A method for simultaneous detection of free testosterone and total testosterone in serum

Abstract

This invention provides a method for simultaneously detecting free and total testosterone in serum. Free testosterone is released from a serum sample using equilibrium dialysis, followed by derivatization. An additive quencher is added to remove excess derivatizing reagent, and then a sample solution containing total testosterone is added for testing. The method provided by this invention is simple to operate, eliminating the need for complex procedures such as nitrogen blowing and liquid-liquid extraction. The quencher chosen is low-toxicity, low-cost pyruvate, which improves detection sensitivity while reducing experimental costs. Furthermore, this invention utilizes the biuret assay to determine whether protein leakage has occurred during equilibrium dialysis, providing a direct and accurate assessment.

Description

Technical Field

[0001] This invention belongs to the field of chemical analysis technology, specifically relating to a method for simultaneously detecting free testosterone and total testosterone in serum. Background Technology

[0002] Testosterone exists in two forms in the blood: free testosterone and protein-bound testosterone, collectively referred to as total testosterone. Approximately 98% of blood testosterone is bound to plasma proteins (mainly sex hormone-binding globulin and albumin), and this bound form is inactive and serves as a reserve. Only about 2% exists in the biologically active free form. A simultaneous decrease in both total and free testosterone levels often indicates testicular disease or hypothalamic-pituitary axis dysfunction. Some patients may also have abnormal protein binding due to obesity, liver disease, or other factors, resulting in normal total testosterone but low / high free testosterone levels. Therefore, combined testing of total and free testosterone can avoid missing cases of abnormal androgen activity. Furthermore, for patients with insufficient testosterone secretion, testosterone replacement therapy can be used clinically. If combined testing of total and free testosterone reveals a decrease in total testosterone but a normal free testosterone level, these patients may not require treatment.

[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. As the most important androgen, the normal range of free testosterone in women is 1.1–6.3 pg / mL. Extremely low levels of free testosterone pose a significant challenge to current detection technologies. Meanwhile, since free testosterone accounts for only about 2% of total testosterone, and the two are the same compound, leakage or interference from total testosterone during the detection of free testosterone will significantly affect the accuracy of free testosterone detection.

[0004] Chinese patent application CN114460199B establishes methods for detecting free testosterone and total testosterone, respectively. The sample processing involves complex operations such as ultrafiltration and magnetic bead extraction. Ultrafiltration requires precise temperature control during centrifugation and carries a potential risk of protein leakage. Furthermore, immunomagnetic beads are expensive and difficult to widely implement. Chinese patent application CN115754070A establishes a method and kit for simultaneously determining the levels of total and free testosterone in blood. Free testosterone is obtained through equilibrium dialysis followed by liquid-liquid extraction, while total testosterone is obtained through protein precipitation followed by concentration. The overall process is complex and uses a large amount of volatile organic reagents, which is unfriendly to personnel and the environment.

[0005] Currently, there is no simple and widely applicable method that can simultaneously detect free testosterone and total testosterone. Therefore, there is an urgent need to provide a method or measure that can accurately detect the levels of free testosterone and total testosterone in the blood while being simple and efficient to operate. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a kit and method for simultaneously detecting free testosterone and total testosterone in serum. One serum sample is subjected to equilibration dialysis, derivatization, and quenching to obtain free testosterone; another serum sample undergoes protein precipitation to obtain total testosterone. The two sample solutions are mixed and the contents of free and total testosterone are detected by LC-MS / MS. The sample pretreatment process is simple, and the quenching agent effectively inhibits further derivatization of total testosterone by excessive derivatizing agent, improving the accuracy and sensitivity of the detection results.

[0007] To achieve the above objectives, the present invention employs the following technical solution:

[0008] On one hand, the present invention provides a kit for simultaneous detection of free testosterone and total testosterone in serum, comprising standards, quality control, internal standard working solution, equilibration dialysate, derivatizing agent, quenching agent, protein precipitant and liquid chromatography mobile phase, wherein the quenching agent is a solution of aldehydes or ketones containing α-hydrogen and their salts.

[0009] Current methods for determining free and total testosterone in serum typically involve separate experiments to measure these levels. This requires the separate preparation of reagents, standards, and quality control materials, resulting in lengthy sample processing and detection cycles, large sample volumes, and high experimental costs. Therefore, to save time and costs, some studies employ methods to simultaneously detect free and total testosterone in a single independent experiment using the same sample. For example, this involves equilibrating the sample and then performing derivatization by heating before detection. However, simultaneously determining free and total testosterone is more challenging than determining them separately. This is because, in determining free testosterone, the derivatizing agent is usually added in excess to ensure complete derivatization. If the derivatized free testosterone sample is directly mixed with the total testosterone sample for analysis, the excess derivatizing agent will further derivatize the testosterone in the total testosterone sample, thus affecting the accuracy of the results. To prevent the total testosterone sample from reacting further with the excess derivatizing agent, this invention innovatively employs a quencher to remove the excess derivatizing agent. After the derivatization reaction is completed, the quencher is added, and the quencher reacts with the derivatizing agent hydroxylamine hydrochloride solution to generate an oxime product containing a carbon-nitrogen double bond, thereby consuming the excess hydroxylamine hydrochloride and preventing the added total testosterone from being derivatized.

