CN110702831A - Kit for detecting serum testosterone hormone by ultra-high performance liquid chromatography-tandem mass spectrometry - Google Patents
Kit for detecting serum testosterone hormone by ultra-high performance liquid chromatography-tandem mass spectrometry Download PDFInfo
- Publication number
- CN110702831A CN110702831A CN201911127530.1A CN201911127530A CN110702831A CN 110702831 A CN110702831 A CN 110702831A CN 201911127530 A CN201911127530 A CN 201911127530A CN 110702831 A CN110702831 A CN 110702831A
- Authority
- CN
- China
- Prior art keywords
- testosterone
- kit
- solution
- extraction liquid
- concentration
- 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.)
- Granted
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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/045—Standards internal
-
- 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
Abstract
The invention provides a kit for detecting serum testosterone hormone by ultra-high performance liquid chromatography-tandem mass spectrometry, which belongs to the technical field of hormone detection, and comprises a standard solution, an internal standard solution, a diluent, an extraction liquid, a dissociating agent, a stabilizer, a quality control product and a chromatographic column; the extraction liquid comprises a first extraction liquid and a second extraction liquid; the first extraction liquid is a mixed liquid of ethyl acetate and n-hexane; the second extraction liquid is n-hexane; 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 kit provided by the invention can be used for detecting the linear range, detection limit, recovery rate and precision of testosterone content, meets the requirements, has high sensitivity and strong specificity, is accurate and reliable, and can be applied to quantitative detection of testosterone hormone in clinical serum samples.
Description
Technical Field
The invention belongs to the technical field of hormone detection, and particularly relates to a kit for detecting serum testosterone hormone by high performance liquid chromatography-tandem mass spectrometry.
Background
Testosterone is a steroid hormone, and has effects of enhancing muscle strength, enhancing immunity, and resisting osteoporosis, and can be used as endogenous hormone and anabolic hormone. The detection of testosterone hormones in serum is of great significance in adult and pediatric endocrinology and oncology. In men, testosterone analysis is used primarily to confirm hypogonadism and also to assess delayed puberty or precocious boys and monitor the appropriateness of testosterone treatment. In women, testosterone analysis can be used to assess hyperandrogenism (e.g., idiopathic hirsutism, congenital adrenal cortical hyperplasia, polycystic ovarian syndrome, and androgen-secreting ovarian or adrenal tumors), as well as androgen deficiency. The circulating concentration of testosterone in women and children is very low (an order of magnitude lower than that in normal men), and is typically less than 0.5ng/mL for adult women, and less than 0.1ng/mL for children and men receiving antiandrogen therapy. Therefore, a method for detecting testosterone hormone is required to have high sensitivity.
At present, the method for measuring endogenous hormone in clinical biological samples mainly adopts immunoassay methods such as chemiluminescence immunoassay and the like, but the sensitivity and the accuracy of the immunoassay method are insufficient because the same antibody can react with various antigens in the immunoassay method. In particular, in the detection of testosterone concentrations in children and women, the accuracy and reproducibility of the results of the immunoassay are poor. 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 spectrometric detection method is still insufficient to meet the requirements of clinical detection, and compared with the conventional clinical mass spectrometric analysis and the research of detection reference standards, the precision and accuracy of the methods are different, so that a new detection method for measuring testosterone hormones of men, women and children based on mass spectrometry with good accuracy and high sensitivity needs to be developed.
For accurate quantitative detection of steroid hormones in serum, pretreatment is crucial, and due to the low content of 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 mu L to meet the signal response of the target object in the serum sample. Therefore, it is required to develop a pretreatment method suitable for low sample amount and capable of efficiently extracting a target and maximally removing unnecessary impurities.
Disclosure of Invention
In view of the above, the invention aims to provide a kit for detecting serum testosterone hormone by using high performance liquid chromatography-tandem mass spectrometry, which has the advantages of high sample extraction efficiency, high impurity removal rate, low sample usage amount and simple and convenient operation.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a kit for detecting serum testosterone hormone by high performance liquid chromatography-tandem mass spectrometry, which comprises a standard solution, an internal standard solution, a diluent, an extraction liquid, a dissociating agent, a stabilizer, a quality control product and a chromatographic column;
the extraction liquid comprises a first extraction liquid and a second extraction liquid; the first extraction liquid is a mixed liquid of ethyl acetate and n-hexane; the second extraction liquid is n-hexane;
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.
Preferably, the volume ratio of the ethyl acetate to the n-hexane in the extract is (2.5-3.5): 2.
Preferably, the standard comprises a first standard and a second standard; the first standard substance is 100ng/mL testosterone standard solution; the second standard is a testosterone standard solution with the concentration of 10 ng/mL.
Preferably, the internal standard solution is a methanol solution containing 8000-12000 pg/mL testosterone-d 3.
