Method and kit for simultaneous determination of total and free testosterone content in blood

Abstract

The application discloses a method for simultaneously determining the content of total testosterone and free testosterone in blood, which comprises the following steps: step one, dividing a blood sample into two sample parts A and B, and obtaining a sample total testosterone solution by processing the sample A; step two, obtaining a sample free testosterone solution by performing equilibrium dialysis and derivative reaction on the sample B; step three, reconstituting the sample total testosterone solution and the sample free testosterone solution with a reconstitution solution and mixing to obtain a sample total solution; and step four, simultaneously detecting the content of total testosterone and free testosterone in the sample total solution by using LC-MS / MS technology, and obtaining the content of total testosterone and free testosterone in the sample total solution. The application has the beneficial effect of simultaneously determining the content of total testosterone and free testosterone. The application provides a kit for simultaneously determining total testosterone and free testosterone, which can rapidly detect total testosterone and trace free testosterone, and the detection limit of the free testosterone can reach 0.5 pg / mL.

Description

Technical Field

[0001] This invention relates to the field of hormone detection technology. More specifically, this invention relates to a method and kit for simultaneously determining the levels of total testosterone and free testosterone in blood. Background Technology

[0002] Testosterone exists in two forms in the blood: free testosterone and total testosterone. Approximately 98% of blood testosterone binds to proteins to form total testosterone, while only about 2% exists as free testosterone. Total testosterone levels are closely related to conditions such as idiopathic precocious puberty in males, familial precocious puberty in males, adrenal hyperplasia, adrenal cortical tumors, male testicular hypoplasia, anorchia, and hypogonadism (hypothalamic or pituitary hypogonadism). Free testosterone is the active form, and measuring its level reflects the level of biologically active testosterone in the body. Studies have shown that free testosterone detection is more suitable for studying female gonadal dysfunction, hyperandrogenism, male sexual development and puberty disorders, and polycystic ovary syndrome. Therefore, free testosterone measurement is of great significance for male fertility and the diagnosis of female diseases.

[0003] Currently, immunoassay and liquid chromatography-tandem mass spectrometry (LC-MS / MS) are commonly used in clinical practice to detect total testosterone concentration. Free testosterone, however, is calculated by measuring total testosterone and sex hormone-binding protein (SHBMP) concentrations separately, and then calculating the free testosterone concentration. However, because SHBMP or albumin binds not only to testosterone but also to other steroid hormones such as cortisol, the calculated free testosterone concentration is difficult to reflect the true free testosterone level. With the development of modern technology, pretreatment methods such as ultrafiltration or balanced dialysis combined with LC-MS / MS can be used to detect free testosterone in the blood, and this is gradually being adopted in clinical practice. However, this technique can only determine the content of free testosterone, not total testosterone.

[0004] Given the crucial role of total and free testosterone in the human body, accurate quantification is essential for the diagnosis of related diseases. However, even single-method detection of free testosterone presents significant challenges, and simultaneous detection of both testosterone and free testosterone would be even more difficult. Therefore, designing a detection method to simultaneously measure the levels of total and free testosterone in the blood is a matter worthy of careful consideration. Summary of the Invention

[0005] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0006] To achieve these objectives and other advantages according to the present invention, a method for simultaneously determining the levels of total testosterone and free testosterone in blood is provided, comprising the following steps:

[0007] Step 1: Divide the blood sample into two parts, A and B. Add the extraction solution to sample A, shake, centrifuge, take the supernatant, and concentrate the supernatant to obtain the total testosterone solution of the sample. The extraction solution contains an isotope internal standard of testosterone.

[0008] Step 2: Sample B is balanced by dialysis to obtain free testosterone dialysate. Testosterone isotope internal standard is added to the free testosterone dialysate, and then derivatization reaction is carried out under heating conditions. After the derivatization reaction is completed, extraction solution is added to the free testosterone dialysate, and liquid-liquid extraction is performed, followed by centrifugation. The supernatant is collected and concentrated to obtain the sample free testosterone solution.

[0009] Step 3: Add a reconstitution solution to reconstitute the total testosterone solution and the free testosterone solution from Step 1 and mix thoroughly to obtain the total sample solution;

[0010] Step 4: Prepare total testosterone standard curve solutions and free testosterone standard curve solutions with multiple standard curve concentration gradients using testosterone standards. Add testosterone isotope internal standards. Measure the peak area of ​​testosterone and its internal standard at each standard curve concentration using LC-MS / MS. Perform linear regression using the ratio of standard curve concentration to the peak area of ​​testosterone and its internal standard to obtain the total testosterone standard curve and the free testosterone standard curve. Simultaneously detect the total testosterone and its internal standard peak area ratio, and the free testosterone and its internal standard peak area ratio in the total sample solution using LC-MS / MS. Substitute these values ​​into the standard curves to calculate the total testosterone and free testosterone content in the total sample solution. The method for processing the testosterone standard when preparing the free testosterone standard curve solution is the same as the method for processing the free testosterone dialysate described in Step 2.

