Method for determining the cortisol content and use thereof

Abstract

This invention relates to the field of in vitro diagnostic technology, and more particularly to a method for determining cortisol levels and its application. This invention provides a pretreatment reagent combination for liquid chromatography, a method for pretreating urine samples in liquid chromatography, and a method for detecting cortisol levels in urine. The method for detecting cortisol levels in urine samples provided by this invention offers convenient and rapid pretreatment, and through method validation, demonstrates excellent performance indicators.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic technology, and in particular to a method for determining cortisol levels and its application. Background Technology

[0002] Cortisol, also known as hydrocortisone, is a glucocorticoid produced by the adrenal glands in response to stress. Its main functions include regulating carbohydrate metabolism and the distribution of electrolytes and water. Cortisol also has immunosuppressive and anti-inflammatory effects. In a normal human body, cortisol secretion is ultimately controlled by the central nervous system.

[0003] Changes in plasma cortisol levels are generally associated with abnormal ACTH levels, depression, and psychological stress, as well as stress factors such as hypoglycemia, illness, fever, trauma, surgery, pain, strenuous physical activity, and extreme temperatures. Pregnancy and estrogen therapy can significantly increase cortisol levels, and other stimuli, such as severe stress, can also increase cortisol secretion. Elevated cortisol is seen in: 1. Pregnancy, and women taking oral estrogen or contraceptives, as increased cortisol steroid-binding globulin can lead to elevated cortisol levels; 2. Patients with functional adrenal disorders and Cushing's syndrome, where serum cortisol is significantly elevated, with a loss of diurnal rhythm and no significant decrease in the afternoon and evening; 3. Patients with ACTH tumors and anterior pituitary hyperfunction are believed to have elevated serum cortisol; 4. Various stress states, such as trauma, surgery, cold, and myocardial infarction, can temporarily increase cortisol levels. Decreased cortisol is seen in: 1. Primary or secondary adrenal insufficiency, such as Addison's disease, adrenal tuberculosis, and adrenalectomy; 2. Anterior pituitary hypofunction, etc. Therefore, cortisol testing can directly detect the state of adrenal function and indirectly observe the state of pituitary function.

[0004] Because serum cortisol concentration exhibits a rhythmic variation, measuring free cortisol in 24-hour urine is the optimal indicator of adrenal function and the preferred test for diagnosing Cushing's syndrome. Cortisol levels can directly monitor adrenal function and indirectly observe pituitary function. For quantitative cortisol detection, immunologically based kits such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and chemiluminescence immunoassay (CLIA) are available on the market. RIA offers advantages such as high sensitivity and specificity, but its short reagent half-life, high cost, and waste disposal issues limit its application to some extent. ELISA and CLIA are widely used clinically due to their rapid and convenient detection methods.

[0005] Cortisol is a small-molecule steroid hormone with various isomers and structural analogs, making it prone to cross-reactivity issues in immunoassays. Gas chromatography-mass spectrometry (GC-MS or GC-MS / MS) and liquid chromatography-mass spectrometry (LC-MS / MS) are rapidly developing chemical analysis methods that can overcome cross-reactivity problems associated with analogs. However, the time and cost of sample pretreatment in these methods limit their clinical application. Furthermore, most techniques for detecting cortisol based on LC-MS / MS platforms primarily use plasma or serum samples, with few reports on its use in detecting cortisol in human urine samples. Therefore, developing a method suitable for detecting cortisol levels in urine has significant practical value. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is a method for determining cortisol content and its application. The present invention is an accurate quantitative technique based on an isotope dilution liquid chromatography-tandem mass spectrometry platform, combined with the bracket method, for accurately determining the concentration level of cortisol in human urine samples.

[0007] This invention provides a pretreatment reagent combination for liquid chromatography, comprising solution A, solution B and solution C; wherein, solution A is an aqueous solution of ammonia and methanol, solution B is an aqueous solution of formic acid and methanol, and solution C is an aqueous solution of acetonitrile and methanol.

[0008] In some embodiments,

[0009] The volume fraction of ammonia in the ammonia-methanol aqueous solution is 0.05%~1%, the volume fraction of methanol is 5%~40%, and the remainder is water;

[0010] The formic acid-methanol aqueous solution has a volume fraction of 0.05% to 1%, a volume fraction of methanol of 5% to 40%, and the remainder is water;

[0011] The acetonitrile-methanol solution contains 60% to 90% acetonitrile by volume and 10% to 40% methanol by volume.

