CN115032380A - Method for detecting thyroxine and triiodothyroxine in dried blood spots - Google Patents
Method for detecting thyroxine and triiodothyroxine in dried blood spots Download PDFInfo
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/534—Production of labelled immunochemicals with radioactive label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
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- G—PHYSICS
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- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
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- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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Abstract
The invention discloses a method for detecting thyroxine and triiodothyroxine in dry blood spots, belonging to the technical field of biochemical analysis and detection; the invention relates to a method for detecting thyroxine and triiodothyroxine in dry blood spots, which comprises the following steps: (1) adding an internal standard extraction liquid into the dry blood spot sample, mixing and oscillating, centrifugally filtering, blowing nitrogen, and redissolving to obtain a sample to be detected; (2) and detecting the sample to be detected by adopting a liquid chromatography-mass spectrometry combined system. The detection method provided by the invention can simultaneously detect the concentration levels of T3 and T4 in the dried blood spots, and has the characteristics of high sensitivity, strong anti-interference capability, no influence by abnormal binding protein, medicines and the like, and high accuracy; meanwhile, dry blood spots are used as a detection basic substance, so that the collection, transportation, detection and preservation of newborn samples can be facilitated, and the kit is suitable for large-scale screening; the pretreatment operation of the dry blood spot specimen is simple, the determination time is short, and the detection efficiency can be obviously improved.
Description
Technical Field
The invention belongs to the technical field of biochemical analysis and detection, and particularly relates to a method for detecting thyroxine and triiodothyroxine in dry blood spots.
Background
Screening congenital hypothyroidism screening for neonatal diseases is mainly characterized in that an immunity method is used for detecting whether the concentration level of Thyrotropin (TSH) in dry blood spots indirectly reflects the level of thyroxine (T4) and triiodothyroxine (T3) in a human body. The immunoassay method for detecting the thyrotropin of the newborn is a solid-phase double-position fluorescence immunoassay method based on a direct sandwich method technology. Two kinds of monoclonal antibodies are directly combined with different point positions of human thyroid stimulating hormone (hTSH) molecules at specific antigen point positions on the hTSH molecules contained in a sample to be detected, and simultaneously react with the solid-phase monoclonal antibody and the europium-labeled monoclonal antibody to form a solid-phase antibody-Eu-labeled monoclonal antibody and an antigen-antibody complex on a sample beta-hTSH subunit, washing and adding a reinforcing solution are carried out, and Eu on the labeled antibody is added with the reinforcing solution + Dissociate and form fluorescent chelates that fluoresce strongly, and the hTSH concentration of each sample is determined by measuring the fluorescence intensity of each well. The immunoassay has low sensitivity and depends on binding protein to some extent, so the immunoassay is easily influenced by various interferences and uncertainties, the interferences can relate to drug interaction, cross reactivity of antibodies, sensitivity to abnormal binding protein, influence of Free Fatty Acid (FFA) and endogenous or exogenous binding inhibitors, so the immunoassay has good sensitivity on primary hypothyroidism and is easy to screen for secondary hypothyroidism to cause false negative.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides the method for detecting thyroxine and triiodothyroxine in the dried blood spots, which has the advantages of high sensitivity, high accuracy, strong anti-interference capability, convenience for large-scale screening and effective reduction of false positive rate.
In order to achieve the purpose, the invention adopts the technical scheme that: a method for detecting thyroxine and triiodothyroxine in dried blood spots comprises the following steps:
(1) adding the internal standard extraction liquid into the dry blood spot sample, mixing and oscillating, centrifugally filtering, blowing nitrogen, and redissolving to obtain a sample to be detected;
(2) and detecting the sample to be detected by adopting a liquid chromatography-mass spectrometry combined system.
