CN113009006A - Method and kit for detecting dehydroepiandrosterone content in saliva - Google Patents
Method and kit for detecting dehydroepiandrosterone content in saliva Download PDFInfo
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- 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
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- 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
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- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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- 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
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- 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
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- 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
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- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
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Abstract
The invention relates to a method and a kit for detecting the content of dehydroepiandrosterone in saliva, wherein the method comprises the following steps: preparing a standard substance: preparing a quality control product: treating a standard substance, and analyzing and detecting by adopting a high performance liquid chromatography tandem mass spectrometry technology: pretreatment of a saliva sample, and analysis and detection by adopting a high performance liquid chromatography tandem mass spectrometry technology: calibration curve: and (3) establishing a standard curve by adopting an isotope internal standard quantitative method and 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 dehydroepiandrosterone in the sample to be detected. The method adopts the high performance liquid chromatography tandem mass spectrometry technology to separate the dehydroepiandrosterone from the interferent, combines an isotope internal standard quantitative method to measure the content of the dehydroepiandrosterone in saliva, obviously improves the detection sensitivity by reacting the dehydroepiandrosterone with the methoxyamine to generate the oxime ester, and ensures the specificity of the detection method by adopting a reaction detection mode (MRM).
Description
Technical Field
The invention relates to the technical field of biological detection, and particularly relates to a method and a kit for detecting the content of dehydroepiandrosterone in saliva.
Background
Dehydroepiandrosterone (DHEA), a steroid hormone secreted by the adrenal cortex, is a precursor substance synthesized from male and female hormones and has a wide range of physiological and pathological effects. DHEA is secreted into the blood and is metabolized to form bound DHEA, which is known as free DHEA, and this type of DHEA is mainly physiologically active. Free DHEA in blood passively diffuses through salivary gland epithelial cells into saliva, and thus the DHEA content in saliva is considered useful for assessing the level of free DHEA in humans.
The traditional methods for detecting DHEA mainly include enzyme-linked immunosorbent assay, chemiluminescence immunoassay and the like, and the methods are easily interfered by DHEA structural analogues in a sample to cause deviation of detection results. In recent years, high performance liquid chromatography tandem mass spectrometry technology is developed and applied to detecting the content of DHEA in human body fluid, however, due to the molecular structure of DHEA, direct detection finds that the ionization efficiency of DHEA is poor, and in addition, the content of DHEA in saliva is low, so that the sensitivity of direct detection cannot meet the requirement.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method and a kit for detecting the content of dehydroepiandrosterone in saliva, and the method and the kit have the advantages of strong specificity and high sensitivity.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a method for detecting the content of dehydroepiandrosterone in saliva comprises the following steps:
preparing a standard substance: taking a dehydroepiandrosterone standard substance, using acetonitrile as a solvent to prepare 1mg/mL of dehydroepiandrosterone standard substance mother liquor, and diluting with acetonitrile to obtain 100ng/mL of dehydroepiandrosterone standard substance solution; taking a d 6-dehydroepiandrosterone standard, preparing 1mg/mL internal standard mother solution by using acetonitrile as a solvent, diluting with acetonitrile to obtain 1 microgram/mL internal standard solution, and diluting to prepare 2.5ng/mL internal standard solution for experiments;
preparing a quality control product: diluting the dehydroepiandrosterone standard solution with saliva blank matrix to constant volume to obtain quality control products with three concentration gradients of 20pg/mL, 100pg/mL and 500 pg/mL;
