A method for simultaneously determining free thyroid hormones T3, rT3, T4 and cortisol in saliva

Abstract

The present invention provides a method for simultaneously measuring free thyroid hormones T3, T4, rT3, and cortisol in saliva. Ultrafiltration and centrifugation are performed using a filter membrane with a suitable molecular weight cutoff range to remove proteins and bound thyroid hormones and cortisol from saliva. Methanol and other anti-adsorption agents are then added in appropriate proportions. This method overcomes the serious adsorption problem that exists during the detection of free T3, rT3, T4, and cortisol in saliva. Utilizing high-performance liquid chromatography-tandem mass spectrometry, the four free hormones, T3, rT3, T4, and cortisol, can be accurately and simultaneously measured in saliva. This method offers the advantages of high throughput, ease of operation, high processing efficiency, low consumables cost, high accuracy, and high sensitivity, making it easy to promote and apply clinically.

Description

[0001] This application is a divisional application of the parent case 202110394022.0. Technical Field

[0002] The present invention belongs to the technical field of biochemical analysis, and in particular relates to a method for simultaneously determining free thyroid hormones T3, rT3, T4 and cortisol in saliva. Background Art

[0003] Thyroid hormones are important endocrine hormones in the human body. Their primary function is to increase the body's metabolic rate, boost oxygen consumption, promote growth and development, and promote the differentiation and maturation of the nervous system. Thyroid hormones include thyroxine (T4), triiodothyronine (T3), and reverse triiodothyronine (rT3). Thyroid hormones are important markers for evaluating thyroid function and are the primary hormones of the thyroid gland for target organs. The total concentration of thyroid hormones in serum reflects the secretory status of the thyroid gland.

[0004] Cortisol is a crucial glucocorticoid in the body, responsible for regulating mood, health, maintaining immune cells and inflammation, and maintaining blood vessels and blood pressure, among other physiological functions. Clinically, cortisol levels in serum or saliva are measured to assess hypothalamic-pituitary-adrenal axis function. Salivary cortisol levels correlate with serum cortisol levels and can be used to assess circulating cortisol levels. Cortisol levels are clinically used to diagnose and screen for conditions such as Cushing's syndrome and adrenal insufficiency.

[0005] Cortisol is also known as the stress hormone, and current research tends to link stress with higher cortisol levels. A study published in the Journal of Clinical Endocrinology and Metabolism showed that cortisol reduces TSH, thereby reducing the production of thyroid hormones. Cortisol inhibits the conversion of T4 to T3, leading to hypothyroidism. Another way in which cortisol aggravates the symptoms of hypothyroidism is through its effect on blood sugar. Both high and low cortisol levels can cause blood sugar imbalance, leading to hypoglycemia and hyperglycemia. At the same time, studies have shown that hypoglycemia and hypothyroidism have a mutual influence. It can be seen that there is a close connection between the level of cortisol and the level of thyroid hormone. It is very necessary to simultaneously detect the levels of thyroid hormone and cortisol in the human body for auxiliary diagnosis and health management of clinical diseases.

[0006] Traditional testing samples are serum, measuring total or free hormone concentrations. Existing research has demonstrated a strong correlation between thyroid hormone and cortisol levels in saliva and serum cortisol levels, and many clinical studies have even confirmed that saliva is a more accurate clinical indicator. If saliva samples could be used to accurately measure both thyroid hormone and cortisol simultaneously, this would greatly facilitate clinical diagnosis due to its non-invasive, easy-to-use, continuous sampling, and minimal protein interference.

[0007] Steroid hormones are partially bound to proteins, while unbound hormones are free. Thyroid hormones are primarily bound to thyroid-binding proteins in peripheral blood, with free T4 (FT4) and free T3 (FT3) exerting their physiological effects. Therefore, T3, T4, FT3, and FT4 are the most commonly used indicators in clinical testing. However, because the levels of free thyroid hormones in serum and saliva are low, at the pg / mL level, detection methods require extremely high sensitivity and specificity.

[0008] Traditional detection methods include immunoassays, electrochemical methods, and fluorescence methods. However, these methods suffer from poor specificity, are unable to address interference from endogenous and exogenous glucocorticoids, and are unable to address low levels of cortisol in saliva, resulting in low detection accuracy. Existing methods are constantly being improved, and studies have explored quantitative analysis of total thyroid hormones in serum using HPLC-MS / MS. However, these methods are unable to simultaneously detect free thyroid hormones T3, rT3, T4, and cortisol, and are not suitable for saliva samples, limiting their scope of application.

[0009] CN107576624B discloses a method for detecting cortisol in saliva. The method removes salivary mucin from saliva by filtering with a composite microfiltration membrane. However, this method cannot remove bound cortisol and thyroid hormones in saliva. The bound hormones remaining in the sample will affect the detection results of free hormones. Therefore, it cannot be used to accurately detect free thyroid hormones T3, rT3, T4 and cortisol in saliva.

