A method for detecting free testosterone and total testosterone concentration

Abstract

The present invention provides a detection method for free testosterone and total testosterone concentration, including antibody-coupled magnetic micro-enrichment of free testosterone and total testosterone in serum samples using a kit, automated pre-treatment and LC-MS / MS detection, the kit includes a testosterone separation and purification reagent strip, a testosterone calibration curve and a testosterone quality control product, and a testosterone separation and purification reagent strip includes testosterone antibody-coupled magnetic particles, testosterone stable isotope internal standard, eluent and eluent that can separate and purify testosterone. The present invention combines the advantages of immunoassay and mass spectrometry, and the required reagents for separating and purifying testosterone are prepared into closed reagent strips, effectively avoiding experimental errors caused by poor contamination of reagents. The sample is added to the reagent strip, and the pre-treatment is automated, and the operation process only requires 12min, completes the extraction of testosterone in the sample, and the treated sample is taken out for detection, without the need for various reagents, consumables, instruments and equipment commonly used in conventional detection methods and their placement space, simple and efficient, easy to clinical detection and application.

Description

Technical Field

[0001] The invention relates to a kit, in particular to a method for detecting the concentrations of free testosterone and total testosterone. Background Art

[0002] Free testosterone is testosterone that exists in a free form in the blood circulation and is in an active state. Testosterone in the blood is divided into two forms: free testosterone and total testosterone. After testosterone is secreted, only about 2% exists in a free form in the blood, about 65% is bound to sex hormone binding protein, and about 33% is bound to albumin. The measurement of free testosterone is of great significance for male fertility and female disease diagnosis. However, the detection method is very challenging. The gold standard equilibrium dialysis method is too complicated for conventional clinical laboratories, the analysis method is not unified, and there is no common reference range to help interpret the results.

[0003] Total testosterone testing is also of great significance in clinical testing. If the testosterone level in the body is elevated, it is seen in diseases such as idiopathic male precocious puberty, familial male precocious puberty, adrenal hyperplasia, adrenal cortical tumors, testicular tumors, etc. Reduced testosterone levels are seen in male testicular dysgenesis, no testicular signs, hypogonadism of the hypothalamus or pituitary, etc. Immunoassay and liquid chromatography tandem mass spectrometry (LC-MS / MS) are commonly used in clinical testing of total testosterone concentration.

[0004] The commonly used clinical method for free testosterone detection is to calculate free testosterone, that is, first measure the total testosterone concentration and the sex hormone binding protein concentration separately, and then calculate the free testosterone concentration. However, this algorithm is inaccurate, because sex hormone binding protein not only binds to testosterone, but also binds to other steroid hormones such as cortisol, and the binding ratio with testosterone is not constant. This method is usually implemented by immunoassay, but the immunoassay for testosterone also has the disadvantage of poor antibody specificity, because there are a large number of other steroid hormones with similar structures to testosterone in the body, such as androstenedione, dihydrotestosterone, progesterone and other steroid hormones, which makes the detection of free testosterone more difficult.

[0005] LC-MS / MS is gradually being used in clinical testing to detect testosterone concentration. It is currently the most reliable detection method. Because the endogenous testosterone content in the human body is very low and there is a complex serum matrix interference, the sample needs to be extracted, purified and concentrated before LC-MS / MS determination. Commonly used methods include liquid-liquid extraction and solid phase extraction (SPE). Liquid-liquid extraction usually requires the use of organic reagents such as methyl tert-butyl ether, diethyl ether, dichloromethane, ethyl acetate or n-hexane to extract testosterone from serum, and then concentrate and re-dissolve before mass spectrometry detection. However, the extract after liquid-liquid extraction has complex components, and macromolecular substances such as proteins are difficult to remove cleanly, which easily clogs the chromatographic column and pipeline, and there is a serious matrix effect. In addition, because the organic reagents used in liquid-liquid extraction are toxic and corrosive, protection is required, and the operation is carried out in a fume hood, and it seriously pollutes the environment. Solid phase extraction is to extract testosterone and a class of compounds with similar physical properties through filler materials to achieve the purpose of separation and purification at the same time. However, solid phase extraction is complicated to operate, and the price of solid phase extraction columns or plates is high. The cost of processing a sample is more than ten or twenty yuan, and the cost of clinical testing is too high, which limits its use. And when solid phase extraction batches multiple samples, the flow rate of samples and various liquids in each column is inconsistent due to the unevenness of the samples. Some columns have been drained, and some columns still have a lot of liquid left. The columns that have been drained will be lost when the eluent is added at the end, making the extraction efficiency of each sample inconsistent, affecting the result detection. Both liquid-liquid extraction and solid phase extraction require complex operations. Each step of the operation requires careful and precise addition of the required liquid. It is very easy to make operational errors. The results may be too low or too high, or there will be no results or the samples will be mixed, which is not conducive to large-scale clinical application. In addition, high-speed centrifugation, vortex oscillation, vacuum concentration or nitrogen blowing are required to complete liquid-liquid extraction or solid phase extraction, and positive pressure devices are also required for solid phase extraction. These operations require the configuration of special equipment and placement space, and require frequent maintenance, which invisibly increases costs.

