A composite extraction agent, a pretreatment method for detecting estrone in blood and a method for detecting estrone in blood

Abstract

The application provides a composite extraction agent, a pretreatment method for detecting estrone in blood and a detection method for estrone in blood, and belongs to the technical field of analysis and detection.The composite extraction agent comprises mixed fillers of C18 fillers and PSA fillers, a lower layer solvent (methanol and / or isopropyl alcohol) and an upper layer solvent (n-hexane); and the mixed fillers are immersed in the lower layer solvent.The application adopts the above-mentioned composite extraction agent to complete the extraction of the sample to be detected through a pretreatment method of 'layered extraction' in only one step, and the operation is convenient; methanol and / or isopropyl alcohol are used as the extraction solvent, the toxicity is lower, the interference with mass spectrometry is less, the matrix effect is reduced, and the accuracy and sensitivity of subsequent detection are improved; liquid chromatography-triple mass spectrometry is used for detection, the interference of daughter ions is avoided by monitoring parent ions, the anti-interference ability is stronger, and quantitative and qualitative analysis of estrone in blood can be accurately and efficiently realized.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology, specifically relating to a composite extractant, a pretreatment method for detecting estrone in blood, and a method for detecting estrone in blood. Background Technology

[0002] Estrone is an endogenous estrogen with the structure shown in Formula I. Methods for detecting estrone in blood mainly include traditional radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), as well as newer techniques such as liquid chromatography-tandem mass spectrometry (LC-MS / MS) and gas chromatography-mass spectrometry (GC-MS). Before detection, sample pretreatment is necessary to reduce matrix effects. Currently, for estrogens in different biological matrices, liquid-liquid extraction (LLE) and solid-phase extraction (SPE) are mostly used for sample pretreatment. Estrone is first extracted with an organic solvent, and then purified using a commercially available solid-phase extraction column. For example, existing technologies (Fan Ruting, Xiao Huaming, Cha Dongmei, et al. Research progress on quantitative analysis of estrogen in biological matrices by liquid chromatography-tandem mass spectrometry [J]. Journal of Analytical Science, 2021, 37(03): 395-402) disclose the extraction of estrogen from biological matrices using organic solvents such as ethyl acetate, methyl tert-butyl ether, chlorobutane, methanol, and acetonitrile, followed by purification using a solid-phase extraction column. However, the above pretreatment methods are relatively complex and cumbersome.

[0003] Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a compound extractant, a pretreatment method for detecting estrone in blood, and a method for detecting estrone in blood, which can complete the extraction of the sample to be tested in only one step and is simple to operate.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] This invention provides a composite extractant, comprising a mixed filler, a lower solvent, and an upper solvent; the mixed filler is immersed in the lower solvent; the mixed filler comprises C 18 The filler and PSA filler; the lower solvent includes methanol and / or isopropanol; the upper solvent includes n-hexane.

[0007] Preferably, the C 18 The mass ratio of the packing material to the PSA packing material is 1:0.1 to 2;

[0008] The C 18 The solid-liquid ratio of the filler and the lower solvent layer is 1g:10-20mL;

[0009] The volume ratio of the upper solvent to the lower solvent is 1:1 to 5.

[0010] Preferably, when the lower solvent is methanol and isopropanol, the volume ratio of methanol to isopropanol is 1 to 20:1.

[0011] This invention provides a pretreatment method for detecting estrone in blood, comprising the following steps:

[0012] The composite extractant described in the above technical solution is used to perform layered extraction on the plasma sample to be tested, thereby obtaining the sample solution to be tested.

[0013] Preferably, the volume ratio of the plasma sample to be tested to the lower solvent is 1:1 to 5.

[0014] Preferably, the layered extraction includes adding the plasma sample to be tested to a composite extractant and sequentially performing shaking extraction and centrifugation separation, with the resulting lower layer solution being the sample solution to be tested.

[0015] Preferably, the shaking extraction speed is 800-1500 rpm and the time is 1-10 min;

[0016] The centrifugation speed is 11641-13273 rpm, and the time is 1-8 min.

[0017] This invention provides a method for detecting estrone in blood, comprising the following steps:

[0018] The composite extractant described in the above technical solution is used to extract plasma samples and estrone- 13 The mixed solution of C3 internal standard was subjected to layer extraction to obtain the test sample solution;

[0019] The sample solution to be tested was subjected to liquid chromatography-level 3 mass spectrometry to obtain the detection results of estrone; the detection results include qualitative detection results and / or quantitative detection results.

[0020] Preferably, the estrone- 13 The concentration of the C3 internal standard solution was 0.1–0.5 μg / mL;

[0021] The plasma sample to be tested and estrone- 13 The liquid-to-solid ratio of the C3 internal standard is 1 mL: 0.01–0.05 μg.