Preferably, the internal standard solution is a methanol solution comprising 10000pg/mL testosterone-d 3.
Preferably, the quality control product is a blank serum matrix solution comprising testosterone.
Preferably, the quality control products comprise a first quality control product, a second quality control product and a third quality control product; the concentration of testosterone in the first quality control product is 80-120 pg/mL, the concentration of testosterone in the second quality control product is 1800-2200 pg/mL, and the concentration of testosterone in the third quality control product is 7500-8500 pg/mL.
Preferably, the concentration of testosterone in the first quality control product is 100pg/mL, the concentration of testosterone in the second quality control product is 2000pg/mL, and the concentration of testosterone in the third quality control product is 8000 pg/mL.
Preferably, the column is a UPLC C18 column; the inner diameter of the chromatographic column is 2.1mm, the length of the chromatographic column is 100mm, and the filler particle size of the chromatographic column is 1.7 mu m.
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 has the beneficial effects that: the kit provided by the invention can be used for quickly and accurately detecting testosterone hormone in serum, and has the advantages of high sensitivity, good specificity and low sample usage amount. According to the invention, ammonium acetate is used as a dissociation agent, testosterone hormone is mainly combined with serum protein, the dissociation agent can completely dissociate testosterone hormone and binding protein, and 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 aggregated on a chromatographic column, the separation effect is improved, and the ion inhibition is reduced. The kit performs secondary extraction in the using process, but the extraction efficiency reaches 80 percent, which is enough to quantitatively determine the testosterone 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 addition, the sample usage amount of the existing testosterone detection method based on mass spectrometry is more than 500 mu L, and the kit of the invention can achieve the detection purpose of low testosterone concentration by only using 100 mu L serum sample.
The kit is used for detecting testosterone in serum, the sample pretreatment is more convenient, and compared with the existing detection means, the detection method of the kit improves the detection sensitivity, increases the recovery rate of the method, has shorter required analysis time, is more beneficial to carrying out large-batch sample detection, and is suitable for screening related diseases in crowds.
Drawings
FIG. 1 is a MRM plot of testosterone hormones in serum;
FIG. 2 is a graph of the MRM of testosterone-d3 in serum.
Detailed Description
The invention provides a kit for detecting serum testosterone hormone by high performance liquid chromatography-tandem mass spectrometry, which comprises a standard solution, an internal standard solution, a diluent, an extraction liquid, a dissociating agent, a stabilizer, a quality control product and a chromatographic column; the extraction liquid comprises a first extraction liquid and a second extraction liquid; the first extraction liquid is a mixed liquid of ethyl acetate and n-hexane; the second extraction liquid is n-hexane; 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.
In the invention, the kit comprises a standard solution, wherein the standard solution comprises a first standard and a second standard; the first standard substance is 100ng/mL testosterone standard solution; the second standard is a testosterone standard solution with the concentration of 10 ng/mL. In the present invention, the solvent of the standard solution is preferably methanol, and the methanol is preferably chromatographically pure. In the present invention, the standard solution is preferably prepared by the following method: mixing testosterone with methanol to prepare testosterone standard substance mother liquor with the concentration of 1.0 mg/mL; and then gradually diluting the concentration of testosterone by 1.0mg/mL 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 as a first high-value concentration calibration point and quality control substances for preparing medium, high and low concentrations.
In the invention, the kit comprises an internal standard solution, wherein the internal standard solution is a methanol solution containing 8000-12000 pg/mL testosterone-d3, preferably a methanol solution containing 10000pg/mL testosterone-d 3; the methanol is preferably chromatographically pure. In the present invention, the internal standard solution is preferably prepared by the following method: testosterone-d3 was mixed with methanol to prepare a mother liquor of 100. mu.g/mL, which was then diluted stepwise to 10000 pg/mL.
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 invention, the kit comprises extraction liquid, wherein the extraction liquid 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 testosterone after two liquid-liquid extractions; in the present invention, the first extract maximizes the separation of testosterone from the matrix and the second extract largely removes polar lipids from the sample extract, thereby improving accuracy and consistency of chromatographic separation.
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 quality control product which is a blank serum matrix solution containing testosterone. In the present invention, the quality control materials preferably include a first quality control material, a second quality control material, and a third quality control material; the concentration of testosterone in the first quality control product is preferably 80-120 pg/mL, more preferably 90-110 pg/mL, and most preferably 100 pg/mL. In the invention, the concentration of testosterone in the second quality control product is preferably 1800-2200 pg/mL, more preferably 1900-2100 pg/mL, and most preferably 2000 pg/mL. In the invention, the concentration of testosterone in the third quality control product is preferably 7500-8500 pg/mL, more preferably 7800-8200 pg/mL, and most preferably 8000 pg/mL. The three control articles are used for evaluating the recovery rate and the precision of the method.