[0010] Because the levels of free thyroid hormones T3, rT3, T4 and cortisol in saliva are very low, there is currently no simple and efficient detection and analysis method that can accurately and simultaneously detect the levels of the four free hormones, free thyroid hormones T3, rT3, T4 and cortisol, in saliva. Summary of the Invention

[0011] To address the above-mentioned problems, the present invention provides an improved high-performance liquid chromatography-tandem mass spectrometry analysis method for simultaneously detecting free thyroid hormones T3, T4, rT3, and cortisol in human saliva samples. Ultrafiltration and centrifugation are used to completely remove all proteins and bound thyroid hormones and cortisol in saliva. Methanol and other anti-adsorption reagents are then added, and high-performance liquid chromatography-tandem mass spectrometry analysis is directly performed. This method can simultaneously and accurately detect free thyroid hormones T3, rT3, T4, and cortisol in saliva. This method has the advantages of high throughput, simple operation, high accuracy, and high sensitivity, and is easy to promote and apply in clinical practice.

[0012] In one aspect, the present invention provides a method for simultaneously determining free thyroid hormones T3, rT3, T4, and cortisol in saliva, comprising the following steps:

[0013] 1) Take a saliva sample and centrifuge it;

[0014] 2) taking the supernatant and placing it in an ultrafiltration centrifuge tube for ultrafiltration centrifugation;

[0015] 3) Collect the filtrate obtained by ultrafiltration and centrifugation, add the internal standard solution and the organic solvent for anti-adsorption, vortex mix, and analyze by liquid chromatography-mass spectrometry system.

[0016] After extensive research, the present invention has confirmed that ultrafiltration centrifugation can completely remove all proteins and bound thyroid hormones and cortisol in saliva. However, when the obtained filtrate is subjected to high-performance liquid chromatography-tandem mass spectrometry analysis, the free thyroid hormones T3, rT3, T4 and cortisol in the sample are easily adsorbed by consumables, resulting in significantly low test results and difficulty in achieving accurate detection.

[0017] Since the concentrations of free thyroid hormones T3, rT3, T4, and cortisol in saliva are at the pg / mL level and have low polarity, which is completely different from the detection of serum samples, interference problems such as consumable adsorption (such as adsorption by 96-well plates during high-throughput detection by high-performance liquid chromatography-tandem mass spectrometry) are prone to occur during the detection process. To prevent adsorption by ultrafiltration membranes, the present invention replaced ultrafiltration membranes from multiple different manufacturers and finally selected Ultracel PL regenerated cellulose ultrafiltration membrane to truly solve the adsorption problem. However, the adsorption problem by 96-well plates has always been difficult to solve. Even after replacing them with low-adsorption 96-well plates, very obvious adsorption problems still exist, resulting in significantly low test results.

[0018] Before ultrafiltration, the consumables may have little adsorption effect on free thyroid hormones T3, rT3, T4 and cortisol due to the presence of proteins and bound thyroid hormones, cortisol, etc. in the saliva sample. However, after ultrafiltration purification, the free thyroid hormones T3, rT3, T4 and cortisol in the ultrafiltrate can easily lead to low test results due to adsorption and other problems during the detection process.

[0019] In order to solve the problem that the free thyroid hormones T3, rT3, T4 and cortisol in the filtrate are easily adsorbed by consumables, the inventors found after a large number of experiments that adding a large amount of organic solvents such as methanol, acetonitrile or isopropanol to the filtrate can effectively prevent the adsorption problem; at the same time, the addition of organic solvents such as methanol may further reduce the matrix effect in the ultrafiltrate, making the four free thyroid hormones T3, rT3, T4 and cortisol more stable, significantly improving the detection sensitivity, and thus truly realizing the simultaneous and accurate detection of the four free hormones of free thyroid hormones T3, rT3, T4 and cortisol in saliva.

[0020] Furthermore, the organic solvent used for anti-adsorption is any one or two selected from methanol, acetonitrile, and isopropanol; and the volume ratio of the filtrate obtained by ultrafiltration and centrifugation to the anti-adsorption organic solvent is 1:1 to 1:5.

[0021] The amount of organic solvents such as methanol added must reach 1 to 5 times the volume of the sample itself to have a significant anti-adsorption effect.

[0022] Furthermore, studies have shown that methanol has a better anti-adsorption effect and can significantly improve detection sensitivity.

[0023] Furthermore, the specification of the ultrafiltration centrifuge tube in step 2) is to use Ultracel PL regenerated cellulose ultrafiltration membrane, and the molecular weight cut-off limit range of the membrane is 10K-100K.

[0024] Ultrafiltration is a commonly used method for laboratory testing of free substances. Ultrafiltration can separate small molecules and proteins. It uses high pressure or centrifugal force to allow free small molecules to pass through the semi-permeable membrane, while large molecular proteins and small molecules bound to proteins remain on the membrane. By selecting a filter membrane of appropriate specifications, proteins of different molecular weights can be retained.

[0025] A filter membrane with an appropriate molecular weight cutoff can help completely remove all proteins in saliva, including proteins bound to the bound hormones T3, rT3, and T4, thereby further improving the detection sensitivity of free hormones in saliva.

