Method for simultaneously detecting five corticoids in blood sample by liquid chromatography-mass spectrometry

Abstract

The invention provides a method for simultaneously detecting five corticoids in a blood sample. The five corticoids are cortisol, cortisone, corticosterone, 21-deoxycortisol and 11-deoxycortisol respectively. The method comprises the following steps: pre-treating a blood sample by adopting the steps of precipitating protein, adding a specific buffer solution and the like, then detecting a target sample solution by adopting a high performance liquid chromatography-tandem mass spectrometry, establishing a standard curve by adopting an internal standard method, and calculating the contents of the five corticoids in the blood. The detection method has the advantages of being high in detection sensitivity, high in flux, simple, convenient, rapid, high in detection efficiency and the like, cortisol, cortisone, corticosterone, 21-deoxycortisol and 11-deoxycortisol in blood can be detected at the same time, accurate qualitative and quantitative analysis can be conducted, clinical application and scientific research cost is reduced, and high application value is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and specifically relates to a method for simultaneously detecting five kinds of corticosteroids in a blood sample by liquid chromatography-mass spectrometry. Background Art

[0002] Corticosteroids refer to adrenal cortical hormones, which are divided into glucocorticoids and mineralocorticoids. They are important hormones in the growth and development of the body. The main corticosteroids are cortisol, cortisone, corticosterone, 21-deoxycortisol, 11-deoxycortisol, 11-deoxycorticosterone and aldosterone.

[0003] Among them, cortisol is a type of hormone produced by the adrenal glands in stress response. Cortisol can increase blood pressure and blood sugar levels and produce immunosuppressive effects. Hypercortisolism is also known as Cushing's syndrome, and one of its clinical diagnostic bases is the abnormal increase in cortisol levels. When Addison's disease, hypopituitarism and other symptoms appear clinically, the level of cortisol in the blood will decrease. Cortisone is a glucocorticoid secreted by the adrenal cortex. It is inactive itself and needs to be metabolized into cortisol in the body to work. 21-Deoxycortisol is a precursor for the biosynthesis of cortisol and cortisone. 11-Deoxycortisol is an intermediate product of the conversion of cholesterol to cortisol. The level of 11-deoxycortisol is measured to diagnose impaired cortisol synthesis, find out the enzyme deficiency that causes impaired cortisol pathways, and distinguish adrenal diseases. Corticosterone is a corticosteroid hormone of the 21-carbon steroid type, produced by the cortex of the adrenal glands. Its content will increase when there are diseases such as hypercortisolism (Cushing's disease), adrenal tumors, myocardial infarction, etc., and its content will decrease when the adrenal cortex function is low. Therefore, in clinical practice, corticosteroids are important diagnostic indicators of clinical diseases, and their detection is very important.

[0004] At present, the main methods for detecting corticosteroids are immunoassay, gas chromatography-mass spectrometry and high performance liquid chromatography-tandem mass spectrometry. Traditional immunoassays have many defects, such as low sensitivity, poor applicability, low analytical efficiency, etc. At the same time, each immunoassay can only measure one corticosteroid and the detection time is long. Although gas chromatography-mass spectrometry has high sensitivity, it often requires cumbersome derivatization steps and long analysis time.

[0005] High performance liquid chromatography tandem mass spectrometry technology utilizes the efficient separation ability of liquid chromatography and the detection ability of mass spectrometry to achieve specific separation and detection of corticosteroids. At present, there are reports on the use of high performance liquid chromatography tandem mass spectrometry to detect corticosteroids. For example, patent CN111175419A discloses a method and kit for simultaneously detecting multiple steroid hormones in blood samples. The blood sample pretreatment method is to use sodium bicarbonate combined with methanol to remove proteins, and then use a solid phase support extraction method to treat the blood sample. Patent CN111366671 A discloses a chemical derivatization-ultra-high performance liquid chromatography-tandem mass spectrometry method for simultaneously detecting 18 types of steroid hormones in serum. The pretreatment method requires derivatization of serum samples. Patent CN112198265 A discloses a pretreatment method, detection method and kit for simultaneously detecting multiple steroid hormones in blood samples. The pretreatment method is to perform liquid-liquid extraction on blood samples. The above pretreatment methods have the problems of cumbersome operation, long time consumption, high personnel requirements and high initial investment.

[0006] Secondly, in liquid chromatography-tandem mass spectrometry detection, calibrants with long-term stability are also a key factor in improving detection efficiency and accuracy. In current research, freeze drying is a method to extend the long-term stability of calibrants, but there are usually problems such as long freeze-dried samples, high costs, and poor uniformity of freeze-dried samples. Therefore, it is necessary to develop a fast and efficient freeze-drying process for trace samples to ensure the uniformity and stability of freeze-dried samples.