In the present invention, the kit comprises a chromatography column, preferably a UPLC C18 chromatography column; the inner diameter of the chromatographic column is preferably 2.1mm, the length of the chromatographic column is preferably 100mm, and the packing particle size of the chromatographic column is preferably 1.7 mu m.
The method of using the kit of the present invention preferably comprises the steps of: 1) mixing and extracting the serum sample internal standard solution, the ammonium acetate solution and the first extraction liquid, collecting supernatant, and drying by nitrogen to obtain a pretreated sample; 2) performing secondary extraction on the pretreated sample by using a second extraction liquid, collecting supernatant, drying by using nitrogen, and redissolving by using methanol to obtain a sample to be detected; 3) and carrying out chromatographic separation and mass spectrum detection on the sample to be detected to obtain the content of testosterone in the serum sample.
In the invention, the sampling volume of chromatographic separation is preferably 1-10 mu L, the flow rate of chromatographic separation is preferably 0.2-0.5 mL/min, the column temperature of a chromatographic column is preferably 35-40 ℃, and the temperature of a sample chamber of chromatographic separation is preferably 12-17 ℃; gradient elution for 5 min; wherein the mobile phase A is 0.1-0.3% v/v formic acid water solution, and the mobile phase B is acetonitrile. In the present invention, the elution gradient procedure for the chromatographic separation is preferably as follows: 0-0.5 min, 10% B; increasing the phase B from 10% to 95% in 0.5-2.5 min; 2.5-3.5 min, 95% B; 3.5-3.6 min, reducing the phase B from 95% to 10%; 3.6-5.0 min, 10% B.
TABLE 1 elution procedure
In the invention, the mass spectrum detection selects ESI negative ion mode to load sample, and adopts multi-reaction monitoring technology to detect testosterone; 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 the multiple reaction monitoring technique to detect testosterone are preferably as shown in Table 2.
TABLE 2 parameters of testosterone determination by multiple reaction monitoring techniques
In the invention, the standard curve is prepared by using standard solutions with a series of concentrations; and 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 to obtain the content of testosterone in the serum sample.
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
According to an embodiment of detecting testosterone hormone in serum by ultra performance liquid chromatography-tandem mass spectrometry, the method for detecting testosterone hormone in a serum sample by adopting isotope dilution liquid chromatography-tandem mass spectrometry comprises the following steps:
1. material
The samples referred to in this example were fresh normal human serum, clear in appearance, without hemolysis, jaundice and lipemia.
(1) The instrument comprises the following steps: ultra performance liquid chromatography-triple quadrupole mass spectrometer (UPLC-MS/MS, Waters corporation, usa); vortex mixer (IKA, germany); centrifuge (sairmer fly, usa); nitrogen spargers (borna aiger); electronic analytical balance (Siderelis instruments systems, Inc., Germany).
(2) Reagent consumables: methanol (chromatographically pure, Merck, germany), acetonitrile (chromatographically pure, Merck, germany), n-hexane (chromatographically pure, tianjin kang science and technology limited), ethyl acetate (chromatographically pure, tianjin kang science and technology limited); formic acid (mass purity, TCI corporation, japan); a blank serum matrix; UPLC C18 column (2.1X 100mm, 1.7 μm); the experimental water was MIlli-Q ultrapure water.
(3) And (3) standard substance: testosterone standard (Testosterone, 99.6%, Hunan province pharmaceutical adjuvant technical research center, Inc.); deuterated Testosterone standards (Testosterone-D3, 100. mu.g/mL, cerilliant standards).
(4) Quality control product: the blank serum matrix solution containing testosterone is divided into three concentrations, namely QC (L)100pg/mL, QC (M)2000pg/mL and QC (H)8000 pg/mL.
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 40 deg.C, sample chamber temperature 15 deg.C, mobile phase A0.1% formic acid water, mobile phase B acetonitrile, gradient elution, elution program as shown in Table 1.
(2) Mass spectrum conditions: mass spectrometry scan mode using Multiple Reaction Monitoring (MRM) in electrospray ionization (ESI) positive ion detection mode. 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. Target testosterone (289.2 → 97) and isotope internal standard testosterone-d 3(292.2 → 97) were monitored and then the Declustering Potential (DP) and collision potential (CE) of the target were systematically optimized (see table 2) to achieve higher stability and sensitivity, respectively.
(3) Preparing a standard substance: accurately weighing 10.0mg of testosterone, adding 10mL of methanol solution, and preparing into testosterone standard substance mother liquor with the concentration of 1.0 mg/mL; then gradually diluting the concentration of testosterone hormone to 100ng/mL from 1.0mg/mL by using methanol to obtain a standard solution A; 100 mu L of the standard solution A is taken and is made into a volume of 1mL by methanol, and the standard solution B with the concentration of 10ng/mL is obtained.