treating a standard substance: taking the dehydroepiandrosterone standard solution, diluting to constant volume with blank saliva matrix solution to obtain seven concentration gradient dehydroepiandrosterone calibration solutions of 10pg/mL, 20pg/mL, 50pg/mL, 100pg/mL, 200pg/mL, 500pg/mL and 1000pg/mL, respectively taking 500 uL of the dehydroepiandrosterone calibration solutions with various concentrations in a 2mL centrifugal tube, adding 20 uL of the internal standard solution and 1mL of extract liquid into each tube, performing vortex oscillation for 5min, performing centrifugation for 10min at the rotating speed of 13000r/min, transferring an organic phase to another 2mL centrifugal tube, performing nitrogen blow-drying, adding a derivatization agent into a blow-drying centrifugal tube, performing a dark water bath reaction, adding 100 uL of water into the centrifugal tube after the reaction is finished, adding 1mL of extract liquid, performing vortex oscillation for 5min, transferring the organic phase to another 2mL centrifugal tube, performing blow-drying, redissolving 100 mu L of 60% acetonitrile water, carrying out vortex oscillation for 1min, centrifuging for 5min at the rotating speed of 13000r/min, and taking 20 mu L of supernatant to carry out analysis and detection by adopting a high performance liquid chromatography tandem mass spectrometry technology;
pretreatment of a saliva sample: adding 20 mu L of the internal standard solution and 1mL of extraction liquid into 500 mu L of saliva sample in a 2mL centrifuge tube, carrying out vortex oscillation for 5min, centrifuging for 10min at the rotation speed of 13000r/min, transferring an organic phase to another 2mL centrifuge tube, drying by blowing nitrogen, adding a derivatizing agent into a drying centrifuge tube, carrying out a light-resistant water bath reaction, adding 100 mu L of water into the centrifuge tube after the reaction is finished, adding 1mL of extraction liquid, carrying out vortex oscillation for 5min, transferring the organic phase to another 2mL centrifuge tube, drying by blowing nitrogen, redissolving by 100 mu L of 60% acetonitrile, carrying out vortex oscillation for 1min, centrifuging for 5min at 13000r/min, and taking 20 mu L of supernatant for analysis and detection by adopting a high performance liquid chromatography tandem mass spectrometry technology;
calibration curve: and (3) establishing a standard curve by adopting an isotope internal standard quantitative method and 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 dehydroepiandrosterone in the sample to be detected.
As a preferred embodiment of the present invention, the chromatographic conditions when the analysis and detection are performed by using the hplc tandem mass spectrometry technology are as follows: the mobile phase A adopts 0.1 percent of formic acid aqueous solution by volume ratio, and the mobile phase B adopts 100 percent of acetonitrile.
As a preferred embodiment of the present invention, the mass spectrometry conditions when the analysis and detection are performed by using the hplc tandem mass spectrometry technology are as follows: in an electrospray ionization positive ion detection mode, a mass spectrum scanning mode of multi-reaction monitoring is adopted, the spraying voltage is 5500V, the collision gas is 8, the gas curtain gas is 30psi, the ion source temperature is 550 ℃, the ion source gas flow GS1 is 70psi, and the GS2 is 50 psi.
As a preferable scheme of the invention, in the standard substance processing step and the saliva sample pretreatment step, the reaction temperature of the dark water bath is 60 ℃, and the reaction time is 30 min.
As a preferred embodiment of the present invention, the blank saliva base solution is prepared by dissolving 3g bovine serum albumin in water to a volume of 1L.
As a preferred embodiment of the present invention, the extract is methyl tert-butyl ether.
In a preferred embodiment of the present invention, the derivatizing agent is a methoxylamine hydrochloride solution with a concentration of 50mmol/l using 50% methanol water as a solvent.
As a preferred embodiment of the present invention, the method further comprises a precision testing step, which comprises:
taking the same saliva sample as a first test sample group, equally dividing the saliva sample into 5-8 parts, sequentially measuring the content of dehydroepiandrosterone in the first test sample group by an isotope internal standard quantitative method, and calculating to obtain the batch precision;
taking the same saliva sample as a second test sample group, equally dividing the same saliva sample into 5-8 parts, sequentially measuring the content of the dehydroepiandrosterone in the second test sample group by an isotope internal standard quantitative method according to the same time interval for 3-5 days, and calculating to obtain the batch precision.
