Method for simultaneously detecting various hormones in saliva based on high performance liquid chromatography-tandem mass spectrometry

Abstract

The invention relates to the technical field of analytical chemistry, in particular to a method for simultaneously detecting multiple hormones in saliva based on high performance liquid chromatography-tandem mass spectrometry. The method for simultaneously detecting various hormones in saliva based on high performance liquid chromatography-tandem mass spectrometry provided by the invention is simple to operate, can realize qualitative and quantitative detection of various hormones with lower content in saliva by using the same pretreatment condition and instrument condition, can simultaneously detect melatonin, cortisol, dehydroepiandrosterone sulfate and other substances, and has a wide application prospect. The detection working steps and time are obviously reduced, and the detection sensitivity and accuracy are improved. In addition, the tedious procedure of collection in a hospital is avoided, a subject can collect samples at home, the samples are convenient to obtain and transport, and the device is used for evaluating the psychological health condition of the subject and treating and preventing clinically related mental and psychological diseases and has important significance in reducing the burden of patients and families and improving the population quality.

Description

Technical Field

[0001] The invention relates to the technical field of analytical chemistry, and in particular to a method for simultaneously detecting multiple hormones in saliva based on high performance liquid chromatography tandem mass spectrometry. Background Art

[0002] With the rapid development of social economy, new business forms and new social models are constantly emerging under the background of scientific and technological innovation. While enjoying the dividends of development, the majority of participants in social production are also facing huge work and life pressure. As the psychological pressure gradually increases, the risk of mental and psychological diseases is also on the rise. For example, psychological diseases such as depression, stress disorders, and anxiety are becoming increasingly prominent. According to the statistical report of the World Health Organization, among patients with mental and psychological diseases worldwide, the number of patients with depression is as high as about 350 million, with a prevalence rate of about 11%. Depression is likely to exceed some common chronic diseases in the next few years and become one of the most important diseases that endanger people's health. In addition, mental health problems are gradually showing a "younger age" trend. Therefore, it is urgent to apply modern scientific and technological means to play an important role in the prevention and treatment of mental and psychological diseases.

[0003] Hormones are essential active substances for maintaining life. Among them, melatonin (MEL) is an amine hormone secreted by the pineal gland of the brain. Its secretion has a circadian rhythm, less during the day and more at night. Studies have found that patients with depression often have abnormal melatonin secretion, especially in seasonal affective disorder, the circadian rhythm of melatonin may be disturbed. In addition, under chronic stress or anxiety, the secretion of melatonin will also be affected. Steroid hormones are fat-soluble small molecules produced by a series of enzymes from cholesterol. Among them, cortisol and dehydroepiandrosterone sulfate are very important adrenal cortical hormones. Cortisol (Cortisol), as the main glucocorticoid secreted by the adrenal gland, exists in the hypothalamus-pituitary-adrenal axis. In addition to its important functions in maintaining the connection between immune cells and inflammation, blood vessels and blood pressure, cortisol is also known as a stress hormone. Its rhythmic production enables the body to adapt to stressful environments and plays an important role in regulating emotions and health. Studies have shown that patients with depression are often accompanied by elevated cortisol levels, especially in chronic stress or severe depression, so it can be used as an important indicator for assessing psychological stress. Like cortisol, dehydroepiandrosterone sulfate (DHEAS), as an adrenal cortex hormone, has a strong correlation with psychological activities or psychological diseases such as long-term stress, anxiety, and depression. It is reported that the level of dehydroepiandrosterone sulfate is positively correlated with healthy psychological conditions, and the level of dehydroepiandrosterone sulfate in patients with depression may be low. The abnormality of dehydroepiandrosterone sulfate is related to the severity of depression and the response to treatment. In addition, dehydroepiandrosterone sulfate is believed to have anti-stress effects. Individuals with low levels of dehydroepiandrosterone sulfate may be more likely to experience adverse reactions during stress reactions. It can also be used as an indicator to assess mental health. Therefore, accurate detection and quantification of melatonin, cortisol, and dehydroepiandrosterone sulfate are of great significance for assessing mental health and preventing and treating clinically related mental and psychological diseases.

[0004] At present, the commonly used detection methods in clinical practice are mainly enzyme-linked immunosorbent assay, electrochemiluminescence immunoassay, etc., which can be automated, have fast detection and low cost, but this method will be interfered by the structurally similar substances in the detection matrix, resulting in large deviations in the results, low sensitivity, poor specificity, and only one hormone can be detected at a time, and the detection takes a long time, which cannot meet the clinical requirements for detection efficiency and detection accuracy. In recent years, high performance liquid chromatography tandem mass spectrometry (LC MS) has been popularized in the field of clinical diagnosis, and the advantages of high sensitivity and high specificity of this technology have been widely recognized. This detection method can effectively solve the problem of low detection sensitivity and specificity, and through method optimization, multiple substances can be detected simultaneously, significantly improving detection efficiency, and the test results are more referenceable.

