CN116298023A - Method for simultaneously measuring free thyroid hormones T3, rT3, T4 and cortisol in saliva - Google Patents
Method for simultaneously measuring free thyroid hormones T3, rT3, T4 and cortisol in saliva Download PDFInfo
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N30/02—Column chromatography
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Abstract
The invention provides a method for simultaneously measuring free thyroid hormones T3, T4, rT3 and cortisol in saliva, which comprises the steps of removing protein and bound thyroid hormone and cortisol in saliva by ultrafiltration and centrifugation and selecting a filter membrane with a proper molecular weight cut-off limit range, and adding anti-adsorption agents such as methanol and the like in a proper proportion, so that the serious adsorption problem existing in the detection process of free thyroid hormones T3, rT3, T4 and cortisol in saliva is solved. By utilizing a high performance liquid chromatography tandem mass spectrometry analysis technology, four free-state hormones of free thyroid hormones T3, rT3, T4 and cortisol in saliva are detected simultaneously and accurately at one time. The method has the advantages of high flux, simple operation, high treatment efficiency, low consumable cost, high accuracy and high sensitivity, and is convenient for clinical popularization and application.
Description
This application is a divisional application of the parent application 202110394022.0.
Technical Field
The invention belongs to the technical field of biochemical analysis, and particularly relates to a method for simultaneously measuring free thyroid hormones T3, rT3, T4 and cortisol in saliva.
Background
Thyroid hormone is an important endocrine hormone for human body, and has the main functions of improving the metabolism rate of the organism, increasing oxygen consumption, promoting growth and development and promoting differentiation and maturation of nervous system. Thyroid hormones include thyroxine (T4), triiodothyronine (T3), and trans-triiodothyronine (rT 3). Thyroid hormone is an important marker for evaluating thyroid function, is the main hormone of thyroid gland to target organ, and the total concentration of thyroid hormone in serum reflects the secretion state of thyroid gland.
Cortisol is an important glucocorticoid in the body and is responsible for regulating emotion, keeping healthy, maintaining immune cells and physiological functions such as inflammation, blood vessels, blood pressure and the like. Hypothalamic-pituitary-adrenal axis function is evaluated clinically by measuring the level of cortisol in serum or saliva. Wherein the level of cortisol in saliva is correlated with the level of serum cortisol, and can be used to evaluate cortisol levels in the circulatory system of the human body. Clinically, cortisol levels are used to diagnose and screen for cushing's syndrome, adrenocortical insufficiency and other diseases.
Cortisol is also known as stress hormone, and current research tends to correlate stress with higher cortisol levels. One study published in journal of clinical endocrine and metabolism shows that cortisol reduces TSH and thus thyroid hormone production. Cortisol inhibits the conversion of T4 to T3, resulting in hypothyroidism. Another way cortisol exacerbates hypothyroidism is by its effect on blood glucose, which is a disturbance in blood glucose balance, resulting in hypoglycemia and hyperglycemia, and studies have shown that hypoglycemia interacts with hypothyroidism. It can be seen that there is a close relationship between cortisol levels and thyroid hormone levels, and that it is highly desirable to use them for the assisted diagnosis and health management of clinical disease by simultaneously detecting thyroid hormone and cortisol levels in humans.
The traditional test sample is serum, and the total or free concentration of hormone in the serum is determined. The existing studies demonstrate that levels of thyroid hormone and cortisol in saliva have a good correlation with serum cortisol levels, even many clinical studies have demonstrated that saliva is a more accurate clinical indicator. If saliva samples are adopted to accurately detect thyroid hormone and cortisol simultaneously, the saliva samples have the advantages of no wound, simple and convenient operation, continuous sampling, less interference by protein and the like, and more convenience is brought to clinical diagnosis.
The steroid hormone is partially bound to the protein, and unbound hormone is in a free state, wherein most of the thyroid hormone is bound to thyroid binding protein in peripheral blood, and the physiological effects are mainly free T4 (FT 4) and free T3 (FT 3). Therefore, in clinical tests, the most common indicators are T3, T4, FT3, FT4. However, the free thyroid hormone content in serum and saliva is low and is in pg/mL level, so the sensitivity and specificity of the detection method are extremely high.
Traditional detection methods include an immunological method, an electrochemical method, a fluorescence method and the like, but have poor specificity, can not solve the problem of interference of endogenous and exogenous glucocorticoids, and have lower cortisol level in saliva and lower detection accuracy. In a continual improvement of the existing method, quantitative analysis methods for detecting total thyroid hormone in serum by an HPLC-MS/MS method have been studied. However, free thyroid hormones T3, rT3, T4 and cortisol cannot be detected simultaneously, and are not suitable for saliva samples, limiting the range of applications.
CN107576624B discloses a method for detecting cortisol in saliva, which removes salivary mucin in saliva by filtering with a composite microfiltration membrane, but the method cannot remove cortisol and thyroid hormone in a bound state in saliva, and the residual bound state hormone in a sample will affect the detection result of free state hormone, so that it cannot be used for accurately detecting free thyroid hormones T3, rT3, T4 and cortisol in saliva.