As a preferred embodiment of the present invention, the method further comprises a recovery rate test step comprising:
taking a saliva sample to divide into 4 parts serving as a third test sample group, wherein each part is 500 mu l, 1 part is not added with a standard substance, the other 3 parts are respectively added with a low-concentration standard substance, a medium-concentration standard substance and a high-concentration standard substance, the 4 parts of test samples are subjected to saliva sample pretreatment and then are subjected to computer measurement for 3 times by adopting a high performance liquid chromatography tandem mass spectrometry technology, and the standard addition recovery rate is calculated.
On the other hand, the invention also provides a kit for detecting the content of dehydroepiandrosterone in saliva, which comprises:
a mobile phase comprising equal volumes of 0.1% by volume formic acid water and 100% acetonitrile;
dehydroepiandrosterone standard solution, wherein the standard mother solution is acetonitrile solution with the concentration of 100ng/ml dehydroepiandrosterone;
an internal standard solution, wherein the internal standard solution is an acetonitrile solution of dehydroepiandrosterone with the concentration of 2.5ng/mld 6-t;
a diluent, the diluent comprising: 3g/L bovine serum albumin aqueous solution and acetonitrile;
an extraction liquid which is methyl tert-butyl ether;
the quality control product comprises a dehydroepiandrosterone solution with three concentration gradients of 20pg/mL, 100pg/mL and 500pg/mL which is obtained by diluting with a blank saliva substrate and fixing the volume.
In conclusion, the invention has the following beneficial effects:
the method adopts a high performance liquid chromatography tandem mass spectrometry technology to separate the dehydroepiandrosterone from the interferent, and combines an isotope internal standard quantitative method to determine the content of the dehydroepiandrosterone in saliva, so that the dehydroepiandrosterone reacts with methoxyamine to generate oxime ester, the detection sensitivity is obviously improved, the specificity of the detection method is ensured by adopting a reaction detection mode (MRM), and the potential interference and the matrix effect are well eliminated and corrected by the online chromatographic separation and the application of the isotope internal standard;
meanwhile, proved by methodology, analysis after blank saliva matrix treatment shows that the method is high in specificity, and dehydroepiandrosterone and internal standard d 6-dehydroepiandrosterone in a spectrogram of a blank sample do not have obvious interference at the peak emergence time; the precision test result shows that the intra-batch precision and the inter-batch precision are respectively 6.88 percent and 8.92 percent, and meet the acceptable standard (CV is less than or equal to 15 percent), which indicates that the method has good stability; the standard adding recovery rate test is carried out by adding standard substances with low, medium and high concentrations, and the result shows that the standard adding recovery rate is 84.9-105.5%, and the standard adding recovery rate meets the acceptable standard (80-120%);
the method has the advantages of strong specificity, high sensitivity, simple pretreatment process, and high precision and standard recovery rate, can be used for detecting the dehydroepiandrosterone with low content in saliva, and provides a reliable quantitative detection method for evaluating the content of the free dehydroepiandrosterone in human body.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a total ion chromatogram of a standard dehydroepiandrosterone and an internal standard d 6-dehydroepiandrosterone according to an example of the present invention.
FIG. 2 is a total ion chromatogram of dehydroepiandrosterone and internal standard d 6-dehydroepiandrosterone in saliva according to an example of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example one
A method for detecting the content of dehydroepiandrosterone in saliva comprises the following steps:
preparing a standard substance: accurately weighing 10mg of dehydroepiandrosterone standard substance, dissolving with acetonitrile as solvent to a constant volume of 10mL, preparing to obtain 1mg/mL of dehydroepiandrosterone standard substance mother liquor, and diluting with acetonitrile to a constant volume of 100mL to obtain 100ng/mL of dehydroepiandrosterone standard substance solution; accurately weighing 10mgd 6-dehydroepiandrosterone standard, dissolving with acetonitrile as solvent to a constant volume of 10mL, preparing to obtain 1mg/mL internal standard mother solution, diluting 10 μ l of internal standard mother solution with acetonitrile to a constant volume of 10mL to obtain 1 μ g/mL internal standard solution, diluting 25 μ l of internal standard solution with acetonitrile to a constant volume of 10mL, and preparing to obtain 2.5ng/mL internal standard solution for experiments.