[0022] Preferably, the liquid chromatography-tertiary mass spectrometry detection includes liquid chromatography detection and tertiary mass spectrometry detection;

[0023] The conditions for the liquid chromatography detection include:

[0024] Column: Waters UPLC Peptide BEH C18 Chromatographic column;

[0025] Column temperature: 20~50℃;

[0026] Injection volume: 1–10 μL;

[0027] Mobile phase: Mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of ammonium fluoride with a concentration of 0.1–1.5 mmol / L; mobile phase B is an isopropanol-acetonitrile solution with a volume fraction of isopropanol of 1–10%; the flow rate of the mobile phase is 0.1–0.6 μL / min;

[0028] Elution method: gradient elution;

[0029] The gradient elution procedure is as follows:

[0030] The volume percentage of the mobile phase B is 10-30% for 0-2 minutes.

[0031] Over 2 to 10 minutes, the volume percentage of the mobile phase B increases uniformly from 10 to 30% to 70 to 100%.

[0032] The volume percentage of the mobile phase B is 70-100% over 10-13 minutes.

[0033] During the 13-13.1 min period, the volume percentage of the mobile phase B decreased uniformly from 70-100% to 10-30%.

[0034] 13.1–15 min, the volume percentage of the mobile phase B is 10–30%.

[0035] The conditions for the three-stage mass spectrometry detection include:

[0036] Ion source: Electrospray ion source, negative ion mode;

[0037] Ionization voltage: 3500~5500V;

[0038] Ion source temperature: 350~550℃;

[0039] Spray gas: flow rate 25–45 psi;

[0040] Auxiliary heating gas: flow rate 35–75 psi;

[0041] Air curtain gas: flow rate 35-75 psi;

[0042] Collision air: Pressure is High;

[0043] Detection method: Level 3 mass spectrometry multiple reaction detection;

[0044] Level 3 monitoring ions: Estrone: 269.1>145>130.1 and 269.1>253.1>145.2;

[0045] Estrone- 13 C3 internal standard: 272.1>148>133.1.

[0046] This invention provides a composite extractant, comprising a mixed filler, a lower solvent, and an upper solvent; the mixed filler is immersed in the lower solvent; the mixed filler comprises C 18 The filler and PSA filler; the lower solvent includes methanol and / or isopropanol; the upper solvent includes n-hexane. In the composite extractant provided by this invention, C... 18 The packing material can adsorb impurities of nonpolar organic compounds in the sample. The PSA packing material absorbs fatty acids (such as oleic acid, palmitic acid, and linoleic acid), metal ions, and sugar interfering substances, reducing matrix effects and improving the accuracy and sensitivity of subsequent detections. Methanol and / or isopropanol are used as the lower-layer solvent for extraction, which has low toxicity and minimal interference with mass spectrometry, further reducing matrix effects and improving the accuracy and sensitivity of subsequent detections. Hexane is used as the upper-layer solvent to dissolve nonpolar substances in the blood, such as triglycerides. This invention uses the above-mentioned composite extractant to dissolve substances in the blood according to their polarity in different layers of the composite extractant, achieving "layered extraction" of estrone in the blood. This method is convenient and significantly shortens the pretreatment time.

[0047] This invention provides a pretreatment method for detecting estrone in blood. The composite extractant described in the above technical solution allows for one-step extraction of estrone from the sample, making the operation convenient. Furthermore, this invention uses methanol and / or isopropanol as extraction solvents, which have low toxicity, minimal interference with mass spectrometry, reduced matrix effects, and improved accuracy and sensitivity of subsequent detection.

[0048] This invention also provides a method for detecting estrone in blood. In traditional secondary mass spectrometry multiple reaction monitoring (MRM) with a mother ion-daughter ion channel, the matrix interference cannot be eliminated when the daughter ion is affected by the sample matrix. This invention employs liquid chromatography-tertiary mass spectrometry (MS / MS / MS, or MS...). 3 It can monitor the mother ion-daughter ion-grandchild ion channel, and avoid interference from daughter ions by monitoring grandchild ions, thus having stronger anti-interference ability and enabling accurate and efficient quantitative and qualitative analysis of estrone in blood.

