A method for the determination of free testosterone based on liquid chromatography-tandem mass spectrometry based on ultrafiltration technology

Abstract

The present invention provides a method for determining free testosterone by liquid chromatography-tandem mass spectrometry based on ultrafiltration technology. After adding corresponding concentrations of ammonium chloride and ammonium fluoride to reagents in a pretreatment method of serum samples, the extraction efficiency can be significantly improved, the quantitative lower limit can be reduced, the detection sensitivity and stability can be improved, and the actual sample detection requirements can be met. At the same time, 0.1mM ammonium chloride and 0.1mM ammonium fluoride are added to the mobile phase, which can significantly improve the peak shape and increase the chromatographic response. Compared with the common mobile phase addition in the prior art, the quantitative lower limit is significantly reduced. The types of ultrafiltration buffer, extraction agent and resolvent in the pretreatment method are also compared and screened. A HEPES ultrafiltration buffer containing 3mM ammonium chloride and 1mM ammonium fluoride, an ethanol-ether extraction agent containing 2mM ammonium chloride and 1mM ammonium fluoride, and a 30% methanol water reconstitution containing 2.5mM ammonium chloride and 2.5mM ammonium fluoride are selected, which promotes chromatographic detection, improves mass spectrometry response, and significantly reduces the quantitative lower limit to 0.05pg / ml.

Description

Technical Field

[0001] The present invention belongs to the field of medical detection technology, and in particular relates to a method for determining free testosterone by liquid chromatography-tandem mass spectrometry based on ultrafiltration technology. Background Art

[0002] Free testosterone is the unbound testosterone in the blood, representing the portion of testosterone that can directly act on body tissues and produce physiological effects. Currently, traditional methods for free testosterone detection rely primarily on radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). These methods are complex, time-consuming, susceptible to interference from the sample matrix, and lack sensitivity and accuracy. With the advancement of technology, equilibrium dialysis and ultrafiltration pretreatment followed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) for quantitative analysis have become the primary methods for free testosterone detection. Chinese patent publication number CN114720704B, entitled "A kit and method for determining free testosterone in serum," utilizes ultrafiltration and liquid-liquid extraction pretreatment, combined with ultra-performance liquid chromatography-tandem mass spectrometry, and improves detection sensitivity by adding additives to mobile phase A. Chinese patent publication number CN116297998A, entitled "A kit and method for detecting free testosterone in serum with high sensitivity mass spectrometry," utilizes a pretreatment process that utilizes extraction followed by derivatization, thereby enhancing the response of the derivative product in mass spectrometry detection and improving the sensitivity of the detection method. The methods in the aforementioned patents enhance detection sensitivity by adding reagents or adding a derivatization step to the mobile phase, achieving a lower limit of quantification of free testosterone of 0.25 to 0.5 pg / ml. The technology still has room for further improvement.

[0003] Therefore, providing an efficient, accurate, and low-cost method for determining free testosterone in serum and further improving the accuracy and sensitivity of free testosterone detection in serum are crucial to improving the clinical application value of free testosterone detection. Summary of the Invention

[0004] To address the limited detection sensitivity of existing methods for determining free testosterone in serum, the present invention provides a method for determining free testosterone based on ultrafiltration technology using liquid chromatography-tandem mass spectrometry. By adding certain concentrations of ammonium chloride and ammonium fluoride to the reagents in the pretreatment method and the mobile phase in the chromatographic analysis, the detection accuracy and sensitivity are significantly improved, and the lower limit of quantification can reach 0.05 pg / mL.

[0005] On the one hand, the present invention provides a method for determining free testosterone in serum based on liquid chromatography-tandem mass spectrometry, comprising pretreatment of a serum sample, wherein the pretreatment of the serum sample comprises ultrafiltration, extraction, and re-dissolution, wherein reagents containing ammonium chloride and ammonium fluoride are used in the ultrafiltration, extraction, and re-dissolution steps.

[0006] Through extensive experimental screening and theoretical research, the present invention has discovered that adding ammonium chloride and ammonium fluoride throughout the reagents of the pretreatment step and the mobile phase of the liquid phase system can significantly improve the chromatographic response and reduce the lower limit of quantification. This is because the target analyte in the sample can be exposed to the same level of ionization conditions throughout the entire process. In addition, the use of ammonium chloride to precipitate proteins and other macromolecular substances can significantly reduce matrix effects, nonspecific adsorption, or inhibition of the ionization of free testosterone, thereby improving the accuracy of the test results and enabling the lower limit of quantification to reach 0.05 pg / mL. Other pretreatment additives cannot achieve this effect.

[0007] Furthermore, the ultrafiltration step uses a HEPES ultrafiltration buffer containing ammonium chloride and ammonium fluoride, the extraction step uses an ethanol-ether extractant containing ammonium chloride and ammonium fluoride, and the re-dissolution step uses 30% methanol water containing ammonium chloride and ammonium fluoride.