[0006] The Chinese patent application publication number is CN 113049719 A, and the invention name is "A method for detecting free testosterone and a kit thereof", which uses a solid phase extraction method to detect the concentration of free testosterone. The Chinese patent application publication number is CN 113390978A, and the invention name is "An analytical method for determining the content of free testosterone in human serum samples by equilibrium dialysis combined with LC-MS / MS technology", which uses a derivatization method to detect the concentration of free testosterone. The methods disclosed in these two patents require complex operations and long processing time, and cannot be automated.

[0007] 10kDa ultrafiltration tube is concentrated by ultrafiltration, and the macromolecular protein greater than 10kDa in serum can be separated from other components, sex hormone binding protein molecular weight 95kDa, albumin molecular weight 66kDa, and bound testosterone is mainly combined with these two proteins. Through ultrafiltration tube concentration, protein greater than 10kDa is trapped on the ultrafiltration tube, and testosterone combined with albumin and sex hormone binding protein is trapped on the ultrafiltration tube, and the liquid below the ultrafiltration tube is a component less than 10kDa, including free testosterone. Ultrafiltration tube concentration can be used as an effective method for separating free testosterone, which is common in literature reports, such as free hormone detection status and the clinical application of liquid chromatography tandem mass spectrometry (Chinese Journal of Laboratory Medicine, October 2017, Vol. 40, No. 10, 748-755 pages), it is pointed out that ultrafiltration and equilibrium dialysis combined with LC-MS / MS method are two main methods for detecting free testosterone, but the free testosterone serum samples separated by these two methods need complicated sample processing, and still cannot be automated. Summary of the invention

[0008] In order to solve the problems that the existing detection methods for free testosterone and total testosterone concentrations are complicated and the immunoassay method for detecting testosterone has the defects of antibody specificity, the present invention provides a detection method for free testosterone and total testosterone concentrations. The present invention is based on the combination of immunoassay and mass spectrometry, and can automatically separate and purify free testosterone or total testosterone in serum samples as needed. The entire pretreatment operation process only takes 12 minutes to complete the extraction of testosterone in the sample, and then mass spectrometry detection is performed, which is simple and efficient and easy for clinical detection and application.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] A method for detecting free testosterone and total testosterone concentrations, the method comprising using a kit to perform antibody-coupled magnetic microenrichment of free testosterone and total testosterone on a serum sample, automated pre-treatment, and LC-MS / MS detection;

[0011] The kit comprises a testosterone separation and purification reagent strip, a testosterone calibration curve and a testosterone quality control product. The testosterone separation and purification reagent strip comprises a testosterone antibody-coupled magnetic particle capable of separating and purifying testosterone, a testosterone stable isotope internal standard, a eluent and an eluting solution.

[0012] In the present invention, the present invention prepares antibody-coupled magnetic particles, internal standards, washing solutions and eluents, which are added to single-portion reagent strips as testosterone separation and purification reagent strips. Free testosterone is pre-separated by a 10kDa ultrafiltration tube, and then extracted by a reagent strip combined with LC-MS / MS to detect the free testosterone concentration. Total testosterone is directly extracted by a reagent strip combined with LC-MS / MS to detect the total testosterone concentration. Through a series of performance verification experiments, it is proved that the method of the present invention can realize automated, accurate and stable detection of testosterone concentration.

