Mass spectrum detection method and system for on-site rapid quantification of steroid hormone

Abstract

The invention provides a mass spectrum detection system and a mass spectrum detection method for on-site rapid quantification of steroid hormones. The detection system comprises: a pretreatment module for pretreating a biological sample to obtain a first sample; the derivatization module is used for carrying out derivatization treatment on the first sample to obtain a second sample; and the capillary electrophoresis-mass spectrometry analysis module is used for sequentially carrying out electrophoretic separation and mass spectrometry detection on the second sample and carrying out qualitative and quantitative analysis on the steroid hormone in the second sample. Therefore, by adopting the detection system disclosed by the invention, the ionization efficiency and the analysis sensitivity of the steroid hormone can be improved. The capillary electrophoresis-mass spectrometry module is adopted, so that the whole analysis process is efficient and rapid, the analysis time is effectively controlled within 10 minutes, and the requirement of instant field detection can be fully met due to the high efficiency.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of mass spectrometry analysis, and in particular, the present application relates to a mass spectrometry detection method and system for rapid on-site quantification of steroid hormones. BACKGROUND

[0002] Steroid hormones are a class of liposoluble hormones derived from cholesterol, which play a wide and key physiological role in the human body, and have an important regulatory role in the life processes such as metabolic regulation, reproductive development, immune anti-inflammatory, etc. Quantitative detection of steroid hormones will help to provide key diagnostic basis for endocrine-related diseases (such as Cushing syndrome, adrenal tumor, precocious puberty, infertility), and optimize the treatment effect through dynamic monitoring, reduce the risk of metabolic abnormalities, osteoporosis and other side effects caused by hormone abuse.

[0003] Mass spectrometry analysis technology has excellent performance in the field of clinical detection of biomarkers, especially in the qualitative and quantitative analysis of low-concentration analytes in complex biological samples, due to its high sensitivity, fast analysis speed and wide application range, and has been widely used in the detection of various biomarkers including steroid hormones. However, the current method for detecting steroid hormones using mass spectrometry technology has many problems such as long analysis process, large instrument system volume, inability to integrate, high requirements for site environment and supporting equipment, etc. These problems seriously restrict the efficiency and accuracy of steroid hormone detection, and cannot realize the rapid on-site detection of steroid hormones, which greatly hinders the timeliness of clinical diagnosis and treatment.

[0004] Therefore, it is necessary to develop a method that can be rapidly and conveniently detected on the clinical site to meet the urgent needs of clinical detection of steroid hormones. SUMMARY

[0005] The present application aims to at least partially solve at least one of the problems in the prior art.

[0006] The present application is based on the following findings of the inventors:

[0007] Due to the relatively low content of steroid hormones in the human body and the low ionization efficiency, in order to improve the sensitivity of mass spectrometric detection of steroid hormones, the steroid hormones are usually derivatized to introduce ionizable groups, such as hydroxylamine and other aminooxy reagents to form oxime derivatives, and the ionization efficiency in positive polarity electrospray ionization is improved. However, excessive derivatization reagents will inhibit the ionization of the target, in order to reduce the influence of matrix effect, usually chromatography-mass spectrometry technology is used to separate the excessive derivatization reagents from the target analyte, and then the mass spectrometric detection is carried out respectively, which greatly improves the sensitivity of mass spectrometric detection. Among them, capillary electrophoresis technology is a liquid phase separation technology with capillary as separation channel and high-voltage direct current electric field as driving force, which can separate each component in the sample according to the difference of electric charge. Compared with liquid chromatography technology, it has the advantages of short analysis time, simple structure, small size, small sample amount and good separation effect, and is very suitable for rapid separation and on-site quantitative detection of steroid hormones.

[0008] In the previous study, the inventor successfully developed a small mass spectrometer (patent number CN109003875A). The mass spectrometer has significant advantages, which can significantly improve the utilization rate of parent ions in each sampling point in each working period, and accurately realize the selective cleavage of parent ions. In the sample analysis process, it can fully and efficiently utilize the parent ions of each sampling point, and then effectively complete the mass spectrometric imaging of isomers on the sample. On this basis, the inventor further developed a small capillary electrophoresis instrument, which can be detachably combined with the aforementioned small mass spectrometer. In addition, the capillary electrophoresis instrument also has a heating function, which can meet the special needs of some samples that need heating treatment. By combining the small capillary electrophoresis instrument with the small mass spectrometer, on-site detection of steroid hormones can be realized. After simple sample pretreatment, the combined system can quickly complete the derivatization of steroid hormones, greatly simplifying the on-site detection process, and the overall analysis time is about 10 minutes, meeting the needs of real-time detection. At the same time, the system effectively separates the excessive derivatization reagents from the derivatized steroid hormones, avoiding the interference of matrix effect on ionization, and significantly improving the sensitivity of on-site mass spectrometric detection.