Because the free thyroid hormones T3, rT3, T4 and cortisol in saliva have very low content, no simple and efficient detection and analysis method is available at present, and the content of the free thyroid hormones T3, rT3, T4 and cortisol 4 in saliva can be accurately detected at the same time.
Disclosure of Invention
In order to solve the problems, the invention provides an improved high performance liquid chromatography tandem mass spectrometry method for simultaneously detecting free thyroid hormones T3, T4, rT3 and cortisol in a human saliva sample, which is characterized in that all proteins and bound thyroid hormones and cortisol in saliva are completely removed by ultrafiltration and centrifugation, then anti-adsorption reagents such as methanol are added for direct high performance liquid chromatography tandem mass spectrometry, and the free thyroid hormones T3, rT3, T4 and cortisol in saliva can be simultaneously and accurately detected.
In one aspect, the invention provides a method for simultaneously measuring free thyroid hormones T3, rT3, T4 and cortisol in saliva, which mainly comprises the following steps:
1) Taking saliva samples, and centrifuging;
2) Placing the supernatant into an ultrafiltration centrifuge tube, and performing ultrafiltration centrifugation;
3) And collecting filtrate obtained by ultrafiltration and centrifugation, adding an internal standard solution and an organic solvent for preventing adsorption, mixing uniformly by vortex, and analyzing by a liquid chromatography-mass spectrometry system.
According to the invention, a large number of researches prove that all proteins and bound thyroid hormone and cortisol in saliva can be completely removed through ultrafiltration centrifugation, but when the obtained filtrate is subjected to high performance liquid chromatography tandem mass spectrometry, free thyroid hormone T3, rT3, T4 and cortisol in a sample are easily adsorbed by consumable materials, so that the detection result is obviously lower, and accurate detection is difficult to realize.
Because the concentration of free thyroid hormones T3, rT3, T4 and cortisol in saliva is in pg/mL level, the polarity is smaller, and the detection process is completely different from that of serum samples, the interference problems such as consumable adsorption (such as adsorption to a 96-pore plate in the high-throughput detection process of high-performance liquid tandem mass spectrometry) and the like easily occur in the detection process, in order to prevent the adsorption of the ultrafiltration membrane, the ultrafiltration membranes of various different manufacturers are replaced, and finally the ultrafiltration membrane of Ultracel PL regenerated cellulose is selected to really solve the adsorption problem, but the adsorption problem of a 96-pore plate is still difficult to solve, even if the ultrafiltration membrane is replaced by a low-adsorption 96-pore plate, the very obvious adsorption problem still exists, and the detection result is obviously lower.
When ultrafiltration is not performed, the consumable has small adsorption effect on free thyroid hormones T3, rT3, T4 and cortisol due to the existence of protein, bound thyroid hormone, cortisol and the like in saliva samples, but after ultrafiltration purification, the free thyroid hormones T3, rT3, T4 and cortisol in ultrafiltrate are easy to cause low detection results due to adsorption and other problems in the detection process.
In order to solve the problem that free thyroid hormones T3, rT3, T4 and cortisol in filtrate are easily adsorbed by consumable materials, the inventor finds that a large amount of organic solvents such as methanol, acetonitrile or isopropanol are added into the filtrate after a large number of experiments, so that the adsorption problem can be effectively prevented; meanwhile, possibly due to the addition of organic solvents such as methanol and the like, the matrix effect in the ultrafiltrate is further reduced, so that the four free thyroid hormones T3, rT3, T4 and cortisol are more stable, the detection sensitivity is obviously improved, and the simultaneous and accurate detection of the free thyroid hormones T3, rT3, T4 and cortisol 4 in saliva is truly realized.
Further, the organic solvent for preventing adsorption is any one or two selected from methanol, acetonitrile and isopropanol; the volume ratio of the filtrate obtained by ultrafiltration and centrifugation to the anti-adsorption organic solvent is 1:1-1:5.
The added organic solvents such as methanol and the like need to be 1 to 5 times of the volume of the sample to play an obvious anti-adsorption role.
Further, the research proves that the methanol has better anti-adsorption effect, and can obviously improve the detection sensitivity.
Further, the specification of the ultrafiltration centrifuge tube in the step 2) is that an Ultracel PL regenerated cellulose ultrafiltration membrane is adopted, and the limit range of the molecular weight cut-off of the membrane is 10K-100K.
Ultrafiltration is a common method for detecting free substances in a laboratory, and can separate small molecular substances from proteins by using high pressure or centrifugal force, so that the free small molecules pass through a semipermeable membrane, and the large molecular proteins and the small molecules combined with the proteins are remained on the membrane, and the proteins with different molecular weights can be intercepted by selecting a filter membrane with proper specification.
The filter membrane with proper molecular weight cut-off can help to completely remove all proteins in saliva, including proteins combined with the combined hormone T3, rT3 and T4, so as to further improve the detection sensitivity of the free hormone in saliva.