[0010] Current methods for simultaneously detecting free and total testosterone in serum using equilibrium dialysis, derivatization combined with liquid-liquid extraction, require multiple steps including organic solvent extraction, concentration, and reconstitution. This process is cumbersome and costly. Furthermore, factors such as reagents, temperature, and pH during liquid-liquid extraction significantly affect the recovery rate and accuracy of free testosterone. The derivatization followed by quenching method employed in this invention offers significant advantages. Firstly, after the derivatization reaction, the reaction can be directly terminated by adding a quencher, avoiding the loss of target analytes caused by organic phase transfer and evaporation concentration during liquid-liquid extraction, resulting in a higher and more stable recovery rate. Secondly, it eliminates the time-consuming liquid-liquid extraction, solvent evaporation, and reconstitution steps, simplifying the process and significantly reducing the overall sample pretreatment time, thus improving detection efficiency.

[0011] Since the derivatizing reagent hydroxylamine hydrochloride is a nitrogen-affinity reagent, aldehydes and ketones can be selected to undergo oxime reactions with hydroxylamine hydrochloride, thereby consuming excess hydroxylamine hydrochloride. In some embodiments, this invention compared the effects of four α-hydrogen-containing aldehydes and ketones—pyruvate, potassium pyruvate, α-ketoglutarate, and citral—and two α-hydrogen-free aldehydes and ketones—urea and benzaldehyde. The results showed that only α-hydrogen-containing aldehydes and ketones could prevent the reaction between hydroxylamine hydrochloride and total testosterone, among which pyruvate was more effective.

[0012] Furthermore, the equilibrium dialysate is a HEPES solution, the derivatizing agent is a hydroxylamine hydrochloride solution, the quenching agent is a pyruvate solution, and the protein precipitant is a zinc sulfate solution.

[0013] Further, the concentration of the pyruvate solution is 250-500 mg / mL, the concentration of the hydroxylamine hydrochloride solution is 50-100 mg / mL, and the concentration of the zinc sulfate solution is 5-10 mg / mL.

[0014] In some embodiments, the present invention screened and optimized the concentrations of the derivatizing agent and the quenching agent. The results showed that when the concentrations of hydroxylamine hydrochloride in the derivatizing agent were 10 mg / mL, 20 mg / mL, and 50 mg / mL, the free testosterone content detected from the same set of standard curve samples and clinical samples was basically consistent. However, at concentrations of 10 mg / mL and 20 mg / mL, the response values ​​of free testosterone were significantly lower than at 50 mg / mL, resulting in decreased sensitivity. Therefore, the preferred concentration of the derivatizing agent was 50 mg / mL. Furthermore, for the same set of standard curve samples and clinical samples, the detection results at quenching agent concentrations of 100 mg / mL, 250 mg / mL, and 500 mg / mL differed significantly. When the quenching agent concentration was 100 mg / mL, the amount of quenching agent was insufficient, leading to the derivatization of some total testosterone and a higher detected free testosterone content. Conversely, when the quenching agent concentration was 500 mg / mL, it inhibited the response of free testosterone, reducing the sensitivity of free testosterone detection. Therefore, the concentration of the quencher is preferably 250 mg / mL.

[0015] Preferably, the concentration of the pyruvate solution is 250 mg / mL, and the concentration of the hydroxylamine hydrochloride solution is 50 mg / mL.

[0016] Furthermore, the standard is a solution containing free testosterone and total testosterone at a standard concentration, the quality control is a serum matrix sample containing low and high concentrations, the internal standard working solution contains internal standards of the free testosterone and total testosterone to be tested, and the A phase of the liquid chromatography mobile phase is an ammonium acetate-water solution, and the B phase is an ammonium acetate-methanol solution.

[0017] Furthermore, it also includes a colorimetric agent and a pH adjuster, wherein the colorimetric agent is a copper sulfate pentahydrate solution and the pH adjuster is a NaOH solution.

[0018] When separating free testosterone from serum using balanced dialysis, it is essential to assess protein leakage in the dialyzed sample. Common protein staining methods include the biuret method and Coomassie brilliant blue staining. This invention experimentally compared the effectiveness of these two methods. The results showed that the biuret method allowed for a clear color transition, distinguishing different amounts of protein leakage. In contrast, the Coomassie brilliant blue staining method did not show a significant change in blue color under varying levels of protein leakage, failing to meet the required standards. Therefore, the biuret method was the only viable option.

[0019] On the other hand, the present invention provides a method for simultaneously detecting free testosterone and total testosterone in serum, using a kit as described in any of the preceding claims.