[0012] In some specific embodiments,

[0013] In solution A, the volume fraction of ammonia is 0.1%, the volume fraction of methanol is 35%, and the remainder is water;

[0014] In solution B, the volume fraction of formic acid is 0.1%, the volume fraction of methanol is 35%, and the remainder is water;

[0015] In solution C, the volume fraction of acetonitrile is 85% and the volume fraction of methanol is 15%.

[0016] In some specific embodiments, the pretreatment reagent combination also includes water.

[0017] Detecting cortisol levels in samples using chemical analysis methods requires pretreatment of biological samples. Existing pretreatment methods are mostly applicable to plasma or serum, but not to urine samples. Therefore, this invention provides the application of the aforementioned pretreatment reagent combination in the preparation of products for detecting cortisol levels in urine. Compared to pretreatment reagents with other components or concentrations, or other treatment schemes, using the reagent combination provided by this invention, containing ammonia-methanol aqueous solution, formic acid-methanol aqueous solution, and acetonitrile-methanol solution, to pretreat urine samples, combined with the use of a solid-phase extraction device, can increase sample throughput. Furthermore, solid-phase extraction eliminates the need for an activation step, making the pretreatment process more convenient and faster.

[0018] This invention provides a pretreatment kit for cortisol content detection, comprising the pretreatment reagents described herein and other reagents. The other reagents include water and an extraction plate. The packing material in the extraction plate comprises a hydrophilic-lipophilic balanced reversed-phase adsorbent. In some embodiments, the extraction plate is an Oasis PRiME HLB μElution Plate solid-phase extraction plate, containing 3 mg of packing material per well.

[0019] This invention provides a pretreatment method for detecting cortisol in urine, comprising diluting the sample with water and loading it onto the sample, then washing the sample sequentially with solution A and solution B as described above, and then eluting the sample with solution C.

[0020] In the dilution step, the volume ratio of sample to water is (1~20):(15~34).

[0021] The volume ratio of the sample, solution A, and solution B is (20~400):150:150.

[0022] The elution step also includes a pre-addition of water step, wherein the volume ratio of the pre-addition of water to solution C is 70:30;

[0023] The volume ratio of solution C to the sample is (20~400):30.

[0024] In an embodiment of the present invention, the pretreatment method for the urine sample includes diluting the sample and calibration solution to an appropriate concentration, followed by sequential centrifugation at 13000 r / min for 10 s, vortexing at 2000 r / min for 50 min, and centrifugation at 13000 r / min for 10 min. The resulting supernatant is then subjected to solid-phase extraction using a 96-well plate. This includes sequentially washing the corresponding wells with solution A and solution B, and then eluting the sample with solution C.

[0025] This invention employs a 96-well plate combined with a 96-well positive pressure extraction device for urine pretreatment, which can accelerate sample throughput and make the pretreatment process convenient and fast. Compared with other pretreatment methods, it can improve the accuracy of detection results.

[0026] The present invention also provides a method for detecting cortisol in urine by liquid chromatography-mass spectrometry, comprising pretreatment with the method described in the present invention, and then detecting the eluent by UPLC-MS.

[0027] Specifically, in the UPLC, the eluent is eluted with mobile phase A and mobile phase B, where mobile phase A is methanol and mobile phase B is a 0.1% (v / v) formic acid aqueous solution, and the flow rate of the mobile phase is 3 mL / min. The UPLC time is 8 min. The injection volume of the eluent is 5 μL.

[0028] In the UPLC, the gradient elution conditions are as follows:

[0029] 0~0.5min, 45 vol% mobile phase A + 55 vol% mobile phase B;

[0030] 3.5 min, 80 vol % mobile phase A + 20 vol % mobile phase B;

[0031] 4~5 min, 95 vol % mobile phase A + 5 vol % mobile phase B;

[0032] 5.5~8 min, 45 vol % mobile phase A + 55 vol % mobile phase B.

[0033] In some specific embodiments, the UPLC column is a BEH type column and / or a T3 type column, and the column temperature is 45°C.