[0009] In a first aspect, the present application provides a detection system for a steroid hormone. According to an embodiment of the present application, the detection system comprises: a pretreatment module configured to pretreat a biological sample to obtain a first sample; a derivatization module configured to derivatize the first sample to obtain a second sample; and a capillary electrophoresis-mass spectrometry module configured to sequentially perform electrophoretic separation and mass spectrometry detection on the second sample, and to qualitatively and quantitatively analyze the steroid hormone in the second sample. Thus, the present application integrates sample pretreatment, offline derivatization and capillary electrophoresis-mass spectrometry analysis into one system, greatly simplifying the operation process of on-site detection. After sample pretreatment is completed, the derivatization of the steroid hormone can be quickly realized, and the excess derivatization reagent and the derivatized steroid hormone can be effectively separated, thereby avoiding the adverse effects of matrix effect on the ionization of the analyte, and significantly improving the sensitivity of on-site mass spectrometry detection of the steroid hormone. The entire analysis process only takes about 10 minutes, fully meeting the needs of instant on-site detection.

[0010] According to an embodiment of the present application, the capillary electrophoresis-mass spectrometry module comprises: an electrophoretic separation-electrospray ionization module configured to separate each component in the second sample according to different electrophoretic mobilities, and to ionize each separated component to form charged ions; and an analysis module configured to detect the mass-to-charge ratio and intensity of the charged ions of the steroid hormone, and to obtain qualitative or quantitative analysis results based on the charged ions of the steroid hormone.

[0011] According to an embodiment of the present application, the electrophoretic separation-electrospray ionization module further comprises: a sample injection module configured to inject the second sample into a separation module; the separation module configured to separate each component in the second sample, and to send each separated component to a capillary electrophoresis-mass spectrometry interface; and the capillary electrophoresis-mass spectrometry interface configured to ionize each separated component to generate charged ions.

[0012] According to an embodiment of the present application, the pretreatment module comprises at least one of a protein precipitation module, a dried blood spot module, and a liquid-liquid microextraction module.

[0013] According to an embodiment of the present application, the protein precipitation module is configured to sequentially mix and filter the biological sample with a solution containing an internal standard to obtain the first sample.

[0014] According to an embodiment of the present application, the dried blood spot module is configured to drop the biological sample on a paper-based material in a paper-based sampling kit to obtain the first sample.

[0015] According to an embodiment of the present application, the liquid-liquid microextraction module is configured to sequentially mix and extract the biological sample with an extraction solvent containing an internal standard to obtain the first sample.

[0016] According to an embodiment of the present application, the derivatization module comprises a mixing module configured to mix the first sample with a derivatization reagent to obtain a mixture.

[0017] According to an embodiment of the present application, the sample injection module comprises a heating module configured to heat the mixture to obtain a second sample.

[0018] Therefore, in a second aspect, the present application provides a method for detecting a steroid hormone. According to an embodiment of the present application, the method comprises using the detection system of the first aspect to detect a biological sample so as to qualitatively and quantitatively analyze the steroid hormone in the biological sample. Thus, the ionization efficiency and analysis sensitivity of the steroid hormone can be improved by using the detection method of the present application. By using the detection system of the present application, the entire analysis process is efficient and fast, and the analysis time is effectively controlled within 10 minutes. This high efficiency can fully meet the requirements of instant on-site detection.

[0019] According to an embodiment of the present application, in the protein precipitation module, the concentration of the internal standard in the acetonitrile solution is 1 ng / mL-1000 ng / mL.

[0020] According to an embodiment of the present application, in the protein precipitation module, the volume ratio of the biological sample to the acetonitrile solution containing the internal standard is 1:1-1:3.

[0021] According to an embodiment of the present application, in the liquid-liquid microextraction module, the concentration of the internal standard in the extraction solvent is 1 ng / mL-1000 ng / mL.

[0022] According to an embodiment of the present application, the volume ratio of the biological sample to the extraction solvent is 1:1-1:3.

[0023] According to an embodiment of the present application, in the mixing module, the derivatization reagent comprises a quaternary amine aminooxy reagent.

[0024] According to an embodiment of the present application, the derivatization reagent comprises O-(3-trimethylammonium propyl) hydroxylamine.

[0025] According to an embodiment of the present application, in the mixture, the concentration of the derivatization reagent is 0.5-10 mg / mL.

