CN110187043A - Method that is a kind of while detecting 13 kinds of steroid hormones in serum - Google Patents
Method that is a kind of while detecting 13 kinds of steroid hormones in serum Download PDFInfo
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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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
- G01N2030/009—Extraction
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
Abstract
The present invention provides a kind of methods for detecting 13 kinds of steroid hormones in serum simultaneously, comprising: mixes test serum sample with inner mark solution, obtains detection liquid by liquid-liquid extraction, detected using ultra performance liquid chromatography-tandem mass spectrum；Liquid phase chromatogram condition: mobile phase A is 0.1% aqueous formic acid, and Mobile phase B is 0.1% formic acid methanol solution；Mass Spectrometry Conditions: positive ion mode, the scanning of the mass spectrum mode of multiple-reaction monitoring；13 kinds of steroid hormones are pregnenolone, progesterone, 17-Hydroxypregnenolone, 17 α-hydroxyprogesterone, 11 α-hydroxyprogesterone, 21- deoxy-cortisol, 11-deoxycorticosterone, cortisone, hydrocortisone, Compd S 11-deoxycortisol, cortisone, aldosterone and oestrone.This method detects detection time only 6 minutes of 13 kinds of steroid hormones simultaneously, and easy to operate, time-consuming is few；High sensitivity, accuracy is good, and high specificity, detection range is wide, has a extensive future in medical treatment and biochemistry detection field.
Description
Technical field
The present invention relates to medical analysis detection technique fields, detect 13 kinds of steroids in serum simultaneously more particularly, to a kind of
The method of body hormone.
Background technique
Endocrine system directly controls the important physiology course of the mankind, such as growth, development, reproduction and metabolism.Steroidal swashs
Plain class drug is a large family in endocrine system, refers specifically to the hormone medicine in molecular structure containing steroidal structure,
It is clinically a kind of important drug, mainly includes cortex hormone of aadrenaline and sex hormone two major classes.Steroid hormone is by cholesterol
Trunk synthesis, is different types of steroids through various enzymatic conversions, they are a kind of Fourth Ring aliphatic hydrocarbon compounds, has ring penta
The more hydrogen phenanthrene parent nucleus of alkane, the slight change of structure, these difference cause usually in the quantity and position of carbonyl and hydroxy functional group
The greatest differences of their physiological functions.Steroid sustain life, adjusting sexual function, adjust body metabolism etc. and play important work
With.
Wherein progestational hormone is mainly pregnenolone, progesterone (progesterone), 17-Hydroxypregnenolone, 17 α-hydroxyprogesterone and 11 α-
Hydroxyprogesterone.Cortin is mainly cortisone, hydrocortisone, cortisone, Compd S 11-deoxycortisol, 11-deoxycorticosterone, 21-
Deoxy-cortisol and aldosterone, oestrone are estrogen.
The information table of above 13 kinds of steroid hormones is as shown in table 1 below:
1 13 kinds of steroid hormone information tables of table
Wherein, pregnenolone (Pregnenolone) is positioned at the upstream of steroid hormone synthesis access, is to synthesize other steroids
The precursor of body hormone.Pregnenolone is referred to collectively as forth generation neurotransmitter with neurosteroid downstream, has and promotes choline
Serotonergic neuron function promotes microtubule polymerization, adjusts synaptic plasticity and adjust nerve cell calcium homeostasis, free radical resisting damage, promote
Into multi-efficiencies such as cognitive functions, there is extensive and significant effect in terms of reconciling nervous function.
It is a kind of natural progestogen secreted by corpus luteum that progesterone (progesterone), which is also known as progesterone, right in vivo
The endometrium that estrogen excited has significant Morphology Effects, to maintain the required hormone of gestation institute.Progesterone concentrations are assisting
Have great importance in Diagnosis of Ectopic Pregnancy, value is also extensively used for threatened abortion, habitual abortion, No-clay weak interbed type and closes
Through the diagnosis with the diseases such as climacteric syndrome.
17 α-hydroxyprogesterone (17- α-hydroxyprogesterone, 17-OHP) is that congenital adrenal cortical hyper plasia is examined
Disconnected important evidence, when internal 21-hydroxylase defect cortisol (hydrocortisone) and cortisone formed completely or partially by
Resistance, low-level cortisol promote the corticotropin (ACTH) of hypothalamus-pituitary-adrenal axis through negative feedback
Secretion increase, enzyme block preposition compound 17-OHP accumulation, therefore 17-OHP content detection be in biochemistry detection one it is important
Index.