Preparing a quality control product: taking 2 mu L, 10 mu L and 50 mu L of 100ng/mL dehydroepiandrosterone standard solution, respectively, diluting with blank saliva substrate to constant volume to 10mL, and obtaining quality control products of QC (L), QC (M) and QC (H) with three concentration gradients, wherein the concentration of QC (L) is 20pg/mL, the concentration of QC (M) is 100pg/mL, and the concentration of QC (H) is 500pg/mL, wherein the blank saliva substrate solution is prepared by dissolving 3g of bovine serum albumin with water to constant volume to 1L.
Treating a standard substance: respectively taking 1 mu l, 2 mu l, 5 mu l, 10 mu l, 20 mu l, 50 mu l and 100 mu l of dehydroepiandrosterone standard solution with the concentration of 100ng/mL in 7 10mL volumetric flasks, and fixing the volume to the scale by using blank saliva matrix solution to obtain seven concentration-gradient dehydroepiandrosterone calibration solutions of 10pg/mL, 20pg/mL, 50pg/mL, 100pg/mL, 200pg/mL, 500pg/mL and 1000 pg/mL; respectively putting 500 mu L of dehydroepiandrosterone calibration solution with each concentration into 2mL centrifuge tubes, adding 20 mu L of internal standard solution and 1mL of extraction liquid into each centrifuge tube, carrying out vortex oscillation for 5min by using a multi-tube vortex oscillator, centrifuging for 10min at the rotating speed of 13000r/min in a temperature-controlled high-speed centrifuge, transferring an organic phase to another 2mL centrifuge tube, drying by using a nitrogen blowing instrument for drying nitrogen, adding a derivative into a drying centrifuge tube, carrying out a dark water bath reaction in a water bath kettle, wherein the temperature of the dark water bath reaction is 60 ℃, and the reaction time is 30 min; and after the reaction is finished, adding 100 mu L of water into a centrifuge tube, then adding 1mL of extract liquor, carrying out vortex oscillation for 5min, transferring an organic phase to another 2mL centrifuge tube, carrying out nitrogen blow-drying, redissolving with 100 mu L of 60% acetonitrile water, carrying out vortex oscillation for 1min, then centrifuging for 5min at the rotating speed of 13000r/min, and taking 20 mu L of supernatant to carry out analysis and detection by adopting a high performance liquid chromatography tandem mass spectrometry technology.
Pretreatment of a saliva sample: adding 20 mu L of internal standard solution and 1mL of extract liquor into 500 mu L of saliva sample in a 2mL centrifuge tube, carrying out vortex oscillation for 5min by using a multi-tube vortex oscillator, centrifuging for 10min at the rotating speed of 13000r/min in a temperature-controlled high-speed centrifuge, transferring an organic phase to another 2mL centrifuge tube, drying by using nitrogen blowing of a nitrogen blowing instrument, adding a derivatization agent into a drying centrifuge tube, carrying out a light-resistant water bath reaction at the temperature of 60 ℃, wherein the reaction time is 30 min; and after the reaction is finished, adding 100 mu L of water into a centrifuge tube, then adding 1mL of extract liquor, carrying out vortex oscillation for 5min, transferring an organic phase to another 2mL centrifuge tube, carrying out nitrogen blow-drying, redissolving with 100 mu L of 60% acetonitrile water, carrying out vortex oscillation for 1min, then centrifuging for 5min at 13000r/min, and taking 20 mu L of supernatant to carry out analysis and detection by adopting a high performance liquid chromatography tandem mass spectrometry technology.
Wherein the extract is methyl tert-butyl ether, the derivatizing agent is a methoxylamine hydrochloride solution with the concentration of 50mmol/l and the solvent of 50% methanol water, the blow-drying temperature of nitrogen is 50 ℃, and the API5500 triple quadrupole mass spectrometer and the LC-20ADXR liquid chromatography system are adopted in the high performance liquid chromatography tandem mass spectrometry technology in the embodiment.