The detection method provided by the invention adopts a high performance liquid chromatography-mass spectrometry method, so that the concentration levels of thyroxine (T3) and triiodothyroxine (T4) in dry blood spots can be simultaneously detected, and on the one hand, the detection method provided by the invention adopts a liquid chromatography-mass spectrometry technology, has the characteristics of high sensitivity, high accuracy and strong anti-interference capability, is not influenced by abnormal binding protein, specimen hemolysis, jaundice, lipemia, medicines and the like, and can obviously improve the accuracy of results; in the second aspect, the technical scheme adopted by the invention takes the dried blood spots as the detection basic material, so that the collection, transportation, detection and storage of newborn samples can be facilitated, and the method is suitable for large-scale screening; compared with the LC-MS/MS detection method at the present stage, the pretreatment operation of the dried blood spot specimen in the technical scheme provided by the invention is simple, the determination time is short, and the detection efficiency can be obviously improved.
In a preferred embodiment of the detection method of the present invention, in the step (1), the dried blood spot sample is a treated dried blood spot, and the treatment is performed by punching the dried blood spot with a punch having a diameter of 3mm to obtain the dried blood spot sample.
As a preferred embodiment of the detection method of the present invention, in the step (1), the internal standard extract contains 13 C 6 -triiodothyroxine and 13 C 6 -thyroxine.
As a preferred embodiment of the detection method of the present invention, the internal standard extract is 13 C 6 -triiodothyroxine and 13 C 6 and the mass concentration of the thyroxine is 1-10 ng/mL respectively.
As a preferred embodiment of the detection method of the present invention, in the step (1), the internal standard extraction liquid is obtained by diluting an internal standard stock solution with an extraction liquid; the internal standardThe stock solution contains 13 C 6 -triiodothyroxine and 13 C 6 -thyroxine; in the internal standard extraction liquid 13 C 6 -triiodothyroxine and 13 C 6 the mass concentrations of thyroxine were all 100ng/mL, respectively.
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: will be provided with 13 C 6 Dissolving triiodothyroxine in methanol-water (50: 50 volume ratio) mixed solution containing 0.1% formic acid to obtain standard solution with mass concentration of 1mg/mL, storing at-80 deg.C, and mixing with methanol-acetonitrile (50: 50 volume ratio) 13 C 6 -triiodothyroxine Standard stock solution and 13 C 6 and mixing and diluting the thyroxine internal standard solution into internal standard stock solutions with the mass concentration of 100ng/mL respectively.
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 the methanol to the acetonitrile in the mixed solution of the methanol and the acetonitrile is 3: 2.
As a preferred embodiment of the detection method of the present invention, in the step (1), after the internal standard extraction liquid is added, before mixing and shaking, a film sealing process using a heat sealing instrument aluminum foil paper is further included.
As a preferred embodiment of the detection method of the present invention, in the step (1), the temperature of mixing and shaking is 35-45 ℃, the rotation speed of mixing and shaking is 700-950rpm, and the time of mixing and shaking is 45-90 minutes.
In a preferred embodiment of the detection method of the present invention, in the step (1), after the mixing and shaking are completed, the supernatant is extracted and filtered and centrifuged.
As a preferred embodiment of the detection method of the present invention, the filter plate is a 96-well micro-filter plate having a pore size of 45. mu.M.
As a preferred embodiment of the detection method of the present invention, in the step (1), the rotation speed of centrifugation is 1400-1600rpm/min, and the time of centrifugation is 2.5-3.5 minutes.
In the step (1), the supernatant after centrifugal filtration is placed in a nitrogen blowing instrument, and is blown for 20min to 30min to be dried under the condition of the room temperature nitrogen flow rate of 5 to 15 mL/min.
In a preferred embodiment of the detection method of the present invention, in the step (1), the solution used for re-dissolution is a methanol aqueous solution, and the volume percentage of methanol in the methanol aqueous solution is 30 to 45%.
