A kit for extracting free testosterone in serum, as well as an extraction method and a detection method

Abstract

The present invention provides a kit for extracting free testosterone from serum, an extraction method and a detection method, which relate to the field of medical detection technology, including magnetic bead liquid, balancing liquid, internal standard liquid, eluent, eluent and diluent. One or more magnetic beads are selected to improve the sensitivity of separating free testosterone; and the eluent contains 0.1-1.0% of a derivatization reagent to achieve in-situ derivatization, which is suitable for a fully automatic magnetic solid phase extraction device to achieve automated extraction of free testosterone; different derivatization reagents are selected to separately derive samples and combine them for loading, so as to achieve one-shot multi-sample detection and improve the detection efficiency of liquid chromatography tandem mass spectrometry. The magnetic bead liquid, balancing liquid, internal standard liquid, eluent, eluent and diluent cooperate with each other to achieve rapid and efficient separation and purification of free testosterone, thereby achieving the purpose of improving detection sensitivity and accuracy. The technical problem that the prior art cannot achieve efficient, accurate, low-cost and automated operation is solved.

Description

Technical Field

[0001] The present invention relates to the field of medical detection technology, and in particular to a kit for extracting free testosterone in serum, an extraction method and a detection method. Background Art

[0002] In human blood circulation, conventional detection methods have many limitations and challenges because the content of free testosterone in serum is extremely low and needs to be separated and detected.

[0003] At present, traditional free testosterone detection methods mainly rely on radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), which have disadvantages such as complex operation, time-consuming, susceptibility to interference from sample matrix, insufficient sensitivity and accuracy, etc., which limit the accuracy and repeatability of free testosterone detection.

[0004] With the development of science and technology, quantitative analysis using liquid chromatography tandem mass spectrometry (LC-MS / MS) technology after equilibrium dialysis (ED) and ultrafiltration (UF) pre-treatment has become the main method for detecting free hormones. Although ultrafiltration can save a certain amount of time compared to equilibrium dialysis, its actual operation has poor repeatability. For example, the temperature control steps are cumbersome, the viscosity of undiluted samples is high and requires extended centrifugation time, there is potential equipment-related protein leakage, and centrifugation equipment is required. The results obtained using different control conditions vary greatly. Therefore, it is currently recognized in the industry that equilibrium dialysis combined with liquid chromatography tandem mass spectrometry is the gold standard for free hormone detection.

[0005] However, the detection using LC-MS / MS technology requires complex sample pretreatment steps, including protein precipitation, centrifugation, solid phase extraction, liquid-liquid extraction and concentration. The Chinese patent application publication number CN 116297998A, the invention name is "A high-sensitivity mass spectrometry detection kit and detection method for free testosterone in serum", which uses sample dilution, ultrafiltration, extraction, centrifugation, and the supernatant is blown dry with nitrogen for derivatization and then re-dissolved. The Chinese patent with authorization announcement number CN114720704B is a kit and method for determining free testosterone in serum, which uses sample dilution, ultrafiltration, extraction, and the supernatant is blown dry with nitrogen for re-dissolution, and then centrifuged to obtain the supernatant. The sample pretreatment method in the above patent involves centrifugation, extraction and multiple sampling operations, which is relatively complicated, not only time-consuming and labor-intensive, but also inefficient, and easily has a significant impact on the accurate quantification of extremely low concentrations of free testosterone (picogram level).

[0006] Application publication number is the detection method of free testosterone of Chinese patent CN115343402A, adopts balanced dialysis, extraction, centrifugation, takes the detection process of redissolving after supernatant nitrogen blows and dries.This method not only relates to the steps such as centrifugation, extraction, nitrogen blowing, making operation complex and inefficient, but also directly detects without derivatization reaction, causing the situation that low response and interference peak occur when detecting the sample containing separation gel blood collection tube, although the method provided can separate the interference peak by selecting suitable ion pair, but the response is reduced by nearly ten times, indicating that there is a serious absolute matrix effect, which may cause the quantitative inaccuracy of free testosterone.Therefore, for the sample detection of extremely low concentration of free testosterone, although some operating steps and time can be saved without derivatization, it may cause certain deviation in the test result.

[0007] In addition, most existing free testosterone detection methods require professional technicians to operate manually throughout the process, which requires high operator skills. At the same time, expensive mass spectrometers can only detect one sample concentration per injection, which limits its wide application in clinical laboratories. Therefore, developing an efficient, accurate, low-cost and easy-to-automate detection method is crucial to improving the clinical application value of free testosterone detection.

[0008] In view of this, the present invention is proposed. Summary of the invention

[0009] One of the purposes of the present invention is to provide a kit for extracting free testosterone in serum, which can efficiently extract free testosterone and improve the sensitivity of testosterone detection. The kit can also be applied to a fully automatic magnetic solid phase extraction device to realize automatic extraction of free testosterone, so as to solve the technical problem that the prior art cannot achieve high efficiency, accuracy, low cost and automatic operation.

[0010] A second object of the present invention is to provide the use of the above-mentioned kit in extracting or detecting free testosterone in serum.

[0011] A third object of the present invention is to provide a method for extracting free testosterone in serum.

[0012] A fourth object of the present invention is to provide a method for detecting free testosterone in serum.

[0013] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0014] In a first aspect, the present invention provides a kit for extracting free testosterone in serum, comprising a magnetic bead solution, a balancing solution, an internal standard solution, a washing solution, an eluent and a diluent;

[0015] The concentration of the magnetic beads in the magnetic bead solution is 2.5-10 mg / ml, the solvent of the magnetic bead solution is methanol aqueous solution, and the concentration of methanol in the magnetic bead solution is 0-100%. The magnetic beads include at least one of hydrophilic-hydrophobic balance magnetic beads, weak anion exchange magnetic beads, weak cation exchange magnetic beads, mixed strong anion exchange magnetic beads or mixed strong cation exchange magnetic beads;

[0016] The balancing solution, eluent and diluent are all methanol-water solutions, the concentration of methanol in the balancing solution is 0-15%, the concentration of methanol in the eluent is 5-30%, and the concentration of methanol in the diluent is 0-50%;

[0017] The internal standard solution is a methanol or acetonitrile solution containing a testosterone internal standard, and the concentration of the testosterone internal standard in the internal standard solution is 10-200 pg / mL;

[0018] The eluent includes at least one of isonicotinoyl chloride hydrochloride eluent, methoxyamine hydrochloride eluent, 3-pyridinemethoxyamine eluent or hydroxylamine hydrochloride eluent, and the concentrations of the isonicotinoyl chloride hydrochloride eluent, the methoxyamine hydrochloride eluent, the 3-pyridinemethoxyamine eluent and the hydroxylamine hydrochloride eluent are independently 0.1-1.0%.

[0019] Furthermore, the magnetic beads include at least one of hydrophilic-hydrophobic balance magnetic beads, mixed strong anion exchange magnetic beads or mixed strong cation exchange magnetic beads, preferably hydrophilic-hydrophobic balance magnetic beads and mixed strong anion exchange magnetic beads.

