Preparation method of steroid hormone blank matrix serum

Abstract

The invention relates to a preparation method of steroid hormone blank matrix serum. The invention particularly relates to a preparation method of steroid hormone blank matrix serum. The preparation method comprises the following steps: activating HLB (Hydrophile Lipophile Balance) magnetic beads by using an ammonium acetate methanol solution; the method comprises the following steps: mixing serum with activated HLB magnetic beads, and extracting steroid hormones in the serum; and applying a magnetic field to adsorb the HLB magnetic beads, and collecting a first supernatant. The invention also relates to a method for extracting the steroid hormone in the serum and a method for regenerating the HLB magnetic beads for extracting the steroid hormone. The method is simple, rapid and safe to operate; the requirements of automatic equipment can be met, and the method is particularly suitable for automatic large-scale preparation; meanwhile, the prepared blank matrix serum is suitable for being used as a blank control or a blank diluent in detection.

Description

Technical Field

[0001] The invention belongs to the field of pre-treatment for steroid hormone detection, and particularly relates to a method for preparing steroid hormone blank matrix serum and a method for extracting steroid hormones. Background Art

[0002] Steroid hormones, also known as steroid hormones, are a variety of lipophilic small molecule compounds. They can be divided into two major categories based on the receptors they bind to: sex hormones (including estrogens, androgens, and progestogens) and corticosteroids (including glucocorticoids and mineralocorticoids). Steroid hormones play an important regulatory role in human metabolism, maintaining internal environmental stability, growth, development, and reproductive behavior. In clinical diagnosis, steroid hormones are associated with a variety of endocrine diseases such as congenital adrenal hyperplasia, polycystic ovary syndrome, primary aldosteronism, Cushing's syndrome, and precocious puberty, and their auxiliary role in clinical testing is becoming increasingly prominent.

[0003] Traditional immunological methods currently used to detect steroid hormones have low automation, poor specificity, and poor anti-interference capabilities, making accurate quantitative detection difficult. However, with the development of mass spectrometry technology, isotope dilution liquid chromatography-tandem mass spectrometry has become a powerful tool for clinical steroid hormone testing due to its high specificity and ability to simultaneously detect and accurately quantify dozens of steroid hormones. Consequently, an increasing number of in vitro diagnostic manufacturers have launched corresponding mass spectrometry products.

[0004] Blank matrix serum refers to serum that contains a small amount or almost no analyte (e.g., steroid hormones) after processing, but retains the basic serum matrix. It is often used as a blank control or blank diluent in clinical practice. In mass spectrometry detection, blank matrix serum can eliminate the matrix effect and ensure accurate and stable results because its characteristics are similar to those of clinical samples. It is widely used in the preparation of calibrators and quality control products. However, steroid hormones are endogenous substances and are often combined with proteins in the body. How to remove steroid hormones and other analytes while retaining the original serum matrix to the maximum extent and prepare blank matrix serum is a key issue that the industry focuses on.

[0005] Currently, blank matrix serum is prepared using activated carbon adsorption and liquid-liquid extraction, which presents challenges such as complex preparation, high toxicity, and significant impact on the serum matrix. For example, one approach provides a method for preparing steroid hormone-negative serum using methyl tert-butyl ether extraction, which is complex to operate and difficult to automate. Furthermore, methyl tert-butyl ether is flammable, explosive, and corrosive, posing a high risk. Another approach provides a method for preparing fat-soluble vitamin-negative serum using liquid-liquid extraction to remove fat-soluble vitamins, but without steroid hormones. This method requires two liquid-liquid extractions, resulting in complex operations and the high toxicity of dichloromethane and n-hexane. Another approach provides a method for preparing a blank blood matrix and its application, using activated carbon and montmorillonite as adsorbents. Activated carbon has a strong adsorption capacity and can significantly impact the blood matrix.

[0006] Therefore, there is an urgent need for a method for preparing blank matrix serum that is simple to operate, efficient and safe. Summary of the Invention

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

[0008] Based on this, the present invention proposes a method for preparing a steroid hormone blank serum matrix. In the process of preparing steroid hormone blank serum using magnetic beads as a solid phase carrier, it was found that using specific ammonium acetate methanol as a reagent for activating the magnetic beads (and eluting the magnetic beads) can improve the extraction recovery rate of steroid hormones while retaining the main components in the serum, thereby completing the present invention.