17-Hydroxypregnenolone (17-hydroxypregnenolone) and 11 α-hydroxyprogesterone (11 α-
It hydroxyprogesterone) is all steroid intermediate, the former is in vivo generally by NSC 37741 in P45c17 enzyme
It is transformed under effect, 17 α-hydroxyprogesterone can be converted into again under the action of 3beta-Hydroxysteroid dehydrogenase.
Cortisone (Corticosterone) is a kind of 21 carbon steroid hormone of corticoid, by adrenal cortex
It generates.Its content can increase when clinically there are the diseases such as hypercortisolism (Cushing disease), adrenal tumor, myocardial infarction
More, when working as hypoadrenocorticism, such as Addison disease, seat Chinese disease disease occurs, content is reduced.Therefore clinically
The hormone is important clinical disease diagnosis index.
Hydrocortisone (cortisol) is also known as cortisol, is a kind of parahormone that adrenal gland generates in stress reaction.
Cortisol can make blood pressure, blood glucose level increase and generate immunosuppressive action.Hypercortisolism is also known as Cushing syndrome, faces
One of bed diagnosis basis is exactly that Determination of cortisol increases extremely.Clinically there is A Disen (Addison) family name disease, hypophysis function subtracts
When moving back equal diseases, Determination of cortisol can be reduced in blood.
Cortisone (cortisone) is the glucocorticoid of adrenocortical secretion, itself is inactive, need to be metabolized in vivo
It just works at hydrocortisone (cortisol).21- deoxy-cortisol (21-deoxycortisol) is raw by 17 α-hydroxyprogesterone
At being the precursor of hydrocortisone and cortisone biosynthesis.11-deoxycorticosterone (11-deoxycorticosterone) is used
In the replacement therapy of primary adrenal cortical hypofunction.The two and 17-OHP are all congenital adrenal cortical hyper plasia
One of diagnosis index.
Aldosterone (Aldosterone) mainly acts on kidney, and primary efficacy is the re-absorption for promoting sodium ion and moisture content,
Maintain water salt balance.Clinically primary aldosteronism, primary cyclic edema, Bartter Cotard, by kidney ball
Device hyperplasia etc. can all cause the content of aldosterone to increase, and to lower (such as Addison disease), primary single for adrenal cortex function
Hypoaldosteronism, high-sodium diet, autonomic nervous dysfunction etc. will lead to the reduction of its content.
Oestrone (Estrone) is mainly synthesized by gonad granulocyte in female body, is converted on a small quantity from androstenedione
After generate, male's oestrone is mainly from androstenedione, on a small quantity directly from testicular secretion, detection oestrone to understand ovary in
Secreting function, judging the women of child-bearing age, whether there is or not ovulation functions important value.Normal women of child-bearing age is female in entire menstrual cycle blood plasma
Ketone is with estradiol concentration in parallel variation, and after climacteric, decrease in estrogen, plasma estradiol fall is greater than oestrone.
Clinically, normal pregnancy (after 12 weeks), hepatopathy, Stein-Leventhal syndrome, adrenal gland or orchioncus are more common in oestrone raising
With Ovarian granukna tumor etc.；Oestrone reduces, and is more common in abnormal pregnancy, hypo-ovaria, amenorrhoea, pituitary gonadotropic hormone
Cell function is low, high prolactin disease etc..