The specific chromatographic conditions are as follows:
mobile phase A: formic acid water with the volume ratio of 0.1 percent;
mobile phase B: acetonitrile with purity of chromatographic purity; the concentration is 100%;
the type of the chromatographic column: waters XBridgeBEHC18, 3.5 μm,2.1mm 150 mm;
a gradient elution mode was used, as shown in table 1;
the flow rate was 0.2ml/min, the column temperature was 40 ℃ and the injection volume was 20. mu.l.
TABLE 1 mobile phase gradient elution parameters
The specific mass spectrum conditions were: in an electrospray ionization positive ion detection mode, a mass spectrum scanning mode of multi-reaction monitoring (MRM) is adopted, the spraying voltage is 5500V, the collision gas is 8, the gas curtain gas is 30psi, the ion source temperature is 550 ℃, the ion source gas flow GS1 is 70psi, GS2 is 50psi, the target dehydroepiandrosterone m/z318.1 → 286.1, and the isotope internal standard d 6-dehydroepiandrosterone m/z324.1 → 292.1; the dehydroepiandrosterone and internal standard declustering voltage (DP), injection voltage (EP), collision voltage (CE), and collision cell injection voltage (CXP) are shown in table 2.
TABLE 2 Mass Spectrometry parameters for dehydroepiandrosterone and internal standards
Calibration curve: establishing a standard curve by adopting an isotope internal standard quantitative method and utilizing Analyst software with 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 dehydroepiandrosterone in the sample to be detected; the specific detection results are shown in fig. 1 and fig. 2, and in the range of 10-1000pg/ml, the linear equation obtained by fitting has good linearity, the linear correlation coefficient is greater than 0.999, and the quantitative requirement is met.
The method also comprises a precision testing step, which comprises the following steps:
and (3) calculating the internal precision: taking 5-8 parts of the same saliva sample as a first test sample group, sequentially measuring the content of dehydroepiandrosterone in the first test sample group by an isotope internal standard quantitative method, and calculating to obtain the internal precision, wherein the saliva sample is 7 parts in the embodiment, and the specific calculation result is shown in table 3.
TABLE 3 internal precision (unit: pg/ml)
Batch precision calculation: taking the same saliva sample as a second test sample group, equally dividing into 5-8 parts, sequentially measuring the content of dehydroepiandrosterone in the second test sample group by an isotope internal standard quantitative method according to the same time interval for 3-5 consecutive days, and calculating to obtain batch precision, wherein in the embodiment, the saliva sample is equally divided into 7 parts, continuously testing for 3 days, and the specific calculation result is shown in table 4.
TABLE 4 precision between batches (unit: pg/ml)
The method also comprises a recovery rate test step, which comprises the following steps:
taking a saliva sample to divide into 4 parts serving as a third test sample group, wherein each part is 500 mu l, 1 part is not added with a standard substance, the other 3 parts are respectively added with a low-concentration standard substance, a medium-concentration standard substance and a high-concentration standard substance, after the 4 parts of test samples are pretreated by the saliva sample, the standard adding recovery rate is calculated after the test samples are measured for 3 times by adopting a high performance liquid chromatography tandem mass spectrometry technology, according to the calculation result, the standard adding recovery rate is between 84.9% and 105.5%, and the specific result is shown in table 5.