[0026] Furthermore, the ultrafiltration centrifugation time in step 2) is 15 minutes to 1 hour, the centrifugal speed is 2000 to 4000 rpm, and the centrifugal temperature of the centrifuge is 36 to 38° C.; the centrifugal speed in step 1) is 3000 to 4000 rpm.

[0027] Furthermore, the ultrafiltration centrifuge tube described in step 2) includes an inner tube and a collection tube. The supernatant is first placed in the inner tube, and then the inner tube is inserted into the collection tube, and placed in a centrifuge for ultrafiltration centrifugation. The filtrate obtained by ultrafiltration centrifugation is located in the collection tube; the centrifuge start-up and stop process adopts a slow acceleration and slow deceleration mode to slowly increase and decrease the speed.

[0028] Furthermore, the internal standard solution in step 3) contains T3- 13 C6, rT3- 13 C6, T4- 13 C6 and Cortisol-d4.

[0029] Furthermore, the method also includes comparison of quality control sample results and evaluation of detection error in step 4); the quality control sample preparation method in step 4) is: the filtrate obtained by filtering the saliva sample through an ultrafiltration tube is added with an equal volume of organic solvent, mixed, and a mixed standard solution containing different concentrations of thyroid hormones T3, rT3, T4 and cortisol is added to obtain saliva matrix samples containing three different levels of concentration: low, medium and high as quality control samples.

[0030] In addition, existing research and literature do not yet have a system and method for systematic quality control of the detection error of this detection method, making it difficult to ensure the reproducibility and consistency of the detection results of different batches, as well as to evaluate the reliability of the method. In order to solve this problem, the inventors attempted to establish a set of quality control sample configuration methods, comprehensively considering the ultrafiltration operation and adsorption issues in the pretreatment of the four hormones.

[0031] The present invention ensures precision control of each test by configuring salivary matrix samples containing three different concentration levels: low, medium, and high as quality control samples. The use of quality control samples also helped the inventors discover the serious adsorption problem that exists during the detection of free T3, rT3, T4, and cortisol. The levels of free thyroid hormones T3, rT3, T4, and cortisol in saliva are already very low, and the presence of adsorption problems will greatly affect the detection results of the four free hormones in saliva. It was also with the help of quality control samples that the inventors, after extensive research, discovered and confirmed the efficacy of anti-adsorption reagents for free thyroid hormones T3, rT3, T4, and cortisol. This truly overcomes the serious adsorption problem that exists during the detection of free T3, rT3, T4, and cortisol in saliva, greatly improving detection sensitivity and bringing more convenience to auxiliary diagnosis of clinical diseases and health management. This method facilitates the promotion and application of this method, helps researchers immediately identify problems in detection, and makes inter-laboratory comparison research possible.

[0032] Furthermore, in the liquid chromatography-mass spectrometry system described in step 3), the liquid chromatography adopts a gradient elution mode, the chromatographic column is a C18 or phenyl filler column, the mobile phase A is a 0.05-0.2 volume% formic acid aqueous solution, the mobile phase B is a 0.05-0.2 volume% formic acid acetonitrile solution, and gradient elution is adopted, and the volume ratio of mobile phase A to mobile phase B is 60-0%:40-100%.

[0033] Furthermore, the mass spectrometer used in the liquid chromatography-mass spectrometry system is a triple quadrupole mass spectrometer, and mass spectrometry detection is performed using an electrospray ionization source and positive ion mode (ESI+) and multiple reaction monitoring (MRM) mode; the quality control sample results of the step 4) are compared and the detection error is evaluated. By comparing the deviation between the detection value of the quality control sample and the theoretical target value, the accuracy and error of the batch detection process are evaluated. The detection results include the measured levels of four hormones, T3, rT3, T4 and cortisol, in the quality control samples.

[0034] On the other hand, the present invention provides a detection kit for simultaneously determining free thyroid hormones T3, rT3, T4 and cortisol in saliva, the kit comprising a standard working solution, a quality control sample, an internal standard solution, an anti-adsorption reagent and a liquid chromatography mobile phase; the standard working solution is a solution containing any one or more of free thyroid hormones T3, rT3, T4 or cortisol at standard concentrations; the quality control sample is a saliva matrix sample containing three different concentration levels of low, medium and high; the anti-adsorption reagent is methanol; the liquid chromatography mobile phase comprises mobile phase A and mobile phase B, mobile phase A is a 0.05-0.2 volume % formic acid aqueous solution, and mobile phase B is an acetonitrile solution of 0.05-0.2 volume % formic acid.

[0035] In another aspect, the present invention provides use of methanol for preparing an anti-adsorption reagent for preventing thyroid hormones in saliva from being adsorbed by consumables.

