CN104807921B - The method of 10 kinds of steroid hormones in using high performance liquid chromatography tandem mass spectrum technology for detection serum - Google Patents
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Abstract
The invention discloses the method for 10 kinds of steroid hormones in using high performance liquid chromatography tandem mass spectrum technology for detection serum, 10 kinds of described steroid hormones are respectively: dehydrobenzene, testosterone, protona, androstenedione, oestrone, cortisone, progesterone, pregnenolone, 17 α-hydroxyprogesterone and 17-Hydroxypregnenolone; Adopt using high performance liquid chromatography tandem mass spectrum technology for detection through the above-mentioned 10 kinds of steroid hormones in pretreated serum, first utilize high performance liquid chromatography that 10 kinds of steroid hormones are separated, recycling mass spectrum Isotopic Internal Standard sizing technique, taking the concentration ratio of standard items and internal standard compound as X-axis, the peak area ratio of standard items and internal standard compound is Y-axis, set up calibration curve, calculate the content of above-mentioned 10 kinds of steroid hormones. The inventive method is highly sensitive, high specificity, accurately and pretreatment process simpler, within 10min, can complete the separation and detection of multiple hormone, precision and recovery of standard addition meet the demands substantially.
Description
Technical field
The invention belongs to blood testing technical field, be specifically related in using high performance liquid chromatography tandem mass spectrum technology for detection serumThe method of 10 kinds of steroid hormones.
Background technology
Steroid hormone is the fat-soluble little molecule hormone of a class, is through a series of enzymatics by cholesterol. The present inventionMainly for wherein to human body grow, sexal maturity and metabolism necessary 10 kinds of steroid hormones analyze, bagDraw together dehydrobenzene (dehydroepiandrosterone, DHEA), testosterone (testosterone, T), protona(dihydrotestosterone, DHT), androstenedione (androstenedione, AD), oestrone (estrone, E1), cortexKetone (corticosterone, CORT), progesterone (progesterone, P4), pregnenolone (pregnenolone, P5), 17 α-Hydroxyprogesterone (17 α-Hydroxyprogesterone, 17 α-OHP4) and 17-Hydroxypregnenolone (17-hydroxylpregnenolone,17-OHP5)。
Wherein, DHEA is a kind of very important hormone, can make people improve memory, and restorative ability reduces bloodIn cholesterol, and can resist fat, heart disease and pressure, can also strengthening immune system. Dehydrobenzene is too low may be drawnPlay the major diseases such as diabetes, immunity deficiency, cancer, hypertension and heart disease. 17 α-OHP4 and CORT increase and may causeCongenital adrenal cortical hyper plasia, adenoma,adrenal or ovarian neoplasm. T can promote memory, increases muscle strength, reduce fatFat, increase bone hardness. The effect of DHT is mainly to maintain the sperm effect that produces, and stimulates the growth of reproductive organs, remains normalSexual desire, can also promote deposition and the erythrocytic generation of bone growth and calcium phosphorus simultaneously. AD raises can cause acne, congenital kidneyUpper gland corticohyperplassia, adrenal cortical tumor etc.; AD reduces can make male sex's development delay, nanism, adrenal cortex functionThe disease of going down, meniscocytosis.
In a word, the rising of human body steroid hormone or reduction and some clinical diseases are as adrenal,congenital hyperplasia, manyThe diseases such as capsule ovary syndrome, adrenal insufficiency are closely bound up, and the height of its content in serum is for evaluatorBody health is vital.