The metabolism of steroid hormone is as shown in Figure 1, steroid hormone is metabolized closely bound up, shortage or increasing with people's normal growth and development
Gao Douhui causes the even other diseases of endocrine disturbance, therefore their detection is highly important.It is clinically biochemical at present
Detection steroid hormone generally uses chemiluminescence (CLIA) or radio immunoassay (RIA), these method sensitivity are low, accurately
It spends low, the problems such as poor specificity, generally existing cross reactivity, is especially measuring low concentration steroidal in some children and old man's body
When hormone, these problems are even more serious.Mass spectrometry has always been considered as being steroid hormone sizing technique " goldstandard ", HPLC-MS/
MS has high specificity no cross contamination, and high sensitivity, analysis speed is fast, the level of energy actual response hormone in vivo, and can be same
When separation determination a variety of hormones the advantages of.In order to reduce matrix interference, sensitivity is improved, at present sample early period in mass spectroscopic assays
Product processing method is extraction sample (liquid-liquid extraction or leaching), then performs the derivatization processing, finally passes through superelevation
Effect liquid phase chromatogram, Liquid Chromatography-Tandem Mass Spectrometry are enriched with, are purified and are detected.This pre-treating method is complicated for operation, derivatization
The time needed is long, has side reaction product interference, and equipment investment is big, on-line preconcentration purifying time-consuming and consumptive material, at high cost.
Summary of the invention
The purpose of the present invention is to provide a kind of methods for detecting 13 kinds of steroid hormones in serum simultaneously, before this method has
Processing is simple, and detection speed is fast, high sensitivity, and accuracy is high, flux greatly can batch detection the advantages of.
The present invention adopts the following technical scheme:
Method that is a kind of while detecting 13 kinds of steroid hormones in serum,
13 kinds of steroid hormones are respectively as follows: pregnenolone, progesterone, 17-Hydroxypregnenolone, 17 α-hydroxyprogesterone, 11 α-hydroxyl
Progesterone, 21- deoxy-cortisol, 11-deoxycorticosterone, cortisone, hydrocortisone, Compd S 11-deoxycortisol, cortisone, aldehyde are solid
Ketone and oestrone；
Specifically comprise the following steps:
S1, test serum sample is mixed with inner mark solution, detection liquid is obtained by liquid-liquid extraction pre-treatment；
S2, the detection liquid is detected using ultra performance liquid chromatography-tandem mass spectrometry；
The inner mark solution is the-D containing progesterone9, 17 α-hydroxyprogesterone-D8, 11-deoxycorticosterone-13C3, aldosterone-D4, cortex
Ketone-D4, hydrocortisone-D4With Compd S 11-deoxycortisol-D5The methanol solution of Isotopic Internal Standard object；
The ultra performance liquid chromatography condition are as follows:
Mobile phase A is 0.1% (v/v) aqueous formic acid；
Mobile phase B is 0.1% (v/v) formic acid methanol solution；
Eluent gradient elution parameters are as follows:
The Mass Spectrometry Conditions are as follows:
Mode is positive ion mode, using the scanning of the mass spectrum mode of multiple-reaction monitoring.
Wherein, the ratio of mobile phase A and Mobile phase B is volume ratio.
The present invention passes through largely research and obtains, can be non-in a short time under above-mentioned eluent gradient elution parameters
Often efficiently and accurately determine 13 kinds of steroid hormones, high sensitivity.
Ultra performance liquid chromatography can be used in this field in ultra performance liquid chromatography-tandem mass spectrometry of the invention
Common chromatographic column, in order to improve separating effect and separating degree, it is preferable to use model Shim-pack GIST C12 (2 μm,
100*2.1mm) chromatographic column.
In a preferred embodiment of the invention, in order to keep testing result more accurate, the used time is shorter, the ultra high efficiency liquid phase
Chromatography-tandem mass spectrometry uses triple quadrupole mass spectrometer, the Mass Spectrometry Conditions specifically:
Ionization source is the source electrospray ionisation ESI, and wherein atomization gas flow is 1.5-3L/min, dry gas stream amount 3-16L/
Min, heating throughput are 10-24L/min, and 200-400 DEG C of interface temperature, 130-180 DEG C of DL temperature heats deblocking temperature 300-
380℃。
The MS detection parameters are as follows:
Preferably, in the above-mentioned technical solutions, the Mass Spectrometer Method uses 8050 triple quadrupole mass spectrometer of Shimadzu, simultaneously
It is spraying at a distance from DL pipe to adjust sample.
Specifically, the installation site of electric spray ion source nozzle needle is adjusted to+3 place of optimum position, to improve detection
Sensitivity.
In a preferred embodiment of the invention, the eluent gradient elution parameters are as follows:
Wherein, the flow velocity of the mobile phase is preferably 0.4ml/min, and the column temperature of chromatographic column is preferably 40 DEG C.