In a preferred embodiment of the detection method of the present invention, in the step (2), the liquid chromatography conditions during detection are: a chromatographic column: watts ACQUITY UPLC HST 3 Column, 1.8 μm × 2.1mm × 50 mm; column temperature: 37-45 ℃; flow rate: 0.3-0.4 mL/min; sample introduction amount: 10 mu L of the solution; mobile phase A: 0.1-0.5% formic acid, 1-5 mmol/L ammonium acetate water solution; mobile phase B: 0.1-0.5% formic acid, 1-5 mmol/L ammonium acetate methanol solution; gradient elution procedure: 0-0.01 min, the volume percentage of the mobile phase A is 80-70%, and the volume percentage of the mobile phase B is 20-30%; the volume percentage of the mobile phase A is reduced to 8-2% from 80-70% in 0.01-3 min, and the volume percentage of the mobile phase B is increased to 92-98% from 20-30%; 3.01-4 min, the volume percentage of the mobile phase A is 8-2%, and the volume percentage of the mobile phase B is 92-98%; 4.01-6 min, the volume percentage of the mobile phase A is 80-70%, and the volume percentage of the mobile phase B is 20-30%.
As a preferred embodiment of the detection method of the present invention, in the step (2), the mass spectrum parameters during detection are: an ion source: an electrospray ionization ion source; ion mode: a multiple reaction monitoring mode positive ion mode; ion source temperature: 350 ℃; ion source voltage: 4500V; ion transport tube temperature: 350 ℃; sheath gas: 35 mL; auxiliary gas: 10 mL.
In addition, the invention also provides a detection kit for simultaneously detecting thyroxine and triiodothyroxine in dry blood spots, wherein the kit comprises standard working solution, internal standard stock solution, extraction liquid and a liquid chromatogram mobile phase; the standard working solution is a thyroid solution and a triiodothyronine solution with standard mass concentration; the liquid chromatography mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is 0.1-0.5% of formic acid and 1-5 mmol/L of ammonium acetate aqueous solution, and the mobile phase B is 0.1-0.5% of formic acid and 1-5 mmol/L of ammonium acetate methanol solution.
Compared with the prior art, the invention has the beneficial effects that:
firstly, the method comprises the following steps: the detection method provided by the invention uses a liquid chromatography-mass spectrometry technology, has the characteristics of high sensitivity, high accuracy and strong anti-interference capability, and is not influenced by abnormal binding protein, specimen hemolysis, jaundice, lipemia, medicines and the like, and the obtained result has higher accuracy;
secondly, the method comprises the following steps: according to the technical scheme, the dry blood spots are used as detection basic substances, so that the collection, transportation, detection and preservation of newborn samples can be facilitated, and the method is suitable for large-scale screening;
thirdly, the method comprises the following steps: the technical scheme provided by the invention has the advantages of simple pretreatment operation of the dried blood spot specimen, short determination time and high detection efficiency.
Drawings
FIG. 1 is a total ion current chromatogram subflows of T3 and T4 obtained in example 1;
FIG. 2 is a line graph of the T3 standard in example 1;
FIG. 3 is a line graph of the T4 standard in example 1;
FIG. 4 is a total ion current chromatogram of T3 and T4 obtained in test group 1 of example 2;
FIG. 5 is a total ion current chromatogram of T3 and T4 obtained from test group 2 in example 2;
FIG. 6 is a chromatogram of T3 and T4 obtained in test group 3 of example 3;
FIG. 7 is a chromatogram of T3 and T4 obtained in test group 4 of example 3;
FIG. 8 is a chromatogram of T3 and T4 obtained in test group 5 of example 3;
FIG. 9 is a chromatogram of T3 and T4 obtained in test group 6 of example 3;
FIG. 10 is a summary graph of peak area data of T3 and T4 measured on samples of example 4 at a concentration of 1.56 ng/mL;
FIG. 11 is a summary graph of peak area data of T3 and T4 measured on samples at a concentration of 25ng/mL in example 4;
FIG. 12 is a summary of peak area data for T3 and T4 from the test of 100ng/mL samples from example 4.
Detailed Description
To better illustrate the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to specific examples.
