CN114674961A - Kit for synchronously detecting 17 steroid hormones in serum without derivatization and application thereof - Google Patents
Kit for synchronously detecting 17 steroid hormones in serum without derivatization and application thereof Download PDFInfo
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- CN114674961A CN114674961A CN202210382756.1A CN202210382756A CN114674961A CN 114674961 A CN114674961 A CN 114674961A CN 202210382756 A CN202210382756 A CN 202210382756A CN 114674961 A CN114674961 A CN 114674961A
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- 239000003270 steroid hormone Substances 0.000 title claims abstract description 87
- 210000002966 serum Anatomy 0.000 title claims abstract description 54
- 238000001212 derivatisation Methods 0.000 title claims abstract description 17
- 238000001514 detection method Methods 0.000 claims abstract description 68
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical group CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 42
- 238000000034 method Methods 0.000 claims abstract description 35
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims abstract description 27
- 239000003480 eluent Substances 0.000 claims abstract description 26
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 claims abstract description 20
- 239000003085 diluting agent Substances 0.000 claims abstract description 9
- 238000003908 quality control method Methods 0.000 claims abstract description 9
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 79
- -1 17 alpha-hydroxyprogesterone ketone Chemical class 0.000 claims description 58
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 claims description 51
- MUMGGOZAMZWBJJ-DYKIIFRCSA-N Testostosterone Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 MUMGGOZAMZWBJJ-DYKIIFRCSA-N 0.000 claims description 51
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 claims description 51
- DBPWSSGDRRHUNT-UHFFFAOYSA-N 17alpha-hydroxy progesterone Natural products C1CC2=CC(=O)CCC2(C)C2C1C1CCC(C(=O)C)(O)C1(C)CC2 DBPWSSGDRRHUNT-UHFFFAOYSA-N 0.000 claims description 40
- 229950000801 hydroxyprogesterone caproate Drugs 0.000 claims description 40
- DOMWKUIIPQCAJU-LJHIYBGHSA-N Hydroxyprogesterone caproate Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@@](C(C)=O)(OC(=O)CCCCC)[C@@]1(C)CC2 DOMWKUIIPQCAJU-LJHIYBGHSA-N 0.000 claims description 35
- 150000002500 ions Chemical class 0.000 claims description 33
- FUFLCEKSBBHCMO-UHFFFAOYSA-N 11-dehydrocorticosterone Natural products O=C1CCC2(C)C3C(=O)CC(C)(C(CC4)C(=O)CO)C4C3CCC2=C1 FUFLCEKSBBHCMO-UHFFFAOYSA-N 0.000 claims description 27
- PQSUYGKTWSAVDQ-UHFFFAOYSA-N Aldosterone Natural products C1CC2C3CCC(C(=O)CO)C3(C=O)CC(O)C2C2(C)C1=CC(=O)CC2 PQSUYGKTWSAVDQ-UHFFFAOYSA-N 0.000 claims description 27
- MFYSYFVPBJMHGN-UHFFFAOYSA-N Cortisone Natural products O=C1CCC2(C)C3C(=O)CC(C)(C(CC4)(O)C(=O)CO)C4C3CCC2=C1 MFYSYFVPBJMHGN-UHFFFAOYSA-N 0.000 claims description 27
- 229960002478 aldosterone Drugs 0.000 claims description 27
- 229960003473 androstanolone Drugs 0.000 claims description 27
- 229960005471 androstenedione Drugs 0.000 claims description 27
- AEMFNILZOJDQLW-UHFFFAOYSA-N androstenedione Natural products O=C1CCC2(C)C3CCC(C)(C(CC4)=O)C4C3CCC2=C1 AEMFNILZOJDQLW-UHFFFAOYSA-N 0.000 claims description 27
- 229960004544 cortisone Drugs 0.000 claims description 27
- 229960000890 hydrocortisone Drugs 0.000 claims description 27
- 239000000186 progesterone Substances 0.000 claims description 27
- 229960003387 progesterone Drugs 0.000 claims description 27
- 229960003604 testosterone Drugs 0.000 claims description 27
- ORNBQBCIOKFOEO-YQUGOWONSA-N Pregnenolone Natural products O=C(C)[C@@H]1[C@@]2(C)[C@H]([C@H]3[C@@H]([C@]4(C)C(=CC3)C[C@@H](O)CC4)CC2)CC1 ORNBQBCIOKFOEO-YQUGOWONSA-N 0.000 claims description 25
- ZESRJSPZRDMNHY-UHFFFAOYSA-N de-oxy corticosterone Natural products O=C1CCC2(C)C3CCC(C)(C(CC4)C(=O)CO)C4C3CCC2=C1 ZESRJSPZRDMNHY-UHFFFAOYSA-N 0.000 claims description 25
- 229960000249 pregnenolone Drugs 0.000 claims description 25
- NVKAWKQGWWIWPM-ABEVXSGRSA-N 17-β-hydroxy-5-α-Androstan-3-one Chemical compound C1C(=O)CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CC[C@H]21 NVKAWKQGWWIWPM-ABEVXSGRSA-N 0.000 claims description 24
- PQSUYGKTWSAVDQ-ZVIOFETBSA-N Aldosterone Chemical compound C([C@@]1([C@@H](C(=O)CO)CC[C@H]1[C@@H]1CC2)C=O)[C@H](O)[C@@H]1[C@]1(C)C2=CC(=O)CC1 PQSUYGKTWSAVDQ-ZVIOFETBSA-N 0.000 claims description 24
- OMFXVFTZEKFJBZ-UHFFFAOYSA-N Corticosterone Natural products O=C1CCC2(C)C3C(O)CC(C)(C(CC4)C(=O)CO)C4C3CCC2=C1 OMFXVFTZEKFJBZ-UHFFFAOYSA-N 0.000 claims description 24
- MFYSYFVPBJMHGN-ZPOLXVRWSA-N Cortisone Chemical compound O=C1CC[C@]2(C)[C@H]3C(=O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 MFYSYFVPBJMHGN-ZPOLXVRWSA-N 0.000 claims description 24
- FMGSKLZLMKYGDP-UHFFFAOYSA-N Dehydroepiandrosterone Natural products C1C(O)CCC2(C)C3CCC(C)(C(CC4)=O)C4C3CC=C21 FMGSKLZLMKYGDP-UHFFFAOYSA-N 0.000 claims description 24
- AEMFNILZOJDQLW-QAGGRKNESA-N androst-4-ene-3,17-dione Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CCC2=C1 AEMFNILZOJDQLW-QAGGRKNESA-N 0.000 claims description 24
- OMFXVFTZEKFJBZ-HJTSIMOOSA-N corticosterone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@H](CC4)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 OMFXVFTZEKFJBZ-HJTSIMOOSA-N 0.000 claims description 24
- 229960002847 prasterone Drugs 0.000 claims description 24
- ZESRJSPZRDMNHY-YFWFAHHUSA-N 11-deoxycorticosterone Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 ZESRJSPZRDMNHY-YFWFAHHUSA-N 0.000 claims description 22
- HFSXHZZDNDGLQN-ZVIOFETBSA-N 18-hydroxycorticosterone Chemical compound C([C@]1(CO)[C@@H](C(=O)CO)CC[C@H]1[C@@H]1CC2)[C@H](O)[C@@H]1[C@]1(C)C2=CC(=O)CC1 HFSXHZZDNDGLQN-ZVIOFETBSA-N 0.000 claims description 22
- ORNBQBCIOKFOEO-QGVNFLHTSA-N pregnenolone Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 ORNBQBCIOKFOEO-QGVNFLHTSA-N 0.000 claims description 22
- 239000000243 solution Substances 0.000 claims description 22
- QADHLRWLCPCEKT-UHFFFAOYSA-N Androstenediol Natural products C1C(O)CCC2(C)C3CCC(C)(C(CC4)O)C4C3CC=C21 QADHLRWLCPCEKT-UHFFFAOYSA-N 0.000 claims description 21
- 229950009148 androstenediol Drugs 0.000 claims description 21
- FMGSKLZLMKYGDP-USOAJAOKSA-N dehydroepiandrosterone Chemical compound C1[C@@H](O)CC[C@]2(C)[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CC=C21 FMGSKLZLMKYGDP-USOAJAOKSA-N 0.000 claims description 21
- 238000001819 mass spectrum Methods 0.000 claims description 21
- DDFHBQSCUXNBSA-UHFFFAOYSA-N 5-(5-carboxythiophen-2-yl)thiophene-2-carboxylic acid Chemical compound S1C(C(=O)O)=CC=C1C1=CC=C(C(O)=O)S1 DDFHBQSCUXNBSA-UHFFFAOYSA-N 0.000 claims description 18
- QADHLRWLCPCEKT-LOVVWNRFSA-N androst-5-ene-3beta,17beta-diol Chemical compound C1[C@@H](O)CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CC=C21 QADHLRWLCPCEKT-LOVVWNRFSA-N 0.000 claims description 18
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- LKQDFQLSEHWIRK-UHFFFAOYSA-N (3alpha,5alpha,17alphaOH)-3,17-Dihydroxypregnan-20-one Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(=O)C)(O)C1(C)CC2 LKQDFQLSEHWIRK-UHFFFAOYSA-N 0.000 claims description 7
- LKQDFQLSEHWIRK-UKBVDAKRSA-N 3alpha,17alpha-Dihydroxy-5beta-pregnan-20-one Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@@](C(=O)C)(O)[C@@]2(C)CC1 LKQDFQLSEHWIRK-UKBVDAKRSA-N 0.000 claims description 7
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- JERGUCIJOXJXHF-UHFFFAOYSA-N 17alpha-Hydroxypregnenolone Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(=O)C)(O)C1(C)CC2 JERGUCIJOXJXHF-UHFFFAOYSA-N 0.000 claims description 5
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- AEMFNILZOJDQLW-QEENUVPPSA-N (8R,9S,10R,13S,14S)-10-methyl-13-(113C)methyl-2,6,7,8,9,11,12,14,15,16-decahydro-1H-cyclopenta[a]phenanthrene-3,17-dione Chemical compound [13CH3][13C@@]12[13C](=O)CC[C@H]1[C@@H]1CCC3=CC(=O)CC[C@]3(C)[C@H]1CC2 AEMFNILZOJDQLW-QEENUVPPSA-N 0.000 claims description 3
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- JERGUCIJOXJXHF-TVWVXWENSA-N 17alpha-hydroxypregnenolone Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@@](C(=O)C)(O)[C@@]1(C)CC2 JERGUCIJOXJXHF-TVWVXWENSA-N 0.000 description 2
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- 239000005695 Ammonium acetate Substances 0.000 description 1
- QGXBDMJGAMFCBF-UHFFFAOYSA-N Etiocholanolone Natural products C1C(O)CCC2(C)C3CCC(C)(C(CC4)=O)C4C3CCC21 QGXBDMJGAMFCBF-UHFFFAOYSA-N 0.000 description 1
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- JUPQTSLXMOCDHR-UHFFFAOYSA-N benzene-1,4-diol;bis(4-fluorophenyl)methanone Chemical compound OC1=CC=C(O)C=C1.C1=CC(F)=CC=C1C(=O)C1=CC=C(F)C=C1 JUPQTSLXMOCDHR-UHFFFAOYSA-N 0.000 description 1
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- QGXBDMJGAMFCBF-LUJOEAJASA-N epiandrosterone Chemical compound C1[C@@H](O)CC[C@]2(C)[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CC[C@H]21 QGXBDMJGAMFCBF-LUJOEAJASA-N 0.000 description 1
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- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 description 1
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Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- 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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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Steroid Compounds (AREA)
Abstract
The invention relates to the technical field of hormone detection, in particular to a kit for synchronously detecting 17 steroid hormones in serum without derivatization and application thereof. The kit comprises eluent, diluent, an extracting agent, a quality control product and a chromatographic column, wherein the extracting agent is ethyl acetate/n-hexane; the kit has the advantages of simplicity, convenience, high flux, and remarkably shortened time consumption, and the detection result is better and more accurate; the method for detecting the 17 steroid hormones in the serum by using the ID-LC-MS/MS (high performance liquid chromatography-tandem mass spectrometry) has the advantages that the pretreatment of the sample is simpler and more convenient, the 17 steroid hormones in the sample can be effectively extracted by only one liquid-liquid extraction process, the time is greatly shortened, derivatization and/or solid phase extraction steps are not needed, the 17 steroid hormones existing in a living body can be subjected to baseline separation by optimizing chromatographic conditions, and the quantitative specificity and accuracy are improved.