[0005] Traditional test samples are often serum, plasma, urine, etc., but in practical applications, these test matrices also have certain limitations. Among them, the hormone content in the blood fluctuates greatly and is easily interfered by environmental factors, and the process of collecting blood samples may cause stress to the subjects, making the results unreliable and not referenceable. Although urine samples can avoid stress reactions during sampling, the hormone content in urine samples will be affected by uncontrollable factors such as the amount of water the subjects drink, resulting in large differences in concentrations between individuals, requiring de-dilution treatment, and sample collection is not as convenient as saliva samples. Studies have shown that the hormone levels in saliva are well correlated with those in blood, and can be used as clinical indicator test samples. Saliva sampling is non-invasive and can avoid stress reactions. It is easy to operate and can be sampled continuously. At the same time, as a fluid sample, it has the characteristics of strong immediacy, and can continuously monitor the health status of the subjects. In addition, compared with traditional sample collection methods, the room temperature transportation and storage of saliva samples can bring greater convenience to clinical applications.

[0006] In summary, there are few reports on the determination of melatonin, cortisol and dehydroepiandrosterone sulfate by HPLC-MS / MS. Most of the currently available methods only detect one of the substances, and there is a lack of methods for the simultaneous qualitative and quantitative determination of multiple hormones such as melatonin, cortisol and dehydroepiandrosterone sulfate in saliva. Therefore, it is urgent to find a method that can achieve simultaneous qualitative, quantitative and accurate determination of the above three substances, which is of great significance for further understanding and evaluation of mental health status and prevention and treatment of related mental and psychological diseases in clinical practice. Summary of the invention

[0007] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a method for simultaneously detecting multiple hormones in saliva based on high performance liquid chromatography tandem mass spectrometry, aiming to solve the problem of the lack of a method for simultaneously performing qualitative and quantitative determination of melatonin, cortisol and dehydroepiandrosterone sulfate in saliva.

[0008] The technical solution of the present invention is as follows:

[0009] The present invention provides a method for simultaneously detecting multiple hormones in saliva based on high performance liquid chromatography tandem mass spectrometry, which comprises:

[0010] Step A, using the ratio of the peak areas of melatonin, cortisol and dehydroepiandrosterone sulfate in the standard working solution of the standard substance to the peak areas of the melatonin isotope, cortisol isotope and dehydroepiandrosterone sulfate isotope in the corresponding mixed isotope standard internal standard solution as the ordinate of the standard curve, and using the ratio of the concentrations of melatonin, cortisol and dehydroepiandrosterone sulfate in the above standard working solution to the concentrations of the melatonin isotope, cortisol isotope and dehydroepiandrosterone sulfate isotope in the corresponding mixed isotope standard internal standard solution as the abscissa of the standard curve to obtain a standard curve;

[0011] Step B, collecting saliva samples;

[0012] Step C, mixing the collected saliva sample with a mixed isotope standard internal standard solution, and then processing to obtain a sample to be tested;

[0013] Step D, using high performance liquid chromatography tandem mass spectrometry to detect the sample to be tested, and obtaining chromatograms of melatonin, cortisol and dehydroepiandrosterone sulfate in the sample to be tested and chromatograms of melatonin isotopes, cortisol isotopes and dehydroepiandrosterone sulfate isotopes in their corresponding mixed isotope standard internal standard solutions;

[0014] Step E: Calculate the concentrations of melatonin, cortisol and dehydroepiandrosterone sulfate in the sample to be tested according to the chromatogram and the standard curve.

[0015] Preferably, step A specifically comprises: using the peak area of ​​melatonin, cortisol and dehydroepiandrosterone sulfate in the standard working solution of the standard substance and the peak area ratio of melatonin isotope, cortisol isotope and dehydroepiandrosterone sulfate isotope in the corresponding mixed isotope standard internal standard solution as the ordinate y1, y2 and y3 of the standard curve, using the concentration of melatonin, cortisol and dehydroepiandrosterone sulfate in the above-mentioned standard working solution and the concentration of melatonin isotope, cortisol isotope and dehydroepiandrosterone sulfate isotope in the corresponding mixed isotope standard internal standard solution as the abscissa x1, x2 and x3 of the standard curve, performing linear regression on the data obtained by the above detection, fitting to obtain the standard curve, and obtaining the standard curve equations of y1=a*x1+b, y2=c*x2+d and y3=e*x3+f, and obtaining weight coefficients a, b, c, d, e and f.

[0016] Among them, the concentration range of melatonin in the standard working solution of melatonin standard is 5-250pg / mL, the concentration range of cortisol in the standard working solution of cortisol standard is 1-100ng / mL, and the concentration range of dehydroepiandrosterone sulfate in the standard working solution of dehydroepiandrosterone sulfate standard is 1-100ng / mL.

