A method for detecting thyroxine and triiodothyronine in dried blood spots

Abstract

The present invention discloses a method for detecting thyroxine and triiodothyronine in dried blood spots, and belongs to the technical field of biochemical analysis and detection; a method for detecting thyroxine and triiodothyronine in dried blood spots of the present invention comprises the following steps: (1) adding an internal standard extract to a dried blood spot sample, mixing and shaking, centrifugal filtering, nitrogen blowing, and re-dissolving to obtain a sample to be tested; (2) using a liquid chromatography-mass spectrometry system to detect the sample to be tested. The detection method provided by the present invention can simultaneously detect the concentration levels of T3 and T4 in dried blood spots, and has the characteristics of high sensitivity, strong anti-interference ability, and is not affected by abnormal binding proteins, drugs, etc., and has high accuracy; at the same time, using dried blood spots as the basic material for detection, it is convenient for neonatal specimen collection, transportation, detection and storage, and is suitable for large-scale screening; and the provided dried blood spot specimen pretreatment operation is simple, the measurement time is short, and the detection efficiency can be significantly improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical analysis and detection, and in particular relates to a method for detecting thyroxine and triiodothyronine in dried blood spots. Background Art

[0002] Neonatal disease screening Congenital hypothyroidism screening is mainly based on the immunological method to detect the concentration level of thyroid stimulating hormone (TSH) in dried blood spots, which indirectly reflects whether the levels of human thyroxine (T4) and triiodothyronine (T3) are low. The immunological method for neonatal thyroid stimulating hormone detection is a solid phase two-site fluorescent immunoassay based on direct sandwich technology. Two monoclonal antibodies directly bind to different sites on the human thyroid stimulating hormone (hTSH) molecule to specific antigen sites on the hTSH molecule contained in the sample to be tested, and at the same time react with the solid phase monoclonal antibody and the europium-labeled monoclonal antibody to form a solid phase antibody-Eu labeled monoclonal antibody and the sample β-hTSH subunit antigen antibody complex. After washing, the enhancement solution is added to the Eu on the labeled antibody. + Dissociate and form a fluorescent chelate with strong fluorescence. By testing the fluorescence intensity of each well, the hTSH concentration of each sample is obtained. The immunoassay has low sensitivity and is dependent on binding proteins to some extent. Therefore, it is susceptible to various interferences and uncertainties. Interferences may involve drug interactions, cross-reactivity of antibodies, sensitivity to abnormal binding proteins, the influence of free fatty acids (FFA), and endogenous or exogenous binding inhibitors. Therefore, the immunoassay has good sensitivity for primary hypothyroidism, but it is easy to miss secondary hypothyroidism, resulting in false negatives. Summary of the invention

[0003] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a method for detecting thyroxine and triiodothyronine in dried blood spots with high sensitivity, high accuracy, strong anti-interference ability, convenient large-scale screening, and effective reduction of false positive rate.

[0004] To achieve the above object, the technical solution adopted by the present invention is: a method for detecting thyroxine and triiodothyronine in dried blood spots, comprising the following steps:

[0005] (1) adding an internal standard extract to a dried blood spot sample, mixing and shaking, centrifuging, nitrogen blowing, and re-dissolving to obtain a sample to be tested;

[0006] (2) Liquid chromatography-mass spectrometry system is used to detect the samples to be tested.

[0007] The detection method provided by the present invention is a method that uses high performance liquid chromatography-mass spectrometry to simultaneously detect the concentration levels of thyroxine (T3) and triiodothyronine (T4) in dried blood spots. On the first hand, the detection method provided by the present invention uses liquid chromatography-mass spectrometry technology, has the characteristics of high sensitivity, high accuracy, strong anti-interference ability, and is not affected by abnormal binding proteins, specimen hemolysis, jaundice, lipemia, drugs, etc., and can significantly improve the accuracy of the results; on the second hand, the technical solution adopted by the present invention uses dried blood spots as the basic material for detection, thereby facilitating the collection, transportation, detection and storage of neonatal specimens, and is suitable for large-scale screening; on the third hand, compared with the current LC-MS / MS detection method, the technical solution provided by the present invention has simple pre-treatment operations for dried blood spot specimens, short measurement time, and can significantly improve the detection efficiency.

[0008] As a preferred embodiment of the detection method of the present invention, in step (1), the dried blood spot sample is a processed dried blood spot, and the processing method is to punch out the dried blood spot through a puncher with a diameter of 3 mm to obtain the dried blood spot sample.