[0007] Thirdly, in current reports, fluoride ions are generally added to improve the sensitivity of liquid chromatography tandem mass spectrometry detection. For example, patent CN111175419A discloses a method using methanol containing 0.2 mM ammonium fluoride as mobile phase B; patent CN114088859 B discloses a method using 0.5 mM ammonium fluoride as a mobile phase additive; patent CN111366671 A discloses a method using trifluoroacetic acid and O-[(3-pyridyl)methyl]hydroxylamine / acetonitrile solution for derivatization reaction during pretreatment. However, fluoride-containing aqueous solutions have a certain corrosive effect on metals due to their acidity, and the high coordination of fluoride ions will increase the electrochemical corrosion rate of metals by reducing the electrode potential of metals. Therefore, in liquid chromatography tandem mass spectrometry detection, the use of fluoride ions will cause damage to the instrument. Summary of the invention

[0008] In view of the deficiencies in the prior art, the technical problem to be solved by the present invention is to provide a method for simultaneously detecting five corticosteroids in a blood sample. The detection method has the advantages of high detection sensitivity, high throughput, simplicity and speed, high detection efficiency, etc. It can simultaneously detect cortisol, cortisone, corticosterone, 21-deoxycortisol, and 11-deoxycortisol in the blood, and perform accurate qualitative and quantitative analysis, and has high application value.

[0009] To achieve the purpose of the present invention, the present invention adopts the following technical solutions:

[0010] The present invention provides a method for simultaneously detecting five corticosteroids in a blood sample, wherein the five corticosteroids are cortisol, cortisone, corticosterone, 21-deoxycortisol and 11-deoxycortisol;

[0011] The method comprises the following steps:

[0012] (1) pre-treating a blood sample to be tested to obtain a target sample solution;

[0013] (2) using high performance liquid chromatography tandem mass spectrometry to detect the target sample solution to obtain the contents of five corticosteroids in the blood sample;

[0014] The pretreatment of the blood sample to be tested specifically includes: collecting the blood sample to be tested, adding an internal standard solution, vortexing, adding a precipitant, vortexing and mixing, centrifuging, taking the supernatant, adding a buffer solution, and mixing to obtain a target sample solution;

[0015] The precipitant is methanol, acetonitrile or a mixed solution of methanol and acetonitrile (the volume ratio of methanol to acetonitrile is 1:1-7:43);

[0016] The buffer solution is an ammonia-ammonium acetate solution, in which the concentration of ammonium acetate is 0.5-1.5 mmol / L, the concentration of ammonia is 0.01%-0.02%, % is mass percentage, and the pH is 8.2-9.8;

[0017] The conditions of the HPLC are:

[0018] Chromatographic column: Waters, BEH C18 column, 1.7μm, 2.1×50mm; column temperature 40℃; injector temperature 4℃; gradient elution with mobile phase A and mobile phase B, the mobile phase A is 0.5mmol / L ammonium acetate aqueous solution, the mobile phase B is methanol, the parameters of the gradient elution are: 0min, mobile phase B: 55%; 0-1.3min, mobile phase B: increased from 55% to 65%; 1.3-1.6min, mobile phase B: increased from 65% to 95%; 1.6-1.7min, mobile phase B: decreased from 95% to 55%; 1.7-2.5min, mobile phase B: 55%; flow rate is 0.3mL / min; injection volume is 2μL.

[0019] Furthermore, in the above step (2), the detection of the target sample solution by high performance liquid chromatography tandem mass spectrometry specifically includes:

[0020] 1) preparing a standard curve working solution containing five kinds of corticosteroids with different known concentrations, and freeze-drying the standard curve working solution and a blank matrix according to the freeze-drying curve to prepare a calibration freeze-dried product, re-dissolving the calibration freeze-dried product to obtain a standard curve sample, and drawing a standard curve of the standard curve sample solution;

[0021] The blank matrix is ​​a 4%-10% (mass volume percentage) bovine serum albumin solution;

[0022] The reconstitution solution is a 0.01M-0.05M PBS aqueous solution;

[0023] 2) Detecting the target sample solution by high performance liquid chromatography tandem mass spectrometry, and obtaining the contents of the five corticosteroids in the target sample solution according to the standard curve, thereby obtaining the contents of the five corticosteroids in the blood sample to be tested.

[0024] Furthermore, the steps of preparing a standard curve sample containing five corticosteroids of different known concentrations and drawing a standard curve of the standard curve sample solution specifically include:

[0025] First, standard curve samples containing five kinds of corticosteroids with different known concentrations are prepared respectively, the internal standard solution is added, vortexed, the precipitant is added, vortexed and mixed, centrifuged, the supernatant is taken, the buffer is added, and mixed, so as to obtain a series of standard curve sample solutions containing five kinds of corticosteroids with different known concentrations;

[0026] Secondly, the standard curve sample solution was detected by high performance liquid chromatography tandem mass spectrometry, the peak area ratios of the five corticosteroids and the internal standard were calculated, and the standard curve was established by the internal standard method.

[0027] Further, the concentration of the internal standard solution is 50 ng / mL;

[0028] The volume ratio of the blood sample to be tested or the standard curve sample to the internal standard solution is 20:1;

[0029] The volume ratio of the blood sample to be tested or the standard curve sample to the buffer solution is 8:1-9:1.