[0034] In the MS method described in this invention, the mass spectrometry detection parameters include an ESI ion source, a positive ion mode, wherein in the positive ion mode, the target quantitative ion pair includes a cortisol ion pair and / or a cortisol internal standard ion pair, the electrospray needle voltage is 2.7~3.0kV, the ion source temperature is 150℃, the desolvation gas temperature is 450~600℃, the collision gas is helium, and the desolvation gas flow rate is 800~1000L / Hr.

[0035] The mass-to-charge ratio conditions for the multiple reaction monitoring (MRM) ion scanning of the target quantitative ion described in this invention include:

[0036] The quantitative ion pair for cortisol is 363 > 121, the cone voltage is 30–45 V, and the collision voltage is 18–25 V. The qualitative ion pair is 363 > 309, the cone voltage is 30–45 V, and the collision voltage is 18–25 V.

[0037] The quantitative ion pair for d4-cortisol is 367 > 331, the cone voltage is 30–45 V, and the collision voltage is 18–25 V. The qualitative ion pair is 367 > 121, the cone voltage is 30–45 V, and the collision voltage is 18–25 V.

[0038] The methods provided by this invention can be for diagnostic purposes or non-diagnostic purposes. For example, they include diagnostic testing methods for human or animal bodies, or ex vivo samples derived from human or animal bodies; they also include scientific or other non-diagnostic testing methods for environmental or simulated samples.

[0039] The method of this invention for detecting cortisol employs the bracket method for quantification, resulting in more precise and accurate results compared to other standard curve methods. This includes adding 20 μL to 400 μL of sample to an internal standard solution, wherein the cortisol content in the sample is quantified using both high and low standard solutions.

[0040] The internal standard solution contains isotopic labels of the analyte. The signal response between the analyte and the internal standard in the sample is 1.0 ± 0.2, the signal response between the analyte and the internal standard in the low standard solution is 0.9 ± 0.2, and the signal response between the analyte and the internal standard in the high standard solution is 1.1 ± 0.2.

[0041] Advantages of this invention:

[0042] 1. The sample pretreatment operation is simple, has a high throughput, and can achieve a certain degree of automation.

[0043] 2. Liquid phase separation is effective and stable, and it effectively avoids potential interfering substances.

[0044] 3. Mass spectrometry detection conditions: Through screening quantitative ions and repeated exploration of conditions, a method with low baseline noise signal and accurate detection was obtained.

[0045] 4. By using the bracket method to quantify the results instead of the commonly used linear calibration method, the quantitative results obtained are more accurate and reliable. Attached Figure Description

[0046] Figure 1 Chromatograms of different extraction methods in sample pretreatment experiments are shown;

[0047] Figure 2 Chromatograms of samples under different elution conditions in the pretreatment test are shown.

[0048] Figure 3 Chromatograms of different gradient elutions in liquid chromatography experiments;

[0049] Figure 4 Chromatograms showing different chromatographic columns, reduced dead volume, and improved pH in liquid chromatography experiments;

[0050] Figure 5 Show the linear graph of the standard curve;

[0051] Figure 6 The results show the limits of detection and limits of quantitation.

[0052] Figure 7 Show the specificity verification chromatogram;

[0053] Figure 8 The results of the recovery experiment are shown;

[0054] Figure 9 Show the results of the accuracy experiment;

[0055] Figure 10 The results of the precision experiment are shown.

[0056] Figure 11 The results of the matrix effect verification are shown.

[0057] Figure 12 The results of the pollution verification are shown. Detailed Implementation

[0058] This invention provides a method for determining cortisol content and its application. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The method and application of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the method and application described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0059] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0060] The certified reference material for cortisol, SRM921, was obtained from the National Institute of Standards and Technology (NIST). Cortisol-d4 (an internal isotope standard) was purchased from Sigma-Aldrich. Methanol, formic acid, and ammonia were purchased from Merck, and the experimental water was prepared using a Millipore pure water system.

[0061] The instrument used in this study was an ACQUITY UPLC I-Class tandem TQ-S mass spectrometer, equipped with an ACQUITY UPLC® HSS T3 column (2.1 × 100, 1.8 µm). A Mettler XPE205 electronic balance was used for the preparation of standard solutions and the weighing of urine and calibration samples. Oasis PRiME HLB μElution Plate solid-phase extraction plates (3 mg / 96-well) were purchased from Waters Corporation.