[0009] As a preferred embodiment of the detection method of the present invention, in step (1), the internal standard extract contains 13 C6-triiodothyronine and 13 C6-Thyroxine.

[0010] As a preferred embodiment of the detection method of the present invention, the internal standard extract 13 C6-triiodothyronine and 13 The mass concentrations of C6-thyroxine are all 1 to 10 ng / mL.

[0011] As a preferred embodiment of the detection method of the present invention, in step (1), the internal standard extract is obtained by diluting the internal standard stock solution with the extract; the internal standard stock solution contains 13 C6-triiodothyronine and 13 C6-thyroxine; the internal standard extract 13 C6-triiodothyronine and 13 The mass concentration of C6-thyroxine was 100 ng / mL respectively.

[0012] As a preferred embodiment of the detection method of the present invention, the preparation method of the internal standard stock solution comprises the following steps: 13 C6-triiodothyronine was dissolved in a methanol-water (volume ratio of 50:50) solution containing 0.1% formic acid to prepare a standard solution with a mass concentration of 1 mg / mL, and stored at -80°C. 13 C6-triiodothyronine standard stock solution and 13The C6-thyroxine internal standard solutions were mixed and diluted to form internal standard stock solutions with a mass concentration of 100 ng / mL.

[0013] As a preferred embodiment of the detection method of the present invention, the extraction liquid includes any one of methanol, n-hexane, ethyl acetate, and a mixed solution of methanol and acetonitrile; the volume ratio of methanol to acetonitrile in the mixed solution of methanol and acetonitrile is 3:2.

[0014] As a preferred embodiment of the detection method of the present invention, in the step (1), after adding the internal standard extract, before mixing and shaking, it also includes sealing the film with aluminum foil paper using a heat sealer.

[0015] As a preferred embodiment of the detection method of the present invention, in the step (1), the temperature of the mixing and shaking is 35°C to 45°C, the speed of the mixing and shaking is 700-950 rpm, and the time of the mixing and shaking is 45-90 minutes.

[0016] As a preferred embodiment of the detection method of the present invention, in the step (1), after the mixing and shaking is completed, the supernatant is extracted and filtered and centrifuged.

[0017] As a preferred embodiment of the detection method of the present invention, the filtration plate is a 96-well microfiltration plate, and the pore size of the microfiltration plate is 45 μM.

[0018] As a preferred embodiment of the detection method of the present invention, in the step (1), the centrifugal speed is 1400-1600 rpm / min, and the centrifugal time is 2.5-3.5 minutes.

[0019] As a preferred embodiment of the detection method of the present invention, in the step (1), the supernatant after centrifugal filtration is placed in a nitrogen blower and blown dry for 20 min to 30 min at room temperature with a nitrogen flow rate of 5-15 mL / min.

[0020] As a preferred embodiment of the detection method of the present invention, in the step (1), the solution used for re-dissolving is a methanol-water solution, and the volume percentage of methanol in the methanol-water solution is 30-45%.

[0021] As a preferred embodiment of the detection method of the present invention, in step (2), the liquid chromatography conditions in the detection are: Chromatographic column: Waters ACQUITY UPLC HSS T3 Column, 1.8μm×2.1mm×50mm; column temperature: 37~45℃; flow rate: 0.3~0.4mL / min; injection volume: 10μL; mobile phase A: 0.1~0.5% formic acid, 1~5mmol / L ammonium acetate aqueous solution; mobile phase B: 0.1~0.5% formic acid, 1~5mmol / L ammonium acetate methanol solution; gradient elution program: 0~0.01min, mobile phase A volume percentage 80~70%, mobile phase B volume percentage 20~30%; 0.01~3min, mobile phase A volume percentage decreased from 80~70% to 8~2%, mobile phase B volume percentage increased from 20~30% to 92~98%; 3.01min~4min, mobile phase A volume percentage 8~2%, mobile phase B volume percentage 92~98%; 4.01~6min, mobile phase A volume percentage 80%~70%, mobile phase B volume percentage 20~30%.

[0022] As a preferred embodiment of the detection method of the present invention, in the step (2), the mass spectrometry parameters in the detection are: ion source: electrospray ionization ion source; ion mode: multiple reaction monitoring mode positive ion mode; ion source temperature: 350°C; ion source voltage: 4500V; ion transfer tube temperature: 350°C; sheath gas: 35mL; auxiliary gas: 10mL.