[0030] The volume ratio of the blood sample to be tested or the standard curve sample to the precipitant is 1:0.6-1:3.

[0031] Furthermore, the freeze-drying of the standard curve working solution and the blank matrix according to the freeze-drying curve specifically includes: quick freezing stage: -50°C duration is 3h; primary sublimation drying stage: -45°C duration is 2h, rising to -40°C duration is 2h, rising to -35°C duration is 2h, rising to -30°C duration is 3h, rising to -25°C duration is 2h, rising to -20°C duration is 1h, rising to -15°C duration is 1h, rising to -10°C duration is 1h, rising to -5°C duration is 1h; secondary sublimation drying stage: 0°C duration is 1h, rising to 10°C duration is 2h, rising to 20°C duration is 2h.

[0032] Furthermore, the mass spectrometry parameters are: ionization mode: ESI+; curtain gas (CUR): 35psi; spray voltage (IS): 5500V; temperature (TEM): 550°C; nebulizer gas (Gas 1): 50psi; auxiliary heating gas (Gas 2): 50psi; multiple reaction monitoring (MRM). The specific mass spectrometry parameters are shown in Table 1.

[0033] Table 1: Specific mass spectrometry parameters

[0034]

[0035] * is the quantification ion, F is cortisol, CORT is corticosterone, S is 11-deoxycortisol, 21-DF is 21-deoxycortisol, CORT is corticosterone, E is cortisone, F-d6 is cortisol-d6, and F-d4 is cortisol-d4.

[0036] The quantitative range of the standard curve is: 10ng / mL-1000ng / mL.

[0037] The beneficial effects of the present invention are:

[0038] (1) The high performance liquid chromatography tandem mass spectrometry provided by the present invention adopts a pretreatment method of protein precipitation and adding a buffer solution, which is not only simple to operate and more in line with actual clinical needs, but also improves the detection sensitivity and efficiency, is less time-consuming, and has low costs for reagents and consumables. It can simultaneously detect cortisol, cortisone, corticosterone, 21-deoxycortisol, and 11-deoxycortisol in the blood, and perform accurate qualitative and quantitative analysis, thereby reducing the cost of clinical application and scientific research.

[0039] (2) The high performance liquid chromatography tandem mass spectrometry provided by the present invention can achieve baseline separation in only 2.5 minutes of liquid-mass analysis without interference from other peaks, which greatly increases the detection throughput of clinical laboratories and is suitable for the detection of large quantities of samples, thus having high application value.

[0040] (3) The HPLC-MS / MS method provided by the present invention further improves the detection sensitivity and accuracy, reduces the matrix effect, and reduces the maintenance and cleaning frequency of the instrument by adopting a specific mobile phase for gradient elution and optimizing the HPLC-MS / MS conditions. The results are objective and easy to analyze.

[0041] (4) The HPLC-MS / MS method provided by the present invention has good uniformity and stability in the preparation of the calibration freeze-dried product. After being reconstituted, it can be used as a calibration product, which is easy for personnel to operate, improves the stability and reproducibility of the detection, increases the consistency of the results, and lays a foundation for the mutual recognition of the detection results of multiple companies. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0043] Figure 1 This is the chromatogram of five corticosteroids in plasma samples;

[0044] Figure 2 is the chromatogram of cortisol in plasma samples;

[0045] Figure 3 is the chromatogram of cortisol in plasma samples after adding FA (formic acid) to the mobile phase;

[0046] Figure 4 is the chromatogram of cortisol in plasma sample after adding 0.5 Mm NH4Ac in the mobile phase;

[0047] Figure 5 is the chromatogram of cortisol in plasma sample after adding 2 Mm NH4Ac in the mobile phase;

[0048] Figure 6is the chromatogram of cortisol in plasma sample after adding NH4Ac to the mobile phase;

[0049] Figure 7 is the chromatogram of cortisol in plasma samples after adding NH4FA to the mobile phase;

[0050] Figure 8 is the chromatogram of cortisol in plasma sample containing buffer in the system;

[0051] Fig. 9 The chromatogram of cortisol in plasma sample without buffer in the system. DETAILED DESCRIPTION

[0052] The embodiments of the present invention are described in detail below. The embodiments are given to better illustrate the contents of the present invention and are only used to explain the present invention, but should not be understood as limiting the present invention.

[0053] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0054] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0055] Example 1: Establishment and validation of the detection method for cortisol and four corticosteroid disruptors in plasma

[0056] 1. Materials and reagents

[0057] Cortisol (F) (China Institute of Metrology), cortisol-d4 (F-d4) (Bepure); 10×0.01M PBS (Solarbio), acetonitrile (chromatographic grade, purity 99.9%, FISHER), water (self-made ultrapure water), acetic acid (FISHER), bovine serum albumin (BSA) (Solarbio), ammonium acetate (Sigma), cortisone (Bepure), corticosterone (Bepure), 11-deoxycortisol (Bepure), 21-deoxycortisol (Bepure), cortisol-d6 (F-d6) (Bepure).