[0011] Preferably, the temperature of the equilibration dialysis operation in step two is 37°C, the equilibration dialysis time is 4 to 16 hours, and the buffer solution used in the equilibration dialysis operation is HEPES buffer with pH = 7.4.

[0012] Preferably, the derivatization reaction in step two is characterized by a temperature of 60–80°C, a reaction time of 40–70 min, and the derivatization reagent used in the reaction being either a methanol aqueous solution of hydroxylamine hydrochloride or a methanol aqueous solution of methoxyamine, wherein the concentration of the derivatization reagent is 0.02–0.06 mol / L, and the methanol mass fraction in the methanol aqueous solution is 20%–80%.

[0013] Preferably, the concentration operation in steps one and two employs either nitrogen blowing or vacuum centrifugal drying and concentration methods.

[0014] Preferably, the liquid-liquid extraction operation in step two uses a mixture of methanol, acetonitrile, and methyl tert-butyl ether, or a combination thereof, as the extraction liquid.

[0015] Preferably, the resolution solution in step two is either methanol or acetonitrile.

[0016] Preferably, the equilibration dialysis in step two uses a 96-well equilibration dialysis plate.

[0017] Preferably, the LC-MS / MS is characterized by the following chromatographic conditions: mobile phase I is an aqueous solution of formic acid, and mobile phase II is a methanol solution of formic acid, with gradient elution, wherein mobile phase I and mobile phase II form a liquid phase elution reagent.

[0018] Preferably, the sample comprises a human blood sample, a quality control sample, and a testosterone standard, wherein the quality control sample uses three quality control concentrations: a high-concentration quality control sample prepared from a mixture of adult male blood samples, a medium-concentration quality control sample prepared from a mixture of adult male and adult female blood samples in a certain proportion, and a low-concentration quality control sample prepared from a mixture of adult female blood samples.

[0019] A kit based on the aforementioned assay method is provided, comprising a standard curve working solution, a quality control sample, an internal standard working solution, a buffer solution, a derivatization reagent, and a liquid chromatography elution reagent, wherein the standard curve working solution is prepared from the testosterone standard using the buffer solution, and the internal standard working solution is prepared from an isotope internal standard of testosterone using the buffer solution.

[0020] The present invention has at least the following beneficial effects:

[0021] First, the determination method provided by this invention can simultaneously determine the content of total testosterone and free testosterone in the blood by injecting the total sample solution once and using LC-MS / MS technology, thereby reducing the number of detections and the time required for determination using a triple tandem LC-MS / MS instrument, and has high clinical application value.

[0022] Secondly, the assay method provided by this invention significantly improves the sensitivity of free testosterone detection to 0.5 pg / ml, meeting the clinical needs for detecting free testosterone levels in women.

[0023] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0024] Figure 1The flowchart illustrates the measurement method according to one of the technical solutions of the present invention.

[0025] Figure 2 This is a total ion chromatogram of total testosterone and free testosterone in blood, representing one of the technical solutions of the present invention.

[0026] Figure 3 An ion chromatogram of total testosterone extraction from blood, representing one of the technical solutions of this invention;

[0027] Figure 4 An ion chromatogram of free testosterone extracted from blood, representing one of the technical solutions of this invention;

[0028] Figure 5 This is a standard curve of total testosterone in blood, representing one of the technical solutions of the present invention.

[0029] Figure 6 This is a standard curve of free testosterone in blood, representing one of the technical solutions of the present invention.

[0030] Figure 7 This is a detection limit diagram of free testosterone in blood, representing one of the technical solutions of the present invention.

[0031] Figure 8 This is a detection limit diagram of total testosterone in blood according to one of the technical solutions of the present invention;

[0032] Figure 9 The graph shows the change in the ratio of the derivatized free testosterone concentration to the initial concentration after the derivatized free testosterone is directly reconstituted and mixed with the total testosterone solution of the sample without extraction, according to one of the technical solutions of the present invention.

[0033] Figure 10 The graph shows the change in the ratio of the derivatized free testosterone concentration to the initial concentration after extraction and reconstitution with the total testosterone solution of the sample, which is one of the technical solutions of the present invention. Detailed Implementation

[0034] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0035] It should be noted that, unless otherwise specified, the experimental methods described in the following embodiments are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified. In the description of this invention, the orientation or positional relationship indicated by the terms is based on the orientation or positional relationship shown in the accompanying drawings, and is only for the convenience of describing this invention and simplifying the description. It does not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this invention.