(4) Preparing a quality control product: taking 10 mu L, 200 mu L and 800 mu L of standard solution B, drying by nitrogen, and respectively metering the volume to 1000 mu L by using blank serum matrix solution to obtain the serum.
(5) Sample processing
1) Treating a standard substance: taking the standard solution B as a first high-value concentration point, respectively transferring 50, 25 and 10 mu L of the 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 standard solution with the concentration of 1000pg/mL as a standard solution C;
respectively transferring 500, 250, 100, 50, 25 and 10 mu L of standard solution C, and fixing the volume to 1mL by using a blank serum matrix to obtain the other six calibration concentration points; a total of 10 calibration concentration points were obtained for the standard line column.
Respectively adding 10 mu L of isotope internal standard solution into 10 blank 1.5mL centrifuge tubes, drying by using nitrogen, respectively adding 100 mu L of 10 standard solutions with calibration concentration, and then adding 100 mu L of ammonium acetate to dissociate at room temperature for 0.5 h; adding 400 mu L of ethyl acetate/n-hexane (v/v, 3:2), uniformly mixing by vortex for 5min, centrifuging for 2min, drying supernatant by nitrogen, adding 100 mu L of sodium carbonate and 400 mu L of n-hexane, centrifuging by vortex, taking supernatant, repeatedly adding 400 mu L of n-hexane for extraction, drying supernatant by a nitrogen blowing instrument, redissolving by 100 mu L of methanol solution, transferring the supernatant into a chromatographic bottle, and performing LC-MS/MS analysis.
2) Pretreatment of a serum sample: adding 10 mu L of 10000pg/mL internal standard solution into a 1.5mL centrifuge tube, drying by nitrogen, adding 100 mu L serum, and adding 100 mu L ammonium acetate to dissociate at room temperature for 0.5 h; adding 400 mu L of ethyl acetate/n-hexane (v/v, 3:2), uniformly mixing by vortex for 5min, centrifuging for 2min, drying supernatant by nitrogen, adding 100 mu L of sodium carbonate and 400 mu L of n-hexane, centrifuging by vortex, taking supernatant, repeatedly adding 400 mu L of n-hexane for extraction, drying supernatant by a nitrogen blowing instrument, redissolving by 100 mu L of methanol solution, transferring the supernatant into a chromatographic bottle, and performing LC-MS/MS analysis.
3) Pretreatment of quality control products: 10 mu L of 10000pg/mL internal standard solution is added into 1.5mL centrifuge tube, after nitrogen is dried, 100 mu L of quality control solution QC (L), QC (M) and QC (H) are respectively taken and put into 1.5mL centrifuge tube, which is consistent with the pretreatment method of serum sample.
The components of the assay kit are shown in Table 3.
TABLE 3 preparation of Testosterone hormone assay kit Components
3. Method of detection
1) Total ion current chromatogram: the peak patterns of the testosterone hormone standard product, the internal standard product and the serum sample are symmetrical, and basically no impurity interference exists when the concentration is not more than 50pg/mL, so that the testosterone can be well detected under the condition, and the condition can be used for quantitative analysis of the serum testosterone hormone.
2) Calibration curve: and establishing a curve by adopting an isotope internal standard quantitative method for the calibration curve, 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 concentration of the substance to be detected in the serum. The testosterone has good linearity within the range of 10-10000 pg/mL, the correlation coefficient is above 0.99, the internal standard peak area stability is good, the relative standard deviation is less than 10%, and the quantitative requirement is met. The linear equation: Y0.000436931X +0.0666518, linear coefficient (r)2) Is 0.996.
3) Detection sensitivity: the limit of detection (LOD) of testosterone hormone is 5pg/mL, the limit of quantitation (LOQ) is 10pg/mL, the CVs detected by 5 times of repetition are 8.7% and 10.6%, respectively, the LOD meets the signal-to-noise ratio (S/N) >3, the LOQ meets the signal-to-noise ratio (S/N) >10, and the CVs detected by 5 times of repetition is less than 20%.
4) And (3) precision experiment: taking testosterone hormone quality control products with low, medium and high concentrations, repeatedly processing 6 batches in one day, quantitatively measuring the concentration of testosterone hormone by using an isotope internal standard method, wherein the batch precision (n is 6) is as follows: CV of 3.16%, 3.64% and 2.81%; batch precision 6 determinations were made per batch for three consecutive days for a total of 18 determinations, and the batch precision (n ═ 18) was calculated as: CV: 6.22%, 3.15% and 3.60%.