Further, the ultrafiltration centrifugation time in the step 2) is 15 min-1 h, the centrifugation speed is 2000-4000 rpm, and the centrifugation temperature of the centrifuge is 36-38 ℃; the centrifugal rotating speed in the step 1) is 3000-4000 rpm.
Further, the ultrafiltration centrifuge tube in the step 2) comprises an inner tube and a collecting tube, wherein the supernatant is firstly placed in the inner tube, then the inner tube is inserted into the collecting tube, the inner tube is placed into a centrifuge for ultrafiltration centrifugation, and filtrate obtained by ultrafiltration centrifugation is positioned in the collecting tube; the starting and stopping process of the centrifugal machine adopts slow acceleration and slow deceleration modes, and the rotating speed is slowly increased and decreased.
Further, the internal standard solution of step 3) contains T3- 13 C6、rT3- 13 C6、T4- 13 C6 and Cortisol-d4.
Further, the method also comprises the quality control sample result comparison and detection error evaluation of the step 4); the preparation method of the quality control sample in the step 4) comprises the following steps: and filtering the saliva sample through an ultrafiltration tube to obtain filtrate, adding an organic solvent with the same volume, uniformly mixing, and adding mixed standard solutions containing thyroid hormones T3, rT3, T4 and cortisol with different concentrations to obtain saliva matrix samples with low, medium and high concentrations at different levels as quality control samples.
In addition, the existing researches and documents do not have a system and a method for controlling the system quality of the detection error of the detection method, the reproducibility and consistency of detection results of different batches are difficult to ensure, and the reliability of the method is difficult to evaluate.
According to the invention, saliva substrate samples with low, medium and high concentration levels are configured as quality control samples, so that the accuracy control of each detection is ensured. Meanwhile, as a quality control sample is adopted, the inventor is helped to find out the serious adsorption problem existing in the detection process of free state T3, rT3, T4 and cortisol, the content of free thyroid hormones T3, rT3, T4 and cortisol in saliva is very low, and the detection result of 4 free state hormones in saliva is greatly influenced by the existence of the adsorption problem. The inventor finds and confirms the efficacy of the anti-adsorption reagent on free thyroid hormones T3, rT3, T4 and cortisol under the assistance of a quality control sample, truly solves the serious adsorption problem existing in the detection process of free thyroid hormones T3, rT3, T4 and cortisol in saliva, greatly improves the detection sensitivity and brings more convenience to the auxiliary diagnosis and health management of clinical diseases. The method is convenient to popularize and apply, can help scientific researchers to discover problems existing in detection in real time, and enables comparison and research of results among rooms to be possible.
Further, in the liquid chromatography-mass spectrometry system of the step 3), a gradient elution mode is adopted in the liquid chromatography, a chromatographic column is a C18 or phenyl packing column, a mobile phase A is 0.05-0.2% by volume of formic acid aqueous solution, a mobile phase B is 0.05-0.2% by volume of formic acid acetonitrile solution, gradient elution is adopted, and the volume ratio of the mobile phase A to the mobile phase B is 60-0%: 40-100%.
Further, the mass spectrum adopted by the liquid chromatography-mass spectrometry combined system is a triple quadrupole mass spectrometer, and electrospray ion sources, a positive ion mode (ESI+) and a multi-reaction monitoring (MRM) mode are adopted for mass spectrum detection; and 4) comparing the quality control sample results in the step and evaluating the detection errors, wherein the accuracy and the errors of the batch of detection processes are evaluated by comparing the deviation of the detection values of the quality control samples and the theoretical target values, and the detection results comprise the actual measurement levels of T3, rT3 and T4 of the quality control samples and 4 hormones of cortisol.
In another aspect, the invention provides a detection kit for simultaneously determining free thyroid hormones T3, rT3, T4 and cortisol in saliva, wherein 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 as follows: a solution containing any one or more of the free thyroid hormones T3, rT3, T4 or cortisol at standard concentrations; the quality control sample is as follows: saliva matrix samples containing three different levels of concentration, low, medium and high; the anti-adsorption reagent is methanol; the liquid chromatography mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is 0.05-0.2% of formic acid water solution by volume percent, and the mobile phase B is 0.05-0.2% of formic acid acetonitrile solution by volume percent.
In yet another aspect, the invention provides the use of methanol for the preparation of an anti-adsorption agent for preventing thyroid hormone in saliva from being adsorbed by a consumable.