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

[0010] The first aspect of the present invention provides a method for preparing a steroid hormone blank matrix serum, comprising: activating HLB magnetic beads using an 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; and applying a magnetic field to adsorb the HLB magnetic beads and collect a first supernatant.

[0011] In some embodiments, the preparation method further includes: after collecting the first supernatant, eluting the HLB magnetic beads with an ammonium acetate methanol solution; extracting steroid hormones that may be present 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 collect the second supernatant.

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

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

[0014] The method for preparing a steroid hormone blank matrix serum of the present invention comprises the following steps: using a methanol solution containing ammonium acetate for activation (and elution) during serum treatment using magnetic beads as a solid phase carrier, thereby not only separating the target compound steroid hormone from the serum matrix with a high extraction recovery rate, but more importantly, retaining major components such as proteins, carbohydrates, and lipids, thereby obtaining a blank control or blank dilution suitable for use in detection.

[0015] On the other hand, the preparation method of the present invention only uses ammonium acetate as an additive for the activation solution (and eluent) based on the magnetic bead extraction method. The preparation method is simple, fast and safe to operate.

[0016] On the other hand, when ammonium acetate is used as the additive, the preparation method of the present invention not only eliminates the traditional hydration equilibration step, but also utilizes the ionic properties contained in the activation solution to obtain the sample liquid level in real time, thereby realizing automated filling, which is particularly suitable for automated large-scale preparation.

[0017] The second aspect of the present invention provides a method for extracting steroid hormones from serum, comprising: activating HLB magnetic beads with an 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; applying a magnetic field to adsorb the HLB magnetic beads and discarding the supernatant; eluting the HLB magnetic beads with an eluent; and eluting with an ammonium acetate methanol solution, applying a magnetic field to adsorb the HLB magnetic beads, and collecting the supernatant.

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

[0019] In some embodiments, in the step of activating, 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 an elution step and a washing step.

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

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

[0023] On the other hand, the extraction method of the present invention only uses ammonium acetate as an additive for the activation solution (and eluent) based on the magnetic bead extraction method. The operation is simple, fast and safe, and is suitable for large-scale extraction.

[0024] A third aspect of the present invention provides a method for regenerating HLB magnetic beads for extracting steroid hormones, comprising: mixing an ammonium acetate methanol solution with the 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 methanolic ammonium acetate solution is 40 mM to 60 mM.

[0026] The magnetic bead regeneration method of the present invention selects ammonium acetate as an additive for the eluent, and the operation is simple and quick.

[0027] On the other hand, the magnetic bead regeneration method of the present invention can efficiently extract steroid hormones during a single steroid hormone extraction process while making the magnetic beads reusable, thereby reducing the cost of the method.

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

[0029] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, constitute a part of the present invention, and do not constitute a limitation of the embodiments of the present invention, wherein:

[0030] Figure 1 1 is a chromatogram of various steroid hormones detected in Example 1.

[0031] Figure 2 Schematic diagram of a method for preparing steroid hormone blank matrix serum according to one embodiment of the present invention.

[0032] Figure 3 This is a chromatogram of the seven steroid hormones detected in Example 4. DETAILED DESCRIPTION

[0033] The following will be combined with specific embodiments of the present invention to clearly and completely describe the technical solution of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0034] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood in the art to which this invention pertains. The following definitions are supplemental to those in the art and relate to this application, but are not intended to be extrapolated to any related or unrelated context, such as any conventionally used patents or applications. Although any methods and materials similar or equivalent to those described herein may be used in the practice of testing this valve, the materials and methods described herein are preferred. Therefore, the terminology used herein is intended to describe specific embodiments only and is not intended to be limiting of the invention.

[0035] The terms "comprising," "including," and "having" in the present invention are open-ended descriptions, encompassing the specified steps described, as well as other steps that do not substantially affect the described steps, and are optional and not excluded. When used to describe a protein or nucleic acid sequence, the protein or nucleic acid may consist of the described sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, while still having the same or similar activity as the original sequence.