The raw materials and equipment used in the examples of the present invention are all available from conventional purchase routes, and the main instruments and reagents are:
1. the main apparatus is as follows:
high performance liquid chromatography tandem mass spectrometry: 9050MD, china inc, engorge, denna;
constant temperature micro-oscillator: wallac corporation;
Milli-Q ultrapure water meter: millipore, USA;
an ultrasonic cleaner: scientific union KM-23C;
the microporous plate seals membrane appearance: hangzhou Ruicheng MSP 200.
2. The main reagents are as follows:
thyroxine solution (standard): supelco corporation;
triiodothyroxine (standard): TRC Corp Ltd;
13 C 6 thyroxine internal standard solution (internal standard): supelco corporation;
13 C 6 triiodothyroxine (internal standard): TRC Corp;
methanol: merck corporation;
acetonitrile: CNW corporation;
formic acid: sigma;
and (3) amine acetate: shanghai' an spectral company.
Example 1
In the embodiment, thyroxine and triiodothyroxine in the dry blood spot sample are detected and analyzed;
preparation of reagent
(1) Preparation of blank whole blood: collecting 2mL of whole blood of a healthy volunteer, performing anticoagulation by sodium dipotassium citrate, centrifuging for 10min at 3500r/min by a high-speed centrifuge after collection, and separating serum and red blood cells; taking out upper serum solution, adding 0.9% sodium chloride solution with the same volume into erythrocytes for washing, repeatedly rotating and shaking for full cleaning, then placing on a high-speed centrifuge for centrifuging at 3500r/min for 10min, repeatedly cleaning for four times, and then removing the upper solution; sucking the red blood cells precipitated at the bottom of the centrifuge tube, and adding the mixture with the volume ratio of the red blood cells being 60: 40, preparing artificial blank whole blood by using the blank serum washed by the activated carbon;
(2) preparation of standard solution: accurately weighing triiodothyroxine standard, dissolving with methanol-water (volume ratio of 50: 50) mixed solution containing 0.1% formic acid to obtain standard stock solution with mass concentration of 1mg/mL, storing at-80 deg.C, and diluting with methanol-water (volume ratio of 50: 50) mixed solution containing 0.1% formic acid to desired concentration when in use;
(3) preparing an internal standard stock solution: accurately weighing 13 C 6 An internal standard substance of triiodothyroxine, which is dissolved by a methanol-water (volume ratio of 50: 50) mixed solution containing 0.1% formic acid to prepare a standard solution of 1mg/mL, and then diluted by a methanol-acetonitrile (volume ratio of 50: 50) mixed solution 13 C 6 -triiodothyroxine stock and 13 C 6 mixing the thyroxine internal standard substance into a mass concentration of 100ng/mL 13 C 6 -triiodothyroxine and a mass concentration of 100ng/mL 13 C 6 -a mixed internal standard stock solution of thyroxine, stored at-80 ℃;
(4) preparing internal standard extract liquid: diluting the internal standard stock solution by using a mixed solution of methanol and acetonitrile (the volume ratio of the methanol to the acetonitrile is 3:2) as an extraction solution to obtain an internal standard extraction solution with the concentration of 5 ng/mL;
(5) preparing a standard dry blood spot: adding a series of standard solutions with the concentration diluted by 50% methanol aqueous solution by volume fraction into blank whole blood to prepare standard solution with the mass concentration of 0.195, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50 and 100ng/mL respectively, sucking a proper amount of whole blood standard sample by a suction pipe and dripping the whole blood standard sample into a dry and clean American No. 903 filter paper sheet respectively, then placing the filter paper sheet on a blood sheet drying rack with the right side facing upwards, naturally drying the blood sheet at room temperature, placing the dried blood sheet into a sealed plastic bag after drying, and placing the plastic bag into a refrigerator with the temperature of-20 ℃ for storage and standby;
(6) preparing a quality control product:
diluting the methanol aqueous solution with the volume fraction of 50% into quality control solutions with high, medium and low concentrations, adding the quality control solutions into blank whole blood, and preparing the quality control solutions with the mass T3 and the T4 concentrations respectively as follows: 10.5, 45.5, 72.7ng/mL, 0.9, 3.2, 9.1ng/mL whole blood quality control sample, accurately taking 200 microliter of whole blood quality control sample and dripping the whole blood quality control sample into a dry and clean American No. 903 filter paper sheet, then placing the filter paper sheet on a blood sheet drying rack with the right side upward, naturally drying the blood sheet at room temperature, placing the blood sheet on a sealed plastic bag after drying the blood sheet, and placing the sealed plastic bag into a refrigerator at the temperature of-20 ℃ for storage and later use. The preparation of the standard substance and the quality control substance dried blood spot can not be simultaneously prepared, and the preparation should be prepared in different batches.