[0049] Furthermore, this invention uses the estrone ion pair (269.1 > 145 > 130.1, ion-mass-nucleus ratio, where the ion to the right of ">" is the fragment ion to the left) in a three-stage mass spectrometer as the quantitative ion. 13 The C3 ion pair (272.1 > 148 > 133.1) was used as an internal standard correction ion. Detection by liquid chromatography-level 3 mass spectrometry can further improve the accuracy and sensitivity of the detection results. Attached Figure Description

[0050] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0051] Figure 1 A schematic diagram of the composite extractant provided by the present invention;

[0052] Figure 2 Monitoring of estrone ions and estrone- in a blank plasma matrix supplemented with 0.01 μg / L estrone. 13 Chromatograms of C3-monitored ions, where the monitored ion in the upper chromatogram is estrone: 269.1>145>130.1; the monitored ion in the middle chromatogram is estrone: 269.1>253.1>145.2; and the monitored ion in the lower chromatogram is estrone- 13 C3 internal standard: 272.1>148>133.1;

[0053] Figure 3 This is the standard curve obtained in this invention. Detailed Implementation

[0054] Figure 1 This is a schematic diagram of the structure of the compound extractant. The following is in conjunction with... Figure 1 A detailed description of the compound extractant is provided.

[0055] This invention provides a composite extractant, comprising a mixed filler, a lower solvent, and an upper solvent; the mixed filler is immersed in the lower solvent; the mixed filler comprises C 18 The filler and PSA filler; the lower solvent includes methanol and / or isopropanol; the upper solvent includes n-hexane.

[0056] Unless otherwise specified, all reagents used in this invention are commercially available products well known to those skilled in the art.

[0057] In this invention, the C 18The mass ratio of the packing material to the PSA packing material is preferably 1:0.1-2, more preferably 1:0.5-1.5, and most preferably 1:0.8-1. In this invention, the C... 18 The solid-liquid ratio of the filler and the lower solvent is preferably 1g:10-20mL, more preferably 1g:12-18mL, and most preferably 1g:14.5-16mL. In this invention, the volume ratio of the upper solvent to the lower solvent is preferably 1:1-5, more preferably 1:2-4, and most preferably 1:2.9-3.5.

[0058] In this invention, the lower layer solvent is preferably methanol and isopropanol. When the lower layer solvent is methanol and isopropanol, the volume ratio of methanol to isopropanol is preferably 1 to 20:1, more preferably 5 to 15:1, and most preferably 9 to 12:1.

[0059] The present invention does not have any particular limitation on the container used for the composite extractant. Any extraction container known to those skilled in the art can be used. In a specific embodiment of the present invention, centrifuge tubes are preferred as containers.

[0060] This invention provides a pretreatment method for detecting estrone in blood, comprising the following steps:

[0061] The composite extractant described in the above technical solution is used to extract plasma samples and estrone- 13 The mixed solution of C3 internal standard was subjected to layer extraction to obtain the sample solution to be tested.

[0062] In this invention, the volume ratio of the plasma sample to be tested to the lower solvent is preferably 1:1 to 5, more preferably 1:1.5 to 4, and most preferably 1:2.9 to 3.5.

[0063] In this invention, the plasma sample to be tested is preferably obtained by centrifugation of anticoagulated blood. In this invention, the concentration of the anticoagulant in the anticoagulated blood is preferably 0.01–0.05 mol / L, more preferably 0.02–0.04 mol / L, and most preferably 0.02–0.03 mol / L; the anticoagulant preferably includes disodium ethylenediaminetetraacetate (EDTA). In this invention, the centrifugation speed is preferably 4000–8000 rpm, more preferably 5000–7000 rpm, and most preferably 5500–6500 rpm; the centrifugation time is preferably 1–10 min, more preferably 4–6 min, and most preferably 4.5–5.5 min.

[0064] In this invention, the layered extraction preferably includes adding the plasma sample to be tested to a composite extractant and sequentially performing shaking extraction and centrifugation, with the resulting lower layer solution (i.e., the lower solvent containing estrone) being the sample solution to be tested. In this invention, the shaking extraction speed is preferably 800–1500 rpm, more preferably 900–1200 rpm, and most preferably 1000–1100 rpm; the shaking extraction time is preferably 1–10 min, more preferably 2–7 min, and most preferably 4–5 min. In this invention, the shaking extraction is preferably performed using a shaker. In this invention, the centrifugation speed is preferably 11641–13273 rpm, more preferably 12000–13000 rpm, and most preferably 12500–12753 rpm; the centrifugation time is preferably 1–8 min, more preferably 2–7 min, and most preferably 4–5 min.

[0065] This invention utilizes a "layered extraction" pretreatment method, which allows for the extraction of the sample in just one step, making the operation convenient and shortening the pretreatment time.

[0066] This invention also provides a method for detecting estrone in blood, comprising the following steps:

[0067] The composite extractant described in the above technical solution is used to extract plasma samples and estrone- 13 The mixed solution of C3 internal standard was subjected to layer extraction to obtain the test sample solution;

[0068] The sample solution to be tested was subjected to liquid chromatography-level 3 mass spectrometry to obtain the detection results of estrone; the detection results include qualitative detection results and quantitative detection results.