[0062] The present invention will be further illustrated below with reference to the embodiments:

[0063] Example 1: Optimization of Sample Preprocessing Method

[0064] 1. Selection of preprocessing method

[0065] Table 1

[0066]

[0067] MTBE extraction steps:

[0068] 1. Take 200 μL of each sample into a centrifuge tube and add 50 μL of internal standard;

[0069] 2. Add 1 mL of MTBE extraction reagent to each sample;

[0070] 3. Place the centrifuge tubes on a vortex mixer at 2500 rpm for 10 minutes;

[0071] 4. After the vortex oscillation is complete, remove the centrifuge tubes and centrifuge them at 13,000 rpm for 10 minutes.

[0072] 5. After centrifugation, transfer the supernatant to a new centrifuge tube and dry it with nitrogen gas;

[0073] 6. After drying, add 800 μL of 45% methanol-water reconstituted solution to each centrifuge tube and vortex at 2500 rpm for 10 min.

[0074] 7. Then, each sample is passed through a membrane and tested on the machine.

[0075] Ethyl acetate extraction steps:

[0076] 1. Take 200 μL of each sample into a centrifuge tube and add 50 μL of internal standard;

[0077] 2. Add 1 mL of MTBE extraction reagent to each sample;

[0078] 3. Place the centrifuge tubes on a vortex mixer at 2500 rpm for 10 minutes;

[0079] 4. After the vortex oscillation is complete, remove the centrifuge tubes and centrifuge them at 13,000 rpm for 10 minutes.

[0080] 5. After centrifugation, transfer the supernatant to a new centrifuge tube and dry it with nitrogen gas;

[0081] 6. After drying, add 800 μL of 45% methanol-water reconstituted solution to each centrifuge tube and vortex at 2500 rpm for 10 min.

[0082] 7. Then, each sample is passed through a membrane and tested on the machine.

[0083] SPE extraction steps:

[0084] Prepare solutions A, B, and C:

[0085] In solution A, the volume fraction of ammonia is 0.1%, the volume fraction of methanol is 35%, and the remainder is water;

[0086] In solution B, the volume fraction of formic acid is 0.1%, the volume fraction of methanol is 35%, and the remainder is water;

[0087] In solution C, the volume fraction of acetonitrile is 85% and the volume fraction of methanol is 15%.

[0088] Based on the initial concentration of the sample to be tested, weigh appropriate amounts of calibration solution and the sample to be tested; then add 50 μL of an appropriate concentration of internal standard solution to the calibration solution and the sample to be tested and weigh it; next, add pure water to the calibration solution and the sample to be tested (20~400 μL) to bring the total to 700 μL; centrifuge each sample at 13000 r / min for 10 s to ensure that each additive is at the bottom; place the above samples on a multi-tube vortex mixer and vortex for 50 min at a speed of 2000 r / min; after the above operation, centrifuge each sample at 13000 r / min for 10 min; transfer 400 μL of the supernatant from the centrifuged samples to a 96-well plate using a pipette, and seal any unused wells with a plate protector; adjust the gas switch of the positive pressure extraction device to allow the sample supernatant to flow down drop by drop. The extraction device is an Oasis PRiME HLB μElution. The process involves: first, using a solid-phase extraction plate; then, washing the corresponding wells with 150 μL of solution A and solution B, followed by pressing the liquid out of the wells after washing; finally, taking a sample receiving plate, adding 70 μL of pure water to the corresponding wells, placing the 96-well plate on the sample receiving plate, adding 30 μL of solution C to each well to elute the sample, and pressing the liquid out of the wells using a positive pressure device; covering the sample receiving plate with a sealing film and mixing thoroughly, then placing the sample receiving plate in the instrument's sample chamber for instrumental analysis.

[0089] From Table 1, the stability of the experimental data and the chromatograms ( Figure 1 From the perspective of chromatographic peak separation, the precision of solid-phase extraction (SPE) is superior to that of liquid-liquid extraction.

[0090] 2. Optimization of the elution process in solid-phase extraction:

[0091] Group 1: 1 pass of solution A, 1 pass of solution B;

[0092] Group 2: Wash twice with a 10% methanol aqueous solution;

[0093] Group 3: 1 application of 10% methanol aqueous solution and 1 application of 1% ammonia-methanol (40% methanol) aqueous solution.