[0036] The present invention has the following beneficial effects:

[0037] (1) By selecting a filter membrane with an appropriate molecular weight cut-off range, all proteins and bound thyroid hormones T3, rT3, T4, and cortisol in saliva can be completely removed, thereby improving the detection sensitivity of free thyroid hormones T3, rT3, T4, and cortisol in saliva;

[0038] (2) By selecting the best anti-adsorption reagent and the most appropriate addition ratio, the serious adsorption problem existing in the detection of free T3, rT3, T4 and cortisol in saliva was truly overcome, greatly improving the detection sensitivity;

[0039] (3) At the same time, the addition of organic solvents such as methanol may further reduce the matrix effect in the ultrafiltrate, making the four free thyroid hormones T3, rT3, T4 and cortisol more stable and easier to accurately detect, significantly improving the detection sensitivity, thereby truly achieving the simultaneous and accurate detection of the four free hormones of free thyroid hormones T3, rT3, T4 and cortisol in saliva;

[0040] (4) One test can simultaneously quantify the levels of four hormones in saliva: free thyroid hormones T3, T4, rT3, and cortisol;

[0041] (5) The pre-treatment operation is simple, avoiding complex operations such as liquid-liquid extraction, drying and re-dissolution, solid-phase extraction, and derivatization. It has low requirements for operators and saves manpower and working time. It is suitable for high-throughput operation and automation of detection methods, with high processing efficiency and low consumables cost, which will bring more convenience to auxiliary diagnosis of clinical diseases and health management;

[0042] (5) It is equipped with quality control samples and methods, which makes it easier for scientific researchers to find problems in the test and correct them in time, and facilitates the clinical promotion and application of the method. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the liquid phase detection chromatogram of the low concentration quality control (L) sample after pretreatment in Example 1, wherein Figure 1 (Top) is the chromatogram of free thyroid hormone T3 and rT3 in the sample; Figure 1 (Middle) is the chromatogram of free thyroid hormone T4. Figure 1 (Bottom) Chromatogram of free cortisol.

[0044] Figure 2 This is the standard curve of the thyroid hormone T3 in Example 1.

[0045] Figure 3 This is the standard curve of the thyroid hormone T4 in Example 1.

[0046] Figure 4 This is the standard curve of the thyroid hormone rT3 in Example 1.

[0047] Figure 5 This is the standard curve of cortisol in Example 1. DETAILED DESCRIPTION

[0048] The present invention will be further described in detail below with reference to the accompanying drawings and examples. It should be noted that the following examples are intended to facilitate understanding of the present invention and do not have any limiting effect on the present invention.

[0049] Example 1 Sample preparation, pretreatment and detection

[0050] The following preparation is based on the use of solid standards. If commercially purchased standard stock solutions are used, the preparation method must be adjusted according to the actual concentration of the standard stock solution used.

[0051] 1. Sample Preparation

[0052] 1. Preparation of standard series solutions

[0053] 1.1 Preparation of primary stock solutions of the four hormones

[0054] Preparation of primary stock solutions of thyroid hormones T3, T4, rT3, and cortisol: According to Table 1, accurately weigh an appropriate amount of standard substances and add an appropriate volume of methanol to prepare primary stock solutions of the four hormones.

[0055] Table 1. Preparation of primary stock solution

[0056]

[0057] 1.2 Preparation of secondary stock solutions of four hormones

[0058] Appropriate volumes of the primary stock solutions of the four hormones were taken separately, and appropriate volumes of methanol were added to dilute them into secondary stock solutions of the four hormones. The specific dilutions are shown in Table 2.

[0059] Table 2. Preparation of secondary stock solution

[0060]

[0061] 1.3 Preparation of tertiary stock solutions of four hormones

[0062] Appropriate volumes of the secondary stock solutions of the four hormones were taken separately, and appropriate volumes of methanol were added to dilute them into tertiary stock solutions of the four hormones. The specific dilutions are shown in Table 3.

[0063] Table 3. Preparation of tertiary stock solution

[0064]

[0065] 1.4 Preparation of the fourth-level stock solution

[0066] Appropriate volumes of the tertiary stock solutions of the three hormones were taken separately, and diluted with appropriate volumes of 50% methanol to form quaternary stock solutions. The specific dilutions are shown in Table 4.

[0067] Table 4. Preparation of the fourth-level stock solution

[0068]

[0069] 1.5 Preparation of MIX stock solution

[0070] The stock solutions of the four hormones with different concentrations were drawn separately and added into the same centrifuge tube, and then further diluted with 50% methanol solution to obtain a mixed stock solution of the four hormones, as shown in Table 5.

[0071] Table 5. Preparation of MIX stock solution

[0072]

[0073] 1.6 Preparation of standard curve working solution

[0074] The MIX mixed stock solution was diluted with 50% methanol to obtain a series of standard solutions SD1 to SD6; the specific preparation method is shown in Table 6.

[0075] Table 6. Preparation of standard curve working solution

[0076]

[0077] The stock solution prepared above was stored in a -80°C refrigerator; the standard curve working solution was divided into small portions, the temporarily unused portion was stored in a -80°C refrigerator, and the currently used working solution was stored in a 4°C refrigerator.

[0078] 2. Preparation of internal standard working solution

[0079] 2.1 Preparation of internal standard stock solution

[0080] Preparation of primary stock solutions of T3-13C6, rT3-13C6, T4-13C6 and Cortisol-d4: According to Table 7, accurately weigh an appropriate amount of standard and add an appropriate volume of methanol to prepare primary stock solutions of the four hormone internal standards.

