In the invention, the volume of the stabilizing agent is preferably 0.8-1.2 times, and more preferably 1 time of the volume of the sex hormone sample to be detected. In the specific implementation process of the present invention, the volume of the first eluent is preferably 500 to 1000 μ L, more preferably 600 to 800 μ L, and most preferably 700 μ L; the volume of the stabilizer is preferably 80-120 mu L, and more preferably 100 mu L.
After the stabilization, the second extraction liquid is used for carrying out second elution on the stabilized sample to obtain an eluent, and the eluent is redissolved by methanol after being dried. In the present invention, the number of times of the second elution is preferably 1 to 3 times, and more preferably 2 times. In the invention, the volume of the second extraction liquid used in each second elution is preferably 3-5 times, and more preferably 4 times of the volume of the sex hormone sample to be detected. In the specific implementation process of the invention, after the last second elution is finished, preferably, a 96-hole biological sample pretreatment instrument is used for slightly applying positive pressure, so that the eluent is completely transferred to a new 96-hole sample collection plate, and then a 96-hole nitrogen blowing concentrator is used for blowing nitrogen to dry and then is redissolved by methanol. In the invention, the volume of the methanol is preferably 0.8-1.2 times, and more preferably 1 time of the volume of the sex hormone sample to be detected.
The invention also provides a method for simultaneously detecting multiple sex hormones by using the kit, which comprises the following steps: 1) pretreating a serum sample by using the sex hormone sample pretreatment method to obtain a pretreated serum sample; 2) and detecting the pretreated serum sample and the standard substance by adopting an ultra performance liquid chromatography tandem mass spectrometry method, establishing a calibration curve by taking the concentration ratio of the standard substance to the internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and calculating the content of the neutral hormone in the serum sample according to the calibration curve.
In the invention, a serum sample is pretreated by using the sex hormone sample pretreatment method to obtain a pretreated serum sample; the preprocessing method is not described herein again.
The pretreated serum sample is detected by adopting an ultra performance liquid chromatography tandem mass spectrometry method after the pretreated serum sample is obtained.
In the method of the ultra performance liquid chromatography tandem mass spectrometry, the chromatographic conditions are preferably as follows: the chromatographic column is preferably an UPLC C18 chromatographic column (2.1 multiplied by 100mm, 1.7 mu m), the sample injection volume is preferably 1-10 mu L, the flow rate is preferably 0.2-0.5 mL/min, the column temperature of the chromatographic column is preferably 25-40 ℃, and the sample chamber temperature is preferably 2-8 ℃; gradient elution for 6 min; wherein the mobile phase A is 0.05-0.5 mM ammonium fluoride aqueous solution, and the mobile phase B is acetonitrile. In the present invention, the elution gradient procedure for the chromatographic separation is preferably as follows: 0-0.1 min, 25% B; the phase B is increased from 25% to 75% in 0.1-3.5 min; 3.5-3.6 min, increasing the phase B from 75% to 95%; 3.6-4.5 min, 95% B; 4.5-4.6 min, reducing the phase B from 95% to 25%; 4.6-6.0 min, 25% B.
In the method of the ultra performance liquid chromatography tandem mass spectrometry, the mass spectrometry conditions are preferably as follows: selecting ESI positive and negative ion mode switching scanning, and detecting multiple sex hormones by adopting a multi-reaction monitoring technology; the mass spectrum parameters in the invention are preferably as follows: the ion source temperature is 180 ℃, the desolventizing gas temperature is 500 ℃, the desolventizing gas flow is 700L/H, and the cone hole blowback gas flow is 140L/H. In the present invention, the parameters for detecting multiple sex hormones by the Multiple Reaction Monitoring (MRM) technique under mass spectrometry conditions are shown in table 1.