[0069] The composite extractant described in the above technical solution is used to extract plasma samples and estrone- 13 The mixed solution of C3 internal standard was subjected to layer extraction to obtain the sample solution to be tested.

[0070] In this invention, the volume ratio of the plasma sample to be tested to the lower layer solvent is preferably the same as that in the aforementioned pretreatment method, and will not be repeated here.

[0071] In this invention, the estrone- 13 The concentration of the C3 internal standard solution is preferably 0.1–0.5 μg / mL, more preferably 0.2–0.4 μg / mL, and most preferably 0.25–0.35 μg / mL. In this invention, the plasma sample to be tested and estrone- 13 The liquid-to-solid ratio of the C3 internal standard is preferably 1 mL: 0.01–0.05 μg, more preferably 1 mL: 0.02–0.04 μg, and most preferably 0.03–0.04 μg.

[0072] After obtaining the sample solution to be tested, the present invention performs liquid chromatography-level 3 mass spectrometry on the sample solution to obtain the detection results of estrone; the detection results include qualitative detection results and / or quantitative detection results.

[0073] In this invention, the liquid chromatography-tertiary mass spectrometry detection includes liquid chromatography detection and tertiary mass spectrometry detection.

[0074] In this invention, the conditions for the liquid chromatography detection preferably include:

[0075] Column: Waters UPLC Peptide BEH C 18 Chromatographic column;

[0076] Column temperature: 20–50°C, more preferably 30–45°C, and most preferably 35–40°C;

[0077] Injection volume: 1–10 μL, more preferably 3–8 μL, and most preferably 5–7 μL;

[0078] Mobile phase: Mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of ammonium fluoride, the concentration of which is 0.1–1.5 mmol / L, more preferably 0.15–1.0 mmol / L, and most preferably 0.2–0.5 mmol / L; mobile phase B is an isopropanol-acetonitrile solution, wherein the volume fraction of isopropanol in which isopropanol is 1–10%, more preferably 2–7%, and most preferably 3–5%; the flow rate of which isopropanol is 0.1–0.6 μL / min, more preferably 0.2–0.5 μL / min, and most preferably 0.3–0.4 μL / min;

[0079] Elution method: gradient elution;

[0080] The gradient elution procedure is as follows:

[0081] The volume percentage of the mobile phase B is 10-30% for 0-2 minutes.

[0082] Over 2 to 10 minutes, the volume percentage of the mobile phase B increases uniformly from 10 to 30% to 70 to 100%.

[0083] The volume percentage of the mobile phase B is 70-100% over 10-13 minutes.

[0084] During the 13-13.1 min period, the volume percentage of the mobile phase B decreased uniformly from 70-100% to 10-30%.

[0085] 13.1–15 min, the volume percentage of the mobile phase B is 10–30%.

[0086] In this invention, the gradient elution procedure is more preferably as follows:

[0087] From 0 to 2 minutes, the volume percentage of the mobile phase B is 20%.

[0088] Over 2 to 10 minutes, the volume percentage of the mobile phase B increases uniformly from 20% to 99%.

[0089] The volume percentage of the mobile phase B is 99% after 10-13 minutes.

[0090] During the 13-13.1 min period, the volume percentage of the mobile phase B decreased from 99% to 20% at a constant rate.

[0091] 13.1–15 min, the volume percentage of the mobile phase B is 20%.

[0092] In this invention, the preferred conditions for the three-stage mass spectrometry detection include:

[0093] Ion source: Electrospray ion source, negative ion mode;

[0094] Ionization voltage: 3500-5500V, more preferably 4000-5000V, and most preferably 4300-4800V;

[0095] Ion source temperature: 350~550℃, more preferably 400~500℃, and most preferably 450~480℃;

[0096] The flow rate of the spray gas is 25–45 psi, more preferably 30–40 psi, and most preferably 32–35 psi;

[0097] Auxiliary heating gas: flow rate of 35-75 psi, more preferably 40-70 psi, and most preferably 45-55 psi;

[0098] Air curtain gas: flow rate of 35-75 psi, more preferably 40-70 psi, and most preferably 45-55 psi;

[0099] Collision air: Pressure is High;

[0100] Detection method: Level 3 mass spectrometry multiple reaction detection;

[0101] Level 3 monitoring ions: Estrone: 269.1>145>130.1 and 269.1>253.1>145.2;

[0102] Estrone- 13C3 internal standard: 272.1>148>133.1.

[0103] In this invention, the spray gas, auxiliary heating gas, curtain gas, and collision gas are all preferably nitrogen; the nitrogen is preferably generated by a nitrogen generator.