[0008] According to the prior art, the types of buffer, extractant and resolvent in the pre-treatment process of free testosterone have a certain impact on the response of free testosterone.

[0009] The present invention compared and screened the effects of different ultrafiltration buffers, extractants, and resolvents when used in combination with the additive ammonium chloride. The results showed that using an ultrafiltration buffer containing HEPES (ammonium chloride and ammonium fluoride) could achieve a lower limit of quantification of free testosterone of 0.05 pg / mL, improving the stability of the method and enabling efficient detection of samples even after a three-month shelf life. This is likely because HEPES containing ammonium chloride and ammonium fluoride does not form complexes with metal ions like some other buffers. Therefore, it does not affect the behavior of metal ions that may be present in serum, nor does it interfere with metal ion-based detection methods, such as mass spectrometry. Furthermore, HEPES containing ammonium chloride and ammonium fluoride generally does not interfere with most enzymatic reactions or cellular functions. For serum samples, this means that it can reduce the impact on endogenous enzyme activity, thereby protecting free testosterone from unwanted degradation or other changes. Furthermore, it effectively maintains the stability of the solution pH, which is crucial for preserving the structural integrity and activity of free testosterone.

[0010] In some embodiments, the present invention has demonstrated through experiments that adding 3 mM ammonium chloride and 1 mM ammonium fluoride to the HEPES ultrafiltration buffer can improve the detection sensitivity of the method and reduce the quantitative detection limit.

[0011] Experimental studies on extractants have shown that a composite extractant containing ethanol and ether, containing ammonium chloride and ammonium fluoride, exhibits higher detection sensitivity than methyl tert-butyl ether and other extractants, achieving a lower limit of quantification of free testosterone of 0.05 pg / mL. This is because the ethanol-ether mixture provides a higher recovery rate of the target compound, thereby improving detection sensitivity. In some embodiments of the present invention, experimental results also indicate that only the addition of 2 mM ammonium chloride and 1 mM ammonium fluoride to the extractant effectively increases the lower limit of quantitative detection of the method.

[0012] The present invention also finds that only by selecting 30% methanol water containing 3mM ammonium chloride and 1mM ammonium fluoride can the sensitivity of free testosterone detection be significantly improved, and the quantitative limit is reduced. The reason that can play a good effect may be that ammonium chloride and ammonium fluoride promote the release of sample. When the ammonium chloride concentration in the redissolution solvent is higher than the mobile phase, the chloride ions and fluoride ions produced can be exchanged specifically with the cations in the sample molecules. This process makes it easier for the sample molecules to be transferred from the stationary phase to the mobile phase, thereby improving the chromatographic response and detection sensitivity, reducing the quantitative limit, and this specific ion exchange effect is difficult for other salts to achieve. Therefore, the ammonium chloride and ammonium fluoride of appropriate concentrations have played a vital role in the redissolution process.

[0013] Furthermore, the concentration of ammonium chloride in the HEPES ultrafiltration buffer is 3 mM, and the concentration of ammonium fluoride is 1 mM; the concentration of ammonium chloride in the ethanol-ether extractant is 2 mM, and the concentration of ammonium fluoride is 1 mM; the concentration of ammonium chloride in the 30% methanol water is 2.5 mM, and the concentration of ammonium fluoride is 2.5 mM.

[0014] Furthermore, it also includes high performance liquid chromatography tandem mass spectrometry analysis, wherein the mobile phase A of the high performance liquid chromatography tandem mass spectrometry analysis is formic acid water containing ammonium chloride and ammonium fluoride, and the mobile phase B is formic acid acetonitrile containing ammonium chloride and ammonium fluoride.

[0015] Furthermore, the concentrations of ammonium chloride and ammonium fluoride in the formic acid water and formic acid acetonitrile are both 0.1 mM.

[0016] In the prior art, liquid chromatography tandem mass spectrometry is used to detect free testosterone in serum samples, and most common mobile phases are used. The chromatographic peak shape of the free testosterone detected by it is poor, the response is low, the sensitivity is poor, and the quantitative lower limit is high. In order to solve this problem, the present invention has determined that 0.1mM ammonium chloride and ammonium fluoride are added to both phase A and phase B of the mobile phase after a large number of experimental screenings and theoretical studies. Compared with the addition of other reagents such as ammonium fluoride and ammonium acetate, the quantitative lower limit of the target compound is significantly reduced after the addition of ammonium chloride and ammonium fluoride. When the concentration of ammonium chloride and ammonium fluoride is 0.1mM, the quantitative lower limit of the target compound is the lowest, which achieves accurate detection of low-concentration samples and improves the accuracy and sensitivity of detection. The possible mechanistic explanation for the above phenomenon is that chloride ions and fluoride ions can also compete with other anions in the mobile phase for adsorption onto the stationary phase, reducing the nonspecific adsorption of these ions to the stationary phase, thereby further improving the recovery rate and detection sensitivity of free testosterone, thereby improving the peak shape, increasing the chromatographic response, and reducing the quantitative lower limit of the target compound.