[0020] Furthermore, it also includes a balanced dialysis reagent for preparing a serum sample dialysate, wherein the balanced dialysis reagent includes a dialysis buffer and a pH adjustment buffer;

[0021] The dialysis buffer comprises the following components at the following concentrations: HEPES 12.57 g / L, NaOH 0.9 g / L, NaCl 5.23 g / L, KH2PO4 0.22 g / L, MgSO4·7H2O 0.28 g / L, Urea 0.3 g / L, CaCl2·2H2O 0.28 g / L and ProClin TM 300 0.05%(v / v);

[0022] The pH of the dialysis buffer is 7.3-7.5;

[0023] The pH adjustment buffer is a HEPES buffer;

[0024] The concentration of the pH adjustment buffer is 500-1000 mol / L;

[0025] The pH value of the pH adjustment buffer is 7.3-7.5;

[0026] The solvent of the eluent is methanol or acetonitrile.

[0027] In a second aspect, the present invention provides the use of the above-mentioned kit in extracting or detecting free testosterone in serum.

[0028] In a third aspect, the present invention provides a method for extracting free testosterone in serum, comprising using the serum to be tested as a sample and using the reagents in the above-mentioned kit to prepare a testosterone extract.

[0029] Further, the following steps are included:

[0030] A. The magnetic beads in the magnetic bead solution are taken out and placed in a balance solution, a serum sample dialysate containing a testosterone internal standard, and an eluent in sequence, and then eluted with an eluent to obtain a testosterone eluate;

[0031] B. The testosterone eluate is heated to perform an in situ derivatization reaction to obtain a testosterone extract.

[0032] Further, the serum sample dialysate containing testosterone internal standard includes mixing an internal standard solution with a serum sample dialysate, and the volume ratio of the internal standard solution to the serum sample dialysate is 1-4:4-6;

[0033] The volume ratio of the magnetic bead liquid to the serum sample dialysate is 1-3:2-3;

[0034] The volume ratio of the balancing solution to the serum sample dialysate is 1-3:2-3;

[0035] The volume ratio of the eluent to the serum sample dialysate is 2-5:2-3;

[0036] The volume ratio of the eluent to the serum sample dialysate is 1-4:4-6.

[0037] The magnetic beads are placed in the equilibrium solution for 10 to 60 seconds;

[0038] The magnetic beads are placed in the serum sample dialysate containing the testosterone internal standard for 30 to 90 seconds;

[0039] The magnetic beads are placed in the eluent for 10 to 60 seconds;

[0040] The elution time of the eluent is 30 to 90 seconds.

[0041] Furthermore, the heating temperature is 40-80°C;

[0042] The heating time is 2 to 10 minutes;

[0043] The step of heating the testosterone eluate to perform an in-situ derivatization reaction further includes adding a diluent;

[0044] The volume ratio of the diluent to the serum sample dialysate is 0.3-1:2-3;

[0045] The method for preparing the serum sample dialysate comprises adding a dialysis buffer and a serum to be tested containing a pH adjustment buffer into a balanced dialysis system, and performing dialysis after sealing the membrane;

[0046] The balanced dialysis system comprises a bottom plate and a balanced dialysis membrane;

[0047] The material of the bottom plate includes polytetrafluoroethylene, polypropylene or polyvinylidene fluoride, preferably polytetrafluoroethylene;

[0048] The equilibrium dialysis membrane comprises a regenerated cellulose membrane with a molecular weight cut-off of 3.5 to 15 KD;

[0049] The volume concentration of the pH adjusting buffer in the test serum containing the pH adjusting buffer is 1-10%;

[0050] The volume ratio of the dialysis buffer to the test serum containing the pH adjustment buffer is 0.8-1.2:1;

[0051] The dialysis time is 3 to 6 hours.

[0052] Furthermore, when the number of samples was >1, a different eluent was used in the extraction process of each sample;

[0053] After obtaining the testosterone extract, the step further includes mixing the testosterone extracts of each sample in proportion;

[0054] When the number of samples = 2, the volume ratio of the two sample extracts is 1~2:1~2, preferably 1:1;

[0055] When the number of samples is greater than 2, the extracts of each sample are mixed in equal proportions.

[0056] In a fourth aspect, the present invention provides a method for detecting free testosterone in serum, comprising performing liquid chromatography-mass spectrometry detection on the testosterone extract obtained by the above-mentioned kit or the above-mentioned extraction method;

[0057] The conditions of the liquid chromatography include: the chromatographic column is a C18 column, the mobile phase A is an aqueous solution of ammonium formate or ammonium acetate, and the mobile phase B is a methanol or acetonitrile solution;

[0058] The elution procedure of the liquid chromatography is:

[0059] 0~1min, mobile phase A 20~40%, mobile phase B 60~80%;

[0060] 1-3.8 min, mobile phase A 20-40% to 2-5%, mobile phase B 60-80% to 95-98%;

[0061] 3.8~4.1min, mobile phase A 2~5%, mobile phase B 95~98%;

[0062] 4.1~4.5min, mobile phase A 2~5% to 20~40%, mobile phase B 95~98% to 60~80%;

[0063] 4.5~5min, mobile phase A 20~40%, mobile phase B 60~80%;

[0064] The ion source of the mass spectrometry detection is an electrospray ion source, and the multiple reaction monitoring mode is adopted in the positive ion mode.

[0065] The present invention provides a kit, extraction method and detection method for extracting free testosterone in serum. Through the cooperation of magnetic bead liquid, balancing liquid, internal standard liquid, eluent, eluent and diluent, rapid and efficient separation and purification of free testosterone can be achieved, thereby achieving the purpose of improving detection sensitivity and accuracy. According to the sample to be tested, one or more magnetic beads can be selected to improve the sensitivity of separating free testosterone, which can be applicable to low-concentration detection; and the eluent contains 0.1-1.0% of a derivatization reagent, which can be heated to achieve in-situ derivatization, and can be applied to a fully automatic magnetic solid phase extraction device to achieve automated extraction of free testosterone. The technical problem that the prior art cannot achieve efficient, accurate, low-cost and automated operation is solved. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0067] Figure 1 The mass spectrum of the INC derivatization reaction detection of the sample provided in Example 7 of the present invention;

[0068] Figure 2 The mass spectrum of the non-derivatized reaction detection of sample 4 provided in Experiment 5 of the present invention;

[0069] Figure 3 The composite detection mass spectra of two samples provided in Example 10 of the present invention;

[0070] Figure 4 This is a composite detection standard curve for two samples provided in Example 10 of the present invention. DETAILED DESCRIPTION

[0071] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0072] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0073] The present invention provides a kit for extracting free testosterone in serum, comprising a magnetic bead solution, a balancing solution, an internal standard solution, a washing solution, an eluent and a diluent;

[0074] The concentration of magnetic beads in the magnetic bead liquid is 2.5-10 mg / ml, the solvent of the magnetic bead liquid is methanol aqueous solution, the concentration of methanol in the magnetic bead liquid is 0-100%, and the magnetic beads include at least one of hydrophilic-hydrophobic balance magnetic beads (HLB), weak anion exchange magnetic beads (WAX), weak cation exchange magnetic beads (WCX), mixed strong anion exchange magnetic beads (MAX) or mixed strong cation exchange magnetic beads (MCX);

[0075] The balancing solution, eluent and diluent are all methanol aqueous solutions, the concentration of methanol in the balancing solution is 0-15%, the concentration of methanol in the eluent is 5-30%, and the concentration of methanol in the diluent is 0-50%;

[0076] The internal standard solution is a methanol or acetonitrile solution containing a testosterone internal standard, and the concentration of the testosterone internal standard in the internal standard solution is 10-200 pg / mL;

[0077] The eluent includes at least one of isonicotinoyl chloride hydrochloride (INC) eluent, methoxyamine hydrochloride (MOA) eluent, 3-pyridinemethoxyamine (POA) eluent or hydroxylamine hydrochloride (HOA) eluent, and the concentrations of the isonicotinoyl chloride hydrochloride eluent, methoxyamine hydrochloride eluent, 3-pyridinemethoxyamine eluent and hydroxylamine hydrochloride eluent are independently 0.1-1.0%.