TABLE 1 parameter table for detecting multiple sex hormones by mass spectrum multiple reaction monitoring technique
In the invention, the standard substance is detected by adopting an ultra performance liquid chromatography tandem mass spectrometry method, the concentration ratio of the standard substance to the internal standard substance is taken as an X axis, the peak area ratio of the standard substance to the internal standard substance is taken as a Y axis, a calibration curve is established, and the content of the neutral hormone in the serum sample is calculated according to the calibration curve. In the present invention, the sex hormone preferably includes any two or more of testosterone, androstenedione, progesterone, estrone, and estradiol.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The isotope dilution liquid chromatography tandem mass spectrometry is adopted to detect the neutral hormone in the serum sample, the sex hormone comprises testosterone and androstenedione, the sample involved in the embodiment is fresh human serum, and the appearance is clear without hemolysis, jaundice and lipemia.
The embodiment comprises the following steps:
1. main instrument
The instrument used in this example was an ultra performance liquid chromatography-triple quadrupole mass spectrometer (WATERS, USA).
2. Method of producing a composite material
(1) Chromatographic conditions are as follows: UPLC C18 chromatographic column (2.1X 100mM, 1.7 μm), flow rate 0.4mL/min, sample volume 5 μ L, column temperature 30 ℃, sample chamber temperature 8 ℃, mobile phase A0.1 mM ammonium fluoride aqueous solution, mobile phase B acetonitrile, using gradient elution mode, the elution procedure is as follows: 0-0.1 min, 25% B; the phase B is increased from 25% to 75% in 0.1-3.5 min; 3.5-3.6 min, increasing the phase B from 75% to 95%; 3.6-4.5 min, 95% B; 4.5-4.6 min, reducing the phase B from 95% to 25%; 4.6-6.0 min, 25% B.
(2) Mass spectrum conditions: scanning was performed in electrospray ionization (ESI) positive ion mode, and mass spectrometry scan mode using Multiple Reaction Monitoring (MRM). The ion source temperature is 180 ℃, the desolventizing gas temperature is 500 ℃, the desolventizing gas flow is 700L/H, and the cone hole blowback gas flow is 140L/H. The mass spectral parameters of testosterone and androstenedione and their isotopes are shown in table 2.
TABLE 2 Mass Spectrometry parameters for Testosterone and androstenedione and their isotopes
(3) Preparing a standard substance: accurately weighing 10.0mg of each of testosterone and androstenedione standard substance, mixing, adding 10mL of methanol solution, and preparing into mixed standard substance mother liquor with concentration of 1.0 mg/mL; then gradually diluting the concentration of the mixed standard substance from 1.0mg/mL to 100ng/mL by using methanol to obtain a mixed standard solution A; 100 mu L of the mixed standard solution A is taken and is made into a volume of 1mL by methanol, and the mixed standard solution B with the concentration of 10ng/mL is obtained.
Taking the mixed standard solution B as a first high-value concentration point, respectively transferring 50, 25 and 10 mu L of the mixed standard solution A into a 1.5mL centrifuge tube, drying by blowing nitrogen, adding a blank serum substrate into the centrifuge tube to fix the volume to 1mL to obtain calibration concentration points with the concentrations of 1000, 2500 and 5000pg/mL, and taking the mixed standard solution with the concentration of 1000pg/mL as a mixed standard solution C; respectively transferring 500, 250, 100, 50, 25 and 10 mu L of the mixed standard solution C, and fixing the volume to 1mL by using a blank serum substrate to obtain the other six calibration concentration points; a total of 10 mixed standard series of calibration concentration points were obtained.
(4) Preparing a quality control product: and drying the mixed standard solution B10 mu L, 200 mu L and 800 mu L by nitrogen, and respectively metering the volume to 1000 mu L by using blank serum matrix solution to obtain the serum.
(5) Preparing an internal standard solution: preparing 100 mu g/mL mother liquor from testosterone-D3 and androstenedione-D3 by using methanol as a solvent, and then gradually diluting to 10000pg/mL mixed internal standard solution.