[0081] Table 7. Preparation of primary stock solutions of four hormone internal standards

[0082]

[0083] 2.2 Preparation of internal standard secondary stock solutions Take appropriate volumes of the four hormone internal standard primary stock solutions respectively, add appropriate volumes of 50% methanol to dilute them into the secondary stock solutions of the four hormones. The specific dilutions are shown in Table 8.

[0084] Table 8. Preparation of secondary stock solutions of four hormone internal standards

[0085]

[0086] 2.3 Preparation of internal standard tertiary stock solution

[0087] Appropriate volumes of the secondary stock solutions of the three hormone internal standards were taken separately, and appropriate volumes of methanol were added to dilute them into the tertiary stock solutions of the three hormone internal standards. The specific dilutions are shown in Table 9.

[0088] Table 9. Preparation of tertiary stock solutions of three hormone internal standards

[0089]

[0090] 2.4 Preparation of mixed internal standard MIX stock solution

[0091] The stock solutions of the four hormone internal standards at different concentrations were respectively drawn and added into the same centrifuge tube, and further diluted with 0.1% by volume formic acid methanol solution to obtain the internal standard mixed working solution of the four hormones, as shown in Table 10.

[0092] Table 10. Preparation of mixed internal standard MIX stock solution

[0093]

[0094] 2.5 Preparation of internal standard working solution

[0095] Take an appropriate amount of the mixed internal standard MIX stock solution and add 0.1% formic acid methanol solution by volume to dilute it to obtain the internal standard working solution. The specific preparation method is shown in Table 11.

[0096] Table 11. Preparation of internal standard working solution

[0097]

[0098] 3. Preparation of quality control samples

[0099] (1) Take a human saliva sample filtered through an ultrafiltration tube, mix it evenly, and add the same volume of methanol as the background;

[0100] (2) Take 100 μL of MIX mixed stock solution and add 900 μL of background as high concentration quality control (H);

[0101] (3) Take 50 μL of MIX mixed stock solution and add 950 μL of background as the medium concentration quality control (M);

[0102] (4) Take 10 μL of MIX mixed stock solution and add 990 μL of background as low concentration quality control (L).

[0103] 2. Sample Pretreatment

[0104] (1) Use a sample collection tube to collect about 1 ml of saliva sample, standard curve working solution, or quality control sample, place the sample collection tube in a centrifuge, and centrifuge at 3500 rpm for 10 min;

[0105] (2) 0.5 mL of supernatant was taken from the saliva collection tube and slowly added to the inner tube of an ultrafiltration centrifuge tube, which was then inserted into the collection tube. The ultrafiltration centrifuge tube was a Millpore Centrifree ultrafiltration device equipped with an Ultracel PL regenerated cellulose ultrafiltration membrane with a molecular weight limit (NMWL) of 30K.

[0106] (3) placing the ultrafiltration centrifuge tube assembled in step (2) into the rotor slot of a centrifuge and centrifuging for 30 minutes. The parameters of the centrifuge process are as follows: the centrifuge temperature is 37°C; the centrifugal speed is 2800 rpm, the start acceleration is set to 1 (slow start mode), and the stop deceleration is set to 1 (slow deceleration mode); the saliva sample is transferred from the inner tube to the collection tube;

[0107] (4) Take 100 μL of filtrate from the collection tube of the ultrafiltration tube, add 20 μL of internal standard working solution and 100 μL of methanol (anti-adsorption reagent) in sequence, mix well, and prepare for liquid chromatography tandem mass spectrometry system analysis.

[0108] 3. Sample Testing

[0109] Liquid chromatography-tandem mass spectrometry analysis was performed using gradient elution. Separation conditions for the analytes were established using reversed-phase chromatography as follows: a Phenomenex Kinetex 2.6 μm phenyl column (100A, 3×100 mm), a flow rate of 0.6 mL / min, and a column temperature of 40°C. Mobile phase A consisted of a 0.1% by volume formic acid solution in water, and mobile phase B consisted of a 0.1% by volume formic acid solution in acetonitrile. The volume ratio of mobile phase A to mobile phase B was 90-5%:10-95%. The gradient program is shown in Table 12. The retention times of thyroid hormone T3 and its internal standard were 3.46 min, rT3 and its internal standard were 3.63 min, T4 and its internal standard were 3.90 min, and cortisol and its internal standard were 3.08 min.

[0110] Table 12. Gradient elution program

[0111] Time (min) Mobile phase A (%) Mobile phase B (%) 1.2 90 10 1.25 75 25 3 65 35 3.6 45 55 3.65 5 95 4.55 5 95 4.6 90 10 5.3 90 10

[0112] When performing mass spectrometry detection, a triple quadrupole mass spectrometer was used for quantitative detection. The instrument model was SCIEX TripleQuad 6500+. The electrospray ionization source was used in positive ion mode (ESI+) and multiple reaction monitoring (MRM) mode for mass spectrometry detection. The corresponding mass spectrometry detection method settings are shown in Table 13 below, and the mass spectrometry parameter conditions are shown in Table 14.

