Method for preparing a steroid hormone blank matrix serum

Abstract

The present application relates to a preparation method of a steroid hormone blank matrix serum. The present application specifically relates to a preparation method of a steroid hormone blank matrix serum, comprising: activating HLB magnetic beads with an ammonium acetate methanol solution; extracting steroid hormones existing in serum by mixing the serum with the activated HLB magnetic beads; and collecting a first supernatant by applying a magnetic field to adsorb the HLB magnetic beads. The present application also relates to a method for extracting steroid hormones in serum and a method for regenerating HLB magnetic beads for extracting steroid hormones. The method of the present application is simple, fast and safe to operate; can meet the needs of automatic equipment, and is especially suitable for automatic mass production; and the prepared blank matrix serum is suitable for use as a blank control or a blank diluent in detection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pretreatment of steroid hormone detection, and particularly relates to a preparation method of a blank matrix serum of a steroid hormone and an extraction method of a steroid hormone. BACKGROUND

[0002] Steroid hormones, also known as steroidal hormones, are lipophilic small molecule compounds, and are various in types. According to the receptors combined, the steroid hormones can be divided into two categories: sex hormones (including estrogen, androgen and progestogen) and corticosteroids (including glucocorticoids and mineralocorticoids). The steroid hormones play an important role in regulating human metabolism, maintaining the stability of the internal environment, growth, development and reproductive behavior. The steroid hormones are related to many endocrine diseases in clinical diagnosis, such as congenital adrenal hyperplasia, multiple ovarian syndrome, primary aldosterone hyperplasia, Cushing's syndrome and precocious puberty, and the auxiliary role of the steroid hormones in clinical detection is increasingly prominent.

[0003] At present, the traditional immunological method for detecting steroid hormones has low automation degree, poor specificity and poor anti-interference ability, and it is difficult to realize accurate quantitative detection. With the development of mass spectrometry, the isotope dilution liquid chromatography-tandem mass spectrometry method has become a powerful tool for detecting steroid hormones in the clinic, because it has high specificity and can simultaneously detect dozens of steroid hormones for accurate quantification. Therefore, more and more in vitro diagnostic manufacturers have launched corresponding mass spectrometry products.

[0004] The blank matrix serum refers to serum containing a small amount or almost no analyte (for example, a steroid hormone) after treatment, but retaining the basic serum matrix. The blank matrix serum is often used as a blank control or blank diluent in the clinic. In mass spectrometry detection, the blank matrix serum can eliminate matrix effects due to its close characteristics to clinical samples, so as to ensure the accuracy and stability of the results, and is widely used in the preparation of calibrators and quality control samples. However, the steroid hormone is an endogenous substance, and is often combined with proteins in vivo. Therefore, how to eliminate the analyte such as the steroid hormone while retaining the original serum matrix to the greatest extent to prepare the blank matrix serum is a key problem focused by the industry.

[0005] Currently, the preparation of blank matrix serum adopts active carbon adsorption and liquid-liquid extraction extraction mode, which has problems such as complex preparation, strong toxicity or great influence on serum matrix. For example, in one scheme, a preparation method of steroid hormone negative serum is provided, which adopts methyl tert-butyl ether extraction mode, and the operation is complex and not easy to be automatically prepared. In addition, methyl tert-butyl ether is flammable, explosive and corrosive, and the danger degree is high. In another scheme, a preparation method of lipid-soluble vitamin negative serum is provided, which adopts liquid-liquid extraction mode to remove lipid-soluble vitamins, but does not involve steroid hormones, and needs twice liquid-liquid extraction, which is complex in operation. In addition, dichloromethane and n-hexane have strong toxicity. In another scheme, a preparation method of blank blood matrix and application thereof are provided, which adopts active carbon and montmorillonite as adsorbents, and the adsorption capacity of active carbon is strong, which can greatly affect the blood matrix.

[0006] Therefore, there is an urgent need for a preparation method of blank matrix serum which is simple, efficient and safe to operate. SUMMARY

[0007] The technical problem to be solved by the present application is how to quickly, simply and safely prepare a steroid hormone blank matrix serum.

[0008] Accordingly, the present application provides a preparation method of a steroid hormone blank serum matrix, which uses magnetic beads as a solid carrier to prepare a steroid hormone blank serum. It is found that using a specific ammonium acetate methanol as an activating agent for the activated magnetic beads (and eluted magnetic beads) can improve the recovery rate of steroid hormones while retaining the main components in the serum, thereby completing the present application.