[0017] Preferably, the step E specifically comprises:

[0018] Substituting the ratio of the peak areas of melatonin, cortisol and dehydroepiandrosterone sulfate in the chromatogram to the peak areas of the melatonin isotope, cortisol isotope and dehydroepiandrosterone sulfate isotope in the corresponding mixed isotope standard internal standard solution into the standard curve, the ratio of the concentrations of melatonin, cortisol and dehydroepiandrosterone sulfate in the sample to be tested to the concentrations of the melatonin isotope, cortisol isotope and dehydroepiandrosterone sulfate isotope in the corresponding mixed isotope standard internal standard solution is calculated;

[0019] The concentrations of melatonin, cortisol and dehydroepiandrosterone sulfate in the sample to be tested are calculated by calculating the ratio of the concentrations of melatonin, cortisol and dehydroepiandrosterone sulfate in the sample to be tested to the concentrations of melatonin isotopes, cortisol isotopes and dehydroepiandrosterone sulfate isotopes in the corresponding mixed isotope standard internal standard solution.

[0020] Preferably, in step B, the patient fasts and does not brush teeth for at least 1 hour before collecting the saliva sample.

[0021] Because different individuals may have different saliva secretion, collection time and method, which will affect the accuracy and comparability of the test results. The present invention requires fasting and not brushing teeth for at least 1 hour before saliva sample collection, which can ensure the quality and consistency of the sample.

[0022] Preferably, the step C specifically comprises: adding 10 μL of mixed isotope standard internal standard solution to the collected 500 μL saliva sample, and then adding 1 mL of extracting solution, mixing by shaking, and centrifuging to obtain a supernatant; re-dissolving 900 μL of the supernatant with 70 μL of methanol aqueous solution, mixing by shaking again, and centrifuging to obtain a sample to be tested;

[0023] The extract is a mixed solvent of butanol and ethyl acetate (referred to as butanol / ethyl acetate), and the volume ratio of butanol to ethyl acetate in the mixed solvent is 1:1.

[0024] Saliva is a complex biological matrix containing a variety of proteins, enzymes and other biomolecules, which may interfere with the detection and affect the accuracy of the test results. When melatonin, cortisol and dehydroepiandrosterone sulfate are detected in biological matrices, complex matrices may cause ion suppression, affecting the sensitivity and quantitative accuracy of the detection. And different compounds may be affected by matrix effects to different degrees, resulting in inaccurate quantification. In addition, since the content of hormones in biological samples is extremely low, complex pretreatment methods are required to improve the accuracy and sensitivity of the detection. The present invention removes impurities in the sample by optimizing the liquid-liquid extraction (LLE) pretreatment method to reduce the influence of the matrix effect. The mixed solvent of butanol / ethyl acetate has good compatibility with polar and non-polar components, which helps to extract melatonin with weak polarity and cortisol and dehydroepiandrosterone sulfate with medium polarity. Compared with a single solvent, it can cover compounds of different polarities more comprehensively. In addition, since ethyl acetate has a low hydrophilicity, it will not precipitate proteins as violently as acetonitrile when it comes into contact with water in the sample, so the matrix effect and protein interference can be reduced in the subsequent centrifugation and extraction steps, and the purity of the extract can be improved. Moreover, compared with solid phase extraction (a pretreatment method that requires multiple steps), the butanol / ethyl acetate liquid-liquid extraction process is simple, low-cost, and does not require special equipment.

[0025] Melatonin, cortisol and dehydroepiandrosterone sulfate have different concentration ranges in the body, especially the concentration of melatonin is usually low under physiological conditions, while cortisol and dehydroepiandrosterone sulfate may fluctuate in a higher concentration range. This difference in concentration range requires the method to compromise between sensitivity and quantitative linear range. The present invention establishes a matrix-matched calibration curve after adding a mixed isotope standard internal standard solution of known concentration to the sample matrix to improve the accuracy of the quantitative result.

[0026] Preferably, in step D, the high performance liquid chromatography tandem mass spectrometry is a high performance liquid chromatography triple quadrupole tandem mass spectrometry.

[0027] More preferably, the chromatographic conditions of the high performance liquid chromatography triple quadrupole tandem mass spectrometry detection are as follows:

[0028] Chromatographic column: 2.1×50 mm, 1.7 μm ACQUITY UPLC BEH C18;

[0029] Mobile Phase:

[0030] Phase A: 0.05-0.2% formic acid in water (such as 0.1% formic acid in water)

[0031] Phase B: 0.05-0.2% formic acid in acetonitrile (e.g. 0.1% formic acid in acetonitrile)

[0032] The chromatographic column adopts a gradient elution mode, and the gradient elution program is: 0-3.8min: 5% B phase; 3.8-4min: 45% B phase; 4-4.2min: 100% B phase; 4.2-6.2min: 5% B phase;

[0033] The above % are all volume percentages;

[0034] Flow rate: 0.35mL / min.