Wherein, in order to further increase sensitivity, it is preferable that the heating throughput is 15L/min, interface temperature 300
DEG C, 150 DEG C of DL temperature, heat 350 DEG C of deblocking temperature.
In the present invention, it is preferred to carry out liquid-liquid extraction using methyl tertiary butyl ether(MTBE) and methanol solution.
It is described in order to improve stability, accuracy and sensitivity and separative efficiency in a preferred embodiment of the invention
The pre-treatment of liquid-liquid extraction specifically includes:
To in the mixed liquor of test serum sample and inner mark solution be added 800-1000 μ L methyl tertiary butyl ether(MTBE), after concussion
Refrigerated centrifuge 2-6min at 10000-15000r/min, 2-5 DEG C takes supernatant liquor 400-800 μ L, N2Air-blowing is dry, and 80- is added
The 50-70% methanol solution of 150 μ L redissolves, and is uniformly mixed, the refrigerated centrifuge 2-6min at 10000-15000r/min, 2-5 DEG C,
Supernatant liquor is taken, the detection liquid is obtained.
In the present invention, method commonly used in the art can be used to obtain the specific concentration value of each hormone.Specifically:
In conjunction with the standard working curve of step S2 obtained testing result and each steroid hormone, each steroid hormone is obtained in blood
Content value in clear.
In detail, the standard working curve of the steroid hormone is made using Isotopic Internal Standard sizing technique, specific steps packet
It includes,
By known and various concentration pregnenolone, progesterone, 17-Hydroxypregnenolone, 17 α-hydroxyprogesterone, 11 α-hydroxyprogesterone,
21- deoxy-cortisol, 11-deoxycorticosterone, cortisone, hydrocortisone, Compd S 11-deoxycortisol, cortisone, aldosterone and female
The methanol solution of ketone is mixed with inner mark solution and blank serum matrix respectively, and the standard for obtaining various concentration by pre-treatment is to be measured
Liquid is detected respectively using standard prepare liquid of the ultra performance liquid chromatography-tandem mass spectrometry to the various concentration, with mark
Each steroid hormone and internal standard compound peak area ratio are Y-axis in quasi- detection liquid, and each steroid hormone concentration is X-axis in standard detection liquid, is obtained
To the standard working curve of each steroid hormone；Testing conditions in the ultra performance liquid chromatography-tandem mass spectrometry are step
Testing conditions in S2.
Wherein,
Above-mentioned pre-treatment step is identical as in the preparation detection pre-treatment step of liquid；
The relevant parameter phase of all parameters and measurement detection liquid in above-mentioned ultra performance liquid chromatography-tandem mass spectrometry
Together；
Above-mentioned standard prepare liquid is that blank serum matrix, mixing inner mark solution and various concentration are contained 13 kinds of hormones
Standard mixed liquor is mixed to prepare.
In the present invention, it is preferred to the use of 0.1% bovine serum albumin solution be blank serum matrix.
Compared with prior art, the invention has the benefit that
(1) method provided by the present invention can detect 13 kinds of steroid hormones in serum simultaneously, and total time-consuming only has 6 minutes,
Efficiency far is higher than the method and traditional radio immunoassay (RIA) for detecting each hormone-content respectively, greatly shortens
Detection time, increases detection flux, is suitble to the detection of batch samples, application value with higher；
(2) method provided by the present invention passes through liquid-liquid extraction, separation and concentration steroid hormone component to be measured, so that detection
As a result stability greatly improves, and compared with other hormone test pre-treating methods, this method is easy to operate, and time-consuming is few；
(3) method provided by the present invention uses superelevation liquid chromatography-mass spectrography Series detectors technology, high specificity, selection
Property good, high sensitivity and no cross contamination, the detection limit of this method is low, and detection range is wide, and precision is good, can be in accurate quantitative analysis body
The hormone of low concentration, can biochemistry detection with the level of actual response human body internal hormone, suitable for clinical treatment.
Detailed description of the invention
Fig. 1 is the metabolic map of steroid hormone；
Fig. 2 is the chromatogram of 13 kinds of steroid hormones in the blood serum sample of the embodiment of the present invention 1；
Fig. 3 is the peace of 8050 triple quadrupole mass spectrometer electric spray ion source nozzle needle of Shimadzu used in the embodiment of the present invention 1
Holding position figure.