Description
Technical Field
The invention relates to the technical field of hormone detection, in particular to a kit for synchronously detecting 17 steroid hormones in serum without derivatization and application thereof.
Background
Steroid hormones are tetracyclic aliphatic compounds, which have a cyclopentane-polyhydrophenanthrene mother nucleus and are also called steroid hormones. Has important regulation functions in the aspects of life maintenance, endocrine function regulation, body development, immunoregulation, disease treatment and fertility control. The detection of hormone has been a well-recognized problem in medical laboratory testing projects in the past mainly due to its low blood content and numerous structural analogues, and therefore, the sensitivity and specificity of the detection method are high. Extensive studies on the detection of steroid hormones began in the 70's of the 20 th century. The methods used at the time were mainly Radioimmunoassay (RIA) methods. In recent years, methods for quantitatively detecting serum steroid hormones have been developed, and mainly include an enzyme-linked immunosorbent assay (EIA), an enzyme-linked immunosorbent assay (ELSIA), a chemiluminescence assay (CLIA), a mass spectrometry (gas chromatography-tandem mass spectrometry and liquid chromatography-tandem mass spectrometry), and the like.
RIA has large damage to human body caused by radionuclide and causes environmental pollution, EIA, ELSIA and the like are gradually replaced by new CLIA due to low detection sensitivity, poor precision and low automation degree, and the CLIA method is a conventional detection technology in the current clinical laboratory. However, the method based on antigen-antibody immunoreaction inevitably brings the problem of non-specific reaction, so that the measurement result has obvious systematic error, and simultaneously, the use of reagents of different manufacturers in each laboratory leads to poor result comparability, thereby directly influencing the credibility of the detection result in disease screening. The biochemical diagnosis of endocrine diseases based on mass spectrometry technology is a widely applied and mature method at present. Because the concentration of the steroid hormone molecules in the biological sample is similar to the chromatographic property and the mass spectrum fragment property of the steroid hormone molecules, the specificity detection difficulty is increased. At present, the concentration of steroid hormone in serum is detected and analyzed by adopting a gas chromatography-mass spectrometry (GC-MS) analysis method, but the sensitivity is insufficient, a derivatization method is generally adopted, the pretreatment is complex, and the analysis time is long.
Although a few reports of Isotope-Dilution Liquid Chromatography-tandem Mass spectrometry (ID-LC-MS/MS) are used for detecting steroid hormones in plasma abroad, pretreatment steps such as derivatization and solid phase extraction are still required to separate isomers of the steroid hormones. At present, a corresponding mass spectrum detection kit is not applied to clinical detection, so that the application of the mass spectrum detection kit to the clinical detection is limited.
Therefore, the development of a stable method for detecting steroid hormones based on LC-MS/MS technology and the development of a corresponding detection kit can provide a more accurate and reliable detection method for clinic, and have important significance for improving the diagnosis and treatment level of internal endocrine diseases in China.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a kit for synchronously detecting 17 steroid hormones in serum without derivatization and application thereof.
In order to achieve the purpose, the invention adopts the technical scheme that:
in a first aspect, the invention provides a kit for synchronously detecting 17 steroid hormones in serum without derivatization, which comprises an eluent, a diluent, an extracting agent, a quality control product and a chromatographic column, wherein the extracting agent is ethyl acetate/n-hexane.
The kit of the invention improves the sample pretreatment by the extractant, does not need derivatization and/or solid phase extraction processes, has simpler and faster pretreatment, and can realize batch treatment.
Preferably, the volume ratio of ethyl acetate to n-hexane in the extractant is 0.5-2: more preferably, the volume ratio of ethyl acetate to n-hexane is 1: 1.
Preferably, the eluent comprises eluent A and eluent B, wherein the eluent A is ammonium fluoride aqueous solution, and the eluent B is mass-spectrum pure methanol. Preferably, the concentration of the ammonium fluoride buffer solution is 0.1-1.0mmol/L, and more preferably, the concentration of the ammonium fluoride buffer solution is 0.2 mmol/L.
The diluent is a blank serum substrate, and the quality control product is a solution taking the diluent as a solvent.
Preferably, the quality control product has 3 kinds of QC1, QC2 and QC3 with low, medium and high concentration, wherein the solute is respectively: the concentrations are respectively: testosterone at 0.47, 0.94, 4.93 ng/mL; the concentrations are respectively: 2.39, 15.14, 31.79ng/mL of progesterone; the concentrations are respectively: 17 α -hydroxyprogesterone at 0.49, 1.77, 5.71 ng/mL; the concentrations are respectively: 8.00, 95.65, 126.57ng/mL of cortisol; the concentrations are respectively: aldosterone at a concentration of 0.24, 0.32, 0.49 ng/mL; the concentrations are respectively: 0.13, 0.48, 4.51ng/mL dihydrotestosterone; the concentrations are respectively: pregnenolone 0.49, 7.88, 79.98 ng/mL; the concentrations are respectively: 0.47, 3.11, 6.43ng/mL of corticosterone; the concentrations are respectively: 0.17, 0.74, 6.27ng/mL of 11-deoxycorticosterone; the concentrations are respectively: 0.52, 1.02, 4.87ng/mL of 11-deoxycorticol; the concentrations are respectively: androstenedione at 0.54, 1.25, 5.87 ng/mL; the concentrations are respectively: 0.45, 228.93, 1237.99ng/mL dehydroepiandrosterone; the concentrations are respectively: 1.497, 20.68, 151.94ng/mL cortisone; the concentrations are respectively: 2.16, 12.84, 142.24ng/mL of 17 α -hydroxypregnanolone; the concentrations are respectively: 0.91, 4.84, 10.06ng/mL of 18-hydroxycorticosterone; the concentrations are respectively: 0.80, 4.92, 10.16ng/mL of 21-deoxycorticosterol; the concentrations are respectively: 5.45, 44.08, 108.5ng/mL androstenediol.
Preferably, the kit further comprises a complex solution, a standard substance and an internal standard solution, wherein the complex solution is a mixed solution of methanol and water, and the volume ratio of the methanol to the water is 1: 1;
the standard substance is a mixed solution of 17 steroid hormones and the compound solution, wherein the 17 steroid hormones, namely testosterone, 17 alpha-hydroxyprogesterone, aldosterone, dihydrotestosterone, corticosterone, 11-deoxycorticosterone, 11-deoxycorticosterol, androstenedione, 18-hydroxycorticosterone and 21-deoxycorticosterol are respectively 0.1, 0.5, 1, 2, 5, 10 and 20ng/mL, the progesterone and pregnenolone are respectively 0.25, 1.25, 2.5, 5, 12.5, 25 and 50ng/mL, the 17 alpha-hydroxyprogesterone ketone, cortisone, cortisol and androstenediol are respectively 1.5, 7.5, 15, 30, 75, 150 and 300ng/mL, and the dehydroepiandrosterone is 15, 75, 150, 300, 750, 1500 and 3000 ng/mL;
the internal standard solution is a mixed solution of 15 steroid hormone solutions, and the 15 steroid hormone solutions are testosterone-d prepared by pure methanol317 alpha-hydroxyprogesterone-d8Aldosterone-d7Dihydrotestosterone-d3Corticosterone-d411-deoxycorticosterone-d711-deoxycorticosterol-d5Androstenedione-13C318-Hydroxycorticosterone-d4Progesterone-d9Pregnenolone-20, 21-13C2-16,16-d2, cortisol-d417 alpha-hydroxypregnanolone-d3Cortisone-d8Dehydroepiandrosterone-d5The mixed solution of (1).