[0026] Additional aspects and advantages of the present application will be given in part in the following description, become apparent from the following description, or be understood by practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0027] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the appended drawings, wherein:

[0028] Figure 1 is a diagram of a steroid hormone detection system according to an embodiment of the present application;

[0029] Figure 2 is a diagram of a capillary electrophoresis-mass spectrometer according to an embodiment of the present application;

[0030] Figure 3 is a diagram of the internal structure of a replaceable capillary electrophoresis separation module according to an embodiment of the present application;

[0031] Figure 4 is a diagram of the mechanism of a capillary electrophoresis-mass spectrometer according to an embodiment of the present application;

[0032] Figure 5 is a flow chart of an experimental method for steroid hormone detection according to an embodiment of the present application;

[0033] Figure 6 is a diagram of tandem mass spectrometry results of a cortisol derivative according to an embodiment of the present application; wherein (a) is a chromatogram of cortisol; and (b) is a mass spectrum of cortisol;

[0034] Figure 7 is a standard curve for quantitative detection of cortisol according to an embodiment of the present application;

[0035] Figure 8 is a detection spectrum of different steroid hormones according to an embodiment of the present application;

[0036] Figure 9 is a diagram of results of cortisol detection using the method (CE-miniMS) and the NanoESI mass spectrometry method (Direct infusion nanoESI) according to an embodiment of the present application, and a comparison diagram. DETAILED DESCRIPTION

[0037] Embodiments of the present application are described in detail below, with the understanding that the present application is not limited to the embodiments described, but rather can be practiced with the scope of the application.

[0038] Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of the application. The appearances of the phrase "in an embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are they necessarily all directed to the same embodiment, or to a single alternative embodiment. It is explicitly contemplated that embodiments described herein can be combined.

[0039] For the sake of brevity, only some numerical ranges are specifically disclosed herein. However, any upper limit can be combined with any lower limit to make a range not expressly recited; and any lower limit can be combined with any other lower limit to make a range not expressly recited, and the same applies to any upper limit, which can be combined with any other upper limit to make a range not expressly recited. Further, each individual disclosed point or single numerical value can be combined with any other point or single numerical value, or with other lower or upper limits, to make a range not expressly recited.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application; the description and drawings of this application and the terms used therein, including the abstract, are not intended to limit the scope of the present application and the patent rights therein, and are included herein solely by way of example and prov ided in no way limits the present application and patent rights therein. The use of any and all examples, or exemplary language (e.g., "such as" and "preferably"), is intended to merely enrich the disclosure and does not indicate any preference or requirement.

[0041] In this document, the terms "comprise" or "comprising" are open- ended, that is, they mean "including, but not limited to," in the sense of "including, but not limited to".

[0042] In this document, the terms "optionally", "optional" or "option" generally mean that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0043] It should be noted that the terms "first", "second" are used only for the purpose of description, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated. Therefore, the features defined with "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present application, unless otherwise stated, the meaning of "a plurality of" is two or more.

[0044] The present application proposes a detection method of a steroid hormone and a detection system of a steroid hormone, which will be described in detail respectively.

[0045] Detection system of a steroid hormone

[0046] In a first aspect of the present application, the present application proposes a detection system of a steroid hormone. According to an embodiment of the present application, the detection system comprises:

[0047] 100: pretreatment module

[0048] In some embodiments of the present application, the pre-processing module is configured to pre-process the biological sample to obtain a first sample. In this way, the pre-processing module can remove interfering substances such as salts, proteins and other components in the biological sample, enrich the target analyte, and simplify the sample matrix, thereby improving the sensitivity, accuracy and reliability of the detection.

[0049] In some embodiments of the present application, the pre-processing module comprises at least one of a protein precipitation module, a dried blood spot module, and a liquid-liquid microextraction module.

[0050] In some embodiments of the present application, the protein precipitation module is configured to sequentially mix and filter the biological sample with an acetonitrile solution containing an internal standard to obtain the first sample.

[0051] In some embodiments of the present application, the concentration of the internal standard in the acetonitrile is 1 ng / mL to 1000 ng / mL. For example, it can be 1 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL, 700 ng / mL, 1000 ng / mL, or a range consisting of any of the above values. In some embodiments of the present application, the volume ratio of the biological sample to the acetonitrile solution containing the internal standard is 1:1 to 1:3. For example, it can be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, or a range consisting of any of the above values.

[0052] In some embodiments of the present application, the dried blood spot module comprises a paper-based sampling kit. In some embodiments of the present application, the dried blood spot module is configured to drop the biological sample on a paper-based material in the paper-based sampling kit to obtain the first sample. In some embodiments of the present application, the paper-based material has an internal standard deposited thereon.

[0053] In some embodiments of the present application, the liquid-liquid microextraction module is configured to sequentially mix and extract the biological sample with an extraction solvent containing an internal standard to obtain the first sample.

[0054] In some embodiments of the present application, the extraction solvent is selected from ethyl acetate, dichloromethane, trichloromethane, methyl tert-butyl ether, etc. In some embodiments of the present application, the concentration of the internal standard in the extraction solvent is 1 ng / mL to 1000 ng / mL. In some embodiments of the present application, the volume ratio of the biological sample to the extraction solvent containing the internal standard is 1:1 to 1:3. For example, it can be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, or a range consisting of any of the above values.