TABLE 5 recovery normalized to pg/ml
In summary, the invention has the following beneficial effects:
the method adopts a high performance liquid chromatography tandem mass spectrometry technology to separate the dehydroepiandrosterone from the interferent, and combines an isotope internal standard quantitative method to determine the content of the dehydroepiandrosterone in saliva, so that the dehydroepiandrosterone reacts with methoxyamine to generate oxime ester, the detection sensitivity is obviously improved, the specificity of the detection method is ensured by adopting a reaction detection mode (MRM), and the potential interference and the matrix effect are well eliminated and corrected by the online chromatographic separation and the application of the isotope internal standard;
meanwhile, proved by methodology, analysis after treatment by using a blank matrix solution shows that the method has high specificity, and the peak emergence time of dehydroepiandrosterone and internal standard d 6-dehydroepiandrosterone in a spectrogram of a blank sample has no obvious interference; the precision test result shows that the intra-batch precision and the inter-batch precision are respectively 6.88 percent and 8.92 percent, and meet the acceptable standard (CV is less than or equal to 15 percent), which indicates that the method has good stability; the standard adding recovery rate test is carried out by adding standard substances with low, medium and high concentrations, and the result shows that the standard adding recovery rate is 84.9-105.5%, and the standard adding recovery rate meets the acceptable standard (80-120%);
the method has the advantages of strong specificity, high sensitivity, simple pretreatment process, and high precision and standard recovery rate, can be used for detecting the dehydroepiandrosterone with low content in saliva, and provides a reliable quantitative detection method for evaluating the content of the free dehydroepiandrosterone in human body.
Example two
A kit for detecting the content of dehydroepiandrosterone in saliva comprises:
a mobile phase, wherein the mobile phase comprises equal volume of 0.1% formic acid water and 100% acetonitrile by volume;
dehydroepiandrosterone standard solution, wherein the standard mother solution is acetonitrile solution with the concentration of 100ng/ml dehydroepiandrosterone;
an internal standard solution, wherein the internal standard solution is an acetonitrile solution of d6-t dehydroepiandrosterone with the concentration of 2.5 ng/ml;
a diluent comprising: 3g/L bovine serum albumin aqueous solution and acetonitrile;
extracting solution which is methyl tert-butyl ether;
the quality control product comprises dehydroepiandrosterone solution with three concentration gradients of 20pg/mL, 100pg/mL and 500pg/mL which are obtained by diluting with saliva blank matrix and fixing the volume.
The specific composition of the kit is shown in table 6:
TABLE 6 kit composition
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A method for detecting the content of dehydroepiandrosterone in saliva is characterized by comprising the following steps:
preparing a standard substance: taking a dehydroepiandrosterone standard substance, using acetonitrile as a solvent to prepare 1mg/mL of dehydroepiandrosterone standard substance mother liquor, and diluting with acetonitrile to obtain 100ng/mL of dehydroepiandrosterone standard substance solution; taking a d 6-dehydroepiandrosterone standard, preparing 1mg/mL internal standard mother solution by using acetonitrile as a solvent, diluting with acetonitrile to obtain 1 microgram/mL internal standard solution, and diluting to prepare 2.5ng/mL internal standard solution for experiments;
preparing a quality control product: diluting the dehydroepiandrosterone standard solution with saliva blank matrix to constant volume to obtain quality control products with three concentration gradients of 20pg/mL, 100pg/mL and 500 pg/mL;
treating a standard substance: taking the dehydroepiandrosterone standard solution, diluting to constant volume with blank saliva matrix solution to obtain seven concentration gradient dehydroepiandrosterone calibration solutions of 10pg/mL, 20pg/mL, 50pg/mL, 100pg/mL, 200pg/mL, 500pg/mL and 1000pg/mL, respectively taking 500 uL of the dehydroepiandrosterone calibration solutions with various concentrations in a 2mL centrifugal tube, adding 20 uL of the internal standard solution and 1mL of extract liquid into each tube, performing vortex oscillation for 5min, performing centrifugation for 10min at the rotating speed of 13000r/min, transferring an organic phase to another 2mL centrifugal tube, performing nitrogen blow-drying, adding a derivatization agent into a blow-drying centrifugal tube, performing a dark water bath reaction, adding 100 uL of water into the centrifugal tube after the reaction is finished, adding 1mL of extract liquid, performing vortex oscillation for 5min, transferring the organic phase to another 2mL centrifugal tube, performing blow-drying, redissolving 100 mu L of 60% acetonitrile water, carrying out vortex oscillation for 1min, centrifuging for 5min at the rotating speed of 13000r/min, and taking 20 mu L of supernatant to carry out analysis and detection by adopting a high performance liquid chromatography tandem mass spectrometry technology;
pretreatment of a saliva sample: adding 20 mu L of the internal standard solution and 1mL of extraction liquid into a 500 mu L saliva sample in a 2mL centrifuge tube, carrying out vortex oscillation for 5min, centrifuging for 10min at the rotation speed of 13000r/min, transferring an organic phase to another 2mL centrifuge tube, drying by blowing nitrogen, adding a derivatizing agent into a drying centrifuge tube, carrying out a light-resistant water bath reaction, adding 100 mu L of water into the centrifuge tube after the reaction is finished, adding 1mL of extraction liquid, carrying out vortex oscillation for 5min, transferring the organic phase to another 2mL centrifuge tube, drying by blowing nitrogen, redissolving by 100 mu L of 60% acetonitrile water, carrying out vortex oscillation for 1min, centrifuging for 5min at 13000r/min, and analyzing and detecting 20 mu L of supernatant by adopting a high performance liquid chromatography tandem mass spectrometry;
calibration curve: and (3) establishing a standard curve by adopting an isotope internal standard quantitative method and 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 dehydroepiandrosterone in the sample to be detected.