[0009] Specifically, the technical solutions adopted by the present application to solve its technical problems are as follows.

[0010] The first aspect of the present application provides a preparation method of a steroid hormone blank matrix serum, comprising: activating HLB magnetic beads with ammonium acetate methanol solution as activating liquid; removing the activating liquid; extracting steroid hormones existing in the serum by mixing the serum with the activated HLB magnetic beads; and applying a magnetic field to adsorb the HLB magnetic beads and collecting the first supernatant.

[0011] In some embodiments, the preparation method further comprises: after collecting the first supernatant, eluting the HLB magnetic beads with ammonium acetate methanol solution; extracting the steroid hormones possibly existing in the first supernatant by mixing the eluted magnetic beads with the first supernatant; and applying a magnetic field to adsorb the HLB magnetic beads and collecting the second supernatant.

[0012] In some embodiments, the concentration of ammonium acetate in the ammonium acetate methanol solution is 40 mM to 60 mM.

[0013] In some embodiments, the step of extracting lasts for 1 minute to 5 minutes.

[0014] The preparation method of the steroid hormone blank matrix serum of the present application can separate the target compound steroid hormone from the serum matrix with high extraction recovery rate by using the methanol solution containing ammonium acetate for activation (and elution) in the process of treating the serum with magnetic beads as the solid phase carrier, and more importantly, can retain the main components such as proteins, carbohydrates and lipids, thereby obtaining a blank control or blank diluent suitable for use in detection.

[0015] In another aspect, the preparation method of the present application only uses ammonium acetate as an additive of the activation solution (and elution solution) on the basis of the magnetic bead extraction method, and the preparation method is simple, fast and safe to operate.

[0016] In another aspect, in the case of using ammonium acetate as an additive, the preparation method of the present application not only eliminates the traditional hydration balance step, but also can use the ion characteristics contained in the activation solution to obtain the sample liquid level in real time, thereby realizing automatic filling and being particularly suitable for automatic mass production.

[0017] The second aspect of the present application provides a method for extracting steroid hormones in serum, comprising: activating HLB magnetic beads with ammonium acetate methanol solution as an activation solution; removing the activation solution; extracting the steroid hormones present in the serum by mixing the serum with the activated HLB magnetic beads; adsorbing the HLB magnetic beads by applying a magnetic field and discarding the supernatant; washing the HLB magnetic beads with a washing solution; and eluting with ammonium acetate methanol solution, adsorbing the HLB magnetic beads by applying a magnetic field, and collecting the supernatant.

[0018] In some embodiments, the washing solution is selected from water or 1-10% methanol aqueous solution.

[0019] In some embodiments, in the step of activation, the HLB magnetic beads are regenerated HLB magnetic beads.

[0020] In some embodiments, the regenerated magnetic beads are obtained by sequentially treating the used HLB magnetic beads with the elution step and the washing step.

[0021] In some embodiments, the concentration of ammonium acetate in the ammonium acetate methanol solution is 40mM to 60mM.

[0022] The method for extracting steroid hormones of the present application significantly improves the extraction efficiency of steroid hormones by selecting ammonium acetate as an additive of the activation solution and the elution solution, can efficiently extract steroid hormones in serum samples, and thereby improves the accuracy and sensitivity in liquid chromatography tandem mass spectrometry detection.

[0023] In another aspect, the extraction method of the present application only uses ammonium acetate as an additive of the activating solution (and eluting solution) on the basis of the magnetic bead extraction method, and is simple, fast and safe, and suitable for large-scale extraction.

[0024] In a third aspect, the present application provides a method for regenerating HLB magnetic beads for extracting steroid hormones, comprising: mixing ammonium acetate methanol solution with HLB magnetic beads, applying a magnetic field to adsorb the HLB magnetic beads, and discarding the supernatant.

[0025] In some embodiments, the concentration of ammonium acetate in the ammonium acetate methanol solution is 40 mM to 60 mM.

[0026] In the magnetic bead regeneration method of the present application, ammonium acetate is selected as an additive of the eluting solution, and the operation is simple and fast.

[0027] In another aspect, in the extraction process of steroid hormones, the magnetic bead regeneration method of the present application can efficiently extract steroid hormones and make the magnetic beads reusable, thereby reducing the cost of the method.