[0055] It should be noted that the selection of the internal standard is not specifically limited in the present application. Those skilled in the art can select the corresponding isotope internal standard according to the specific type of steroid hormone to be detected. For example, when detecting cortisol, the internal standard is selected from the isotope internal standard of cortisol; when detecting corticosterone, the internal standard is selected from the isotope internal standard of corticosterone; when detecting testosterone, the internal standard is selected from the isotope internal standard of testosterone; when detecting estrone, the internal standard is selected from the isotope internal standard of estrone; when detecting progesterone, the internal standard is selected from the isotope internal standard of progesterone; and when detecting dehydroepiandrosterone, the internal standard is selected from the isotope internal standard of dehydroepiandrosterone.

[0056] In some embodiments of the present application, the biological sample is blood, urine, saliva, etc.

[0057] 200: derivatization module

[0058] In some embodiments of the present application, the derivatization module is used to perform derivatization treatment on the first sample to obtain a second sample. In this way, the first sample can be subjected to derivatization treatment by the derivatization module to introduce easily ionizable groups (such as amino, hydroxyl, etc.) on the steroid hormones in the first sample, thereby improving the ionization efficiency of the steroid hormones in mass spectrometric detection, and further enhancing the signal intensity and improving the detection sensitivity.

[0059] In some embodiments of the present application, the derivatization module comprises a mixing module for mixing the first sample with a derivatization reagent to obtain a mixed solution.

[0060] In some embodiments of the present application, the derivatization reagent comprises a quaternary amine aminooxy reaction reagent. In some embodiments of the present application, the derivatization reagent comprises O-(3-trimethylammonium propyl) hydroxylamine. In this way, the hydroxylamine, aniline, etc. in the derivatization reagent can react with the steroid hormones to introduce charged amine groups or hydroxylamine groups. These groups can significantly improve the ionization efficiency of the molecules during electrospray ionization (ESI).

[0061] In some embodiments of the present application, the volume ratio of the first sample to the derivatization reagent is 3:1 to 5:1. For example, it can be 3:1, 3.5:1, 4:1, 4.5:1, 5:1, etc., or it can be a range composed of any of the above values.

[0062] 300: capillary electrophoresis-mass spectrometry analysis module

[0063] In some embodiments of the present application, the capillary electrophoresis-mass spectrometry analysis module is used to sequentially perform electrophoretic separation and mass spectrometry detection on the second sample, and to perform qualitative and quantitative analysis on the steroid hormones in the second sample. In this way, by performing electrophoretic separation and mass spectrometry detection in the capillary electrophoresis-mass spectrometry analysis module, the operation process of on-site detection can be greatly simplified, and the overall analysis time is about 10 minutes, which can meet the needs of instant on-site detection. At the same time, electrophoretic separation can quickly separate excess derivatization reagents from target steroid hormones, avoiding the influence of excess derivatization reagents on the detection of steroid hormones.

[0064] In some embodiments of the present application, the capillary electrophoresis-mass spectrometry analysis module comprises: an electrophoretic separation-electrospray ionization module for separating each component in the second sample according to different electrophoretic mobilities, and ionizing each component after separation to form charged ions; and an analysis module for detecting the mass-to-charge ratio and intensity of the charged ions of the steroid hormones, and obtaining qualitative or quantitative analysis results based on the charged ions of the steroid hormones.

[0065] In some embodiments of the present application, the electrophoretic separation-electrospray ionization module further comprises: a sample injection module for injecting the second sample into a separation module; the separation module for separating each component in the second sample and sending each component after separation to a capillary electrophoresis-mass spectrometry interface; and the capillary electrophoresis-mass spectrometry interface for ionizing each component after separation to generate charged ions.

[0066] It can be understood that the electrophoretic separation-electrospray ionization module in the embodiments of the present application is composed of a sample injection module, a separation module and a capillary electrophoresis-mass spectrometry interface.

[0067] In some embodiments of the present application, the second sample can be injected into the separation module by the injection module. The injection module uses an electro-injection method. After the capillary and electrode are transferred to the sample bottle, a voltage is applied to inject the sample into the capillary by electroosmotic flow. In some embodiments of the present application, the separation module can separate each component in the target analyte and send each separated component to the capillary electrophoresis-mass spectrometry interface. The separation module includes but is not limited to capillary electrophoresis and chip electrophoresis. The separation module is a quartz capillary with a polyimide coating treated by sodium hydroxide solution, with an inner diameter of 50 μm, an outer diameter of 150 μm, and a length of 40 cm. About 1 cm long polyimide coating is removed from both ends by ablation or cutting to expose the internal quartz capillary, avoiding the influence of polyimide coating on the injection process and interface connection. In some embodiments of the present application, the capillary electrophoresis-mass spectrometry interface can ionize each separated component to generate charged ions. The capillary electrophoresis-mass spectrometry interface uses a sheath flow type interface, and the main body is a standard four-way connector with an inner diameter of 1 / 16 inch. The nanoTIP can be connected by a standard fastening head. The nanoTIP is a borosilicate glass tube with an inner diameter of 0.8 mm and an outer diameter of 1.5 mm. The tip is drawn by a needle puller. After drawing, the tip opening diameter is about 5-10 μm, which can ensure normal spraying and avoid tip blockage. The capillary is inserted into the nanoTIP tip through the four-way connector. The other two ends of the four-way connector are electrically connected to the 2kv power supply through the sheath flow liquid.