[0017] Therefore, the present invention uses ammonium chloride and ammonium fluoride as reagents to improve the sensitivity of high performance liquid chromatography tandem mass spectrometry for detecting free testosterone in serum samples. It should be noted that during use, ammonium chloride and ammonium fluoride are used as additives to ultrafiltration buffer, extraction agent, and resolvent in the pretreatment step and as additive components of the mobile phase in liquid chromatography tandem mass spectrometry analysis. The added concentrations of ammonium chloride and ammonium fluoride are not the same. The present invention selects specific ammonium chloride and ammonium fluoride concentrations for the above reagents to achieve the best effect, and their effects are different in different reagents, and the principles of their effects are also different. Therefore, ammonium chloride and ammonium fluoride participate in the entire pretreatment and chromatographic analysis in different ways.

[0018] Furthermore, the method comprises the following steps:

[0019] (1) Dilute the serum sample with ultrafiltration buffer to obtain a diluted sample;

[0020] (2) ultrafiltration of the diluted sample from step (1) to obtain an ultrafiltrate;

[0021] (3) adding an internal standard to the ultrafiltrate of step (2) and performing extraction to obtain an extract;

[0022] (4) freeze-drying the extract from step (3) and redissolving it to obtain a redissolved solution;

[0023] (5) The supernatant of step (4) is subjected to high performance liquid chromatography tandem mass spectrometry detection.

[0024] Furthermore, the chromatographic conditions of the high performance liquid chromatography tandem mass spectrometry in step (5) are as follows: injection volume: 10 uL; flow rate: 0.4 mL / min; column temperature: 40°C; mobile phase gradient:

[0025] .

[0026] Furthermore, the mass spectrometry conditions of the high performance liquid chromatography tandem mass spectrometry in step (5) are an ESI ion source, and the ion source parameters are:

[0027] ; MRM parameters:

[0028] .

[0029] On the other hand, the present invention provides a kit for determining free testosterone in serum based on liquid chromatography tandem mass spectrometry, comprising an ultrafiltration buffer, an extractant, and a resolvation solvent, wherein the ultrafiltration buffer is HEPES containing ammonium chloride and ammonium fluoride, the extractant is an ethanol-ether extractant containing ammonium chloride and ammonium fluoride, and the resolvation solvent is 30% methanol water containing ammonium chloride and ammonium fluoride.

[0030] In some embodiments, the kit provided by the present invention further comprises a calibrator and an internal standard, wherein the calibrator is prepared from a free testosterone standard solution, and the concentration of the calibrator solution is 2 to 500 pg / mL; the internal standard is prepared from a free testosterone T-d3 standard solution, and the concentration of the internal standard is 0.5 ng / mL.

[0031] In another aspect, the present invention provides the use of ammonium chloride and ammonium fluoride for preparing a reagent for improving the sensitivity of liquid chromatography tandem mass spectrometry for determining free testosterone in serum, wherein ammonium chloride and ammonium fluoride are added to both the pretreatment reagent of the serum sample and the mobile phase of the chromatographic analysis.

[0032] The present invention has the following beneficial effects:

[0033] 1. The present invention improves the method for detecting free testosterone in serum samples based on liquid chromatography-tandem mass spectrometry. By adding ammonium chloride and ammonium fluoride to the reagents in the pretreatment method, the extraction efficiency can be significantly improved, the quantitative limit can be reduced, and the detection accuracy and sensitivity can be improved to meet the actual sample detection needs. The liquid-liquid extraction method adopted is cost-effective, simple, fast, and highly operable.

[0034] 2. Adding 0.1mM ammonium chloride and 0.1mM ammonium fluoride to the mobile phase can significantly improve the peak shape and enhance the chromatographic response. Compared with the common mobile phase additions in the prior art, it significantly reduces the limit of quantification.

[0035] 3. The types of ultrafiltration buffer, extraction agent and resolvent in the pretreatment method were compared and screened. HEPES ultrafiltration buffer containing 3mM ammonium chloride and 1mM ammonium fluoride, ethanol-ether extraction agent containing 2mM ammonium chloride and 1mM ammonium fluoride, and 30% methanol water containing 2.5mM ammonium chloride and 2.5mM ammonium fluoride were selected to promote chromatographic detection, improve chromatographic response and reduce the quantitative limit. DETAILED DESCRIPTION

[0036] In order to make the above-mentioned objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to embodiments. The illustrative embodiments of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention.

[0037] Unless otherwise specified, the raw materials used in the examples were purchased commercially.