[0028] In another aspect, the magnetic bead regeneration method of the present application can regenerate the magnetic beads after extracting steroid hormones, so as to be repeatedly used in the extraction process of steroid hormones of the sample and / or other samples or the preparation process of matrix serum, thereby reducing the cost of these methods. BRIEF DESCRIPTION OF DRAWINGS

[0029] The accompanying drawings, which are included to provide a further understanding of the embodiments of the present application and constitute a part of the present application, do not constitute a limitation to the embodiments of the present application, and illustrate:

[0030] Figure 1 The chromatogram of each steroid hormone detected in Example 1.

[0031] Figure 2 The schematic diagram of the preparation method of the steroid hormone blank matrix serum according to an embodiment of the present application.

[0032] Figure 3 The chromatogram of seven steroid hormones detected in Example 4. DETAILED DESCRIPTION

[0033] The technical solutions of the present application will be described in detail below with reference to specific embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0034] 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 following definitions are supplemental to those above and are intended to apply uniformly throughout this application, unless an otherwise expressly different definition is anticipated to apply. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present application, the preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0035] The terms "comprise", "comprising", "containing", "having" in the present application are open-ended descriptions, containing the specified steps described, and other steps that do not materially affect are optional and not excluded. When used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid can be composed of the sequence, or at one end or both ends of the protein or nucleic acid, additional amino acids or nucleotides can be added, but still have the same or similar activity as the original sequence.

[0036] Unless specifically stated, the use of the terms "first", "second" or the like in the present application is not intended to limit the number of elements to which the terms apply, but is intended to identify those elements in a manner that is otherwise particularly descriptive. Thus, such terms are used in conjunction with specific embodiments but are not intended to limit the application of the application to a single or particular set of embodiments described.

[0037] As described above, in view of the defects of the existing preparation method of blank matrix serum, such as complex operation, large loss of blood matrix and strong solvent toxicity, the present application provides a preparation method of a steroid hormone blank serum matrix.

[0038] Specifically, the present application provides a preparation method of a steroid hormone blank serum matrix, comprising: activating HLB magnetic beads with ammonium acetate methanol solution as an activating solution; removing the activating solution; extracting steroid hormones existing in the serum by mixing the serum with the activated HLB magnetic beads; and collecting a first supernatant by applying a magnetic field to adsorb the HLB magnetic beads.

[0039] The preparation method of the present application is simple, fast and safe, and can meet the needs of automatic equipment, especially suitable for automatic mass production; at the same time, the prepared blank matrix serum has less steroid hormone content, but basically retains the main components such as protein, sugar and lipid, and is suitable for use as a blank control or blank diluent in detection.

[0040] The preparation method of the steroid hormone blank matrix serum of the present application comprises activating HLB magnetic beads with ammonium acetate methanol solution.

[0041] In this application, HLB magnetic beads are hydrophilic-lipophilic balance water-wettable reversed-phase magnetic solid phase extraction agents, which contain hydrophilic and hydrophobic groups on the surface, and can efficiently capture and enrich trace amounts of target analytes in samples.

[0042] In some embodiments, the HLB magnetic beads can be used at a concentration that is conventional for solid phase extraction of serum (for steroid hormones). For example, the concentration of HLB magnetic beads can be 7.5 mg / mL to 12.5 mg / mL, such as 7.5 mg / mL, 8.0 mg / mL, 8.5 mg / mL, 9.0 mg / mL, 9.5 mg / mL, 10.0 mg / mL, 10.5 mg / mL, 11.0 mg / mL, 11.5 mg / mL, 12.0 mg / mL, 12.5 mg / mL.

[0043] It can be understood that, generally, as the concentration of magnetic beads increases, the extraction and detection of steroid hormones first shows an overall upward trend. When the concentration of magnetic beads is too high, some of the compounds to be tested cannot be fully eluted due to the increase in the concentration of magnetic beads, resulting in a decrease in the detection efficiency of steroid hormones. A person skilled in the art can determine a suitable concentration of magnetic beads. For example, in the present application, the concentration of HLB magnetic beads is selected to be 10.0 mg / mL.

[0044] In the activation step of the present application, ammonium acetate methanol solution is used as the activation solution, which can improve the extraction efficiency of various steroid hormones, so that the content of steroid hormones in the prepared blank matrix serum is very low. Compared with the methanol solution, the addition of ammonium acetate in the solution causes the presence of ions in the solution, which can be used to judge the inventory of the solution through the ion concentration, so that the preparation process can meet the needs of automatic equipment, especially suitable for automatic mass production.