2. The method for detecting the content of dehydroepiandrosterone in saliva according to claim 1, wherein the chromatographic conditions for the analysis and detection by high performance liquid chromatography-tandem mass spectrometry are as follows: the mobile phase A adopts 0.1 percent of formic acid aqueous solution by volume ratio, and the mobile phase B adopts 100 percent of acetonitrile.
3. The method for detecting the content of dehydroepiandrosterone in saliva according to claim 1 or 2, wherein the mass spectrometric conditions for the analytical detection by HPLC-MS are as follows: in an electrospray ionization positive ion detection mode, a mass spectrum scanning mode of multi-reaction monitoring is adopted, the spraying voltage is 5500V, the collision gas is 8, the gas curtain gas is 30psi, the ion source temperature is 550 ℃, the ion source gas flow GS1 is 70psi, and the GS2 is 50 psi.
4. The method for detecting dehydroepiandrosterone content in saliva according to claim 3, wherein the standard treatment step and the saliva sample pretreatment step are carried out at a temperature of 60 ℃ in a dark water bath for 30 min.
5. The method for detecting dehydroepiandrosterone content in saliva according to claim 1, wherein the blank saliva matrix solution is prepared by dissolving 3g of bovine serum albumin with water to a volume of 1L.
6. The method for detecting dehydroepiandrosterone content in saliva according to claim 1, wherein the extract is methyl tert-butyl ether.
7. The method of claim 1, wherein the derivatizing agent is a solution of methoxylamine hydrochloride with a concentration of 50mmol/l and 50% methanol water is used as a solvent.
8. The method for detecting dehydroepiandrosterone content in saliva according to claim 1, further comprising a precision testing step comprising:
taking the same saliva sample as a first test sample group, equally dividing the saliva sample into 5-8 parts, sequentially measuring the content of dehydroepiandrosterone in the first test sample group by an isotope internal standard quantitative method, and calculating to obtain the batch precision;
taking the same saliva sample as a second test sample group, equally dividing the same saliva sample into 5-8 parts, sequentially measuring the content of the dehydroepiandrosterone in the second test sample group by an isotope internal standard quantitative method according to the same time interval for 3-5 days, and calculating to obtain the batch precision.
9. The method for detecting dehydroepiandrosterone content in saliva according to claim 2, further comprising a recovery test step comprising:
taking a saliva sample to divide into 4 parts serving as a third test sample group, wherein each part is 500 mu l, 1 part is not added with a standard substance, the other 3 parts are respectively added with a low-concentration standard substance, a medium-concentration standard substance and a high-concentration standard substance, the 4 parts of test samples are subjected to saliva sample pretreatment and then are subjected to computer measurement for 3 times by adopting a high performance liquid chromatography tandem mass spectrometry technology, and the standard addition recovery rate is calculated.