[0113] Table 13. Mass spectrometry detection method parameters

[0114] Analyte / internal standard Q1 Q3 Declustering voltage DP Collision Energy CE Collision cell exit voltage CXP T4 777.6 731.7 135 37 14 T4 3C6-1 783.8 737.8 137 33 13 T3 651.9 605.8 114 30 11 T3 13C6-1 658 611.8 113 29 12 rT3 651.9 605.8 76 31 12 rT3 13C6-1 657.9 611.7 117 31 12 Cortisol-1 363.3 309.2 63 25 4 Cortisol-d4 367.4 313.1 115 25 6

[0115] Table 14. Mass spectrometry parameter conditions

[0116]

[0117] A triple quadrupole mass spectrometer can be used to detect the free thyroid hormones T3, T4, rT3 and cortisol in saliva samples through ion pairs in multiple reaction monitoring (MRM) mode and corresponding retention times. Isotopic internal standards are used to eliminate the matrix effect of the sample, thereby accurately quantifying the content of the free thyroid hormones T3, T4, rT3 and cortisol in saliva samples.

[0118] Figure 1 After pretreatment, the low-concentration quality control (L) sample was separated by liquid chromatography. The four hormones eluted at different times and were detected by MRM mode to quantify their contents. Figure 1 (Top) is the chromatogram of free thyroid hormone T3 and rT3 in the sample; Figure 1 (Middle) is the chromatogram of free thyroid hormone T4. Figure 1 (Bottom) Chromatogram of free cortisol.

[0119] 4. Data Processing and Analysis

[0120] 1. Draw a standard curve

[0121] After the standard curve samples are separated by liquid chromatography, the four hormones elute at different elution times and are detected by mass spectrometry MRM mode to detect their content. According to a certain standard concentration, the test sample is configured for detection, and the concentration of the four hormone standards of free thyroid hormones T3, T4, rT3 and cortisol is used as the horizontal axis, and the peak area ratio of the four hormones of free thyroid hormones T3, T4, rT3 and cortisol to their respective internal standards is used as the vertical axis. Linear regression is performed to obtain a standard curve. The standard curve is prepared as shown in the figure. Figure 2-5 As shown, Figure 2 is the standard curve of thyroid hormone T3, Figure 3 is the standard curve of thyroid hormone T4, Figure 4 is the standard curve of thyroid hormone rT3, Figure 5 is the standard curve of cortisol; the standard curve equation and correlation coefficient are shown in Table 15. It can be seen that within the concentration range shown by the standard curve, the linear relationship is good.

[0122] Table 15. Standard curve regression equation and correlation coefficient

[0123]

[0124]

[0125] 2. Accuracy inspection

[0126] The peak area ratio of the analyte to its internal standard in the quality control sample was substituted into the established standard curves of the four free thyroid hormones T3, T4, rT3, and cortisol to calculate the concentrations of the four free hormones in the quality control sample. The detection accuracy of the three concentrations of quality control samples was then calculated. The test results are shown in Tables 16-19.

[0127] Table 16. Accuracy of T3

[0128] T3 T3 T3 Theoretical value (pg / mL) Measured value (pg / mL) Accuracy % Quality Control H 20 20.06 103 Quality Control 10 10.07 107 Quality Control 2 2.08 109

[0129] Table 17. Accuracy of T4

[0130] T4 T4 T4 Theoretical value (pg / mL) Measured value (pg / mL) Accuracy % Quality Control H 20 20.22 101 Quality Control 10 9.64 96.4 Quality Control 2 1.96 98.0

[0131] Table 18. Accuracy of rT3

[0132] rT3 rT3 rT3 Theoretical value (pg / mL) Measured value (pg / mL) Accuracy % Quality Control H 20 19.52 97.6 Quality Control 10 9.89 98.9 Quality Control 2 2.04 102

[0133] Table 19. Accuracy of Cortisol

[0134] cortisol cortisol cortisol Theoretical value Measured value Accuracy % Quality Control H 2500 2500 100 Quality Control 1250 1250 100 Quality Control 250 252.5 101

[0135] Based on the above data, we investigated the accuracy of free thyroid hormones (T3, T4, rT3, and cortisol) at three quality control levels: low, medium, and high. The results showed that the accuracy of this method for all four hormones was less than 15% at all levels, meeting clinical testing requirements. This quality control system comprehensively controls the experimental process, facilitating the control of test bias and quality in routine clinical testing and enabling timely identification of issues.

[0136] Example 2 Effect of different ultrafiltration membranes on test results

[0137] Because the ultrafiltration membrane has a certain adsorption effect on the free thyroid hormones T3, T4, rT3, and cortisol in the saliva sample, thereby affecting the test results, in this example, after completing the sample preparation according to the steps of Example 1, a low-concentration quality control sample (L) was taken and sample pretreatment was performed according to the sample pretreatment steps in Example 1. Different ultrafiltration membranes were used for ultrafiltration and centrifugation to investigate the adsorption effects of different ultrafiltration membranes on the free thyroid hormones T3, T4, rT3, and cortisol in the saliva sample. Each group was repeated 3 times and the average value was taken. The test results are shown in Table 20.