(6) Sample pretreatment:
taking a 96-well sample collection plate, adding 10 mu L of mixed internal standard solution into each well, drying by nitrogen, adding 100 mu L of serum sample, adding 100 mu L of ammonium acetate aqueous solution, uniformly mixing, and dissociating at room temperature for 30 min; loading the sample into a solid-phase support liquid-liquid extraction 96-well plate, standing for 5min, and adding 700 μ L of first extract (the ratio of ethyl acetate to n-hexane is 3:2) for first elution; and adding 100 mu L of sodium carbonate aqueous solution serving as a stabilizer after the elution liquid completely flows out, standing for 5min, then respectively eluting for 2 times by using a second extracting agent n-hexane, wherein the dosage of the extracting agent is 400 mu L each time, slightly applying positive pressure by using a 96-hole biological sample pretreatment instrument after the last elution to completely transfer the eluent into a new 96-hole sample collection plate, then blowing nitrogen by using a 96-hole nitrogen blowing concentrator for drying, redissolving by using 100 mu L of methanol, and uniformly mixing to be tested. The pretreatment process of the standard substance and the quality control substance is consistent with that of a serum sample.
The components of the assay kit are shown in Table 3.
TABLE 3 sex hormone detection kit Components
3. Methodological validation results
1) The specificity is as follows: the peak patterns of the testosterone and androstenedione standards and each internal standard and serum sample were symmetric (see fig. 6), with essentially no impurity interference, indicating good specificity under these conditions, indicating that these conditions can be used for quantitative analysis of serum sex hormones.
2) Linearity, detection limit and quantitation limit: the method comprises the steps of preparing a standard curve by utilizing sex hormone standard substances with series concentrations by adopting an isotope internal standard method, taking the isotope of the sex hormone as an internal standard substance, taking the concentration ratio of the standard substance to the internal standard substance as an X axis, taking the peak area ratio of the standard substance to the internal standard substance as a Y axis, respectively establishing calibration curves, and calculating the content of the neutral hormone in the serum sample. Determining the detection Limit (LOD) of the target object according to the mass concentration of which the signal-to-noise ratio (S/N) is not less than 3, and determining the quantification Limit (LOQ) of the target object according to the mass concentration of which the S/N is not less than 10. The results are shown in Table 4.
TABLE 42 sex hormone Linearity, detection limits and lower quantitation limits
3) Precision and accuracy of the method
And selecting the quality control products with low, medium and high concentrations to simultaneously carry out precision and accuracy investigation of the method, and treating and measuring the quality control products according to a sample pretreatment method. The precision of the method is usually examined by using the variation Coefficient (CV) of the quality control product between batches (n is 6) and (n is 18), and the CV should be less than 15%. Accuracy is the closeness of the measured concentration of the sample to the real concentration, generally should be in the range of 85% -115%, and the CV should be less than 15%. The results of the precision and accuracy of the method are shown in Table 5.
TABLE 52 sex hormone method precision and accuracy results
As can be seen from the above table, the method of the present embodiment has good reproducibility, stability and recovery rate when being used for simultaneously detecting 2 sex hormones.
Example 2
In another embodiment of the method and the kit for detecting the sex hormone in the serum by the ultra performance liquid chromatography-tandem mass spectrometry, the sex hormone in the serum sample is detected by the isotope dilution liquid chromatography-tandem mass spectrometry, the sex hormone comprises progesterone, estrone and estradiol, the sample involved in the embodiment is fresh human serum, and the appearance is clear without hemolysis, jaundice and lipemia.
The embodiment comprises the following steps:
1. main instrument
The instrument used in this example was an ultra performance liquid chromatography-triple quadrupole mass spectrometer (WATERS, USA).
2. Method of producing a composite material
(1) Chromatographic conditions are as follows: UPLC C18 chromatographic column (2.1X 100mM, 1.7 μm), flow rate 0.3mL/min, sample volume 5 μ L, column temperature 40 ℃, sample chamber temperature 4 ℃, mobile phase A0.2 mM ammonium fluoride aqueous solution, mobile phase B acetonitrile, using gradient elution mode, the elution procedure is as follows: 0-0.1 min, 25% B; the phase B is increased from 25% to 75% in 0.1-3.5 min; 3.5-3.6 min, increasing the phase B from 75% to 95%; 3.6-4.5 min, 95% B; 4.5-4.6 min, reducing the phase B from 95% to 25%; 4.6-6.0 min, 25% B.