[0045] In some embodiments, the concentration of ammonium acetate in the ammonium acetate methanol solution is 40 mM to 60 mM. For example, the concentration of ammonium acetate in the ammonium acetate methanol solution can be 40 mM, 42 mM, 44 mM, 46 mM, 48 mM, 50 mM, 52 mM, 54 mM, 56 mM, 58 mM or 60 mM.

[0046] In a specific embodiment, the activation step comprises, after activating the HLB magnetic beads with ammonium acetate methanol solution, applying a magnetic field to adsorb the HLB magnetic beads, and discarding the supernatant.

[0047] The preparation method of the steroid hormone blank matrix serum of the present application comprises removing the activation solution ammonium acetate methanol after activation. Ammonium acetate methanol can activate magnetic beads and also can be used as an eluent. Therefore, in order to make the magnetic beads "adsorb" more steroid hormones and at the same time avoid the pollution of ammonium acetate methanol, it is necessary to remove the ammonium acetate methanol when preparing the blank matrix serum. The preparation method of the steroid hormone blank matrix serum of the present application comprises extracting the steroid hormones present in the serum by mixing the serum with the activated HLB magnetic beads.

[0048] In the method of the present application, the mixing of the serum with the activated HLB magnetic beads can include continuous and sufficient mixing of the two during the extraction process (e.g. by flicking, shaking, stirring, etc.), or can include incubation of the mixture for a period of time.

[0049] In some embodiments, the duration of the step of extracting can be 1 minute to 5 minutes, for example 1 minute, 2 minutes, 3 minutes, 4 minutes or 5 minutes.

[0050] The method for preparing the steroid hormone blank matrix serum of the present application includes applying a magnetic field to adsorb the HLB magnetic beads, and collecting a first supernatant.

[0051] In some embodiments, the first supernatant is a steroid hormone blank matrix serum.

[0052] In other embodiments, the method for preparing further includes: after collecting the first supernatant, eluting the HLB magnetic beads with an ammonium acetate methanol solution; extracting possible steroid hormones in the first supernatant by mixing the eluted magnetic beads with the first supernatant; and applying a magnetic field to adsorb the HLB magnetic beads and collecting a second supernatant.

[0053] In these embodiments, the second supernatant is a steroid hormone blank matrix serum.

[0054] In the method for preparing of the present application, by using the ammonium acetate methanol solution to elute the extracted HLB magnetic beads, the content of the extracted steroid hormones on the magnetic beads is very low after elution, and the magnetic beads can be reused for extracting steroid hormones in the sample, thereby completing the regeneration of the magnetic beads and reducing the cost of the method.

[0055] In the method for preparing of the present application, mixing the eluted magnetic beads with the first supernatant can further extract possible residual steroid hormones in the first supernatant, further reducing the content of steroid hormones in the prepared blank matrix serum. In the case of using the regenerated magnetic beads to extract the first supernatant, since the eluent has the same composition as the activation solution, the regenerated magnetic beads do not need to be activated before extraction, thereby further simplifying the method.

[0056] In some embodiments, the various methods of the present application do not include the process of using a balancing solution to balance the activated magnetic beads. Illustratively, the activation solution is water or a buffer solution. Illustratively, the balancing step occurs after the magnetic bead activation step. Illustratively, the step of extracting the substance to be detected by the magnetic beads occurs after the balancing step.

[0057] It can be understood that, in general, a balance solution is needed to create a suitable working environment for the magnetic beads after activation and to remove possible residual impurities. Surprisingly, in the case of adding ammonium acetate as an activating agent, even if the activated magnetic beads are not balanced, the extraction effect of the magnetic beads and the retention of the serum matrix are successfully achieved. More importantly, the omission of the balancing step makes it possible to monitor the liquid surface in real time by salt ions, thereby enabling the automation of the method.

[0058] The second aspect of the present application provides a method for extracting steroid hormones in serum, comprising: activating HLB magnetic beads with an ammonium acetate methanol solution as an activating solution; removing the activating solution; extracting steroid hormones present in the serum by mixing the serum with the activated HLB magnetic beads; adsorbing the HLB magnetic beads by applying a magnetic field and discarding the supernatant; eluting the HLB magnetic beads with an elution solution; and eluting with an ammonium acetate methanol solution, adsorbing the HLB magnetic beads by applying a magnetic field, and collecting the supernatant.