Summary of the invention
Technical problem to be solved by this invention is to provide in a kind of using high performance liquid chromatography tandem mass spectrum technology for detection serumThe method of 10 kinds of steroid hormones.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
The method of 10 kinds of steroid hormones in using high performance liquid chromatography tandem mass spectrum technology for detection serum,
10 kinds of described steroid hormones are respectively: dehydrobenzene, testosterone, protona, androstenedione, oestrone, skinMatter ketone, progesterone, pregnenolone, 17 α-hydroxyprogesterone and 17-Hydroxypregnenolone;
Adopt using high performance liquid chromatography tandem mass spectrum technology for detection to swash through the above-mentioned 10 kinds of steroids in pretreated serumElement, first utilizes high performance liquid chromatography that 10 kinds of steroid hormones are separated, and recycling mass spectrum Isotopic Internal Standard sizing technique, with standard itemsWith the concentration ratio of internal standard compound be X-axis, the peak area ratio of standard items and internal standard compound is Y-axis, sets up calibration curve, calculates above-mentioned 10 kindsThe content of steroid hormone, concrete chromatographic condition is:
(1) high-efficient liquid phase chromatogram condition:
Mobile phase A: 0.1%v/v aqueous formic acid;
Mobile phase B: the methanol solution of 0.1%v/v formic acid;
Chromatographic column model: PhenomenexC18100mm × 2.1mm, 2.6 μ m;
Adopt the mode of gradient elution, in table 1;
Flow velocity is 0.5mL/min, and column temperature is 35 DEG C, and sampling volume is 20 μ L;
Table 1 eluent gradient wash-out parameter
(2) mass spectrum condition:
Under electron spray ionisation cation detecting pattern, adopt the scanning of the mass spectrum pattern of multiple-reaction monitoring; Spray voltage is5500V; Collision gas is Medium; Gas curtain gas is 40kPa; Ion gun atomization gas and heating assisted gas are 60kPa; Desolventizing temperatureDegree is 550 DEG C; Monitored simultaneously object progesterone (m/z345.093 → 124.1), pregnenolone (m/z332.0 → 86.2),17 α-hydroxyprogesterone (m/z361.0 → 112.2), 17-Hydroxypregnenolone (m/z348.0 → 330.3), androstenedione (m/z317.0 → 112.2), dehydrobenzene (m/z304.2 → 253.2), testosterone (m/z304.1/112.2), protona (m/z306.2 → 81.1), cortisone (m/z410.0 → 377.2) and oestrone (m/z286.0 → 253.0) and Isotopic Internal StandardD2-oestrone (m/z288.0 → 255.1), d6-dehydrobenzene (m/z310.3 → 219.2), d8-17 α-hydroxyprogesterone (m/z269.2 → 115.1) and d3-testosterone (m/z307.0 → 112.2); Each object go a bunch voltage, collision voltage and collisionPond outlet voltage parameter is in table 2;
Table 210 kind of steroid hormone mass spectrum parameter
Wherein, the serum that described serum is human or animal.
Wherein, the pretreated serum of described process is prepared as follows and obtains: get 200 μ L serum in 1.5mL fromIn core barrel, add wherein 20 μ L100ng/mL to mix interior mark liquid B, then add 600 μ LMTBE, then after the vortex several seconds, vibrate15min, gets 400 μ L supernatant Nitrogen evaporators and dries up, then add 80 μ L1.5mol/L azanols after the centrifugal 5min of 14000r/min,The vortex 15min that vibrates after 5 seconds, puts into 60 DEG C of derivative reaction 1h of insulating box.
Wherein, in described mixing, mark liquid B prepares as follows: accurately take respectively the deuterated isotope of 10.0mgInterior mark product, comprise d6-dehydrobenzene, d3-testosterone, d8-17 α-hydroxyprogesterone, d2-oestrone and d9-progesterone, add respectively 10mLMethyl tertiary butyl ether(MTBE) dissolves completely, obtains concentration and be 5 kinds of Isotopic Internal Standard solution of 1.0mg/mL, then with methyl alcohol by this 5The concentration of planting Isotopic Internal Standard solution is diluted to 1.0 μ g/mL by 1.0mg/mL, and then respectively getting 1mL concentration is 5 of 1.0 μ g/mLKind of Isotopic Internal Standard solution adds water and is settled to 10mL, and preparation obtains mark liquid B in mixing that concentration is 100ng/mL.