[0036] <Example>

[0037] Step 1: Take 100 μL of the blood sample to be tested and add 400 μL of a 1 ng / mL testosterone-containing isotope internal standard (testosterone- 13 The methanol solution of C3 was vortexed at 2000×g and room temperature for 5 min, and then centrifuged at 14000×g and 10℃ for 10 min. The supernatant was collected and dried under vacuum at 1300×g and 30℃ to obtain the total testosterone solution of the sample.

[0038] Step 2: Take 200 μL of the blood sample to be tested into the dialysis sample chamber. Add 400 μL of HEPES buffer (pH = 7.4) to another dialysis buffer chamber separated by a dialysis membrane. Incubate at 37℃ and 250 rpm for 4 hours to reach equilibration. Free testosterone dialysate is obtained in the dialysis buffer chamber. Take 150 μL of the free testosterone dialysate and add 10 μL of testosterone isotope internal standard (testosterone-) to the free testosterone dialysate. 13 C3) 140 μL of a methanol-water solution containing 0.04 mol / L hydroxylamine hydrochloride, wherein the methanol mass fraction in the methanol-water solution is 60%, and the derivatization reaction is carried out at 65℃ and 600 rpm for 60 min. After the derivatization reaction, the free testosterone dialysate after the derivatization reaction is cooled in a refrigerator at 2-8℃ for 10 min, then removed and 100 μL of H2O is added, followed by 600 μL of methyl tert-butyl ether for liquid-liquid extraction. After vortexing at 2000×g at room temperature for 5 min, the solution is centrifuged at 14000×g at 10℃ for 10 min. The supernatant is collected and vacuum dried at 1300×g at 30℃ to obtain the sample free testosterone solution.

[0039] Step 3: Redissolve the total testosterone solution from Step 1 in 200 μL of methanol, and then use it to redissolve the free testosterone solution from Step 2. Vortex the solution at 2000×g until homogeneous, centrifuge at 14000×g and 10℃ for 10 min, and take the supernatant to obtain the total solution.

[0040] Step 4: Dissolve commercially available testosterone standards (without free hormones) in buffer solution to obtain a standard curve working solution. Prepare total testosterone and free testosterone standard curve solutions of various concentrations using this working solution. Add 100 μL of each concentration to a 96-well plate, and add 10 μL of internal standard working solution prepared from the testosterone isotope internal standard using the buffer solution. Add 110 μL of the total sample solution to the 96-well plate, and transfer the plate to an LC-MS / MS instrument (ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometer) for analysis.

[0041] The total testosterone standard solution and the free testosterone standard solution were prepared by diluting the standard curve working solution with buffer solution. The free testosterone standard solution required the same heating derivatization reaction and liquid-liquid extraction operation as in step two. The corresponding standard concentrations are shown in Table 1 below.

[0042] Table 1. Standard Concentrations of Total Testosterone and Free Testosterone

[0043] Standard solution Concentration 1 Concentration 2 Concentration 3 Concentration 4 Concentration 5 Concentration 6 Concentration 7 Concentration 8 Total testosterone (ng / mL) 0 0.2 0.5 2 5 10 20 30 Free testosterone (pg / mL) 0 1.5 5 10 25 100 250 1000

[0044] In this embodiment, the LC-MS / MS chromatographic separation conditions are as follows:

[0045] The chromatographic column is a BEH ultra-high performance chromatography column C18, with a packing particle size of 1.7 μm, an inner diameter of 2.1 mm, and a column length of 50 mm;

[0046] The column temperature is 40℃;

[0047] Mobile phase I was an aqueous solution of 0.1% formic acid, and mobile phase II was a methanol solution of 0.1% formic acid. Reversed-phase chromatography was used for gradient elution, and the gradient elution program is shown in Table 2.

[0048] Table 2 Gradient elution program

[0049]

[0050]

[0051] In this embodiment, the LC-MS / MS mass spectrometry conditions are as follows:

[0052] The electrospray ion source adopts a positive ion mode, with the ion source temperature at 550℃, the atomizer pressure at 50Psi, the auxiliary pressure at 50Psi, the curtain air pressure at 35Psi, and the spray voltage at 5500V.

[0053] The multi-reaction monitoring (MRM) mode was used to detect the mother ion / daughter ion pairs and their corresponding retention times, declustering voltages, inlet voltages, and collision energies, as shown in Table 3 below. Ions marked with "*" are quantitative ions, while those without "*" are qualitative ions.