(II) detection method
The detection method of the embodiment of the invention comprises the following steps:
(1) respectively punching two blood spots of a standard dry blood spot, a quality control dry blood spot and a to-be-detected dry blood spot into a 96-hole microplate hole by using a puncher with the diameter of 3mm to respectively obtain a standard dry blood spot sample, a quality control dry blood spot sample and a to-be-detected dry blood spot sample;
(2) adding 200 mu L of internal standard extract liquor into a standard product, a quality control product and a dry blood spot sample to be detected, sealing a film by using aluminum foil paper of a heat sealing instrument, and placing the film into a constant-temperature oscillator for mixing and oscillating, wherein the temperature of the mixing oscillator is 37 ℃, the rotating speed is 750rpm, and the oscillating time is 60 minutes; after the oscillation is finished, extracting 150 mu L of supernatant to a 45 mu M96-hole micro-filtration plate with the aperture, centrifuging for 3 minutes at the rotating speed of 1500rpm/min, drying the filtrate by nitrogen with the flow rate of 10mL/min, adding 35% methanol water complex solution, and oscillating for 5 minutes at a high speed of 1020rpm/min to obtain a sample to be detected;
(3) detecting a sample to be detected by adopting a liquid chromatography-mass spectrometry combined system; wherein, the liquid phase chromatographic conditions are that a chromatographic column: watts ACQUITY UPLC HSS T3 Column, 1.8 μm × 2.1mm × 50mm, Column temperature: 37 ℃, flow rate: 0.4mL/min, sample size: 10 μ L, mobile phase A: 0.1% formic acid, 2nmol/L aqueous ammonium acetate, mobile phase B: 0.1% formic acid, 2nmol/L ammonium acetate in methanol, gradient elution procedure: 0-0.01 min, the volume percentage of the mobile phase A is 70%, and the volume percentage of the mobile phase B is 30%; 0.01-3 min, the volume percentage of the mobile phase A is reduced from 70% to 2%, and the volume percentage of the mobile phase B is increased from 30% to 98%; 3.01-4 min, the volume percentage of the mobile phase A is 2 percent, and the volume percentage of the mobile phase B is 98 percent; 4.01-6 min, the volume percent of the mobile phase A is 70%, and the volume percent of the mobile phase B is 30%;
the mass spectrum parameters are as follows: an ion source: electrospray ionization ion source, ion mode: multiple reaction monitoring mode positive ion mode, ion source temperature: 350 ℃, ion source voltage: 4500V, ion transfer tube temperature: 350 ℃, sheath gas: 35mL, auxiliary gas: 10 mL;
wherein, in the detection, the total ion chromatogram of the obtained standard substance is shown in fig. 1, and as can be seen from fig. 1, the peak shapes of the T3 and T4 standard substances are symmetrical, and no interference of other impurities exists, which indicates that good detection can be obtained under the conditions;
in mass spectrometric detection, molecular ions, daughter ions, radio frequency voltage, collision voltage and residence time of T3 and T4 mass spectrometric parameters are shown in Table 1:
TABLE 1
(III) data analysis
1. Drawing a standard curve
After the standard curve sample is separated by liquid chromatography, the thyroxine and triiodothyroxine peak at different elution times and are detected by mass spectrometry, thereby detecting the content of the thyroxine and the triiodothyroxine. Configuring a sample to be detected according to a certain standard sample mass concentration for detection, establishing a calibration curve by adopting an isotope internal standard quantitative method and utilizing analysis software to take the concentration ratio of a standard substance and an internal standard substance as an X axis and the peak area ratio of the standard substance and the internal standard substance as a Y axis, and calculating the mass concentration of thyroxine and triiodothyroxine of a dry blood spot sample; the prepared standard curve of T3 is shown in FIG. 2, and the standard curve of T4 is shown in FIG. 3; the standard curve equation and the correlation coefficient are shown in table 2;
TABLE 2
| Test object | Equation of scalar curve | Coefficient of correlation r |
| T3 | Y＝0.636X+0.6738 | 0.9942 |
| T4 | Y＝0.6103X+0.2967 | 0.9970 |
As can be seen from the data in FIGS. 2-3 and Table 2, T3 and T4 exhibited good linearity in dried blood spots over the mass concentration range of 1-100 ng/mL.