[0020] Furthermore, the method includes the following steps:

[0021] S1: The free testosterone serum sample was balanced by dialysis to obtain free testosterone balanced dialysate;

[0022] S2: The free testosterone balanced dialysate described in step S1 undergoes a derivatization and quenching reaction;

[0023] S3: Add protein precipitant to the total testosterone serum sample, and add the supernatant after precipitation and centrifugation to the quenched solution described in step S2;

[0024] S4: The mixed solution obtained in step S3 was analyzed by LC-MS / MS to detect the content of free testosterone and total testosterone.

[0025] Further, a colorimetric agent and a pH adjuster are added to the free testosterone balanced dialysate obtained in step S1 to determine whether there is protein leakage. If there is, step S1 is repeated; if not, step S2 is performed.

[0026] Further, the chromatographic conditions for LC-MS / MS detection in step S4 are as follows: chromatographic column: Phenomenex C18 2.6μm 50*2.1mm, column temperature: 40℃, flow rate: 0.5 mL / min, injection volume: 20 μL.

[0027] In another aspect, the present invention provides the use of pyruvate in the preparation of a reagent for the simultaneous detection of free testosterone and total testosterone in serum, wherein the pyruvate is used to quench the reaction.

[0028] The present invention has the following beneficial effects:

[0029] 1. The method of derivatization followed by quenching is used to pre-treat free testosterone samples, which eliminates the time-consuming liquid-liquid extraction, concentration and reconstitution steps, significantly shortens the overall sample pre-treatment time and reduces costs; at the same time, it reduces sample loss and can obtain a higher and more stable recovery rate.

[0030] 2. Pyruvic acid was chosen as the quencher. Pyruvic acid consumes excess hydroxylamine hydrochloride through an oxime reaction to generate an oxime product containing a carbon-nitrogen double bond, thereby preventing the added total testosterone from being further derivatized and improving the accuracy of detection.

[0031] 3. The concentrations of the derivatizing agent and the quenching agent were optimized. A 50 mg / mL hydroxylamine hydrochloride solution was used as the derivatizing agent, and a 250 mg / mL pyruvate solution was used as the quenching agent, which significantly improved the accuracy and sensitivity of the detection results.

[0032] 4. The biuret method is used to determine the amount of protein leakage. The degree of blue color development can be used to determine the amount of protein leakage, which is intuitive and accurate. Attached Figure Description

[0033] Figure 1 Chromatograms of a standard sample (left) and a clinical sample (right) for free testosterone.

[0034] Figure 2 Chromatograms of a standard sample (left) and a clinical sample (right) for total testosterone.

[0035] Figure 3 The results of the biuret method protein colorimetric assay are shown. From left to right, the protein leakage rates are 0%, 0.1%, 0.2%, and 0.4%, respectively.

[0036] Figure 4 The results of the Coomassie Brilliant Blue colorimetric assay are shown. From left to right, the protein leakage rates are 0%, 2%, 5%, and 10%, respectively. Detailed Implementation

[0037] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. It should be noted that the embodiments described below are intended to facilitate understanding of the present invention and are not intended to limit it in any way. The reagents used in this embodiment are all known products and were obtained by purchasing commercially available products.

[0038] Example 1: This invention provides a method for detecting free testosterone and total testosterone in serum.

[0039] I. Sample and Reagent Preparation

[0040] 1. Preparation of standard solutions: Prepare testosterone standard stock solutions. Take an appropriate amount of the standard stock solution and add it to a 4% BSA aqueous solution to prepare total testosterone standard solutions of different concentrations. Take an appropriate amount of the testosterone standard stock solution and add it to a 50% methanol solution to prepare free testosterone standard solutions of different concentrations (as shown in the table below).

[0041] Table 1. Standard curves for free testosterone and total testosterone

[0042]

[0043] 2. Preparation of quality control materials: Take an appropriate amount of serum samples from healthy women and mix them to obtain a low-value quality control, which can be used as two quality control materials for low total testosterone (TTL) and low free testosterone (FTL); take an appropriate amount of serum samples from healthy men and mix them to obtain a high-value quality control, which can be used as two quality control materials for high total testosterone (TTH) and low free testosterone (FTH).

[0044] 3. Preparation of internal standard working solution: Take an appropriate amount of testosterone-d4 stock solution and dilute it with an appropriate amount of methanol aqueous solution to obtain a total testosterone internal standard solution with a testosterone-d4 concentration of 1500 pg / mL and a free testosterone internal standard solution with a testosterone-d4 concentration of 50 pg / mL.

[0045] 4. Preparation of equilibrium dialysis solution: Weigh 5353 mg sodium chloride, 439 mg tripotassium phosphate trihydrate, 276 mg magnesium sulfate heptahydrate, 12570 mg HEPES, 200 mg urea, 900 mg sodium hydroxide, 200 mg potassium chloride, 200 mg Proclin 300, and 211 mg calcium chloride respectively, dissolve them in 1 L of water, and mix well to obtain the solution.