[0094] Table 2

[0095]

[0096] Based on the responses of the test samples in Table 2, the responses of the test samples and standard solutions in Group 3 were too low. Although Group 2 had the highest response, the baseline of the chromatographic peak was high, and the impurity peaks were obvious (see Table 2). Figure 2 Instead, it was not conducive to accurate detection, so we ultimately chose the elution conditions of Group 1 as the elution conditions for our solid phase extraction.

[0097] 3. Determination of sample pretreatment methods

[0098] Based on the initial concentration of the sample to be tested, weigh appropriate amounts of calibration solution and the sample to be tested; then add 50 μL of an appropriate concentration of internal standard solution to the calibration solution and the sample to be tested and weigh it; next, add pure water to the calibration solution and the sample to be tested to bring the total to 700 μL; centrifuge each sample at 13000 r / min for 10 s to ensure that each additive is at the bottom; place the above samples on a multi-tube vortex mixer and vortex for 50 min at a speed of 2000 r / min; after the above operation, centrifuge each sample at 13000 r / min for 10 min; transfer 400 μL of the supernatant from the centrifuged samples to a 96-well plate using a pipette, and seal any unused wells with a plate label. Apply protective film; adjust the gas switch of the positive pressure extraction device to allow the sample supernatant to flow down drop by drop; then wash the corresponding wells with 150 μL of 0.1% ammonia-methanol aqueous solution (35% methanol) and 0.1% formic acid-methanol aqueous solution (35% methanol), respectively, and finally press the liquid in the wells dry after washing; finally, take a sample receiving plate, add 70 μL of pure water to the corresponding well beforehand, place the above 96-well plate on the sample receiving plate, add 30 μL of 85% acetonitrile-methanol to each well to elute the sample, and press the liquid in the wells dry with the positive pressure device; cover the sample receiving plate with a sealing film and mix well, and then place the sample receiving plate in the sample chamber of the instrument for instrument detection.

[0099] Example 2 Optimization of Liquid Phase Experimental Scheme

[0100] 1. Optimization of gradient elution conditions

[0101] Mobile phase A was methanol, and mobile phase B was 0.1% formic acid aqueous solution; gradient elution conditions are shown in Table 3, injection volume was 5 μL, flow rate was 3 mL / min, column temperature was 45 ℃, and autosampler temperature was 10 ℃.

[0102] Table 3. Liquid phase conditions for cortisol

[0103]

[0104] From the results ( Figure 3 As can be seen, under the elution conditions of gradient 1, the baseline of the liquid chromatography peaks is appropriate, and there are no impurity peaks. Therefore, gradient 1 is selected for liquid chromatography experiments.

[0105] 2. Optimization of chromatographic column, dead volume, and mobile phase pH.

[0106] After screening chromatographic columns, reducing system dead volume, and adjusting mobile phase pH using certain methods, liquid chromatography experiments were conducted. The results showed that ( Figure 4 When selecting the ACQUITY UPLC® HSS T3 Column (2.1 × 100, 1.8 µm) and the mobile phase pH being the pH value of water with 0.1%~0.2% formic acid added, the chromatographic peak baseline is appropriate and there are no impurity peaks.

[0107] Example 3 Optimization of Mass Spectrometry Experimental Protocol

[0108] 1. Optimization of mass spectrometry conditions

[0109] Mass spectrometry conditions 1: Electrospray ionization (ESI) positive ion mode and multiple reaction monitoring (MRM) mode were used for analysis. The capillary voltage was 2.0 kV, the ion source temperature was 150 ℃, the desolvation gas temperature was 400 ℃, and the desolvation gas flow rate was 800 L / Hr. For quantitative detection, the ion conversion to mass-to-charge ratio is as follows: m / z 363.2 ➝ 121.1 (cortisol), with a cone voltage of 38 V and a collision voltage of 20 V; m / z 367.2 ➝ 331.2 (cortisol internal standard), with a cone voltage of 42 V and a collision voltage of 18 V. For qualitative detection, the ion conversion to mass-to-charge ratio is as follows: m / z 363.2 ➝ 309.2 (cortisol), with a cone voltage of 38 V and a collision voltage of 16 V; m / z 367.2 ➝ 121.1 (cortisol internal standard), with a cone voltage of 42 V and a collision voltage of 20 V.