Wherein, described standard items prepare in accordance with the following steps:
Accurately take respectively dehydrobenzene 10.0mg, testosterone 25.0mg, protona 5.2mg, androstenedione 1.5mg,Oestrone 2.5mg, cortisone 2.0mg, progesterone 10mg, pregnenolone 5.1mg, 17 α-hydroxyprogesterone 10mg and 17-Hydroxypregnenolone25mg, adds respectively the methyl-tert fourth of 1mL, 2.5mL, 5.2mL, 1.5mL, 2.5mL, 2mL, 1mL, 5.1mL, 10mL and 2.5mLBase ether, is mixed with the dehydrobenzene that concentration is respectively 10.0mg/mL, the testosterone of 10.0mg/mL, two hydrogen testis of 1.0mg/mLThe cortisone of the androstenedione of ketone, 1.0mg/mL, the oestrone of 1.0mg/mL, 1.0mg/mL, the progesterone of 10.0mg/mL, 1.0mg/The standard items mother liquor of the 17-Hydroxypregnenolone of the pregnenolone of mL, the 17-OH progesterone of 1.0mg/mL and 10.0mg/mL;
From above dehydrobenzene, testosterone, progesterone and 17-Hydroxypregnenolone standard items mother liquor, pipetting respectively 100 μ L usesMethyl tertiary butyl ether(MTBE) is settled to 1mL, and the concentration that obtains these 10 kinds of steroid hormones is all 1.0mg/mL, then with methyl alcohol by this 10Plant the concentration of steroid hormone by 1.0mg/mL stepwise dilution to 1.0 μ g/mL;
Get respectively androstenedione, 17 α-hydroxyprogesterone, pregnenolone, oestrone and two hydrogen testis that 200 μ L concentration are 1.0 μ g/mLKetone, 800 μ L concentration are the dehydrobenzene of 1.0 μ g/mL, testosterone, progesterone and cortisone that each 1.0mL concentration is 1.0 μ g/mL,500 μ L concentration are that the 17-Hydroxypregnenolone of 1.0 μ g/mL all joins in 15mL centrifuge tube, then add deionized water constant volume and arrive10mL, is mixed with hybrid standard liquid A; In hybrid standard liquid A, androstenedione, 17 α-hydroxyprogesterone, pregnenolone, oestrone and two hydrogenThe concentration of testosterone is 20ng/mL, and the concentration of dehydrobenzene is 80ng/mL, and the concentration of progesterone, testosterone and cortisone is 100ng/ML, the concentration of 17-Hydroxypregnenolone is 50ng/mL;
Pipette 100 μ L hybrid standard liquid A in 1.5mL centrifuge tube, add wherein the blank serum matrix of 100 μ L (40mg/The mL bovine serum albumin aqueous solution) as first high value concentration point; Pipette 40 μ L hybrid standard liquid A serum matrix analog fixedHold to 400 μ L and obtain standard mixed liquid B; Get respectively 5,25,50,100,200 μ L standard mixed liquid B and be settled to 200 μ L, obtain itIts five calibration concentration point, these six concentration point volumes are 200 μ L; Then respectively add wherein 20 μ L100ng/mL to mixInterior mark liquid B, then add 600 μ L methyl tertiary butyl ether(MTBE)s, then the vortex 15min that vibrates after 5 seconds, gets after the centrifugal 5min of 14000r/min400 μ L supernatants dry up with Nitrogen evaporator, then add 80 μ L1.5mol/L azanols, the vortex 15min that vibrates after 5 seconds, then put into constant temperature60 DEG C of derivative reaction 1h of case.
Beneficial effect: the inventive method is highly sensitive, high specificity, accurately and pretreatment process simpler, within 10minCan complete the separation and detection of multiple hormone, precision and recovery of standard addition meet the demands substantially, can be used for serum class clinicallyThe quantitative analysis of steroid hormone, for the health evaluating of men and women's hormonal readiness clinically provides a kind of detection method reliably.
Brief description of the drawings
Fig. 1 is the TIC of marking standard items in 10 kinds of steroid hormones and 5 kinds
Fig. 2 is target TIC in 10 kinds of steroid hormones and 5 kinds in serum.
Detailed description of the invention
According to following embodiment, the present invention may be better understood. But, those skilled in the art will readily understand, realExecute routine described content only for the present invention is described, and should also can not limit in claims described in detailInvention.
Embodiment 1:
1. material
The sample of methodological study experiment comes from Dean, Shanghai medical test in September And October, 2014 university student bodyThe serum sample of inspection.
(1) instrument: API4000+Triple level Four bar mass spectrographs (AppliedBiosystem company); Kspert ' ultraLC100 liquid chromatographic system (joining automatic sampler); Medical high speed freezing centrifuge (Beijing Bai Yang Medical Devices Co., Ltd.);Ultra-pure water instrument (the excellent general ultrapure Science and Technology Ltd. in Chengdu); Constant Temp. Oven (the gloomy reliable Co., Ltd that tests in Shanghai); MultitubeVortex mixed instrument (Hangzhou Ao Sheng Instrument Ltd.); Vortex mixer (the safe medical apparatus plant in Jiangyan City) fast; Nitrogen evaporator(MD200, Hangzhou Ao Sheng Instrument Ltd.); Adjustable pipette (ThermoScientific0.5～10 μ L, 10～100 μL, 100～1000 μ L); Glass apparatus, beaker, graduated cylinder etc.
(2) reagent consumptive material: Chromatographic Pure Methanol (TediaHighPuritySolventCompany.Inc);(100mm × 2.1mm, 2.6 μ m) and the pre-guard column of PhenomenexC18 (U.S.'s Féraud for PhenomenexC18 reverse-phase chromatographic columnMen company); Methyl tertiary butyl ether(MTBE) (methyltert-butylether, MTBE) (purity 99.9%, Sigma-Aldrich public affairsDepartment); Azanol (Sigma-Aldrich company).