[0038] Example 1: A method for determining free testosterone in serum

[0039] 1. Solution preparation

[0040] (1) Preparation of standard solution

[0041] A. Preparation of Primary Standard Stock Solution

[0042] Accurately prepare 1 mL of 1 mg / mL first-grade testosterone T standard stock solution using methanol as the preparation solvent and store at -80°C in the dark.

[0043] B. Preparation of Secondary Standard Stock Solution

[0044] Use a 20 μL pipette to transfer 10 μL of 1 mg / mL primary testosterone T standard stock solution into a 10 mL volumetric flask, dilute with methanol and adjust to volume, shake well to obtain 1 μg / mL secondary testosterone T standard stock solution, and store at -80°C in the dark.

[0045] C. Preparation of tertiary standard working solution

[0046] Use a 100 μL pipette to transfer 50 μL of 1 μg / mL primary testosterone T standard stock solution into a 10 mL volumetric flask, dilute with methanol and adjust to volume, shake well to obtain a 5 ng / mL tertiary standard working solution, and store at -80°C in the dark.

[0047] D. Preparation of Standard Curve Solution

[0048] Use the tertiary standard working solution to prepare 8 standard curve solutions S0~S7. The preparation volume and concentration are shown in Table 1 below.

[0049] Table 1 Preparation of standard solution

[0050]

[0051] E. Preparation of Quality Controls

[0052] Use the tertiary standard working solution to prepare two quality control solutions, L and H. Use a 100μL pipette to transfer 20μL of the 5ng / mL first-level testosterone T standard stock solution into a 1.5ml EP tube, dilute with methanol and adjust the volume to 1ml, shake well to obtain a 100pg / mL high-value quality control solution (H); then take 500μL of the high-value quality control solution into a 1.5ml EP tube, dilute with methanol and adjust the volume to 1ml, shake well to obtain a 50pg / mL low-value quality control solution (L), and store at -80℃ in the dark.

[0053] (2) Preparation of internal standard

[0054] A. Preparation of Testosterone T-d3 Internal Standard Stock Solution

[0055] a) Preparation of primary testosterone T-d3 internal standard stock solution

[0056] Accurately prepare 1 μg / mL of primary testosterone T-d3 internal standard stock solution using methanol as the preparation solvent and store at -80°C in the dark.

[0057] b) Preparation of secondary testosterone T-d3 internal standard stock solution

[0058] Use a 1 mL pipette to transfer 900 μL of methanol to a 1.5 mL centrifuge tube. Use a 100 μL pipette to transfer 100 μL of 1 μg / mL primary testosterone T-d3 internal standard stock solution to the centrifuge tube. Vortex mix to obtain a 100 ng / mL secondary testosterone T-d3 stock solution. Store at -80°C in the dark.

[0059] c) Preparation of internal standard working solution

[0060] Accurately pipette 50 μL of 100 ng / mL testosterone T-d3 secondary stock solution into a 15 mL centrifuge tube, add 9950 μL of methanol to obtain 0.5 ng / mL testosterone T-d3 internal standard working solution, and store at -20°C in the dark.

[0061] (3) Reagent preparation

[0062] A. Ultrafiltration Buffer

[0063] Prepare HEPES ultrafiltration buffer according to the ingredients and amounts described in Table 2.

[0064] Table 2 Composition and content of HEPES ultrafiltration buffer

[0065]

[0066] B. Extraction Agent

[0067] According to the volume ratio of ethanol to ether being 1:3, ethanol solution and ether solution were measured separately to prepare 1 L of ethanol-ether extractant.

[0068] C. Reconstitution Solution

[0069] Use a graduated cylinder to measure 300 mL of methanol and 700 mL of ultrapure water, add them to a reagent bottle, and mix them by ultrasonication to obtain 30% methanol water.

[0070] D. Additives

[0071] Accurately weigh ammonium chloride powder and prepare 100 mL of ammonium chloride solution at concentrations of 0.1 mM, 2 mM, 2.5 mM, and 3 mM, respectively.

[0072] Accurately weigh ammonium fluoride powder and prepare 100 mL of ammonium fluoride solution at concentrations of 0.1 mM, 1 mM, and 2.5 mM, respectively.

[0073] E. Mobile Phase

[0074] Mobile phase A: Add 1 mL of formic acid to 1 L of water to prepare a 0.1% formic acid solution.

[0075] Mobile phase B: Add 1 mL of formic acid to 1 L of acetonitrile to prepare a 0.1% formic acid-acetonitrile solution.

[0076] 2. Sample Pretreatment

[0077] (1) Pipette 150 μL of HEPES buffer solution, add 25 μL of 3 mM ammonium chloride and 25 μL of 1 mM ammonium fluoride into a 1.5 mL centrifuge tube, pipette 200 μL of serum sample (standard, quality control) into the centrifuge tube, cover tightly, vortex mix for 1 minute, and then place in a 37°C constant temperature drying oven for 20 minutes. At this time, adjust the centrifuge temperature to 37°C.