[0058] 2. Instruments and equipment

[0059] LCMS-4500MD liquid chromatography-tandem mass spectrometer, with triple quadrupole tandem mass spectrometer as the detector; MiliQ water purifier; vortex mixer; low-temperature high-speed centrifuge; medical low-temperature storage box.

[0060] 3. Preparation of standard curve and quality control basic working solution:

[0061] Prepare various series of standard curves and quality control working solutions as required, vortex mix, and prepare standard curves and quality control samples of various concentrations. Finally, the standard curve concentrations and quality control concentrations are shown in Table 2:

[0062] Table 2: Concentration and preparation process of standard curve and quality control samples

[0063]

[0064]

[0065] 4. Standard curve, quality control, and plasma sample pretreatment process:

[0066] Preparation of standard curve and quality control samples: Take 50 μL of standard curve sample or quality control sample for later use.

[0067] Preparation of plasma samples: Take 50 μL of sample and let it stand for later use.

[0068] Preparation of spiked samples: Take 47.5 μL blank matrix + 2.5 μL working solution and let stand for use.

[0069] Precipitation process: take the standard curve, quality control, spiked sample or plasma sample, add 5 μL internal standard working solution, vortex at 2000rpm for 5min, add 150 μL precipitant, vortex at 2000rpm for 5min, centrifuge at 15000g, 4℃ for 5min, take 90 μL supernatant solution, add 10 μL buffer, vortex at 2000rpm for 30s, take 60 μL supernatant for injection analysis.

[0070] 5. Detection of cortisol and four interfering substances: This experiment uses the following chromatographic and mass spectrometric conditions:

[0071] Liquid chromatography conditions were as follows: chromatographic column: Waters, BEH C181.7μm 2.1×50mm; column temperature 40°C; injector temperature 4°C; mobile phase A was 0.5mmol / L ammonium acetate aqueous solution, B was methanol, gradient elution: 0min, mobile phase B was 55%; 0-1.3min, mobile phase B was 55% to 65%; 1.3-1.6min, mobile phase B was 65% to 95%; 1.6-1.7min, mobile phase B was 95% to 55%; 1.7-2.5min, mobile phase B was 55%; flow rate 0.3mL / min; injection volume 2μL

[0072] The mass spectrometry parameters were: ESI+; curtain gas (CUR): 35 psi; spray voltage (IS): 5500 V; temperature (TEM): 550°C; nebulizer gas (Gas 1): 50 psi; auxiliary heating gas (Gas 2): 50 psi; multiple reaction monitoring (MRM) (reference Table 1);

[0073] 6. Methodological evaluation

[0074] 6.1 The present invention was validated according to the "2020 edition of the Chinese Pharmacopoeia-9012 Validation Guidelines for Quantitative Analysis Methods of Biological Samples" and "Recommendations for the Clinical Application of Liquid Chromatography-Mass Spectrometry (Guidelines and Consensus)" (Chinese Journal of Laboratory Medicine, 2017, 40(10): 770-779) and CNAS-GL037 Clinical Chemistry Quantitative Test Procedure Performance Validation Guidelines: including specificity and selection, linear range, sensitivity, precision, accuracy (spiked recovery), matrix effect and stability.

[0075] (1) Specificity and selectivity

[0076] After the double blank, single blank and LLOQ samples were processed according to the "4. Standard curve, quality control whole blood sample pretreatment process", LC-MS / MS analysis was performed. The results showed that under the experimental conditions, the peak shapes of each analyte and internal standard were good and had high specificity. There was no interference from other peaks on both sides of the chromatographic peak, which met the requirements of no background peak or interference peak area less than 20% of the peak area of ​​the quantification lower limit of the analyte and less than 5% of the peak area of ​​the internal standard.

[0077] Separation of cortisol from cortisone, corticosterone, 11-deoxycortisol, and 21-deoxycortisol Figure 1 , indicating that cortisone, corticosterone, 11-deoxycortisol, 21-deoxycortisol and cortisol are baseline separated and there is no cross-interference between channels.

[0078] Table 3: Selectivity and specificity investigation

[0079]

[0080] (2) Linear range

[0081] After the standard curve whole blood samples were processed according to the "4. Standard curve, quality control whole blood sample pretreatment process", LC-MS / MS analysis was performed. According to the ratio Y of each drug peak area As and internal standard peak area Ai (Y = As / Ai), weighted linear regression was performed on the drug concentration X to fit the standard curve, with a weight of 1 / X 2 (X is the concentration value), the standard curve represented by Y=slope×X+intercept is shown in Table 4.

[0082] Table 4: Standard curve and linear range

[0083]

[0084] (3) Precision and accuracy

[0085] After the quality control samples were processed according to the "4. Standard curve, quality control whole blood sample pretreatment process", LC-MS / MS analysis was performed, and 6 copies of each batch and each concentration were made in parallel for three consecutive days, one batch per day. The ratio Y (Y = As / Ai) of the drug peak area As and the internal standard peak area Ai was substituted into the standard curve of the day to calculate the sample concentration, and the intra-batch / inter-batch precision (CV < 15% is qualified) and accuracy (85% to 115% is qualified) were calculated. The precision and accuracy data of each drug are shown in Table 5.

[0086] Table 5: Precision and accuracy investigation

[0087]

[0088] (4) Matrix effect

[0089] Preparation of pure solution sample: Take a 1.5mL EP tube, take 2.5μL of WS QC M solution, add 47.5μL of blank matrix, and vortex for 5min. Follow the "sample pretreatment" process and take the supernatant for liquid chromatography-tandem mass spectrometry analysis. Make 6 replicates in parallel, and repeat the injection 3 times for each replicate. Calculate the mean value ys of the ratio of the response value of the analyte to the internal standard in the pure solution sample.

[0090] Preparation of plasma samples: Take 1.5mL EP tubes, take 50μL of plasma from 6 different human sources, follow the "sample pretreatment" process, and take the supernatant for liquid chromatography-tandem mass spectrometry analysis. Each sample is processed in parallel 3 times. Calculate the mean yx of the ratio of the response value of the analyte to the internal standard in the plasma sample.

[0091] Preparation of plasma: pure solution mixture (3:7, v / v): Take a 1.5mL EP tube, take 15μL and 35μL of the above two samples respectively, and vortex for 5min. Follow the "sample pretreatment" process and take the supernatant for liquid chromatography-tandem mass spectrometry analysis. Each sample is processed in parallel 3 times. Calculate the average ratio of the response value of the analyte to the internal standard in the mixture sample 7:3ys+x.

[0092] Preparation of plasma: pure solution mixture (7:3, v / v): Take a 1.5mL EP tube, take 35μL and 15μL of the above two samples respectively, and vortex for 5min. Follow the "sample pretreatment" process and take the supernatant for liquid chromatography-tandem mass spectrometry analysis. Each sample is processed in parallel 3 times. Calculate the average ratio of the response value of the analyte to the internal standard in the mixture sample 3:7ys+x.

[0093] Substitute the corresponding yx, ys and ys+x of each group into the formula: Deviation (%) = 1-(ys+x) / (yx×0.3+ys×0.7)×100%, Deviation (%) = 1-(ys+x) / (yx×0.7+ys×0.3)×100% to calculate the relative matrix effect deviation of the analyte. The matrix effect calculation results are shown in Table 6:

[0094] Table 6: Matrix Effects

[0095]

[0096] (5) Sample stability study

[0097] Low and high concentration QC whole blood samples were used, with 4 parallel samples for each concentration. After the sample analysis, the accompanying standard curve on the day of analysis was added to calculate the concentration of each drug. The stability of the sample at room temperature for 3 days, 2-8°C for 15 days, three freeze-thaw cycles, -20°C for 90 days, -70°C for 90 days, and the stability of the sample after processing and placed in an automatic sampler at 4°C for 72 hours were investigated. The results are shown in Table 7:

[0098] Table 7: Stability study

[0099]

[0100]

[0101] The above items comply with the requirements of "2020 Edition of the Chinese Pharmacopoeia-9012 Validation Guidelines for Quantitative Analysis Methods of Biological Samples", "Recommendations for the Clinical Application of Liquid Chromatography-Mass Spectrometry (Guidelines and Consensus)" (Chinese Journal of Laboratory Medicine, 2017, 40(10):770-779) and CNAS-GL037 Clinical Chemistry Quantitative Test Procedure Performance Validation Guidelines, and can be applied to the detection of cortisol.

[0102] Example 2: Application of a freeze-drying method for human serum albumin sample calibrator

[0103] 1. Target

[0104] Take cortisol as an example

[0105] 2. Instruments and equipment

[0106] LCMS-4500MD liquid chromatography-tandem mass spectrometer, with triple quadrupole tandem mass spectrometer as detector; 1 / 10,000 electronic analytical balance; MiliQ water purifier; vortex mixer; low-temperature high-speed centrifuge; medical low-temperature storage box.

[0107] 3. Preparation of calibration freeze-dried products:

[0108] Prepare the target substance according to the required concentration, take 500 μL of the target substance, add 9500 μL of 8% bovine serum albumin, vortex mix for 5 minutes, take 200 μL and dispense into 45 vials, and the freeze-drying method is shown in Table 8:

[0109] Table 8: Lyophilization method

[0110]

[0111]

[0112] 4. Calibrator and sample pretreatment process:

[0113] Preparation of calibrators: Add 400 μL 0.01 M PBS and vortex for pretreatment.

[0114] The pretreatment of calibrators and samples was the same as in Example 1.

[0115] 5. Test results:

[0116] The test results are shown in Table 9:

[0117] Table 9: Test results

[0118] Cortisol Concentration (ng / mL) MEAN-Not Freeze-dried 104.59 Freeze drying 0H001 108.82 Freeze drying 0H002 103.54 Freeze drying 0H003 108.37 Freeze drying 0H004 109.90 MEAN-freeze drying 0h 107.66 RE%-freeze drying 0H 2.9％ Freeze-dried products are stored for 12 months S001 112.75 Freeze-dried products stored for 12 months S002 105.34 Freeze-dried products are stored for 12 months S003 107.21 Freeze-dried products stored for 12 months S004 112.73 MEAN-freeze-dried products are stored for 12 months 109.51 RE% - freeze-dried product for 12 months 1.7％

[0119] 6. Result analysis:

[0120] The results were in compliance with the "General Requirements for Liquid Chromatography-Mass Spectrometry Assay Kits". The results showed that the RE% of the lyophilized product was <±15% before and after freeze-drying, and the RE% of the lyophilized product was <±15% after 12 months of storage, which met the testing requirements.

[0121] Example 3: Comparative analysis of reaction systems

[0122] 1. Effects of mobile phase additives on analytes

[0123] 1. Target

[0124] Taking Cortisol and Cortisol-d6 as an example

[0125] 2. Instruments and equipment

[0126] LCMS-4500MD liquid chromatography-tandem mass spectrometer, with triple quadrupole tandem mass spectrometer as detector; 1 / 10,000 electronic analytical balance; MiliQ water purifier; vortex mixer; low-temperature high-speed centrifuge; medical low-temperature storage box.

[0127] 3. Mobile phase

[0128] 0.5mM NH4Ac-H2O(A); MeOH(B)(1)

[0129] 0.1% FA-H2O(A); 0.1% FA-MeOH(B)(2)

[0130] 4. Plasma sample pretreatment process

[0131] The blood sample is pre-treated to obtain a target sample solution: a blood sample containing cortisol to be tested is collected, an internal standard solution is added, vortexed, a precipitant is added, vortexed and mixed, centrifuged, a supernatant is taken, a buffer solution is added, and mixed to obtain a target sample solution.

[0132] 5. Sample testing

[0133] The mobile phase (1) and the mobile phase (2) were selected respectively, and the target sample solution was tested using high performance liquid chromatography tandem mass spectrometry to examine the effect of adding acid into the system on the response of the target.

[0134] 6. Test results

[0135] The effect of adding acid to the system on the target compound was determined based on the response to the internal standard and the internal standard.

[0136] The response of cortisol in the mobile phase (1) system is 1140 cps, the response of the isotope internal standard cortisol-d6 is 4560 cps, and the retention time is about 1.33 min. Figure 2 shown.

[0137] The response of cortisol in the mobile phase (2) system is 600 cps, the response of the isotope internal standard cortisol-d6 is 1500 cps, and the retention time is 1.08 min. Figure 3 shown.

[0138] 7. Results Analysis

[0139] According to the response investigation of the target in the plasma sample, it was found that when acid was added to the system, the target response was reduced to about 1 / 3 of that of the salt system when the pH was too low. Therefore, choosing salt instead of acid as the additive can improve the sensitivity of the component.

[0140] 2. Effect of salt concentration in mobile phase on analytes

[0141] 1. Target

[0142] Taking Cortisol and Cortisol-d6 as an example

[0143] 2. Instruments and equipment

[0144] LCMS-4500MD liquid chromatography-tandem mass spectrometer, with triple quadrupole tandem mass spectrometer as detector; 1 / 10,000 electronic analytical balance; MiliQ water purifier; vortex mixer; low-temperature high-speed centrifuge; medical low-temperature storage box.

[0145] 3. Mobile phase

[0146] 0.5mM NH4Ac-H2O(A); MeOH(B)(1)

[0147] 2mM NH4Ac-H2O(A);MeOH(B)(2)

[0148] 4. Plasma sample pretreatment process

[0149] The blood sample is pre-treated to obtain a target sample solution: a blood sample containing cortisol to be tested is collected, an internal standard solution is added, vortexed, a precipitant is added, vortexed and mixed, centrifuged, a supernatant is taken, a buffer solution is added, and mixed to obtain a target sample solution.

[0150] 5. Sample testing

[0151] Mobile phase (1) and mobile phase (2) were selected respectively, and the target sample solution was tested using high performance liquid chromatography tandem mass spectrometry to examine the effect of increasing the salt concentration in the system on the response of the target.

[0152] 6. Test results

[0153] The effect of increasing the salt concentration in the system on the target compound is determined based on the response to the internal standard and the internal standard.

[0154] The response of cortisol in the mobile phase (1) system is 1500 cps, the response of the isotope internal standard cortisol-d6 is 4780 cps, and the retention time is 1.38 min. Figure 4 shown.

[0155] The response of cortisol in the mobile phase (2) system is 1200 cps, the response of the isotope internal standard cortisol-d6 is 5360 cps, and the retention time is 1.38 min. Figure 5 shown.

[0156] 7. Results Analysis

[0157] According to the response investigation of the target in the plasma sample, it was found that increasing the salt concentration in the system did not significantly improve the response, and too high a concentration of salt had an inhibitory effect on the response of the target, and the peak shape did not improve significantly. Therefore, a salt concentration of 0.5 mM was selected as the most preferred.

[0158] 3. Effect of salt type in mobile phase on analytes

[0159] 1. Target

[0160] Taking Cortisol and Cortisol-d6 as an example

[0161] 2. Instruments and equipment

[0162] LCMS-4500MD liquid chromatography-tandem mass spectrometer, with triple quadrupole tandem mass spectrometer as detector; 1 / 10,000 electronic analytical balance; MiliQ water purifier; vortex mixer; low-temperature high-speed centrifuge; medical low-temperature storage box.

[0163] 3. Mobile phase

[0164] 0.5mM NH4Ac-H2O(A); MeOH(B)(1)

[0165] 0.5mM NH4FA-H2O(A); MeOH(B)(2)

[0166] 4. Plasma sample pretreatment process

[0167] The blood sample is pre-treated to obtain a target sample solution: a blood sample containing cortisol to be tested is collected, an internal standard solution is added, vortexed, a precipitant is added, vortexed and mixed, centrifuged, a supernatant is taken, a buffer solution is added, and mixed to obtain a target sample solution.

[0168] 5. Sample testing

[0169] Mobile phase (1) and mobile phase (2) were selected respectively, and the target sample solution was tested using high performance liquid chromatography tandem mass spectrometry to examine the effect of increasing the salt concentration in the system on the response of the target.

[0170] 6. Test results

[0171] The effect of the salt type in the system on the response of the target was determined based on the response to the internal standard and the internal standard.

[0172] The response of cortisol in the mobile phase (1) system is 1480 cps, the response of the isotope internal standard cortisol-d6 is 7000 cps, and the retention time is 1.35 min. Figure 6 shown.

[0173] The response of cortisol in the mobile phase (2) system is 1140 cps, the response of the isotope internal standard cortisol-d6 is 4560 cps, and the retention time is 1.33 min. Figure 7 shown.

[0174] 7. Results Analysis

[0175] According to the response investigation of the target substances in the plasma samples, it was found that the response of ammonium acetate in the mobile phase was higher than that of ammonium formate, and there was no significant difference in the peak shape. Therefore, ammonium acetate was selected as the mobile phase additive.

[0176] 4. Effect of buffer in the system on analytes

[0177] 1. Target

[0178] Taking Cortisol and Cortisol-d6 as an example

[0179] 2. Instruments and equipment

[0180] LCMS-4500MD liquid chromatography-tandem mass spectrometer, with triple quadrupole tandem mass spectrometer as detector; 1 / 10,000 electronic analytical balance; MiliQ water purifier; vortex mixer; low-temperature high-speed centrifuge; medical low-temperature storage box.

[0181] 3. Mobile phase

[0182] 0.5mM NH4Ac-H2O(A);MeOH(B)

[0183] 4. Plasma sample pretreatment process

[0184] The blood sample was pretreated to obtain the target sample solution: the blood sample containing cortisol to be tested was collected, the internal standard solution was added, vortexed, a precipitant was added, vortexed and mixed, centrifuged, the supernatant was taken, a buffer solution (1 mmol / L ammonium acetate and 0.01% ammonia solution) was added, and the target sample solution 1 was obtained.

[0185] The blood sample is pre-treated to obtain the target sample solution: a blood sample containing cortisol to be tested is collected, an internal standard solution is added, vortexed, a precipitant is added, vortexed and mixed, centrifuged, the supernatant is taken, the aqueous solution is added, and mixed to obtain the target sample solution 2.

[0186] 5. Sample testing

[0187] The target sample solution 1 and the target sample solution 2 were tested using high performance liquid chromatography tandem mass spectrometry to examine the effect of the buffer in the system on the response of the target.

[0188] 6. Test results

[0189] The effect of the buffer in the system on the response of the target was determined based on the response to the internal standard and the internal standard.

[0190] The response of cortisol added to the buffer system is 1920 cps, the response of the isotope internal standard cortisol-d6 is 7600 cps, and the retention time is 1.31 min. Figure 8 shown.

[0191] The response of cortisol in the buffer-free system is 1200 cps, the response of the isotope internal standard cortisol-d6 is 4000 cps, and the retention time is 1.34 min. Fig. 9 shown.

[0192] 7. Results Analysis

[0193] According to the response investigation of the target in the plasma sample, it was found that after using a specific buffer in the system, the target response was significantly higher than that without the buffer, and there was no significant difference in the chromatographic peak shape, so it was necessary to add a buffer to the system. Adding a buffer to improve the response instead of choosing solid phase extraction concentration is more conducive to personnel operation and reproducibility, greatly reducing the cost of scientific research.