[0036] Unless otherwise specified, the terms "first", "second", etc. in the present invention are only used to distinguish multiple similar elements, and are not intended to indicate any difference in importance or order between the elements.

[0037] As described above, in view of the defects of the current blank matrix serum preparation methods such as complex operation, large blood matrix loss, and strong solvent toxicity, the present invention proposes a method for preparing a steroid hormone blank serum matrix.

[0038] Specifically, the first aspect of the present invention provides a method for preparing a steroid hormone blank matrix serum, comprising: activating HLB magnetic beads using 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; and applying a magnetic field to adsorb the HLB magnetic beads and collect the first supernatant.

[0039] The preparation method of the present invention is simple, fast and safe to operate, and can meet the requirements of automated equipment, and is particularly suitable for automated large-scale preparation. At the same time, the blank matrix serum prepared has a low content of steroid hormones, but basically retains major components such as proteins, carbohydrates, and lipids, and is suitable for use as a blank control or blank diluent in detection.

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

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

[0042] In some embodiments, HLB magnetic beads can be used at a conventional concentration for solid phase extraction (steroid hormones) of serum. For example, the concentration of HLB magnetic beads can be 7.5 mg / mL to 12.5 mg / mL, for example, 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 is understood that, generally, as the magnetic bead concentration increases, the overall steroid hormone extraction and detection efficiency initially shows an upward trend. When the magnetic bead concentration is too high, some test compounds cannot be fully eluted due to the increased magnetic bead concentration, resulting in reduced steroid hormone detection efficiency. Those skilled in the art are able to determine an appropriate magnetic bead concentration. For example, in the present invention, a concentration of 10.0 mg / mL of HLB magnetic beads is selected.

[0044] In the activation step of the present invention, an ammonium acetate methanol solution is used as an 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. In addition, compared with the methanol solution, ions are present in the solution after the addition of ammonium acetate, and the amount of the solution can be determined by the ion concentration, so that the preparation process can meet the requirements of automated equipment and is particularly suitable for automated large-scale preparation.

[0045] In some embodiments, the concentration of ammonium acetate in the methanolic ammonium acetate solution is 40 mM to 60 mM. For example, the concentration of ammonium acetate in the methanolic ammonium acetate 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 includes activating the HLB magnetic beads with an ammonium acetate methanol solution, applying a magnetic field to adsorb the HLB magnetic beads, and discarding the supernatant.

[0047] The method for preparing a steroid hormone blank matrix serum of the present invention includes removing the activation solution, ammonium acetate methanol, after activation. Ammonium acetate methanol can activate magnetic beads and also serve as an eluent. Therefore, in order to allow the magnetic beads to "adsorb" more steroid hormones when in contact with serum while avoiding contamination by ammonium acetate methanol, ammonium acetate methanol needs to be removed during the preparation of the blank matrix serum. The method for preparing a steroid hormone blank matrix serum of the present invention includes extracting the steroid hormones present in the serum by mixing the serum with activated HLB magnetic beads.

[0048] In the method of the present invention, mixing the serum with the activated HLB magnetic beads may include continuously and thoroughly mixing the two during the extraction process (eg, by pipetting, shaking, stirring, etc.), or may include incubating the two for a period of time after mixing.

[0049] In some embodiments, the duration of the extracting step 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 invention comprises applying a magnetic field to adsorb the HLB magnetic beads and collecting a first supernatant.

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

[0052] In other embodiments, the preparation method further includes: after collecting the first supernatant, eluting the HLB magnetic beads with an ammonium acetate methanol solution; extracting steroid hormones that may be present 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 collect the second supernatant.

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

[0054] In the preparation method of the present invention, the extracted HLB magnetic beads are eluted with an ammonium acetate methanol solution, and the content of the steroid hormone on the magnetic beads after elution is extremely low, so the magnetic beads can be reused to extract the steroid hormone in the sample, thereby completing the regeneration of the magnetic beads and reducing the cost of the method.