Wherein testosterone-d317 alpha-hydroxyprogesterone-d811-deoxycorticosterone-d711-deoxycorticosterol-d5Androstenedione-13C3The concentration of (A) is preferably 20 ng/mL; progesterone-d9The concentration of (A) is preferably 100 ng/mL; dihydrotestosterone-d3Corticosterone-d4The concentration of (A) is preferably 200 ng/mL; cortisol-d4Cortisone-d8The concentration of (A) is preferably 300 ng/mL; aldosterone-d7The concentration of (A) is preferably 600 ng/mL; pregnenolone-20, 21-13C2-16,16-d2The concentration of (A) is preferably 1000 ng/mL; dehydroepiandrosterone-d5The concentration of (A) is preferably 2000 ng/mL; 17 alpha-hydroxypregnenolone-d3The concentration of (A) is preferably 3000 ng/mL;
preferably, the chromatographic column is a Waters acquire
C18, 1.7 μm, 50X 2.1mm column. It is to be noted that the kit of the present invention utilizes Waters ACQUITY
C18, 1.7 mu m, 50X 2.1mm chromatographic column and condition optimization realize effective separation of 17 steroid hormones and structural analogues thereof, and the method has good specificity.
In a second aspect, the invention provides an application of the kit in detecting serum 17 steroid hormones, wherein the application comprises the steps of detecting the serum 17 steroid hormones by adopting isotope dilution liquid chromatography tandem mass spectrometry, and the method comprises the following steps:
(1) sample pretreatment: treating a serum sample by using ethyl acetate/normal hexane, taking a supernatant, carrying out vacuum spin drying, and redissolving a methanol water solution;
(2) and (3) chromatographic separation: loading 10-50 mu L of the sample pretreated in the step (1) to Waters ACQUITY
C18, 1.7 mu m, 50 multiplied by 2.1mm chromatographic column, under the conditions of flow rate of 0.2-0.4 mL/min and column temperature of 45 ℃, eluting with gradient, preferably, eluting for 7 min;
wherein the eluent A is 0.1-1.0mmol/L ammonium fluoride buffer solution, and the eluent B is methanol;
the gradient conditions were as follows: 0-1.0 min, 40% B; 1.0-3.5 min, increasing the phase B from 40% to 55%; 4.5-5.5 min, increasing the phase B from 55% to 98%; reducing the phase B from 98% to 40% in 6.0-6.1 min; 6.1-7.0 min, 40% B;
(3) mass spectrum detection: taking the sample pretreated in the step (1), selecting an ESI positive ion mode for sample loading, and detecting 17 steroid hormones by adopting a multi-reaction monitoring technology, wherein mass spectrum parameters are as follows: the collision gas is 0.2mL/Min, the gas curtain gas is 650L/Hr, and the temperature is 450 ℃; monitoring mass spectrum scanning mode by adopting multiple reactions;
(4) a step of preparing a standard curve by using a series of 17 steroid hormones of concentration standard, wherein a calibration curve is established by taking the concentration ratio of the standard to an internal standard as an X axis and the peak area ratio of the standard to the internal standard as a Y axis, and the content of the 17 steroid hormones in a serum sample is calculated; 17 steroid hormones include testosterone, progesterone, 17 alpha-hydroxyprogesterone, cortisol, aldosterone, dihydrotestosterone, pregnenolone, corticosterone, 11-deoxycorticosterone, 11-deoxycorticosterol, androstenedione, dehydroepiandrosterone, cortisone, 17 alpha-hydroxyprogesterone, 18-hydroxycorticosterone, 21-deoxycorticosterol, androstenediol.
The invention adopts a detection method of 17 hormones and utilizes an ESI positive ion mode; mass spectrum conditions are optimized, and the sensitivity of detection signals is greatly improved; the result of methodology investigation shows that the precision, accuracy and stability of the method all meet the requirements of quantitative analysis.
Preferably, step (1) further comprises adding an internal standard compound selected from testosterone-d before performing pretreatment of the serum sample317 alpha-hydroxyprogesterone-d8Aldosterone-d7Dihydrotestosterone-d3Corticosterone-d411-deoxycorticosterone-d711-deoxycorticosterol-d5Androstenedione-13C318-Hydroxycorticosterone-d4Pregnenolone-20, 21-13C2-16,16-d2, progesterone-d9Cortisol-d417 alpha-hydroxypregnanolone-d 31, canPine-d8Dehydroepiandrosterone-d5。
Preferably, the multiple reaction monitoring technique in step (3) detects the parameters of 17 steroid hormones as follows:
ESI + mode:
testosterone ion pair, m/z is 289.4-97, and collision voltage is 28V;
progesterone ion pair, m/z is 315.4-97, and collision voltage is 45V;
17 alpha-hydroxyprogesterone ion pair, wherein the m/z is 331.4-97, and the collision voltage is 26V;
a cortisol ion pair, m/z is 363-121, and the collision voltage is 45V;
aldosterone ion pair, m/z 361-;
dihydrotestosterone ion pair, m/z is 291.4-255, and collision voltage is 10V;
pregnenolone ion pair, m/z:317.2-281.3, and collision voltage of 13V;
the cortical ketone ion pair has the m/z of 347.3-329, and the collision voltage is 10V;
11-deoxycorticosterone ion pair with m/z of 331.3-97 and collision voltage of 20V;
11-deoxycorticosterol ion pair, m/z of 347.2-97, and collision voltage of 20V;
androstenedione ion pair with m/z of 287.2-97 and collision voltage of 26V;
a dehydroepiandrosterone ion pair with m/z of 289.2-253 and a collision voltage of 10V;
cortisone ion pair, m/z is 361.3-163.1, and collision voltage is 15V;
17 alpha-hydroxypregnenolone ion pair, m/z is 333.3-297.2, and collision voltage is 10V;
18-hydroxycorticosterone ion pair, wherein the m/z is 363-121.1, and the collision voltage is 15V;
21-deoxycorticosterol ion pair with m/z of 347.2-121.1 and collision voltage of 28V;
androstene diol ion pair with m/z of 255.3-159.3 and collision voltage of 25V;
testosterone-d3Ion pair, m/z is 292.2-97, and collision voltage is 28V;
progesterone-d9Ion pair, m/z is 324.4-100, and collision voltage is 27V;
17 alpha-hydroxyprogesterone-d8Ion pair, m/z is 339.4-100.1, and collision voltage is 26V;
cortisol-d4Ion pair, m/z:367- > 122, and the collision voltage is 30V;
aldosterone-d7Ion pair, m/z 368-350, collision voltage 15V;
dihydrotestosterone-d3Ion pair, m/z is 294.4-258.2, and collision voltage is 10V;
pregnenolone-20, 21-13C2-16,16-d2Ion pair, m/z is 321.2-285.3, and collision voltage is 13V;
corticosterone-d4Ion pair, m/z 351-;
11-deoxycorticosterone-d7Ion pair, m/z is 338.4-100, and collision voltage is 26V;
11-deoxycorticosterol-d5Ion pair, m/z is 352.2-100, and collision voltage is 26V;
androstenedione-13C3Ion pair, m/z is 290.2-100, and collision voltage is 26V;
dehydroepiandrosterone-d5Ion pair, m/z is 294.3-258.2, and collision voltage is 14V;
cortisone-d8Ion pair with m/z of 369.3-168.2 and collision voltage of 15V;
17 alpha-hydroxypregnenolone-d3Ion pair, m/z is 336.1-300.1, and collision voltage is 10V;
18-Hydroxycorticosterone-d4Ion pair, m/z is 367 to 121.9, and the collision voltage is 45V;
other general parameters: the taper hole voltage is 25V, the capillary tube voltage is 3kV, the taper hole gas pressure is 150L/Hr, and the atomization gas pressure is 7.0 Bar.
Preferably, the step (2) takes 10 μ L of the pretreated sample to be loaded, and elutes at a flow rate of 0.4 ml/min; preferably, eluent A is 0.2mmol/L ammonium fluoride buffer, and eluent B is mass-purified methanol.
Preferably, the step (1) is specifically operated as follows: taking 200. mu.L of plasma and separating in 15mLAdding 20 μ L testosterone-d into cardiac tube3Progesterone-d917 alpha-hydroxyprogesterone-d8Cortisol-d4Aldosterone-d7Dihydrotestosterone-d3Pregnenolone-20, 21-13C2-16,16-d2Corticosterone-d411-deoxycorticosterone-d711-deoxycorticosterol-d5Androstenedione-13C3Dehydroepiandrosterone-d5Cortisone-d817 alpha-hydroxypregnanolone-d318-Hydroxycorticosterone-d4Internal standard mixture, vortex mixing evenly, then balancing for 20min at room temperature, adding ethyl acetate/n-hexane (50/50, v/v), vortex shaking for 10s, 3 times, centrifuging at 13000rpm for 10min, taking supernatant, vacuum spin drying, and redissolving with 600 μ L50% methanol water solution.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention provides a kit for synchronously detecting 17 steroid hormones in serum by non-derivatization and application thereof, wherein ID-LC-MS/MS (high performance liquid chromatography-tandem mass spectrometry) is adopted to detect the 17 steroid hormones in the serum, the pretreatment of a sample is simpler and more convenient, the 17 steroid hormones in the sample can be effectively extracted by only one-step liquid-liquid extraction process, the time is greatly shortened, derivatization and/or solid phase extraction steps are not needed, the baseline separation of the 17 steroid hormones existing in a living body can be realized by optimizing chromatographic conditions, and the quantitative specificity and accuracy are improved; the inventor can provide a more accurate and reliable detection method for clinic by developing a corresponding detection kit of the ID-LC-MS/MS detection method, thereby improving the detection level of steroid hormones in China;
(2) the kit has the characteristics of high accuracy, good reproducibility, stability and reliability, and can be used for synchronous quantitative detection of 17 steroid hormones in serum;
(3) compared with a GC-MS/MS method, the detection method provided by the invention has the advantages that the sensitivity is improved, the operation is simple, the analysis cost is saved, the required analysis time is short, the detection of a large number of samples is facilitated, and the method is suitable for screening related diseases in crowds; provides a feasible method and a detection kit for the detection of serum steroid hormones in China.