Specific embodiment
The present invention is described in further detail combined with specific embodiments below, so that those skilled in the art is more
The present invention is expressly understood.Following embodiment is merely to illustrate the present invention, but is used to limit the scope of the invention incessantly.It is based on
Embodiment in the present invention, those of ordinary skill in the art in the case where not making creative work, it is obtained other
All embodiments, belong to protection scope of the present invention.
Embodiment 1
Ultra performance liquid chromatography-tandem mass spectrometry provided by the present embodiment detects the side of 13 kinds of steroid hormones simultaneously
Method, comprising the following steps:
1, material
2012 year June physical examination of the experiment sample of methodological study from Wuhan Co., Ltd, Kang Shengda medical test institute
Serum sample.
2, instrument and reagent consumptive material
Instrument: 8050 triple quadrupole mass spectrometer of Shimadzu, Shimadzu 30A Ultra Performance Liquid Chromatography instrument, ultrapure water instrument
(Millipore ultrapure water machine), electronic analytical balance (FA1004, Shanghai Sunny Hengping Scientific Instrument Co., Ltd.), high speed cold
Freeze centrifuge (GL-20M high speed freezing centrifuge), vortex blending instrument (SCILOGEX company, the U.S.), GM-12 water-bath nitrogen evaporator
(Beijing at sprout great achievement Science and Technology Ltd.), liquid-transfering gun (big dragon Xing Chuan laboratory apparatus Co., Ltd, Beijing), volumetric flask, EP pipe.
Reagent consumptive material: chromatographic column is Shim-pack GIST C12 (2 μm, 100*2.1mm), Chromatographic Pure Methanol (Sigma-
Aldrich), methyl tertiary butyl ether(MTBE) (Aladdin reagent Co., Ltd, China), (Chinese medicines group chemical reagent is limited for acetic acid
Company).
Standard items and Isotopic Internal Standard object: the standard items of pregnenolone and cortisone are purchased from Dalian Mei Lun company, progesterone, 17
The standard items of α-hydroxyprogesterone and hydrocortisone come from Aladdin company, 17-Hydroxypregnenolone, 11 α-hydroxyprogesterone, 21- deoxidation skin
The standard items of the pure and mild 11-deoxycorticosterone of matter come from Toronto Research Chemicals (TRC) company, 11- deoxidation skin
The standard items of matter alcohol come from TargetMol company, and the standard items of cortisone come from Shanghai Yuan Ye company, the mark of aldosterone and oestrone
Quasi- product come from J&K company, progesterone-D9Internal standard comes from TRC company, 17 α-hydroxyprogesterone-D8, 11-deoxycorticosterone-13C3, 11- deoxidation
Cortisol-D5, aldosterone-D4, cortisone-D4With hydrocortisone-D4Internal standard is all from sigma company.
3, method
(1) chromatographic condition:
Chromatographic column is Shim-pack GIST C12 (2 μm, 100*2.1mm), and mobile phase A is 0.1% formic acid-aqueous solution
(ultrapure water), B are 0.1% formic acid methanol solution, and flow velocity 0.2-0.4ml/min, column temperature is 40 DEG C；
Eluent gradient elution parameters are as follows:
(2) Mass Spectrometry Conditions:
Using cation ionization mode, the scanning of the mass spectrum mode of multiple-reaction monitoring, ionization source is electrospray ionisation ESI
Source, wherein atomization gas flow is 3L/min, and dry gas stream amount 8L/min, heating throughput is 15L/min, interface temperature 300
DEG C, 150 DEG C of DL temperature, heat 350 DEG C of deblocking temperature.
The MS detection parameters are as follows:
In addition, as shown in figure 3, optimize and revise that sample in 8050 triple quadrupole mass spectrometer of Shimadzu is spraying and DL pipe away from
From specifically, the installation site of electric spray ion source nozzle needle is adjusted to+3 place of optimum position, to reach raising detection sensitivity
Purpose.
(3) mixed standard solution preparation:
The standard items for accurately weighing the above-mentioned 13 kinds of hormones of 10mg respectively are separately added into 10ml methanol and are completely dissolved, and obtain concentration
It is 1 × 10-4The standard items mother liquor of g/ml is mixed the mixed standard solution followed by with methanol dilution at various concentration.