[0023] In addition, the present invention also provides a detection kit for simultaneously determining thyroxine and triiodothyronine in dried blood spots, the kit comprising a standard working solution, an internal standard stock solution, an extract and a liquid chromatography mobile phase; the standard working solution is a thyroid solution and a triiodothyronine solution containing standard mass concentrations; the liquid chromatography mobile phase comprises a mobile phase A and a mobile phase B, the mobile phase A is a 0.1-0.5% formic acid, 1-5 mmol / L ammonium acetate aqueous solution, and the mobile phase B is a 0.1-0.5% formic acid, 1-5 mmol / L ammonium acetate methanol solution.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] First: The detection method provided by the present invention uses liquid chromatography-mass spectrometry technology, which has the characteristics of high sensitivity, high accuracy, strong anti-interference ability, and is not affected by abnormal binding proteins, specimen hemolysis, jaundice, lipemia, drugs, etc. The obtained results have high accuracy;

[0026] Second: The technical solution adopted by the present invention uses dried blood spots as the basic substance for detection, which can facilitate the collection, transportation, detection and storage of newborn specimens and is suitable for large-scale screening;

[0027] Third: In the technical solution provided by the present invention, the pretreatment operation of the dried blood spot specimen is simple, the measurement time is short, and the detection efficiency is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The total ion current chromatogram and sub-current chromatogram of T3 and T4 obtained in Example 1;

[0029] Figure 2 It is the linear graph of T3 standard in Example 1;

[0030] Figure 3 It is the linear graph of T4 standard in Example 1;

[0031] Figure 4 The total ion current chromatograms of T3 and T4 obtained in the test group 1 in Example 2;

[0032] Figure 5 The total ion current chromatograms of T3 and T4 obtained in the test group 2 in Example 2;

[0033] Figure 6 The chromatograms of T3 and T4 obtained in Experimental Group 3 in Example 3;

[0034] Figure 7 The chromatograms of T3 and T4 obtained from test group 4 in Example 3;

[0035] Figure 8 The chromatograms of T3 and T4 obtained from test group 5 in Example 3;

[0036] Fig. 9 The chromatograms of T3 and T4 obtained from test group 6 in Example 3;

[0037] Fig.10 This is a summary of the T3 and T4 peak area data obtained from the sample test with a concentration of 1.56 ng / mL in Example 4;

[0038] Fig.11 This is a summary of the T3 and T4 peak area data obtained from the sample test at a concentration of 25 ng / mL in Example 4;

[0039] Fig.12 This is a summary of the T3 and T4 peak area data obtained from the sample test with a concentration of 100 ng / mL in Example 4. DETAILED DESCRIPTION

[0040] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0041] Unless otherwise specified, the raw materials and equipment used in the examples of the present invention can be obtained through conventional purchase channels. The main instruments and reagents are:

[0042] 1. Main instruments:

[0043] High performance liquid chromatography tandem mass spectrometer: Jinan Yingsheng Biological Co., Ltd., 9050MD;

[0044] Constant temperature micro-oscillator: Wallac;

[0045] Milli-Q ultrapure water instrument: Millipore, USA;

[0046] Ultrasonic cleaner: KM-23C;

[0047] Microplate sealing instrument: Hangzhou Ruicheng MSP200.

[0048] 2. Main reagents:

[0049] Thyroxine solution (standard): Supelco;

[0050] Triiodothyronine (standard product): TRC Company;

[0051] 13 C6-thyroxine internal standard solution (internal standard): Supelco;

[0052] 13 C6-triiodothyronine (internal standard): TRC Company;

[0053] Methanol: Merck;

[0054] Acetonitrile: CNW Company;

[0055] Formic acid: Sigma;

[0056] Ammonium acetate: Shanghai Anpu Company.

[0057] Example 1

[0058] This embodiment detects thyroxine and triiodothyronine in dried blood spot samples and performs analysis;

[0059] (I) Preparation of reagents

[0060] (1) Preparation of blank whole blood: 2 mL of whole blood was collected from healthy volunteers and anticoagulated with sodium citrate dipotassium. After collection, the whole blood was centrifuged at 3500 r / min for 10 min in a high-speed centrifuge to separate serum and red blood cells. The upper serum solution was removed and an equal volume of 0.9% sodium chloride solution was added to the red blood cells for washing. The blood was rotated and shaken repeatedly for thorough washing. The blood was then centrifuged at 3500 r / min for 10 min in a high-speed centrifuge. The washing was repeated four times and the upper solution was removed. The red blood cells precipitated at the bottom of the centrifuge tube were aspirated and blank serum washed with activated carbon was added at a volume ratio of 60:40 to the red blood cells to prepare artificial blank whole blood.