[0055] In the preparation method of the present invention, the eluted magnetic beads are mixed with the first supernatant to further extract any residual steroid hormones that may be present in the first supernatant, further reducing the steroid hormone content in the resulting blank matrix serum. When the first supernatant is extracted using regenerated magnetic beads, since the eluent and activation solution have the same composition, the regenerated magnetic beads can be used for extraction without activation, further simplifying the method.

[0056] In some embodiments, the various methods of the present invention do not include equilibrating the activated magnetic beads with an equilibration solution. Exemplarily, the activating solution is water or a buffer. Exemplarily, the equilibration step occurs after the magnetic bead activation step. Exemplarily, the step of extracting the test substance using the magnetic beads occurs after the equilibration step.

[0057] It is understood that, generally speaking, after magnetic bead activation, an equilibration solution is required to create a suitable working environment for the beads and remove any remaining impurities. Surprisingly, even without equilibration of the activated magnetic beads, the extraction effect of the beads and the retention of the serum matrix were successfully achieved when ammonium acetate was used as an additive to the activator. More importantly, the omission of the equilibration step enables real-time monitoring of the liquid level using salt ions, thus enabling automation of the method.

[0058] The second aspect of the present invention provides a method for extracting steroid hormones from serum, comprising: activating HLB magnetic beads with an 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; applying a magnetic field to adsorb the HLB magnetic beads and discarding the supernatant; eluting the HLB magnetic beads with an eluent; and eluting with an ammonium acetate methanol solution, applying a magnetic field to adsorb the HLB magnetic beads, and collecting the supernatant.

[0059] The method for extracting steroid hormones from serum of the present invention has high extraction efficiency and can efficiently extract steroid hormones from serum samples. At the same time, the operation is simple, fast and safe, and is suitable for large-scale extraction.

[0060] The method for extracting steroid hormones from serum of the present invention is identical to the method for preparing blank matrix serum of the first aspect of the present invention in the preceding steps, except that after extracting the steroid hormones from the serum, the supernatant is discarded and the HLB magnetic beads are subsequently processed. Thus, the method for extracting steroid hormones of the present invention also possesses the advantages of the preparation method of the present invention, which are provided by the features prior to the extraction step.

[0061] In the method for extracting steroid hormones from serum of the present invention, the HLB magnetic beads after extraction are eluted to remove impurities such as ions on the surface of the magnetic beads, thereby reducing the impact on subsequent mass spectrometry detection.

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

[0063] In a specific embodiment, the number of rinses is one.

[0064] In a specific embodiment, the eluent is water.

[0065] In the method for extracting steroid hormones from serum of the present invention, ammonium acetate methanol solution is used for elution. After elution, the magnetic beads are regenerated and can be reused for steroid hormone extraction. In this process, the elution solution and the activation solution use the same ammonium acetate methanol solution, so that the elution and activation processes of the magnetic beads can be completed simultaneously, making the operation simpler and faster. The obtained magnetic beads can be directly used for steroid hormone extraction, which is more conducive to large-scale application of the method.

[0066] In some embodiments, in the step of activating, 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 an elution step.

[0068] Alternatively, the present invention provides a pretreatment method for detecting steroid hormones by liquid phase tandem mass spectrometry, comprising the steps of the method for extracting steroid hormones from serum of the present invention.

[0069] In some embodiments, during the liquid phase tandem mass spectrometry detection of steroid hormones, the serum sample solution and the internal standard solution are detected together.

[0070] A third aspect of the present invention provides a method for regenerating HLB magnetic beads for extracting steroid hormones, comprising: mixing an ammonium acetate methanol solution with the 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 invention is simple and quick to operate. During the extraction process of steroid hormones, the steroid hormones can be extracted efficiently while the magnetic beads can be reused, thereby reducing the cost of the method.

[0072] In the magnetic bead regeneration method of the present invention, 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 simultaneously, and the operation is simpler and faster.

[0073] The following are preferred embodiments of the present invention, and the present invention is not limited to the following preferred embodiments. It should be noted that, for those skilled in the art, any modifications and improvements made based on this inventive concept fall within the scope of protection of the present invention. The reagents used, for which the manufacturer is not indicated, are all commercially available conventional products.