[0110] Mass spectrometry conditions 2: Electrospray ionization (ESI) positive ion mode and multiple reaction monitoring (MRM) mode were used for analysis. The capillary voltage was 3 kV, the ion source temperature was 150 ℃, the desolvation gas temperature was 550 ℃, and the desolvation gas flow rate was 1000 L / Hr. For quantitative detection, the ion conversion to mass-to-charge ratio is m / z 363.2 ➝ 121.1 (cortisol), with a cone voltage of 38V and a collision voltage of 20V; for m / z 367.2 ➝ 331.2 (cortisol internal standard), the cone voltage is 42V and the collision voltage is 18V. For qualitative detection, the ion conversion to mass-to-charge ratio is m / z 363.2 ➝ 309.2 (cortisol), with a cone voltage of 38V and a collision voltage of 16V; for m / z 367.2 ➝ 121.1 (cortisol internal standard), the cone voltage is 42V and the collision voltage is 20V.

[0111] Mass spectrometry analysis of the five samples under the above conditions revealed (Table 4) that the samples under mass spectrometry condition 2 showed a more significant improvement in response.

[0112] Table 4

[0113]

[0114] Example 4: Result Calibration and Screening of Calculation Methods

[0115] 1. Before the formal analysis and determination, a preliminary sample was tested to obtain the approximate concentration of cortisol. Based on the initial concentration, the sample volume was determined. According to the sample volume and the concentration of the internal standard used, the appropriate sample volume and the mass of the internal standard solution were weighed and recorded to ensure that the ratio of the sample to the internal standard was approximately 1:1.

[0116] The prepared standard solution and internal standard working solution were used to design sampling quantities with a standard to internal standard ratio of 0.9 and 1.1, respectively. The precise mass of each solution was measured on a balance. Based on the peak area ratio of the sample, the concentration of the standard solution, and the mass of the sample and internal standard, the concentration of cortisol in the sample was calculated.

[0117] 2. Using the standard curve method is a common quantitative method. It is a linear regression method that considers the perpendicular distance from a point to the line. This quantitative method also involves the issue of weighting, with common weights being 1 / X, 1 / X... 2 The 1 / X weight means that the distance from each point to the line is multiplied by 1 / X, then summed, and the minimum value is taken. 2The weighting, reflected on the standard curve, is more decisive at lower concentrations. Based on the premise that low concentrations can be measured very accurately, all concentrations of the standard are measured ten times, the average and variance are calculated, and then the coefficient of variation (CV) is calculated. If the CV is within 5% from high to low concentrations, then the low concentration is assigned a weight of 1 / X. 2 The weighting is acceptable. However, if the CV for high concentration is good, but the CV for low concentration is close to 10%, then the low concentration should not be assigned 1 / X. 2 The weighting is adjusted. This method typically involves preparing two mixed calibration solutions, controlling the concentration ratio of the analyte to the internal standard in the sample to be approximately 1.0. Because the signal ratio of the calibrator is very close to that of the test sample, this method is accurate and precise.

[0118] 2.1 Quantitative analysis using the standard curve method

[0119] Table 5

[0120]

[0121] 2.2. Using the method of quantitative analysis

[0122] Table 6

[0123]

[0124] As can be seen from Tables 5 and 6, for the same sample, when comparing different quantitative methods, the bracket method results in a smaller deviation from the target value and better precision.

[0125] Example 5: Method Performance Validation

[0126] 1. Linear

[0127] Low-value urine samples were used as the matrix, and high-value standard solutions were added to prepare samples with concentrations of 4.49, 9.22, 18.30, 45.90, 93.37, 186.83, 473.81, and 984.87 ng / g. The same amount of internal standard solution was added to each sample for pretreatment. Each sample was injected three times consecutively. The linear equation was obtained by plotting the mean response ratio against the mass ratio. Figure 5 ).

[0128] The linear equation is: y = 0.011139x + 0.179596, R² = 0.9999. The linearity is good in the range of 4.49 ng / g to 984.87 ng / g, which meets the linearity requirement (R² ≥ 0.999).