Drawings
FIG. 1 is an LC-MS/MS spectrum for simultaneous non-derivatization detection of 17 steroid hormones in serum using the kit of the present invention, wherein the ordinate (Intensity) represents relative abundance and the abscissa (time) represents time.
Detailed Description
The invention relates to the field of in-vitro detection of biomarkers, in particular to a detection method and a kit for synchronously detecting 17 steroid hormones by adopting an isotope dilution liquid chromatography-tandem mass spectrometry technology, and particularly relates to a special detection method and a kit for synchronously detecting testosterone, progesterone, 17 alpha-hydroxyprogesterone, cortisol, aldosterone, dihydrotestosterone, pregnenolone, corticosterone, 11-deoxycorticosterone, 11-deoxycorticosterol, androstenedione, dehydroepiandrosterone, cortisone, 17 alpha-hydroxyprogesterone, 18-hydroxycorticosterone, 21-deoxycorticosterol and androstenediol in a serum specimen by adopting non-derivatization.
The invention provides a method and a kit for detecting 17 steroid hormones in serum by high performance liquid chromatography-tandem mass spectrometry, which realize the detection of the 17 steroid hormones in the serum by the optimization selection of pretreatment, chromatographic columns, chromatographic conditions, mass spectrometry types and mass spectrometry parameters, particularly realize the synchronous quantitative detection of the 17 steroid hormones in the serum and realize baseline separation of interferents; the method is simple and rapid when being used for detecting the 17 steroid hormones in the serum, has good specificity, precision, accuracy and stability, and can provide a more accurate and reliable detection method for clinic.
In some embodiments, the present invention provides a method for detecting 17 steroid hormones in serum by isotope dilution liquid chromatography tandem mass spectrometry, comprising the steps of:
synchronously detecting 17 steroid hormones in a pretreated serum sample by adopting an isotope dilution liquid chromatography-tandem mass spectrometry technology, separating target hormones from impurities by utilizing an isotope dilution liquid chromatography, quantifying by utilizing an isotope internal standard method, establishing a calibration curve by taking the concentration ratio of a standard substance to an 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 respectively calculating the contents of the 17 steroids.
In some embodiments, the present invention provides a method for detecting 17 steroid hormones in a serum sample, the method comprising using isotope dilution liquid chromatography tandem mass spectrometry to detect 17 steroid hormones in the serum sample, including testosterone, progesterone, 17 α -hydroxyprogesterone, cortisol, aldosterone, dihydrotestosterone, pregnenolone, corticosterone, 11-deoxycorticosterone, 11-deoxycorticosterol, androstenedione, dehydroepiandrosterone, cortisone, 17 α -hydroxyprogesterone, 18-hydroxycorticosterone, 21-deoxycorticosterol, and androstenediol, comprising the steps of:
(1) sample pretreatment: the serum samples were treated with ethyl acetate/n-hexane (50/50, v/v), the supernatant taken, dried by vacuum spin, and redissolved in aqueous methanol.
(2) And (3) chromatographic separation: loading 10-50 μ L of the sample pretreated in step (1) to Waters ACQUITY
C18, 1.7 μm, 50 × 2.1mm chromatographic column, under the conditions of flow rate of 0.4mL/min and column temperature of 45 deg.C, eluting with gradient for 7 min;
wherein the eluent A is methanol, and the eluent B is ammonium fluoride buffer solution containing 0.1-1.0 mmol/L;
the gradient conditions were as follows: 0-1.0 min, 40% B; 1.0-3.5 min, increasing the phase B from 40% to 55%; 4.5-5.5 min, increasing the phase B from 55% to 98%; reducing the phase B from 98% to 40% in 6.0-6.1 min; 6.1-7.0 min, 40% B.
(3) Mass spectrum detection: the ESI positive ion mode is selected.
The mass spectrum parameters were as follows: the collision gas is 0.2mL/Min, the gas curtain gas is 650L/Hr, and the temperature is 450 ℃.
(4) And (3) preparing a standard curve by using the 17 steroid hormones standard products with the series of concentrations, establishing a calibration curve by taking the concentration ratio of the standard product to the internal standard product as an X axis and the peak area ratio of the standard product to the internal standard product as a Y axis, and calculating the content of the 17 steroid hormones in the plasma sample.
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments. Experimental procedures without specific conditions noted in the following examples, generally according to conventional conditions or according to conditions recommended by the manufacturers. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
The instruments, materials and reagents involved in the examples of the present invention are described below:
tandem mass spectrometer (Waters TQ-S4500), high performance liquid phase (Waters Acquity I UPLC). Testosterone, progesterone, 17 α -hydroxyprogesterone, cortisol, aldosterone, pregnenolone, 18-hydroxycorticosterone, pregnenolone-20, 21-13C2-16,16-d2, corticosterone-d 4, 17 α -hydroxyprogesterone-d 3, 18-hydroxycorticosterone-d 4, dehydroepiandrosterone-d 5 standards, and ammonium fluoride are all purchased from Sigma (Shanghai); dihydrotestosterone, corticosterone, androstenedione standards were purchased from dr. 11-deoxycorticosterol, 17 alpha-hydroxypregnanolone, aldosterone-d711-deoxycorticosterone-d7Cortisone-d8The standard was purchased from TRC, canada; testosterone-d3Progesterone-d9Cortisol-d4Dihydrotestosterone-d311-deoxycorticosterol-d5Androstenedione-13C3Standards were purchased from Cerilliant corporation, usa; 17 alpha-hydroxyprogesterone-d8The standard was purchased from C/D/N isotope, Canada; dehydroepiandrosterone standards were purchased from Dalian biologies; cortisone standards were purchased from Bepure, beijing; 11-deoxycorticosterone, 21-deoxycorticosterol standards were purchased from Supelco, USA; androstenediol standards were purchased from the Shanghai Macklin company. Activated carbon adsorbed serum was purchased from Equitech-Bio, Inc. (USA). LC grade ethanol was purchased from j.t.baker, ms grade methanol from Merck, 15-mL centrifuge tubes from Kirgen, volumetric flask from BRAND GMBH + CO KG (germany). Purified water was purified by Millipore silicon UA water purifier.
The preparation method of the standard substance and the internal standard solution adopts the following steps:
(1) preparation of stock solution of standard product
1. Standard stock solution 1(1.0 mg/mL): respectively and accurately weighing 10mg of testosterone, progesterone, 17 alpha-hydroxyprogesterone, cortisol, aldosterone, dihydrotestosterone corticosterone, 11-deoxycorticosterol, androstenedione, dehydroepiandrosterone, cortisone and 17 alpha-hydroxyprogesterone, dissolving the mixture in 8mL of methanol, and fixing the volume by using a 10mL volumetric flask to obtain 1.0mg/mL of stock solution 1;
2. standard stock 2 (100. mu.g/mL): respectively diluting standard stock solutions 1 of progesterone, cortisol, pregnenolone, cortisone and 17 alpha-hydroxypregnenolone by 10 times by using methanol to obtain 100 mu g/mL stock solutions 2;
3. standard stock 3 (20. mu.g/mL): diluting standard stock solutions 1 of testosterone, 17 alpha-hydroxyprogesterone, aldosterone, dihydrotestosterone, corticosterone, 11-deoxycorticosterol, androstenedione by 50 times with methanol respectively; respectively diluting the standard stock solutions 2 of the deoxycorticosterone and the 18-hydroxycorticosterone by 5 times to obtain a stock solution 3 with the concentration of 20 mu g/mL;
(2) standard mixed mother liquor: respectively measuring 400 mu L of standard stock solutions of testosterone, 17 alpha-hydroxyprogesterone, aldosterone, dihydrotestosterone, corticosterone, 11-deoxycorticosterol, androstenedione, deoxycorticosterone and 18-hydroxycorticosterone by using a liquid transfer device, wherein 3 mu L of progesterone and 2 mu L of pregnenolone standard stock solutions; 1200. mu.L cortisol, 17. alpha. -hydroxypregnanolone, 2. mu.L cortisone stock solution, 1200. mu.L dehydroepiandrosterone stock solution 1, in a 20mL volumetric flask, with a 50% methanol water volume.
(3) Preparation of standard curve
The standard mixed mother liquor was diluted with 50% methanol water to 7 calibrators of different concentrations: testosterone, 17 α -hydroxyprogesterone, aldosterone, dihydrotestosterone, corticosterone, 11-deoxycorticosterone, 11-deoxycorticosterol, androstenedione, 18-hydroxycorticosterone, 21-deoxycorticosterol at 0.1, 0.5, 1, 2, 5, 10 and 20ng/mL, progesterone and pregnenolone at 0.25, 1.25, 2.5, 5, 12.5, 25 and 50ng/mL, 17 α -hydroxyprogesterone, cortisone, cortisol, androstenediol at 1.5, 7.5, 15, 30, 75, 150 and 300ng/mL, dehydroepiandrosterone at 15, 75, 150, 300, 750, 1500 and 3000ng/mL, respectively; the calibrator with different concentrations is respectively 20mL, and is subpackaged and stored at-20 ℃.