5) Recovery rate experiment: taking blank serum matrix, adding standard substance into one part, adding high, medium and low 3 concentrations of standard substance into the other three parts, repeating the treatment with the same steps, and measuring for 3 times. The testosterone concentration of the blank matrix is detected to be lower than 10ppt, and the result shows that the standard recovery rate of testosterone in the serum matrix is between 89.5% and 102.8%, and the CV of 3 repeated experiments is between 1.72% and 5.70%.
Example 2
1. Material
The samples referred to in this example were obtained from outpatients and inpatients of a certain hospital in Tianjin, and were fresh human serum, clear in appearance, and free from hemolysis, jaundice and lipemia.
(1) The instrument comprises the following steps: ultra performance liquid chromatography-triple quadrupole mass spectrometer (UPLC-MS/MS, Waters corporation, usa); vortex mixer (MS 3basic, IKA, germany); centrifuge (eppendorf, germany); nitrogen spargers (borna aiger); one ten thousandth balance (sidoris instruments systems ltd, germany); an adjustable pipettor (0.5-10 muL, 10-100 muL, 100-1000 muL, Eppendorf, Germany); glassware, beaker, graduated cylinder, etc.
(2) Reagent consumables: methanol (chromatographically pure, Merck, germany), acetonitrile (chromatographically pure, Merck, germany), n-hexane (chromatographically pure, tianjin kang science and technology limited), ethyl acetate (chromatographically pure, tianjin kang science and technology limited); formic acid (mass purity, Waters corporation, usa); a blank serum matrix; UPLC C18 column (2.1X 100mm, 1.7 μm); the experimental water was deionized water.
(3) And (3) standard substance: testosterone standard (Testosterone, 99.6%, Hunan province pharmaceutical adjuvant technical research center, Inc.); deuterated Testosterone standards (Testosterone-D3, 100. mu.g/mL, cerilliant standards).
(4) Quality control product: the blank serum matrix solution containing testosterone is divided into three concentrations, namely QC (L)100pg/mL, QC (M)2000pg/mL and QC (H)8000 pg/mL.
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 40 deg.C, sample chamber temperature 15 deg.C, mobile phase A0.1% formic acid water, mobile phase B acetonitrile, gradient elution, elution program as shown in Table 1.
(2) Mass spectrum conditions: mass spectrometry scan mode using Multiple Reaction Monitoring (MRM) in electrospray ionization (ESI) positive ion detection mode. 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. Target testosterone (289.2 → 97) and isotope internal standard testosterone-d 3(292.2 → 97) were monitored and then the Declustering Potential (DP) and collision potential (CE) of the target were systematically optimized (see table 2) to achieve higher stability and sensitivity, respectively.
(3) Preparing a standard substance: accurately weighing 10.0mg of testosterone, adding 10mL of methanol solution, and preparing into testosterone standard substance mother liquor with the concentration of 1.0 mg/mL; then gradually diluting the concentration of testosterone hormone to 100ng/mL from 1.0mg/mL by using methanol to obtain a standard solution A; 100 mu L of the standard solution A is taken and is made into a volume of 1mL by methanol, and the standard solution B with the concentration of 10ng/mL is obtained.
(4) Preparing a quality control product: taking 10 mu L, 200 mu L and 800 mu L of standard solution B, drying by nitrogen, and respectively metering the volume to 1000 mu L by using blank serum matrix solution to obtain the serum.
(5) Sample processing
1) Treating a standard substance: taking the standard solution B as a first high-value concentration point, respectively transferring 50, 25 and 10 mu L of the 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 standard solution with the concentration of 1000pg/mL as a standard solution C;
respectively transferring 500, 250, 100, 50, 25 and 10 mu L of standard solution C, and fixing the volume to 1mL by using a blank serum matrix to obtain the other six calibration concentration points; a total of 10 calibration concentration points were obtained for the standard line column.
Respectively adding 10 mu L of isotope internal standard solution into 10 blank 1.5mL centrifuge tubes, drying by using nitrogen, respectively adding 100 mu L of 10 standard solutions with calibration concentration, and then adding 100 mu L of ammonium acetate to dissociate at room temperature for 0.5 h; adding 400 mu L of ethyl acetate/n-hexane (v/v, 1:1), uniformly mixing by vortex for 5min, centrifuging for 2min, drying supernatant by nitrogen, adding 100 mu L of sodium carbonate and 400 mu L of n-hexane, centrifuging by vortex, taking supernatant, repeatedly adding 400 mu L of n-hexane for extraction, drying supernatant by a nitrogen blowing instrument, redissolving by 100 mu L of methanol solution, transferring the supernatant into a chromatographic bottle, and performing LC-MS/MS analysis.