[0059] The method for extracting steroid hormones in serum of the present application has high extraction efficiency and can efficiently extract steroid hormones in serum samples, while being simple, fast and safe to operate, and suitable for large-scale extraction.

[0060] The method for extracting steroid hormones in serum of the present application has the same first step as the method for preparing a blank matrix serum of the first aspect of the present application, and the difference lies in that after extracting the steroid hormones in the serum, the supernatant is discarded and the HLB magnetic beads are subjected to subsequent treatment. Thus, the method for extracting steroid hormones of the present application also has the advantages brought by the characteristics before the extraction step in the preparation method of the present application.

[0061] In the method for extracting steroid hormones in serum of the present application, the HLB magnetic beads after extraction are subjected to elution, which can remove impurities such as ions on the surface of the magnetic beads and reduce the influence on subsequent mass spectrometric detection.

[0062] In some embodiments, the elution solution is selected from water or a 1-10% methanol aqueous solution.

[0063] In specific embodiments, the elution is performed once.

[0064] In specific embodiments, the elution solution is water.

[0065] In the method for extracting steroid hormones in serum of the present application, ammonium acetate methanol solution is used for elution, and the eluted magnetic beads are regenerated and can be reused for the extraction of steroid hormones. In this process, the elution solution and the activating solution use the same ammonium acetate methanol solution, so that the elution and activation processes of the magnetic beads can be completed at the same time, the operation is more simple and fast, and the obtained magnetic beads can be directly used for the extraction of steroid hormones, which is more conducive to the large-scale application of the method.

[0066] In some embodiments, in the step of activation, the HLB magnetic beads are regenerated HLB magnetic beads.

[0067] In some embodiments, the regenerated magnetic beads are obtained by treating the used HLB magnetic beads with the step of elution.

[0068] Alternatively, the present application provides a pre-treatment method for detecting steroid hormones by liquid chromatography tandem mass spectrometry, comprising the steps of the method for extracting steroid hormones from serum of the present application.

[0069] In some embodiments, the serum sample solution is detected together with the internal standard solution in the process of detecting steroid hormones by liquid chromatography tandem mass spectrometry.

[0070] The third aspect of the present application provides a method for regenerating HLB magnetic beads for extracting steroid hormones, comprising: mixing ammonium acetate methanol solution with HLB magnetic beads, applying a magnetic field to adsorb the HLB magnetic beads, and discarding the supernatant.

[0071] The magnetic bead regeneration method of the present application is simple and fast, and in the process of extracting steroid hormones, the magnetic beads can be reused while efficiently extracting steroid hormones, thereby reducing the cost of the method.

[0072] In the magnetic bead regeneration method of the present application, the ammonium acetate methanol solution mixed with the HLB magnetic beads is the same ammonium acetate methanol solution as the activation solution, so that the elution and activation processes of the magnetic beads can be completed at the same time, and the operation is more simple and fast.

[0073] The following is a preferred embodiment of the present application, and the present application is not limited to the following preferred embodiment. It should be noted that, for those skilled in the art, on the basis of the inventive concept, several modifications and improvements made, all belong to the protection scope of the present application. The reagents used are not specified by the manufacturer, and are conventional products that can be obtained from the market.

[0074] Example 1: Extraction and detection of steroid hormones in serum

[0075] Method:

[0076] The HLB magnetic bead filler dry powder was prepared into a suspension of 100 mg / mL with methanol containing 50 mM ammonium acetate, and activated for 2 minutes;

[0077] 20 μL of the activated suspension was taken, and the ammonium acetate methanol liquid was removed by applying an external magnetic field to obtain activated HLB magnetic bead solids;

[0078] The sample to be tested (200 μL of serum and 100 μL of isotope internal standard solution) was added to the activated magnetic beads and mixed, and the activated HLB magnetic beads were used to extract steroid hormones for 2 minutes, a magnetic field was applied to adsorb the HLB magnetic beads, and the supernatant was removed;

[0079] Use 1 mL of ultrapure water to rinse the HLB magnetic beads, apply a magnetic field to adsorb the HLB magnetic beads, and remove the supernatant;

[0080] Add 300 μL of 50 mM ammonium acetate methanol solution to elute the HLB magnetic beads for 2 minutes, apply a magnetic field to adsorb the HLB magnetic beads, collect the supernatant, and use LC-MS / MS to detect steroid hormones. The obtained magnetic beads are reused for extracting steroid hormones.