[0078] Through the cooperation of magnetic bead liquid, balance liquid, internal standard liquid, eluent, eluent and diluent, it is possible to achieve rapid and efficient separation and purification of free testosterone, so as to improve the detection sensitivity and accuracy. Among them, according to the sample to be tested, one or more magnetic beads can be selected to improve the sensitivity of separating free testosterone, which can be suitable for low-concentration detection; and 0.1~1.0% of the derivatization reagent is pre-added to the eluent, and no nitrogen blowing and derivatization are required. The temperature can be directly increased in situ to start derivatization to realize the integration of elution and derivatization, reducing the experimental operation steps; at the same time, derivatization can enhance the anti-interference ability of the detection and significantly improve the ionization efficiency and detection sensitivity of free testosterone at the picogram level, and the quantitative lower limit can reach 0.5pg / mL; it is suitable for fully automatic magnetic solid phase extraction device to realize automatic extraction of free testosterone. It solves the technical problem that the existing technology cannot achieve efficient, accurate, low-cost and automated operation.

[0079] In some specific embodiments, the magnetic beads include at least one of HLB, MAX or MCX, preferably HLB and MAX.

[0080] Experiments have shown that the average testosterone detection peaks corresponding to HLB, MAX and MCX magnetic solid phase extraction materials are higher than those of WAX and WCX, among which HLB is the highest. However, further experiments have found that the combination of HLB and MAX can further increase the average testosterone detection peak to improve sensitivity.

[0081] Wherein, the particle size of the magnetic beads is selected from the range of 15~50μm.

[0082] In some specific embodiments, a balanced dialysis reagent for preparing a serum sample dialysate is also included, wherein the balanced dialysis reagent includes a dialysis buffer and a pH adjustment buffer.

[0083] The dialysis buffer included the following components at the following concentrations: HEPES 12.57 g / L, NaOH 0.9 g / L, NaCl 5.23 g / L, KH2PO4 0.22 g / L, MgSO4·7H2O 0.28 g / L, Urea 0.3 g / L, CaCl2·2H2O 0.28 g / L, and ProClin TM 300 0.05% (v / v); the pH of the dialysis buffer was 7.3~7.5.

[0084] In some specific embodiments, the pH adjustment buffer is a HEPES buffer.

[0085] The concentration of the pH adjustment buffer is 500-1000 mol / L; the pH value of the pH adjustment buffer is 7.3-7.5.

[0086] In some specific embodiments, the solvent of the eluent is methanol or acetonitrile.

[0087] According to another aspect of the present invention, there is also provided the use of the above-mentioned kit in extracting or detecting free testosterone in serum.

[0088] According to another aspect of the present invention, a method for extracting free testosterone in serum is also provided, comprising using the serum to be tested as a sample and using the reagents in the above-mentioned kit to prepare a testosterone extract.

[0089] The reagents in the kit can be used to efficiently separate testosterone from serum samples with a testosterone content as low as 0.5 pg / mL to obtain a testosterone extract.

[0090] In some specific embodiments, the steps include:

[0091] A. The magnetic beads in the magnetic bead solution are taken out and placed in a balance solution, a serum sample dialysate containing a testosterone internal standard, and an eluent in sequence, and then eluted with an eluent to obtain a testosterone eluate;

[0092] B. The testosterone eluate is heated to perform an in situ derivatization reaction to obtain a testosterone extract.

[0093] The free testosterone in the serum sample is extracted by equilibrium dialysis to obtain an equilibrium dialysate containing free testosterone, and the free testosterone content in the dialysate is detected by combining LC-MS / MS technology. During the process, a derivatization agent is pre-added to the eluent, and no nitrogen blowing is required for derivatization. Derivatization can be started by directly raising the temperature in situ. By integrating elution and derivatization, not only can the experimental operation steps be reduced, but derivatization can also enhance the anti-interference ability of the detection and significantly improve the ionization efficiency and detection sensitivity of picogram-level free testosterone, and the quantitative lower limit can reach 0.5pg / mL. This method can be used for fully automatic magnetic solid phase extraction devices by pre-configuring reagents, and can realize automatic pipetting, automatic extraction and magnetic bead extraction according to the set program, so that the whole process does not require cumbersome liquid-liquid extraction, centrifugation, nitrogen blowing and other operations.

[0094] In some specific embodiments, the serum sample dialysate containing testosterone internal standard includes mixing an internal standard solution with a serum sample dialysate, and the volume ratio of the internal standard solution to the serum sample dialysate is 1:1-6.

[0095] The volume ratio of the internal standard solution to the serum sample dialysate may be, but is not limited to, 1:1, 1:2, 1:3, 1:4, 1:5 or 1:6, or any value between 1:1 and 6.

[0096] In some specific embodiments, the volume ratio of the magnetic bead solution to the serum sample dialysate is 1-3:2-3.

[0097] The volume ratio of the magnetic bead solution to the serum sample dialysate may be, but is not limited to, 1:3, 1:2, 2:3, 1:1 or 3:2, or any value between 1-3:2-3.

[0098] In some specific embodiments, the volume ratio of the balancing solution to the serum sample dialysate is 1-3:2-3.

[0099] The volume ratio of the balancing solution to the serum sample dialysate may be, but is not limited to, 1:3, 1:2, 2:3, 1:1 or 3:2, or any value between 1-3:2-3.

[0100] In some specific embodiments, the volume ratio of the eluent to the serum sample dialysate is 2-5:2-3.

[0101] The volume ratio of the eluent to the serum sample dialysate may be, but is not limited to, 2:3, 1:1, 3:2, 4:3, 2:1 or 5:2, or any value between 2 and 5:2 and 3.

[0102] In some specific embodiments, the volume ratio of the eluent to the serum sample dialysate is 1:1-6.

[0103] The volume ratio of the eluent to the serum sample dialysate may be, but is not limited to, 1:1, 1:2, 1:3, 1:4, 1:5 or 1:6, or any value between 1:1 and 6.