(2) Mass spectrum conditions: scanning was performed in electrospray ionization (ESI) positive and negative ion switching mode, and mass spectrometry scan mode using Multiple Reaction Monitoring (MRM). The ion source temperature is 180 ℃, the desolventizing gas temperature is 500 ℃, the desolventizing gas flow is 700L/H, and the cone hole blowback gas flow is 140L/H. The mass spectral parameters of progesterone, estrone and estradiol and their isotopes are shown in table 6.
TABLE 6 Mass Spectrometry parameters for Progesterone, estrone and estradiol and their isotopes
(3) Preparing a standard substance: accurately weighing 10.0mg of each of progesterone, estrone and estradiol standards, mixing, adding 10mL of methanol solution, and preparing into mixed standard mother liquor with the concentration of 1.0 mg/mL; then gradually diluting the concentration of the mixed standard substance from 1.0mg/mL to 100ng/mL by using methanol to obtain a mixed standard solution A; 100 mu L of the mixed standard solution A is taken and is made into a volume of 1mL by methanol, and the mixed standard solution B with the concentration of 10ng/mL is obtained.
Taking the mixed standard solution B as a first high-value concentration point, respectively transferring 50, 25 and 10 mu L of the mixed standard solution A into a 1.5mL centrifuge tube, drying by blowing nitrogen, adding a blank serum substrate into the centrifuge tube to fix the volume to 1mL to obtain calibration concentration points with the concentrations of 1000, 2500 and 5000pg/mL, and taking the mixed standard solution with the concentration of 1000pg/mL as a mixed standard solution C; respectively transferring 500, 250, 100, 50, 25 and 10 mu L of the mixed standard solution C, and fixing the volume to 1mL by using a blank serum substrate to obtain the other six calibration concentration points; a total of 10 mixed standard series of calibration concentration points were obtained.
(4) Preparing a quality control product: and drying the mixed standard solution B10 mu L, 200 mu L and 800 mu L by nitrogen, and respectively metering the volume to 1000 mu L by using blank serum matrix solution to obtain the serum.
(5) Preparing an internal standard solution: respectively preparing 100 mu g/mL mother liquor from progesterone-D9, estrone-D4 and estradiol-D5 by using methanol as a solvent, and then gradually diluting to 10000pg/mL mixed internal standard solution.
(6) Sample pretreatment: taking a 96-well sample collection plate, adding 10 mu L of mixed internal standard solution into each well, drying by nitrogen, adding 100 mu L of serum sample, adding 100 mu L of ammonium acetate aqueous solution, uniformly mixing, and dissociating at room temperature for 30 min; loading the sample into a solid-phase support liquid-liquid extraction 96-well plate, standing for 5min, and adding 700 μ L of first extract (the ratio of ethyl acetate to n-hexane is 3:2) for first elution; and adding 100 mu L of sodium carbonate aqueous solution serving as a stabilizer after the elution liquid completely flows out, standing for 5min, then respectively eluting for 2 times by using a second extracting agent n-hexane, wherein the dosage of the extracting agent is 400 mu L each time, slightly applying positive pressure by using a 96-hole biological sample pretreatment instrument after the last elution to completely transfer the eluent into a new 96-hole sample collection plate, then blowing nitrogen by using a 96-hole nitrogen blowing concentrator for drying, redissolving by using 100 mu L of methanol, and uniformly mixing to be tested.
The pretreatment process of the standard substance and the quality control substance is consistent with that of a serum sample.
The components of the assay kit are shown in Table 7.
TABLE 7 sex hormone assay kit Components
3. Methodological validation results
1) The specificity is as follows: the peak patterns of the progesterone, estrone and estradiol standards and the internal standard standards and the serum sample are symmetrical, and basically have no impurity interference, which indicates that the specificity is good under the condition, and indicates that the condition can be used for quantitative analysis of the serum sex hormone.
2) Linearity, detection limit and quantitation limit: the method comprises the steps of preparing a standard curve by utilizing sex hormone standard substances with series concentrations by adopting an isotope internal standard method, taking the isotope of the sex hormone as an internal standard substance, taking the concentration ratio of the standard substance to the internal standard substance as an X axis, taking the peak area ratio of the standard substance to the internal standard substance as a Y axis, respectively establishing calibration curves, and calculating the content of the neutral hormone in the serum sample. Determining the detection Limit (LOD) of the target object according to the mass concentration of which the signal-to-noise ratio (S/N) is not less than 3, and determining the quantification Limit (LOQ) of the target object according to the mass concentration of which the S/N is not less than 10. The results are shown in Table 8.