[0054] Table 3 Monitoring data of each substance during the reaction process

[0055]

[0056] Based on the detection results of the total testosterone standard curve solution and the free testosterone standard curve solution, linear regression was performed with the standard curve solution concentration as the x-axis and the ratio of the peak area of ​​total testosterone or free testosterone and its internal standard as the y-axis to obtain the total testosterone standard curve, as shown in the attached figure. Figure 5The standard curves for free testosterone are shown in the attached figure. Figure 6 As shown, total testosterone exhibited a good linear relationship within the concentration range of 0–30 ng / mL, with a correlation coefficient >0.998. The relevant linear regression equations are detailed in the appendix. Figure 5 Free testosterone showed good linearity in the concentration range of 0–1000 pg / mL, with a correlation coefficient >0.997. The relevant linear regression equations are detailed in the appendix. Figure 6 .

[0057] The ratio of total testosterone peak area to its internal standard peak area and the ratio of free testosterone peak area to its internal standard peak area in the total solution of the sample were detected by mass spectrometry. The ratios were then substituted into the corresponding standard curve equations to calculate the content of total testosterone and free testosterone in the blood. The detection method for total testosterone and free testosterone provided in this embodiment achieves the beneficial effect of simultaneously determining the content of total testosterone and free testosterone in human blood samples, and has the beneficial effect of detection limits of 0.05 ng / mL and 0.5 pg / mL for total testosterone and free testosterone, respectively.

[0058] <Method Validation of Quality Control Samples>

[0059] I. Detection Accuracy and Precision

[0060] Three quality control samples with different concentration levels (high, medium, and low) were selected and processed according to the method described in the example. Each batch of quality control samples at each concentration level was measured in parallel 10 times. The total and free testosterone content was detected in each single injection, and the accuracy and precision of the test results were calculated. The results are shown in Tables 4 and 5 below. The high-concentration quality control sample was prepared from a mixture of adult male blood samples; the medium-concentration quality control sample was prepared from a 1:1 mixture of adult male and adult female blood samples; and the low-concentration quality control sample was prepared from a mixture of adult female blood samples.

[0061] Table 4. Accuracy and Precision Testing of Total Testosterone

[0062]

[0063]

[0064] Table 5. Accuracy and Precision Testing of Free Testosterone

[0065]

[0066]

[0067] As shown in Tables 4 and 5, the CV of the test results obtained by the method of the embodiment for quality control samples of different concentrations is less than 15%, which is within the acceptable range, indicating that the accuracy and precision of the evaluation test method fully meet the requirements.

[0068] <Comparative Example>

[0069] The total and free testosterone levels in the blood were determined using the same method as in the previous example. The difference was that, after the derivatization reaction in step two, the free testosterone dialysate was not added to methyl tert-butyl ether for liquid-liquid extraction. Instead, it was directly shaken, centrifuged, and vacuum dried to obtain the sample free testosterone solution.

[0070] The total solutions of the two samples obtained from the examples and comparative examples were placed at 10°C, and the ratio of the derivatized free testosterone concentration to the initial free testosterone concentration was monitored. It was found that the ratio of the derivatized free testosterone concentration to the initial free testosterone concentration in the total solution of the sample obtained from the comparative example continuously increased after 3 hours of placement, while the ratio of the derivatized free testosterone concentration to the initial free testosterone concentration in the total solution of the sample obtained from the examples did not change significantly within 6 days (144 hours). The reason for this may be that the derivatizing reagent in the comparative example, which was not extracted with methyl tert-butyl ether, would further react with the total testosterone solution after redissolving and mixing, generating more derivatized free testosterone, thus affecting the ratio. Therefore, extracting the free testosterone dialysate after the derivatization reaction in step two can remove excess derivatizing reagent, thereby achieving the beneficial effect of accurately detecting the content of testosterone and free testosterone.

[0071] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

[0072] like Figure 2 , Figure 3 , Figure 4 , Figure 7 , Figure 8 As shown, intensity, cps represents the ion intensity in counts per second, and time, min represents the time in minutes; for example... Figure 5 and Figure 6 As shown, the English explanations are as follows: Area Ratio is the area ratio, and Concentration Ratio is the concentration ratio.