[0035] The chemical differences between melatonin, cortisol and dehydroepiandrosterone sulfate are large, which makes it difficult to separate them well in liquid chromatography. Because melatonin is a molecule with relatively high polarity, and cortisol and dehydroepiandrosterone sulfate have different degrees of fat solubility, especially the sulfate group of dehydroepiandrosterone sulfate makes it more polar. The present invention ensures that melatonin, cortisol and dehydroepiandrosterone sulfate are well separated in liquid chromatography by using a chromatographic column (C18 bonded silica gel reverse phase chromatographic column) with suitable polarity, a mobile phase and a gradient elution procedure.

[0036] In addition, the substances in saliva are usually at a low concentration, so a highly sensitive detection method is required for accurate measurement. The present invention adopts an UPLC (ultra-high performance liquid chromatography) system, which has the following advantages compared to HPLC (high performance liquid chromatography): 1. High separation efficiency: the UPLC system adopts filler particles of 1.7 microns or even smaller, and smaller filler particles mean more theoretical plates and higher separation efficiency; 2. Higher sensitivity: The separation efficiency brought by smaller filler particles is improved, so that UPLC can obtain narrower chromatographic peaks, thereby improving the resolution and sensitivity of detection, which is particularly important for the analysis of complex samples; 3. Faster analysis speed: Due to smaller filler particles and efficient separation ability, the analysis time is significantly shortened, and the experimental throughput can be improved; 4. Lower sample and solvent consumption: The UPLC system uses a small inner diameter chromatographic column (such as 2.1 mm), and combines small particle fillers to make the chromatographic peak narrower, the peak height increases, and the detector's response signal to the target compound is enhanced, thereby reducing the consumption of samples and solvents.

[0037] More preferably, the mass spectrometry conditions of the high performance liquid chromatography triple quadrupole tandem mass spectrometry detection are as follows:

[0038] The scanning method adopted the multiple ion reaction monitoring mode;

[0039] The ionization method adopted was electrospray ionization;

[0040] The spray voltage in the positive ion mode is 2500-3500 V (such as 2500 V, 3000 V, 3500 V, etc.), and the spray voltage in the negative ion mode is 2000-3000 V (such as 2000 V, 2500 V, 3000 V, etc.);

[0041] The flow rate of sheath gas was 10 L / min.

[0042] Melatonin, cortisol and dehydroepiandrosterone sulfate have different ionization efficiencies in mass spectrometry, which may result in weak signals of some compounds under the same detection conditions, or even ineffective detection. For example, dehydroepiandrosterone sulfate has a low ionization efficiency in electrospray ionization (ESI) due to its sulfate group. The present invention improves the ionization efficiency of all target compounds by adjusting ion source parameters such as spray voltage, gas flow rate, etc., thereby ensuring effective detection of all target compounds in mass spectrometry.

[0043] Preferably, the mixed isotope standard internal standard solution is prepared by the following method: 5 μL of melatonin standard isotope internal standard solution, 50 μL of cortisol standard isotope internal standard solution and 100 μL of dehydroepiandrosterone sulfate standard isotope internal standard solution are respectively pipetted into a 100 mL volumetric flask, and diluted with a methanol aqueous solution with a volume ratio of 1:1 and fixed to the scale line of the volumetric flask to obtain a mixed isotope standard internal standard solution;

[0044] Wherein, the concentration of the melatonin standard isotope internal standard solution is 100 μg / mL, the concentration of the cortisol standard isotope internal standard solution is 100 μg / mL, and the concentration of the dehydroepiandrosterone sulfate standard isotope internal standard solution is 100 μg / mL.

[0045] Beneficial effects: The method for simultaneously detecting multiple hormones in saliva based on high performance liquid chromatography tandem mass spectrometry provided by the present invention is simple to operate. Using the same pretreatment conditions and instrument conditions, it can achieve the qualitative and quantitative detection of multiple hormones with low content in saliva, including the simultaneous detection of substances such as melatonin, cortisol and dehydroepiandrosterone sulfate, which significantly reduces the detection steps and time, and improves the detection sensitivity and accuracy. In addition, the present invention avoids the cumbersome procedures of going to the hospital for collection. The subject can collect samples at home by himself. The samples are easy to obtain and transport. They are used to evaluate the mental health status of the subject, treat and prevent clinically related mental and psychological diseases, and are of great significance to reducing the burden on patients and families and improving the quality of the population. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is the chromatogram of melatonin in the standard curve; top: melatonin internal standard isotope; bottom: melatonin standard.

[0047] Figure 2 Chromatogram of cortisol in the standard curve; top: cortisol internal standard isotope; bottom: cortisol standard.

[0048] Figure 3 This is the chromatogram of dehydroepiandrosterone sulfate in the standard curve; top: dehydroepiandrosterone sulfate internal standard isotope; bottom: dehydroepiandrosterone sulfate standard.