[0081] The parameters used in liquid chromatography are as follows:

[0082] ACQUITY UPLC BEH C18 column (2.1 x 50 mm, 1.7 μm, Waters, USA), sample volume 10 μL, column temperature 40℃, mobile phase A 0.2 mmol / L ammonium fluoride solution, mobile phase B methanol gradient elution, flow rate 0.3 mL / min, see Table 1 below for details.

[0083] Table 1

[0084]

[0085] The mass spectrometry parameters are as follows: using electrospray ionization source (ESI) positive ion mode and multiple reaction monitoring mode (MRM) analysis, high-purity nitrogen as the desolvation gas, desolvation gas temperature 400℃, desolvation gas flow rate 900 L / h, high-purity argon as the collision gas, collision gas flow rate 0.15 mL / min, source temperature 150℃, and the multiple reaction monitoring ion pair parameters of steroid hormones and internal standards are shown in Table 2.

[0086] Table 2

[0087]

[0088] Results: The chromatograms of various steroid hormones are shown in Figure 1 It can be seen that each type of steroid hormone has a distinct characteristic peak, indicating that each type of steroid hormone has been successfully extracted and detected.

[0089] Example 2: Effect of added reagents in eluent and activation solution on extraction recovery

[0090] The samples to be tested in this example are prepared as follows: 7 kinds of steroid hormone standard and internal standard are dissolved in methanol, diluted and prepared into steroid hormone standard stock solution and internal standard stock solution; the steroid hormone stock solution is further diluted with methanol to prepare the steroid hormone standard working solution and the internal standard working solution.

[0091] Meanwhile, 50 mM potassium iodide methanol, 50 mM ammonium formate methanol, 0.1% formic acid methanol, and 50 mM ammonium acetate 0.1% formic acid methanol and pure methanol were configured as eluents and activation liquids, and the method in Example 1 was used for processing, with the only difference being that the eluents and activation liquids were different, and then the detection of steroid hormones was performed according to the LC-MS / MS in Example 1, and the influence of different additives on the extraction recovery was screened. The standard working solution was also detected on the machine as a control.

[0092] The calculation method of the extraction recovery is as follows:

[0093]

[0094] The peak area and the test results of the extraction recovery of different activation liquids and eluents are shown in Table 3.

[0095] Table 3

[0096]

[0097] From the extraction recovery results in Table 3, 50 mM ammonium acetate methanol as the activation liquid and the eluent has a relatively balanced extraction recovery for all steroid hormones and reaches more than 90%, while other types of activation liquids and eluents have different extraction efficiencies for various steroid hormones, and cannot achieve efficient extraction of all steroid hormones. It can be seen that 50 mM ammonium acetate methanol as the activation liquid and the eluent has a significantly excellent effect on the extraction of steroid hormones.

[0098] Example 3: Influence of activation liquid additive components on extraction recovery

[0099] The sample to be tested in this example is prepared as follows: 7 kinds of steroid hormone standard and internal standard are dissolved with methanol, and are diluted to prepare steroid hormone standard stock solution and internal standard stock solution; the steroid hormone stock solution is further diluted with methanol to prepare steroid hormone standard working solution and internal standard working solution.

[0100] The method in Example 1 was used for processing, with the only difference being that the activation step used ammonium acetate methanol activation, methanol activation or no activation, and then the detection of steroid hormones was performed, the extraction effect of the magnetic beads without activation and using different reagents for activation on the sample was evaluated, the extraction recovery was calculated in the manner in Example 2, and the test results are shown in Table 4. The standard working solution was also detected on the machine as a control.

[0101] Table 4

[0102]

[0103] As can be seen from Table 4, the extraction recovery rates of various steroid hormones after activation are all above 90%, the extraction recovery rate of the magnetic beads without the activation step is generally lower than that of the magnetic beads after activation, and the extraction recovery rate of the magnetic beads activated by ammonium acetate methanol is higher than that of the magnetic beads activated by methanol.

[0104] Example 4: Preparation of a steroid hormone blank matrix serum

[0105] After the above optimization steps, it is confirmed that 50 mM ammonium acetate methanol is selected for activating and eluting the magnetic beads.

[0106] Preparation method of the steroid hormone blank matrix serum:

[0107] Take 0.1 g of HLB magnetic beads, add 10 mL of methanol containing 50 mM ammonium acetate for activation, apply a magnetic field to adsorb the HLB magnetic beads, and remove the supernatant.