[0138] Table 20. Effects of different ultrafiltration membranes on test results

[0139]

[0140] As can be seen from Table 20, the adsorption of free thyroid hormones T3, T4, rT3, and cortisol in saliva samples by different ultrafiltration membranes will seriously affect the accuracy of the test results. Among them, Ultracel PL regenerated cellulose ultrafiltration membrane can better solve this problem and significantly improve the accuracy of the test results.

[0141] Example 3 Effect of Ultrafiltration Membrane Molecular Weight Limit on Test Results

[0142] After completing the sample preparation according to the steps in Example 1, a low-concentration quality control sample (L) was taken and pre-treated according to the sample pre-treatment steps in Example 1. During the ultrafiltration and centrifugation process, ultrafiltration membranes with different molecular weight limits were selected for ultrafiltration and centrifugation. After the pre-treatment was completed, the liquid chromatography-tandem mass spectrometry conditions in Example 1 were used for detection to investigate the effect of different molecular weight limits of the ultrafiltration membrane on the detection accuracy of free thyroid hormones T3, T4, rT3, and cortisol in the saliva samples. Each group was repeated 3 times, and the average value was taken. The test results are shown in Table 21.

[0143] Table 21. Effects of different molecular weight limits of ultrafiltration membranes on test results

[0144]

[0145] As can be seen from Table 21, the different molecular weight limits of ultrafiltration membranes have significant differences in the detection results of free thyroid hormones T3, T4, rT3 and cortisol in saliva samples, resulting in large differences in the accuracy of the final test results.

[0146] When the molecular weight is 150K, the measured results for free thyroid hormones T3, T4, rT3, and cortisol are significantly higher than the theoretical values. This may be because the protein filtration removal in the saliva sample is not sufficient, resulting in some bound hormones not being effectively removed, causing the measured results to include some bound hormones, not just free hormones, resulting in higher test results. When the molecular weight is 1K, the measured results for free thyroid hormones T3, T4, rT3, and cortisol are slightly lower than the theoretical values. This may be because the ultrafiltration pressure increases, causing the detection error to increase. When the molecular weight is 10K-100K, the measured results for free thyroid hormones T3, T4, rT3, and cortisol are more accurate, fully meeting clinical testing requirements. The most preferred molecular weight is 30K, which can significantly improve detection sensitivity and accuracy.

[0147] Example 4 Selection of anti-adsorption reagents

[0148] In this example, after completing sample preparation according to the steps of Example 1, a low-concentration quality control sample (L) was pretreated according to the sample pretreatment steps of Example 1. Different types of organic solvents were used as anti-adsorption reagents, and one group without organic solvent served as the control group. The effect of different organic solvents on the detection accuracy of free thyroid hormones T3, T4, rT3, and cortisol in saliva samples was examined, thereby investigating the effect of different organic solvents on the anti-adsorption effect of free thyroid hormones T3, T4, rT3, and cortisol in saliva samples. Each group was repeated three times, and the average value was taken. The test results are shown in Table 22.

[0149] Table 22. Effects of different anti-adsorption reagents on test results

[0150]

[0151] As can be seen from Table 22, there is a very obvious adsorption phenomenon in the control group, and the test results are significantly lower than the theoretical values; different organic solvents have completely different anti-adsorption effects on the free thyroid hormones T3, T4, rT3 and cortisol in saliva samples, among which ethanol and propanol almost have no anti-adsorption effect, and the test values ​​are significantly lower than the theoretical values; methanol, acetonitrile and isopropanol can have a certain anti-adsorption effect, among which methanol has the best effect, which can significantly improve the detection sensitivity and accuracy of the free thyroid hormones T3, T4, rT3 and cortisol in saliva samples.

[0152] At the same time, the addition of organic solvents such as methanol may further reduce the matrix effect in the ultrafiltrate, making the four free thyroid hormones T3, rT3, T4 and cortisol more stable, without affecting each other, easier to be accurately detected, and significantly improving the detection sensitivity, thereby truly realizing the simultaneous and accurate detection of the four free hormones of free thyroid hormones T3, rT3, T4 and cortisol in saliva.

[0153] Example 5 Effect of the Volume Ratio of Sample Filtrate and Methanol on the Detection Results

[0154] In this example, after completing the sample preparation according to the steps of Example 1, a low-concentration quality control sample (L) was taken and pre-treated according to the sample pretreatment steps in Example 1, wherein the anti-adsorption reagent was methanol, and the volume ratios of the filtrate and methanol were 1:0.5, 1:1, 1:3, 1:5, and 1:7, respectively. The effects of different volume ratios of the sample filtrate and methanol on the detection accuracy of free thyroid hormones T3, T4, rT3, and cortisol in the saliva samples were investigated, thereby investigating the effects of different volume ratios of the sample filtrate and methanol on the anti-adsorption effect of free thyroid hormones T3, T4, rT3, and cortisol in the saliva samples. Each group was repeated 3 times, and the average value was taken. The test results are shown in Table 23.