[0104] In some specific embodiments, the magnetic beads are placed in the equilibration solution for 10 to 60 seconds. If a fully automatic magnetic solid phase extraction device is used, the mixing equilibration well position can be set for 10 to 60 seconds.

[0105] In some specific embodiments, the magnetic beads are placed in the serum sample dialysate containing the testosterone internal standard for 30 to 90 seconds. If a fully automatic magnetic solid phase extraction device is used, the sample mixing well can be set for 30 to 90 seconds.

[0106] In some specific embodiments, the magnetic beads are placed in the eluent for 10 to 60 seconds. If a fully automatic magnetic solid phase extraction device is used, the mixing and elution wells can be set for 10 to 60 seconds.

[0107] In some specific embodiments, the elution time of the eluent is 30 to 90 seconds. If a fully automatic magnetic solid phase extraction device is used, the mixing and elution hole position can be set for 30 to 90 seconds, and the magnetic beads can be removed and discarded.

[0108] In some specific embodiments, the heating temperature is 40-80° C., and the heating time is 2-10 min.

[0109] In some specific embodiments, the heating of the testosterone eluate for in-situ derivatization reaction further includes adding a diluent, and the diluent can be directly used for subsequent free testosterone detection.

[0110] Wherein, the volume ratio of the diluent to the serum sample dialysate is 0.3~1:2~3.

[0111] In some specific embodiments, the method for preparing the serum sample dialysate comprises adding a dialysis buffer and a serum to be tested containing a pH adjustment buffer into a balanced dialysis system containing a bottom plate and a balanced dialysis membrane, and performing dialysis after sealing the membrane;

[0112] Wherein, the material of the bottom plate includes polytetrafluoroethylene, polypropylene or polyvinylidene fluoride, preferably polytetrafluoroethylene; the equilibrium dialysis membrane includes a regenerated cellulose membrane with a molecular weight cutoff of 3.5-15KD; the volume concentration of the pH adjusting buffer in the test serum containing the pH adjusting buffer is 0-10%; the volume ratio of the dialysis buffer to the test serum containing the pH adjusting buffer is 0.8-1.2:1;

[0113] In some specific embodiments, the dialysis time is 3 to 6 hours.

[0114] In some specific embodiments, when the number of samples is greater than 1, the eluent used in the extraction process of each sample is different.

[0115] The different eluents specifically refer to different samples using different in-situ derivatization reagents in the eluents, which may be isonicotinoyl chloride hydrochloride eluent, methoxyamine hydrochloride eluent, 3-pyridinemethoxyamine eluent or hydroxylamine hydrochloride eluent, respectively.

[0116] After obtaining the testosterone extract, the method further comprises mixing the testosterone extracts of each sample in proportion.

[0117] When the number of samples = 2, the volume ratio of the two sample extracts is 1~2:1~2.

[0118] Experiments have shown that 1~2:1~2 is better than 1:3 or 3:1. Considering that the mixing order of the extracts can be set arbitrarily, 1:1 is preferred.

[0119] When the number of samples is greater than 2, the extracts of each sample are mixed in equal proportions.

[0120] By reasonably combining different derivatization reagents, different samples are treated with different derivatization reagents, which are then combined and loaded onto the machine after derivatization. Multiple samples can be tested simultaneously using one injection. This method is accurate and reliable, improves detection efficiency, and reduces detection costs.

[0121] In some specific embodiments, the testosterone extract obtained by the above-mentioned kit or the above-mentioned extraction method is subjected to liquid chromatography-mass spectrometry detection.

[0122] The testosterone extract obtained by the above-mentioned kit or the above-mentioned extraction method realizes the integration of elution and derivatization, and can be directly used for liquid chromatography-mass spectrometry detection.

[0123] In some specific embodiments, the liquid chromatography conditions include: the chromatographic column is a C18 column, the mobile phase A is an aqueous solution of ammonium formate or ammonium acetate, and the mobile phase B is a methanol or acetonitrile solution.

[0124] The elution procedure of the liquid chromatography is:

[0125] 0~1min, mobile phase A 20~40%, mobile phase B 60~80%;

[0126] 1-3.8 min, mobile phase A 20-40% to 2-5%, mobile phase B 60-80% to 95-98%;

[0127] 3.8~4.1min, mobile phase A 2~5%, mobile phase B 95~98%;

[0128] 4.1~4.5min, mobile phase A 2~5% to 20~40%, mobile phase B 95~98% to 60~80%;

[0129] 4.5~5min, mobile phase A 20~40%, mobile phase B 60~80%.

[0130] Set the elution program of liquid chromatography to further improve the detection efficiency of liquid chromatography.

[0131] In some specific embodiments, the ion source of the mass spectrometry detection is an electrospray ion source, and a multiple reaction monitoring mode is used in a positive ion mode.

[0132] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0133] Example 1

[0134] A kit for extracting free testosterone in serum, comprising the following reagents:

[0135] (1) Preparation of magnetic bead solution: Prepare HLB magnetic bead suspension with a concentration of 5 mg / mL using 50% methanol aqueous solution.

[0136] (2) Preparation of equilibrium solution: 5% methanol aqueous solution.

[0137] (3) Preparation of internal standard solution: dilute testosterone with a concentration of 100 μg / mL with methanol. 13 C3 stock solution was prepared into an internal standard solution with a concentration of 100 pg / mL.

[0138] (4) Preparation of eluent: 20% methanol in water.

[0139] (5) Preparation of eluent: 0.5% isonicotinoyl chloride hydrochloride methanol solution.

[0140] (6) Preparation of diluent: 10% methanol aqueous solution.

[0141] (7) Preparation of dialysis buffer: Weigh 12.57 g HEPES, 0.9 g NaOH, 5.23 g NaCl, 0.22 g KH2PO4, 0.28 g MgSO4·7H2O, 0.3 g Urea and 0.28 g CaCl2·2H2O into 1 L purified water, and add 500 μL ProClinTM 300; after full dissolution, adjust the pH to 7.40 with HCl.

[0142] (8) Preparation of pH adjustment buffer: Weigh 9.25 g of HEPES into 50 mL of purified water. After fully dissolving, adjust the pH to 7.40 with HCl.

[0143] Example 2

[0144] A kit for extracting free testosterone in serum, comprising the following reagents:

[0145] (1) Preparation of magnetic bead solution: Prepare HLB magnetic bead suspension with a concentration of 2.5 mg / mL using 0% methanol aqueous solution.

[0146] (2) Preparation of equilibrium solution: 0% methanol aqueous solution.

[0147] (3) Preparation of internal standard solution: dilute testosterone with a concentration of 100 μg / mL with methanol. 13 C3 stock solution was prepared into an internal standard solution with a concentration of 10 pg / mL.

[0148] (4) Preparation of eluent: 5% methanol in water.

[0149] (5) Preparation of eluent: 0.1% isonicotinoyl chloride hydrochloride methanol solution.

[0150] (6) Preparation of diluent: 0% methanol aqueous solution.

[0151] (7) Preparation of dialysis buffer: Weigh 12.57 g HEPES, 0.9 g NaOH, 5.23 g NaCl, 0.22 g KH2PO4, 0.28 g MgSO4·7H2O, 0.3 g Urea and 0.28 g CaCl2·2H2O into 1 L purified water, and add 500 μL ProClinTM 300; after full dissolution, adjust the pH to 7.40 with NaOH.