TABLE 83 Linear, detection and quantitation limits of sex hormones
3) Precision and accuracy of the method
And selecting the quality control products with low, medium and high concentrations to simultaneously carry out precision and accuracy investigation of the method, and treating and measuring the quality control products according to a sample pretreatment method. The precision of the method is usually examined by using the variation Coefficient (CV) of the quality control product between batches (n is 6) and (n is 18), and the CV should be less than 15%. Accuracy is the closeness of the measured concentration of the sample to the real concentration, generally should be in the range of 85% -115%, and the CV should be less than 15%. The results of the precision and accuracy of the method are shown in Table 9.
TABLE 93 results of accuracy and precision of sex hormone method
As can be seen from the above table, the detection method of the present embodiment has good reproducibility, stability and recovery rate when being used for simultaneously detecting 3 sex hormones.
From the above examples, it can be seen that the method for sex hormone detection using the kit provided by the present invention overcomes the problems of lack of specificity and poor sensitivity caused by cross reaction and matrix interference in the chemical immunoassay, and the isotope internal standard method greatly eliminates the interference of the matrix, and largely eliminates the influence of conditions such as pretreatment process, sample loading volume and flow, thereby achieving the purpose of accurate quantification. The methodological research in the embodiment shows that the kit for detecting the sex hormone content meets the requirements on linear range, detection limit, accuracy and precision, has high sensitivity, strong specificity, accuracy and reliability, and can be applied to simultaneous quantitative detection of multiple sex hormones in clinical serum samples.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A kit for simultaneously detecting multiple sex hormones is characterized by comprising the following components: the device comprises a solid-phase supporting liquid extraction container, a dissociation agent, a stabilizing agent, a first extraction liquid, a second extraction liquid, an internal standard solution, a diluent, a quality control product and a chromatographic column;
the medium filled in the solid-phase supporting liquid extraction container is modified diatomite;
the modified diatomite is obtained by calcining diatomite at 400-800 ℃ for 2-6 h and purifying;
the particle size of the modified diatomite is 30-600 mu m, and the pore diameter of the modified diatomite is
The internal standard solution is a methanol solution containing more than two sex hormone isotope internal standards;
the dissociating agent is 0.4-0.6 mol/L ammonium acetate water solution;
the stabilizer is 0.1-0.3 mol/L sodium carbonate aqueous solution;
the first extraction liquid is a mixed liquid of ethyl acetate and n-hexane; the second extraction liquid is n-hexane.
2. The kit according to claim 1, wherein the solid-phase-supporting liquid-liquid extraction container is a multi-well plate for solid-phase-supporting liquid-liquid extraction, the particle size of the modified diatomaceous earth is 50 to 200 μm, and the modified diatomaceous earth isThe pore diameter of the soil is
3. The kit according to claim 1, wherein the standard is a mixed solution comprising two or more sex hormones, and the standard comprises a first standard and a second standard; the concentration of the first standard substance is 80-120 ng/mL by a concentration meter of a single sex hormone; and the concentration of the second standard substance is 8-12 ng/mL by using a concentration meter of a single sex hormone.
4. The kit of claim 1, wherein the concentration of each sex hormone isotope internal standard in the internal standard solution is independently 8000-12000 pg/mL.
5. The kit of claim 4, wherein said sex hormone isotope internal standard comprises testosterone-D3, androstenedione-D3, progesterone-D9, estrone-D4, or estradiol-D5.
6. The kit of claim 1, wherein the quality control product is a blank serum matrix solution comprising a standard; the quality control products comprise a first quality control product, a second quality control product and a third quality control product; the concentration of the standard substance in the first quality control substance is 80-120 pg/mL by a single sex hormone concentration meter, the concentration of the standard substance in the second quality control substance is 1800-2200 pg/mL by the single sex hormone concentration meter, and the concentration of the standard substance in the third quality control substance is 7500-8500 pg/mL by the single sex hormone concentration meter.