The invention has the following beneficial effects:
(1) Through selecting a filter membrane with a proper molecular weight cut-off limit range, all proteins and bound thyroid hormones T3, rT3, T4 and cortisol in saliva are completely removed, so that the detection sensitivity of free thyroid hormones T3, rT3, T4 and cortisol in saliva is improved;
(2) The optimal anti-adsorption reagent and the optimal adding proportion are selected, so that the serious adsorption problem existing in the detection process of free T3, rT3, T4 and cortisol in saliva is truly solved, and the detection sensitivity is greatly improved;
(3) Meanwhile, possibly due to the addition of organic solvents such as methanol and the like, the matrix effect in the ultrafiltrate is further reduced, so that the four free thyroid hormones T3, rT3, T4 and cortisol are more stable and are easier to accurately detect, the detection sensitivity is obviously improved, and the simultaneous accurate detection of the free thyroid hormones T3, rT3, T4 and 4 free hormones of cortisol in saliva is truly realized;
(4) The levels of free thyroid hormones T3, T4, rT3 and cortisol 4 in saliva can be simultaneously quantified by one-time detection;
(5) The pretreatment operation is simple and convenient, complex operations such as liquid-liquid extraction, blow-drying and re-dissolution, solid-phase extraction, derivatization and the like are avoided, the requirements on operators are low, meanwhile, the manpower and the working time are saved, the method is suitable for automation of high-throughput operation and detection modes, the treatment efficiency is high, the consumable cost is low, and more convenience is brought to auxiliary diagnosis and health management of clinical diseases;
(5) The quality control sample and the quality control method are provided, so that scientific researchers can find problems in detection in time and correct the problems, and the clinical popularization and application of the quality control method are facilitated.
Drawings
FIG. 1 is a liquid phase detection chromatogram of a pretreated low concentration quality control (L) sample in example 1, wherein FIG. 1 (top) is a chromatogram of free thyroid hormones T3, rT3 in the sample; FIG. 1 (middle) is a chromatogram of thyroid hormone T4 in the free state, and FIG. 1 (bottom) is a chromatogram of cortisol in the free state.
FIG. 2 is a standard curve of thyroid hormone T3 in example 1.
FIG. 3 is a standard curve of thyroid hormone T4 in example 1.
FIG. 4 is a standard curve of thyroid hormone rT3 in example 1.
Fig. 5 is a standard curve of cortisol in example 1.
Detailed Description
The invention will be described in further detail below with reference to the drawings and examples, it being noted that the examples described below are intended to facilitate an understanding of the invention and are not intended to limit the invention in any way.
Example 1 sample preparation, pretreatment and detection
The following formulation is based on the use of solid standards, such as standard stock solutions commercially available, with the formulation method being adjusted to the actual standard stock solution concentration used.
1. Sample preparation
1. Preparation of standard series solutions
1.1 Preparation of primary stock solution of 4 kinds of hormone
Preparation of thyroid hormones T3, T4, rT3 and cortisol primary stock: according to Table 1, a proper amount of standard substances were precisely weighed, and a proper amount of methanol was added to prepare a first-order stock solution of 4 kinds of hormones.
TABLE 1 preparation of primary stock solutions
1.2 Preparation of secondary stock solutions of 4 kinds of hormone
The 4 kinds of hormone primary stock solutions are respectively absorbed in proper volumes, and are diluted into the 4 kinds of hormone secondary stock solutions by adding proper volumes of methanol, wherein the specific dilution is shown in table 2.
TABLE 2 preparation of secondary stock solutions
1.3 Preparation of tertiary stock solutions of 4 kinds of hormones
The appropriate volumes of the secondary stock solutions of 4 hormones are respectively absorbed, and an appropriate volume of methanol is added to dilute the secondary stock solutions of 4 hormones into tertiary stock solutions of 4 hormones, and the specific dilutions are shown in table 3.
TABLE 3 preparation of tertiary stock solutions
1.4 preparation of four-stage stock solution
The appropriate volumes of three-stage stock solutions of 3 hormones are respectively absorbed, and 50% methanol with appropriate volumes is added for dilution to form four-stage stock solution, and the specific dilutions are shown in Table 4.
TABLE 4 preparation of four-stage stock solutions
1.5 preparation of Mixed MIX stock solution
Stock solutions of 4 hormones with different concentrations are respectively absorbed, added into the same centrifuge tube, and further added with 50% methanol solution for dilution to obtain mixed mix stock solution of 4 hormones, and the specific details are shown in table 5.
TABLE 5 preparation of Mixed MIX stock solution
1.6 preparation of working solution with Standard Curve
Diluting the MIX mixed stock solution with 50% methanol to obtain serial standard solutions SD 1-SD 6; the specific preparation method is shown in Table 6.
TABLE 6 preparation of standard curve working solutions
The stock solution prepared above is stored in a refrigerator at the temperature of minus 80 ℃; the standard curve working solution is split into small parts, the temporarily unused part is stored in a refrigerator at-80 ℃, and the existing working solution is stored in a refrigerator at 4 ℃.
2. Preparation of internal standard working solution
2.1 preparation of internal Standard stock solution
Preparation of primary stock solutions of T3-13C6, rT3-13C6, T4-13C6 and Cortisol-d 4: according to Table 7, a proper amount of standard substances are precisely weighed, and a proper amount of methanol is added to prepare a first-order stock solution of the internal standards of 4 hormones.
Table 7, preparation of primary stock solutions of 4 hormone internal standards
2.2 preparation of internal standard secondary stock solution A proper amount of 4 kinds of internal standard primary stock solutions of hormones are respectively absorbed, and 50% methanol with proper amount is added for dilution to obtain 4 kinds of secondary stock solutions of hormones, and the specific dilution is shown in Table 8.