[0152] (8) Preparation of pH adjustment buffer: Weigh 5.96 g of HEPES into 50 mL of purified water. After fully dissolving, adjust the pH to 7.40 with NaOH.

[0153] Example 3

[0154] A kit for extracting free testosterone in serum, comprising the following reagents:

[0155] (1) Preparation of magnetic bead solution: Prepare HLB magnetic bead suspension with a concentration of 10 mg / mL using 100% methanol aqueous solution.

[0156] (2) Preparation of equilibrium solution: 15% methanol aqueous solution.

[0157] (3) Preparation of internal standard solution: dilute testosterone with a concentration of 100 μg / mL with methanol. 13 C3 stock solution was prepared into an internal standard solution with a concentration of 200 pg / mL.

[0158] (4) Preparation of eluent: 30% methanol in water.

[0159] (5) Preparation of eluent: 1.0% isonicotinoyl chloride hydrochloride methanol solution.

[0160] (6) Preparation of diluent: 50% methanol aqueous solution.

[0161] (7) Preparation of dialysis buffer: Weigh 12.57 g HEPES, 0.9 g NaOH, 5.23 g NaCl, 0.22 g KH2PO4, 0.28 g MgSO4·7H2O, 0.3 g Urea and 0.28 g CaCl2·2H2O into 1 L purified water, and add 500 μL ProClinTM 300; after full dissolution, adjust the pH to 7.40 with NaOH.

[0162] (8) Preparation of pH adjustment buffer: Weigh 11.92 g of HEPES into 50 mL of purified water. After fully dissolving, adjust the pH to 7.40 with NaOH.

[0163] Embodiment 4-6

[0164] A method for extracting free testosterone in serum, using the reagents in the kit provided in Examples 1 to 3 respectively, specifically following the steps below:

[0165] 1. Sample equilibrium dialysis: Take the cleaned and dried polytetrafluoroethylene base plate and install the equilibrium dialysis membrane with a molecular weight cutoff of 10kd. After the device is assembled, immediately add 400μL of dialysis buffer to one side of the membrane, and add 380μL of the serum sample to be tested (human serum sample collected in the laboratory) and 20μL of pH adjustment buffer to the other side of the membrane. Then seal the membrane and incubate the dialysis device at 150rpm and 37℃ for 4h.

[0166] 2. After dialysis, take out 300 μL of dialysate and place it in a deep well plate.

[0167] 3. Take 300 μL of standard solution (100 μg / mL testosterone stock solution was diluted with dialysis buffer, including standard solutions with concentrations of 0.5 pg / mL, 1.0 pg / mL, 4.0 pg / mL, 10.0 pg / mL, 25.0 pg / mL, 100.0 pg / mL and 250.0 pg / mL) and 300 μL of quality control solution (100 μg / mL testosterone stock solution was diluted with dialysis buffer, including quality control solutions with concentrations of 5.0 pg / mL, 50.0 pg / mL and 200.0 pg / mL) in deep well plates.

[0168] 4. Place the magnetic bead solution, balancing solution, internal standard solution, eluent, elution solution, diluent and syringe cleaning solution in the fully automatic magnetic solid phase extraction device (iMS-5000, Baichen Medical) in turn, and place the deep-well plate in the deep-well plate slot of the fully automatic magnetic solid phase extraction device.

[0169] 5. Set the automated extraction program as shown in Table 1.

[0170] Table 1

[0171]

[0172] Among them, the needle cleaning fluid is 80% methanol aqueous solution.

[0173] The loading solution is the testosterone extract, which is placed in a 96-well loading plate to be used for liquid chromatography-mass spectrometry detection.

[0174] Embodiment 7~9

[0175] A method for detecting free testosterone in serum, wherein the testosterone extract obtained by the method of Examples 4 to 6 is subjected to liquid chromatography-mass spectrometry detection, and the operation is specifically performed according to the following steps:

[0176] 1. Sample testing

[0177] (1) Liquid chromatography conditions:

[0178] The chromatographic column used was Venusil MP C18 (2.1*50 mm, 3μm); the mobile phase A was 20mM ammonium formate aqueous solution, and the mobile phase B was methanol solution; the column temperature was 40°C; the injection volume was 20μL; and the gradient elution program was shown in Table 2.

[0179] Table 2 Chromatographic conditions

[0180] Serial number time Flow rate (mL / min) Mobile phase A% Mobile phase B% 1 0.00 0.3 40 60 2 1.00 0.3 40 60 3 3.80 0.3 5 95 4 4.10 0.3 5 95 5 4.50 0.3 40 6 6 5.00 0.3 40 60

[0181] (2) Mass spectrometry conditions:

[0182] The ion source was an electrospray ion source (ESI), in positive ion mode, multiple reaction monitoring mode (MRM), capillary voltage 2.0 kV, desolvation temperature 500 °C, desolvation gas 600 L / Hr, cone gas 150 L / Hr; the mass spectrometry parameters such as parent ion, daughter ion, cone voltage, collision energy, etc. of each compound are shown in Table 3.

[0183] Table 3 Mass spectrometry parameters

[0184]

[0185] 2. Linearity test

[0186] The concentration of free testosterone was 0.5-250 pg / mL as the horizontal axis, and the peak area ratio of the compound detected by mass spectrometry at each concentration to the internal standard was used as the vertical axis to draw a standard curve, and regression calculation was performed. The results are shown in Table 4.

[0187] Table 4 Linear regression equation for free testosterone

[0188]

[0189] As shown in Table 4, the linearity is good within the detection range of 0.5-250pg / mL, and the correlation coefficients are all above 0.990. When detecting serum samples with the quantitative lower limit concentration (0.5pg / mL), the CVs of the detection results of Examples 7-9 are all less than 10%, and the relative deviations are all within the range of [-10%, 10%], meeting the quantitative requirements. The mass spectrum of the sample INC derivatization reaction detection in Example 7 is shown in Figure 1 shown.

[0190] Test 1 Precision Verification

[0191] Human serum sample dialysate was used as a matrix, and an appropriate amount of testosterone standard was added to prepare three samples of low concentration, medium concentration and high concentration. The reagent provided in Example 1 was used to perform the test according to the methods of Examples 4 and 7 to verify its precision. Each concentration sample was repeated 6 times per day for 3 consecutive days. The results calculated according to the linear regression equation of Example 7 in Table 4 are shown in Table 5:

[0192] Table 5 Precision verification results (concentration unit: pg / mL)

[0193]

[0194] As shown in Table 5, the detection method has good precision, and the three-day precision test results are all less than 5%.