[0078] (2) Transfer all the liquid in the 1.5 mL centrifuge tube to a 0.5 mL ultrafiltration centrifuge tube and centrifuge at 1800 g and 37°C for 1 h.

[0079] (3) After centrifugation, transfer 200 μL of the filtrate to a new 2 mL centrifuge tube, add 20 μL of 0.5 ng / mL testosterone-d3 internal standard working solution, vortex for 1 min to mix, and let it stand at room temperature for 5 min.

[0080] (4) Add 1.2 mL of ethanol-ether extractant, 50 μL of 2 mM ammonium chloride, and 50 μL of 1 mM ammonium fluoride to the above 2 mL centrifuge tube, close the lid tightly, vortex for 10 minutes, and centrifuge in a desktop high-speed centrifuge at 13,000 rpm and 4°C for 5 minutes.

[0081] (5) Transfer 1000 μL of the supernatant to a 1.5 mL centrifuge tube and freeze it in a vacuum freeze dryer until it is completely dry. After freeze-drying, add 70 μL of 30% methanol aqueous solution and 30 μL of 2.5 mM ammonium chloride and 2.5 mM ammonium fluoride solution to the 1.5 mL centrifuge tube for reconstitution. Tighten the lid and vortex on a vortex shaker for 5 minutes. Then transfer the tube to a sample vial with a 300 μL inner liner for analysis.

[0082] 3. Liquid chromatography tandem mass spectrometry analysis

[0083] (1) Chromatographic conditions

[0084] Liquid chromatography system: ACQUITY UPLC I-Class IVD; mobile phase A: 0.1% formic acid in water containing 0.1 mM ammonium chloride and 0.1 mM ammonium fluoride; mobile phase B: 0.1% formic acid in acetonitrile containing 0.1 mM ammonium chloride and 0.1 mM ammonium fluoride; column: ACQUITY UPLC BEH (C18 Column, 2.1 × 50 mm, 1.7 μm); injection volume: 10 μL; flow rate: 0.4 mL / min; column temperature: 40°C;

[0085] The mobile phase gradient is shown in Table 3:

[0086] Table 3 Gradient elution parameters

[0087]

[0088] (2) Mass spectrometry conditions

[0089] Mass spectrometry system: Waters Xevo TQ-S IVD; ESI source, positive ion mode; multiple reaction monitoring (MRM) scanning mode.

[0090] The ion source parameters are shown in Table 4, and the MRM parameters are shown in Table 5.

[0091] Table 4 Ion source parameters

[0092]

[0093] Table 5 MRM parameters

[0094]

[0095] 4. Data Processing and Analysis

[0096] (1) Draw a standard curve

[0097] The standard curve was linearly fitted with the concentration of testosterone T calibrator as the horizontal axis and the ratio of testosterone T peak area to its corresponding internal standard peak area as the vertical axis. It was found that the samples had a good linear relationship in the concentration range of 0.05~50pg / mL, with a correlation coefficient of 0.9997.

[0098] The samples of the standard solution S0 in Table 1 were measured in parallel, and the test was repeated 6 times. The standard deviation and coefficient of variation were calculated, as shown in Table 6 below.

[0099] Table 6 Precision and accuracy verification of the lower limit of quantification (LLOQ)

[0100]

[0101] As shown in Table 6, the accuracy deviation of the lower limit of quantification was within ±20%, and the precision was within 20%, both meeting the requirements.

[0102] The stock solution of the mixed standard working solution was prepared into serum quality control samples at three concentration levels: low, medium, and high. The samples were pretreated according to the method of this example. The spiked recovery and precision data of testosterone T are shown in Table 7.

[0103] Table 7 Spiked recovery and precision results

[0104]

[0105] Conclusion: The spiked recovery rate was within 85%~115%, and the coefficient of variation was within 10%, and the results met the requirements.

[0106] The separation and determination method provided by the present invention can maintain an effective detection period of 1-3 months. The serum sample after ultrafiltration separation can still reach the detection limit of 0.05 pg / mL after being stored for 3 months, with high stability and repeatability.

[0107] Example 2, screening and optimization of ultrafiltration buffer

[0108] The present invention evaluates the effects of additives in ultrafiltration buffer in combination with other components on the peak area and signal intensity of free testosterone. First, different additives were studied, such as ammonium chloride, ammonium fluoride, ammonium formate, and ammonium acetate. The remaining reagents and method steps were the same as in Example 1. The peak area and quantitative lower limit detection results of free testosterone are shown in Table 8.