[0049] Figure 4 This is the chromatogram of melatonin in the sample to be tested; top: melatonin internal standard isotope; bottom: melatonin detection in the sample to be tested.

[0050] Figure 5 This is the chromatogram of cortisol in the sample to be tested; top: cortisol internal standard isotope; bottom: cortisol detection in the sample to be tested.

[0051] Figure 6 This is the chromatogram of dehydroepiandrosterone sulfate in the sample to be tested; top: internal standard isotope of dehydroepiandrosterone sulfate; bottom: detection of dehydroepiandrosterone sulfate in the sample to be tested. DETAILED DESCRIPTION

[0052] The present invention provides a method for simultaneously detecting multiple hormones in saliva based on high performance liquid chromatography tandem mass spectrometry. In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0053] (I) Establishment of standard curve

[0054] (a) Preparation of standard working solution

[0055] 1.0 mg of melatonin (MEL) standard was accurately weighed on an analytical balance and dissolved in 1 mL of methanol to obtain a melatonin standard stock solution with a final concentration of 1 mg / mL. 1.0 mg of cortisol and dehydroepiandrosterone sulfate (DHEAS) standard were weighed and dissolved in 1 mL of methanol to obtain a cortisol standard stock solution with a final concentration of 1 mg / mL and a dehydroepiandrosterone sulfate standard stock solution with a final concentration of 1 mg / mL, respectively. Subsequently, 5 μL of melatonin standard stock solution, 25 μL of cortisol standard stock solution and 25 μL of dehydroepiandrosterone sulfate standard stock solution were taken and placed in 100 mL volumetric flasks respectively, and the volume was fixed to the scale line of the volumetric flask with diluent (methanol aqueous solution, v:v=50:50) to obtain 500 pg / mL melatonin standard stock solution, 250 ng / mL cortisol standard stock solution and 250 ng / mL dehydroepiandrosterone sulfate standard stock solution, as shown in STD8 in Table 1.

[0056] Prepare 7 10mL volumetric flasks, labeled as STD1-7 respectively. According to Table 1, first, transfer 0.1, 0.2, 0.5, 1, 2, 3, 5mL of melatonin standard stock solution to STD1-7 volumetric flasks in sequence; then transfer 0.04, 0.1, 0.2, 0.4, 1, 2, 4mL of cortisol and dehydroepiandrosterone sulfate standard stock solutions to STD1-7 volumetric flasks in sequence, and finally dilute with methanol aqueous solution (v:v=50:50) and make up to the scale line of the volumetric flask to prepare standard working solutions of various concentrations for preparing standard curves; the concentration range of melatonin is 5-250pg / mL, and the concentration range of cortisol and dehydroepiandrosterone sulfate is 1-100ng / mL, and the specific concentrations are shown in Table 1.

[0057] Table 1. Standard working solution

[0058]

[0059] (b) Preparation of mixed isotope standard internal standard solution

[0060] Accurately pipette 5 μL of melatonin standard isotope internal standard solution (100 μg / mL), 50 μL of cortisol standard isotope internal standard solution (100 μg / mL) and 100 μL of dehydroepiandrosterone sulfate standard isotope internal standard solution (100 μg / mL) (Table 2) into a 100 mL volumetric flask, dilute with methanol-water solution (v:v=50:50) and make up to the scale to obtain a mixed isotope standard internal standard solution, as shown in Table 3.

[0061] Table 2. Information of standard isotope internal standard solution

[0062]

[0063] Table 3. Mixed isotope standard internal standard solution

[0064]

[0065] (c) Calibrate with standard solution to obtain a standard curve

[0066] Use a pipette to take 50 μL of eight different concentrations of standard working solutions (Table 1) and add them to the corresponding centrifuge tubes, then add 450 μL of phosphate buffered saline (PBS) and mix thoroughly; add 10 μL of mixed isotope standard internal standard solution, then add 1 mL of extraction solution (butanol / ethyl acetate = 1:1, v / v), vortex and mix at room temperature for 30 minutes, and centrifuge at 12,500 rpm for 10 minutes; aspirate 900 μL of the supernatant and transfer it to a new centrifuge tube, blow dry with nitrogen; re-dissolve with 70 μL of methanol aqueous solution (v:v = 50:50), vortex and shake for 30 seconds; centrifuge at 4°C, 12,500 rpm for 15 minutes; the supernatant is used for LC-MS / MS detection. The chromatograms of melatonin, cortisol and dehydroepiandrosterone sulfate in the above three standard working solutions (STD1-8) and the chromatograms of melatonin isotopes, cortisol isotopes and dehydroepiandrosterone sulfate isotopes in the corresponding mixed isotope standard internal standard solutions were obtained respectively ( Figure 1-3). The peak areas of melatonin, cortisol and dehydroepiandrosterone sulfate in the above three standard working solutions and the peak areas of melatonin isotope, cortisol isotope and dehydroepiandrosterone sulfate isotope in the corresponding mixed isotope standard internal standard solution were used as the ordinates y1, y2 and y3 of the standard curve, and the concentrations of melatonin, cortisol and dehydroepiandrosterone sulfate in the above standard working solutions and the concentrations of melatonin isotope, cortisol isotope and dehydroepiandrosterone sulfate isotope in the corresponding mixed isotope standard internal standard solution were used as the abscissas x1, x2 and x3 of the standard curve. The above test data were subjected to linear regression, and the fitting obtained standard curve equations were y1=a*x1+b, y2=c*x2+d and y3=e*x3+f, and the weight coefficients a, b, c, d, e and f were obtained (Table 4). That is, the standard curve was obtained by plotting the concentration ratio of the three standard working solutions and the corresponding mixed isotope standard internal standard solution as the horizontal axis, and the quantitative chromatographic peak area ratio of the three standard working solutions and the corresponding mixed isotope standard internal standard solution as the vertical axis. The results showed that the linear correlation coefficient (R 2 ) were all greater than 0.999, and the limits of quantification (LOQ) were shown in Table 4.