[0155] Table 23. Effects of different volume ratios of filtrate and methanol on test results

[0156]

[0157] Table 23 shows that different volume ratios of sample filtrate to methanol significantly affect the anti-adsorption effect on free thyroid hormones T3, T4, rT3, and cortisol in saliva samples. When the volume ratios of filtrate to methanol were 1:0.1 and 1:0.5, there was almost no anti-adsorption effect. However, when the volume ratio reached 1:1, the anti-adsorption effect and the improvement in detection sensitivity were very significant. When the volume ratio of filtrate to methanol was between 1:1 and 1:5, the anti-adsorption effect was ideal for free thyroid hormones T3, T4, rT3, and cortisol in saliva samples, and the detection accuracy was high. However, when the volume ratio of filtrate to methanol reached 1:7, the anti-adsorption effect decreased. This may be due to the high methanol ratio, which increased the detection error and made it difficult to accurately detect low levels of free hormones. Therefore, the volume ratio of filtrate to methanol should be between 1:1 and 1:5 to achieve the best anti-adsorption effect.

[0158] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the scope defined by the claims.

Claims

1. A kit for simultaneously determining free thyroid hormones T3, rT3, T4 and cortisol in saliva, characterized in that: The following steps are involved: 1) Take a saliva sample and centrifuge it; 2) Take the supernatant and place it in an ultrafiltration centrifuge tube for ultrafiltration centrifugation; 3) collecting the filtrate obtained by ultrafiltration and centrifugation, adding an internal standard solution and an anti-adsorption agent, vortex mixing, and analyzing the mixture using a liquid chromatography-mass spectrometry system; the volume ratio of the filtrate obtained by ultrafiltration and centrifugation to the anti-adsorption agent is 1:1 to 1:5; The kit comprises a standard working solution, a quality control sample, an internal standard solution, an anti-adsorption reagent, and a liquid chromatography mobile phase; the standard working solution is a solution containing standard concentrations of thyroid hormones T3, rT3, T4, and cortisol; the quality control sample is a saliva quality control sample containing three different concentration levels: low, medium, and high; the anti-adsorption reagent is methanol; and the liquid chromatography mobile phase comprises mobile phase A and mobile phase B, mobile phase A is a 0.05-0.2% by volume formic acid aqueous solution, and mobile phase B is an acetonitrile solution of 0.05-0.2% by volume formic acid.

2. The use according to claim 1, characterized in that The ultrafiltration centrifuge tube in step 2) uses Ultracel PL regenerated cellulose ultrafiltration membrane, and the molecular weight cutoff limit of the membrane is within the range of 10K-100K.

3. The use according to claim 2, characterized in that The ultrafiltration centrifugation time in step 2) is 15 minutes to 1 hour, the centrifugal speed is 2000 to 4000 rpm, and the centrifugal temperature of the centrifuge is 36 to 38° C.; the centrifugal speed in step 1) is 3000 to 4000 rpm.

4. The use according to claim 3, characterized in that The ultrafiltration centrifuge tube described in step 2) includes an inner tube and a collection tube. The supernatant is first placed in the inner tube, and then the inner tube is inserted into the collection tube. The tube is placed in a centrifuge for ultrafiltration centrifugation. The filtrate obtained by ultrafiltration centrifugation is located in the collection tube. The centrifuge is started and stopped in a slow acceleration and slow deceleration mode, and the speed is slowly increased and decreased.

5. The use according to any one of claims 1 to 4, characterized in that Step 3) The internal standard solution contains T3- 13 C6, rT3- 13 C6, T4- 13 C6 and Cortisol-d4; also includes comparison of quality control sample results and evaluation of detection error in step 4); the quality control sample preparation method of step 4) is: adding an equal volume of organic solvent to the filtrate obtained by ultrafiltration of the saliva sample, mixing, using this mixed solution as a matrix, adding a mixed standard solution containing different concentrations of thyroid hormones T3, rT3, T4 and cortisol, to obtain saliva matrix samples containing three different levels of concentration: low, medium and high as quality control samples.

6. The use according to claim 5, characterized in that Step 3) The liquid chromatography-mass spectrometry system, wherein the liquid chromatography adopts a gradient elution mode, the chromatographic column is a C18 or phenyl packing column, the mobile phase A is a 0.05-0.2 volume% formic acid aqueous solution, and the mobile phase B is a 0.05-0.2 volume% formic acid acetonitrile solution. The gradient elution mode is adopted, and the volume ratio of mobile phase A to mobile phase B is 60-0%:40-100%.

7. The use according to claim 6, characterized in that The liquid chromatography-mass spectrometry system uses a triple quadrupole mass spectrometer, which uses an electrospray ionization source in positive ion mode (ESI+) and multiple reaction monitoring (MRM) mode for mass spectrometry detection. The quality control sample result comparison and detection error evaluation in step 4) evaluate the accuracy and error of the batch detection process by comparing the deviation between the detection value of the quality control sample and the theoretical target value. The detection results include the measured levels of four hormones, T3, rT3, T4, and cortisol, in the quality control sample.

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