[0046] 5. Preparation of the derivatizing agent: Take 50 mg of hydroxylamine hydrochloride, add 1 mL of 90% methanol-water solution, and mix well.

[0047] 6. Preparation of quenching agent: Take 250 mg of pyruvic acid, add 1 mL of water, and mix well.

[0048] 7. Preparation of protein precipitant: Take 90 mg of zinc sulfate, add 1 mL of water and 9 mL of methanol, and mix well.

[0049] 8. Preparation of colorimetric reagent: Take 0.1 g of copper sulfate pentahydrate, add 10 mL of water, and mix well.

[0050] 9. Preparation of pH adjuster: Take 0.4 g of sodium hydroxide, add 10 mL of water, and mix well.

[0051] II. Sample Pretreatment

[0052] 1. Add 200 μL of the sample / control to be tested to the red well of the equilibration dialysis apparatus, and add 400 μL of dialysis buffer to the adjacent well;

[0053] 2. The equilibration dialysis apparatus was placed at 37°C for 4 hours;

[0054] 3. Take 100 μL of the dialyzed solution from one side of the dialysate, add 20 μL of pH adjuster and 10 μL of color reagent, vortex to mix, let stand, and observe whether there is a color change to determine whether there is protein leakage during the equilibration dialysis process. If there is, repeat the equilibration dialysis step; if not, proceed to the next step.

[0055] 4. Take 100 μL of the dialyzed solution from one side of the dialysate, and take another 100 μL of free testosterone standard solutions of different concentrations. Add 20 μL of free testosterone internal standard solution and 30 μL of derivatizing agent to each solution, vortex to mix, and then place at 60°C for 10 minutes.

[0056] 5. Take 80 μL of the derivatized solution, add 10 μL of quenching agent, and let stand at 60°C for 10 minutes.

[0057] 6. Take 100 μL of total testosterone standard solution / quality control / sample to be tested, add 100 μL of protein precipitant containing total testosterone internal standard solution, vortex to mix, centrifuge, and take 80 μL of the supernatant to add to the above quenched solution, mix well and then test.

[0058] III. LC-MS / MS Detection

[0059] (1) Chromatographic conditions

[0060] The chromatographic column was a Phenomenex C18 2.6μm 50*2.1mm. Mobile phase A was 2 mM ammonium acetate-water solution; mobile phase B was 2 mM ammonium acetate-methanol solution. The column temperature was 40℃, the flow rate was 0.5 mL / min, the injection volume was 20 μL, and the elution gradient is shown in Table 2.

[0061] Table 2 Elution gradient

[0062]

[0063] (2) Mass spectrometry conditions

[0064] Mass spectrometry system: AB SCIEX Triple Quad TM 4500MD; ESI source, positive ion mode; multiple reaction monitoring (MRM) scan mode.

[0065] The ion source parameters are shown in Table 3, and the MRM parameters are shown in Table 4.

[0066] Table 3 Ion source parameters

[0067]

[0068] Table 4 MRM Parameters

[0069]

[0070] IV. Data Processing and Analysis

[0071] (1) Specificity analysis

[0072] according to Figure 1 and Figure 2 As can be seen, the retention times of the analytes in the standard curve and clinical samples are consistent. The retention time of free testosterone is 2.65 min, and that of total testosterone is 2.45 min. The two are completely separated in both liquid chromatography and mass spectrometry, and there is no interference near the peaks of each analyte, indicating good specificity.

[0073] (2) Establishment of standard curve

[0074] Based on retention time and ion pairs, the mass spectrometry response signals of the standard curve and the analyte in the sample, along with the corresponding internal standard peak area values, were read. A fitted curve was plotted using the ratio of the peak area of ​​the standard curve to the peak area of ​​the internal standard as the Y-value and the standard curve concentration as the X-value. The standard curve equations for total testosterone and free testosterone are shown in Table 5. The peak area ratios of the analyte and the internal standard in the sample were substituted into the corresponding curve equations to calculate the concentration of the analyte in the corresponding serum sample.

[0075] Table 5 Standard curves for total testosterone (TT) and free testosterone (FT)

[0076]

[0077] It can be seen that the linear fitting equations for total testosterone and free testosterone within their respective concentration ranges show good linearity, with correlation coefficients above 0.990.

[0078] (3) Spike recovery rate

[0079] Testosterone standard solution of known concentration was added to the serum after dialysis to prepare two spiked samples with high and low concentrations. Five samples were processed in parallel, and the recovery rate was calculated. The results are shown in Table 6.

[0080] Table 6. Results of free testosterone spike recovery

[0081]

[0082] Testosterone standard solution of known concentration was added to serum to prepare two spiked samples with high and low concentrations. Five samples were processed in parallel, and the recovery rate was calculated. The results are shown in Table 7.