2) Pretreatment of a serum sample: adding 10 mu L of 10000pg/mL internal standard solution into a 1.5mL centrifuge tube, drying by nitrogen, adding 100 mu L serum, and adding 100 mu L ammonium acetate to dissociate at room temperature for 0.5 h; adding 400 mu L of ethyl acetate/n-hexane (v/v, 1:1), uniformly mixing by vortex for 5min, centrifuging for 2min, drying supernatant by nitrogen, adding 100 mu L of sodium carbonate and 400 mu L of n-hexane, centrifuging by vortex, taking supernatant, repeatedly adding 400 mu L of n-hexane for extraction, drying supernatant by a nitrogen blowing instrument, redissolving by 100 mu L of methanol solution, transferring the supernatant into a chromatographic bottle, and performing LC-MS/MS analysis.
3) Pretreatment of quality control products: 10 mu L of 10000pg/mL internal standard solution is added into 1.5mL centrifuge tube, after nitrogen is dried, 100 mu L of quality control solution QC (L), QC (M) and QC (H) are respectively taken and put into 1.5mL centrifuge tube, which is consistent with the pretreatment method of serum sample.
The components of the assay kit are shown in Table 3.
3. Method of detection
1) Total ion current chromatogram: the peak patterns of the testosterone hormone standard product, the internal standard product and the serum sample are symmetrical, and basically no impurity interference exists when the concentration is not more than 50pg/mL, so that the testosterone can be well detected under the condition, and the condition can be used for quantitative analysis of the serum testosterone hormone.
2) Calibration curve: and establishing a curve by adopting an isotope internal standard quantitative method for the calibration curve, 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 concentration of the substance to be detected in the serum. The testosterone has good linearity within the range of 10-10000 pg/mL, the correlation coefficient is above 0.99, the internal standard peak area stability is good, the relative standard deviation is less than 10%, and the quantitative requirement is met. The linear equation: y0.000458751 × X +0.514436, linear coefficient (r2) 0.999.
3) Detection sensitivity: the limit of detection (LOD) of testosterone hormone is 5pg/mL, the limit of quantitation (LOQ) is 10pg/mL, the CVs detected by 5 times of repetition are 7.9% and 11.2%, respectively, the LOD meets the signal-to-noise ratio (S/N) >3, the LOQ meets the signal-to-noise ratio (S/N) >10, and the CVs detected by 5 times of repetition is less than 20%.
4) And (3) precision experiment: taking testosterone hormone quality control products with low, medium and high concentrations, repeatedly processing 6 batches in one day, quantitatively measuring the concentration of testosterone hormone by using an isotope internal standard method, wherein the batch precision (n is 6) is as follows: CV of 5.53%, 1.92%, 3.43%; batch precision 6 determinations were made per batch for three consecutive days for a total of 18 determinations, and the batch precision (n ═ 18) was calculated as: CV: 5.01%, 3.04%, 3.53%.
5) Recovery rate experiment: taking blank serum matrix, adding standard substance into one part, adding high, medium and low 3 concentrations of standard substance into the other three parts, repeating the treatment with the same steps, and measuring for 3 times. The result of detecting that the concentration of testosterone in the blank matrix is lower than 10ppt shows that the standard recovery rate of testosterone in the serum matrix is between 88.1 and 105.5 percent, and the CV of 3 repeated experiments is between 3.15 and 6.22 percent.
From the above examples, it can be seen that the method for detecting testosterone by using the kit provided by the invention overcomes the problems of lack of specificity and insufficient sensitivity caused by cross reaction and matrix interference in a chemical immunoassay, greatly eliminates the interference of a matrix by using an isotope internal standard method, and largely eliminates the influence of conditions such as a pretreatment process, a sample loading volume and flow, thereby achieving the purpose of accurate quantification. The methodology research in the embodiment 1 shows that the kit for detecting the testosterone content meets the requirements on linear range, detection limit, recovery rate and precision, has high sensitivity, strong specificity, accuracy and reliability, and can be applied to quantitative detection of testosterone hormone 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 detecting serum testosterone hormone by high performance liquid chromatography-tandem mass spectrometry is characterized by comprising a standard solution, an internal standard solution, a diluent, an extraction liquid, a dissociation agent, a stabilizer, a quality control material and a chromatographic column;
the extraction liquid comprises a first extraction liquid and a second extraction liquid; the first extraction liquid is a mixed liquid of ethyl acetate and n-hexane; the second extraction liquid is n-hexane;
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.
2. The kit according to claim 1, wherein the volume ratio of ethyl acetate to n-hexane in the extract is (2.5-3.5): 2.
3. The kit of claim 1, wherein the standards comprise a first standard and a second standard; the first standard substance is 100ng/mL testosterone standard solution; the second standard is a testosterone standard solution with the concentration of 10 ng/mL.
4. The kit of claim 1, wherein the internal standard solution is a methanol solution comprising 8000-12000 pg/mL testosterone-d 3.