[0061] (2) Preparation of standard solution: Accurately weigh triiodothyronine standard and dissolve it in a mixed solution of methanol and water (volume ratio of 50:50) containing 0.1% formic acid to prepare a standard stock solution with a mass concentration of 1 mg / mL. Store it at -80°C and dilute it to the desired concentration with a mixed solution of methanol and water (volume ratio of 50:50) containing 0.1% formic acid before use.

[0062] (3) Preparation of internal standard stock solution: Accurately weigh 13 C6-triiodothyronine internal standard was prepared by dissolving in a methanol-water (volume ratio 50:50) solution containing 0.1% formic acid to prepare a 1 mg / mL standard solution, and then diluted with a methanol-acetonitrile (volume ratio 50:50) solution. 13 C6-triiodothyronine stock solution and 13 C6-thyroxine internal standard was mixed to a mass concentration of 100 ng / mL 13 C6-triiodothyronine and mass concentration is 100ng / mL 13 C6-thyroxine mixed internal standard stock solution was stored at -80°C;

[0063] (4) Preparation of internal standard extract: A mixed solution of methanol and acetonitrile (volume ratio of methanol to acetonitrile was 3:2) was used as an extract to dilute the internal standard stock solution to obtain a 5 ng / mL internal standard extract;

[0064] (5) Preparation of standard dried blood spots: A series of standard solutions diluted with 50% methanol aqueous solution were added to blank whole blood to prepare standard solutions with mass concentrations of 0.195, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, and 100 ng / mL, respectively. Appropriate amounts of whole blood standard samples were respectively drawn with a pipette and dropped onto a dry and clean American 903# filter paper. The filter paper was then placed face up on a blood spot drying rack and allowed to dry naturally at room temperature. After drying, the solutions were placed in a sealed plastic bag and stored in a -20°C refrigerator for later use.

[0065] (6) Preparation of quality control products:

[0066] A 50% methanol aqueous solution was diluted into three concentrations of high, medium and low quality control solutions and added to blank whole blood to make quality T3 and T4 concentrations of 10.5, 45.5, 72.7 ng / mL, 0.9, 3.2, 9.1 ng / mL whole blood quality control solutions, and 200 μL of whole blood quality control samples were accurately dropped into a dry and clean American 903# filter paper, and then the filter paper was placed on the blood spot drying rack with the front side facing up, and dried naturally at room temperature. After drying, it was placed in a sealed plastic bag and stored in a -20℃ refrigerator for later use. The preparation of standard and quality control dried blood spots cannot be prepared at the same time, and should be prepared in different batches.

[0067] (II) Detection method

[0068] The detection method of the embodiment of the present invention comprises the following steps:

[0069] (1) Punch two blood spots of the standard dried blood spot, the quality control dried blood spot, and the test dried blood spot into a 96-well microplate using a 3 mm diameter puncher to obtain the standard dried blood spot sample, the quality control dried blood spot sample, and the test dried blood spot sample, respectively;

[0070] (2) Add 200 μL of internal standard extract to the standard, quality control, and dry blood spot samples to be tested, seal the film with aluminum foil paper of a heat sealer, and place it in a constant temperature shaker for mixing and shaking, wherein the temperature of the mixing shaker is 37° C., the speed is 750 rpm, and the shaking time is 60 minutes; after the shaking, extract 150 μL of supernatant to a 45 μM pore size 96-well microfiltration plate, centrifuge at a speed of 1500 rpm / min for 3 minutes, blow dry the filtrate with nitrogen at a flow rate of 10 mL / min, then add 35% methanol water solution, shake at a high speed of 1020 rpm / min for 5 minutes to obtain the sample to be tested;