[0067] Table 4. Standard curve and correlation coefficient

[0068] Analytes Linear equations <![CDATA[Correlation coefficient (R 2 )]]> LOQ Melatonin Y=0.068834x+0.030438 0.9997 0.5(pg / mL) Cortisol Y=22.014145x-0.232598 0.9994 0.25 (ng / mL) Dehydroepiandrosterone Sulfate Y=23.320610x+0.007505 0.9998 0.25 (ng / mL)

[0069] (II) Collection of saliva samples

[0070] The saliva samples were collected by natural flow method, and the subjects spit saliva directly into the collection tube. The saliva was collected at least 3 mL, and the collected saliva samples were stored in a -80℃ refrigerator. Before the experiment, the samples were thawed in a 4℃ refrigerator. After thawing, the samples were vortexed for 30 seconds and centrifuged at 500 rpm for 2 minutes for later use.

[0071] (III) Processing of samples to be tested

[0072] Use a pipette to transfer 500 μL of the sample to be tested in step (ii) and add them to the corresponding centrifuge tubes. After adding 10 μL of mixed isotope standard internal standard solution, add 1 mL of extract solution (butanol / ethyl acetate = 1:1, v / v), vortex and mix at room temperature for 30 min, and centrifuge at 12,500 rpm for 10 min; transfer 900 μL of the supernatant to a new centrifuge tube and blow dry with nitrogen; re-dissolve with 70 μL of methanol-water solution (v:v = 50:50), vortex and shake for 30 s; centrifuge at 4°C, 12,500 rpm for 15 min; use for LC-MS / MS detection.

[0073] (IV) Testing of samples to be tested

[0074] Take the sample to be tested in step (iii), and use a high performance liquid chromatography triple quadrupole tandem mass spectrometer to detect the sample to be tested, and obtain the chromatograms of melatonin, cortisol and dehydroepiandrosterone sulfate in the sample to be tested and the chromatograms of melatonin isotopes, cortisol isotopes and dehydroepiandrosterone sulfate isotopes in the corresponding mixed isotope standard internal standard solution ( Figure 4-6 ). The ratios Y1, Y2 and Y3 of the peak areas of melatonin, cortisol and dehydroepiandrosterone sulfate in the above chromatogram and the peak areas of melatonin isotopes, cortisol isotopes and dehydroepiandrosterone sulfate isotopes in the corresponding mixed isotope standard internal standard solution are substituted into the standard curve equation of the above step (c) (Table 4), and the ratios X1, X2 and X3 of the concentrations of melatonin, cortisol and dehydroepiandrosterone sulfate in the sample to be tested and the concentrations of melatonin isotopes, cortisol isotopes and dehydroepiandrosterone sulfate isotopes in the corresponding mixed isotope standard internal standard solution are obtained by calculation. The concentrations of melatonin isotopes, cortisol isotopes and dehydroepiandrosterone sulfate isotopes in the mixed isotope standard internal standard solution are known, and the concentrations of melatonin, cortisol and dehydroepiandrosterone sulfate in the sample to be tested are obtained by calculation.

[0075] The conditions for the high performance liquid chromatography triple quadrupole tandem mass spectrometry detection are as follows:

[0076] 1) Chromatographic conditions:

[0077] Chromatographic column: ACQUITY UPLC BEH C18 (2.1×50mm, 1.7um) Column temperature: 40℃

[0078] Mobile Phase:

[0079] Phase A: 0.1% formic acid in water

[0080] Phase B: 0.1% formic acid in acetonitrile

[0081] Injection volume: 10uL

[0082] The chromatographic column adopts gradient elution mode, and the gradient elution program parameters are shown in Table 5 below.