[0104] In this invention, the step of obtaining the qualitative detection result preferably includes:

[0105] The liquid chromatography-mass spectrometry (LC-MS) data of the sample obtained by the above technical solution are compared with the LC-MS data of the predetermined standard to obtain the qualitative detection result. Specifically, the chromatographic retention time is compared. When a chromatographic peak with a retention time consistent with that of estrone at a certain concentration in the mixed standard working solution is detected in the sample solution (within ±2.5%), and the relative abundance ratio of the selected monitoring ion pair in the chromatogram of the sample solution deviates from the relative abundance ratio (k) of the ion in the equivalent concentration standard solution within the range specified in Table 1, the corresponding compound is confirmed to be detected in the sample.

[0106] Table 1. Maximum permissible deviation of relative ion abundance for qualitative analysis.

[0107] Permissible relative deviation / % ±20 ±25 ±30 ±50

[0108] In this invention, the mixed standard working solution is preferably estrone standard and estrone- 13 A mixed solution of C3 internal standard; the preferred concentrations of estrone standard in the mixed standard working solution are 0.01 μg / L, 0.05 μg / L, 0.1 μg / L, 0.2 μg / L, 0.5 μg / L, 0.8 μg / L, and 1 μg / L, respectively. 13 The concentration of the C3 internal standard is preferably 0.05 μg / L; the solvent in the mixed standard working solution is preferably methanol and water, and the volume fraction of methanol in the solvent is preferably 0.1-0.8%, more preferably 0.2-0.7%, and most preferably 0.3-0.6%.

[0109] In this invention, the method for preparing the mixed standard working solution preferably includes:

[0110] Prepare a 1 mg / mL estrone standard stock solution and a 1 mg / mL estrone- 13 C3 internal standard stock solution;

[0111] Prepare 10 μg / mL estrone intermediate stock solution and 10 μg / mL estrone- 13 C3 internal standard intermediate stock solution;

[0112] Using the aforementioned estrone intermediate stock solution and estrone- 13Prepare a mixed standard working solution from C3 internal standard intermediate stock solution.

[0113] In an embodiment of the present invention, the method for preparing the mixed standard working solution is as follows:

[0114] Accurately weigh 0.010 g of estrone standard (accurate to 0.001 g), dilute to 10 mL of methanol, and prepare a 1 mg / mL estrone standard stock solution; weigh 0.010 g (accurate to 0.001 g) of estrone... 13 C3 internal standard was diluted to 10 mL with methanol to prepare a 1 mg / mL estrone solution. 13 C3 internal standard stock solution was stored at -20°C.

[0115] Accurately pipette 0.1 mL of estrone standard stock solution into a 10 mL volumetric flask, and dilute to the mark with methanol to obtain a 10 μg / mL estrone intermediate stock solution. 13 C3 internal standard stock solution was transferred to a 10 mL volumetric flask and diluted to the mark with methanol to obtain 10 μg / mL estrone. 13 C3 internal standard intermediate stock solution was stored at -20℃.

[0116] Accurately aspirate the intermediate stock solution of estrone and estrone- 13 The C3 internal standard intermediate stock solution is diluted to volume with an aqueous methanol solution and shaken well to obtain a mixed standard working solution. In this invention, the volume fraction of methanol in the aqueous methanol solution is preferably 35%. There are no special requirements for the volume of estrone intermediate stock solution taken or the final volume of dilution with the aqueous methanol solution, as long as the mixed standard working solution of the above concentration can be obtained.

[0117] In this invention, obtaining the quantitative detection result preferably includes: comparing the liquid chromatography-mass spectrometry detection data of the sample to be tested obtained by the above technical solution with a predetermined standard curve to obtain the quantitative detection result, wherein the predetermined standard curve is estrone standard and estrone- 13 A linear relationship curve between the concentration ratio of C3 internal standard and the ratio of the peak area of ​​quantitative ion mass chromatography to the peak area of ​​internal standard correction ion chromatography.

[0118] In this invention, the method for preparing the standard curve preferably includes the following steps:

[0119] The mixed standard working solution was subjected to liquid chromatography-level 3 mass spectrometry (LC-MS / MS) to obtain the chromatographic peak areas of estrone at different concentrations. The concentrations of estrone standard and estrone-... 13The concentration ratio of C3 internal standard is used as the x-axis, and the ratio of the peak area of ​​the quantitative ion mass chromatography to the peak area of ​​the internal standard correction ion chromatography is used as the y-axis to plot a standard curve, thus obtaining a linear regression equation. In this invention, the preferred linear regression equation is y = 0.16843x + 0.10387, the correlation coefficient r = 0.99610, the preferred linear range is 0.01–1.0 μg / L, and the preferred limit of quantitation is 0.005 μg / L.

[0120] In this invention, the conditions for liquid chromatography-third-level mass spectrometry detection are preferably the same as those for liquid chromatography-third-level mass spectrometry detection of the sample solution to be tested described above, and will not be repeated here.