[0013] As a preferred embodiment of the present invention, the method for preparing the testosterone antibody-coupled magnetic particles comprises the following steps:

[0014] 1) Testosterone antibodies were centrifuged multiple times, ultrafiltered, and stored in PBS buffer;

[0015] 2) Mixing the magnetic particles uniformly;

[0016] 3) adding the magnetic particles from step 2) into deionized water, mixing well, and removing the supernatant by magnetic separation, and repeating the process multiple times;

[0017] 4) resuspending the magnetic particles obtained in step 3) with PBS buffer, removing the supernatant by magnetic separation, and repeating multiple times;

[0018] 5) resuspending the magnetic particles obtained in step 4) with PBS buffer, adding the testosterone antibody in step 1), and mixing at room temperature; adding carbodiimide solution to make up the volume of the system, vortexing at room temperature, and removing the supernatant by magnetic separation;

[0019] 6) resuspending the antibody-coupled magnetic particles obtained in step 5) using PBS buffer, removing the supernatant by magnetic separation, and repeating multiple times;

[0020] 7) resuspending the antibody-coupled magnetic particles obtained in step 6) to remove endogenous testosterone and other steroid hormones;

[0021] 8) Using monohydric alcohol or acetonitrile aqueous solution with gradient concentration from high to low, the activity of the antibody-coupled magnetic particles obtained in step 7) is restored, and the particles are stored in a methanol solution with a volume fraction of 1-20%.

[0022] As a preferred embodiment of the present invention, the gradient concentration from high to low in step 8) is: 80-100% monoalcohol or acetonitrile, 60-79% monoalcohol or acetonitrile, 50-59% monoalcohol or acetonitrile, 40-49% monoalcohol or acetonitrile, 30-39% monoalcohol or acetonitrile, 20-29% monoalcohol or acetonitrile and 0-19% monoalcohol or acetonitrile solution.

[0023] As a preferred embodiment of the present invention, the reagent strip is provided with holes No. 1 to No. 15; wherein, holes No. 3, No. 4 and No. 5 are used for placing eluent; hole No. 6 is used for placing testosterone stable isotope internal standard; hole No. 7 is used for placing testosterone antibody-coupled magnetic particles; and hole No. 9 is used for placing eluent.

[0024] As a preferred embodiment of the present invention, the eluent is 100-400 μL of PBS solution.

[0025] As a preferred embodiment of the present invention, the testosterone stable isotope internal standard is 5 ng / mL testosterone-D3 internal standard prepared in 30-100 μL of 2-30% methanol by volume.

[0026] As a preferred embodiment of the present invention, the eluent is 100-300 μL of a monohydric alcohol or acetonitrile solution with a volume fraction of 70-100%.

[0027] As a preferred embodiment of the present invention, the testosterone calibration curve is prepared by mixing testosterone with 2% monohydric alcohol or acetonitrile solution 98% PBS-30% monohydric alcohol or acetonitrile solution 70% PBS to 0.005ng / mL, 0.01ng / mL, 0.02ng / mL, 0.05ng / mL, 0.2ng / mL, 0.5ng / mL, 1ng / mL, 5ng / mL and 15ng / mL.

[0028] As a preferred embodiment of the present invention, the testosterone quality control product includes a low-concentration quality control product of 0.15 ng / mL and a high-concentration quality control product of 10.5 ng / mL.

[0029] As a preferred embodiment of the present invention, free testosterone is pre-separated by a 10 kDa ultrafiltration tube, and then extracted by a reagent strip and combined with LC-MS / MS to detect the free testosterone concentration; total testosterone is directly extracted by a reagent strip and combined with LC-MS / MS to detect the total testosterone concentration.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention combines the advantages of immunoassay and mass spectrometry, and prepares the reagents required for separating and purifying testosterone into closed reagent strips, effectively avoiding experimental errors caused by poor reagent contamination. It only takes a simple addition of the sample to the reagent strip, and the pretreatment can be completed automatically by the iMS-1000 equipment of Hangzhou Baichen Medical Laboratory. The operation process only takes 12 minutes to complete the extraction of testosterone in the sample, and then the treated sample is taken out for LC-MS / MS detection. There is no need for various reagents, consumables, instruments and equipment and their placement space required for conventional liquid chromatography tandem mass spectrometry detection, such as liquid-liquid extraction and solid phase extraction. It is simple and efficient, and easy for clinical detection and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the testosterone calibration curve of the present invention. DETAILED DESCRIPTION

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

[0034] In the present invention, unless otherwise specified, % refers to volume fraction, and the reagents used can be purchased from the market.