[0109] Table 8 Effect of ultrafiltration buffer additives on testosterone response

[0110]

[0111] According to the test results in Table 8, compared with the control (no additives), the addition of ammonium formate, ammonium fluoride, ammonium acetate, and ammonium formate + ammonium acetate additives had no significant effect on the improvement of the chromatographic response of the target compound of the present invention, and the lower limit of quantification was higher than 0.5 pg / mL, which could not meet the detection requirements of clinical female specimens. After the addition of 3 mM ammonium chloride and 3 mM ammonium chloride + 1 mM ammonium fluoride, the peak area of free testosterone increased significantly. Among them, the simultaneous addition of ammonium chloride and ammonium fluoride can make the lower limit of quantification of free testosterone reach 0.05 pg / mL, which is the best effect.

[0112] At the same time, the main buffer substance in the ultrafiltration buffer was further verified to be used in combination with ammonium chloride + ammonium fluoride additives. The effect on the peak area and signal intensity of free testosterone was compared and analyzed by the present invention on different ultrafiltration buffer substance types. The method of Example 1 was used for testing. The peak area and quantitative lower limit detection results of free testosterone are shown in Table 9.

[0113] Table 9 Effect of ultrafiltration buffer on testosterone response

[0114]

[0115] As shown in the results in Table 9, by comparing the peak area and signal intensity of testosterone, it can be seen that the detection signal intensity is HEPES>0.5% formic acid>0.5% acetic acid>PBS. Among them, HEPES as an additive has the largest peak area, which can make the quantitative limit of free testosterone reach 0.05 pg / mL. When other ultrafiltration buffers are used to detect free testosterone, their quantitative limits are all higher than 0.5 pg / mL, which cannot meet the detection requirements of clinical female specimens.

[0116] The present invention further optimizes the concentration of the additive in the ultrafiltration buffer, and selects four additives of different concentrations shown in Table 10. The rest are carried out in the same manner as in Example 1. The peak area and quantitative lower limit detection results of free testosterone are shown in Table 10.

[0117] Table 10 Effect of ammonium chloride concentration in ultrafiltration buffer on testosterone response

[0118]

[0119] According to experimental results, the peak area was maximized after adding 3mM ammonium chloride and 1mM ammonium fluoride, and the lower limit of quantification of free testosterone reached 0.05pg / mL. Therefore, 3mM ammonium chloride and 1mM ammonium fluoride were used as ultrafiltration buffer additives.

[0120] Furthermore, the present invention evaluated the stability of the sample treated by ultrafiltration. The 50 pg / mL low-value quality control sample prepared in Example 1 was used as a sample. The sample was pre-treated according to the following ultrafiltration methods and then analyzed by liquid chromatography-tandem mass spectrometry: ① The ultrafiltration method of Example 1 was used to separate free testosterone in serum; ② No additives were added to the ultrafiltration buffer, and the rest of the operation was the same as in Example 1; ③ 3mM ammonium formate and 1mM ammonium acetate were added to the ultrafiltration buffer, and the rest of the operation was the same as in Example 1; ④ The ultrafiltration method in the Chinese patent with the same publication number CN114720704B and the invention name "A kit and method for determining free testosterone in serum" was used. The samples after pretreatment by different methods were analyzed by liquid chromatography-tandem mass spectrometry before storage, 15 days, 1 month, 2 months, and 3 months, and the analysis method was the same as in Example 1. The test results are shown in Table 11.

[0121] Table 11 Stability test of serum samples pre-treated by ultrafiltration

[0122]

[0123] According to the results in Table 11, it can be seen that only the ultrafiltration pretreatment method provided in Example 1 can achieve a stable detection effect, and the free testosterone content in the serum can still be accurately detected within a period of 3 months; and after the samples ultrafiltrated by the method of Groups ②-④ are stored for a certain period of time, the detected free testosterone content gradually decreases, and even free testosterone cannot be detected after the third month of storage, indicating that only by adding 3mM ammonium chloride and 1mM ammonium fluoride to the ultrafiltration buffer can the storage period of the sample be effectively extended and stable detection of free testosterone be achieved, proving that the method provided by the present invention significantly improves the stability of free testosterone determination compared with the prior art.

[0124] Example 3: Screening and Optimization of Extraction Agents

[0125] According to the prior art, different extractants have a significant effect on the detection response of free testosterone. Therefore, the present invention conducted experimental research on the types of extractants and compared the detection sensitivity of methyl tert-butyl ether, dichloromethane, 70% acetonitrile water, and ethanol-ether (volume ratio 1:3). The other method steps are basically the same as those in Example 1. The test results are shown in Table 12.

[0126] Table 12 Effect of extraction agents on testosterone response

[0127]

[0128] As shown in the results in Table 12, by comparing the peak area and signal intensity of testosterone, it can be seen that the extractant containing 2mM ammonium chloride and 1mM ammonium fluoride additives can detect a lower limit of quantification than the extractant without additives, indicating that the detection results of the extractant containing additives are more accurate. Among them, among the extractants containing additives, the detection signal intensity is ethanol-ether>methyl tert-butyl ether>dichloromethane>70% acetonitrile water, among which ethanol-ether as an additive has the largest peak area, which can make the lower limit of quantification of free testosterone reach 0.05pg / mL. When methyl tert-butyl ether detects free testosterone, its lower limit of quantification reaches 0.25pg / mL, which is not as sensitive as ethanol-ether. When other extractants are used to detect free testosterone, their lower limits of quantification are all higher than 0.6pg / mL.