10. A kit for detecting the content of dehydroepiandrosterone in saliva is characterized by comprising:
a mobile phase comprising equal volumes of 0.1% by volume formic acid water and 100% acetonitrile;
dehydroepiandrosterone standard solution, wherein the standard mother solution is acetonitrile solution with the concentration of 100ng/ml dehydroepiandrosterone;
an internal standard solution, wherein the internal standard solution is an acetonitrile solution of d6-t dehydroepiandrosterone with the concentration of 2.5 ng/ml;
a diluent, the diluent comprising: 3g/L bovine serum albumin aqueous solution and acetonitrile;
an extraction liquid which is methyl tert-butyl ether;
the quality control product comprises a dehydroepiandrosterone solution with three concentration gradients of 20pg/mL, 100pg/mL and 500pg/mL which is obtained by diluting with a blank saliva substrate and fixing the volume.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040235193A1 (en) * | 2003-04-14 | 2004-11-25 | Soldin Steven J. | Steroid hormone analysis by mass spectrometry |
| CN104807921A (en) * | 2015-05-21 | 2015-07-29 | 上海迪安医学检验所有限公司 | Method for detecting 10 kinds of steroid hormones in serum through high performance liquid chromatography tandem mass spectrum technique |
| WO2016019037A1 (en) * | 2014-07-29 | 2016-02-04 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Free hormone and hormone metabolite workup and analysis by mass spectrometry |
| US20170328921A1 (en) * | 2016-05-02 | 2017-11-16 | Sanis Biomedical, LLC | Methods for detecting hormones and other analytes |
| CN109828073A (en) * | 2019-04-01 | 2019-05-31 | 成都益康谱科技有限公司 | The method for qualitative and quantitative detection of several steroid hormones in saliva |
| CN110927289A (en) * | 2019-12-17 | 2020-03-27 | 杭州度安医学检验实验室有限公司 | Steroid hormone detection kit |
| CN111398447A (en) * | 2020-03-12 | 2020-07-10 | 南京品生医学检验实验室有限公司 | Kit for detecting 12 kinds of steroid hormones in serum by ultra-high performance liquid chromatography tandem mass spectrometry technology |
| CN111487328A (en) * | 2019-01-27 | 2020-08-04 | 上海歌特维生物科技集团有限公司 | Method for detecting multiple trace hormones in human body by mass spectrometry |
-
2021
- 2021-01-20 CN CN202110075247.XA patent/CN113009006A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040235193A1 (en) * | 2003-04-14 | 2004-11-25 | Soldin Steven J. | Steroid hormone analysis by mass spectrometry |
| WO2016019037A1 (en) * | 2014-07-29 | 2016-02-04 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Free hormone and hormone metabolite workup and analysis by mass spectrometry |
| CN104807921A (en) * | 2015-05-21 | 2015-07-29 | 上海迪安医学检验所有限公司 | Method for detecting 10 kinds of steroid hormones in serum through high performance liquid chromatography tandem mass spectrum technique |
| US20170328921A1 (en) * | 2016-05-02 | 2017-11-16 | Sanis Biomedical, LLC | Methods for detecting hormones and other analytes |
| CN111487328A (en) * | 2019-01-27 | 2020-08-04 | 上海歌特维生物科技集团有限公司 | Method for detecting multiple trace hormones in human body by mass spectrometry |
| CN109828073A (en) * | 2019-04-01 | 2019-05-31 | 成都益康谱科技有限公司 | The method for qualitative and quantitative detection of several steroid hormones in saliva |
| CN110927289A (en) * | 2019-12-17 | 2020-03-27 | 杭州度安医学检验实验室有限公司 | Steroid hormone detection kit |
| CN111398447A (en) * | 2020-03-12 | 2020-07-10 | 南京品生医学检验实验室有限公司 | Kit for detecting 12 kinds of steroid hormones in serum by ultra-high performance liquid chromatography tandem mass spectrometry technology |
Non-Patent Citations (1)
| Title |
|---|
| 周亚飞等: "HPLC-MS/MS同位素稀释法测定人体血清中类固醇激素的研究", 《检验医学》, no. 05 * |
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