[0035] The mass spectrometer used in the present invention is Waters TQS, and the instrument used for sample pretreatment is iMS-1000 equipment of Hangzhou Baichen Medical Laboratory.

[0036] Example 1

[0037] This embodiment provides a method for detecting the concentration of free testosterone and total testosterone, including a testosterone separation and purification reagent strip, a testosterone calibration curve and a testosterone quality control product. The testosterone separation and purification reagent strip includes a testosterone antibody-coupled magnetic particle that can separate and purify testosterone, a testosterone stable isotope internal standard, a eluent and an eluting solution.

[0038] The method for preparing testosterone antibody-coupled magnetic particles comprises the following steps:

[0039] 1) Antibody solution pretreatment: Use a 30kD ultrafiltration tube to ultrafilter 1 mg of testosterone antibody at 4°C, 12000g for 10 min, add 500 μL PBS, and ultrafilter at 12000g for 10 min. Repeat 3 times to replace the testosterone antibody storage solution with PBS buffer.

[0040] 2) The magnetic particles were mixed evenly, 1000 μL (10 mg) of magnetic particles were added to a 2 mL EP tube, and the supernatant was removed by magnetic separation on a magnetic stand;

[0041] 3) Add deionized water, mix well, remove the supernatant by magnetic separation, and repeat 2 more times;

[0042] 4) Add 100 μL PBS to resuspend the magnetic particles, remove the supernatant by magnetic separation, and repeat twice;

[0043] 5) Add 50 μL PBS to resuspend the magnetic particles, add 1 mg testosterone antibody (dissolved in PBS in advance), and rotate and mix at room temperature for 30 minutes;

[0044] 6) Add 10 μL of freshly prepared 10 mg / mL carbodiimide solution, add PBS to make the total volume of the system 1000 μL, vortex couple at room temperature for 1 to 3 hours, and remove the supernatant by magnetic separation;

[0045] 7) Add 100 μL of quenching buffer (50 mmol / L Tris-HCl, pH 7.4) to resuspend the magnetic particles, vortex mix for 30 min, and remove the supernatant by magnetic separation;

[0046] 8) Add 1000 μL PBS, mix and resuspend the magnetic particles, remove the supernatant by magnetic separation, and repeat twice;

[0047] 9) The coupled antibody-coupled magnetic particles are resuspended in 1 mL of 80-100% methanol and mixed by inversion for 3 minutes to remove endogenous testosterone and other steroid hormones. The antibody-coupled magnetic particles are then washed several times with 80-100% methanol until testosterone and other steroid hormones are no longer detected in the supernatant, and the 80-100% methanol is discarded;

[0048] 10) Add 60-79% methanol to resuspend the antibody-coupled magnetic microparticles, mix by inversion for 3 minutes, and discard the 60-79% methanol. Then use 50-59% methanol, 40-49% methanol, 30-39% methanol, 20-29% methanol, and 0-19% methanol in a gradient to restore the antibody activity, and store the antibody-coupled magnetic microparticles in a 1-20% methanol solution to make the concentration of the antibody-coupled magnetic microparticles reach 0.5-3 mg / mL.

[0049] Testosterone test strip preparation

[0050] The reagent strip includes wells 1 to 15, and wells 3, 4, and 5 are respectively added with 100-400 μL PBS as a washing solution. Well 6 is added with 30-100 μL 10% methanol-prepared 5 ng / mL testosterone-D3 (Testosterone-D3) internal standard. Well 7 is added with 5-50 μL 1 mg / mL testosterone antibody-coupled magnetic particles. Well 9 is added with 100-300 μL 70-100% methanol as an eluent. Then the reagent strip is sealed with a sealing aluminum film to prepare a testosterone purification reagent strip, which is stored at 4°C.

[0051] Testosterone calibration curve preparation

[0052] Testosterone was prepared with 2% methanol 98% PBS-30% methanol 70% PBS to prepare a calibration curve as shown in Table 1. The unit is ng / mL. Each tube was packaged with 250 μL and stored at -80°C.

[0053] Table 1 Testosterone calibration curve concentration

[0054]

[0055] Testosterone quality control preparation

[0056] The serum was dialyzed several times with a dialysis bag until the testosterone concentration was lower than 0.15 ng / mL. Testosterone standard was added to prepare low and high concentration quality control products. The theoretical value of the low concentration quality control product was 0.15 ng / mL, and the high concentration quality control product was 10.5 ng / mL. Each tube was packaged in 250 μL and stored at -80°C.