[0129] Therefore, the present invention further experiments on the additives added to the ethanol-ether extractant, and adjusts the concentrations of ammonium chloride and ammonium fluoride. The method of Example 1 is used for testing. The peak area and quantitative lower limit detection results of free testosterone are shown in Table 13.

[0130] Table 13 Effects of different additives and different concentrations of ammonium chloride on the response to testosterone

[0131]

[0132] According to the test results in Table 13, when different additives are used in combination with ethanol-ether, the response effects of the target compound vary greatly. Only when ammonium chloride and ammonium fluoride are added to the ethanol-ether extractant can the detection limit of 0.05 pg / mL be reached. At the same time, the peak area and quantitative limit of free testosterone also change with the concentration of ammonium chloride and ammonium fluoride. Only when 2 mM ammonium chloride and 1 mM ammonium fluoride are added, the peak area of the target compound shows excellent performance, and the quantitative limit of free testosterone can reach 0.05 pg / mL, at this time the detection sensitivity is the highest.

[0133] Example 4: Screening and Optimization of Resolvents

[0134] The present invention further evaluated the detection sensitivity of resolubilization agents containing additives. By comparing the effects of different resolubilization agents and resolubilization agents containing different additives on the peak area and signal intensity of free testosterone, the four resolubilization agents shown in Table 14 were first selected. The others were carried out in the manner of Example 1. The test results are shown in Table 14.

[0135] Table 14 Effect of reconstitution solvent on testosterone response

[0136]

[0137] As shown in the results in Table 14, by comparing the peak area and signal intensity of testosterone, it can be seen that the detection signal intensity is 30% methanol water > 0.1% formic acid water > 70% methanol water > pure water. Among them, 30% methanol water as a resolvent has the largest peak area, which can make the quantitative limit of free testosterone reach 0.05 pg / mL. When other resolvents are used to detect free testosterone, their quantitative limits are all higher than 0.5 pg / mL, which cannot meet the detection requirements of clinical female specimens.

[0138] Therefore, the present invention further conducted a screening test on additives added to 30% methanol water, and compared the detection sensitivity of four ammonium salts of ammonium chloride, ammonium fluoride, ammonium formate and ammonium acetate after adding 30% methanol water. The concentration was 2.5 mM and the method of Example 1 was used for testing. The peak area and quantitative lower limit detection results of free testosterone are shown in Table 15.

[0139] Table 15 Effect of Resolvent Additives on Testosterone Response

[0140]

[0141] According to the test results in Table 15, compared with the control (no additives), the quantitative limit of free testosterone did not decrease significantly after the addition of ammonium formate, ammonium fluoride, and ammonium acetate to the resolvent, and all were higher than 0.7 pg / mL, which could not meet the detection requirements of clinical female specimens; after adding 2.5 mM ammonium chloride or 2.5 mM ammonium formate + 2.5 mM ammonium acetate, the quantitative limit could reach 0.45 pg / mL and 0.50 pg / mL, respectively; after adding 2.5 mM ammonium chloride and ammonium fluoride, the peak area of free testosterone increased significantly, and the quantitative limit of free testosterone could reach 0.05 pg / mL, which is the best effect.

[0142] After determining that the additive in the resolvent is a combination of ammonium chloride and ammonium fluoride, the present invention further optimized the concentrations of the two and compared them according to the combination shown in Table 16. The rest was carried out in the manner of Example 1. The test results are shown in Table 16.

[0143] Table 16 Effect of ammonium chloride concentration in resolvent on testosterone response

[0144]

[0145] According to experimental results, the peak area of free testosterone was maximized after adding 2.5mM ammonium chloride and ammonium fluoride, which could reduce the limit of quantification of free testosterone to 0.05pg / mL. Therefore, 2.5mM ammonium chloride and ammonium fluoride were used as ultrafiltration buffer additives.

[0146] Example 5, screening and optimization of mobile phase

[0147] According to the prior art, adding ammonium fluoride to the mobile phase can further improve the chromatographic response of free testosterone. Therefore, the present invention evaluates the effect of adding different additives to the mobile phase on improving the chromatographic response of free testosterone, such as 0.1 mM ammonium chloride, 0.1 mM ammonium formate, 0.1 mM ammonium fluoride and 0.1 mM ammonium acetate, as well as combinations of additives at different concentrations. Others are carried out in the manner of Example 1. The test results are shown in Table 17.