7. The kit of claim 1, wherein the diluent comprises a first diluent and a second diluent; the first diluent is a blank serum matrix solution, and the second diluent is methanol.
8. A sex hormone sample pretreatment method comprises the following steps:
1) mixing and dissociating the internal standard solution, the sex hormone sample to be detected and the dissociating agent to obtain a dissociation solution;
2) transferring the dissociation solution to the solid-phase supporting solution extraction container of claim 1, performing first elution by using a first extraction solution, and then stabilizing by using a stabilizing agent to obtain a stabilized sample;
3) and carrying out second elution on the stabilized sample by using a second extraction liquid to obtain an eluent, drying the eluent, and then re-dissolving the eluent by using methanol.
9. A method for simultaneously detecting multiple sex hormones using the kit according to any one of claims 1 to 7, comprising the steps of:
1) pretreating a serum sample by the method for pretreating a sex hormone sample according to claim 8 to obtain a pretreated serum sample;
2) and detecting the pretreated serum sample and the standard substance by adopting an ultra performance liquid chromatography tandem mass spectrometry method, establishing a calibration curve by taking the concentration ratio of the standard substance to the internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and calculating the content of the neutral hormone in the serum sample according to the calibration curve.
10. The method of claim 9, wherein the sex hormone comprises any two or more of testosterone, androstenedione, progesterone, estrone, and estradiol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110243850.4A CN113009036A (en) | 2021-03-05 | 2021-03-05 | Kit for detecting sex hormone, sex hormone sample pretreatment method and method for simultaneously detecting multiple sex hormones |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110243850.4A CN113009036A (en) | 2021-03-05 | 2021-03-05 | Kit for detecting sex hormone, sex hormone sample pretreatment method and method for simultaneously detecting multiple sex hormones |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113009036A true CN113009036A (en) | 2021-06-22 |
Family
ID=76406380
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110243850.4A Pending CN113009036A (en) | 2021-03-05 | 2021-03-05 | Kit for detecting sex hormone, sex hormone sample pretreatment method and method for simultaneously detecting multiple sex hormones |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113009036A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113504379A (en) * | 2021-07-28 | 2021-10-15 | 北京乐普诊断科技股份有限公司 | Sex hormone dissociation agent and detection kit |
| CN113960224A (en) * | 2021-09-26 | 2022-01-21 | 裕菁科技(上海)有限公司 | Additive applied to liquid-liquid extraction pretreatment of biological sample and extraction method |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109470791A (en) * | 2018-11-29 | 2019-03-15 | 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院） | A kind of method and kit of high performance liquid chromatography-tandem mass detection serum estradiol |
| WO2019232381A1 (en) * | 2018-06-01 | 2019-12-05 | Laboratory Corporation Of America Holdings | Simplified biosample processing for lc-ms/ms |
| CN110702831A (en) * | 2019-11-18 | 2020-01-17 | 天津汉科生物科技有限公司 | Kit for detecting serum testosterone hormone by ultra-high performance liquid chromatography-tandem mass spectrometry |
| CN110927264A (en) * | 2019-11-08 | 2020-03-27 | 河北省医疗器械与药品包装材料检验研究院(河北省医疗器械技术审评中心) | Method and kit for detecting glucocorticoid in serum |
| CN111175419A (en) * | 2020-03-02 | 2020-05-19 | 山东英盛生物技术有限公司 | Method and kit for simultaneously detecting multiple steroid hormones in blood sample |
-
2021
- 2021-03-05 CN CN202110243850.4A patent/CN113009036A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019232381A1 (en) * | 2018-06-01 | 2019-12-05 | Laboratory Corporation Of America Holdings | Simplified biosample processing for lc-ms/ms |
| CN109470791A (en) * | 2018-11-29 | 2019-03-15 | 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院） | A kind of method and kit of high performance liquid chromatography-tandem mass detection serum estradiol |