[0057] Preparation of testosterone separation and purification kit by immunomass spectrometry

[0058] The prepared testosterone separation and purification reagent strip, testosterone calibration curve and testosterone quality control product are combined into a testosterone separation and purification kit.

[0059] Example 2

[0060] This embodiment adopts the kit described in Example 1, and includes the following steps:

[0061] Sample processing

[0062] Free testosterone detection method: add 200μL ultrapure water to a 10kD ultrafiltration tube, centrifuge at 12000g for 10min, discard the waste liquid at the bottom of the ultrafiltration tube, and remove the liquid in the ultrafiltration tube. Add 400μL human serum sample to the ultrafiltration tube, centrifuge at 12000g for 20min, take the serum 220 at the bottom of the ultrafiltration tube and add it to well 1 of the testosterone separation pure reagent strip, put the reagent strip and sample into the iMS-1000 instrument, put in the corresponding number of pipette tips, select the testosterone method, click the start button to process, and the processing is completed after about 12 minutes. Take out 100μL of pre-treated testosterone and androstenedione lines 1-6, low and high quality control products and samples to be tested from well 14 into a 96-well plate and detect with a Waters TQS mass spectrometer.

[0063] For the total testosterone detection method, serum 220 was directly added to well 1 of the testosterone separation pure reagent strip. The reagent strip and sample were placed in the iMS-1000 instrument, and the corresponding number of pipette tips were placed. The testosterone method was selected, and the start button was clicked for processing. The processing was completed after about 12 minutes. 100 μL of pre-treated testosterone and androstenedione lines 1-6, low and high quality control products, and samples to be tested were taken from well 14 and placed in a 96-well plate for detection by the Waters TQS mass spectrometer.

[0064] Chromatographic parameters

[0065] 1. Chromatographic column: Waters CORTECS C18 (2.1×50mm, 2.7μm).

[0066] 2. Mobile phase A: 0.1% formic acid, 2mmol / L ammonium acetate aqueous solution; mobile phase B: 0.1% formic acid, 2mmol / L ammonium acetate methanol solution.

[0067] 3. Liquid chromatography elution gradient:

[0068] Table 2 Liquid chromatography elution gradient

[0069]

[0070]

[0071] 4. Flow rate: 0.5 mL / min; column temperature: 45°C; injection: 15 μL.

[0072] Mass spectrometry parameters

[0073] 1. Mass spectrometer model and scanning mode: I-class Waters TQS, positive ion mode

[0074] 2. Capillary voltage: 0.8kv; desolvation temperature: 350℃; desolvation gas: 800L / hr; cone gas: 150L / hr;

[0075] 3. Scanning mode: multiple reaction monitoring (MRM), the mass spectrometry parameters of each compound are as follows:

[0076] Table 3 Testosterone multiple reaction monitoring (MRM) mass spectrometry parameters

[0077]

[0078] Precision

[0079] The sample concentrations at two levels, low and high, were tested respectively, with three repetitions per day for five consecutive days.

[0080] Correctness Verification

[0081] The actual patient serum was selected as the basic sample for the recovery experiment. 100 μL of low, medium and high standard samples were mixed with 900 μL of the basic sample, respectively, as shown in Table 4. The requirements are: the low concentration is 3 times the lower limit of the linear range; the medium concentration is the middle concentration of the linear range; the high concentration is 75% of the upper limit of the linear range. The above "basic sample" and "experimental sample" were repeated and measured in parallel 6 times, and the experimental results were recorded and statistically analyzed for the spiked recovery of the three concentration levels of low, medium and high. The recovery rate = (detection value - basic sample value) / theoretical value × 100%, 85% < recovery rate < 115%.

[0082] Table 4 Spike recovery preparation table

[0083]

[0084] Lower limit of quantitation

[0085] The lower limit of quantification is the lowest value of the target analyte that can be detected by the LC-MS / MS method under the premise of meeting the laboratory's requirements for accuracy and precision. Five samples with concentrations close to the detection limit were selected for the lower limit of quantification. Each concentration sample was divided into five parts for processing. Each sample was repeated for three times, and three batches were measured continuously. The total precision (CV) of each concentration sample and the deviation of its mean concentration measurement from the theoretical concentration were evaluated respectively. The mean measurement value of the lowest concentration sample with CV≤20% and bias<15% was taken as the lower limit of quantification of the method.