[0121] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0122] In embodiments of the present invention, the specifications of the specific instruments, reagents, and samples to be tested are as follows:

[0123] The instruments specifically included: a liquid chromatography-triple quadrupole / linear ion trap mass spectrometer (AB Qtrap6500+, SCIEX, USA); a Millipore Q ultrapure water system; a Multi Reax multi-functional shaker (Heidolph Instruments, Germany); and a Sigma 1-14 mini benchtop centrifuge (SIGMA, Germany). Acetonitrile, methanol, and formic acid were of chromatographic grade and were purchased from Merck, Germany.

[0124] The reagents were: nitrogen (99.99% purity) purchased from Hangzhou Jingong Special Gases Co., Ltd.; C18 packing material, 40–63 μm, purchased from Shanghai Anpu Experimental Technology Co., Ltd.; ethylenediamine-N-propylsilylated silica gel (PSA), 40–60 μm, purchased from Beijing Bona Ager Technology Co., Ltd.; and estrone, C... 18 H 22 O2, CAS: 53-16-7, Estrone- 13 C3, C 15 13 C3H 22 O2, CAS: 1241684-29-6, all with a purity greater than 99%, were purchased from Beijing Manhage Technology Co., Ltd.; all liquid reagents were of chromatographic purity.

[0125] The conditions for liquid chromatography detection in the liquid chromatography-tertiary mass spectrometry detection are as follows:

[0126] Column: Waters UPLC Peptide BEH C 18 Chromatographic column (1.7 μm, 2.1 mm × 100 mm);

[0127] Column temperature: 40℃;

[0128] Injection volume: 5 μL;

[0129] Mobile phase: Mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of ammonium fluoride with a concentration of 0.2 mmol / L; mobile phase B is an isopropanol-acetonitrile solution with a volume fraction of 3% isopropanol; the flow rate of the mobile phase is 0.4 μL / min;

[0130] Elution method: gradient elution;

[0131] The gradient elution procedure is as follows:

[0132] From 0 to 2 minutes, the volume percentage of the mobile phase B is 20%.

[0133] Over 2 to 10 minutes, the volume percentage of the mobile phase B increases uniformly from 20% to 99%.

[0134] The volume percentage of the mobile phase B is 99% after 10-13 minutes.

[0135] During the 13-13.1 min period, the volume percentage of the mobile phase B decreased from 99% to 20% at a constant rate.

[0136] 13.1–15 min, the volume percentage of the mobile phase B is 20%.

[0137] The conditions for mass spectrometry detection in the liquid chromatography-tertiary mass spectrometry detection are as follows:

[0138] Ion source: Electrospray ion source, negative ion mode;

[0139] Ionization voltage: 5000V;

[0140] Ion source temperature: 450℃;

[0141] Aerosol: Nitrogen, flow rate 35 psi;

[0142] Auxiliary heating gas: nitrogen, flow rate 55 psi;

[0143] Curtain gas: Nitrogen, flow rate 55 psi;

[0144] Collision gas: Nitrogen, pressure High;

[0145] Detection method: Level 3 mass spectrometry multiple reaction detection;

[0146] Level 3 monitoring ions: Estrone: 269.1>145>130.1 and 269.1>253.1>145.2;

[0147] Estrone- 13 C3 internal standard: 272.1>148>133.1.

[0148] The ion pairs and mass spectrometry parameters for the three-level monitoring are shown in Table 2.

[0149] Table 2 Main mass spectrometry parameters

[0150]

[0151] Example 1

[0152] Method limit of quantitation, recovery rate and precision

[0153] (1) Preparation of standard solutions

[0154] Accurately weigh 0.010 g of estrone standard (accurate to 0.001 g), dilute to 10 mL of methanol, and prepare a 1 mg / mL estrone standard stock solution; weigh 0.010 g (accurate to 0.001 g) of estrone... 13 C3 internal standard was diluted to 10 mL with methanol to prepare a 1 mg / mL estrone solution. 13 C3 internal standard stock solution was stored at -20°C.

[0155] Accurately pipette 0.1 mL of estrone standard stock solution into a 10 mL volumetric flask, and dilute to the mark with methanol to obtain a 10 μg / mL estrone intermediate stock solution. 13 C3 internal standard stock solution was transferred to a 10 mL volumetric flask and diluted to the mark with methanol to obtain 10 μg / mL estrone. 13 C3 internal standard intermediate stock solution was stored at -20℃.