[0071] (3) The sample was tested using a liquid chromatography-mass spectrometry system; wherein the liquid chromatography conditions were as follows: chromatographic column: Waters ACQUITY UPLC HSS T3 Column, 1.8 μm×2.1 mm×50 mm, column temperature: 37°C, flow rate: 0.4 mL / min, injection volume: 10 μL, mobile phase A: 0.1% formic acid, 2 nmol / L ammonium acetate aqueous solution, mobile phase B: 0.1% formic acid, 2 nmol / L ammonium acetate methanol solution, gradient elution program: 0-0.01 min, mobile phase A volume percentage 70%, mobile phase B volume percentage 30%; 0.01-3 min, mobile phase A volume percentage decreased from 70% to 2%, mobile phase B volume percentage increased from 30% to 98%; 3.01 min-4 min, mobile phase A volume percentage 2%, mobile phase B volume percentage 98%; 4.01-6 min, mobile phase A volume percentage 70%, mobile phase B volume percentage 30%;

[0072] The mass spectrometry parameters were as follows: ion source: electrospray ionization ion source, ion mode: multiple reaction monitoring mode positive ion mode, ion source temperature: 350°C, ion source voltage: 4500 V, ion transfer tube temperature: 350°C, sheath gas: 35 mL, auxiliary gas: 10 mL;

[0073] Among them, in the detection, the total ion chromatogram of the standard product obtained is as follows Figure 1 As shown, from Figure 1 It can be seen that the peaks of T3 and T4 standards are symmetrical and there is no interference from other impurities, indicating that good detection can be obtained under this condition;

[0074] Among them, in mass spectrometry detection, T3 and T4 mass spectrometry parameters molecular ion, product ion, radio frequency voltage, collision voltage and dwell time are shown in Table 1:

[0075] Table 1

[0076]

[0077] 3. Data Analysis

[0078] 1. Draw a standard curve

[0079] After the standard curve samples are separated by liquid chromatography, thyroxine and triiodothyronine peak at different elution times and are detected by mass spectrometry to detect their contents. According to a certain standard mass concentration, the sample to be tested is configured for detection, and the isotope internal standard quantitative method is adopted. The analysis software is used to establish a calibration curve with the concentration ratio of the standard and the internal standard as the X-axis and the peak area ratio of the standard and the internal standard as the Y-axis, and the mass concentration of thyroxine and triiodothyronine in the dried blood spot sample is calculated; the prepared T3 standard curve is as follows Figure 2The standard curve of T4 is shown in Figure 3 The standard curve equation and correlation coefficient are shown in Table 2;

[0080] Table 2

[0081] Object to be tested Standard curve equation Correlation coefficient r T3 Y=0.636X+0.6738 0.9942 T4 Y=0.6103X+0.2967 0.9970

[0082] from Figure 2-3 As can be seen from the data in Table 2, T3 and T4 in the dried blood spots show a good linear relationship in the mass concentration range of 1-100 ng / mL.

[0083] 2. Intra-batch precision

[0084] Three quality control samples of high, medium and low concentrations were taken, 3 copies of each concentration were taken, and each copy was tested 3 times, and each concentration was tested 9 times in total. The intra-batch precision of T3 is shown in Table 3, and the intra-batch precision of T4 is shown in Table 4;

[0085] Table 3

[0086]

[0087] Table 4

[0088]

[0089] As can be seen from Tables 3-4, the detection of T3 and T4 obtained by the method of the present invention has better intra-batch precision.

[0090] 3. Batch-to-batch precision

[0091] Take three quality control samples with high, medium and low concentrations, repeat the measurement 3 times per day for 5 consecutive days, and record the results. The inter-batch precision of T3 is shown in Table 5, and the inter-batch precision of T4 is shown in Table 6;

[0092] Table 5

[0093]

[0094] Table 6

[0095]

[0096] It can be seen from Tables 5-6 that the method of the present invention has better inter-batch precision for the detection of T3 and T4.

[0097] 4. Accuracy

[0098] Collect three clinical blood samples, mix them and divide them into four parts, one of which is the matrix blood sample, and the other three parts are added with high, medium and low standard solutions to obtain mixed samples. The high, medium and low concentrations of spiked amounts T3 and T4 are 80ng / mL, 10ng / mL; 40ng / mL, 5ng / mL; 15ng / mL, 1ng / mL. The above four samples were made into dried blood spot samples, and each sample was processed in 3 parts and tested at the same time. The accuracy (spiked recovery rate) of T3 is shown in Table 7, and the accuracy (spiked recovery rate) of T4 is shown in Table 8;

[0099] Table 7

[0100]

[0101] Table 8

[0102]

[0103] It can be seen from Tables 7-8 that the accuracy of T3 is between 96.78% and 107.64%, and the accuracy of T4 is between 91.33% and 111.13%, indicating that the accuracy of the detection of T3 and T4 using the technical solution of the present invention is relatively high.