[0068] In some embodiments of the present application, the injection module includes a heating module for heating the mixed liquid in the mixing module to obtain the second sample.

[0069] In some embodiments of the present application, the injection module in the electrophoretic separation-electrospray ionization module has a heating module, which can be used for heating the mixed liquid to promote the derivatization reaction. In some embodiments of the present application, the temperature range of the heating treatment is 30-70℃. For example, it can be 30℃, 40℃, 50℃, 60℃, 70℃, etc., or it can be a range composed of any of the above values. Thus, the temperature in this range can shorten the reaction time, and the reaction between the derivatization reagent and the steroid hormone can reach equilibrium faster, thereby improving the efficiency of derivatization.

[0070] In some embodiments of the present application, the capillary electrophoresis-mass spectrometry analysis module is a capillary electrophoresis-mass spectrometry analyzer. The electrophoretic separation-electrospray ionization module is a capillary electrophoresis instrument, which can separate each component in the second sample according to different electrophoretic mobilities and ionize each separated component to form charged spray. The analysis module is a mass spectrometry analyzer for detecting the mass-to-charge ratio and intensity of the charged ion steroid hormone, and obtaining qualitative or quantitative analysis results based on the charged ion.

[0071] In some embodiments of the present application, the schematic diagram of the capillary electrophoresis-mass spectrometer is as shown in Figure 2 The capillary electrophoresis-mass spectrometer 1000 of the present application is composed of two parts, including a mass spectrometer 01 and a capillary electrophoresis instrument 02, and the capillary electrophoresis instrument 02 includes a sample inlet 021, a buffer storage tank 022, and a replaceable capillary electrophoresis separation module 023. The sample inlet 021 is used to heat the sample mixed with the derivatization reagent, so as to realize the derivatization of the sample, and at the same time, the sample after derivatization is sent into the capillary electrophoresis instrument 02, and the steroid hormone in the sample after derivatization is separated from the excess derivatization reagent and other components in the sample. The buffer storage tank 022 is used to place the buffer and sheath flow liquid required by capillary electrophoresis, and the replaceable capillary electrophoresis separation module 023 is used to realize the separation of the target analyte and the matrix, and at the same time, the integrated replacement of the capillary consumables is realized.

[0072] In some embodiments of the present application, the internal structure diagram of the replaceable capillary electrophoresis separation module 023 is as shown in Figure 3 The replaceable capillary electrophoresis separation module 023 includes an electrode 0231, a fused quartz capillary microchannel 0232, a sheath flow tube 0233, and an electrospray emitter 0234. The electrode 0231 is used to apply a high-voltage electric field to drive the migration of charged molecules; the fused quartz capillary microchannel 0232 is used to guide and control the flow of liquid; the sheath flow tube 0233 is used to transport the sheath flow liquid; and the electrospray emitter 0234 is used to convert the liquid sample into charged droplets, which is usually a capillary or nozzle with a tip, and by applying a high voltage, the liquid forms charged droplets at the tip, and further evaporates to form charged ions, so as to realize the ionization of the sample.

[0073] In some embodiments of the present application, the capillary electrophoresis instrument 02 is detachably connected with the mass spectrometer 01. In some embodiments of the present application, the capillary electrophoresis instrument 02 is connected with the mass spectrometer 01 through a nanoliter electrospray interface, as shown in Figure 4As shown, the buffer and the sheath flow liquid are pumped into the capillary microchannel and the electrospray emitter under the control of the micro-pump and the micro-electromagnetic valve respectively, to complete the connection of the flow path and the current loop; under the action of the voltage, the electroosmotic flow is formed in the capillary, driving the liquid in the capillary to flow forward, and at the same time, the analyte in the capillary also moves forward at different migration speeds under the action of the electric field force, so as to realize the separation between different substances. After the analyte reaches the outlet of the capillary, it enters the nanoliter electrospray interface tip, wherein the nanoliter electrospray interface is connected with the nanoESI high-voltage source through the sheath flow liquid, so that the nanoliter electrospray interface can form charged droplets of the separated steroid hormone at the capillary tip by applying a high voltage, and these droplets are further evaporated to form charged ions under the action of the electric field, so as to realize the ionization of the steroid hormone. The mass spectrometer detects and analyzes the ionized steroid hormone, and presents the detection and analysis result on the display screen carried by the mass spectrometer.