[0083] Table 7. Results of total testosterone spike recovery

[0084]

[0085] The results in Tables 6 and 7 show that the spiked recoveries of total testosterone and free testosterone were between 85% and 115%, both meeting the requirements.

[0086] (4) Precision

[0087] Samples of high and low concentrations of free testosterone and total testosterone were taken and processed in parallel for 3 consecutive days. The intra-batch precision and inter-batch precision over the three days were calculated. The results are shown in Tables 8 and 9.

[0088] Table 8. Intra-batch and inter-batch precision results of free testosterone

[0089]

[0090] Table 9. Intra-assay and inter-assay precision results for total testosterone.

[0091]

[0092] The results showed that the intra-batch and inter-batch precision of total testosterone and free testosterone were both within 15%, which met the requirements.

[0093] (5) Accuracy

[0094] Six samples of free testosterone and total testosterone were taken and processed in parallel. The accuracy results are shown in Table 10.

[0095] Table 10 Results of limit of quantitation tests for each compound

[0096]

[0097] The results showed that the accuracy of the quantification limits for free testosterone and total testosterone was between 95% and 105%, and the relative standard deviation was within 10%, both of which met the requirements.

[0098] (6) Method reliability

[0099] Free testosterone and total testosterone samples were detected separately using individual methods: Free testosterone samples were subjected to equilibrium dialysis, and the resulting equilibrium dialysis solution was further derivatized to obtain a free testosterone sample, which was then detected using LC-MS / MS. Total testosterone serum samples were treated with a protein precipitant, precipitated, centrifuged, and the supernatant was collected to obtain the total testosterone sample, which was then detected using LC-MS / MS. The results were compared with the combined test results of this embodiment, as shown in Tables 11 and 12.

[0100] Table 11 Comparison of results from individual and combined tests for free testosterone

[0101]

[0102] Table 12 Comparison of Total Testosterone Results from Individual and Combined Tests

[0103]

[0104] The results showed that the deviation between simultaneous testing of free testosterone and total testosterone and separate testing was within ±10%, indicating that the results of simultaneous detection of free testosterone and total testosterone using the combined testing method provided by this invention are reliable and meet the requirements.

[0105] Example 2: The Necessity of "Derivation Before Quenching"

[0106] Simultaneous determination of free and total testosterone is more challenging than determining free testosterone alone. To ensure complete derivatization of free testosterone, an excess of derivatizing agent is often added during free testosterone sample processing. However, this excess agent can react with subsequently added total testosterone, producing derivatives identical to those in the free testosterone sample. This can lead to erroneous results, with higher free testosterone readings and lower total testosterone readings. Therefore, excess derivatizing agent must be removed before adding the total testosterone sample to avoid interference with free testosterone detection. Existing studies typically use liquid-liquid extraction to separate excess derivatizing agent, a complex and cumbersome process. To improve the sensitivity of the combined detection method for free and total testosterone while simplifying the procedure, this invention innovatively introduces a quenching reaction to remove excess derivatizing agent.

[0107] For the two methods of "derivative followed by liquid-liquid extraction" and "derivative followed by quenching", this embodiment uses the same set of standard curve samples and clinical samples to examine the differences in detection accuracy, sensitivity and other aspects between the two methods. The experimental steps of "derivative followed by liquid-liquid extraction" are as described in the invention application with publication number CN115754070A and invention title "Method and kit for simultaneous determination of total testosterone and free testosterone in blood". The experimental steps of "derivative followed by quenching" are the same as in Example 1. In addition, the control group is tested separately for free testosterone and total testosterone. The test method is the same as the reliability step of method (6) in Example 1. The test method of the "only adding derivatizing agent" group is basically the same as the sample pretreatment in Example 1. The difference is that no quenching agent is added in step 5. Instead, an equal volume of purified water is used. The ratios (Ratio) and response values ​​(Area) of free testosterone, total testosterone and their internal standard in the standard curve and clinical samples are compared. The response values ​​are expressed in scientific notation.

[0108] Table 13 Test results for "Derivatization followed by liquid-liquid extraction" and "Derivatization followed by quenching"

[0109]