[0083] Table 5. Gradient elution program

[0084] Time(min) A(％) B(％) Flow rate (mL / min) 0 95 5 0.35 3.8 55 45 0.35 4 0 100 0.35 4.2 95 5 0.35 6.2 95 5 0.35

[0085] 2) Mass spectrometry conditions

[0086] Triple quadrupole tandem mass spectrometer: Agilent, 6495C, USA

[0087] Scanning mode: Multiple ion reaction monitoring (Dynamic MRM) mode Ionization mode: Electrospray ionization, positive ion (ESI +) and negative ions (ESI - )Spray voltage: 3500V(+), 3000V(-)

[0088] Atomizing gas: 20psi

[0089] Sheath gas: 10L / min

[0090] Sheath gas temperature: 400℃

[0091] The MRM ion pair parameters are shown in Table 6 below.

[0092] Table 6. Multiple reaction monitoring parameters

[0093]

[0094] (V) Precision and recovery of this method

[0095] Saliva samples of three concentration levels, low, medium and high, were prepared. Within one batch, the saliva samples of three concentration levels, low, medium and high, were tested six times in a row, as intra-batch precision test data; saliva samples of two concentration levels, low and high, were selected for inter-batch precision verification. The samples of the two levels were tested six times a day for three consecutive days, as inter-batch precision test data (precision <15% was considered normal). The results are shown in Table 7 below:

[0096] Table 7. Precision test results

[0097]

[0098]

[0099] High, medium and low concentration standard solutions were added to the sample matrix (the volume of the added standard solution did not exceed 5% of the total volume), and the final concentration after spiking needed to cover the entire medical decision level. The matrix and each concentration after spiking were processed in parallel for 3 samples, and the average value of the matrix and the recovery rate (R) of each sample corresponding to the matrix after spiking were calculated. R = (measured value after spiking - average value of unspiked matrix) / theoretical value of spiking * 100% (R values ​​between 85% and 115% are considered normal), and the results are shown in Table 8 below:

[0100] Table 8. Spiked recovery test results

[0101]

[0102] Based on the above experimental results, the technical indicators of this experimental method, such as the limit of quantification, precision and spike recovery rate, all meet the requirements. This method can simultaneously detect the contents of melatonin, cortisol and dehydroepiandrosterone sulfate in saliva, with good reproducibility and high spike recovery rate, which improves the accuracy of the test results.

[0103] In summary, the method provided by the present invention for simultaneously detecting multiple hormones in saliva based on high performance liquid chromatography tandem mass spectrometry can realize the qualitative and quantitative analysis of three hormones in saliva, namely melatonin, cortisol and dehydroepiandrosterone sulfate, which significantly improves the detection efficiency and accuracy. The samples are easy to obtain and can be transported at room temperature, which is more suitable for detection and risk screening assessment of large-scale populations.

[0104] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A method for simultaneously detecting multiple hormones in saliva based on high performance liquid chromatography tandem mass spectrometry, characterized in that: include: Step A, using the ratio of the peak areas of melatonin, cortisol and dehydroepiandrosterone sulfate in the standard working solution of the standard substance to the peak areas of the melatonin isotope, cortisol isotope and dehydroepiandrosterone sulfate isotope in the corresponding mixed isotope standard internal standard solution as the ordinate of the standard curve, and using the ratio of the concentrations of melatonin, cortisol and dehydroepiandrosterone sulfate in the above standard working solution to the concentrations of the melatonin isotope, cortisol isotope and dehydroepiandrosterone sulfate isotope in the corresponding mixed isotope standard internal standard solution as the abscissa of the standard curve to obtain a standard curve; Step B, collecting saliva samples; Step C, mixing the collected saliva sample with a mixed isotope standard internal standard solution, and then processing to obtain a sample to be tested; Step D, using high performance liquid chromatography tandem mass spectrometry to detect the sample to be tested, and obtaining chromatograms of melatonin, cortisol and dehydroepiandrosterone sulfate in the sample to be tested and chromatograms of melatonin isotopes, cortisol isotopes and dehydroepiandrosterone sulfate isotopes in their corresponding mixed isotope standard internal standard solutions; Step E: Calculate the concentrations of melatonin, cortisol and dehydroepiandrosterone sulfate in the sample to be tested according to the chromatogram and the standard curve.

2. The method for simultaneously detecting multiple hormones in saliva based on high performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: In the step D, the high performance liquid chromatography tandem mass spectrometry is a high performance liquid chromatography triple quadrupole tandem mass spectrometry.

3. The method for simultaneously detecting multiple hormones in saliva based on high performance liquid chromatography tandem mass spectrometry according to claim 2, characterized in that: The chromatographic conditions of the high performance liquid chromatography triple quadrupole tandem mass spectrometry detection are as follows: Chromatographic column: 2.1×50 mm, 1.7 μm ACQUITY UPLC BEH C18; Mobile Phase: Phase A: 0.05%-0.2% formic acid in water Phase B: 0.05%-0.2% formic acid in acetonitrile The chromatographic column adopts a gradient elution mode, and the gradient elution program is: 0-3.8min: 5% B phase; 3.8-4min: 45% B phase; 4-4.2min: 100% B phase; 4.2-6.2min: 5% B phase; The above % are all volume percentages; Flow rate: 0.35mL / min.