(4) mixing inner mark solution is prepared:
The deuterated isotope mark product of 10mg are accurately weighed respectively, comprising: progesterone-D9, 17 α-hydroxyprogesterone-D8, 11- deoxidation cortex
Ketone-13C3, aldosterone-D4, cortisone-D4, hydrocortisone-D4With Compd S 11-deoxycortisol-D5, it is complete to be separately added into 10ml methanol
Fully dissolved, obtaining concentration is 1 × 10-47 kinds of internal standard product mother liquors of g/ml, then use methanol dilution at the mixing of required concentration it
Inner mark solution.
(5) standard prepare liquid is prepared:
A certain amount of 0.1% bovine serum albumin solution is taken, the hybrid standard for being separately added into inner mark solution and various concentration is molten
Liquid obtains the standard prepare liquid of various concentration.
(6) detection liquid is prepared:
A certain amount of test serum sample is taken, mixing inner mark solution is added, obtains detection liquid.
(7) sample pre-treatments:
To standard prepare liquid and detection liquid in carry out pre-treatment respectively: to standard prepare liquid and detection liquid in be separately added into
800 μ L methyl tertiary butyl ether(MTBE)s (MTBE) shake, and 10000r/min refrigerated centrifuge 6min at 4 DEG C takes 400 μ L of supernatant liquor, uses N2
Drying, 50% methanol solution that 100 μ L are added redissolve, 15000r/min refrigerated centrifuge at mixing 15 seconds, 4 DEG C on turbine mixer
2min draws 100 μ L supernatant liquors in ultra high efficiency liquid-phase inlet bottle, upper machine testing.
4, testing result
Testing result is as shown in Fig. 2, using Isotopic Internal Standard sizing technique, using reference substance and internal standard compound peak area ratio as Y-axis,
Reference substance concentration is X-axis, establishes standard working curve.
Concentration calculation result in the linear equation and detection liquid of obtained 13 kinds of steroid hormones is as shown in table 2 below.
Concentration calculation result in the linear equation and detection liquid of 2 13 kinds of steroid hormones of table
Specific testing result is as shown in Fig. 2, component representated by each number in Fig. 2 are as follows: and 1 is aldosterone, and 2 be cortisone, 3
It is 21- deoxy-cortisol for hydrocortisone, 4,5 be cortisone, and 6 be Compd S 11-deoxycortisol, and 7 be 11 α-hydroxyprogesterone, and 8 be 11-
Deoxycortone, 9 be 17 α-hydroxyprogesterone, and 10 be progesterone, and 11 be 17-Hydroxypregnenolone, and 12 be pregnenolone, and 13 be oestrone.
Embodiment 2
Ultra performance liquid chromatography-tandem mass spectrometry provided by the present embodiment detects the side of 13 kinds of steroid hormones simultaneously
The step of method, step is with embodiment 1, is identical, and difference is only that:
Eluent gradient elution parameters are as follows:
Compared with Example 1, MRM mass spectrum response signal and sensitivity are declined embodiment 2, and sensitivity is (according to minimum
Detection quantitative limit) reduce 15%.
Embodiment 3
Ultra performance liquid chromatography-tandem mass spectrometry provided by the present embodiment detects the side of 13 kinds of steroid hormones simultaneously
The step of method, step is with embodiment 1, is identical, and difference is only that: carrying out matter using 8050 triple quadrupole mass spectrometer of Shimadzu
When spectrum detection, electric spray ion source nozzle needle installation site is default setting (at 0), rather than after the adjustment installation in embodiment 1
+ 3 place positions.
MRM mass spectrum response signal intensity in embodiment 3 reduces 35-45% relative to embodiment 1, to reduce it
The sensitivity of detection, and then influence the lowest detection quantitative limit of steroid hormone.
Comparative example 1
Ultra performance liquid chromatography-tandem mass spectrometry provided by this comparative example detects the side of 13 kinds of steroid hormones simultaneously
The step of method, step is with embodiment 1, is identical, and difference is only that:
Eluent gradient elution parameters are as follows:
Compared with Example 1, detection time extended to 9 minutes by 6 minutes to comparative example 1, but sensitivity and accuracy do not have
It has clear improvement.