[0156] Accurately aspirate the intermediate stock solution of estrone and estrone- 13 The C3 internal standard intermediate stock solution was diluted to volume with a methanol-water solution (methanol volume fraction 35%), and shaken well to obtain a series of mixed standard working solutions with concentrations of 0.01, 0.05, 0.1, 0.2, 0.5, 0.8, and 1 μg / L. Each of these mixed standard working solutions contained 0.05 μg / L estrone. 13 C3 was used as an internal standard for isotopes.

[0157] (2) Three-stage mass spectrometry (MS) 3 Determination of ion collection

[0158] Using a 35% methanol aqueous solution as the diluent, a 100 μg / L estrone standard working solution was prepared using estrone intermediate stock solution. 13 C3 internal standard intermediate stock solution is prepared with 100 μg / L estrone. 13 The C3 internal standard working solution was used for constant flow injection via a syringe pump in a third-stage mass spectrometer to optimize the monitoring parameters. In positive ion mode, a first-stage mass spectrometry scan (Q1 MS) was performed to obtain the precursor ion information of the target compound. A second-stage fragment ion scan (Production MS) was then performed. 2 The declustering voltage DP, the inlet voltage EP, and the collision energy CE were optimized to obtain the corresponding secondary daughter ions. Finally, a grandchild ion scan was performed using tertiary ion trap mass spectrometry (MS). 3 ), and optimize the excitation energy AF2 to obtain information about tertiary ions.

[0159] The monitored ions were: estrone: 269.1>145>130.1 and 269.1>253.1>145.2, estrone- 13 C3 internal standard: 272.1>148>133.1. Among them, 269.1>145>130.1 is the quantitative ion, and 272.1>148>133.1 is the internal standard correction ion.

[0160] (3) Determination of the method limit of quantitation

[0161] The method of adding estrone to a plasma blank matrix was adopted. Estrone at a concentration of 0.01 μg / L was added to the plasma blank matrix and detected by liquid chromatography-mass spectrometry. The limit of quantitation was determined based on the signal-to-noise ratio (S / N) of the chromatographic peak of the quantitative ion, which was 10.

[0162] Figure 2 Monitoring of estrone ions and estrone- in a blank plasma matrix supplemented with 0.01 μg / L estrone. 13 Chromatogram of C3-monitored ions. (From...) Figure 2 It can be seen that the limit of quantification for estrone in the plasma samples tested by this method is 0.005 μg / L.

[0163] (4) Recovery and precision of the method

[0164] Estrone at concentrations of 0.05 μg / L, 0.1 μg / L, and 0.5 μg / L was added to a blank plasma matrix, and the samples were detected by liquid chromatography-mass spectrometry (LC-MS). The recovery and precision of the method were analyzed. The experiment was repeated 6 times (n=6). The recoveries of the three concentration levels of 0.05 μg / L, 0.1 μg / L, and 0.5 μg / L were 89.3±6.7%, 92.6±5.8%, and 95.1±6.5%, respectively (mean ± relative standard deviation RSD).

[0165] Example 2

[0166] Establishment of preprocessing methods

[0167] Take a 1.5 mL centrifuge tube and add 0.02 g of C. 18 The following were prepared: packing material, 0.02 g PSA packing material, 0.29 mL methanol-isopropanol mixture (methanol to isopropanol volume ratio 9:1), 0.1 mL n-hexane, and 0.01 mL 0.2 μg / mL estrone. 13 The solution of internal standard C3 was allowed to stand for 2 minutes.

[0168] A schematic diagram of the compound extractant is shown below. Figure 1 As shown, the packing material is C. 18 The packing material and PSA packing material have a lower layer solvent of methanol-isopropanol mixture and an upper layer solvent of n-hexane.

[0169] Take 1 mL of anticoagulated blood and centrifuge at 6000 rpm to obtain the plasma sample to be tested. Take 0.1 mL of the plasma sample to be tested and place it in the composite extractant. Shake at 1000 rpm for 1 min in a shaker. Then centrifuge at 12753 rpm for 5 min. Take 0.15 mL of the lower layer solution into the sample vial to obtain the sample solution to be tested.

[0170] Example 3

[0171] Construction of standard curve

[0172] Accurately aspirate the intermediate stock solution of estrone and estrone- 13 C3 internal standard intermediate stock solution was diluted to volume with methanol-water solution (methanol volume fraction 35%), and shaken well to obtain mixed standard working solutions with estrone standard concentrations of 0.01, 0.05, 0.1, 0.2, 0.5, 0.8 and 1 μg / L, respectively. Each mixed standard working solution contained estrone at a concentration of 0.05 μg / L. 13 C3 internal standard.