[0110] As shown in Table 13, regardless of whether the method involved "derivation followed by liquid-liquid extraction" or "derivation followed by quenching," the ratios of free testosterone and total testosterone to their internal standards in the standard and clinical samples were generally consistent and close to the results of individual tests. However, in the test method that only performed derivatization without liquid-liquid extraction or quenching, the ratio of free testosterone was significantly higher than that of individual tests, while the ratio of total testosterone was significantly lower. This indicates that excessive derivatization reagent will cause free testosterone to be further derivatized into total testosterone. Liquid-liquid extraction or quenching after derivatization can prevent further derivatization of total testosterone. The ratios of free testosterone in the "derivation followed by liquid-liquid extraction" and "derivation followed by quenching" methods were similar, indicating that "liquid-liquid extraction" and "quenching" had comparable removal effects on the derivatizing agent. However, when using the "derivative-then-liquid extraction" method, the response value (Area) of free testosterone was significantly lower than that of the "derivative-then-quenching" method. The "derivative-then-quenching" method showed significantly better sensitivity for detecting free testosterone than the "derivative-then-liquid extraction" method, suggesting that some free testosterone is lost during liquid-liquid extraction and concentration. Furthermore, the experiment revealed that the "derivative-then-liquid extraction" method requires steps such as liquid-liquid extraction, concentration, and reconstitution, which is cumbersome, time-consuming, and costly. In contrast, the "derivative-then-quenching" method only requires adding a quencher after derivatization to terminate further derivatization, making the process simpler. In conclusion, only the "derivative-then-quenching" method can improve the sensitivity of free testosterone detection while simplifying experimental procedures and reducing costs.

[0111] Example 3: Screening and Optimization of Quenching Agents

[0112] I. Selection of Quenching Agent

[0113] This embodiment further investigated the differences in efficacy of different quenchers for the simultaneous detection of free testosterone and total testosterone. Test samples included standard curve S5 and clinical samples. Control groups without quenchers, control groups containing only free testosterone, control groups containing only total testosterone, and test groups with different quenchers were established. The processing methods for each test group were as follows:

[0114] 1. Control group without quenching agent: The test method is the same as the "only derivatizing agent added" group in Example 2.

[0115] 2. Control group containing only free testosterone (FT): The sample does not contain total testosterone and quencher. The free testosterone sample is prepared according to steps 1 to 4 of the sample pretreatment in Example 1. Step 5 is adjusted as follows: Take 80 μL of the derivatized solution, add 10 μL of purified water and place at 60°C for 10 minutes. Step 6 is adjusted as follows: Take 100 μL of 4% BSA solution, add 100 μL of protein precipitant (without internal standard), vortex to mix, centrifuge and take 80 μL of the supernatant to add to the solution obtained in step 5.

[0116] 3. Control group containing only total testosterone (TT): The sample was added with a protein precipitant containing an internal standard, precipitated, centrifuged, and 80 μL of the supernatant was added to 90 μL of purified water and mixed to obtain a total testosterone-only sample.

[0117] 4. Test groups with different quenchers: Pyruvic acid, potassium pyruvate, α-ketoglutarate, citral (aldehydes and ketones containing α-hydrogen) and urea and benzaldehyde (aldehydes and ketones without α-hydrogen) were used as quenchers, and quenching reactions were carried out respectively. Other pretreatment steps were the same as in Example 1.

[0118] The ratios (Ratios) and response values ​​(Areas) of free testosterone and its internal standard in the standard curve S5 and clinical samples are compared in Table 14, where the response values ​​are expressed in scientific notation.

[0119] Table 14 Test results of different quenching agents

[0120]

[0121] Note: " / " indicates no quencher was added, and "-" indicates it was not detected.

[0122] As shown in Table 14, compared with the control group containing only free testosterone and total testosterone, the Raito values ​​of free testosterone were significantly higher than those of the control when no quencher was added to terminate the derivatization reaction in the standard curve S5 and clinical samples, while the Raito value of total testosterone was significantly lower than that of the control. This is consistent with the test results in Example 2. Excessive derivatizing agent causes total testosterone to be derivatized into free testosterone, resulting in a large error between the test results and the actual results. When the quencher is an aldehyde or ketone containing α-hydrogen, such as pyruvate, potassium pyruvate, α-ketoglutarate, or citral, the Raito values ​​of free testosterone measured in the standard curve S5 sample and the clinical sample are close to those of the control sample containing only FT, and the Raito value of total testosterone measured is close to that of the control sample containing only TT. This indicates that the use of aldehydes or ketones containing α-hydrogen can remove excess derivatizing agent hydroxylamine hydrochloride. The reason is that hydroxylamine hydrochloride is a nitrogen affinity reagent. Aldehydes and ketones can react with nitrogen affinity reagents through oxime reactions to form oxime products containing carbon-nitrogen double bonds, thereby consuming excess hydroxylamine hydrochloride and preventing further derivatization reactions between the subsequently added total testosterone and hydroxylamine hydrochloride. When quenchers are chosen that do not contain α-hydrogen aldehydes or ketones, such as urea or benzaldehyde, the Raito values ​​of free testosterone measured in the standard curve S5 sample and clinical samples are significantly different from those of the control sample containing only FT, and the Raito value of total testosterone measured is significantly different from that of the control sample containing only TT. This indicates that using aldehydes or ketones without α-hydrogen cannot remove excess derivatizing agent hydroxylamine hydrochloride. Therefore, aldehydes or ketones containing α-hydrogen should be chosen as quenchers. Furthermore, comparing the four standard curves and clinical samples treated with α-hydrogen aldehydes or ketones, the Ratio values ​​of the four quenchers were found to be similar, suggesting that the quenching performance of these four quenchers is comparable. Among them, pyruvate has a larger Area value, indicating that pyruvate has the weakest signal suppression and the highest detection sensitivity in subsequent liquid chromatography-tandem mass spectrometry analysis. Therefore, pyruvate is the preferred quencher among the four quenchers.