[0195] Test 2: Correctness Verification

[0196] Human serum dialysate was used as the basic sample for the recovery experiment. 30 μL of the low-concentration, medium-concentration and high-concentration samples prepared in Test 1 were mixed into each 270 μL of the basic sample. The reagents provided in Example 1 were used to detect the basic sample and the spiked sample according to the methods of Examples 4 and 7, respectively. The determination was repeated 6 times, and the spiked recovery at the three levels of low concentration, medium concentration and high concentration was statistically analyzed. The recovery rate = (spiked sample detection value-basic sample value) / theoretical value × 100%. The results calculated according to the linear regression equation of Example 7 in Table 4 are shown in Table 6:

[0197] Table 6 Correctness verification results (concentration unit: pg / mL)

[0198]

[0199] It can be seen from Table 6 that the recovery rates of this method are all in the range of 85%-115%, which meets the requirements.

[0200] Test 3: Verification of the lower limit of quantification

[0201] An appropriate amount of testosterone standard was added to the dialysis buffer to prepare a quantitative lower limit sample with a concentration of 0.5 pg / mL. The reagent provided in Example 1 was used, and the detection was repeated 10 times according to the methods of Example 4 and Example 7. The results calculated according to the linear regression equation of Example 7 in Table 4 are shown in Table 7.

[0202] Table 7 Quantitative lower limit verification results (concentration unit pg / mL)

[0203]

[0204] As shown in Table 7, the method provided in this embodiment can reach a lower limit of quantification of 0.5 pg / mL when detecting free testosterone.

[0205] Experiment 3 Screening of magnetic beads

[0206] (1) Effect of magnetic bead type on detection

[0207] This example uses the detection methods provided in Example 4 and Example 7 to detect free testosterone in the sample, and uses HLB, WAX, WCX, MAX and MCX magnetic solid phase extraction materials and combined magnetic beads HLB-WAX (1:1), HLB-MAX (1:1), HLB-MCX (1:1) to extract free testosterone in the sample, and detects the content of free testosterone by LC-MS / MS. All magnetic beads are prepared into a magnetic bead suspension with a concentration of 5 mg / mL using 50% methanol aqueous solution, and the volume of the magnetic bead liquid used is 200 μL. Each group is measured 5 times, and the average value is taken. The results are shown in Table 8:

[0208] Table 8 Effects of different types of magnetic beads on detection

[0209]

[0210] As shown in Table 8, different magnetic beads have different extraction effects on free testosterone, which affects the final detection sensitivity of free testosterone. Compared with other types of magnetic beads, when HLB magnetic beads are used for extraction among single magnetic beads, the detected free testosterone response peak area is the highest; and when HLB-MAX combination magnetic beads are used for extraction, the detected free testosterone response peak area is about 15% higher than that of single HLB magnetic beads, which is particularly important when the instrument response is poor and low-concentration free testosterone samples are detected.

[0211] Further, two low-concentration samples from women collected in the laboratory were tested according to the methods of Examples 4 and 7, wherein the magnetic beads were used to extract free testosterone in the samples using HLB and combined magnetic beads HLB-MAX, respectively. Specific magnetic bead liquid preparation: 50% methanol aqueous solution was used to prepare HLB magnetic bead suspension and HLB-MAX combined magnetic bead magnetic bead liquid with a concentration of 5 mg / mL, wherein the ratio of HLB and MAX magnetic beads in the HLB-MAX combined magnetic bead liquid was 1:1. The results are shown in Table 9.

[0212] Table 9 Detection of low concentration samples by different types of magnetic beads

[0213]

[0214] It can be seen from Table 9 that when testing low-concentration samples, the overall CV of the extraction using HLB-MAX combination magnetic beads is smaller than that of the single HLB magnetic beads extraction, which is more conducive to the quantitative detection of low-concentration samples.

[0215] (2) Effect of magnetic bead solution concentration on detection

[0216] HLB magnetic bead suspensions with concentrations of 2.5 mg / mL, 5 mg / mL, 10 mg / mL, and 20 mg / mL were prepared with 50% methanol aqueous solution, and 200 μL of magnetic bead solution was taken to extract free testosterone in the sample, and the content of free testosterone was detected by LC-MS / MS. Each group was measured 5 times, and the average value was taken. The test results are shown in Table 10.

[0217] Table 10 Effects of different magnetic beads concentration on detection

[0218]

[0219] As shown in Table 10, the magnetic bead concentration has reached a saturated extraction state at 5-10 mg / mL. Considering the cost of magnetic beads for practical applications, 5 mg / mL is preferred.

[0220] Experiment 4 Effects of different pre-treatment methods on test results

[0221] Human serum collected in the laboratory was selected and divided into 4 parts, numbered as samples 1 to 4, and free testosterone in samples 1 to 4 was extracted using the method of Example 4, a solid phase extraction plate SPE filled with commercially available HLB material, and a conventional derivatization method, respectively.

[0222] Among them, the solid phase extraction plate SPE extraction filled with commercially available HLB material: ① obtain serum dialysate through equilibrium dialysis; ② take 300μL serum dialysate in a centrifuge tube and add 100μL internal standard solution, mix and centrifuge for 5min; ③ take 200μL methanol and add it to SPE, and pressurize it for 2min through a positive pressure device; ④ take 200μL purified water and add it to SPE, and pressurize it for 2min through a positive pressure device; ⑤ take 250μL of the supernatant of serum dialysate and internal standard solution in SPE, and pressurize it for 3min through a positive pressure device; ⑥ take 200μL 20% methanol water was added to SPE, and positive pressure was applied for 2 minutes through a positive pressure device; ⑦ 70 μL of acetonitrile was added to SPE, and positive pressure was applied for 2 minutes through a positive pressure device to elute the liquid into a 96-well sample plate; ⑧ 50 μL of derivatization reagent was added to the 96-well sample plate eluate, and the solution was placed in a constant temperature oscillator at 55°C for 2 minutes; ⑨ After the reaction, 60 μL of purified water was added, the plate was covered with a plate mat, and mixed for 2 minutes, and then the sample was tested. In the experiment, manual pretreatment was used for the solid phase extraction plate SPE.

[0223] Conventional derivatization method extraction: ① Obtain serum dialysate through equilibrium dialysis; ② Take 200μL magnetic bead solution to the magnetic bead well of the deep-well plate; ③ Pipette 200μL equilibrium solution to the equilibrium well of the deep-well plate; ④ Pipette 100μL internal standard solution to the sample well of the deep-well plate; ⑤ Pipette 400μL eluent to the eluent well of the deep-well plate; ⑥ Pipette 100μL acetonitrile to the elution well of the deep-well plate; ⑦ Place in the deep-well plate slot of the fully automatic magnetic solid phase extraction device for extraction; ⑧ After the end, take out the eluent in the deep-well plate into a 1.5mL centrifuge tube and blow dry with nitrogen for 20min; ⑨ Then add 100μL of derivatization reagent respectively, place in a constant temperature oscillator at 55℃ for reaction for 2min; ⑩ After the reaction, add 60μL purified water, mix for 2min, then add the sample to the 96-well sample plate, cover with a plate mat, and wait for sample loading and detection. The conventional derivatization method uses elution first, then nitrogen blow dry, and finally add the derivatization reagent for derivatization reaction.

[0224] Each group was tested three times according to the method of Example 7. The results are shown in Table 11.