2. In-batch precision
Taking three quality control product concentration samples of high, medium and low, wherein each concentration is 3 parts, each part is repeatedly subjected to sample injection detection for 3 times, and each concentration is measured for 9 times, wherein the batch precision of T3 is shown in Table 3, and the batch precision of T4 is shown in Table 4;
TABLE 3
TABLE 4
As can be seen from tables 3-4, the test of both T3 and T4 obtained by the method of the present invention has superior in-batch precision.
3. Inter-batch precision
Taking three quality control substance concentration samples of high, medium and low, repeatedly measuring for 3 times per case every day, continuously measuring for 5 days, and recording the result, wherein the batch-to-batch precision of T3 is shown in Table 5, and the batch-to-batch precision of T4 is shown in Table 6;
TABLE 5
TABLE 6
As can be seen from tables 5-6, the test of both T3 and T4 using the method of the present invention has superior batch-to-batch precision.
4. Accuracy of
Collecting three clinical blood samples, uniformly mixing, and evenly dividing into four parts, wherein one part is a matrix blood sample, the other three parts are respectively added with high, medium and low standard substance solutions to be mixed to obtain a mixed sample, and scalar quantities T3 and T4 are added, and the high, medium and low concentrations are 80ng/mL and 10ng/mL respectively; 40ng/mL and 5 ng/mL; 15ng/mL and 1 ng/mL. The four samples are prepared into dry blood spot samples, each sample is divided into 3 parts for treatment and detection, wherein the accuracy (standard adding recovery rate) of T3 is shown in table 7, and the accuracy (standard adding recovery rate) of T4 is shown in table 8;
TABLE 7
TABLE 8
From tables 7-8, it can be seen that the accuracy of T3 is between 96.78 and 107.64%, and the accuracy of T4 is between 91.33 and 111.13%, which indicates that the detection accuracy of T3 and T4 is higher by using the technical solution of the present invention.
5. Detection limit
Selecting 3 concentration levels of calibrator blood spots, respectively taking a signal-to-noise ratio (S/N) >3 and an S/N >10 as a method detection limit and a quantification limit, processing 3 parts of each concentration by the quantification limit, repeatedly measuring 3 times for each sample, connecting and measuring 3 batches, calculating the deviation of the total precision (CV) and the concentration measurement mean value of each concentration sample from a theoretical concentration, taking the lowest concentration calibrator blood spot with the CV being less than or equal to 20% and the accuracy being less than 15% as a quantification lower limit, wherein the detection limit and the quantification limit of T3 are shown in Table 9, and the detection limit and the quantification limit of T4 are shown in Table 10;
TABLE 9
| |
|
|
|
| Theoretical value (ng/mL) | 0.1 | 0.4 | 0.8 |
| Measured mean value (ng/mL) | 0.12 | 0.45 | 0.87 |
| Number of |
9 | 9 | 9 |
| |
6 | 33 | 58 |
| CV(％) | 18.93 | 14.62 | 11.83 |
| Offset (%) | 20 | 12.5 | 8.75 |
| |
|
|
|
| Theoretical value (ng/mL) | 0.2 | 0.6 | 1.0 |
| Measured mean value (ng/mL) | 0.26 | 0.67 | 1.09 |
| Number of |
9 | 9 | 9 |
| SN | 5 | 42 | 71 |
| CV(％) | 17.23 | 11.38 | 10.69 |
| Offset (%) | 30 | 11.67 | 9 |
As can be seen from tables 9-10, the detection limit and the quantification limit of T3 obtained by the test are respectively 0.1ng/mL and 0.4 ng/mL; the detection limit and the quantification limit of T4 were 0.2ng/mL and 0.6ng/mL, respectively.