[0074] Method for detecting steroid hormone

[0075] In a second aspect of the present application, a method for detecting a steroid hormone is provided. According to an embodiment of the present application, the method comprises: detecting a biological sample by using the detection system of the first aspect, so as to qualitatively and quantitatively analyze the steroid hormone in the biological sample.

[0076] In some embodiments of the present application, as shown in Figure 5 The method comprises:

[0077] S100: pretreatment

[0078] In this process, the pretreatment module in the detection system of the first aspect is used to pretreat the biological sample, to obtain a first sample.

[0079] In some embodiments of the present application, the pretreatment comprises at least one of a protein precipitation method, a dried blood spot method and a liquid-liquid microextraction method. In this way, through the pretreatment, the interfering substances such as salt, protein and other components in the biological sample can be removed, the target analyte can be enriched, and the sample matrix can be simplified, so as to improve the sensitivity, accuracy and reliability of the detection.

[0080] In some embodiments of the present application, the protein precipitation method comprises: mixing the biological sample with an acetonitrile solution containing an internal standard, and filtering to obtain the first sample. In some embodiments of the present application, the protein precipitation method is performed in the protein precipitation module in the detection system of the first aspect.

[0081] In some embodiments of the present application, the concentration of the internal standard in the acetonitrile solution is 1 ng / mL-1000 ng / mL. For example, it can be 1 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL, 700 ng / mL, 1000 ng / mL, or a range consisting of any of the above values. In some embodiments of the present application, the volume ratio of the biological sample to the acetonitrile solution containing the internal standard is 1:1-1:3. For example, it can be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, or a range consisting of any of the above values.

[0082] In some embodiments of the present application, the dried blood spot method is performed using a paper-based sampling kit. In some embodiments of the present application, the dried blood spot method comprises: dropping the biological sample on a paper-based material in the paper-based sampling kit, the paper-based material having an internal standard deposited thereon; and drying the paper-based material with the biological sample dropped thereon to obtain the first sample. In some embodiments of the present application, the dried blood spot method is performed in the dried blood spot module in the detection system of the first aspect.

[0083] In some embodiments of the present application, the liquid-liquid microextraction method comprises: sequentially mixing and extracting the biological sample with an extraction solvent containing an internal standard to obtain the first sample. In some embodiments of the present application, the liquid-liquid microextraction method is performed in the liquid-liquid microextraction module in the detection system of the first aspect.

[0084] In some embodiments of the present application, the extraction solvent is selected from ethyl acetate, dichloromethane, trichloromethane, methyl tert-butyl ether, etc. In some embodiments of the present application, the concentration of the internal standard in the extraction solvent is 1 ng / mL-1000 ng / mL. In some embodiments of the present application, the volume ratio of the biological sample to the extraction solvent containing the internal standard is 1:1-1:3. For example, it can be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, or a range consisting of any of the above values.

[0085] S200: Derivatization treatment

[0086] In this process, the derivatization module in the detection system of the first aspect is used to perform derivatization treatment on the first sample obtained after pretreatment to obtain a second sample. Thus, through derivatization treatment, the first sample can be derivatized to introduce easily ionizable groups (such as amino, hydroxyl, etc.) on the steroid hormones in the first sample, thereby improving the ionization efficiency of the steroid hormones in mass spectrometric detection, and further enhancing the signal intensity and improving the detection sensitivity.

[0087] In some embodiments of the present application, the derivatization process comprises mixing the first sample with a derivatization reagent, and heating the mixture to obtain a second sample. In some embodiments of the present application, the mixing is performed in a mixing module in the detection system of the first aspect. In some embodiments of the present application, the heating is performed in a heating module in the detection system of the first aspect.

[0088] In some embodiments of the present application, the derivatization reagent comprises a quaternary amine aminooxy reagent. In some embodiments of the present application, the derivatization reagent comprises O-(3-trimethylammonium propyl)hydroxylamine. In this way, the hydroxylamine, aniline, etc. in the derivatization reagent can be chemically reacted with the steroid hormone to introduce a charged amine group or hydroxylamine group. These groups can significantly improve the ionization efficiency of the molecule during electrospray ionization (ESI).

[0089] In some embodiments of the present application, the volume ratio of the first sample to the derivatization reagent is 3:1 to 5:1. For example, it can be 3:1, 3.5:1, 4:1, 4.5:1, 5:1, etc., or it can be a range composed of any of the above values.

[0090] In some embodiments of the present application, the heating temperature is 30°C to 70°C. For example, it can be 30°C, 40°C, 50°C, 60°C, 70°C, etc., or it can be a range composed of any of the above values. In this way, the temperature in this range can shorten the reaction time, so that the reaction between the derivatization reagent and the steroid hormone reaches equilibrium faster, thereby improving the efficiency of derivatization.