[0148] Table 17 Effect of mobile phase additives on the response of testosterone

[0149]

[0150] According to the results in Table 17, only the addition of 0.1 mM ammonium chloride and ammonium fluoride can improve the response of free testosterone, and the lower limit of quantification of free testosterone can reach 0.05 pg / mL, at which time the peak area is the largest; however, the peak area of free testosterone does not show a regular change with the change of ammonium chloride concentration. When the concentration of ammonium chloride and ammonium fluoride is lower than 0.1 mM or higher than 0.1 mM, the detection sensitivity is reduced.

[0151] This example further compares the effects of different mobile phases containing ammonium chloride and ammonium fluoride on the peak area and signal intensity of free testosterone. Mobile phase A is 0.1% formic acid water, 0.1% trifluoroethylamine water, and pure water. Mobile phase B is 0.1% formic acid acetonitrile, 0.1% formic acid methanol, and 0.1% formic acid isopropanol. The remaining reagents, methods, and steps are the same as in Example 1. The test results are shown in Table 18.

[0152] Table 18 Effect of mobile phase on testosterone response

[0153]

[0154] As shown in the results of Table 18, by comparing the peak area and signal intensity of testosterone, it can be seen that using 0.1% formic acid water as mobile phase A and 0.1% formic acid acetonitrile as mobile phase B, the peak area is the largest, and the quantitative limit of free testosterone can reach 0.05pg / mL; while when other mobile phase reagents are used to detect free testosterone, their quantitative limits are all higher than 0.6pg / mL. Therefore, 0.1% formic acid water is preferred as mobile phase A and 0.1% formic acid acetonitrile is preferred as mobile phase B.

[0155] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for determining free testosterone in serum based on liquid chromatography tandem mass spectrometry, characterized in that: The method includes pretreatment of serum samples, which includes ultrafiltration, extraction and re-dissolution. The ultrafiltration step uses a HEPES ultrafiltration buffer containing ammonium chloride and ammonium fluoride, the extraction step uses an ethanol-ether extractant containing ammonium chloride and ammonium fluoride, and the re-dissolution step uses 30% methanol water containing ammonium chloride and ammonium fluoride.

2. The method according to claim 1, wherein The concentration of ammonium chloride in the HEPES ultrafiltration buffer is 3 mM, and the concentration of ammonium fluoride is 1 mM; the concentration of ammonium chloride in the ethanol-ether extractant is 2 mM, and the concentration of ammonium fluoride is 1 mM; the concentration of ammonium chloride in the 30% methanol water is 2.5 mM, and the concentration of ammonium fluoride is 2.5 mM.

3. The method according to claim 2, wherein The method comprises high performance liquid chromatography tandem mass spectrometry analysis, wherein the mobile phase A of the high performance liquid chromatography tandem mass spectrometry analysis is formic acid water containing ammonium chloride and ammonium fluoride, and the mobile phase B is formic acid acetonitrile containing ammonium chloride and ammonium fluoride.

4. The method according to claim 3, wherein The concentrations of ammonium chloride and ammonium fluoride in the formic acid water and formic acid acetonitrile are both 0.1 mM.

5. The method according to claim 4, wherein The following steps are involved: (1) Dilute the serum sample with ultrafiltration buffer to obtain a diluted sample; (2) ultrafiltration of the diluted sample from step (1) to obtain an ultrafiltrate; (3) adding an internal standard to the ultrafiltrate of step (2) and performing extraction to obtain an extract; (4) freeze-drying the extract from step (3) and redissolving it to obtain a redissolved solution; (5) The reconstituted solution of step (4) is subjected to high performance liquid chromatography tandem mass spectrometry detection.

6. The method according to claim 5, wherein The chromatographic conditions of the high performance liquid chromatography tandem mass spectrometry in step (5) are as follows: injection volume: 10 uL; flow rate: 0.4 mL / min; column temperature: 40°C; mobile phase gradient: 。 7. The method according to claim 6, wherein The mass spectrometry conditions of the high performance liquid chromatography tandem mass spectrometry in step (5) are ESI ion source, and the ion source parameters are: ； MRM parameters: 。 8. A kit for determining free testosterone in serum based on liquid chromatography tandem mass spectrometry, characterized in that: The method comprises an ultrafiltration buffer, an extractant and a resolvent, wherein the ultrafiltration buffer is HEPES containing ammonium chloride and ammonium fluoride, the extractant is an ethanol-ether extractant containing ammonium chloride and ammonium fluoride, and the resolvent is 30% methanol water containing ammonium chloride and ammonium fluoride.

9. Use of ammonium chloride and ammonium fluoride for preparing a reagent for improving the sensitivity of liquid chromatography tandem mass spectrometry for determining free testosterone in serum, characterized in that: The reagents include an ultrafiltration buffer, an extractant, and a resolvent. The ultrafiltration buffer is HEPES containing ammonium chloride and ammonium fluoride. The extractant is an ethanol-ether extractant containing ammonium chloride and ammonium fluoride. The resolvent is 30% methanol water containing ammonium chloride and ammonium fluoride.

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