Claims

1. A method for simultaneously determining the levels of total testosterone and free testosterone in blood, characterized in that, Includes the following steps: Step 1: Divide the blood sample into two parts, A and B. Add the extraction solution to sample A, shake, centrifuge, take the supernatant, and concentrate the supernatant to obtain the total testosterone solution of the sample. The extraction solution contains an isotope internal standard of testosterone. Step 2: Sample B is dialyzed to obtain free testosterone dialysate. An isotopic internal standard of testosterone is added to the free testosterone dialysate, followed by a derivatization reaction under heating conditions. The derivatization reaction conditions are: temperature 60-80℃, time 40-70 min, and the derivatization reagent is either a methanol-water solution of hydroxylamine hydrochloride or a methanol-water solution of methoxyamine, with a concentration of 0.02-0.06 mol / L and a methanol mass fraction of 20%-80%. After the derivatization reaction, an extraction solvent is added to the free testosterone dialysate, followed by liquid-liquid extraction, centrifugation, and concentration of the supernatant to obtain a sample free testosterone solution. Before adding the extraction solvent, the free testosterone dialysate after the derivatization reaction is cooled to 2-8℃ and diluted with water. Step 3: Add a reconstitution solution to reconstitute the total testosterone solution and the free testosterone solution from Step 1 and mix thoroughly to obtain the total sample solution; Step 4: Prepare total testosterone standard curve solutions and free testosterone standard curve solutions with multiple standard curve concentration gradients using testosterone standards, and add testosterone isotope internal standards. Use LC-MS / MS to determine the peak area of ​​testosterone and its internal standard at each standard curve concentration. Perform linear regression on the ratio of standard curve concentration, testosterone and its internal standard peak area to obtain the total testosterone standard curve and the free testosterone standard curve. Simultaneously detect the total testosterone and its internal standard peak area ratio and the free testosterone and its internal standard peak area ratio in the total sample solution using LC-MS / MS, and substitute them into the standard curves to calculate the content of total testosterone and free testosterone in the total sample solution. The method of processing testosterone standards when preparing free testosterone standard curve solutions is the same as the method of processing free testosterone dialysate in Step 2. The LC-MS / MS used the following chromatographic conditions: mobile phase I was an aqueous solution of formic acid, and mobile phase II was a methanol solution of formic acid, with gradient elution. Mobile phase I and mobile phase II formed a liquid eluent. The gradient elution program was as follows: at 0 min, mobile phase I was 60%, mobile phase II was 40%, and the flow rate was 0.2 mL / min; at 1.2 min, mobile phase I was 60%, mobile phase II was 40%, and the flow rate was 0.2 mL / min; at 1.5 min, mobile phase I was 0%, mobile phase II was 100%, and the flow rate was 0.2 mL / min; at 5.2 min, mobile phase I was 0%, mobile phase II was 100%, and the flow rate was 0.2 mL / min; at 5.5 min, mobile phase I was 60%, mobile phase II was 40%, and the flow rate was 0.2 mL / min; at 7 min, mobile phase I was 60%, mobile phase II was 40%, and the flow rate was 0.2 mL / min.

2. The method for simultaneously determining the levels of total testosterone and free testosterone in blood as described in claim 1, characterized in that, The equilibration dialysis operation in step two is performed at a temperature of 37°C for 4 to 16 hours, and the buffer solution used in the equilibration dialysis operation is HEPES buffer with a pH of 7.

4.

3. The method for simultaneously determining the levels of total testosterone and free testosterone in blood as described in claim 1, characterized in that, The concentration operations in steps one and two can be performed using either nitrogen blowing or vacuum centrifugal drying and concentration methods.

4. The method for simultaneously determining the levels of total testosterone and free testosterone in blood as described in claim 1, characterized in that, In step two, the liquid-liquid extraction operation uses methanol, acetonitrile, methyl tert-butyl ether, or a mixture of multiple of these as the extraction liquid.

5. The method for simultaneously determining the levels of total testosterone and free testosterone in blood as described in claim 1, characterized in that, The reconstitution solution in step two is either methanol or acetonitrile.

6. The method for simultaneously determining the levels of total testosterone and free testosterone in blood as described in claim 1, characterized in that, The equilibrium dialysis in step two uses a 96-well equilibrium dialysis plate.

7. The method for simultaneously determining total testosterone and free testosterone in blood as described in claim 1, characterized in that, Three quality control concentrations were used for the quality control samples: a high-concentration quality control sample prepared from mixed adult male blood samples, a medium-concentration quality control sample prepared from mixed adult male and adult female blood samples in a certain proportion, and a low-concentration quality control sample prepared from mixed adult female blood samples.

8. A kit based on the method described in claims 1-7, comprising a standard curve working solution, quality control samples, an internal standard working solution, a buffer solution, a derivatization reagent, and a liquid chromatography elution reagent, wherein, The standard curve working solution is prepared by using the testosterone standard with the buffer solution, and the internal standard working solution is prepared by using the isotope internal standard of testosterone with the buffer solution.

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