[0086] Matrix Effects

[0087] Six human serum samples were processed separately, and each sample was repeated 3 times. 10% methanol was used to prepare three concentration levels of testosterone: low concentration (0.15 ng / mL), medium concentration (7.5 ng / mL) and high concentration (11.25 ng / mL). The three concentrations of low, medium and high were mixed with six human serum samples in a 1:1 ratio for mass spectrometry analysis. Matrix effect = (serum sample testosterone peak area + 10% methanol sample peak area) / 1:1 mixture peak area × 100%. If the matrix effect is <15%, it means there is no matrix effect.

[0088] Dilution reliability

[0089] Blood samples from patients near the upper limit of the linear range were collected, diluted 2, 4 and 8 times with water, respectively, and analyzed by mass spectrometry with the requirement: 85% < average recovery rate < 115%.

[0090] 72-hour stability of the sample tray

[0091] Ten samples with low to high concentrations were selected, and after pretreatment, they were placed in an LC-MS / MS injection plate (8°C), and the stability of the treated samples in the injection plate for 72 hours was analyzed at 0, 24, 48 and 72 hours, respectively.

[0092] Carrying pollution

[0093] First, repeat the injection of low-concentration samples, and then alternately inject high-concentration samples and low-concentration samples. Compare the difference between the test results of the low-concentration sample injected for the first time and the test results of the low-concentration samples injected subsequently. If it is lower than the predetermined standard (20%), it is considered that there is no obvious carryover contamination when measuring the high-concentration and lower-concentration samples.

[0094] Actual sample testing

[0095] Actual clinical samples tested in our laboratory were selected, 8 female samples and 2 male samples, and free testosterone and total testosterone were tested respectively.

[0096] Experimental reagents and materials

[0097] Formic acid was purchased from Aladdin, dialysis bags, 10kD and 30kD ultrafiltration tubes, methanol, testosterone and testosterone-13C3 were purchased from Merck, ultrapure water was purchased from Watsons, testosterone antibody was purchased from Thermo, and magnetic particles and magnetic racks were purchased from Yingruicheng Biochemical Technology Co., Ltd.

[0098] Example 3

[0099] This embodiment provides the results obtained by the method of embodiment 2, specifically:

[0100] Calibration curve linearity test results

[0101] See also Figure 1 , calibration curve range 0.005~15ng / mL, R 2 =0.9998, meets the requirements (requires R 2 ≥0.99).

[0102] Precision

[0103] Table 5 Testosterone 5-day precision

[0104]

[0105] Correctness Verification

[0106] Table 6 Testosterone accuracy verification results

[0107]

[0108]

[0109] Lower limit of quantitation

[0110] Table 7 Testosterone quantitative lower limit results

[0111]

[0112] Matrix Effects

[0113] Table 8 Testosterone low concentration matrix effect results

[0114]

[0115]

[0116] Table 9 Testosterone concentration matrix effect results

[0117]

[0118] Table 10 Testosterone high concentration matrix effect results

[0119]

[0120]

[0121] Dilution reliability

[0122] Table 11 Testosterone dilution reliability results

[0123]

[0124] 3-day stability of processed sample tray

[0125] Table 12 Testosterone treated sample injection plate (8°C) 3-day stability results

[0126]

[0127]

[0128] Carrying pollution

[0129] Table 13 Testosterone carryover contamination results

[0130]

[0131]

[0132] Note: H is a high concentration sample; L is a low concentration sample.

[0133] Actual sample free testosterone and total testosterone test results

[0134] The results in Table 14 show that the method of the present invention can detect extremely low testosterone concentrations in samples, especially free testosterone concentrations in most women, and the proportion of free testosterone to total testosterone is consistent with theoretical expectations (theoretical proportion is about 2%).