| CN110927264A (en) * | 2019-11-08 | 2020-03-27 | 河北省医疗器械与药品包装材料检验研究院(河北省医疗器械技术审评中心) | Method and kit for detecting glucocorticoid in serum |
| CN110702831A (en) * | 2019-11-18 | 2020-01-17 | 天津汉科生物科技有限公司 | Kit for detecting serum testosterone hormone by ultra-high performance liquid chromatography-tandem mass spectrometry |
| CN111175419A (en) * | 2020-03-02 | 2020-05-19 | 山东英盛生物技术有限公司 | Method and kit for simultaneously detecting multiple steroid hormones in blood sample |
Non-Patent Citations (2)
| Title |
|---|
| YUESONG WANG ET AL.: "Total testosterone quantitative measurement in serum by LC-MS/MS", 《CLINICA CHIMICA ACTA》 * |
| 张双庆 主编: "《毒代动力学》", 30 September 2014 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113504379A (en) * | 2021-07-28 | 2021-10-15 | 北京乐普诊断科技股份有限公司 | Sex hormone dissociation agent and detection kit |
| CN113960224A (en) * | 2021-09-26 | 2022-01-21 | 裕菁科技(上海)有限公司 | Additive applied to liquid-liquid extraction pretreatment of biological sample and extraction method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111366671B (en) | Chemical derivatization-ultra-high performance liquid chromatography-tandem mass spectrometry for simultaneously detecting 18 steroid hormones in serum | |
| CN111398446B (en) | Method for detecting 12 steroid hormones in serum by ultra-high performance liquid chromatography tandem mass spectrometry technology | |
| CN111398447B (en) | Kit for detecting 12 steroid hormone in serum by ultra-high performance liquid chromatography tandem mass spectrometry technology | |
| CN111175419B (en) | Method and kit for simultaneously detecting multiple steroid hormones in blood sample | |
| Chang et al. | Quantitative measurement of male steroid hormones using automated on-line solid phase extraction-liquid chromatography-tandem mass spectrometry and comparison with radioimmunoassay | |
| CN110702831B (en) | Kit for detecting serum testosterone hormone by ultra-high performance liquid chromatography-tandem mass spectrometry | |
| WO2004090553A1 (en) | Thyroid hormone analysis by mass spectrometry | |
| CN114674961A (en) | Kit for synchronously detecting 17 steroid hormones in serum without derivatization and application thereof | |
| CN113009036A (en) | Kit for detecting sex hormone, sex hormone sample pretreatment method and method for simultaneously detecting multiple sex hormones | |
| CN112611827B (en) | Chemical derivation-ultra high performance liquid chromatography-tandem mass spectrometry for detecting 3 estrogens | |
| CN111323507A (en) | Detection method for simultaneously detecting 11 steroids in serum based on solid phase extraction method | |
| CN112485341A (en) | Method for detecting hormone in blood plasma by liquid chromatography-tandem mass spectrometry technology | |
| US11536733B2 (en) | Methods and systems for the detection of 11-oxo androgens by LC-MS/MS | |
| CN112986433A (en) | Method for detecting steroid content in human serum sample | |
| CN114414696B (en) | Kit and method for determining multiple estrogens in dried blood slices | |
| CN116519850B (en) | Method for rapidly detecting 16 hormone concentrations in serum sample | |
| CN109828043A (en) | The quantitative analysis method of Acotiamide in a kind of human plasma | |
| Liu et al. | A sensitive approach for simultaneous quantification of carbonyl and hydroxyl steroids using 96-well SPE plates based on stable isotope coded-derivatization-UPLC-MRM: method development and application | |
| CN115343398A (en) | Kit and method for simultaneously measuring multiple steroid hormones in serum | |
| CN116593628B (en) | LC-MS/MS-based method and kit for simultaneously detecting multiple steroid hormones in serum | |
| Pan et al. | Quantification of 37 glucocorticoids in chicken muscle by UHPLC-Q-Orbitrap-MS with parallel reaction monitoring | |
| CN114720570B (en) | Method for detecting 8 estrogens in fish meat | |
| CN114609297B (en) | Method and kit for simultaneously detecting 5-steroid hormone and application of kit | |
| CN114720571B (en) | Method for detecting 15 antibiotics in fish body | |
| Vlase et al. | New LC-MS/MS method for testosterone and dihydrotestosterone analysis in male rat plasma |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210622 |