5. The kit of claim 4, wherein the internal standard solution is a methanol solution comprising 10000pg/mL testosterone-d 3.
6. The kit of claim 1, wherein the quality control product is a blank serum matrix solution comprising testosterone.
7. The kit of claim 6, wherein the quality controls comprise a first quality control, a second quality control, and a third quality control; the concentration of testosterone in the first quality control product is 80-120 pg/mL, the concentration of testosterone in the second quality control product is 1800-2200 pg/mL, and the concentration of testosterone in the third quality control product is 7500-8500 pg/mL.
8. The kit of claim 7, wherein the concentration of testosterone in the first control substance is 100pg/mL, the concentration of testosterone in the second control substance is 2000pg/mL, and the concentration of testosterone in the third control substance is 8000 pg/mL.
9. The kit of claim 1, wherein the chromatography column is a UPLC C18 chromatography column; the inner diameter of the chromatographic column is 1.0-2.1 mm, the length of the chromatographic column is 30-150 mm, and the particle size of a filler of the chromatographic column is 1.4-2.0 mu m.
10. 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911127530.1A CN110702831B (en) | 2019-11-18 | 2019-11-18 | Kit for detecting serum testosterone hormone by ultra-high performance liquid chromatography-tandem mass spectrometry |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911127530.1A CN110702831B (en) | 2019-11-18 | 2019-11-18 | Kit for detecting serum testosterone hormone by ultra-high performance liquid chromatography-tandem mass spectrometry |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110702831A true CN110702831A (en) | 2020-01-17 |
| CN110702831B CN110702831B (en) | 2020-06-16 |
Family
ID=69207078
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911127530.1A Active CN110702831B (en) | 2019-11-18 | 2019-11-18 | Kit for detecting serum testosterone hormone by ultra-high performance liquid chromatography-tandem mass spectrometry |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110702831B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113009036A (en) * | 2021-03-05 | 2021-06-22 | 天津汉科生物科技有限公司 | Kit for detecting sex hormone, sex hormone sample pretreatment method and method for simultaneously detecting multiple sex hormones |
| CN114441678A (en) * | 2022-01-26 | 2022-05-06 | 苏州药明泽康生物科技有限公司 | Detection method and detection kit for high-stability 25-hydroxyvitamin D |
| CN114460199A (en) * | 2022-02-09 | 2022-05-10 | 杭州佰辰医疗器械有限公司 | Method for detecting concentration of free testosterone and total testosterone |
| CN114720704A (en) * | 2022-04-15 | 2022-07-08 | 合肥歆智医疗器械有限公司 | Kit and method for measuring free testosterone in serum |
| CN114740107A (en) * | 2022-03-25 | 2022-07-12 | 武汉生物样本库有限公司 | Method for determining content of short-chain fatty acids in feces, plasma and urine samples based on GC-MS or GC-MS/MS |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150031581A1 (en) * | 2011-11-29 | 2015-01-29 | Rijksuniversiteit Groningen | Methods and kits for determining protein-bound biomarkers |
| CN105675788A (en) * | 2016-04-11 | 2016-06-15 | 北京洛奇临床检验所股份有限公司 | Method and kit for detecting progesterone and testosterone in saliva through high performance liquid chromatography-tandem mass spectrometry technique |
| CN109196109A (en) * | 2016-04-06 | 2019-01-11 | 绿光生物科技股份有限公司 | Cell-free generation ribonucleic acid |
| 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 |
| CN110291207A (en) * | 2017-01-27 | 2019-09-27 | 豪夫迈·罗氏有限公司 | Bar coded DNA for long-range sequencing |
-
2019
- 2019-11-18 CN CN201911127530.1A patent/CN110702831B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150031581A1 (en) * | 2011-11-29 | 2015-01-29 | Rijksuniversiteit Groningen | Methods and kits for determining protein-bound biomarkers |
| CN109196109A (en) * | 2016-04-06 | 2019-01-11 | 绿光生物科技股份有限公司 | Cell-free generation ribonucleic acid |
| CN105675788A (en) * | 2016-04-11 | 2016-06-15 | 北京洛奇临床检验所股份有限公司 | Method and kit for detecting progesterone and testosterone in saliva through high performance liquid chromatography-tandem mass spectrometry technique |
| CN110291207A (en) * | 2017-01-27 | 2019-09-27 | 豪夫迈·罗氏有限公司 | Bar coded DNA for long-range sequencing |
| 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 |
Non-Patent Citations (4)
| Title |
|---|
| THERMOFISHER SCIENTIFIC: "动物源性样品中合成类固醇类激素的检测（SPE-LC/MS）", 《THERMO SCIENTIFIC样品前处理应用手册》 * |