Preparation of secondary stock solutions of internal standards for 4 hormones in Table 8
2.3 preparation of internal Standard three-stage stock solution
And respectively sucking proper volumes of secondary stock solutions of the internal standards of 3 hormones, and adding proper volumes of methanol to dilute the secondary stock solutions into the tertiary stock solutions of the internal standards of 3 hormones, wherein the specific dilutions are shown in Table 9.
Preparation of tertiary stock solutions of internal standards for Table 9, 3 hormones
2.4 preparation of Mixed internal standard MIX stock solution
Stock solutions of the internal standards of 4 hormones with different concentrations are respectively absorbed, added into the same centrifuge tube, and further added with 0.1% methanolic formate solution in volume ratio for dilution to obtain an internal standard mixed mix working solution of 4 hormones, and the specific details are shown in table 10.
TABLE 10 preparation of Mixed internal standard MIX stock
2.5 configuration of internal Standard working fluid
And adding a proper amount of mixed internal standard MIX stock solution into a 0.1% methanolic formate solution to dilute to obtain an internal standard working solution, wherein the specific preparation method is shown in Table 11.
TABLE 11 preparation of internal standard working fluids
3. Preparation of quality control samples
(1) Taking a human saliva sample filtered by a ultrafiltration tube, uniformly mixing, and adding methanol with the same volume as background;
(2) Taking 100 microliters of MIX mixed stock solution, adding 900 microliters of background, and taking the mixed stock solution as high-concentration quality control (H);
(3) Taking 50 microliters of MIX mixed stock solution, adding 950 microliters of background, and taking the background as medium concentration quality control (M);
(4) 10 microliters of MIX stock was taken and a background of 990 microliters was added as a low concentration quality control (L).
2. Sample pretreatment
(1) Collecting saliva sample or standard curve working solution or quality control sample about 1ml by using a sample collecting tube, placing the sample collecting tube in a centrifuge, and centrifuging at 3500rpm for 10min;
(2) Taking 0.5mL of supernatant from a saliva collection tube, slowly adding the supernatant into an inner tube of an ultrafiltration centrifuge tube, and inserting the inner tube into the collection tube, wherein the ultrafiltration centrifuge tube is a Millpore Centrifree ultrafiltration device equipped with an Ultracel PL regenerated cellulose ultrafiltration membrane having a molecular weight limit (NMWL) of 30K;
(3) Putting the ultrafiltration centrifuge tube assembled in the step (2) into a rotor slot of a centrifuge, and centrifuging for 30min, wherein the parameters of the centrifugation process are as follows: the temperature of the centrifuge is 37 ℃; centrifugal speed is 2800rpm, start acceleration is set to 1 (slow start mode), stop deceleration is set to 1 (slow deceleration mode); osmotic transfer of saliva sample from the inner tube into the collection tube;
(4) 100 microliters of filtrate was taken from the collection tube of the ultrafiltration tube, and 20 microliters of internal standard working solution and 100 microliters of methanol (anti-adsorption reagent) were added in sequence, and mixed well, ready for analysis by a liquid chromatography tandem mass spectrometry system.
3. Sample detection
When the liquid chromatography tandem mass spectrometry analysis is carried out, the liquid chromatography adopts gradient elution, and the reverse phase chromatography establishes the separation conditions of the to-be-detected object as follows: the chromatographic column is a Phenomenex Kinetex 2.6.6 μm phenyl column (100A, 3X 100 mm), the flow rate is 0.6mL/min, and the column temperature is 40 ℃; wherein the mobile phase A is formic acid aqueous solution with the volume ratio of 0.1%, the mobile phase B is acetonitrile solution with the volume ratio of 0.1%, and the volume ratio of the mobile phase A to the mobile phase B is 90-5%: 10-95%. The gradient procedure is shown in Table 12, with a retention time of 3.46min for thyroid hormone T3 and internal standard, 3.63min for thyroid hormone rT3 and internal standard, 3.90min for thyroid hormone T4 and internal standard, and 3.08min for cortisol and internal standard.
TABLE 12 gradient elution procedure
| 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 |
In mass spectrometry detection, a triple quadrupole mass spectrometer is adopted for detection and quantification, the model of the instrument is SCIEX Triple Quad 6500+, and a positive ion mode (ESI+) and a multi-reaction monitoring MRM mode of an electrospray ion source are adopted for mass spectrometry detection, wherein the corresponding mass spectrometry detection method is set as shown in the following table 13, and the mass spectrometry parameter conditions are shown in the table 14.
Table 13, mass spectrometry detection method parameters
| Analyte/internal standard | Q1 | Q3 | Declustering voltage DP | Collision energy CE | Collision cell outlet 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 |
Table 14, mass spectrum parameter conditions
By adopting a triple quadrupole mass spectrometer, the ion pairs in MRM mode and the corresponding retention time can be monitored through multiple reactions to detect free thyroid hormones T3, T4, rT3 and cortisol 4 hormones in saliva samples, and the matrix effect of the samples is eliminated by using the internal standard of isotopes, so that the content of the free thyroid hormones T3, T4, rT3 and cortisol 4 hormones in the saliva samples can be accurately quantified.