4. The method for simultaneously detecting multiple hormones in saliva based on high performance liquid chromatography tandem mass spectrometry according to claim 2, characterized in that: The mass spectrometry conditions of the high performance liquid chromatography triple quadrupole tandem mass spectrometry detection are as follows: The scanning method adopted the multiple ion reaction monitoring mode; The ionization method adopted was electrospray ionization; The spray voltage in positive ion mode was 2500-3500 V, and the spray voltage in negative ion mode was 2000-3000 V; The flow rate of sheath gas was 10 L / min.

5. The method for simultaneously detecting multiple hormones in saliva based on high performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The step A specifically includes: using the peak area of ​​melatonin, cortisol and dehydroepiandrosterone sulfate in the standard working solution of the standard substance and the peak area ratio of melatonin isotope, cortisol isotope and dehydroepiandrosterone sulfate isotope in the corresponding mixed isotope standard internal standard solution as the ordinates y1, y2 and y3 of the standard curve, using the concentration of melatonin, cortisol and dehydroepiandrosterone sulfate in the above standard working solution and the concentration of melatonin isotope, cortisol isotope and dehydroepiandrosterone sulfate isotope in the corresponding mixed isotope standard internal standard solution as the abscissas x1, x2 and x3 of the standard curve, performing linear regression on the data obtained by the above detection, fitting to obtain the standard curve, and obtaining the standard curve equations of y1=a*x1+b, y2=c*x2+d and y3=e*x3+f, and obtaining weight coefficients a, b, c, d, e and f.

6. The method for simultaneously detecting multiple hormones in saliva based on high performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The step E specifically comprises: Substituting the ratio of the peak areas of melatonin, cortisol and dehydroepiandrosterone sulfate in the chromatogram to the peak areas of the melatonin isotope, cortisol isotope and dehydroepiandrosterone sulfate isotope in the corresponding mixed isotope standard internal standard solution into the standard curve, the ratio of the concentrations of melatonin, cortisol and dehydroepiandrosterone sulfate in the sample to be tested to the concentrations of the melatonin isotope, cortisol isotope and dehydroepiandrosterone sulfate isotope in the corresponding mixed isotope standard internal standard solution is calculated; The concentrations of melatonin, cortisol and dehydroepiandrosterone sulfate in the sample to be tested are calculated by calculating the ratio of the concentrations of melatonin, cortisol and dehydroepiandrosterone sulfate in the sample to be tested to the concentrations of melatonin isotopes, cortisol isotopes and dehydroepiandrosterone sulfate isotopes in the corresponding mixed isotope standard internal standard solution.

7. The method for simultaneously detecting multiple hormones in saliva based on high performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The step C specifically comprises: adding 10 μL of mixed isotope standard internal standard solution to the collected 500 μL saliva sample, and then adding 1 mL of extract solution, shaking and mixing, and centrifuging to obtain a supernatant; re-dissolving 900 μL of the supernatant with 70 μL of methanol aqueous solution, shaking and mixing again, and centrifuging to obtain a sample to be tested; Wherein, the extract is a mixed solvent composed of butanol and ethyl acetate, and the volume ratio of butanol to ethyl acetate in the mixed solvent is 1:

1.

8. The method for simultaneously detecting multiple hormones in saliva based on high performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The mixed isotope standard internal standard solution is prepared by the following method: 5 μL of melatonin standard isotope internal standard solution, 50 μL of cortisol standard isotope internal standard solution and 100 μL of dehydroepiandrosterone sulfate standard isotope internal standard solution are respectively taken into a 100 mL volumetric flask, and diluted with a methanol aqueous solution with a volume ratio of 1:1 and fixed to the scale line of the volumetric flask to obtain a mixed isotope standard internal standard solution; Wherein, the concentration of the melatonin standard isotope internal standard solution is 100 μg / mL, the concentration of the cortisol standard isotope internal standard solution is 100 μg / mL, and the concentration of the dehydroepiandrosterone sulfate standard isotope internal standard solution is 100 μg / mL.

9. The method for simultaneously detecting multiple hormones in saliva based on high performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The concentration range of melatonin in the melatonin standard working solution is 5-250 pg / mL, the concentration range of cortisol in the cortisol standard working solution is 1-100 ng / mL, and the concentration range of dehydroepiandrosterone sulfate in the dehydroepiandrosterone sulfate standard working solution is 1-100 ng / mL.

10. The method for simultaneously detecting multiple hormones in saliva based on high performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: In step B, the patient fasts and does not brush teeth for at least 1 hour before collecting the saliva sample.

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