[0135] Table 14 Actual sample testosterone and total testosterone test results

[0136]

[0137] The above is only a preferred embodiment of the present invention, and is not any formal or substantial limitation of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, can make some changes, modifications and evolutions of the technical content disclosed above, which are equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for detecting free testosterone and total testosterone concentrations, characterized in that, The detection method comprises using a kit to perform antibody-coupled magnetic microenrichment of free testosterone and total testosterone on serum samples, automated pre-treatment, and LC-MS / MS detection; The kit comprises a testosterone separation and purification reagent strip, a testosterone calibration curve and a testosterone quality control product, wherein the testosterone separation and purification reagent strip comprises a testosterone antibody-coupled magnetic particle capable of separating and purifying testosterone, a testosterone stable isotope internal standard, a eluent and an eluent; the reagent strip is provided with holes No. 1 to No. 15; wherein holes No. 3, No. 4 and No. 5 are used for placing the eluent; hole No. 6 is used for placing the testosterone stable isotope internal standard; hole No. 7 is used for placing the testosterone antibody-coupled magnetic particle; and hole No. 9 is used for placing the eluent; The preparation method of the testosterone antibody-coupled magnetic particles comprises the following steps: 1) Testosterone antibody was centrifuged multiple times, ultrafiltered, and stored in PBS buffer; 2) Mixing the magnetic particles uniformly; 3) adding the magnetic particles from step 2) into deionized water, mixing well, and removing the supernatant by magnetic separation, and repeating the process multiple times; 4) resuspending the magnetic particles obtained in step 3) with PBS buffer, removing the supernatant by magnetic separation, and repeating multiple times; 5) resuspending the magnetic particles obtained in step 4) with PBS buffer, adding the testosterone antibody in step 1), and mixing at room temperature; adding carbodiimide solution to make up the volume of the system, vortexing at room temperature, and removing the supernatant by magnetic separation; 6) resuspending the antibody-coupled magnetic particles obtained in step 5) using PBS buffer, removing the supernatant by magnetic separation, and repeating multiple times; 7) resuspending the antibody-coupled magnetic particles obtained in step 6) to remove endogenous testosterone and other steroid hormones; 8) using a monohydric alcohol or acetonitrile solution with a gradient concentration from high to low to restore the activity of the antibody-coupled magnetic particles obtained in step 7) and store them in a 1-20% methanol solution; Free testosterone was pre-separated by 10kDa ultrafiltration tube, and then extracted by reagent strips and combined with LC-MS / MS to detect the free testosterone concentration; total testosterone was directly extracted by reagent strips and combined with LC-MS / MS to detect the total testosterone concentration; chromatographic parameters were as follows: chromatographic column: Waters CORTECS C18, 2.1×50mm, 2.7μm; mobile phase A: 0.1% formic acid, 2mmol / L ammonium acetate aqueous solution; mobile phase B: 0.1% formic acid, 2mmol / L ammonium acetate methanol solution; flow rate: 0.5mL / min; column temperature: 45℃; injection: 15μL; Mass spectrometry parameters: mass spectrometry model and scanning mode: I-class Waters TQS, positive ion mode; capillary voltage: 0.8 kv; desolvation temperature: 350°C; desolvation gas: 800 L / hr; cone gas: 150 L / hr.

2. A method for detecting free testosterone and total testosterone concentration according to claim 1, characterized in that, The gradient concentrations from high to low in step 8) are: 80-100% monoalcohol or acetonitrile, 60-79% monoalcohol or acetonitrile, 50-59% monoalcohol or acetonitrile, 40-49% monoalcohol or acetonitrile, 30-39% monoalcohol or acetonitrile, 20-29% monoalcohol or acetonitrile and 0-19% monoalcohol or acetonitrile solution.

3. A method for detecting free testosterone and total testosterone concentration according to claim 1, characterized in that, The eluent is 100-400 μL of PBS solution.

4. The method for detecting free testosterone and total testosterone concentration according to claim 1, characterized in that: The testosterone stable isotope internal standard is a 5 ng / mL testosterone-D3 internal standard prepared in 30-100 μL of 2-30% methanol by volume.

5. A method for detecting free testosterone and total testosterone concentration according to claim 1, characterized in that, The eluent is 100-300 μL of a monohydric alcohol or acetonitrile solution with a volume fraction of 70-100%.

6. The method for detecting free testosterone and total testosterone concentration according to claim 1, characterized in that: The testosterone calibration curve is prepared by sequentially preparing testosterone into 0.005 ng / mL, 0.01 ng / mL, 0.02 ng / mL, 0.05 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL and 15 ng / mL using 2% monohydric alcohol or acetonitrile solution 98% PBS-30% monohydric alcohol or acetonitrile solution 70% PBS.

7. The method for detecting free testosterone and total testosterone concentration according to claim 1, characterized in that: The testosterone quality control product includes a low-concentration quality control product of 0.15 ng / mL and a high-concentration quality control product of 10.5 ng / mL.

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