Comparative example 2
Ultra performance liquid chromatography-tandem mass spectrometry provided by this comparative example detects the side of 13 kinds of steroid hormones simultaneously
The step of method, step is with embodiment 1, is identical, and difference is only that:
The MS detection parameters are as follows:
Compared with Example 1, sensitivity (according to lowest detection quantitative limit) reduces 30% to comparative example 2, accuracy
3.5%.
Experimental example 1
Methodology validation experiment is carried out to the detection method of 13 kinds of steroid hormones in embodiment 1.
1, Precision Experiment
Practical serum sample is taken, low middle high three horizontal quality-control products are separately added into, carries out Precision Experiment, each concentration
Detection 5 times, calculates the average value and relative standard deviation (RSD%) of testing result.Methodology validation experimental data is respectively such as
Shown in table 3-14.
3 progesterone precision data of table tests (unit: ng/mL)
4 pregnenolone precision data of table tests (unit: ng/mL)
5 17-Hydroxypregnenolone precision data of table tests (unit: ng/mL)
6 17 α of table-hydroxyprogesterone precision data experiment (unit: ng/mL)
7 11 α of table-hydroxyprogesterone precision data experiment (unit: ng/mL)
8 cortisone precision data of table tests (unit: ng/mL)
9 hydrocortisone precision data of table tests (unit: ng/mL)
10 cortisone precision data of table tests (unit: ng/mL)
11 Compd S 11-deoxycortisol precision data of table tests (unit: ng/mL)
12 11-deoxycorticosterone precision data of table tests (unit: ng/mL)
13 21- deoxy-cortisol precision data of table tests (unit: ng/mL)
14 aldosterone precision data of table tests (unit: ng/mL)
15 oestrone precision data of table tests (unit: ng/mL)
2, recovery of standard addition
A collection of pooled serum sample is prepared, base concentration is measured, is separately added into low, medium and high three horizontal samples, into
Row recovery of standard addition experiment, wherein each concentration is measured in parallel 3 times.
Each hormone rate of recovery summary sheet of table 16
The present invention uses the pre-treating method of liquid-liquid extraction easy to operate, utilizes superelevation liquid phase series connection triple quadrupole bar
Mass spectrometry method (UPLC-MS/MS) has detected 13 kinds of steroid hormones in serum simultaneously, and this method can substantially improve radio-immunity point
The problems such as cross reactivity is strong in analysis method (RIA), and poor specificity, sensitivity is low, and accuracy is low；Quantitatively may be used using Isotopic Internal Standard
Greatly to eliminate the interference of matrix, keep result more accurate；And the method detection speed of the invention is fast, when flux can be saved greatly
Between, batch detection.
The detection method low concentration of 13 kinds of steroid hormones in serum is detected while above-mentioned inspection result shows of the invention
Lower precision≤20%, middle concentration and high concentration≤15% illustrate that this method precision is good.Recovery of standard addition is in 80-120%
Between, meet requirement of the FDA for the rate of recovery of biological sample.
In conclusion this method precision is good, high specificity, accuracy are high, and the low and sample pre-treatments of detection limit are simple, 6
The detection of 13 kinds of progestational hormone and glucocorticoid steroid hormone can be completed in minute, precision, recovery of standard addition and matrix are imitated
The quantitative detection requirement of clinical human's blood steroid hormone should be met, prepare detection and monitoring human body steroidal for clinical precisely medical treatment
Hormonal readiness, the diagnosing and treating of adrenal gland related disease provide a kind of reliable analyzing detecting method.
Finally, method of the invention is only preferable embodiment, it is not intended to limit the scope of the present invention.It is all
Within the spirit and principles in the present invention, any modification, equivalent replacement, improvement and so on should be included in protection of the invention
Within the scope of.