FIG. 1 shows the content of 4 kinds of hormones, which are detected by MRM mode and quantified, after pretreatment and liquid chromatography separation of low-concentration quality control (L) samples, wherein FIG. 1 (above) shows the chromatograms of thyroid hormones T3 and rT3 in free form in the samples; FIG. 1 (middle) is a chromatogram of thyroid hormone T4 in the free state, and FIG. 1 (bottom) is a chromatogram of cortisol in the free state.
4. Data processing and analysis
1. Drawing a standard curve
After separation of the standard curve samples by liquid chromatography, the 4 hormones peak at different elution times and are detected by mass spectrometry MRM mode, so that the content of the hormones is detected. According to a certain standard sample concentration, preparing a sample to be detected, detecting, taking the free thyroid hormone T3, T4, rT3 and cortisol 4 hormone standard substance concentrations as the abscissa, carrying out linear regression with the free thyroid hormone T3, T4, rT3 and cortisol 4 hormones, respectively, taking the peak area ratio of the internal standard substances as the ordinate, and obtaining a standard curve, wherein the standard curve is shown in figures 2-5, and the figures 2 are the standard curve of the thyroid hormone T3, the figures 3 are the standard curve of the thyroid hormone T4, the figures 4 are the standard curve of the thyroid hormone rT3, and the figures 5 are the standard curve of the cortisol; the standard curve equation and the correlation coefficient are shown in table 15, and it can be seen that the linear relationship is good in the concentration range shown by the standard curve.
TABLE 15 Standard Curve regression equation and correlation coefficient
2. Accuracy investigation
Substituting the area ratio of the to-be-detected object to the internal standard peak of the to-be-detected object in the quality control sample into the established standard curves of the free thyroid hormones T3, T4, rT3 and cortisol 4 hormones, calculating to obtain the concentrations of the 4 free hormones in the quality control sample, and calculating the detection accuracy of the quality control sample with the three concentrations, wherein the detection results are shown in tables 16-19.
Accuracy of tables 16, T3
| T3 | T3 | T3 | |
| Theoretical value (pg/mL) | Actual measurement value (pg/mL) | Accuracy% | |
| |
20 | 20.06 | 103 |
| |
10 | 10.07 | 107 |
| Quality control L | 2 | 2.08 | 109 |
Accuracy of Table 17, T4
| T4 | T4 | T4 | |
| Theoretical value (pg/mL) | Actual measurement value (pg/mL) | Accuracy% | |
| |
20 | 20.22 | 101 |
| |
10 | 9.64 | 96.4 |
| Quality control L | 2 | 1.96 | 98.0 |
Table 18 accuracy of rT3
| rT3 | rT3 | rT3 | |
| Theoretical value (pg/mL) | Actual measurement value (pg/mL) | Accuracy% | |
| |
20 | 19.52 | 97.6 |
| |
10 | 9.89 | 98.9 |
| Quality control L | 2 | 2.04 | 102 |
TABLE 19 accuracy of cortisol
| Cortisol | Cortisol | Cortisol | |
| Theoretical value | Actual measurement value | Accuracy% | |
| Quality control H | 2500 | 2500 | 100 |
| Quality control M | 1250 | 1250 | 100 |
| Quality control L | 250 | 252.5 | 101 |
By combining the data, we examine the accuracy of the free thyroid hormones T3, T4, rT3 and cortisol 4 at three quality control levels, and the results show that the accuracy of the method for quality control of the 4 hormones at different levels is less than 15%, which meets the clinical detection requirements. The quality control system comprehensively controls the experimental process, is favorable for controlling the detection deviation and the detection quality in daily clinical detection, and can find problems in time.
Example 2 Effect of different Ultrafiltration membranes on detection results
Since ultrafiltration membranes have a certain adsorption effect on free thyroid hormones T3, T4, rT3 and cortisol in saliva samples, so that detection results are affected, in this example, after sample preparation is completed according to the steps of example 1, a low-concentration quality control (L) sample is taken to perform sample pretreatment according to the sample pretreatment steps of example 1, wherein ultrafiltration centrifugation is performed by using different ultrafiltration membranes respectively, the adsorption effect of different ultrafiltration membranes on free thyroid hormones T3, T4, rT3 and cortisol in saliva samples is examined, each group is repeated 3 times, average values are obtained, and detection results are shown in Table 20.
TABLE 20 influence of different Ultrafiltration membranes on detection results
As can be seen from table 20, the accuracy of the detection results can be seriously affected by the adsorptivity of the free thyroid hormones T3, T4, rT3 and cortisol in the saliva sample by different ultrafiltration membranes, wherein the ultrafiltration membrane of the Ultracel PL regenerated cellulose can better solve the problem, and the accuracy of the detection results is obviously improved.