[0173] The above mixed standard working solution was sequentially analyzed by liquid chromatography-level 3 mass spectrometry. The estrone standard and estrone-...13 The concentration ratio of C3 internal standard is used as the abscissa (x), and the ratio of the peak area of ​​quantitative ion mass chromatography to the peak area of ​​internal standard correction ion chromatography is used as the ordinate (y). A standard curve is plotted to obtain the linear regression equation. Figure 3 The standard curve obtained in this invention is derived from... Figure 3 It can be seen that estrone exhibits a good linear relationship within a series of concentrations ranging from 0.01 to 1.0 μg / L. The linear regression equation is y = 0.16843x + 0.10387 (r = 0.99610).

[0174] Example 4

[0175] Detection of estrone content in plasma samples to be tested

[0176] The pretreatment method and liquid chromatography-mass spectrometry detection method established according to the present invention were used to pretreat and detect 9 plasma samples to be tested and 1 quality control plasma sample. The results are shown in Table 3. Among them, the quality control plasma sample contained 0.60 μg / L of estrone, and the detection value was 0.58 μg / L. The detection values ​​of estrone content in the 9 plasma samples to be tested are as follows: estrone was not detected in 1 sample, and the estrone content in the other 8 plasma samples to be tested ranged from 0.07 to 0.95 μg / L.

[0177] Table 3. Detection values ​​of estrone content in plasma samples to be tested and quality control plasma samples.

[0178]

[0179]

[0180] As shown in Table 3, the pretreatment method and liquid chromatography-tertiary mass spectrometry detection method established in this invention can accurately and efficiently achieve quantitative and qualitative analysis of estrone in blood.

[0181] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for detecting estrone in blood, characterized in that, Includes the following steps: The plasma sample to be tested was added to the composite extractant and subjected to shaking extraction and centrifugation in sequence. The lower layer solution obtained was the sample solution to be tested. The composite extraction agent includes a mixed packing, a lower layer solvent, and an upper layer solvent; the mixed packing is immersed in the lower layer solvent; the mixed packing includes C 18 packing and PSA packing; the lower layer solvent includes methanol and isopropyl alcohol; and the upper layer solvent includes n-hexane. The C 18 The mass ratio of the packing material to the PSA packing material is 1:0.1 to 2; The C 18 The solid-liquid ratio of the filler and the lower solvent layer is 1g:10-20mL; The volume ratio of the upper solvent to the lower solvent is 1:1 to 5; The volume ratio of methanol to isopropanol is 1 to 20:1; The sample solution to be tested was subjected to liquid chromatography-level 3 mass spectrometry to obtain the detection results of estrone; the detection results include qualitative detection results and / or quantitative detection results; The liquid chromatography-level 3 mass spectrometry detection includes liquid chromatography detection and level 3 mass spectrometry detection; The conditions for the liquid chromatography detection include: Column: Waters UPLC Peptide BEH C 18 Chromatographic column; Column temperature: 20~50℃; Injection volume: 1–10 μL; Mobile phase: Mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of ammonium fluoride with a concentration of 0.1–1.5 mmol / L; mobile phase B is an isopropanol-acetonitrile solution with a volume fraction of isopropanol of 1–10%; the flow rate of the mobile phase is 0.1–0.6 μL / min; Elution method: gradient elution; The gradient elution procedure is as follows: The volume percentage of the mobile phase B is 10-30% for 0-2 minutes. Over 2 to 10 minutes, the volume percentage of the mobile phase B increases uniformly from 10 to 30% to 70 to 100%. The volume percentage of the mobile phase B is 70-100% over 10-13 minutes. During the 13-13.1 min period, the volume percentage of the mobile phase B decreased uniformly from 70-100% to 10-30%. 13.1–15 min, the volume percentage of the mobile phase B is 10–30%; The conditions for the three-stage mass spectrometry detection include: Ion source: Electrospray ion source, negative ion mode; Ionization voltage: 3500~5500V; Ion source temperature: 350~550℃; Spray gas: flow rate 25–45 psi; Auxiliary heating gas: flow rate 35–75 psi; Air curtain gas: flow rate 35-75 psi; Collision air: Pressure is High; Detection method: Level 3 mass spectrometry multiple reaction detection; Level 3 monitoring ions: Estrone: 269.1>145>130.1 and 269.1>253.1>145.2; Estrone- 13 C3 internal standard: 272.1>148>133.

1.

2. The detection method according to claim 1, characterized in that, The volume ratio of the plasma sample to be tested to the lower solvent is 1:1 to 5.

3. The detection method according to claim 1, characterized in that, The shaking extraction speed is 800-1500 rpm, and the time is 1-10 min; The centrifugation speed is 11641-13273 rpm, and the time is 1-8 min.

4. The detection method according to claim 1, characterized in that, The estrone- 13 The concentration of the C3 internal standard solution was 0.1–0.5 μg / mL; The plasma sample to be tested and estrone- 13 The liquid-to-solid ratio of the C3 internal standard is 1 mL: 0.01–0.05 μg.

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