[0108] Add 10 mL of serum to be treated to the activated magnetic beads and mix well, extract the steroid hormones using the activated HLB magnetic beads for 2 minutes, apply a magnetic field to adsorb the HLB magnetic beads, and collect the first supernatant.

[0109] After obtaining the first supernatant, add 10 mL of 50 mM ammonium acetate methanol solution to elute the HLB magnetic beads, apply a magnetic field to adsorb the HLB magnetic beads, and the obtained magnetic beads are reused for extracting steroid hormones; the first supernatant is reused for extraction as the serum to be treated, and the second supernatant is obtained as the steroid hormone blank matrix serum.

[0110] The schematic diagram of the preparation method of the steroid hormone blank matrix serum of the present embodiment is shown in Figure 2 .

[0111] The obtained steroid hormone blank matrix serum is detected for 7 kinds of steroid hormones by using the same LC-MS / MS method as in Example 1.

[0112] Results:

[0113] The chromatograms of the 7 kinds of steroid hormones are shown in Figure 3 . The peak area of the chromatogram of the blank matrix serum obtained by the treatment of the present embodiment is compared with that of the untreated serum sample, and the residual amount of the 7 kinds of steroid hormones is less than 0.5%, which indicates that the preparation method of the present application can effectively remove the steroid hormones in the serum.

[0114] Example 5: Preparation of a calibration sample using a blank matrix serum

[0115] The blank matrix serum of steroid hormones was prepared according to the method of Example 4, the seven steroid hormone standard samples were dissolved with methanol, and were diluted to prepare a steroid hormone stock solution. The steroid hormone stock solution was diluted with the blank matrix serum to prepare six calibration samples with different concentrations. The seven steroid hormones were detected by using the same LC-MS / MS method as in Example 1, and a calibration curve was drawn.

[0116] The concentrations of the steroid hormone calibration samples are shown in Table 5.

[0117] Table 5

[0118]

[0119]

[0120] The concentrations of the standard curve samples were taken as the X-axis, and the ratio of the peak area of the analyte to the peak area of the corresponding internal standard was taken as the Y-axis. Linear regression was performed by using the least squares method with 1 / x weighting, and the calibration curves of the steroid hormone calibration samples are shown in Table 6.

[0121] Table 6

[0122]

[0123] As can be seen from Table 6, the correlation coefficients R 2 of the seven steroid hormone standard curves are all greater than 0.99, indicating that the blank matrix serum prepared by the method has very little steroid content and can be used as a blank diluent.

[0124] Example 6: Preparation of steroid hormone quality control samples using blank matrix serum

[0125] The blank matrix serum was prepared by using the same method as in Example 4, and different concentrations of steroid standard sample stock solution were added to prepare serum matrix quality control samples with low, medium and high concentrations. The quality control samples were stored at -80°C, and the detection of steroid hormones was performed by using the same LC-MS / MS method as in Example 1 at 0 months, 3 months, 6 months, 9 months and 12 months. The stability of the quality control samples prepared using the blank matrix serum was observed.

[0126] The deviations of the detection results of each group from the detection results of the standard sample stock solution are shown in Table 7.

[0127] Table 7

[0128]

[0129]

[0130] As can be seen from Table 7, the fluctuation range is between -4.5% and 1.9% compared with just after placement, and there is no significant fluctuation. It is shown that the stability of the quality control prepared by the method of the present application meets the clinical requirements, and the matrix is the same as the matrix of the clinical sample, and has good interchangeability, which can greatly reduce the matrix effect.

[0131] Example 7: Changes of main components such as proteins, sugars, lipids, etc. in blank matrix serum

[0132] The blank matrix serum is prepared by the same method in Example 4, and the obtained first supernatant is recorded as blank matrix serum 1, and the obtained second supernatant is recorded as blank matrix serum 2. The sugars, total proteins, lipids, etc. in the blank matrix serum 1 and the blank matrix serum 2 are determined on the automatic biochemical analyzer, and the differences of the main components are compared and analyzed. The untreated serum sample in Example 4 is also taken as a control sample and is detected in the same way.

[0133] The detection results are shown in Table 8.

[0134] Table 8

[0135] Test item Untreated serum Methanol activation Ammonium acetate methanol activation Glucose (g / L) 5.82 5.75 5.65 Total protein (mmol / L) 72.82 71.30 70.40 Total cholesterol (mmol / L) 1.76 1.76 1.72 Triglycerides (mmol / L) 4.23 4.12 4.05

[0136] As can be seen from Table 8, the sugars, total proteins, lipids, etc. in the serum treated by the method of the present application are reduced to a certain extent, but the overall difference is less than 5%, which shows that there is no obvious difference in the main components between the prepared blank matrix serum and the untreated serum.