(4) Preparation of 15 internal standard mixed working solutions
Preparing 15 internal standard mixed working solutions according to the preparation steps of the standard solution: wherein testosterone-d317 alpha-hydroxyprogesterone-d811-deoxycorticosterone-d711-deoxycorticosterol-d5Androstenedione-13C3The final concentration of (a) is 20 ng/mL; progesterone-d9The final concentration of (a) is 100 ng/mL; dihydrotestosterone-d3Corticosterone-d4The final concentration of (a) is 200 ng/mL; cortisol-d4Cortisone-d8The final concentration of (a) is 300 ng/mL; aldosterone-d7The final concentration of (2) is 600 ng/mL; pregnenolone-20, 21-13C2-16,16-d2The final concentration of (a) is 1000 ng/mL; dehydroepiandrosterone-d5The final concentration of (a) is 2000 ng/mL; 17 alpha-hydroxypregnenolone-d3The final concentration of (2) was 3000 ng/mL.
Example 1 optimization of assay conditions
(1) Optimization of chromatographic conditions: chromatographic column selection
Use of PEEK in preliminary experiments
zwitterionic column,3.5 μm, 100mm × 2.1mm column; phenomenex Kinetex-C18, 2.6 μm, 100X 2.1mm column; saimer Feishale Hypersil gold, 5 μm, 50X 2.1mm column; waters ACQUITY
C18, 1.7 μm, 50X 2.1mm column, etc. 4 different chromatographic columns were used for sample analysis.
The results show that Waters ACQUITY
A C18, 1.7 μm, 50X 2.1mm column was best for the isolation of 17 steroid hormones and their structural analogues. The other three LC columns had poor separation. Waters ACQUITY
C18, 1.7 mu m, 50 multiplied by 2.1mm has better retention and higher column efficiency on 17 target compounds, and can separate 17 hormones and isomers thereof, thus being beneficial to improving the specificity and sensitivity of detection, and therefore, the method is selected as a chromatographic column of the method.
(2) And (3) sample injection quantity selection:
comparing the sample volumes of 5, 10, 20, 25, 40 and 50 μ L, it is shown that high sample volumes result in solvent effects at the sample peak, leading to poor peak shape and even to a split peak. Too low a sample size may result in reduced detection sensitivity. When 10 mu L of sample is injected, the peak shape is good, and the sensitivity can also meet the detection requirement. Therefore, a sample size of 10. mu.L was selected for the experiment.
(3) Kinds and concentrations of buffer salts:
by comparing ammonium formate (0.2mmol/L), ammonium acetate (0.2mmol/L) and ammonium fluoride (0.2 mmol/L). The analyte peaks were comparable in width, compared in peak height.
The results show that ammonium fluoride with proper concentration can improve the ionization efficiency. Ammonium fluoride was therefore chosen as the buffer salt for the analysis.
Respectively preparing ammonium fluoride buffer solutions with different concentrations: 0.02, 0.05, 0.1, 0.2 and 0.5mmol/L, and samples were injected with 1.0ng/mL standard, and the peak response values and S/N were compared at different ammonium fluoride concentrations.
The results showed that the response and S/N were maximum when the concentration of ammonium fluoride was 0.2 mmol/L. Therefore, this concentration was chosen to be performed as the buffer salt concentration.
(4) Gradient of mobile phase:
the determination of the mobile phase gradient is obtained under the conditions of proper sample retention, good chromatographic peak shape and good chromatographic separation capacity, and under the condition that the flow rate is 0.4mL/min, the gradient elution mode shown in the table 2 is adopted, the elution is carried out for 7min, the target substance has good peak shape, the separation effect is good, and the peak-appearance time is relatively fast, so that the condition is selected for elution.
(5) Optimizing mass spectrum conditions:
the determination of the mass spectrometric conditions is obtained according to the following steps: the ion responses of the same concentration of analyte in both the Atmospheric Pressure Chemical Ionization (APCI) and electrospray ionization (ESI) ionization modes were compared and the ESI mode was selected, with the response found to be high in the ESI mode. Then, the positive ion mode response of 17 steroid hormones was found to be high by comparing the positive and negative ion intensities in the ESI mode, so that the ESI positive ion mode was selected.
After the ion mode is determined, optimizing the conditions of a multi-reaction monitor (MRM), and specifically comprising the following steps: determining the parent ion of the analyte according to the molecular weight of the analyte, and performing parent ion scanning by injecting standard solution by using a needle pump of 7 mu L/min under an ESI positive ion mode, wherein the scanning range is as follows: 50-500Da, scanning speed: 200 Da/s. The analyte parent ion is looked for in the pattern and then the particular parent ion is selected for selection of the daughter ion. The selection of the daughter ions is obtained according to the following steps: selecting specific parent ions and then scanning the child ions, wherein the scanning range is as follows: 50-500Da, scanning speed: 200 Da/s. CE was adjusted to find the highest responding daughter ion. And (4) after the parent ions and the daughter ions are determined, optimizing mass spectrum parameters and MRM conditions.
(6) Optimization of mass spectrometry parameters and MRM conditions requires optimization of the following parameters: capillary voltage (Capillary pressures), Cone voltage (Cone pressures), atomization temperature (atomization temperature), atomization Gas Flow rate (atomization Gas Flow), Cone Gas Flow rate (Cone Gas Flow), atomization Gas pressure (Nebuliser), collisional Gas Flow rate (Collision Gas Flow), residence time (Dwell time).
The mass spectrum condition is that a mass spectrum scanning mode of multi-reaction monitoring (MRM) is adopted under an electrospray ionization (ESI) positive ion detection mode; the capillary voltage is 3.00 kV; the voltage of the taper hole is 30V; the atomization temperature is 450 ℃; flow rate of atomizing gas: 650L/Hr; taper hole air flow velocity: 150L/Hr; atomizing air pressure: 7.0Bar, impinging air flow rate: 0.2 mL/Min; the residence time is: 0.008 s; the parent-child ion mass-to-charge ratio (m/z) and the collision voltage parameters of the target 17 steroid hormone and the internal standard are shown in Table 1.
TABLE 117 Mass Spectrometry parameters for steroid hormones and their internal standards
Example 2A method for detecting 17 steroid hormones in a serum sample
The invention adopts an isotope dilution liquid chromatography tandem mass spectrometry method to detect 17 steroid hormones in a serum sample, wherein the hormones comprise testosterone, progesterone, 17 alpha-hydroxyprogesterone, cortisol, aldosterone, dihydrotestosterone, pregnenolone, corticosterone, 11-deoxycorticosterone, 11-deoxycorticosterol, androstenedione, dehydroepiandrosterone, cortisone, 17 alpha-hydroxyprogesterone, 18-hydroxycorticosterone, 21-deoxycorticosterol and androstenediol, and the method comprises the following steps:
(1) sample pretreatment: treating a serum sample with ethyl acetate/n-hexane (50/50, v/v), performing liquid-liquid extraction, taking a supernatant, performing vacuum spin-drying (spin-drying temperature is 40-50 ℃), and redissolving with a methanol aqueous solution.
(2) LC chromatographic separation: loading 10 μ L of the pretreated sample from step (1) to Waters ACQUITY
C18, 1.7 μm, 50 × 2.1mm chromatographic column, under the conditions of flow rate of 0.4mL/min and column temperature of 45 deg.C, eluting with gradient for 7 min;
wherein the eluent A is ammonium fluoride buffer solution containing 0.2mmol/L, and the eluent B is methanol;
the gradient conditions were as follows: 0-1.0 min, 40% B; 1.0-3.5 min, increasing the phase B from 40% to 55%; 4.5-5.5 min, increasing the phase B from 55% to 98%; reducing the phase B from 98% to 40% in 6.0-6.1 min; 6.1-7.0 min, 40% B.
(3) Mass spectrum detection: the ESI positive ion mode is selected.
The mass spectrum parameters were as follows: the collision gas is 0.2mL/Min, the gas curtain gas is 650L/Hr, and the temperature is 450 ℃.
(4) And (3) preparing a standard curve by using the 17 steroid hormones standard products with the series of concentrations, establishing a calibration curve by taking the concentration ratio of the standard product to the internal standard product as an X axis and the peak area ratio of the standard product to the internal standard product as a Y axis, and calculating the content of the 17 steroid hormones in the plasma sample.
Wherein, the step (1) also comprises adding testosterone-d before the pretreatment of the serum sample317 alpha-hydroxyprogesterone-d8Aldosterone-d7Dihydrotestosterone-d3Corticosterone-d411-deoxycorticosterone-d711-deoxycorticosterol-d5Androstenedione-13C318-Hydroxycorticosterone-d4Pregnenolone-20, 21-13C2-16,16-d2Progesterone-d9Cortisol-d417 alpha-hydroxypregnanolone-d3Cortisone-d8Dehydroepiandrosterone-d5Manipulation of the internal standard compound.
The step (1) is specifically operated as follows: 200 μ L of plasma was placed in a 15mL centrifuge tube and 20 μ L,20ng/mL testosterone-d was added317 alpha-hydroxyprogesterone-d811-deoxycorticosterone-d711-deoxycorticosterol-d5Androstenedione-13C3100ng/mL progesterone-d9200ng/mL dihydrotestosterone-d3Corticosterone-d4300ng/mL cortisol-d4Cortisone-d8600ng/mL aldosterone-d71000ng/mL pregnenolone-20, 21-13C2-16,16-d22000ng/mL dehydroepiandrosterone-d53000ng/mL of 17 alpha-hydroxypregnanolone-d3Internal standard compound, balance 20min at room temperature after vortex mixing, add ethyl acetate/n-hexane (50/50, v/v), vortex at room temperature 10s, 3 times, centrifuge at 13000rpm for 10min, take the supernatant, vacuum spin dry, redissolve with 600 μ L50% methanol water solution.