Example 3 influence of UF molecular weight Limit on detection results
After sample preparation was completed according to the procedure of example 1, a low concentration quality control (L) sample was taken and subjected to sample pretreatment according to the sample pretreatment procedure of example 1, wherein during ultrafiltration centrifugation, ultrafiltration membranes with different molecular weight limits were selected for ultrafiltration centrifugation, detection was performed after pretreatment was completed using the lc tandem mass spectrometry conditions of example 1, the different molecular weight limits of the ultrafiltration membranes were examined, the influence on the detection accuracy of free thyroid hormone T3, T4, rT3 and cortisol in saliva samples was examined, each group was repeated 3 times, the average was taken, and the detection results are shown in table 21.
TABLE 21 influence of different molecular weight limits of Ultrafiltration films on detection results
As can be seen from table 21, the different molecular weight limits of the ultrafiltration membrane significantly differ in the detection results of free thyroid hormones T3, T4, rT3 and cortisol in saliva samples, resulting in a large difference in the accuracy of the final detection results.
When the molecular weight is 150K, the measured results of free thyroid hormones T3, T4, rT3 and cortisol are significantly higher than the theoretical values, probably because the protein in the saliva sample is insufficiently filtered and removed, so that part of the bound hormone is not effectively removed, and the measured results include part of the bound hormone, which is not only free hormone, and therefore the detection results are higher. At a molecular weight of 1K, the measured results for the free thyroid hormones T3, T4, rT3 and cortisol were slightly below the theoretical values, probably due to the increased ultrafiltration pressure, resulting in increased detection errors. When the molecular weight is 10K-100K, the accuracy of the actual measurement results of the free thyroid hormones T3, T4, rT3 and cortisol is higher, and the clinical detection requirements are completely met, wherein the most preferable molecular weight is 30K, and the detection sensitivity and accuracy can be remarkably improved.
Example 4 selection of anti-adsorption Agents
In this example, after sample preparation was completed according to the procedure of example 1, a low concentration quality control (L) sample was taken and subjected to sample pretreatment according to the sample pretreatment procedure of example 1, wherein the anti-adsorption reagents were respectively selected from different types of organic solvents, one group was not added with an organic solvent as a control group, the influence on the detection accuracy of free thyroid hormones T3, T4, rT3 and cortisol in saliva samples was examined, the influence of different organic solvents on the anti-adsorption effect of free thyroid hormones T3, T4, rT3 and cortisol in saliva samples was examined, each group was repeated 3 times, and the average was taken, and the detection results are shown in table 22.
TABLE 22 influence of different anti-adsorption reagents on detection results
As can be seen from table 22, the control group has a very significant adsorption phenomenon, and the detection result is significantly lower than the theoretical value; the anti-adsorption effects of the different organic solvents on free thyroid hormones T3, T4, rT3 and cortisol in saliva samples are completely different, wherein ethanol and propanol almost cannot have the anti-adsorption effect, and the detection value is obviously lower than the theoretical value; methanol, acetonitrile and isopropanol can play a certain role in preventing adsorption, wherein the best effect is methanol, and the sensitivity and accuracy of detecting free thyroid hormone T3, T4, rT3 and cortisol in saliva samples can be obviously improved.
Meanwhile, possibly due to the addition of organic solvents such as methanol and the like, the matrix effect in the ultrafiltrate is further reduced, so that the four free thyroid hormones T3, rT3, T4 and cortisol are more stable, cannot influence each other, are easier to accurately detect, and remarkably improve the detection sensitivity, thereby truly realizing the simultaneous accurate detection of the free thyroid hormones T3, rT3, T4 and the 4 free hormones of cortisol in saliva.
Example 5 influence of the volume ratio of sample filtrate and methanol on the detection results
In this example, after sample preparation was completed according to the procedure of example 1, a low-concentration quality control (L) sample was taken and subjected to sample pretreatment according to the sample pretreatment procedure of example 1, wherein the anti-adsorption agent was methanol, the volume ratio of filtrate to methanol was 1:0.5, 1:1, 1:3, 1:5, and 1:7, respectively, the different volume ratios of the sample filtrate to methanol were examined, the influence of the different volume ratios of the sample filtrate to methanol on the detection accuracy of free thyroid hormones T3, T4, rT3, and cortisol in the saliva sample was examined, the influence of the different volume ratios of the sample filtrate to the anti-adsorption effect of free thyroid hormones T3, T4, rT3, and cortisol in the saliva sample was repeated 3 times per group, and the detection results were averaged as shown in table 23.