[0137] Further, the serum prepared by adopting ammonium acetate methanol activation and methanol activation in Example 3 is also detected for sugars, total proteins, lipids, etc. on the automatic biochemical analyzer, and the differences of the main components are compared and analyzed. The untreated serum sample in Example 4 is also taken as a control sample and is detected in the same way.

[0138] The detection results are shown in Table 9.

[0139] Table 9

[0140] Test item Untreated serum Methanol activation Ammonium acetate methanol activation Glucose (g / L) 6.12 6.05 6.01 Total protein (mmol / L) 78.55 76.91 77.34 Total cholesterol (mmol / L) 2.33 2.33 2.28 Triglycerides (mmol / L) 3.25 3.17 3.18

[0141] As can be seen from Table 9, the sugars, total proteins, lipids, etc. in the serum treated by different activation methods are reduced to a certain extent, but the overall difference is less than 5%, which shows that there is no obvious difference in the main components between the treated serum and the untreated serum.

[0142] Example 8: Magnetic bead regeneration experiment

[0143] The used magnetic beads are incubated with 50 mM ammonium acetate methanol for 5 minutes, and then the magnetic field is applied to adsorb the magnetic beads, and the supernatant is discarded. The regeneration of the magnetic beads is completed and is ready for use.

[0144] The samples were detected by LC-MS / MS in the same manner as in Example 1 after the magnetic beads were regenerated in the above manner using 0, 2, 4, 6, 8, 10 times. The extraction recovery of the magnetic beads of each group to the samples is shown in Table 10.

[0145] Table 10

[0146]

[0147] As can be seen from Table 10, the extraction recovery of the regenerated magnetic beads to the samples is all above 90%, and the extraction recovery effect of the regenerated magnetic beads has no obvious difference with that of the new magnetic beads.

[0148] The principles and implementation manners of the present application are described by applying specific examples in the present application, and the above examples are only used to help understand the method and the central idea of the present application, but not to limit the process method. It should be pointed out that, for those skilled in the art, some improvements and modifications can be made to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection of the claims of the present application.

Claims

1. A method for preparing steroid hormone blank matrix serum, characterized in that, include: HLB magnetic beads were activated using ammonium acetate methanol solution as the activating solution. Remove the activation solution; Steroid hormones present in the serum were extracted by mixing the serum with activated HLB magnetic beads. A magnetic field is applied to attract the HLB magnetic beads, and the first supernatant is collected. After collecting the first supernatant, the HLB magnetic beads were eluted with ammonium acetate methanol solution; By mixing the eluted magnetic beads with the first supernatant, steroid hormones that may be present in the first supernatant can be extracted; and A magnetic field is applied to attract the HLB magnetic beads, and the second supernatant is collected. The preparation method does not include the process of equilibrating the activated magnetic beads with an equilibration solution. The concentration of ammonium acetate in the ammonium acetate methanol solution is 40 mM to 60 mM.

2. The preparation method according to claim 1, characterized in that, The extraction process lasts from 1 to 5 minutes.

3. A method for extracting steroid hormones from serum, characterized in that, include: HLB magnetic beads were activated using ammonium acetate methanol solution as the activating solution. Remove the activation solution; Steroid hormones present in the serum were extracted by mixing the serum with activated HLB magnetic beads. A magnetic field is applied to attract the HLB magnetic beads, and the supernatant is discarded. The HLB magnetic beads were rinsed with a rinsing solution. as well as The HLB magnetic beads were eluted with ammonium acetate methanol solution, and a magnetic field was applied to adsorb the HLB magnetic beads. The supernatant was then collected. The method does not include the process of equilibrating the activated magnetic beads with an equilibration solution. The concentration of ammonium acetate in the ammonium acetate methanol solution is 40 mM to 60 mM.

4. The method according to claim 3, characterized in that, The rinsing solution is selected from water or a 1%-10% methanol aqueous solution.

5. The method according to claim 3, characterized in that, In the activation step, the HLB beads are regenerated HLB beads.

6. The method of claim 5, characterized in that, The regenerated magnetic beads are obtained by treating the used HLB magnetic beads using the elution step.

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