The parameters of the steroid hormones in the multiple reaction detector (MRM)17 in step (3) are as follows:
testosterone ion pair, m/z is 289.4-97, and collision voltage is 28V;
progesterone ion pair, m/z is 315.4-97, and collision voltage is 45V;
17 alpha-hydroxyprogesterone ion pair, wherein m/z is 331.4-97, and collision voltage is 26V;
a cortisol ion pair, m/z is 363-121, and the collision voltage is 45V;
aldosterone ion pair, m/z 361-;
dihydrotestosterone ion pair, m/z is 291.4-255, and collision voltage is 10V;
pregnenolone ion pair, m/z:317.2-281.3, and collision voltage of 13V;
the cortical ketone ion pair has the m/z of 347.3-329, and the collision voltage is 10V;
11-deoxycorticosterone ion pair with m/z of 331.3-97 and collision voltage of 20V;
11-deoxycorticosterol ion pair, m/z of 347.2-97, and collision voltage of 20V;
androstenedione ion pair with m/z of 287.2-97 and collision voltage of 26V;
a dehydroepiandrosterone ion pair with m/z of 289.2-253 and a collision voltage of 10V;
cortisone ion pair, m/z is 361.3-163.1, and collision voltage is 15V;
17 alpha-hydroxypregnenolone ion pair, m/z is 333.3-297.2, and collision voltage is 10V;
18-hydroxycorticosterone ion pair with m/z of 363-121.1 and collision voltage of 15V;
21-deoxycorticosterol ion pair with m/z of 347.2-121.1 and collision voltage of 28V;
androstene diol ion pair with m/z of 255.3-159.3 and collision voltage of 25V;
testosterone-d3Ion pair, m/z is 292.2-97, and collision voltage is 28V;
progesterone-d9Ion pair, m/z is 324.4-100, and collision voltage is 27V;
17 alpha-hydroxyprogesterone-d8Ion pair, m/z is 339.4-100.1, and collision voltage is 26V;
cortisol-d4Ion pair, m/z:367- > 122, and the collision voltage is 30V;
aldosterone-d7Ion pair, m/z 368-350, collision voltage 15V;
dihydrotestosterone-d3Ion pair, m/z 294.4-258.2, the collision voltage is 10V;
pregnenolone-20, 21-13C2-16,16-d2Ion pair, m/z is 321.2-285.3, and collision voltage is 13V;
corticosterone-d4Ion pair, m/z 351-;
11-deoxycorticosterone-d7Ion pair, m/z is 338.4-100, and collision voltage is 26V;
11-deoxycorticosterol-d5Ion pair, m/z is 352.2-100, and collision voltage is 26V;
androstenedione-13C3Ion pair, m/z is 290.2-100, and collision voltage is 26V;
dehydroepiandrosterone-d5Ion pair, m/z is 294.3-258.2, and collision voltage is 14V;
cortisone-d8Ion pair, m/z is 369.3-168.2, and collision voltage is 15V;
17 alpha-hydroxypregnenolone-d3Ion pair, m/z is 336.1-300.1, and collision voltage is 10V;
18-Hydroxycorticosterone-d4Ion pair, m/z is 367 to 121.9, and the collision voltage is 45V; .
Wherein, the mass spectrum conditions are as follows:
adopting a mass spectrum scanning mode of multi-reaction monitoring (MRM) in an electrospray ionization positive ion detection mode; the capillary voltage is 3.00 kV; the voltage of the taper hole is 30V; the atomization temperature is 450 ℃; flow rate of atomizing gas: 650L/Hr; taper hole air flow rate: 150L/Hr; atomizing air pressure: 7.0Bar, impinging air flow rate: 0.2 mL/Min; the residence time is: 0.008 s; the parent-child ion mass-to-charge ratio (m/z) and the collision voltage parameters of the target 17 steroid hormone and the internal standard are shown in Table 1.
The chromatographic conditions are as follows:
mobile phase A: 0.2mmol/L ammonium fluoride aqueous solution;
mobile phase B: methanol with purity of mass spectrometry;
the type of the chromatographic column: waters ACQUITY
C18，1.7μm，50×2.1mm；
Eluting for 7min by gradient elution, wherein the gradient conditions are shown in Table 2;
TABLE 217 gradient conditions for steroid hormone detection
Example 3 kit for simultaneous non-derivatization detection of 17 steroid hormones in serum
The invention provides a kit for synchronously detecting 17 steroid hormones in serum without derivatization, which comprises the following specific components shown in Table 3,
the method for detecting the 17 steroid hormones in the serum sample by adopting an isotope dilution liquid chromatography tandem mass spectrometry technology comprises the following reagents:
(1) eluent:
eluent A: 0.2mmol/L ammonium fluoride aqueous solution;
eluent B: methanol, the purity is mass spectrum purity;
(2) and (3) standard substance:
the preparation of the standard substance mainly comprises the preparation of mother liquor of each standard substance, mixed mother liquor and mixed working solution of the standard substance:
standard mixed mother liquor: 17 steroid hormone in 50% aqueous methanol;
standard mixed working solution: 7 concentrations of 17 steroid hormones in 50% methanol in water;
the preparation method comprises the following steps:
1) standard stock solution 1(1.0 mg/mL): respectively and accurately weighing 10mg of testosterone, progesterone, 17 alpha-hydroxyprogesterone, cortisol, aldosterone, dihydrotestosterone corticosterone, 11-deoxycorticosterol, androstenedione, dehydroepiandrosterone, cortisone and 17 alpha-hydroxyprogesterone, dissolving the mixture in 8mL of methanol, and fixing the volume by using a 10mL volumetric flask to obtain 1.0mg/mL of stock solution 1;
2) standard stock 2 (100. mu.g/mL): respectively diluting standard stock solutions 1 of progesterone, cortisol, pregnenolone, cortisone and 17 alpha-hydroxypregnenolone by 10 times by using methanol to obtain 100 mu g/mL stock solutions 2;
3) standard stock 3 (20. mu.g/mL): diluting standard stock solutions 1 of testosterone, 17 alpha-hydroxyprogesterone, aldosterone, dihydrotestosterone, corticosterone, 11-deoxycorticosterol, androstenedione by 50 times with methanol respectively; respectively diluting the standard stock solutions 2 of the deoxycorticosterone and the 18-hydroxycorticosterone by 5 times to obtain a stock solution 3 with the concentration of 20 mu g/mL;
4) standard mixed mother liquor: respectively measuring 400 mu L of standard stock solutions of testosterone, 17 alpha-hydroxyprogesterone, aldosterone, dihydrotestosterone, corticosterone, 11-deoxycorticosterol, androstenedione, deoxycorticosterone and 18-hydroxycorticosterone by using a liquid transfer device, wherein 3 mu L of progesterone and 2 mu L of pregnenolone standard stock solutions; 1200. mu.L cortisol, 17. alpha. -hydroxypregnanolone, 2. mu.L cortisone stock solution, 1200. mu.L dehydroepiandrosterone stock solution 1, in a 20mL volumetric flask, with a 50% methanol water volume.
5) Standard mixed working solution: the standard mixed mother liquor was diluted with 50% methanol water to 7 calibrators of different concentrations: concentrations of testosterone, 17 alpha-hydroxyprogesterone, aldosterone, dihydrotestosterone, corticosterone, 11-deoxycorticosterone, 11-deoxycorticosterol, androstenedione, 18-hydroxycorticosterone, 21-deoxycorticosterol were 0.1, 0.5, 1, 2, 5, 10 and 20ng/mL, respectively, concentrations of progesterone and pregnenolone were 0.25, 1.25, 2.5, 5, 12.5, 25 and 50ng/mL, concentrations of 17 alpha-hydroxyprogesterone, cortisone, cortisol, androstenediol were 1.5, 7.5, 15, 30, 75, 150 and 300ng/mL, respectively, and concentrations of dehydroepiandrosterone were 15, 75, 150, 300, 750, 1500 and 3000 ng/mL.
The calibrator with different concentrations is respectively 20mL, and is subpackaged and stored at-20 ℃.
(3) Internal standard solution: 15 internal standard mixed solutions;
preparing 15 internal standard mixed working solutions according to the preparation steps of the standard solution: wherein testosterone-d317 alpha-hydroxyprogesterone-d811-deoxycorticosterone-d711-deoxycorticosterol-d5Androstenedione-13C3The final concentration of (a) is 20 ng/mL; progesterone-d9The final concentration of (a) is 100 ng/mL; dihydrotestosterone-d3Corticosterone-d4The final concentration of (a) is 200 ng/mL; cortisol-d4Cortisone-d8Final concentration ofThe degree is 300 ng/mL; aldosterone-d7The final concentration of (a) is 600 ng/mL; pregnenolone-20, 21-13C2-16,16-d2The final concentration of (a) is 1000 ng/mL; dehydroepiandrosterone-d5The final concentration of (a) is 2000 ng/mL; 17 alpha-hydroxypregnenolone-d3The final concentration of (2) was 3000 ng/mL.
(4) Liquid-liquid extracting agent: ethyl acetate/n-hexane (50/50, v/v).
(5) Quality control product: 17 steroid hormone serum matrix solution. And (3) respectively preparing low-concentration and medium-concentration QC1, QC2 and QC3 by using the diluent, wherein the concentration of testosterone is as follows: 0.47, 0.94 and 4.93 ng/mL; the concentrations of progesterone were: 2.39, 15.14, 31.79 ng/mL; the concentrations of 17 α -hydroxyprogesterone were: 0.49, 1.77, 5.71 ng/mL; the concentrations of cortisol were: 8.00, 95.65, 126.57 ng/mL; the concentrations of aldosterone were: 0.24, 0.32, 0.49 ng/mL; the concentrations of dihydrotestosterone were: 0.13, 0.48, 4.51 ng/mL; the concentrations of pregnenolone were: 0.49, 7.88, 79.98 ng/mL; the concentrations of corticosterone were: 0.47, 3.11, 6.43 ng/mL; the concentrations of 11-deoxycorticosterone were: 0.17, 0.74, 6.27 ng/mL; the concentrations of 11-deoxycorticosterol were: 0.52, 1.02, 4.87 ng/mL; the concentrations of androstenedione were: 0.54, 1.25, 5.87 ng/mL; the concentrations of dehydroepiandrosterone were: 0.45, 228.93, 1237.99 ng/mL; the concentrations of cortisone were: 1.497, 20.68, 151.94 ng/mL; the concentrations of 17 alpha-hydroxypregnanolone are respectively: 2.16, 12.84, 142.24 ng/mL; the concentrations of 18-hydroxycorticosterone were: 0.91, 4.84, 10.06 ng/mL; the concentrations of 21-deoxycorticosterol were: 0.80, 4.92 and 10.16 ng/mL; the concentrations of androstenediol were: 5.45, 44.08 and 108.5 ng/mL.