[0104] 5. Detection limit

[0105] Three concentration levels of calibrator blood spots were selected, and the signal-to-noise ratio (S / N)>3 and S / N>10 were used as the detection limit and quantification limit of the method, respectively. Each concentration of the quantification limit was processed in 3 parts, and each sample was repeated 3 times. Three batches were connected and measured. The total precision (CV) of each concentration sample and the deviation between the mean concentration and the theoretical concentration were calculated. The lowest concentration of calibrator blood spots with CV≤20% and accuracy<15% was used as the lower limit of quantification. The detection limit and quantification limit of T3 are shown in Table 9, and the detection limit and quantification limit of T4 are shown in Table 10.

[0106] Table 9

[0107] Calibrator 1 Calibrator 2 Calibrator 3 Theoretical value (ng / mL) 0.1 0.4 0.8 Measured mean value (ng / mL) 0.12 0.45 0.87 Number of samples 9 9 9 SN 6 33 58 CV(%) 18.93 14.62 11.83 Offset(%) 20 12.5 8.75

[0108] Table 10

[0109] Calibrator 1 Calibrator 2 Calibrator 3 Theoretical value (ng / mL) 0.2 0.6 1.0 Measured mean value (ng / mL) 0.26 0.67 1.09 Number of samples 9 9 9 SN 5 42 71 CV(%) 17.23 11.38 10.69 Offset(%) 30 11.67 9

[0110] As can be seen from Tables 9 to 10, the detection limit and quantification limit of T3 were 0.1 ng / mL and 0.4 ng / mL respectively; the detection limit and quantification limit of T4 were 0.2 ng / mL and 0.6 ng / mL respectively.

[0111] Example 2

[0112] This example explores the influence of liquid chromatography conditions, wherein the flow rate of the chromatographic column of the test group 1 of this example is 0.3 mL / min, and the column temperature is 45°C, and the flow rate of the chromatographic column of the test group 2 is 0.4 mL / min, and the column temperature is 45°C. Except that the flow rate and column temperature of the chromatographic column are different from those of Example 1, the rest are completely consistent, and the chromatogram obtained is as follows Figure 1 , Figure 4-5 shown; from Figure 1 , Figure 4-5 It can be seen that although increasing the column flow rate and column temperature can shorten the peak time of T3 and T4, the two have tailing and there is a possibility of mutual interference, so the separation effect is better when the column flow rate is 0.3mL / min and the column temperature is 37℃.

[0113] Example 3

[0114] This example explores the influence of the extracting solution used in the preparation process of the internal standard extracting solution. The test groups 3 to 6 respectively use a mixed solution of methanol and acetonitrile (the volume ratio of methanol to acetonitrile is 3:2) (i.e., the same as the extracting solution used in Example 1), methanol, ethyl acetate, and n-hexane as the extracting solution, and the rest is completely consistent with Example 1. The obtained T3 and T4 peak area spectra are as follows: Figures 6 to 9 As shown;

[0115] from Figures 6 to 9 It can be seen that when a mixed solution of methanol and acetonitrile (the volume ratio of methanol to acetonitrile is 3:2) is used as the extracting liquid to prepare the internal standard extracting liquid, the peak areas of T3 and T4 are the highest, the instrument response is the strongest, and the extraction efficiency is the highest, followed by methanol. When ethyl acetate and n-hexane are used as the extracting liquid, the extraction efficiency is poor.

[0116] Example 4

[0117] This example explores the effect of the time and speed of mixing and shaking on the results, specifically including the following steps: three concentration standard dried blood spots of 1.56 ng / mL, 25 ng / mL, and 100 ng / mL were taken and divided into five groups for study, each sample was processed in parallel 3 times, the time and speed of the first group were 45 min and 750 rpm, the time and speed of the second group were 45 min and 950 rpm, the time and speed of the third group were 60 min and 750 rpm (consistent with the time and speed of Example 1), the time and speed of the fourth group were 60 min and 950 rpm, the time and speed of the fifth group were 90 min and 750 rpm, and the time and speed of the sixth group were 90 min and 950 rpm. Except for the different time and speed of the above-mentioned mixing and shaking, the rest were completely consistent with Example 1; the results obtained are as follows Figures 10-12 shown; Figures 10-12These are the summary graphs of peak area data of T3 and T4 at three concentrations of 1.56 ng / mL, 25 ng / mL, and 100 ng / mL;