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

[0075] method:

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

[0077] Take 20 μL of the activated suspension and remove the ammonium acetate methanol liquid through an external magnetic field to obtain the activated HLB magnetic bead solid;

[0078] The sample to be tested (200 μL serum and 100 μL isotope internal standard solution) was added to the activated magnetic beads and mixed evenly. The steroid hormones were extracted using the activated HLB magnetic beads 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] 300 μL of 50 mM ammonium acetate methanol solution was added to elute the HLB magnetic beads for 2 minutes, a magnetic field was applied to adsorb the HLB magnetic beads, and the supernatant was collected and used to detect steroid hormones using LC-MS / MS. The obtained magnetic beads were reused for extracting steroid hormones.

[0081] The parameters used for liquid chromatography were:

[0082] An ACQUITY UPLC BEH C18 column (2.1×50 mm, 1.7 μm, Waters, USA) was used, with an injection volume of 10 μL, a column temperature of 40°C, mobile phase A consisting of 0.2 mmol / L ammonium fluoride solution, and mobile phase B consisting of methanol gradient elution at a flow rate of 0.3 mL / min. Details are shown in Table 1 below.

[0083] Table 1

[0084]

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

[0086] Table 2

[0087]

[0088] Results: The chromatograms of various steroid hormones are as follows Figure 1 As shown, it can be seen that various steroid hormones have obvious characteristic peaks, indicating that various steroid hormones have been successfully extracted and detected.

[0089] Example 2: Effect of Adding Reagents to Eluent and Activation Solution on Extraction Recovery

[0090] The test samples in this example were prepared as follows: seven steroid hormone standards and an internal standard were dissolved in methanol and diluted to prepare a steroid hormone standard stock solution and an internal standard stock solution; the steroid hormone stock solution was further diluted with methanol to prepare a steroid hormone standard working solution and an internal standard working solution.

[0091] Simultaneously, 50 mM potassium iodide in methanol, 50 mM ammonium formate in methanol, 0.1% formic acid in methanol, 50 mM ammonium acetate in 0.1% formic acid in methanol, and pure methanol were prepared as eluents and activation solutions. Treatment was performed according to the method in Example 1, differing only in the selection of the eluent and activation solution. Steroid hormones were then detected by LC-MS / MS as in Example 1 to screen for the effects of different additives on extraction recovery. A standard working solution was also tested on the instrument as a control.

[0092] The extraction recovery rate is calculated as follows:

[0093]

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

[0095] Table 3

[0096]

[0097] The extraction recovery results in Table 3 show that 50 mM ammonium acetate methanol, used as both the activation solution and eluent, achieved a relatively balanced extraction recovery of over 90% for all steroid hormones. However, the extraction efficiency of other activation solutions and eluents varied, failing to achieve efficient extraction of all steroid hormones. This demonstrates that 50 mM ammonium acetate methanol, used as both the activation solution and eluent, exhibits significantly superior steroid hormone extraction.

[0098] Example 3: Effect of Additives in Activation Solution on Extraction Recovery

[0099] The test samples in this example were prepared as follows: seven steroid hormone standards and an internal standard were dissolved in methanol and diluted to prepare a steroid hormone standard stock solution and an internal standard stock solution; the steroid hormone stock solution was further diluted with methanol to prepare a steroid hormone standard working solution and an internal standard working solution.

[0100] Treatment was performed according to the method of Example 1, with the only difference being the use of ammonium acetate methanol, methanol, or no activation in the activation step. Steroid hormone detection was then performed to evaluate the sample extraction efficiency of the magnetic beads after activation with different reagents and without activation. The extraction recovery was calculated according to the method of Example 2. The test results are shown in Table 4 below. A separate standard working solution was also tested on the instrument 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 rates of the samples by the magnetic beads that have not undergone the activation step are generally lower than those of the magnetic beads that have undergone activation. In addition, the extraction recovery rate of the samples by the magnetic beads activated with ammonium acetate and methanol is higher than that of the magnetic beads activated with methanol.