(6) Diluting liquid: the diluent is a blank serum matrix solution, and the blank serum matrix solution is human serum adsorbed by activated carbon.
TABLE 3 kit Components for the detection of 17 steroid hormones in serum
Example 4 Effect verification of kit
The MRM monitoring chromatogram obtained using the isotope dilution liquid chromatography tandem mass spectrometry method of steroid hormone of example 217 and the kit of example 3 is shown in fig. 1: the 17 steroid hormones, the internal standard substance and the plasma sample have symmetrical peak shapes and basically have no impurity interference, and can realize baseline separation. Indicating that the conditions can be used for quantitative analysis of serum 17 steroid hormones.
1. Determination of the steroid hormone content: the calibration curve adopts an isotope internal standard quantitative method, and utilizes Analyst software to establish a curve by taking the concentration ratio of the standard substance to the internal standard as an X axis and the peak area ratio of the standard substance to the internal standard as a Y axis and calculate the concentration of various steroid hormones in plasma; testosterone, 17 alpha-hydroxyprogesterone, aldosterone, dihydrotestosterone, corticosterone, 11-deoxycorticosterone, 11-deoxycorticosterol, androstenedione, 18-hydroxycorticosterone and 21-deoxycorticosterol are 0.1-20 ng/mL, progesterone and pregnenolone are 0.25-50 ng/mL, 17 alpha-hydroxyprogesterone, cortisone, cortisol and androstenediol are 1.5-300 ng/mL, dehydroepiandrosterone is better linear in the range of 15-3000 ng/mL, the correlation coefficient is above 0.99, and the quantitative requirement is met.
2. Detection sensitivity: the kit of example 3 had a limit of detection (LOD) for testosterone of 0.01ng/mL and a limit of quantitation (LOQ) of 0.05 ng/mL; the limit of detection (LOD) for progesterone is: 0.02ng/mL, limit of quantitation (LOQ) is 0.1 ng/mL; the detection Limit (LOD) of 17 alpha-hydroxyprogesterone is 0.01ng/mL, and the quantification Limit (LOQ) is 0.05 ng/mL; the limit of detection (LOD) for cortisol is: 0.2ng/mL, limit of quantitation (LOQ) is 1.0 ng/mL; the limit of detection (LOD) of aldosterone is 0.01ng/mL, and the limit of quantitation (LOQ) is 0.05 ng/mL; the limit of detection (LOD) for dihydrotestosterone is: 0.01ng/mL, limit of quantitation (LOQ) is 0.05 ng/mL; the detection Limit (LOD) of pregnenolone is 0.2ng/mL, and the quantification Limit (LOQ) is 1.0 ng/mL; the limit of detection (LOD) for corticosterone was: 0.01ng/mL, limit of quantitation (LOQ) is 0.05 ng/mL; the limit of detection (LOD) of 11-deoxycorticosterone is 0.01ng/mL, and the limit of quantitation (LOQ) is 0.05 ng/mL; the limit of detection (LOD) for 11-deoxycorticosterol is: 0.01ng/mL, limit of quantitation (LOQ) is 0.05 ng/mL; the detection Limit (LOD) of androstenedione is 0.01ng/mL, and the quantification Limit (LOQ) is 0.05 ng/mL; the limit of detection (LOD) for dehydroepiandrosterone was: 4.0ng/mL, limit of quantitation (LOQ) is 15 ng/mL; the detection Limit (LOD) of cortisone is 0.1ng/mL, and the quantification Limit (LOQ) is 1.0 ng/mL; the limit of detection (LOD) for 17 alpha-hydroxypregnanolone is: 0.5ng/mL, limit of quantitation (LOQ) is 1.0 ng/mL; the limit of detection (LOD) of 18-hydroxycorticosterone is 0.05ng/mL, and the limit of quantitation (LOQ) is 0.1 ng/mL; the limit of detection (LOD) for 21-deoxycorticosterol is: 0.05ng/mL, limit of quantitation (LOQ) is 0.1 ng/mL; the limit of detection (LOD) of androstenediol is 1.0ng/mL, and the limit of quantitation (LOQ) is 1.5 ng/mL; . The LOD satisfies signal-to-noise ratio (S/N) >3, LOQ satisfies signal-to-noise ratio (S/N) >10, and detection CVs < 20% is repeated 4 times.
3. And (3) precision test: taking 200 mu L of 17 steroid hormone quality control products with low, medium and high concentrations, wherein the concentrations of testosterone are respectively as follows: 0.47, 0.94 and 4.93 ng/mL; the concentrations of progesterone were: 2.39, 15.14, 31.79 ng/mL; the concentrations of 17 α -hydroxyprogesterone were: 0.49, 1.77, 5.71 ng/mL; the concentrations of cortisol were: 8.00, 95.65, 126.57 ng/mL; the concentrations of aldosterone were: 0.24, 0.32, 0.49 ng/mL; the concentrations of dihydrotestosterone were: 0.13, 0.48, 4.51 ng/mL; the concentrations of pregnenolone were: 0.49, 7.88, 79.98 ng/mL; the concentrations of corticosterone were: 0.47, 3.11, 6.43 ng/mL; the concentrations of 11-deoxycorticosterone were: 0.17, 0.74, 6.27 ng/mL; the concentrations of 11-deoxycorticosterol were: 0.52, 1.02, 4.87 ng/mL; the concentrations of androstenedione were: 0.54, 1.25, 5.87 ng/mL; the concentrations of dehydroepiandrosterone were: 0.45, 228.93, 1237.99 ng/mL; the concentrations of cortisone were: 1.497, 20.68, 151.94 ng/mL; the concentrations of 17 alpha-hydroxypregnanolone are respectively: 2.16, 12.84, 142.24 ng/mL; the concentrations of 18-hydroxycorticosterone were: 0.91, 4.84, 10.06 ng/mL; the concentrations of 21-deoxycorticosterol were: 0.80, 4.92 and 10.16 ng/mL; the concentrations of androstenediol were: 5.45, 44.08, 108.5 ng/mL; the batch precision is 10 replicates per sample. The batch-to-batch precision was measured 5 times per batch, and a standard curve was plotted for each batch for 3 consecutive days for 15 measurements. Result in-batch precision (n ═ 10) CV: 0.48% -9.93%; batch-to-batch precision (n ═ 15) CV: 0.92% to 9.49%.
4. Recovery rate test: taking 200 mu L serum blank matrix, and detecting that the concentration of 17 steroid hormones in the blank matrix is lower than the detection limit of each hormone; adding testosterone, 17 alpha-hydroxyprogesterone, aldosterone, dihydrotestosterone, corticosterone, 11-deoxycorticosterone, androstenedione, 18-hydroxycorticosterone standard solutions to concentrations of 0.5, 2.0 and 8.0ng/mL, progesterone standard solutions to concentrations of 2.5, 10.0 and 40.0ng/mL, cortisol, pregnenolone standard solutions to concentrations of 7.5, 30.0 and 120.0ng/mL, cortisone standard solutions to concentrations of 1.5, 30.0 and 150.0ng/mL, 17 alpha-hydroxyprogesterone standard solutions to concentrations of 1.5, 15.0 and 150.0ng/mL, androstenediol standard solutions to concentrations of 5.0, 30.0 and 100.0ng/mL, 21-deoxycorticosterol standard solutions to concentrations of 0.4, 2.0 and 10.0ng/mL, epiandrosterone standard solutions to concentrations of 1.5, 15.0 and 150.0ng/mL, 3 samples per sample, respectively, to a blank serum matrix, the operation was repeated 3 times.
The result shows that the standard recovery rate of testosterone in the serum matrix is between 93.2 and 97.5 percent; the standard recovery rate of the progesterone is between 94.1 and 101.6 percent; the recovery rate of the 17 alpha-hydroxyprogesterone is between 91.7% and 100.4%; the standard recovery rate of the cortisol is between 99.8 and 109.8 percent; the standard recovery rate of aldosterone is between 96.2 and 104.7 percent; the standard recovery rate of the dihydrotestosterone is between 96.2 and 99.8 percent; the standard recovery rate of pregnenolone is 92.8-99.8%; the standard recovery rate of corticosterone is between 94.3 and 97.5 percent; the recovery rate of the 11-deoxycorticosterone by adding standard is between 97.0 and 109.0 percent; the recovery rate of the 11-deoxycorticosterol by adding standard is between 96.5 and 104.8 percent; the recovery rate of androstenedione is 95.6-109.2%; the recovery rate of dehydroepiandrosterone is 99.0-108.5%; the recovery rate of the cortisone by adding standard is 92.3-101.3%; the standard recovery rate of the 17 alpha-hydroxypregnanolone is 94.9-106.7%; the recovery rate of androstenediol is 96.3-108.9%; the recovery rate of the 18-hydroxycorticosterone is between 94.4 and 108.9 percent; the recovery rate of the 21-deoxycorticosterol by adding standard is between 92.5 and 102.0 percent.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. A kit for synchronously detecting 17 steroid hormones in serum without derivatization is characterized by comprising an eluent, a diluent, an extracting agent, a quality control product and a chromatographic column, wherein the extracting agent is ethyl acetate/n-hexane.
2. The kit of claim 1, wherein the volume ratio of ethyl acetate to n-hexane in the extractant is (0.5-2): more preferably, the volume ratio of ethyl acetate to n-hexane is 1: 1.
3. The kit of claim 1, wherein the eluent comprises eluent A and eluent B, wherein the eluent A is an aqueous ammonium fluoride solution and the eluent B is mass-purified methanol.