[0091] S300: electrophoretic separation and mass spectrometric detection

[0092] In this process, the capillary electrophoresis-mass spectrometry module in the detection system of the first aspect is used to sequentially perform electrophoretic separation and mass spectrometric detection on the second sample obtained after derivatization, and to perform qualitative and quantitative analysis on the steroid hormone in the second sample. In this way, by performing electrophoretic separation and mass spectrometric detection in the capillary electrophoresis-mass spectrometry module, the operation process of on-site detection can be greatly simplified, and the overall analysis time is about 10 minutes, which can meet the needs of instant on-site detection. At the same time, electrophoretic separation can quickly separate excess derivatization reagent from the target steroid hormone, avoiding the influence of excess derivatization reagent on the detection of the steroid hormone.

[0093] Examples of the present application are described in detail below. The examples described below are exemplary only and are not intended to be limiting of the present application. Unless otherwise defined, scientific and technical terms used in the examples have the meanings commonly understood by one of ordinary skill in the art in the field of the present application. The examples are not intended to be limiting of the present application. Unless otherwise indicated, the techniques and procedures used to characterize and / or measure the results of the examples are those that are conventional in the art to which the present application pertains. The reagents or instruments used in the examples are conventional products available on the market unless otherwise indicated.

[0094] Example 1: Quantitative determination of cortisol in blood using steroid hormone field rapid quantitative mass spectrometry detection system

[0095] 1. 100 μL blood was added to a container containing 200 μL of acetonitrile solution in which 10 ng / mL cortisol-d7 was dissolved, mixed, and then a filter dropper was attached to filter the supernatant;

[0096] 2. The supernatant obtained in the previous step was dropped into a reaction container containing 100 μL of methanol solution in which 10 mg / mL of derivatization reagent (O-(3-trimethylammonium propyl) hydroxylamine) was dissolved, and 5% acetic acid was added;

[0097] 3. The reaction container was placed in a mixing and heating device and reacted at 60°C for 5 min;

[0098] 4. The sample after reaction was introduced into the capillary electrophoresis separation module of the portable capillary electrophoresis-mass spectrometry analyzer using electrospray (20 kV, 30 s), and a 20 kV electrophoresis separation voltage was applied to achieve separation within 3 min;

[0099] 5. The small mass spectrometry system simultaneously performed tandem mass spectrometry analysis on the cortisol derivative (i.e. the product after the reaction of cortisol and derivatization reagent) and the cortisol-d7 derivative (as shown in Figure 6 ). Among them Figure 6 a is the analysis chromatogram of the cortisol derivative, indicating that the separation of the cortisol derivative can be achieved within 3 min, Figure 6 b is the analysis mass spectrum of 0.1 ng / mL cortisol, indicating that the method can achieve high-sensitivity analysis of sub-ppb level cortisol.

[0100] The isotopic internal standard method (i.e. the ratio of analyte to internal standard in a series of samples with known concentrations is determined, and a standard curve is fitted) was used to determine the standard curve of cortisol, as shown in Figure 7 , indicating that the method of the present application can achieve accurate quantification of cortisol, and the linear range can meet the detection requirements.

[0101] In summary, it is shown that the method of the present application can achieve rapid field quantitative analysis of steroid hormones in about 10 min, and the results are accurate.

[0102] Example 2: Highly sensitive qualitative detection of multiple steroid hormones in blood using a steroid hormone on-site rapid mass spectrometry detection system

[0103] In addition to quantitative detection of cortisol, this system is also applicable to multiple types of steroid hormones containing carbonyl groups, including corticosterone, dehydroepiandrosterone, testosterone, progesterone, estrogen, etc.

[0104] Sample pretreatment, derivatization and capillary electrophoresis-mass spectrometry detection were performed in a similar manner to Example 1. The spectra of various steroid hormones were as follows: Figure 8 As shown, samples containing a series of analyte concentrations were measured to determine the analyte concentration at which the signal-to-noise ratio was 3, which was the detection limit. The detection limits of the analytes were as follows: testosterone—0.15 ng / mL, progesterone—0.79 ng / mL, estrone—3.39 ng / mL, corticosterone—4.99 ng / mL, and dehydroepiandrosterone—5.1 ng / mL.

[0105] This demonstrates that the method of the present invention can greatly improve the sensitivity of on-site detection of steroid hormones and achieve multi-target high-sensitivity detection.