[0225] Table 11 Comparison of extraction by different pre-treatment methods

[0226]

[0227] As can be seen from Table 11, the extraction effect of Example 4 of the present invention is greatly improved compared with SPE, especially in terms of the coefficient of variation CV, the CV of Example 4 of the present invention is much smaller, which shows that the repeatability of the extraction of Example 4 is better than that of SPE extraction, and the cost of magnetic beads used for a single sample is much lower than that of SPE.

[0228] Example 4 of the present invention innovatively adopts a method of adding a derivatization reagent to the eluent to achieve an integrated process of in-situ elution and derivatization. Compared with conventional derivatization methods, not only does it eliminate the cumbersome operation steps of nitrogen blowing and back-and-forth pipetting, but it also improves the detection response, especially in terms of the coefficient of variation CV, which is much smaller in Example 4 of the present invention.

[0229] Experiment 5 Effect of derivatization reagents on detection

[0230] The methods provided in Example 4 and Example 7 were used to detect free testosterone in the sample, wherein isonicotinoyl chloride hydrochloride, methoxyamine hydrochloride, 3-pyridinemethoxyamine and hydroxylamine hydrochloride were respectively selected as derivatization reagents in the eluent, and no derivatization reagent was added, and the content of free testosterone was detected by LC-MS / MS. In addition, the concentration of isonicotinoyl chloride hydrochloride as a derivatization reagent was investigated. The volume of the eluent used was 100 μL, and each group was measured 5 times, and the average value was taken. The test results are shown in Table 12. The mass spectrum of sample 4 non-derivatization reaction detection is shown in Table 12. Figure 2 shown.

[0231] Table 12 Effects of different derivatization reagents on detection

[0232]

[0233] As shown in Table 12, different derivatization reagents have different derivatization effects, which affects the final detection sensitivity of free testosterone. In this experiment, isonicotinoyl chloride hydrochloride is used as a derivatization reagent. Compared with the reagents of hydroxylamine and methoxyamine derivatization reactions, it is a method based on acylation derivatization, which can be applied to the derivatization of various steroid hormones including testosterone. The derivatization temperature of this derivatization is 50~55℃ and the derivatization reaction can be completed in 2 minutes, which can save reaction time. From the results, the use of isonicotinoyl chloride hydrochloride as a derivatization reagent has the best effect, which may be due to the difference in the efficiency of derivatization and the ionization efficiency of the derivatization product. At the same time, from the results of different concentrations of isonicotinoyl chloride hydrochloride methanol solutions, it can be seen that the derivatization effect is best at a concentration of 0.5%, and the detection sensitivity of free testosterone is the highest.

[0234] In addition, the analysis found that the response of the non-derivatized group was the lowest, and it was found that interference peaks would be generated when the serum samples were tested without derivatization, affecting the accurate quantification of the product.

[0235] Example 10

[0236] 1. Two sample composite detection detection reagents, which are different from Example 1 in that the eluents are prepared with 0.5% hydroxylamine hydrochloride methanol solution (HOA) and 0.5% methoxyamine hydrochloride methanol solution (MOA) respectively.

[0237] 2. Extract free testosterone from the two samples according to the following steps:

[0238] (1) Performing balanced dialysis treatment on the sample according to Example 4;

[0239] (2) Take two sets of deep-well plates, and put 300 μL of standard solution (7 concentration levels), 300 μL of quality control solution (3 concentration levels), and 300 μL of sample dialysate into each set of deep-well plates;

[0240] (3) Use a pipette to add 200 μL of magnetic bead solution, 200 μL of balancing solution, 100 μL of internal standard solution, 400 μL of eluent, and 100 μL of elution solution to the corresponding wells of the deep-well plate. The elution solution added to one set of deep-well plates is 0.5% hydroxylamine hydrochloride methanol solution, and the other set is 0.5% methoxyamine hydrochloride methanol solution.

[0241] (4) Place the deep-well plate into the deep-well plate slot of the fully automatic magnetic solid phase extraction device and set the automated extraction program as shown in Table 13.

[0242] Table 13

[0243]

[0244] (5) After the program is completed, use a pipette to transfer 100 μL of each of the standard solution, quality control solution, and sample solution into a 96-well sample plate. Then transfer 100 μL of the other standard solution, quality control solution, and sample solution into the corresponding 96-well sample plate. Combine the samples. Cover with a plate mat, mix for 30 seconds, and prepare for testing.

[0245] 3. Two types of sample testing

[0246] The relevant conditions of liquid chromatography and liquid mass spectrometry were set according to Example 7, wherein the mass spectrometry parameters are shown in Table 14. The test results are shown in Table 14. Figure 3 As shown, A is a HOA derivative and B is a MOA derivative.

[0247] Table 14 Mass spectrometry parameters

[0248]

[0249] 4. Linearity test

[0250] The standard curve was drawn with the concentration of free testosterone 0.5-250 pg / mL as the horizontal axis and the peak area ratio of the compound detected by mass spectrometry at each concentration to the internal standard as the vertical axis. Figure 4 As shown (where A is the MOA derivatization reaction standard curve, and B is the HOA derivatization reaction standard curve), regression calculation was performed and the results are shown in Table 15.

[0251] Table 15 Linear regression equation for multi-sample composite detection

[0252]

[0253] From the results, we can see that the linearity is good in the detection range of 0.5-250pg / mL, and the correlation coefficients of the two groups are both above 0.990, which meets the quantitative requirements.

[0254] 5. Correctness verification

[0255] Human serum dialysate was used as the basic sample for the recovery experiment. 270 μL of the basic sample was mixed with 30 μL of the low-concentration, medium-concentration and high-concentration samples prepared in Experiment 1. The basic sample and the spiked sample were measured 6 times, and the spiked recoveries at the low, medium and high concentration levels were statistically analyzed. The recovery rate = (spike-sample detection value - basic sample value) / theoretical value × 100%. The results are shown in Table 16.

[0256] Table 16 Verification results of accuracy of multiple sample composite detection (concentration unit: pg / mL)

[0257]

[0258] As can be seen from Table 16, the recovery rates of this method are all in the range of 85%-115%, indicating that two derivatization reagents are used to derivatize the samples separately, and finally the sample loading solutions obtained by the two derivatization reagents are combined for mass spectrometry detection, and the detection results meet the requirements.

[0259] Embodiment 11

[0260] Two low-concentration female serum samples collected in the laboratory were divided into five parts, and the free testosterone in the samples was detected by the method provided in Example 10. Each test was performed three times. The difference from Example 10 was that the volume ratios of the HOA-derived and MOA-derived extracts were 1:1, 1:2, 1:3, 2:1, and 3:1, respectively. The test results are shown in Table 17.

[0261] Table 17 Effect of different extract mixing ratios on detection

[0262]

[0263] As can be seen from Table 17, when two derivatization reagents were used to derivatize the samples separately, and the volume ratio of the extracts was 1~2:1~2, the CVs of the two derivative product detections were all within 10%; when the mixing ratio exceeded this ratio, when detecting low-concentration samples, one of the products may have a low detection response, causing detection errors, and the CV of the test results was also large, which was not conducive to quantitative detection.