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

[0105] After the above optimization steps, it was confirmed that 50 mM ammonium acetate methanol was selected for activation and elution of the magnetic beads.

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

[0107] Take 0.1 g of HLB magnetic beads and activate them by adding them to 10 mL of methanol containing 50 mM ammonium acetate. Apply a magnetic field to adsorb the HLB magnetic beads and remove the supernatant.

[0108] 10 mL of serum to be processed was added to the activated magnetic beads and mixed evenly. Steroid hormones were extracted using the activated HLB magnetic beads for 2 minutes. A magnetic field was applied to adsorb the HLB magnetic beads, and the first supernatant was collected.

[0109] After obtaining the first supernatant, 10 mL of 50 mM ammonium acetate methanol solution was added to elute the HLB magnetic beads, and a magnetic field was applied to adsorb the HLB magnetic beads. The obtained magnetic beads were reused to extract steroid hormones; the first supernatant was re-extracted as the serum to be processed to obtain the second supernatant as the steroid hormone blank matrix serum.

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

[0111] The obtained steroid hormone blank matrix serum was used to detect seven steroid hormones using the same LC-MS / MS method as in Example 1.

[0112] result:

[0113] The chromatograms of seven steroid hormones were obtained as follows Figure 3 The blank matrix serum treated in this example showed that the peak areas of the steroid hormone chromatograms were less than 0.5% compared with those of the untreated serum sample, indicating that the preparation method of the present invention can effectively remove steroid hormones from serum.

[0114] Example 5: Preparation of Calibrators Using Blank Matrix Serum

[0115] Steroid hormone blank matrix serum was prepared according to the method of Example 4. Seven steroid hormone standards were dissolved in methanol and diluted to prepare steroid hormone stock solutions. The steroid hormone stock solutions were diluted with blank matrix serum to prepare six levels of calibrators at different concentrations. The seven steroid hormones were detected using the same LC-MS / MS method as in Example 1, and calibration curves were drawn.

[0116] The concentrations of various steroid hormone calibrators are shown in Table 5.

[0117] Table 5

[0118]

[0119]

[0120] The concentration of the standard curve sample analyte was used as the X-axis, and the ratio of the analyte peak area to the corresponding internal standard peak area was used as the Y-axis. The least squares method with a 1 / x weight was used for linear regression to obtain the calibration curves of various steroid hormone calibrators as shown in Table 6.

[0121] Table 6

[0122]

[0123] As can be seen from Table 6, the correlation coefficients R 2 The linear relationship was good, indicating that the blank matrix serum prepared by the method of the present invention contained very little steroid content and could be used as blank dilution.

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

[0125] Blank matrix serum was prepared using the same method as in Example 4, and different concentrations of steroid standard stock solutions were added to prepare low, medium, and high concentration serum matrix quality control products, which were stored at -80°C. Steroid hormones were detected using the same LC-MS / MS method as in Example 1 at 0, 3, 6, 9, and 12 months to observe the stability of the quality control products prepared using blank matrix serum.

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

[0127] Table 7

[0128]

[0129]

[0130] As shown in Table 7, the stability of the control samples after 12 months of storage at -80°C fluctuated between -4.5% and 1.9% compared to the initial storage time, with no significant fluctuation. This indicates that the stability of the control samples prepared using the method of the present invention meets clinical requirements, and the matrix is ​​identical to that of clinical samples, offering good interchangeability and significantly reducing matrix effects.

[0131] Example 7: Changes in the main components such as proteins, carbohydrates, and lipids in blank matrix serum

[0132] Blank matrix serum was prepared using the same method as in Example 4. The first supernatant obtained was designated as blank matrix serum 1, and the second supernatant was designated as blank matrix serum 2. Carbohydrates, total protein, lipids, etc., in blank matrix serum 1 and blank matrix serum 2 were measured on an automatic biochemical analyzer, and the differences in the main components were compared and analyzed. The untreated serum sample from Example 4 was also tested as a control.