[0106] Comparative Example 1

[0107] The blood was pre-treated and derivatized according to steps 1-3 in Example 1, and the derivatized sample was detected by NanoESI mass spectrometry. That is, the sample was directly infused into the NanoESI mass spectrometer. The mass spectrum of 10 ng / mL cortisol in the blood matrix was as follows: Figure 9 As shown, while this method can also detect steroid hormones, the method of the present invention has an analyte intensity improvement of more than 30 times compared to the method of the present invention. This shows that the method of the present invention has the advantage of high sensitivity compared to the existing technology.

[0108] Comparative Example 2

[0109] Steroid hormones were detected using the LC-MS method. The specific steps are as follows:

[0110] 1. Add 200 μL of ethyl acetate to 100 μL of blood and mix for 3 minutes;

[0111] 2. Place the sample in a centrifuge, centrifuge at 10,000 rpm for 3 minutes, and collect the supernatant;

[0112] 3. Place the supernatant in a nitrogen drying apparatus and dry it for about 10 minutes.

[0113] 4. Add 100 μL of methanol to the dried sample, place it in a vortex instrument, and re-dissolve for 5 minutes;

[0114] 5. The sample is put into LC-MS system, and LC-MS detection is carried out, and the required time is about 15 min.

[0115] The total time consumption is about 30-40 min, and the support of vortex instrument, centrifuge, nitrogen blowing and other equipment is required, the LC-MS equipment is huge in size, and the analysis process operation is complex, and the equipment has high requirements on the environment.

[0116] Therefore, it is illustrated that the method of the application has the advantages of portability, simple process and fast analysis speed compared with the prior art.

[0117] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. Furthermore, the different embodiments or examples described in the present specification and the features of the different embodiments or examples can be combined and combined by those skilled in the art without contradiction.

[0118] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.

Claims

1. A steroid hormone detection system, characterized in that: The detection system comprises: A pre-processing module, used for pre-processing the biological sample to obtain a first sample; a derivatization module, configured to perform a derivatization treatment on the first sample to obtain a second sample; The capillary electrophoresis-mass spectrometry analysis module is used to sequentially perform electrophoresis separation and mass spectrometry detection on the second sample, and perform qualitative and quantitative analysis on the steroid hormones in the second sample.

2. The detection system according to claim 1, characterized in that The capillary electrophoresis-mass spectrometry analysis module includes: an electrophoretic separation-electrospray ionization module, for separating the components in the second sample according to their electrophoretic mobility, and ionizing the separated components to form charged ions; The analysis module is used to detect the mass-to-charge ratio and intensity of the steroid hormone charged ions, and obtain qualitative or quantitative analysis results based on the steroid hormone charged ions.

3. The detection system according to claim 2, characterized in that The electrophoresis separation-electrospray ionization module further comprises: an injection module, configured to inject the second sample into the separation module; The separation module is used to separate the components in the second sample and send the separated components to the capillary electrophoresis-mass spectrometry interface; The capillary electrophoresis-mass spectrometry interface is used to ionize the separated components to generate charged ions.

4. The detection system according to claim 3, characterized in that The pre-processing module includes at least one of a protein precipitation module, a dried blood spot module, and a liquid-liquid microextraction module.

5. The detection system according to claim 4, characterized in that: The protein precipitation module is used to sequentially mix and filter the biological sample and an acetonitrile solution containing an internal standard to obtain the first sample; Optionally, the dried blood spot module is used to drop the biological sample onto a paper-based material in a paper-based sampling kit to obtain the first sample; Optionally, the liquid-liquid microextraction module is used to sequentially mix and extract the biological sample with an extraction solvent containing an internal standard to obtain the first sample.

6. The detection system according to any one of claims 1 to 5, characterized in that: The derivatization module includes: The mixing module is used to mix the first sample with a derivatization reagent to obtain a mixed solution.

7. The detection system according to claim 6, characterized in that The injection module comprises: The heating module is used to heat the mixed liquid to obtain a second sample.

8. A method for detecting steroid hormones, characterized in that: include: A biological sample is detected using the detection system according to any one of claims 1 to 7, so as to perform qualitative and quantitative analysis of the steroid hormones in the biological sample.

9. The detection method according to claim 8, characterized in that In the protein precipitation module, the concentration of the internal standard in the acetonitrile solution is 1 ng / mL to 1000 ng / mL; optionally, the volume ratio of the biological sample to the acetonitrile solution containing the internal standard is 1:1 to 1:3; Optionally, in the liquid-liquid microextraction module, the concentration of the internal standard in the extraction solvent is 1 ng / mL to 1000 ng / mL; optionally, the volume ratio of the biological sample to the extraction solvent containing the internal standard is 1:1 to 1:

3.

10. The detection method according to claim 8, characterized in that In the mixing module, the derivatization reagent includes a quaternary amine aminooxy reactive reagent; Optionally, the derivatization reagent comprises O-(3-trimethylammoniumpropyl)hydroxylamine; Optionally, in the mixed solution, the concentration of the derivatization reagent is 0.5-10 mg / mL.

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