[0123] II. Optimization of Quenching Agent Concentration

[0124] This embodiment further investigated the effect of different concentrations of quencher on the detection results of free testosterone. The quencher concentrations in Example 1 were set to 100 mg / mL, 250 mg / mL, and 500 mg / mL, respectively. The remaining operations were the same as in Example 1, and the standard curve samples and clinical samples used were the same as those in Table 14. The detection results are shown in Table 15, where the response values ​​are expressed in scientific notation.

[0125] Table 15 Test results of quenchers with different concentrations

[0126]

[0127] As shown in Table 15, when the quencher concentration was 100 mg / mL, the ratio of free testosterone in the standard curve and clinical samples was several tens of times higher than that of the control samples containing only free testosterone in Table 14, while the ratio of total testosterone was significantly lower than that of the control samples containing only total testosterone in Table 14. This is because insufficient quencher led to the derivatization of some total testosterone, affecting the results for free testosterone. When the quencher concentration was 250 mg / mL and 500 mg / mL, the ratio values ​​of the standard curve and clinical samples were basically consistent with the corresponding control sample results in Table 14, indicating that the excess derivatizer in the free testosterone samples was completely quenched, and the subsequent addition of total testosterone samples did not affect the quantitative results of free testosterone. However, when the quencher concentration was 500 mg / mL, it inhibited the response of free testosterone (the area of ​​free testosterone decreased), thus affecting the sensitivity of free testosterone detection. In conclusion, the preferred quencher concentration is 250 mg / mL.

[0128] Example 4: Optimization of conditions for the derivatization reaction

[0129] To ensure that free testosterone in the sample can be completely derivatized and to improve the sensitivity of its detection, this embodiment further screened and optimized the conditions in the derivatization reaction process.

[0130] I. Derivatizing agent concentration

[0131] The concentrations of the derivatizing agent in Example 1 were set to 10 mg / mL, 20 mg / mL, 50 mg / mL, and 100 mg / mL. The same standard curve and clinical sample were tested respectively. The remaining operations were the same as those in the "free testosterone only" group in Example 3: the sample did not contain total testosterone and quencher. The free testosterone sample was prepared according to steps 1 to 4 of the sample pretreatment in Example 1. Step 5 was adjusted as follows: 80 μL of the derivatized solution was taken and 10 μL of purified water was added. The solution was placed at 60°C for 10 minutes. Step 6 was adjusted as follows: 100 μL of 4% BSA solution was taken and 100 μL of protein precipitant (without internal standard) was added. The mixture was vortexed and centrifuged. 80 μL of the supernatant was added to the solution obtained in step 5.

[0132] The results of the detection of the ratio of free testosterone to its internal standard and the response value (Area) are shown in Table 16, where the response value is expressed in scientific notation.

[0133] Table 16 Detection results of different concentrations of derivatizing agents

[0134]

[0135] As shown in Table 16, the ratio of free testosterone to its internal standard in the standard and clinical samples was generally consistent across different concentrations of the derivatizing agent. However, the response area of ​​free testosterone varied considerably. The highest response value for free testosterone was observed at a derivatizing agent concentration of 50 mg / mL, indicating that this concentration provided the highest sensitivity for free testosterone. Therefore, a derivatizing agent concentration of 50 mg / mL is optimal.

[0136] II. Derivative Temperature and Time

[0137] The derivatization temperatures and times in Example 1 were set to 60℃ for 10 min, 60℃ for 30 min, and 70℃ for 30 min. The same standard curve samples and clinical samples as in Table 16 were tested, with the remaining procedures identical to the "free testosterone only" group in Example 3. The results of the ratio and area of ​​free testosterone and its internal standard are shown in Table 17, where the area is expressed in scientific notation.

[0138] Table 17 Detection results at different derivation temperatures and times

[0139]

[0140] As shown in Table 17, for the same standard curve sample and clinical sample, the peak area and ratio value of detected free testosterone were basically consistent when using derivatization temperatures and times of 60℃ for 10 min, 60℃ for 30 min, or 70℃ for 30 min, indicating that the response sensitivity and detection accuracy were similar under the three conditions. Therefore, to improve pretreatment efficiency and save experimental costs, the optimal derivatization temperature and time is 60℃ for 10 min.