[0129] 2. Limit of Detection and Limit of Quantification (LOD & LOQ)

[0130] The analytical sensitivity of the method was evaluated by preparing a series of diluted samples using low-value samples as the matrix. Three samples were processed in parallel for each level of the test sample, with two batches tested, resulting in six data points. The deviation from the theoretical value and the variation were calculated. The concentration with an average signal-to-noise ratio (S / N) ≥ 3 was used as the limit of detection (LOD). The lowest concentration that met the conditions of S / N ≥ 10, total variability (CV) ≤ 20% (imprecision), and deviation of the mean concentration from the theoretical concentration < 15% (accuracy) was used as the LOD. The concentration of 0.021 ng / g was used as the LOD, and 0.040 ng / g was used as the LOD. Figure 6 ).

[0131] 3. Specificity test

[0132] Cortisol in urine is a type of glucocorticoid, and its synthesis and metabolism in the body involve many intermediate metabolites. It also has many isomers and analogs. These substances can interfere with normal quantitative detection of cortisol. Typically, interference detection is first addressed through liquid chromatography, by changing the mobile phase or selecting a suitable column to separate the target substance from the interfering substance. Alternatively, mass spectrometry can be used to select specific ion pairs for separation.

[0133] In this experiment, based on the properties of the target analyte and the characteristics of the detection instrument, we selected the following six substances that may interfere with the target analyte to validate the established target analyte detection method. Based on the liquid chromatography separation conditions for cortisol, we established mass spectrometry conditions for the six potential interfering substances to be validated, and then detected these substances.

[0134] From Table 7 and Figure 7 It can be seen that the peaks of Cortisol overlap with those of 5α-dihydrocortisone and prednisone. However, according to the MRM model, the potential interfering substances and analytes can be distinguished by the differentiation of daughter ions.

[0135] Table 7 Mass spectrometry conditions for 6 potential interfering substances

[0136]

[0137] 4. Recycling Experiment

[0138] Spiked recovery rate is calculated by adding standards of different concentration gradients to a fixed amount of urine and then measuring the recovery rate using an instrument. It can verify the accuracy of the established method, indicate to some extent whether there is interference from other substances, and also serve as a simple and direct method to reflect the magnitude of matrix effects.

[0139] A low-value sample of 8.8 ng / g was selected as the basal sample. Standard solutions at low, medium, and high concentrations were added at levels of 45.1 ng / g, 87.39 ng / g, and 178.83 ng / g, respectively. Spike recovery experiments were conducted on two batches, with three replicates per batch. The corresponding recoveries were 99.65%, 100.37%, and 99.90%, respectively, ranging from 99.65% to 100.37%, which is better than the general recovery requirements (85%–115%) for clinical testing method validation. Results are shown below. Figure 8 .

[0140] 5. Accuracy

[0141] Because no accuracy verification material was available for urine samples, we selected ERM-DA192 and ERM-DA193, reference materials for cortisol serum measurement, as accuracy verification materials for the accuracy confirmation experiment. Two parallel samples were tested for each verification material, with three batches tested, and the average of the six values ​​obtained was used as the final target value. The detection offset of the two verification materials at all four levels was within 2%, meeting the offset requirements for accuracy control materials. The test unit is nmol / L. Results are shown below. Figure 9 .

[0142] 6. Precision

[0143] Precision experiments were conducted using samples at three concentration levels: low, medium, and high. Each concentration level was tested over three days, with five repeated tests per day. One-way ANOVA was used to analyze the data. The experimental data are presented below. Figure 10 As shown.

[0144] The variation results during the precision test of the three concentrations of samples were 1.27%, 1.06% and 1.42%, respectively, indicating excellent method precision (≤5%).

[0145] 7. Matrix effect

[0146] The relative matrix effect was investigated using a matrix mixing experiment. Five matrix samples and one mixed matrix sample were randomly selected. Five matrix samples were prepared: a standard solution for the matrix, a serum matrix sample, a 20:80 mixture of serum and solution, a 1:1 mixture of serum and solution, and an 80:20 mixture of serum and solution. The same amount of internal standard solution was added to each sample. After sample pretreatment, the samples were injected for analysis, with three consecutive injections. Results... Figure 11 It can be seen that the matrix deviation meets the requirements (≤5%).

[0147] Carryover (%) = (L3-L2) / (H-L2) The two high-value samples H1: 473.81ng / g and H2: 984.97ng / g were tested respectively, and the carryover rate of both samples met the requirements (≤1%).