4. The kit according to claim 1, wherein the diluent is a blank serum substrate and the quality control substance is a solution using the diluent as a solvent.
5. The kit of claim 4, wherein the quality control product comprises 3 kinds of QC1, QC2 and QC3 with low concentration and medium concentration, wherein the solute is: the concentrations are respectively: testosterone at 0.47, 0.94, 4.93 ng/mL; the concentrations are respectively: 2.39, 15.14, 31.79ng/mL of progesterone; the concentrations are respectively: 17 α -hydroxyprogesterone at 0.49, 1.77, 5.71 ng/mL; the concentrations are respectively: 8.00, 95.65, 126.57ng/mL of cortisol; the concentrations are respectively: aldosterone at a concentration of 0.24, 0.32, 0.49 ng/mL; the concentrations are respectively: 0.13, 0.48, 4.51ng/mL dihydrotestosterone; the concentrations are respectively: pregnenolone 0.49, 7.88, 79.98 ng/mL; the concentrations are respectively: 0.47, 3.11, 6.43ng/mL of corticosterone; the concentrations are respectively: 0.17, 0.74, 6.27ng/mL of 11-deoxycorticosterone; the concentrations are respectively: 0.52, 1.02, 4.87ng/mL of 11-deoxycorticol; the concentrations are respectively: androstenedione at 0.54, 1.25, 5.87 ng/mL; the concentrations are respectively: 0.45, 228.93, 1237.99ng/mL dehydroepiandrosterone; the concentrations are respectively: 1.497, 20.68, 151.94ng/mL cortisone; the concentrations are respectively: 2.16, 12.84, 142.24ng/mL of 17 α -hydroxypregnanolone; the concentrations are respectively: 0.91, 4.84, 10.06ng/mL of 18-hydroxycorticosterone; the concentrations are respectively: 0.80, 4.92, 10.16ng/mL of 21-deoxycorticosterol; the concentrations are respectively: 5.45, 44.08, 108.5ng/mL androstenediol.
6. The kit of claim 1, further comprising a double solution, a standard substance and an internal standard solution, wherein the double solution is a mixed solution of methanol and water, and the volume ratio of methanol to water is 1: 1;
the standard substance is a mixed solution of 17 steroid hormones and the compound solution, wherein the 17 steroid hormones, namely testosterone, 17 alpha-hydroxyprogesterone, aldosterone, dihydrotestosterone, corticosterone, 11-deoxycorticosterone, 11-deoxycorticosterol, androstenedione, 18-hydroxycorticosterone and 21-deoxycorticosterol are respectively 0.1, 0.5, 1, 2, 5, 10 and 20ng/mL, the progesterone and pregnenolone are respectively 0.25, 1.25, 2.5, 5, 12.5, 25 and 50ng/mL, the 17 alpha-hydroxyprogesterone ketone, cortisone, cortisol and androstenediol are respectively 1.5, 7.5, 15, 30, 75, 150 and 300ng/mL, and the dehydroepiandrosterone is 15, 75, 150, 300, 750, 1500 and 3000 ng/mL;
the internal standard solution is a mixed solution of 15 steroid hormone solutions, and the 15 steroid hormone solutions are testosterone-d prepared by pure methanol317 alpha-hydroxyprogesterone-d8Aldosterone-d7Dihydrotestosterone-d3Corticosterone-d411-deoxycorticosterone-d711-deoxycorticosterol-d5Androstenedione-13C318-Hydroxycorticosterone-d4Progesterone-d9Pregnenolone-20, 21-13C2-16,16-d2Cortisol-d417 alpha-hydroxypregnanolone-d3Cortisone-d8Dehydroepiandrosterone-d5The mixed solution of (1).
7. The kit of claim 1, wherein the chromatography column is a Waters acquisition
BEH C18，1.7μm，50×2.1mm。
8. Use of a kit according to any one of claims 1 to 7 for the detection of serum 17 steroid hormones, said use comprising the detection of serum 17 steroid hormones by isotope dilution liquid chromatography tandem mass spectrometry, comprising the steps of:
(1) sample pretreatment: treating a serum sample with ethyl acetate/n-hexane, taking a supernatant, carrying out vacuum spin drying, and re-dissolving with a methanol aqueous solution;
(2) and (3) chromatographic separation: loading 10-50 mu L of the sample pretreated in the step (1) to a chromatographic column, and performing gradient elution at the flow rate of 0.2-0.4 mL/min and the column temperature of 45 ℃;
wherein the eluent A is 0.1-1.0mmol/L ammonium fluoride buffer solution, and the eluent B is methanol;
the gradient conditions were as follows: 0-1.0 min, 40% B; 1.0-3.5 min, increasing the phase B from 40% to 55%; 4.5-5.5 min, increasing the phase B from 55% to 98%; reducing the phase B from 98% to 40% in 6.0-6.1 min; 6.1-7.0 min, 40% B;
(3) mass spectrum detection: taking the sample pretreated in the step (1), selecting an ESI positive ion mode for sample loading, and detecting 17 steroid hormones by adopting a multi-reaction monitoring technology, wherein mass spectrum parameters are as follows: the collision gas is 0.2mL/Min, the gas curtain gas is 650L/Hr, and the temperature is 450 ℃;
(4) a step of preparing a standard curve by using a series of 17 steroid hormones of concentration standard, wherein a calibration curve is established by taking the concentration ratio of the standard to an internal standard as an X axis and the peak area ratio of the standard to the internal standard as a Y axis, and the content of the 17 steroid hormones in a serum sample is calculated; 17 steroid hormones include testosterone, progesterone, 17 alpha-hydroxyprogesterone, cortisol, aldosterone, dihydrotestosterone, pregnenolone, corticosterone, 11-deoxycorticosterone, 11-deoxycorticosterol, androstenedione, dehydroepiandrosterone, cortisone, 17 alpha-hydroxyprogesterone, 18-hydroxycorticosterone, 21-deoxycorticosterol, androstenediol.
9. The use of claim 8, wherein step (1) further comprises adding an internal standard compound selected from the group consisting of testosterone-d prior to performing the pretreatment of the serum sample317 alpha-hydroxyprogesterone-d8Aldosterone-d7Dihydrotestosterone-d3Corticosterone-d411-deoxycorticosterone-d711-deoxycorticosterol-d5Androstenedione-13C318-Hydroxycorticosterone-d4Pregnenolone-20, 21-13C2-16,16-d2, progesterone-d9Cortisol-d417 alpha-hydroxypregnanolone-d3Cortisone-d8Dehydroepiandrosterone-d5。
10. The use of claim 8, wherein the multiple response monitoring technique in step (3) detects the parameters of 17 steroid hormones as follows:
ESI + mode:
testosterone ion pair, m/z is 289.4-97, and collision voltage is 28V;
progesterone ion pair with m/z of 315.4-97 and collision voltage of 45V;
17 alpha-hydroxyprogesterone ion pair, wherein m/z is 331.4-97, and collision voltage is 26V;
a cortisol ion pair, m/z is 363-121, and the collision voltage is 45V;
aldosterone ion pair, m/z 361-;
dihydrotestosterone ion pair, m/z is 291.4-255, and collision voltage is 10V;
pregnenolone ion pair, m/z:317.2-281.3, and collision voltage of 13V;
the cortical ketone ion pair has the m/z of 347.3-329, and the collision voltage is 10V;
11-deoxycorticosterone ion pair with m/z of 331.3-97 and collision voltage of 20V;
11-deoxycorticosterol ion pair, m/z of 347.2-97, and collision voltage of 20V;
androstenedione ion pair with m/z of 287.2-97 and collision voltage of 26V;
a dehydroepiandrosterone ion pair with m/z of 289.2-253 and a collision voltage of 10V;
cortisone ion pair, m/z is 361.3-163.1, and collision voltage is 15V;
17 alpha-hydroxypregnenolone ion pair, m/z is 333.3-297.2, and collision voltage is 10V;
18-hydroxycorticosterone ion pair with m/z of 363-121.1 and collision voltage of 15V;
21-deoxycorticosterol ion pair, wherein the m/z is 347.2-121.1, and the collision voltage is 28V;
androstene diol ion pair with m/z of 255.3-159.3 and collision voltage of 25V;
testosterone-d3Ion pair, m/z is 292.2-97, and collision voltage is 28V;
progesterone-d9Ion pair, m/z is 324.4-100, and collision voltage is 27V;
17 alpha-hydroxyprogesterone-d8Ion pair, m/z is 339.4-100.1, and collision voltage is 26V;
cortisol-d4Ion pair, m/z:367- > 122, and the collision voltage is 30V;
aldosterone-d7Ion pair, m/z 368-350, collision voltage 15V;
dihydrotestosterone-d3Ion pair, m/z is 294.4-258.2, and collision voltage is 10V;
pregnenolone-20, 21-13C216,16-d2 ion pair, m/z 321.2-285.3, and the collision voltage is 13V;
corticosterone-d4Ion pair, m/z 351-;
11-deoxycorticosterone-d7Ion pair, m/z is 338.4-100, and collision voltage is 26V;
11-deoxycorticosterol-d5Ion pair, m/z:352.2-100, collision voltage is 26V;
androstenedione-13C3Ion pair with m/z of 290.2-100 and collision voltage of 26V;
dehydroepiandrosterone-d5Ion pair, m/z is 294.3-258.2, and collision voltage is 14V;
cortisone-d8Ion pair, m/z is 369.3-168.2, and collision voltage is 15V;
17 alpha-hydroxypregnenolone-d3Ion pair, m/z is 336.1-300.1, and collision voltage is 10V;
18-Hydroxycorticosterone-d4Ion pair, m/z is 367 to 121.9, and the collision voltage is 45V;
other general parameters: the taper hole voltage is 25V, the capillary tube voltage is 3kV, the taper hole gas pressure is 150L/Hr, and the atomization gas pressure is 7.0 Bar.
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