TABLE 23 influence of different volume ratios of filtrate and methanol on detection results
As can be seen from table 23, the different volume ratios of the sample filtrate and methanol have a significant effect on the anti-adsorption effect of free thyroid hormones T3, T4, rT3 and cortisol in saliva samples, and hardly play any role in anti-adsorption when the volume ratio of filtrate to methanol is 1:0.1 and 1:0.5; when the volume ratio of the filtrate to the methanol reaches 1:1, the anti-adsorption effect and the effect of improving the detection sensitivity are very obvious; when the volume ratio of the filtrate to the methanol is in the range of 1:1 to 1:5, the ideal anti-adsorption effect can be achieved on free thyroid hormones T3, T4, rT3 and cortisol in saliva samples, and the detection accuracy is high; however, when the volume ratio of the filtrate to the methanol reaches 1:7, the anti-adsorption effect is reduced, which is probably due to the fact that the methanol ratio is too high, so that the detection error is increased, and accurate detection of low-content free hormone is difficult to realize. Therefore, the volume ratio of the filtrate to the methanol should be in the range of 1:1 to 1:5 to exert the best anti-adsorption effect.
Although the present invention is disclosed above, the present invention is not limited thereto. Various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention, and the scope of the invention should be assessed accordingly to that of the appended claims.
Claims (10)
1. The detection kit for simultaneously determining free thyroid hormones T3, rT3, T4 and cortisol in saliva is characterized by 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 as follows: solutions containing standard concentrations of thyroid hormones T3, rT3, T4 and cortisol; the quality control sample is as follows: saliva quality control samples containing three different concentration levels, low, medium, and high; the anti-adsorption reagent is methanol; the liquid chromatography mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is 0.05-0.2% of formic acid water solution by volume percent, and the mobile phase B is 0.05-0.2% of formic acid acetonitrile solution by volume percent.
2. Use of methanol for the preparation of an anti-adsorption agent for preventing free thyroid hormones T3, rT3, T4 in saliva from being adsorbed by a consumable.
3. A method for simultaneously measuring free thyroid hormones T3, rT3, T4 and cortisol in saliva, comprising the steps of:
1) Taking saliva samples, and centrifuging;
2) Placing the supernatant into an ultrafiltration centrifuge tube, and performing ultrafiltration centrifugation;
3) And collecting filtrate obtained by ultrafiltration and centrifugation, adding an internal standard solution and an organic solvent for preventing adsorption, mixing uniformly by vortex, and analyzing by a liquid chromatography-mass spectrometry system.
4. A method according to claim 3, wherein the organic solvent for anti-adsorption is any one or two selected from methanol, acetonitrile, isopropanol; the volume ratio of the filtrate obtained by ultrafiltration and centrifugation to the anti-adsorption organic solvent is 1:1-1:5.
5. The method of claim 3 or 4, wherein the ultrafiltration centrifuge tube of step 2) employs an Ultracel PL regenerated cellulose ultrafiltration membrane having a molecular weight cut-off in the range of 10K to 100K.
6. The method of claim 5, wherein the ultrafiltration centrifugation time of step 2) is 15min to 1h, the centrifugation speed is 2000 to 4000rpm, and the centrifugation temperature of the centrifuge is 36 to 38 ℃; the centrifugal rotating speed in the step 1) is 3000-4000 rpm.
7. The method of claim 6, wherein the ultrafiltration centrifuge tube in step 2) comprises an inner tube and a collecting tube, wherein the supernatant is placed in the inner tube, then the inner tube is inserted into the collecting tube, the inner tube is placed in a centrifuge for ultrafiltration centrifugation, and the filtrate obtained by ultrafiltration centrifugation is placed in the collecting tube; the starting and stopping process of the centrifugal machine adopts slow acceleration and slow deceleration modes, and the rotating speed is slowly increased and decreased.
8. The process of any one of claims 3 to 7, wherein the internal standard solution of step 3) comprises T3- 13 C6、rT3- 13 C6、T4- 13 C6 and Cortisol-d4; the method further comprises the steps of quality control sample result comparison and detection error evaluation in the step 4); the preparation method of the quality control sample in the step 4) comprises the following steps: adding an equal volume of organic solvent into a filtrate obtained by ultrafiltration of a saliva sample, uniformly mixing, taking the mixed solution as a matrix, and adding mixed standard solutions containing thyroid hormones T3, rT3, T4 and cortisol with different concentrations to obtain saliva matrix samples with three different levels of concentrations of low, medium and high as quality control samples.
9. The method of claim 8, wherein the liquid chromatography-mass spectrometry system of step 3) employs a gradient elution mode, wherein the chromatographic column is a C18 or phenyl packed column, mobile phase a is an aqueous solution of 0.05-0.2% by volume formic acid, mobile phase B is an acetonitrile solution of 0.05-0.2% by volume formic acid, and the volume ratio of mobile phase a to mobile phase B is 60-0% by gradient elution mode: 40-100%.
10. The method of claim 9, wherein the mass spectrum employed by the liquid chromatography-mass spectrometry system is a triple quadrupole mass spectrometer and mass spectrometry is performed using an electrospray ion source and positive ion mode (esi+) and Multiple Reaction Monitoring (MRM) modes; and 4) comparing the quality control sample results in the step and evaluating the detection errors, wherein the accuracy and the errors of the batch of detection processes are evaluated by comparing the deviation of the detection values of the quality control samples and the theoretical target values, and the detection results comprise the actual measurement levels of T3, rT3 and T4 of the quality control samples and 4 hormones of cortisol.
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