(3) standard items: E1, P4, P5, CORT, 17 α-OHP4,17-OHP5, AD, DHEA, DHT and T are purchased from Sigma-Aldrich, mark d in stable isotope2-E1, d9-P4, d6-DHEA, d8-17OHP4 and d3-T are purchased from TorontoResearchChemicalInc. company, purity all >=98%.
(4) quality-control product: the blank serum that contains T, DHEA, DHT, AD, E1, CORT, P4, P5,17-OHP4 and 17-OHP5Matrix solution, point high, normal, basic three concentration are respectively QC (L), QC (M), QC (H), are shown in Table 3.
Table 310 kind of steroid hormone quality-control product corresponding concentration (ng/mL of unit)
2. method
(1) chromatographic condition: mobile phase A: 0.1%v/v aqueous formic acid; Mobile phase B: the methyl alcohol of 0.1%v/v formic acid is moltenLiquid. Chromatographic column model: PhenomenexC18100mm × 2.1mm, 2.6 μ m, the mode of employing gradient elution, refers to table 1. StreamSpeed is 0.5mL/min, and column temperature is 35 DEG C, and sampling volume is 20 μ L.
(2) mass spectrum condition: at electron spray ionisation (electrosprayionization, ESI) cation detecting patternUnder, the scanning of the mass spectrum pattern of employing multiple-reaction monitoring (multiplereactionmonitoring, MRM). Spray voltage(ionspray, IS) is 5500V; Collision gas (collisiongas, CAD) is Medium; Gas curtain gas (curtaingas,CUR) be 40kPa; Ion gun atomization gas (ionsourceGS1, GS1) and heating assisted gas (GS2) are 60kPa; DesolventizingTemperature is 550 DEG C. Object P4 (m/z345.093 → 124.1), P5 (m/z332.0 → 86.2), 17-have been monitored simultaneouslyOHP4(m/z361.0→112.2)、17-OHP5(m/z348.0→330.3)、AD(m/z317.0→112.2)、DHEA(m/z304.2→253.2)、T(m/z304.1/112.2)、DHT(m/z306.2→81.1)、CORT(m/z410.0→377.2)And E1 (m/z286.0 → 253.0) and Isotopic Internal Standard d2-E1(m/z288.0→255.1)、d6-DHEA(m/z310.3→ 219.2), d8-17OHP4 (m/z269.2 → 115.1) and d3-T (m/z307.0 → 112.2). Again respectively to each targetThing go a bunch voltage (declusteringpotential, DP), collision voltage (collisionenergy, CE) and collide pondThe conditions such as outlet voltage (collisioncellexitpotential, CXP) have been carried out system optimization (in table 2), to reachTo higher stability and sensitivity.
(3) standard items preparation:
Accurately take respectively dehydrobenzene 10.0mg, testosterone 25.0mg, protona 5.2mg, androstenedione 1.5mg,Oestrone 2.5mg, cortisone 2.0mg, progesterone 10mg, pregnenolone 5.1mg, 17 α-hydroxyprogesterone 10mg and 17-Hydroxypregnenolone25mg, adds respectively the methyl-tert fourth of 1mL, 2.5mL, 5.2mL, 1.5mL, 2.5mL, 2mL, 1mL, 5.1mL, 10mL and 2.5mLBase ethereal solution, is mixed with the dehydrobenzene that concentration is respectively 10.0mg/mL, the testosterone of 10.0mg/mL, two hydrogen of 1.0mg/mLThe cortisone of the androstenedione of testosterone, 1.0mg/mL, the oestrone of 1.0mg/mL, 1.0mg/mL, the progesterone of 10.0mg/mL,The standard items mother liquor of the 17-Hydroxypregnenolone of the pregnenolone of 1.0mg/mL, the 17-OH progesterone of 1.0mg/mL and 10.0mg/mL;From above dehydrobenzene, testosterone, progesterone and 17-Hydroxypregnenolone standard items mother liquor, pipette respectively 100 μ L methyl-tert fourthsBase ether is settled to 1mL, and the concentration that obtains these 10 kinds of steroid hormones is all 1.0mg/mL, then with methyl alcohol by these 10 kinds of steroidsThe concentration of hormone is by 1.0mg/mL stepwise dilution to 1.0 μ g/mL; Then getting respectively 200 μ L concentration is the androstene two of 1.0 μ g/mLKetone, 17 α-hydroxyprogesterone, pregnenolone, oestrone and protona, 800 μ L concentration are the dehydrobenzene of 1.0 μ g/mL, each 1.0mLConcentration is testosterone, progesterone and the cortisone of 1.0 μ g/mL, and 500 μ L concentration are that the 17-Hydroxypregnenolone of 1.0 μ g/mL all joinsIn 15mL centrifuge tube, then add deionized water constant volume to 10mL, be mixed with hybrid standard liquid A, obtain androstenedione, 17 α-hydroxyl is pregnantThe concentration of ketone, pregnenolone, oestrone and protona is 20ng/mL, and the concentration of dehydrobenzene is 80ng/mL, progesterone, testosteroneWith the concentration of cortisone be 100ng/mL, the concentration of 17-Hydroxypregnenolone is 50ng/mL.