Claims (9)
1. a kind of method for detecting 13 kinds of steroid hormones in serum simultaneously, which is characterized in that
13 kinds of steroid hormones are respectively as follows: pregnenolone, progesterone, 17-Hydroxypregnenolone, 17 α-hydroxyprogesterone, 11 α-hydroxyprogesterone,
21- deoxy-cortisol, 11-deoxycorticosterone, cortisone, hydrocortisone, Compd S 11-deoxycortisol, cortisone, aldosterone and female
Ketone；
Specifically comprise the following steps:
S1, test serum sample is mixed with inner mark solution, detection liquid is obtained by liquid-liquid extraction pre-treatment；
S2, the detection liquid is detected using ultra performance liquid chromatography-tandem mass spectrometry；
The inner mark solution is the-D containing progesterone9, 17 α-hydroxyprogesterone-D8, aldosterone-D4, cortisone-D4, hydrocortisone-D4、11-
Deoxycortone-13C3With Compd S 11-deoxycortisol-D5The methanol solution of Isotopic Internal Standard object；
The ultra performance liquid chromatography condition are as follows:
Mobile phase A is 0.1% (v/v) aqueous formic acid；
Mobile phase B is 0.1% (v/v) formic acid methanol solution；
Eluent gradient elution parameters are as follows:
The Mass Spectrometry Conditions are as follows:
Mode is positive ion mode, using the scanning of the mass spectrum mode of multiple-reaction monitoring.
2. the method according to claim 1, wherein the Mass Spectrometry Conditions specifically:
Ionization source be the source electrospray ionisation ESI, wherein atomization gas flow be 1.5-3L/min, dry gas stream amount 3-16L/min,
Heating throughput is 10-24L/min, and 200-400 DEG C of interface temperature, 130-180 DEG C of DL temperature heats 300-380 DEG C of deblocking temperature；
The MS detection parameters are as follows:
Preferably, the Mass Spectrometer Method uses 8050 triple quadrupole mass spectrometer of Shimadzu, while adjusting that sample is spraying and DL pipe
Distance, specifically, the installation site of electric spray ion source nozzle needle is adjusted to+3 place of optimum position.
3. method according to claim 1 or 2, which is characterized in that the eluent gradient elution parameters are as follows:
4. method according to claim 1-3, which is characterized in that the flow velocity of the mobile phase is 0.4ml/min,
The column temperature of chromatographic column is 40 DEG C.
5. according to the method described in claim 2, it is characterized in that, the heating throughput is 15L/min, interface temperature 300
DEG C, 150 DEG C of DL temperature, heat 350 DEG C of deblocking temperature.
6. method according to claim 1-5, which is characterized in that in step S1, the pre-treatment is to use first
Base tertbutyl ether and methanol solution carry out liquid-liquid extraction pre-treatment；
Preferably, the pre-treatment specifically includes: to addition 800-1000 μ in the mixed liquor of test serum sample and inner mark solution
L methyl tertiary butyl ether(MTBE), after concussion at 10000-15000r/min, 2-5 DEG C refrigerated centrifuge 2-6min, take supernatant liquor 400-
800 μ L, N2Air-blowing is dry, and the 50-70% methanol solution that 80-150 μ L is added redissolves, and is uniformly mixed, 10000-15000r/min,
Refrigerated centrifuge 2-6min, takes supernatant liquor at 2-5 DEG C, obtains the detection liquid.
7. method according to claim 1-6, which is characterized in that further include the detection obtained in conjunction with step S2
As a result with the standard working curve of each steroid hormone, content value of each steroid hormone in serum is obtained.
8. the method according to the description of claim 7 is characterized in that the standard working curve of the steroid hormone uses isotope
Internal standard quanitation is made, and specific steps include,
By known and various concentration pregnenolone, progesterone, 17-Hydroxypregnenolone, 17 α-hydroxyprogesterone, 11 α-hydroxyprogesterone, 21- are de-
Oxygen cortisol, 11-deoxycorticosterone, cortisone, hydrocortisone, Compd S 11-deoxycortisol, cortisone, aldosterone and oestrone
Methanol solution is mixed with inner mark solution and blank serum matrix respectively, obtains the standard prepare liquid of various concentration by pre-treatment,
It is detected respectively using standard prepare liquid of the ultra performance liquid chromatography-tandem mass spectrometry to the various concentration, with standard
Detecting each steroid hormone and internal standard compound peak area ratio in liquid is Y-axis, and each steroid hormone concentration is X-axis in standard detection liquid, is obtained
The standard working curve of each steroid hormone；Testing conditions in the ultra performance liquid chromatography-tandem mass spectrometry are step S2
In testing conditions.
9. according to the method described in claim 8, it is characterized in that, the blank serum matrix is 0.1% bovine serum albumin(BSA)
Solution.
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