[0118] from Figures 10-12 It can be seen that the peak areas of T3 and T4 of the three concentrations increase with the increase of extraction time under the same rotation speed conditions. The peak areas of the 45min, 750rpm group (the first group) are lower than those of the 60min, 750rpm group (the third group). After non-parametric rank sum test, the two-to-two comparisons showed P < 0.5, and the difference was statistically significant; while the peak area ratio of the 60min, 750rpm group (the third group) and the 90min, 750rpm group (the fifth group) was P> 0.5, and the difference was not statistically significant. For the same extraction time, the peak area of ​​the 750rpm group was higher than that of the 950rpm group. This may be because the high rotation speed caused the extract to overflow or volatilize, thereby reducing the extraction efficiency. In the present invention, the optimal extraction time is 60min and the optimal rotation speed is 750rpm through oscillation heating extraction, and the optimal extraction efficiency is achieved in a shorter time, ensuring the efficiency and reliability of the experiment.

[0119] Finally, it should be noted that the above embodiments are intended to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for detecting thyroxine and triiodothyronine in dried blood spots, characterized in that: The following steps are involved: (1) Add the internal standard extract to the dried blood spot sample, mix and shake, centrifuge, filter, blow with nitrogen, and re-dissolve to obtain the sample to be tested; (2) Using a liquid chromatography-mass spectrometry system to detect the sample to be tested; In the step (1), the temperature of the mixing and shaking is 35°C to 45°C, the speed of the mixing and shaking is 700-950 rpm, and the time of the mixing and shaking is 45-90 minutes; In the step (1), the internal standard extract is obtained by diluting the internal standard stock solution with the extract; The internal standard stock solution contains 13 C6-triiodothyronine and 13 C6-thyroxine; the internal standard stock solution 13 C6-triiodothyronine and 13 The mass concentrations of C6-thyroxine were all 100 ng / mL; The extract comprises any one of methanol, n-hexane, ethyl acetate, and a mixed solution of methanol and acetonitrile; the volume ratio of methanol to acetonitrile in the mixed solution of methanol and acetonitrile is 3:2; In step (2), the liquid chromatography conditions in the detection are: Chromatographic column: Waters ACQUITY UPLC HSS T3 Column, 1.8µm × 2.1mm × 50mm; Column temperature: 37~45℃; Flow rate: 0.3-0.4 mL / min; Injection volume: 10 µL; Mobile phase A: 0.1-0.5% formic acid, 1-5 mmol / L ammonium acetate aqueous solution; Mobile phase B: 0.1-0.5% formic acid, 1-5 mmol / L ammonium acetate methanol solution; Gradient elution program: 0-0.01 min, mobile phase A volume percentage 80-70%, mobile phase B volume percentage 20-30%; From 0.01 to 3 min, the volume percentage of mobile phase A decreased from 80 to 70% to 8 to 2%, and the volume percentage of mobile phase B increased from 20 to 30% to 92 to 98%; 3.01min~4min, mobile phase A volume percentage 8~2%, mobile phase B volume percentage 92~98%; 4.01~6min, mobile phase A volume percentage 80%~70%, mobile phase B volume percentage 20~30%.

2. The detection method according to claim 1, characterized in that: In the step (1), the internal standard extract contains 13 C6-triiodothyronine and 13 C6-thyroxine; the internal standard extract 13 C6-triiodothyronine and 13 The mass concentrations of C6-thyroxine are all 1 to 10 ng / mL.

3. The detection method according to claim 1, characterized in that: In the step (1), after the mixing and shaking is completed, the supernatant is extracted and centrifuged and filtered.

4. The detection method according to claim 1, characterized in that: In the step (1), the centrifugal speed is 1400-1600 rpm / min, and the centrifugal time is 2.5-3.5 minutes.

5. The detection method according to claim 1, characterized in that: In the step (1), the solution used for re-dissolving is a methanol-water solution, and the volume percentage of methanol in the methanol-water solution is 30-45%.

6. The detection method according to claim 1, characterized in that: In step (2), the mass spectrometry parameters in the detection are: Ion source: electrospray ionization ion source; Ion mode: Multiple reaction monitoring mode positive ion mode; Ion source temperature: 350°C; Ion source voltage: 4500V; Ion transfer tube temperature: 350°C; Sheath gas: 35mL; Auxiliary gas: 10mL.

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