Accurately take respectively the deuterated Isotopic Internal Standard product of 10.0mg, comprise that dehydrobenzene-d6, testosterone-d3,17 α-hydroxyl are pregnantKetone-d8, oestrone-d2 and progesterone-d9, add respectively 10mL methyl tertiary butyl ether(MTBE) to dissolve completely, obtains concentration and be 1.0mg/mL5 kinds of Isotopic Internal Standard solution. Then with methyl alcohol, the concentration of these 5 kinds of Isotopic Internal Standard solution is diluted to by 1.0mg/mL1.0 μ g/mL, 5 kinds of Isotopic Internal Standard solution then respectively getting 1mL concentration and be 1.0 μ g/mL add water and are settled to 10mL, and preparation obtainsConcentration is the interior mark of the mixing liquid B of 100ng/mL.
(4) preparation of quality-control product: get standard items mother liquor 20 μ L, 100 μ L, 500 μ L fixed with blank serum matrix solution respectivelyHold to 1000 μ L and obtain.
The upper and lower surrounding blooming of kit, anti-shock, thermal insulating, eluent A and B are placed in upper left side, and 4*1mL is placed respectively in lower leftAmpoule bottle, is respectively titer and quality-control product; 10mL dilution 1,20mL dilution 2 and 100mL extract are placed respectively in the right.
(5) sample treatment
1) standard items processing: pipette 100 μ L hybrid standard liquid A in 1.5mL centrifuge tube, add wherein 100 μ L serumMatrix analog is as first high value concentration point; Pipette 40 μ L hybrid standard liquid A serum matrix analogs and be settled to 400 μ LObtain standard mixed liquid B; Get respectively 5,25,50,100,200 μ L standard mixed liquid B and be settled to 200 μ L, obtain other five schoolsAccurate concentration point, these six concentration point volumes are 200 μ L. Then respectively add wherein 20 μ L100ng/mL to mix interior mark liquid B,Add 600 μ LMTBE, the 15min that then vibrates after the vortex several seconds, gets 400 μ L supernatants and uses after the centrifugal 5min of 14000r/min againNitrogen evaporator dries up, then adds 80 μ L1.5mol/L azanols, the 15min that vibrates after the vortex several seconds, then put into 60 DEG C of derivatizations of insulating boxReaction 1h, then move into loading in chromatogram bottle and carry out LC-MS/MS analysis.
2) blood serum sample pre-treatment: get 200 μ L serum in 1.5mL centrifuge tube, add wherein 20 μ L100ng/mL mixedClose interior mark liquid B, then add 600 μ LMTBE, the 15min that then vibrates after the vortex several seconds, gets 400 μ after the centrifugal 5min of 14000r/minL supernatant dries up with Nitrogen evaporator, then adds 80 μ L1.5mol/L azanols, and the 15min that vibrates after the vortex several seconds, puts into insulating box 60DEG C derivative reaction 1h, finally moves into loading in chromatogram bottle and carries out LC-MS/MS analysis.
(3) quality-control product pre-treatment: get respectively quality-control product solution QC (L), QC (M), the each 200 μ L of QC (H) are in 1.5mL centrifuge tubeIn, then consistent with blood serum sample pre-treating method, repeat no more herein.
In assay kit, each component is in table 4.
The preparation of table 410 kind of steroid hormone assay kit component
3, the Establishment and optimization of method
Experiment, in order more to be stablized and highly sensitive object signal, is used azanol by the carbonyl in steroid hormoneBase, through oximation reaction, adopts the ion pair (Q1/Q3) of its derivatization product of MRM mode monitoring, uses " Ramp " to select differentDP, CE and the CXP of ion pair are optimized, and from optimize chromatogram, can see the corresponding the best of ion highest signal strengthAs a result, record optimization the results are shown in Table 2. In addition, some other parameter provides according to instrument as IS, CAD, CUR, GS1 and GS2 etc.Reference range is selected suitable numerical value, to ensure stronger signal strength signal intensity.
Four, method validation
1. TIC: the peak type of the standard items of 10 kinds of steroid hormones and blood serum sample is more symmetrical, denseWhen degree is not more than 50ng/mL, substantially not assorted peak disturbs, and illustrates and can obtain with this understanding good detection, and Fig. 1 is 10 kindsTarget TIC in steroid hormone and 5 kinds, Fig. 2 is the total ion current chromatogram of 10 kinds of steroid hormones in serumFigure, although these 10 kinds of materials are not separated completely, is enough to meet quantitative requirement for the detection of LC-MS/MS, without completelySeparate.
2. calibration curve: adopt Isotopic Internal Standard sizing technique, utilize the concentration of Analyst software with reference material and internal standard compoundThan being X-axis, reference material and internal standard compound peak area ratio are Y-axis, set up calibration curve, and calculate the concentration of determinand in serum.10 kinds of steroid hormones linear fit equation in concentration range separately, linear good, coefficient correlation is more than 0.999, fullFoot quantitative requirement, in table 5.
Table 510 kind of steroid hormone equation of linear regression and linearly dependent coefficient
3. precision test: get in normal human serum sample one day 8 batches of reprocessings, measure with Isotopically labelled internal standardThe concentration of 10 kinds of steroid hormones, withinrun precision scope is 1.61%～15.42%, the results are shown in Table 6; Point 8 batches of places in three daysReason, calculating betweenrun precision scope is 4.27%～17.02%, the results are shown in Table 7.
Table 6 withinrun precision result of the test (ng/mL of unit)
Table 7 betweenrun precision result of the test (ng/mL of unit)
3. recovery test: routine patients serum's sample is chosen in experiment at random, and wherein 1 does not add standard items, other 3 pointsDo not add the standard items of basic, normal, high 3 concentration, with same steps reprocessing and measure 3 times, result is as calculated as table 8Show. Result demonstration, the recovery of standard addition of 10 kinds of hormones, between 80%-130.6%, repeats the RSD of test for 3 times at 0.6%-12.7% scope.
The recovery of standard addition result (ng/mL of unit) of table 810 kind of steroid hormone
Five, discuss
This research adopts ID-HPLC-MS/MS method to measure 10 kinds of steroid hormones in human serum simultaneously. At present, behaviourMaking simple and cost low is the advantage of immunization, but causes lacking specificity because it exists cross reaction and matrix interference, andAnd sensitivity is lower. LC-MS/MS detects for appearance time and the ion pair of object simultaneously, highly sensitive, can the utmost pointThe large interference of avoiding cross reaction. Meanwhile, adopt Isotopically labelled internal standard can eliminate greatly the interference of matrix,And be not subject to preprocessing process, loading volume and the mobile impact that equates condition, can reach accurate quantitative analysis.
In order to improve the Ionization Efficiency of analyte, utilize pre-column derivatization method, adopt in azanol and steroid hormoneCarbonyl generation derivative reaction, has greatly improved sensitivity and the stability of analyte, minimum quantitative limit LLOQ (with S/N >=10 is standard) can be low to moderate tens pg/mL, substantially meet the testing requirement of some trace hormones in serum.
Investigate reappearance and the rate of recovery of the detection method of these 10 kinds of hormones, withinday precision is 1.61%～15.42%, day to day precision is 4.27%～17.02%, except the betweenrun precision of E1 is large, and other 9 kinds of steroid hormonesPrecision be all less than 15% (acceptable standard is CV≤15%), but also relevant with the concentration of determinand, if concentration is too low(ppb level is following) can relax to CV≤20%, and therefore the method has good stability. In addition, experiment acquisition recovery of standard addition exists80%-130.6% scope, acceptable standard is 80%-120%, equally also relevant with concentration, if the lower tolerance interval of concentrationCan relax to 70%-130%, and the rate of recovery of E1 is 130.6%, therefore, except E1 is large, other hormone meets substantially to be wantedAsk. Compared with other several hormones, precision and the rate of recovery of E1 are larger, may be lower due to its content in human serum,Instrument is larger to its noise comparatively speaking, large thereby the error of testing result can become; Larger precision also may be followed samplePreservation, preprocessing process and matrix in other material relevant to its interference. Thereby, can be by periodic cleaning instrument orESI spray needle reduces ambient interferences as far as possible, or it is too low to select more high performance mass spectrometer to carry out detection levelMaterial, as E1 etc.
In a word, the method is highly sensitive, high specificity, accurately and pretreatment process simpler, within 10min, can complete manyThe separation and detection of planting hormone, precision and recovery of standard addition meet the demands substantially, can be used for Seram steroid clinicallyQuantitative analysis, for the health evaluating of men and women's hormonal readiness clinically provides a kind of detection method reliably.
Claims (6)
1. the method for 10 kinds of steroid hormones in using high performance liquid chromatography tandem mass spectrum technology for detection serum, is characterized in that,
10 kinds of described steroid hormones are respectively: dehydrobenzene, testosterone, protona, androstenedione, oestrone, cortisone,Progesterone, pregnenolone, 17 α-hydroxyprogesterone and 17-Hydroxypregnenolone;
Adopt using high performance liquid chromatography tandem mass spectrum technology for detection through the above-mentioned 10 kinds of steroid hormones in pretreated serum, firstUtilize high performance liquid chromatography that 10 kinds of steroid hormones are separated, recycling mass spectrum Isotopic Internal Standard sizing technique, with standard items with inThe concentration ratio of mark thing is X-axis, and the peak area ratio of standard items and internal standard compound is Y-axis, sets up calibration curve, calculates above-mentioned 10 kinds solidThe content of alcohol hormone, concrete chromatographic condition is:
(1) high-efficient liquid phase chromatogram condition:
Mobile phase A: 0.1%v/v aqueous formic acid;
Mobile phase B: the methanol solution of 0.1%v/v formic acid;
Chromatographic column model: PhenomenexC18100mm × 2.1mm, 2.6 μ m;
Adopt the mode of gradient elution, in table 1;
Flow velocity is 0.5mL/min, and column temperature is 35 DEG C, and sampling volume is 20 μ L;
Table 1 eluent gradient wash-out parameter
(2) mass spectrum condition:
Under electron spray ionisation cation detecting pattern, adopt the scanning of the mass spectrum pattern of multiple-reaction monitoring; Spray voltage is5500V; Collision gas is Medium; Gas curtain gas is 40kPa; Ion gun atomization gas and heating assisted gas are 60kPa; Desolventizing temperatureDegree is 550 DEG C; Monitored simultaneously object progesterone m/z345.093 → 124.1, pregnenolone m/z332.0 → 86.2,17 α-Hydroxyprogesterone m/z361.0 → 112.2,17-Hydroxypregnenolone m/z348.0 → 330.3, androstenedione m/z317.0 →112.2, dehydrobenzene m/z304.2 → 253.2, testosterone m/z304.1/112.2, protona m/z306.2 → 81.1,Cortisone m/z410.0 → 377.2 and oestrone m/z286.0 → 253.0 and Isotopic Internal Standard d2-oestrone m/z288.0 →255.1, d6-dehydrobenzene m/z310.3 → 219.2, d8-17 α-hydroxyprogesterone m/z269.2 → 115.1 and d3-testosterone m/z307.0 → 112.2; Go a bunch voltage, collision voltage and the collision pond of each object export voltage parameter in table 2;
Table 210 kind of steroid hormone mass spectrum parameter
2. the side of 10 kinds of steroid hormones in using high performance liquid chromatography tandem mass spectrum technology for detection serum according to claim 1Method, is characterized in that, the serum that described serum is human or animal.
3. the side of 10 kinds of steroid hormones in using high performance liquid chromatography tandem mass spectrum technology for detection serum according to claim 1Method, is characterized in that, the pretreated serum of described process is prepared as follows and obtains: get 200 μ L serum in 1.5mL fromIn core barrel, add wherein 20 μ L100ng/mL to mix interior mark liquid B, then add 600 μ LMTBE, then after the vortex several seconds, vibrate15min, gets 400 μ L supernatant Nitrogen evaporators and dries up, then add 80 μ L1.5mol/L azanols after the centrifugal 5min of 14000r/min,The vortex 15min that vibrates after 5 seconds, puts into 60 DEG C of derivative reaction 1h of insulating box.
4. the side of 10 kinds of steroid hormones in using high performance liquid chromatography tandem mass spectrum technology for detection serum according to claim 3Method, is characterized in that, in described mixing, mark liquid B prepares as follows: accurately take respectively the deuterated coordination of 10.0mgMark product in element, comprise d6-dehydrobenzene, d3-testosterone, d8-17 α-hydroxyprogesterone, d2-oestrone and d9-progesterone, add respectively10mL methyl tertiary butyl ether(MTBE) dissolves completely, obtains concentration and be 5 kinds of Isotopic Internal Standard solution of 1.0mg/mL, then will with methyl alcoholThe concentration of these 5 kinds of Isotopic Internal Standard solution is diluted to 1.0 μ g/mL by 1.0mg/mL, and then respectively getting 1mL concentration is 1.0 μ g/mL5 kinds of Isotopic Internal Standard solution add water and be settled to 10mL, preparation obtains mark liquid B in mixing that concentration is 100ng/mL.
5. the side of 10 kinds of steroid hormones in using high performance liquid chromatography tandem mass spectrum technology for detection serum according to claim 3Method, is characterized in that, described standard items prepare in accordance with the following steps:
Accurately take respectively dehydrobenzene 10.0mg, testosterone 25.0mg, protona 5.2mg, androstenedione 1.5mg, oestrone2.5mg, cortisone 2.0mg, progesterone 10mg, pregnenolone 5.1mg, 17 α-hydroxyprogesterone 10mg and 17-Hydroxypregnenolone 25mg, pointDo not add the methyl tertiary butyl ether(MTBE) of 1mL, 2.5mL, 5.2mL, 1.5mL, 2.5mL, 2mL, 1mL, 5.1mL, 10mL and 2.5mL, joinMake dehydrobenzene, the testosterone of 10.0mg/mL, the protona of 1.0mg/mL, 1.0mg/ that concentration is respectively 10.0mg/mLThe pregnene alcohol of the cortisone of the androstenedione of mL, the oestrone of 1.0mg/mL, 1.0mg/mL, the progesterone of 10.0mg/mL, 1.0mg/mLThe standard items mother liquor of 17 α-hydroxyprogesterone of ketone, 1.0mg/mL and the 17-Hydroxypregnenolone of 10.0mg/mL;
From above dehydrobenzene, testosterone, progesterone and 17-Hydroxypregnenolone standard items mother liquor, pipette respectively 100 μ L methylTertbutyl ether is settled to 1mL, and the concentration that obtains these 10 kinds of steroid hormones is all 1.0mg/mL, then with methyl alcohol by this 10 kindThe concentration of steroid hormone is by 1.0mg/mL stepwise dilution to 1.0 μ g/mL;
Getting respectively 200 μ L concentration is androstenedione, 17 α-hydroxyprogesterone, pregnenolone, oestrone and the protona of 1.0 μ g/mL,800 μ L concentration are the dehydrobenzene of 1.0 μ g/mL, testosterone, progesterone and cortisone that each 1.0mL concentration is 1.0 μ g/mL, 500 μL concentration is that the 17-Hydroxypregnenolone of 1.0 μ g/mL all joins in 15mL centrifuge tube, then adds deionized water constant volume to 10mL, joinsMake hybrid standard liquid A; In hybrid standard liquid A, androstenedione, 17 α-hydroxyprogesterone, pregnenolone, oestrone and protona denseDegree is 20ng/mL, and the concentration of dehydrobenzene is 80ng/mL, and the concentration of progesterone, testosterone and cortisone is 100ng/mL, 17-hydroxylThe concentration of pregnenolone is 50ng/mL;
Pipette 100 μ L hybrid standard liquid A in 1.5mL centrifuge tube, add wherein the blank serum matrix of 100 μ L as firstHigh value concentration point; Pipetting 40 μ L hybrid standard liquid A serum matrix analogs is settled to 400 μ L and obtains standard mixed liquid B; RespectivelyGet 5,25,50,100,200 μ L standard mixed liquid B and be settled to 200 μ L, obtain other five calibration concentration point, these six concentration pointVolume is 200 μ L; Then respectively add wherein 20 μ L100ng/mL to mix interior mark liquid B, then add 600 μ L methyl tertbutylsEther, then the vortex 15min that vibrates after 5 seconds, gets 400 μ L supernatant Nitrogen evaporators and dries up, then add after the centrifugal 5min of 14000r/minEnter 80 μ L1.5mol/L azanols, the vortex 15min that vibrates after 5 seconds, then put into 60 DEG C of derivative reaction 1h of insulating box.
6. the side of 10 kinds of steroid hormones in using high performance liquid chromatography tandem mass spectrum technology for detection serum according to claim 5Method, is characterized in that, described blank serum matrix is the 40mg/mL bovine serum albumin aqueous solution.
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Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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