| U.TURPEINEN ET AL.: "Determination of testosterone in serum by liquid chromatography-tandem mass spectrometry", 《THE SCANDINAVIAN JOURNAL OF CLINICAL & LABORATORY INVESTIGATION》 * |
| 任玉琴 等: "超高效液相色谱-串联质谱法测定猪肉和猪肝中烯丙孕素残留", 《食品安全质量检测学报》 * |
| 张天娇 等: "同位素稀释液相色谱串联质谱法测定血清睾酮", 《临床检验杂志》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113009036A (en) * | 2021-03-05 | 2021-06-22 | 天津汉科生物科技有限公司 | Kit for detecting sex hormone, sex hormone sample pretreatment method and method for simultaneously detecting multiple sex hormones |
| CN114441678A (en) * | 2022-01-26 | 2022-05-06 | 苏州药明泽康生物科技有限公司 | Detection method and detection kit for high-stability 25-hydroxyvitamin D |
| CN114441678B (en) * | 2022-01-26 | 2024-04-19 | 苏州药明泽康生物科技有限公司 | High-stability 25-hydroxy vitamin D detection method and detection kit |
| CN114460199A (en) * | 2022-02-09 | 2022-05-10 | 杭州佰辰医疗器械有限公司 | Method for detecting concentration of free testosterone and total testosterone |
| CN114740107A (en) * | 2022-03-25 | 2022-07-12 | 武汉生物样本库有限公司 | Method for determining content of short-chain fatty acids in feces, plasma and urine samples based on GC-MS or GC-MS/MS |
| CN114720704A (en) * | 2022-04-15 | 2022-07-08 | 合肥歆智医疗器械有限公司 | Kit and method for measuring free testosterone in serum |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110702831B (en) | 2020-06-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110702831B (en) | Kit for detecting serum testosterone hormone by ultra-high performance liquid chromatography-tandem mass spectrometry | |
| CN111366671B (en) | Chemical derivatization-ultra-high performance liquid chromatography-tandem mass spectrometry for simultaneously detecting 18 steroid hormones in serum | |
| Gómez-Pérez et al. | Analysis of pesticide and veterinary drug residues in baby food by liquid chromatography coupled to Orbitrap high resolution mass spectrometry | |
| Guo et al. | Ultra-trace analysis of 12 β2-agonists in pork, beef, mutton and chicken by ultrahigh-performance liquid-chromatography–quadrupole-orbitrap tandem mass spectrometry | |
| CN104807920A (en) | Kit for detecting 10 kinds of steroid hormones in serum through high performance liquid chromatography tandem mass spectrum technique | |
| EP2939024B1 (en) | C peptide detection by mass spectrometry | |
| CN110927289A (en) | Steroid hormone detection kit | |
| CA3000178C (en) | Amyloid beta detection by mass spectrometry | |
| CN114674961A (en) | Kit for synchronously detecting 17 steroid hormones in serum without derivatization and application thereof | |
| CN113049719A (en) | Method and kit for detecting free testosterone | |
| US9012835B2 (en) | Methods for simultaneous quantification of thyroid hormones and metabolites thereof by mass spectrometry | |
| US11536733B2 (en) | Methods and systems for the detection of 11-oxo androgens by LC-MS/MS | |
| CN114720704B (en) | Kit and method for measuring free testosterone in serum | |
| CN113655132A (en) | Method and kit for detecting content of 25 hydroxy vitamin D in human serum | |
| CN113030293A (en) | Tandem mass spectrometry method for rapidly detecting nine free amino acids in plasma | |
| Qin et al. | Quantitative determination of dipyridamole in human plasma by high‐performance liquid chromatography–tandem mass spectrometry and its application to a pharmacokinetic study | |
| CN112014509A (en) | Method for synchronously determining angiotensin I and aldosterone in sample | |
| CN113341027A (en) | Method and kit for detecting testosterone in saliva by high performance liquid chromatography tandem mass spectrometry | |
| Vicente et al. | Measurement of serum testosterone using high-performance liquid chromatography/tandem mass spectrometry | |
| CN113720946A (en) | Method and kit for detecting multiple steroid hormones in blood | |
| CN108593790B (en) | Method for simultaneously detecting 24,25(OH)2D and 25OHD of serum | |
| CN113009036A (en) | Kit for detecting sex hormone, sex hormone sample pretreatment method and method for simultaneously detecting multiple sex hormones | |
| CN113063866A (en) | Method for detecting content of DHEA (dehydroepiandrosterone) in human body fluid | |
| CN108982703B (en) | Liquid chromatography-mass spectrometry detection method for polyphenol substances | |
| Yang et al. | Determination of palonosetron in human plasma by ultra performance liquid chromatography–tandem mass spectrometry and its application to a pharmacokinetic study |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |