Method for detecting aldosterone, cortisol, deoxycorticosterone and cortisone in plasma by magnetic bead assisted LC-MS / MS

Abstract

The invention relates to the technical field of medical detection, in particular to a method for detecting aldosterone, cortisol, deoxycorticosterone and cortisone in plasma through LC-MS / MS. The method comprises the following steps: (a) mixing a plasma sample with an internal standard solution to obtain a mixed solution; (b) adding the mixed solution into the magnetic bead suspension, sequentially carrying out adsorption, weak washing, strong washing and elution, and collecting an eluent; (c) carrying out liquid nitrogen blowing and redissolving on the eluent, and then carrying out LC-MS / MS detection; wherein the surfaces of the magnetic beads are modified with steroid adsorption groups, and the target detection objects are aldosterone, cortisol, deoxycorticosterone and cortisone. The detection method is convenient and simple, the detection efficiency is higher than that of a traditional method, and a feasible solution is provided for promoting popularization and application of the LC-MS / MS technology in clinical practice.

Description

Technical Field

[0001] This invention relates to the field of medical testing technology, and in particular to a method for detecting aldosterone, cortisol, deoxycorticosterone, and cortisol in plasma using magnetic bead-assisted LC-MS / MS. Background Technology

[0002] Primary aldosteronism (PA) is a common and etiologically known secondary hypertension in hypertensive individuals. PA is caused by excessive autonomous secretion of aldosterone by the adrenal cortex, significantly increasing the risk of damage to target organs such as the heart, brain, and kidneys, leading to serious complications and severely impacting patient prognosis and quality of life. However, with early diagnosis and intervention (pharmacological or surgical treatment), PA patients can achieve good blood pressure control; therefore, PA screening in hypertensive populations has significant clinical value.

[0003] The diagnosis of PA involves three stages: screening, confirmation, and subtyping. Adrenal venous sampling (AVS) is the gold standard for subtyping diagnosis, providing crucial information for individualized treatment plans by measuring aldosterone levels in both adrenal veins. Besides aldosterone, the metabolic profiles of other steroid hormones such as deoxycorticosterone, cortisol, and cortisol also differ in PA patients. Excessive secretion of deoxycorticosterone can lead to hypertension and hypokalemia, while the detection of cortisol and cortisol levels helps differentiate hyporenin hypertension caused by excess aldosterone, cortisol, deoxycorticosterone, and cortisol, such as primary glucocorticoid resistance and Cushing's syndrome. Therefore, accurate analysis of the differences in steroid hormone profiles not only helps deepen the understanding of PA pathogenesis but may also provide new research directions for PA subtype classification.

[0004] In terms of detection technologies, commonly used clinical methods include radioimmunoassay (RIA), chemiluminescent immunoassay (CLIA), and liquid chromatography-tandem mass spectrometry (LC-MS / MS). RIA carries the risk of radioactive contamination, while CLIA, although avoiding radioactive hazards, suffers from insufficient specificity and sensitivity. LC-MS / MS, currently considered the "gold standard" for hormone detection, offers high specificity and sensitivity and effectively avoids cross-reactivity; however, its traditional pretreatment methods are complex, limiting its widespread clinical application. Summary of the Invention

[0005] The purpose of this invention is to establish a magnetic bead-assisted LC-MS / MS method for the simultaneous detection of aldosterone, cortisol, deoxycorticosterone, and cortisol in plasma. This detection method is convenient, simple, and more efficient than traditional methods, providing a feasible solution for promoting the widespread application of LC-MS / MS technology in clinical practice.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A method for simultaneous detection of aldosterone, cortisol, deoxycorticosterone, and cortisol in plasma using magnetic bead-assisted LC-MS / MS includes the following steps: (a) Mix the plasma sample with the internal standard solution to obtain a mixture; (b) Add the mixture to the magnetic bead suspension and perform adsorption, weak washing with deionized water, strong washing with 20% methanol aqueous solution and elution in sequence, and collect the eluent; the elution is performed with methanol:isopropanol = 1:1 (v / v); (c) After nitrogen blowing and reconstitution of the eluent, LC-MS / MS analysis was performed. Mobile phase A: aqueous solution containing 2 mM ammonium acetate, mobile phase B: methanol solution containing 2 mM ammonium acetate; The magnetic beads are magnetic beads with surface-modified steroid adsorption groups, and the target analytes are aldosterone, cortisol, deoxycorticosterone, and cortisone.

[0007] Preferably, the chromatographic conditions for LC-MS / MS detection are as follows: Chromatographic column: DISIGNS column-004, 50mm × 2.1mm; Gradient elution procedure: 0-0.6 min: Mobile phase B is maintained at 30%; 0.6–2.0 min: Mobile phase B increases from 30% to 60%; 2.0-2.9 min: Mobile phase B maintained at 60%; 2.9–2.95 min: Mobile phase B increases from 60% to 95%; 2.95-3.95 min: Mobile phase B is maintained at 95%.

[0008] Preferably, the magnetic bead is an MSi050 / Steroid magnetic bead.

[0009] Preferably, step (b) specifically includes: (i) Activate the magnetic beads by mixing them with a 40% methanol aqueous solution; (ii) Add 20% methanol aqueous solution to equilibrate; (iii) Load the sample mixture and allow it to adsorb for 5 min; (iv) Wash gently with deionized water and then wash strongly with 20% methanol aqueous solution in sequence; (v) Elute with elution buffer and collect the elution buffer.

[0010] Preferably, the gradient elution program is set to a flow rate of 0.5 mL / min.

[0011] Preferably, the internal standard is an isotope label, including: aldosterone-d8, cortisol-d4, deoxycorticosterone-d8, and cortisone-d8.

[0012] Preferably, the limits of quantitation for the method are: aldosterone ≤ 5 pg / mL, deoxycorticosterone ≤ 10 pg / mL, cortisone ≤ 0.5 ng / mL, and cortisol ≤ 1.1 μg / mL.

[0013] The present invention also provides applications of the above method in diagnostic products for primary aldosteronism, such as diagnostic kits.

[0014] This invention also provides a diagnostic kit for primary aldosteronism, comprising: Magnetic bead solution: A mixture of MSi050 / Steroid magnetic beads and a 40% methanol aqueous solution; Internal standard solutions: aldosterone-d8, cortisol-d4, deoxycorticosterone-d8, cortisone-d8; Equilibrium solution: 20% methanol aqueous solution; Mild cleaning solution: Deionized water; Strong cleaning solution: 20% methanol aqueous solution; Eluent: A mixture of methanol and isopropanol in a volume ratio of 1:1.

[0015] Preferably, the above-mentioned kit also includes: Calibration solution: containing gradient concentrations of aldosterone, cortisol, deoxycorticosterone, and cortisone; Quality control solution: contains low, medium and high concentrations of aldosterone, cortisol, deoxycorticosterone and cortisone.

[0016] In the calibrator solutions, the concentrations ranged from 17.20 to 3790.00 pg / mL, cortisol from 1.85 to 369.77 μg / mL, deoxycorticosterone from 80.30 to 17140.00 pg / mL, and cortisone from 0.96 to 191.16 ng / mL. For example, the concentrations for aldosterone were 17.20 pg / mL, 37.20 pg / mL, 95.20 pg / mL, 188.00 pg / mL, 920.00 pg / mL, and 3790.00 pg / mL; for cortisol, the concentrations were 1.85 μg / mL, 4.02 μg / mL, 10.13 μg / mL, 20.07 μg / mL, 95.81 μg / mL, and 369.77 μg / mL; for deoxycorticosterone, the concentrations were 80.30 pg / mL, 163.00 pg / mL, 395.00 pg / mL, 768.00 pg / mL, 4196.00 pg / mL, and 17140.00 pg / mL; and for cortisone, the concentrations were 0.96 ng / mL, 2.05 ng / mL, and 5.12 ng / mL. ng / mL, 10.01ng / mL, 47.44 ng / mL and 191.16 ng / mL.

[0017] By implementing the above technical solution, the present invention has the following beneficial effects: This invention provides a simple and convenient method for the simultaneous detection of aldosterone, cortisol, deoxycorticosterone, and cortisol in plasma. This automated extraction method performs comparably to traditional SLE in key parameters such as linear range, limit of quantitation, accuracy, and precision, while significantly improving operational efficiency. Analysis of AVS patient samples, confirmed by Passing-Bablok regression and Bland-Altman consistency tests, demonstrates good agreement between the magnetic bead-assisted extraction method and the traditional SLE method, confirming the clinical feasibility of this method in the diagnosis of primary aldosteronism and potentially promoting the wider application of LC-MS / MS technology in clinical testing. (See attached figures.) Figure 1 This represents a linear regression for aldosterone; Figure 2 This represents a linear regression of cortisol. Figure 3 This represents a linear regression of deoxycorticosterone. Figure 4 This is a representative linear regression of cortisol; Figure 5 Representative chromatograms of aldosterone were analyzed using the automated magnetic bead-assisted liquid chromatography-tandem mass spectrometry (LC-MS / MS) method of this invention. Figure 6 To analyze a representative chromatogram of aldosterone-d8 using the automated magnetic bead-assisted liquid chromatography-tandem mass spectrometry (LC-MS / MS) method of this invention; Figure 7 To analyze representative chromatograms of cortisol using the automated magnetic bead-assisted liquid chromatography-tandem mass spectrometry (LC-MS / MS) method of this invention; Figure 8 To analyze a representative chromatogram of cortisol-d4 using the automated magnetic bead-assisted liquid chromatography-tandem mass spectrometry (LC-MS / MS) method of this invention; Figure 9 A representative chromatogram of deoxycorticosterone was analyzed using the automated magnetic bead-assisted liquid chromatography-tandem mass spectrometry (LC-MS / MS) method of this invention; Figure 10 To analyze a representative chromatogram of deoxycorticosterone-d8 using the automated magnetic bead-assisted liquid chromatography-tandem mass spectrometry (LC-MS / MS) method of this invention; Figure 11 To analyze representative chromatograms of cortisone using the automated magnetic bead-assisted liquid chromatography-tandem mass spectrometry (LC-MS / MS) method of this invention; Figure 12 To analyze the representative chromatogram of cortisone-d8 using the automated magnetic bead-assisted liquid chromatography-tandem mass spectrometry (LC-MS / MS) method of this invention; Figure 13 Passing-Bablok regression analysis plot for the detection of aldosterone using SLE and magnetic bead-assisted extraction methods; Figure 14 Bland-Altman plot for the detection of aldosterone using SLE and magnetic bead-assisted extraction methods; Figure 15 Passing-Bablok regression analysis plot for the detection of cortisol using SLE and magnetic bead-assisted extraction methods; Figure 16 Bland-Altman plot for the detection of cortisol using SLE and magnetic bead-assisted extraction method; Figure 17 Passing-Bablok regression analysis plot for the detection of deoxycorticosterone using SLE and magnetic bead-assisted extraction methods; Figure 18 Bland-Altman plot for the detection of deoxycorticosterone using SLE and magnetic bead-assisted extraction methods; Figure 19 Passing-Bablok regression analysis plot for detecting cortisone using SLE and magnetic bead-assisted extraction methods; Figure 20To detect the Bland-Altman plot of cortisone using SLE and magnetic bead-assisted extraction methods; Figure 21 This is a comparison chart of the internal standard peak areas of aldosterone under the detection methods of the examples and comparative examples; Figure 22 This is a comparison chart of the internal standard peak areas of cortisol under the detection methods of the examples and comparative examples; Figure 23 This is a comparison chart of the internal standard peak areas of deoxycorticosterone under the detection methods of the examples and comparative examples; Figure 24 This is a comparison chart of the internal standard peak area of ​​cortisone under the detection methods of the embodiment and comparative examples. Detailed Implementation

[0018] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0019] The reagents and equipment used in the specific embodiments of this invention can all be purchased commercially.

[0020] 1. Chemicals and Reagents: Standards of aldosterone, cortisol, deoxycorticosterone, and cortisone, and their isotope-labeled internal standards (ISs), were purchased from Zhejiang Disai Diagnostics Technology Co., Ltd. (Hangzhou, China). MSi050 / Steroid magnetic beads were purchased from Yingruicheng Biochemical Technology Co., Ltd. (Shanghai, China). High-performance liquid chromatography grade methanol and isopropanol solutions were purchased from Thermo Fisher Scientific (Fairlawn, New Jersey, USA). Deionized water was purchased from Watson Group (Hong Kong, China).

[0021] 2. Main instruments and equipment Ultra-high performance liquid chromatograph: Zhejiang CALIBRA CalQuant-S, Zhejiang Kailai Spectrum Technology Co., Ltd.

[0022] Triple quadrupole tandem mass spectrometer: Zhejiang CALIBRA CalQuant-S, Zhejiang CALIBRA Spectrometer Technology Co., Ltd.

[0023] Sample pretreatment system: Sansure Biotech Natch 96, Hunan Shengxiang Biotechnology Co., Ltd.

[0024] Example 1: Determination of the concentrations of aldosterone, cortisol, deoxycorticosterone, and cortisol in plasma (I) Solution Preparation Aldosterone, cortisol, deoxycorticosterone and cortisone standard solutions and internal standard working solutions were all purchased commercially available. Conditioning solution: Take MSi050 / Steroid magnetic beads and add 40% methanol aqueous solution to prepare magnetic bead solution; Equilibrium solution: Take methanol, add water to prepare a 20% methanol aqueous solution, and use it as the equilibrium solution; Mild cleaning solution: Use deionized water as the mild cleaning solution; Strong cleaning solution: Take methanol, add water to prepare a 20% methanol aqueous solution, and use it as a strong cleaning solution; Eluent: Use methanol and isopropanol (1:1) as the eluent; (II) Establishment of standard curves for aldosterone-d8, cortisol-d4, deoxycorticosterone-d8, and cortisone-d8 a. Preparation of standard curve and quality control solution: Six standard solutions of different concentrations were used to prepare a series of concentration standard curves. For aldosterone, the concentrations were 17.20 pg / mL, 37.20 pg / mL, 95.20 pg / mL, 188.00 pg / mL, 920.00 pg / mL, and 3790.00 pg / mL; for cortisol, the concentrations were 1.85 μg / mL, 4.02 μg / mL, 10.13 μg / mL, 20.07 μg / mL, 95.81 μg / mL, and 369.77 μg / mL; and for deoxycorticosterone, the concentrations were 80.30 pg / mL, 163.00 pg / mL, 395.00 pg / mL, 768.00 pg / mL, 4196.00 pg / mL, and 17140.00 pg / mL. pg / mL; for cortisone, the concentrations were 0.96 ng / mL, 2.05 ng / mL, 5.12 ng / mL, 10.01 ng / mL, 47.44 ng / mL and 191.16 ng / mL, respectively.

[0025] Take 300 μL of each series of concentration standard curve solutions, add 80 μL of internal standard working solution and mix. Load the mixture into a Sansure Biotech Natch 96 sample pretreatment system for high-throughput magnetic extraction. Collect the eluent after extraction, dry it under nitrogen for 20 minutes, redissolve it with 200 μL of 40% methanol, vortex for 3 minutes, centrifuge for 10 minutes and collect the liquid as the series of concentration standard curve working solutions.

[0026] The operating procedure of the Sansure Biotech Natch 96 sample pretreatment system is as follows: Mix 100 μL of magnetic beads with 150 μL of conditioning solution for 1 min, add 500 μL of equilibration solution and mix for 1 min, load 380 μL of the mixture, adsorb for 5 mins, add 500 μL of weak washing solution for 1 min, add 500 μL of strong washing solution for 1 min, add 300 μL of elution buffer for elution, and collect the eluent. b. Inject the working solutions of the series of concentration standard curves into the LC-MS / MS instrument and measure the peak area. Plot the concentrations of aldosterone-d8, cortisol-d4, deoxycorticosterone-d8, and cortisone-d8 of the working solutions of the series of concentration standard curves as the abscissa (X). Correspondingly, plot the peak area ratio of aldosterone-d8, cortisol-d4, deoxycorticosterone-d8, and cortisone-d8 to the internal standard as the ordinate (Y). Perform regression calculation using the weighted least squares method (W = 1 / X2) to obtain the linear regression equation, which is the standard curve of aldosterone-d8, cortisol-d4, deoxycorticosterone-d8, and cortisone-d8.

[0027] Chromatographic conditions are shown in Table 1, and gradient elution programs are shown in Table 2.

[0028]

[0029]

[0030] Mass spectrometry conditions: Ion source parameters are shown in Table 3, and compound parameters are shown in Table 4.

[0031]

[0032]

[0033] (III) Determination of aldosterone, cortisol, deoxycorticosterone and cortisol content in the sample to be tested c. Preparation of biological samples and quality control samples Take 300 μL of plasma sample into a 96-well plate, add 80 μL of internal standard solution and mix. Load the mixture into the Sansure Biotech Natch 96 sample pretreatment system and perform high-throughput magnetic extraction according to the same procedure as step a). Collect the eluent, reconstitute and centrifuge to obtain the biological sample. Take 300 μL of each of the following quality control working solutions: aldosterone, cortisol, deoxycorticosterone and cortisone into a 96-well plate, add 80 μL of internal standard solution and mix. Load the mixture into a Sansure Biotech Natch 96 sample pretreatment system and perform high-throughput magnetic extraction according to the same procedure as step a). Collect the eluent, reconstitute and centrifuge to obtain the quality control sample. d. Determination of biological samples Take 25 μL of quality control sample and biological sample respectively, inject them into LC-MS / MS instrument for detection, and detect them under the same conditions as in step b. Use the quality control sample to monitor whether the instrument responds to aldosterone-d8 and aldosterone, cortisol-d4 and cortisol, deoxycorticosterone-d8 and deoxycorticosterone, and cortisone-d8 and cortisone consistently. Obtain the content of aldosterone, cortisol, deoxycorticosterone and cortisone in the biological sample according to the standard curve in step (ii).

[0034] Example 2: Verification of the method shown in Example 1 1. The chromatographic conditions, gradient elution program, and mass spectrometry conditions are the same as in Example 1.

[0035] 2. Preparation of working solution and sample 2.1 Preparation of standard solutions, quality control solutions, and internal standard working solutions for aldosterone, cortisol, deoxycorticosterone, and cortisone. Aldosterone, cortisol, deoxycorticosterone and cortisone standard solutions, quality control solutions and internal standard working solutions were all purchased from Zhejiang Disais Diagnostics Technology Co., Ltd.

[0036] 2.1.1 Standard solutions of aldosterone, cortisol, deoxycorticosterone, and cortisone A series of concentration standard curve solutions were prepared using six standard solutions of different concentrations. For aldosterone, the concentrations were 17.20 pg / mL, 37.20 pg / mL, 95.20 pg / mL, 188.00 pg / mL, 920.00 pg / mL, and 3790.00 pg / mL; for cortisol, the concentrations were 1.85 μg / mL, 4.02 μg / mL, 10.13 μg / mL, 20.07 μg / mL, 95.81 μg / mL, and 369.77 μg / mL; for deoxycorticosterone, the concentrations were 80.30 pg / mL, 163.00 pg / mL, 395.00 pg / mL, 768.00 pg / mL, 4196.00 pg / mL, and 17140.00 pg / mL; and for cortisone, the concentrations were 0.96 ng / mL, 2.05 ng / mL, and 2.05 ng / mL. ng / mL, 5.12 ng / mL, 10.01 ng / mL, 47.44 ng / mL and 191.16 ng / mL.

[0037] The standard solution should be stored at 2–8°C.

[0038] 2.1.2 Quality control solutions for aldosterone, cortisol, deoxycorticosterone, and cortisone The quality control solutions were prepared in two concentrations: 199.00 pg / mL and 1168.00 pg / mL for aldosterone; 39.12 μg / mL and 221.30 μg / mL for cortisol; 191.00 pg / mL and 1216.00 pg / mL for deoxycorticosterone; and 5.01 ng / mL and 27.66 ng / mL for cortisone. The quality control solutions were stored at 2–8 °C.

[0039] 2.1.3 Internal Standard (IS) Working Solution The internal standard working solution was obtained by purchase, and the stock solution was stored at 2–8°C.

[0040] 3. Sample processing (1) All matrix samples were thawed at 10–30°C; (2) Add samples to the Sansure Biotech Natch 96 sample pretreatment system according to Table 5 below:

[0041] *Magnetic bead type: Steroid purification microspheres MSi050 / Steroid. Source: Yingruicheng Biochemical Technology Co., Ltd.

[0042] (3) Set up the sample pretreatment system according to the procedure in Table 6 below*:

[0043] (4) Place the 96-well plate treated by the sample pretreatment system on the magnetic plate, take 25 μL of the supernatant from well 6, dry it under nitrogen for 20 minutes, redissolve it with 200 μL of 40% methanol, vortex for 3 minutes, centrifuge for 10 minutes and collect the liquid in the injection plate for analysis.

[0044] 4. Methodological Validation Results 4.1 Linear range and LOQ of the standard curve Calibration curves were generated using six different concentrations of calibrators, and linearity was evaluated by analyzing five replicates for each concentration. Calibration curves were generated using six different concentrations of non-zero calibration standards by plotting the peak area ratio of the analyte to the internal standard (Y) against the concentration (X).

[0045] The LOQ was defined as the lowest concentration at which the signal-to-noise ratio (S / N) was greater than 10, the precision deviation was less than 15%, and the accuracy CV was less than 20%. The LOQ of this method was determined by selecting four low-concentration samples, each sample being processed in five replicates, with each replicate measured three times. Analysis was performed in three consecutive batches. The overall precision for each concentration level was evaluated, and the deviation of the measured mean concentration from the theoretical concentration was assessed.

[0046] The results showed that aldosterone exhibited good linearity in the range of 17.20–3790.00 pg / mL; cortisol showed good linearity in the range of 1.85–369.77 μg / mL; deoxycorticosterone showed good linearity in the range of 80.30–17140.00 pg / mL; and cortisone showed good linearity in the range of 0.96–191.16 ng / mL, essentially covering the concentration range of clinical samples. Representative linear regression results are shown below. Figure 1-4 The final determined LOQ values ​​were: aldosterone 4.09 pg / mL, cortisol 1.08 μg / mL, deoxycorticosterone 8.2 pg / mL, and cortisone 0.45 ng / mL. Detailed results for the four analytes are shown in Table 7.

[0047]

[0048] 4.2 Precision and Accuracy Precision was assessed using quality control (QC) samples at three concentration levels (low, medium, and high). Each concentration level was divided into five replicates and measured in three consecutive batches to evaluate intra-batch precision, inter-batch precision, and overall precision. A coefficient of variation (CV) ≤15% was considered acceptable.

[0049] Spiking experiments were conducted using standards at low, medium, and high concentrations in a mixed sample of known concentrations. Relative recovery and accuracy were evaluated through three repeated measurements. A tolerance of ±15% was set, and recoveries were required to be within the range of 85% to 115%. Accuracy and relative recovery were assessed by comparing the measured concentrations with the theoretical concentrations.

[0050] The results showed that the magnetic bead extraction method had good reproducibility, with intra-assay CVs of 1.99% to 4.46%, 1.41% to 3.50%, 1.54% to 3.41%, and 2.08% to 4.27% for aldosterone, cortisol, deoxycorticosterone, and cortisone, respectively. The total CVs for these analytes were 1.87% to 5.22%, 1.66% to 3.38%, 1.87% to 6.29%, and 2.41% to 14.07%, respectively, comparable to the performance of the conventional SLE method (see Table 8 for details). The relative recoveries of aldosterone were 87.79% to 97.92%, cortisol 85.69% to 114.37%, deoxycorticosterone / cortisone 85.54% to 112.89%, and cortisone 90.58% to 112.27%. All deviations were within 15%, meeting the accuracy acceptance criteria (see Table 9 for details).

[0051]

[0052]

[0053] 4.3 Matrix effect In this study, matrix effects were assessed using a post-addition method after sample processing. Plasma samples were collected from five individuals, each divided into two aliquots. After processing the plasma samples using a magnetic bead-assisted extraction method, a mixture of high- and low-concentration standard solutions was added to the extracted plasma samples to measure matrix effects. Matrix effects were assessed by comparing the absolute response (peak area) and relative response (analyte peak area / internal standard peak area ratio) of the standards in the solvent with the response values ​​in the extracted plasma samples. The ability of the isotope-labeled internal standard to compensate for potential matrix effects was evaluated, with an acceptable range set at 80% to 120%.

[0054] The results showed that the matrix factors (MF) of aldosterone and cortisone ranged from 85.37% to 103.8% and 99.38% to 119.31%, respectively (see Table 10), both within the range of 80% to 120%. Without internal standard correction, the absolute responses of deoxycorticosterone and cortisol exhibited ion suppression. However, by introducing an internal standard, the matrix effect was effectively compensated, with calculated matrix effects of 80.23% to 97.34% and 98.88% to 118.87%, respectively. The absolute responses of the analytes indicate that ion suppression effects exist in some samples without internal standard correction. However, internal standard correction can compensate for the observed matrix effect.

[0055]

[0056] 4.4 Method Comparison In this study, 40 adrenal vein samples collected from Zhejiang Provincial People's Hospital between August 2024 and January 2025 were analyzed comparatively to evaluate the clinical application value of magnetic bead-assisted extraction (SLE). Plasma samples were collected and stored at -80°C until analysis. Before measurement, plasma samples were thawed to room temperature and analyzed using both SLE and magnetic bead-assisted extraction methods. Method comparisons were evaluated using Passing-Bablok regression and Bland-Altman plots. P < 0.05 was considered statistically significant.

[0057] The results showed that the 95% confidence interval (CI) for the slope included 1, while the 95% CI for the intercept included 0, indicating comparability between the two methods. Furthermore, the Bland-Altman analysis showed good agreement among all four analytes, with over 95% of the differences within the agreement limit (±1.96 SD). The Passing-Bablok regression and Bland-Altman plots for the four analytes are shown below. Figure 13-20 .

[0058] 5. Conclusion The present invention provides a highly specific, accurate, and reproducible method for quantifying aldosterone, cortisol, deoxycorticosterone, and cortisol in plasma.

[0059] In summary, the method of this invention is simple to operate and can simultaneously detect aldosterone, cortisol, deoxycorticosterone, and cortisol in plasma. This automated extraction method performs comparably to traditional SLE in key parameters such as linear range, limit of quantitation, accuracy, and precision, while significantly improving operational efficiency. Analysis of samples from 40 AVS patients, confirmed by Passing-Bablok regression and Bland-Altman consistency tests, showed good agreement between the magnetic bead-assisted extraction method and the traditional SLE method, demonstrating the clinical feasibility of this method in the diagnosis of primary aldosteronism and potentially promoting the wider application of LC-MS / MS technology in clinical testing.

[0060] Comparison Examples In this comparative example, the mobile phases were replaced as follows: mobile phase A was an aqueous solution containing 1 mM ammonium fluoride, and mobile phase B was a methanol solution containing 1 mM ammonium fluoride (containing 5% isopropanol). All other conditions were the same as in the examples. The chromatographic conditions for the examples and comparative examples are shown in Tables 11 and 12.

[0061]

[0062]

[0063] The samples consisted of six standard solutions of different concentrations, as well as low and high quality control values. The concentrations of the six standard solutions for the four substances are as follows: For aldosterone, the concentrations are 17.20 pg / mL, 37.20 pg / mL, 95.20 pg / mL, 188.00 pg / mL, 920.00 pg / mL, and 3790.00 pg / mL; for cortisol, the concentrations are 1.85 μg / mL, 4.02 μg / mL, 10.13 μg / mL, 20.07 μg / mL, 95.81 μg / mL, and 369.77 μg / mL; for deoxycorticosterone, the concentrations are 80.30 pg / mL, 163.00 pg / mL, 395.00 pg / mL, 768.00 pg / mL, 4196.00 pg / mL, and 17140.00 pg / mL; and for cortisone, the concentrations are 0.96 ng / mL, 2.05 ng / mL, and 2.05 ng / mL. The concentrations of aldosterone were 199.00 pg / mL, 5.12 ng / mL, 10.01 ng / mL, 47.44 ng / mL, and 191.16 ng / mL. The lower and higher control values ​​for the four substances were as follows: for aldosterone, the concentrations were 199.00 pg / mL and 1168.00 pg / mL; for cortisol, the concentrations were 39.12 μg / mL and 221.30 μg / mL; for deoxycorticosterone, the concentrations were 191.00 pg / mL and 1216.00 pg / mL; and for cortisone, the concentrations were 5.01 ng / mL and 27.66 ng / mL.

[0064] The internal standard peak areas of the examples and comparative examples were compared, and the results are shown in [link to relevant documentation]. Figure 21-24 The results show that, among the four substances, the internal standard peak area response of the mobile phase containing ammonium acetate used in the examples is higher than that of the mobile phase containing ammonium fluoride used in the comparative examples.

Claims

1. A method for simultaneous detection of aldosterone, cortisol, deoxycorticosterone, and cortisol in plasma using magnetic bead-assisted LC-MS / MS, characterized in that, Includes the following steps: (a) The plasma sample was mixed with the internal standard solution to obtain a mixture; (b) The mixture was added to the magnetic bead suspension and subjected to adsorption, weak washing with deionized water, strong washing with 20% methanol aqueous solution, and elution in sequence, and the eluent was collected; the elution was performed using methanol:isopropanol = 1:1 (v / v); (c) The eluent was purged with nitrogen and reconstituted, and then detected by LC-MS / MS. Mobile phase A: aqueous solution containing 2 mM ammonium acetate, mobile phase B: methanol solution containing 2 mM ammonium acetate; the magnetic beads were magnetic beads with surface-modified steroid adsorption groups, and the target analytes were aldosterone, cortisol, deoxycorticosterone, and cortisone.

2. The method according to claim 1, characterized in that, The chromatographic conditions for LC-MS / MS detection are as follows: Chromatographic column: DISIGNS column-004, 50mm × 2.1mm; Gradient elution procedure: 0-0.6 min: Mobile phase B is maintained at 30%; 6-2.0 min: Mobile phase B increases from 30% to 60%; 0-2.9 min: Mobile phase B is maintained at 60%; 9-2.95 min: Mobile phase B increases from 60% to 95%; 95-3.95 min: Mobile phase B is maintained at 95%.

3. The method according to claim 2, characterized in that, Step (b) specifically includes: (i) activating the magnetic beads by mixing them with a 40% methanol aqueous solution; (ii) equilibrating the mixture by adding a 20% methanol aqueous solution; (iii) loading the mixture onto the sample and allowing it to adsorb for 5 min; (iv) washing the mixture weakly with deionized water and then strongly with a 20% methanol aqueous solution; and (v) eluting with an eluent and collecting the eluent.

4. The method according to claim 1, characterized in that, The gradient elution program was set to a flow rate of 0.5 mL / min.

5. The method according to claim 1, characterized in that, The internal standard is an isotope label, including: aldosterone-d8, cortisol-d4, deoxycorticosterone-d8, and cortisone-d8.

6. The method according to claim 1, characterized in that, The limits of quantitation for the method are: aldosterone ≤ 5 pg / mL, deoxycorticosterone ≤ 10 pg / mL, cortisone ≤ 0.5 ng / mL, and cortisol ≤ 1.1 μg / mL.

7. The application of the method as described in any one of claims 1-6, characterized in that, In diagnostic products used for primary aldosteronism.

8. A diagnostic kit for primary aldosteronism, characterized in that, include: Magnetic bead solution: A mixture of MSi050 / Steroid magnetic beads and a 40% methanol aqueous solution; Internal standard solutions: aldosterone-d8, cortisol-d4, deoxycorticosterone-d8, cortisone-d8; Equilibrium solution: 20% methanol aqueous solution; Mild cleaning solution: Deionized water; Strong cleaning solution: 20% methanol aqueous solution; Eluent: A mixture of methanol and isopropanol in a volume ratio of 1:

1.

9. The reagent kit according to claim 8, characterized in that, Also includes: Calibration solution: containing gradient concentrations of aldosterone, cortisol, deoxycorticosterone, and cortisone; Quality control solution: contains low, medium and high concentrations of aldosterone, cortisol, deoxycorticosterone and cortisone.

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