[0264] Example 12

[0265] Three female serum samples and three male serum samples collected in the laboratory were respectively subjected to composite detection. The difference from Example 10 was that the eluents were prepared with concentrations of 0.5% isonicotinoyl chloride hydrochloride methanol solution, 0.5% hydroxylamine hydrochloride methanol solution (HOA) and 0.5% methoxyamine hydrochloride methanol solution (MOA), respectively; the sample extracts obtained by the automated extraction procedure were mixed in equal proportions (1:1:1), and the test results are shown in Table 18.

[0266] Table 18 Detection of three derivatization reagents

[0267]

[0268] It can be seen from Table 18 that the use of three derivatization reagents to derivatize the samples separately can accurately detect the concentration of each sample; at the same time, it can be seen that the peak area values ​​of male samples (high concentration) are all relatively high, which has certain advantages for composite detection using multiple derivatization reagents.

[0269] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A kit for extracting free testosterone in serum, characterized in that: Including magnetic bead solution, balancing solution, internal standard solution, eluent, eluting solution and diluent; The concentration of the magnetic beads in the magnetic bead liquid is 2.5-10 mg / ml, the solvent of the magnetic bead liquid is methanol aqueous solution, the concentration of methanol in the magnetic bead liquid is 0-100%, and the magnetic beads include at least two of hydrophilic-hydrophobic balance magnetic beads, mixed strong anion exchange magnetic beads or mixed strong cation exchange magnetic beads; The balancing solution, eluent and diluent are all methanol-water solutions, the concentration of methanol in the balancing solution is 0-15%, the concentration of methanol in the eluent is 5-30%, and the concentration of methanol in the diluent is 0-50%; The internal standard solution is a methanol or acetonitrile solution containing a testosterone internal standard, and the concentration of the testosterone internal standard in the internal standard solution is 10-200 pg / mL; The eluent includes at least one of isonicotinoyl chloride hydrochloride eluent, methoxyamine hydrochloride eluent, 3-pyridine methoxyamine eluent or hydroxylamine hydrochloride eluent, and the concentrations of the isonicotinoyl chloride hydrochloride eluent, methoxyamine hydrochloride eluent, 3-pyridine methoxyamine eluent and hydroxylamine hydrochloride eluent are independently 0.1-1.0%; Also included is a balanced dialysis reagent for preparing a serum sample dialysate, wherein the balanced dialysis reagent includes a dialysis buffer and a pH adjustment buffer; The dialysis buffer comprises the following components at the following concentrations: HEPES 12.57 g / L, NaOH 0.9 g / L, NaCl 5.23 g / L, KH2PO4 0.22 g / L, MgSO4·7H2O 0.28 g / L, Urea 0.3 g / L, CaCl2·2H2O 0.28 g / L and ProClin TM 300 0.05% volume concentration; The pH of the dialysis buffer is 7.3-7.5; The pH adjustment buffer is a HEPES buffer; The concentration of the pH adjustment buffer is 500-1000 mol / L; The pH value of the pH adjustment buffer is 7.3-7.5; The solvent of the eluent is methanol or acetonitrile.

2. Use of the kit according to claim 1 in extracting or detecting free testosterone in serum.

3. A method for extracting free testosterone in serum, characterized in that: The method comprises taking the serum to be tested as a sample and using the reagents in the kit according to claim 1 to prepare a testosterone extract.

4. The extraction method according to claim 3, characterized in that The following steps are involved: A. The magnetic beads in the magnetic bead solution are taken out and placed in a balance solution, a serum sample dialysate containing a testosterone internal standard, and an eluent in sequence, and then eluted with an eluent to obtain a testosterone eluate; B. The testosterone eluate is heated to perform an in situ derivatization reaction to obtain a testosterone extract.

5. The extraction method according to claim 4, characterized in that The serum sample dialysate containing the testosterone internal standard comprises a mixture of an internal standard solution and a serum sample dialysate, wherein the volume ratio of the internal standard solution to the serum sample dialysate is 1-4:4-6; The volume ratio of the magnetic bead liquid to the serum sample dialysate is 1-3:2-3; The volume ratio of the balancing solution to the serum sample dialysate is 1-3:2-3; The volume ratio of the eluent to the serum sample dialysate is 2-5:2-3; The volume ratio of the eluent to the serum sample dialysate is 1-4:4-6; The magnetic beads are placed in the equilibrium solution for 10 to 60 seconds; The magnetic beads are placed in the serum sample dialysate containing the testosterone internal standard for 30 to 90 seconds; The magnetic beads are placed in the eluent for 10 to 60 seconds; The elution time of the eluent is 30 to 90 seconds.

6. The extraction method according to claim 4, characterized in that The heating temperature is 40-80°C; The heating time is 2 to 10 minutes; The step of heating the testosterone eluate to perform an in-situ derivatization reaction further includes adding a diluent; The volume ratio of the diluent to the serum sample dialysate is 0.3-1:2-3; The method for preparing the serum sample dialysate comprises adding a dialysis buffer and a serum to be tested containing a pH adjustment buffer into a balanced dialysis system, and performing dialysis after sealing the membrane; The balanced dialysis system comprises a bottom plate and a balanced dialysis membrane; The material of the bottom plate includes polytetrafluoroethylene, polypropylene or polyvinylidene fluoride; The equilibrium dialysis membrane comprises a regenerated cellulose membrane with a molecular weight cut-off of 3.5 to 15 KD; The volume concentration of the pH adjusting buffer in the test serum containing the pH adjusting buffer is 1-10%; The volume ratio of the dialysis buffer to the test serum containing the pH adjustment buffer is 0.8-1.2:1; The dialysis time is 3 to 6 hours.

7. The extraction method according to any one of claims 4 to 6, characterized in that When the number of samples was > 1, a different eluent was used during the extraction of each sample; After obtaining the testosterone extract, the step further includes mixing the testosterone extracts of each sample in proportion; When the number of samples = 2, the volume ratio of the two sample extracts is 1~2:1~2; When the number of samples is greater than 2, the extracts of each sample are mixed in equal proportions.

8. A method for detecting free testosterone in serum, characterized in that: The method comprises performing liquid chromatography-mass spectrometry detection on a testosterone extract obtained by using the kit according to claim 1 or the extraction method according to any one of claims 3 to 7; The conditions of the liquid chromatography include: the chromatographic column is a C18 column, the mobile phase A is an aqueous solution of ammonium formate or ammonium acetate, and the mobile phase B is a methanol or acetonitrile solution; The elution procedure of the liquid chromatography is: 0~1min, mobile phase A 20~40%, mobile phase B 60~80%; 1-3.8 min, mobile phase A 20-40% to 2-5%, mobile phase B 60-80% to 95-98%; 3.8~4.1min, mobile phase A 2~5%, mobile phase B 95~98%; 4.1~4.5min, mobile phase A 2~5% to 20~40%, mobile phase B 95~98% to 60~80%; 4.5~5min, mobile phase A 20~40%, mobile phase B 60~80%; The ion source of the mass spectrometry detection is an electrospray ion source, and the multiple reaction monitoring mode is adopted in the positive ion mode.

Images