[0194] Obviously, the above embodiments are only examples for clearly explaining the present invention, and are not intended to limit the implementation methods of the present invention. Those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for simultaneously detecting five corticosteroids in blood samples. It is characterized in that The five corticosteroids are cortisol, cortisone, corticosterone, 21-deoxycortisol and 11-deoxycortisol; The method comprises the following steps: (1) pre-treating a blood sample to be tested to obtain a target sample solution; (2) using high performance liquid chromatography tandem mass spectrometry to detect the target sample solution to obtain the contents of five corticosteroids in the blood sample; The pretreatment of the blood sample to be tested specifically includes: collecting the blood sample to be tested, adding an internal standard solution, vortexing, adding a precipitant, vortexing and mixing, centrifuging, taking the supernatant, adding a buffer solution, and mixing to obtain a target sample solution; The precipitant is methanol, acetonitrile or a mixed solution of methanol and acetonitrile (the volume ratio of methanol to acetonitrile is 1:1-7:43); The buffer solution is an ammonia-ammonium acetate solution, in which the concentration of ammonium acetate is 0.5-1.5 mmol / L, the concentration of ammonia is 0.01%-0.02%, % is mass percentage, and the pH is 8.2-9.8; The conditions of the HPLC are: Chromatographic column: Waters, BEH C18 column; column temperature 40°C; injector temperature 4°C; gradient elution with mobile phase A and mobile phase B, the mobile phase A is 0.5mmol / L ammonium acetate aqueous solution, the mobile phase B is methanol, and the parameters of the gradient elution are: 0min, mobile phase B: 55%; 0-1.3min, mobile phase B: increased from 55% to 65%; 1.3-1.6min, mobile phase B: increased from 65% to 95%; 1.6-1.7min, mobile phase B: decreased from 95% to 55%; 1.7-2.5min, mobile phase B: 55%; flow rate is 0.3mL / min; injection volume is 2μL.

2. The method according to claim 1, Features: In step (2), the detection of the target sample solution by high performance liquid chromatography tandem mass spectrometry specifically includes: 1) preparing a standard curve working solution containing five kinds of corticosteroids with different known concentrations, and freeze-drying the standard curve working solution and a blank matrix according to the freeze-drying curve to prepare a calibration freeze-dried product, re-dissolving the calibration freeze-dried product to obtain a standard curve sample, and drawing a standard curve of the standard curve sample solution; The blank matrix is ​​a 4%-10% (mass volume percentage) bovine serum albumin solution; The reconstitution solution is a 0.01M-0.05M PBS aqueous solution; 2) Detecting the target sample solution by high performance liquid chromatography tandem mass spectrometry, and obtaining the contents of the five corticosteroids in the target sample solution according to the standard curve, thereby obtaining the contents of the five corticosteroids in the blood sample to be tested.

3. The method according to claim 2, Features: The steps of preparing a standard curve sample containing five corticosteroids of different known concentrations and drawing a standard curve of the standard curve sample solution specifically include: First, standard curve samples containing five kinds of corticosteroids with different known concentrations are prepared respectively, the internal standard solution is added, vortexed, the precipitant is added, vortexed and mixed, centrifuged, the supernatant is taken, the buffer is added, and mixed, so as to obtain a series of standard curve sample solutions containing five kinds of corticosteroids with different known concentrations; Secondly, the standard curve sample solution was detected by high performance liquid chromatography tandem mass spectrometry, the peak area ratios of the five corticosteroids and the internal standard were calculated, and the standard curve was established by the internal standard method.

4. The method according to claim 2, Features: The freeze-drying of the standard curve working solution and the blank matrix according to the freeze-drying curve specifically includes: a quick freezing stage: a duration of 3 hours at -50°C; a primary sublimation drying stage: a duration of 2 hours at -45°C, a duration of 2 hours at -40°C, a duration of 2 hours at -35°C, a duration of 3 hours at -30°C, a duration of 2 hours at -25°C, a duration of 1 hour at -20°C, a duration of 1 hour at -15°C, a duration of 1 hour at -10°C, and a duration of 1 hour at -5°C; a secondary sublimation drying stage: a duration of 1 hour at 0°C, a duration of 2 hours at 10°C, and a duration of 2 hours at 20°C.

5. The method according to claim 1 or 3, Features: The concentration of the internal standard solution is 50 ng / mL; The volume ratio of the blood sample to be tested or the standard curve sample to the internal standard solution is 20:1; The volume ratio of the blood sample to be tested or the standard curve sample to the buffer solution is 8:1-9:

1.

6. The method according to claim 1 or 3, Features: The volume ratio of the blood sample to be tested or the standard curve sample to the precipitant is 1:0.6-1:

3.

7. The method according to claim 1, Features: The mass spectrometry parameters are: ionization mode: ESI+; curtain gas (CUR): 35 psi; spray voltage (IS): 5500 V; temperature (TEM): 550° C.; nebulizer gas (Gas 1): 50 psi; auxiliary heating gas (Gas 2): 50 psi; and multiple reaction monitoring (MRM).

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