[0141] Example 5: Selection of Protein Colorimetric Method

[0142] When separating free testosterone from serum via equilibrium dialysis, if the dialysis membrane is damaged, defective, or poorly sealed, serum proteins may leak into the "free fraction," which should only contain free testosterone. In subsequent LC-MS / MS measurements of free testosterone concentration, this portion of testosterone, which should be in the bound state, will be incorrectly counted as free testosterone, leading to a significantly falsely elevated measured free testosterone concentration. Therefore, it is essential to assess protein leakage in post-dialysis samples. Common protein staining methods include the biuret method (i.e., Example 1) and the Coomassie brilliant blue staining method, both of which can determine the presence of protein based on color changes. This example further examines the difference in effectiveness between the biuret method and the Coomassie brilliant blue staining method in assessing protein leakage.

[0143] First, after completing the serum sample equilibration dialysis according to sample pretreatment steps 1-2 in Example 1, take out 100 μL of the dialyzed solution and add 2 μL of dialysate, 2 μL of diluted serum 1 (serum diluted 20 times with dialysate), 2 μL of diluted serum 2 (serum diluted 10 times with dialysate), and 2 μL of diluted serum 3 (serum diluted 5 times with dialysate) to simulate protein leakage rates of 0, 0.1%, 0.2%, and 0.4%. Add 20 μL of sodium hydroxide solution and 10 μL of copper sulfate solution to the above solutions in sequence, vortex to mix, let stand, and observe the color.

[0144] Take another 100 μL of the dialyzed solution and add 2 μL of dialysate, 2 μL of serum, 5 μL of serum and 10 μL of serum to simulate protein leakage rates of 0%, 2%, 5% and 10%. Add 20 μL of Coomassie Brilliant Blue solution to each of the above solutions in turn, vortex to mix, let stand and observe the color.

[0145] like Figure 3 As shown, under protein leakage rates of 0%, 0.1%, 0.2%, and 0.4%, the color transitions from light blue to dark purple. The higher the leakage rate, the darker the color. A protein leakage rate of 0.1% is visible to the naked eye, while protein leakage rates of 0.2% and above are clearly observable. From Figure 4 It can be seen that under the conditions of protein leakage rates of 0%, 2%, 5%, and 10%, all samples showed a pale blue color, and the blue color did not change significantly under different protein leakage rates. It can be concluded that the Coomassie luminescence method has a significantly different sensitivity compared to the biuret method for protein luminescence, and therefore cannot meet the requirements for use. Thus, the biuret method is preferred.

[0146] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A kit for simultaneous detection of free and total testosterone in serum, characterized in that, The kit comprises a standard, a quality control, an internal standard working solution, a balanced dialysis solution, a derivatizing agent, a quencher, a protein precipitant, and a liquid chromatography mobile phase, wherein the quencher is any one of pyruvic acid, potassium pyruvate, alpha-ketoglutaric acid, or citral, and the derivatizing agent is a hydroxylamine hydrochloride-methanol solution.

2. The kit of claim 1, wherein The balanced dialysis solution is a HEPES solution, the quencher is pyruvic acid, and the protein precipitant is a zinc sulfate-methanol solution.

3. The kit of claim 2, wherein The concentration of the pyruvic acid solution is 250-500 mg / mL, the concentration of the hydroxylamine hydrochloride solution is 50-100 mg / mL, and the concentration of the zinc sulfate solution is 5-10 mg / mL.

4. The kit of claim 3, wherein The standard is a solution containing standard concentrations of free testosterone and total testosterone, the quality control is a serum matrix sample containing low and high concentrations, the internal standard working solution contains an internal standard for the free testosterone and total testosterone to be measured, and the liquid chromatography mobile phase A is an ammonium acetate-water solution, and B is an ammonium acetate-methanol solution.

5. The kit of claim 4, wherein The kit further comprises a color developing agent and a pH adjuster, wherein the color developing agent is a copper sulfate pentahydrate solution, and the pH adjuster is a NaOH solution.

6. A method for simultaneous detection of free and total testosterone in serum, characterized by, The kit is used for LC-MS / MS detection, and the chromatographic conditions of the LC-MS / MS detection are as follows: the chromatographic column is a Phenomenex C18 2.6 μm 50*2.1 mm, the column temperature is 40℃, the flow rate is 0.5 mL / min, and the injection volume is 20 μL.

7. The method of claim 6, wherein, The kit comprises the following steps: S1: performing balanced dialysis on a free testosterone serum sample to obtain a free testosterone balanced dialysis solution; S2: performing derivatization and quenching on the free testosterone balanced dialysis solution obtained in step S1; S3: adding a protein precipitant to a total testosterone serum sample, precipitating and centrifuging, and then adding the supernatant to the quenched solution obtained in step S2; S4: detecting the contents of free testosterone and total testosterone in the mixed solution obtained in step S3 by LC-MS / MS.

8. The method of claim 7, wherein, The free testosterone balanced dialysis solution obtained in step S1 is added with a color developing agent and a pH adjuster to determine whether there is protein leakage, and if there is, step S1 is repeated; if not, step S2 is performed.

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