[0148] 8. Carrying pollution

[0149] Considering the range of cortisol concentrations in human serum, high-value samples were added at concentrations of H1: 200 ng / mL and H2: 500 ng / mL; and low-value samples (preferably near the lower limit of quantitation) were diluted to a concentration of L: 5 ng / mL. The same amount of internal standard solution was added, and after sample pretreatment, the low-concentration samples were injected repeatedly, followed by alternating injections of high-concentration and low-concentration samples in the order of low-value, low-value, high-value, low-value. From the results... Figure 12 As can be seen, the two high-value samples, 200 PPB and 500 PPB, were tested respectively, and the contamination rate of both samples met the requirements (≤1%).

[0150] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting cortisol in urine using liquid chromatography coupled with mass spectrometry, characterized in that, The process includes diluting the sample with water and loading it onto an extraction plate, washing the sample sequentially with solution A and solution B, eluting the sample with solution C, and then detecting the eluent using UPLC-MS. The extraction plate is an Oasis PRiME HLB μElution Plate solid-phase extraction plate. The potential interfering substances in the sample are 5α-dihydrocortisone, 5β-dihydrocortisone, Tetrahydrocortisone, aldosterone, cortisone, and prednisolone; Solution A is an ammonia-methanol aqueous solution, wherein the volume fraction of ammonia is 0.05%~1%, the volume fraction of methanol is 5%~40%, and the remainder is water; Solution B is an aqueous solution of formic acid and methanol, wherein the volume fraction of formic acid is 0.05%~1%, the mass fraction of methanol is 5%~40%, and the remainder is water; Solution C is an acetonitrile-methanol solution, wherein the volume fraction of acetonitrile is 60%~90% and the volume fraction of methanol is 10%~40%. The chromatographic conditions for UPLC include: The chromatographic column is a T3 type column; Mobile phase A is methanol. Mobile phase B is a 0.1% (v / v) formic acid aqueous solution. The conditions for gradient elution are: At 0 min, the volume fraction of mobile phase A is 45%. At 0.5 min, the volume fraction of mobile phase A is 45%. At 3.5 min, the volume fraction of mobile phase A is 80%. After 4 minutes, the volume fraction of mobile phase A is 95%. The volume fraction of mobile phase A is 95% after 5 minutes; At 5.5 min, the volume fraction of mobile phase A was 45%. After 8 minutes, the volume fraction of mobile phase A was 45%. The mass spectrometry detection parameters include an ESI ion source and a positive ion mode. In the positive ion mode, the target quantitative ion pair includes cortisol ion pairs and / or cortisol internal standard ion pairs. The electrospray needle voltage is 2.7~3.0kV, the ion source temperature is 150℃, the desolvation gas temperature is 450~600℃, the collision gas is helium, and the desolvation gas flow rate is 800~1000L / Hr.

2. The method according to claim 1, characterized in that, The column temperature was 45℃ and the flow rate of the mobile phase was 3 mL / min.

3. The method according to claim 1, characterized in that, In the dilution step, the volume ratio of the sample to water is (1~20):(15~34). The volume ratio of the sample, solution A, and solution B is (20~400):150:

150. The elution step also includes a pre-addition of water step, wherein the volume ratio of the pre-addition of water to solution C is 70:30; The volume ratio of solution C to the sample is (20~400):

30.

4. The method according to claim 1, characterized in that, The mass-to-charge ratio conditions for multiple reaction monitoring (MRM) of target ions include: The quantitative ion pair for cortisol is 363 > 121, the cone voltage is 30–45 V, and the collision voltage is 18–25 V. The qualitative ion pair is 363 > 309, the cone voltage is 30–45 V, and the collision voltage is 18–25 V. The quantitative ion pair for d4-cortisol is 367 > 331, the cone voltage is 30–45 V, and the collision voltage is 18–25 V. The qualitative ion pair is 367 > 121, the cone voltage is 30–45 V, and the collision voltage is 18–25 V.

5. The method according to any one of claims 1 to 4, characterized in that, After the UPLC-MS, the results are quantitatively detected using the bracket method, which includes adding 20 μL to 400 μL of sample to an internal standard solution, wherein the cortisol content in the sample is quantified using high and low standard solutions; The internal standard solution contains isotopic labels of the analyte. The signal response between the analyte and the internal standard in the sample is 1.0 ± 0.2, the signal response between the analyte and the internal standard in the low standard solution is 0.9 ± 0.2, and the signal response between the analyte and the internal standard in the high standard solution is 1.1 ± 0.2.

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