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

[0134] Table 8

[0135] Test items Untreated serum Blank matrix serum 1 Blank matrix serum 2 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, after the serum was treated with the method of the present invention, the carbohydrates, total protein, lipids, etc. in the serum were all reduced to a certain extent, but the overall difference was less than 5%, indicating that there was no significant difference in the main components of the blank matrix serum prepared by the present invention and the serum before treatment.

[0137] Furthermore, the sera prepared by ammonium acetate methanol activation and methanol activation in Example 3 were also measured for carbohydrates, total protein, lipids, etc. on an automatic biochemical analyzer to compare and analyze the differences in the main components. The untreated serum sample in Example 4 was also taken as a control and tested in the same manner.

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

[0139] Table 9

[0140] Test items 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, after serum was treated with different activation methods, the sugars, total proteins, lipids, etc. in the serum were all reduced to a certain extent, but the overall difference was less than 5%, indicating that there was no significant difference in the main components of the serum after treatment and the serum before treatment.

[0142] Example 8: Magnetic bead regeneration experiment

[0143] The used magnetic beads were incubated with 50 mM ammonium acetate methanol for 5 minutes, and then a magnetic field was applied to adsorb the magnetic beads. The supernatant was discarded and the magnetic beads were regenerated and ready for use.

[0144] The magnetic beads were regenerated as described above after being used 0, 2, 4, 6, 8, and 10 times, and the samples were analyzed by LC-MS / MS in the same manner as in Example 1. The recovery rates of the magnetic beads for each group of samples are shown in Table 10.

[0145] Table 10

[0146]

[0147] As can be seen from Table 10, the extraction recovery rates of the regenerated magnetic beads for samples are all above 90%, and there is no significant difference in the extraction recovery rate between the regenerated magnetic beads and the new magnetic beads.

[0148] Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The above examples are only intended to facilitate understanding of the method and its central concept, and are not intended to limit the process. It should be noted that those skilled in the art may make various improvements and modifications to the present invention without departing from the principles of the present invention, and such improvements and modifications are also protected by the claims of the present invention.

Claims

1. A method for preparing a steroid hormone blank matrix serum, characterized in that: include: Activate HLB magnetic beads using ammonium acetate methanol solution as activation solution; Remove the activation solution; extracting steroid hormones present in the serum by mixing the serum with activated HLB magnetic beads; as well as A magnetic field is applied to adsorb the HLB magnetic beads, and the first supernatant is collected.

2. The preparation method according to claim 1, characterized in that The preparation method further comprises: After collecting the first supernatant, eluting the HLB magnetic beads with ammonium acetate methanol solution; extracting steroid hormones that may be present in the first supernatant by mixing the eluted magnetic beads with the first supernatant; and A magnetic field is applied to adsorb the HLB magnetic beads, and the second supernatant is collected.

3. The preparation method according to claim 1 or 2, characterized in that The concentration of ammonium acetate in the ammonium acetate methanol solution is 40 mM to 60 mM.

4. The preparation method according to claim 1, characterized in that The extraction step lasts from 1 minute to 5 minutes.

5. A method for extracting steroid hormones from serum, characterized in that: include: Activate HLB magnetic beads using ammonium acetate methanol solution as activation solution; Remove the activation solution; extracting steroid hormones present in the serum by mixing the serum with activated HLB magnetic beads; Applying a magnetic field to adsorb the HLB magnetic beads, and discarding the supernatant; Eluent is used to elute the HLB magnetic beads; as well as The mixture was eluted with ammonium acetate methanol solution, a magnetic field was applied to adsorb the HLB magnetic beads, and the supernatant was collected. Preferably, the eluent is selected from water or a 1-10% methanol aqueous solution.

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

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

8. The method according to claims 5 to 7, characterized in that The concentration of ammonium acetate in the ammonium acetate methanol solution is 40 mM to 60 mM.

9. A method for regenerating HLB magnetic beads for extracting steroid hormones, characterized in that: The method comprises: The ammonium acetate methanol solution was mixed with the HLB magnetic beads, a magnetic field was applied to adsorb the HLB magnetic beads, and the supernatant was discarded.

10. The method according to claim 9, characterized in that The concentration of ammonium acetate in the ammonium acetate methanol solution is 40 mM to 60 mM.

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