Example 2
The present example investigated the influence of liquid chromatography conditions, wherein the flow rate of the chromatographic column of test group 1 of the present example was 0.3mL/min, the column temperature was 45 ℃, the flow rate of the chromatographic column of test group 2 was 0.4mL/min, the column temperature was 45 ℃, the rest remained completely consistent except that the flow rate and the column temperature of the chromatographic column were not consistent with those of example 1, and the obtained chromatograms were shown in fig. 1, fig. 4 to fig. 5; as can be seen from FIGS. 1 and 4 to 5, the peak emergence times of T3 and T4 can be shortened by increasing the flow rate and the column temperature of the chromatographic column, but the flow rate and the column temperature of the chromatographic column are 0.3mL/min and the separation effect at 37 ℃ is better because the two have tails and mutual interference is possible.
Example 3
In this example, the influence of the extraction liquid used in the preparation process of the internal standard extraction liquid is studied, wherein test groups 3 to 6 respectively adopt a mixed solution of methanol and acetonitrile (the volume ratio of methanol to acetonitrile is 3:2) (namely, the volume ratio is consistent with that of the extraction liquid used in example 1), 4 organic solvents of methanol, ethyl acetate and n-hexane as extraction liquids, and the rest are completely consistent with that of example 1, and the peak area maps of T3 and T4 obtained are respectively shown in fig. 6 to 9;
as can be seen from FIGS. 6 to 9, when the mixed solution of methanol and acetonitrile (the volume ratio of methanol to acetonitrile is 3:2) is used as the extraction liquid to prepare the internal standard extraction liquid, the peak areas of T3 and T4 are the highest, the instrument has the strongest response, the extraction efficiency is the highest, and the extraction efficiency is the next highest when methanol is used, while the extraction efficiency is poor when ethyl acetate and n-hexane are used as the extraction liquid.
Example 4
The embodiment researches the influence of the time and the rotating speed of the mixing oscillation on the result, and specifically comprises the following steps: taking three concentration standard substance dry blood spots of 1.56ng/mL, 25ng/mL and 100ng/mL respectively, dividing the standard substance dry blood spots into five groups for research, carrying out parallel treatment on each sample for 3 times, wherein the time and the rotating speed of the first group are 45min and 750rpm, the time and the rotating speed of the second group are 45min and 950rpm, the time and the rotating speed of the third group are 60min and 750rpm (consistent with the time and the rotating speed of the example 1), the time and the rotating speed of the fourth group are 60min and 950rpm, the time and the rotating speed of the fifth group are 90min and 750rpm, and the time and the rotating speed of the sixth group are 90min and 950rpm, and the rest are completely consistent with the example 1 except that the time and the rotating speed of the mixing oscillation are different; the obtained results are shown in FIGS. 10 to 12; wherein, FIGS. 10 to 12 are respectively a summary chart of peak area data of the 1.56ng/mL, 25ng/mL and 100ng/mL concentrations T3 and T4;
from fig. 10 to 12, it can be seen that peak areas of T3 and T4 at three concentrations increase with the extraction time under the same rotation speed condition, the peak areas increase with the increase, and P is less than 0.5 through nonparametric rank sum test and pairwise comparison in the 45min and 750rpm group (the first group) than the 60min and 750rpm group (the third group), and the difference has statistical significance; the peak area ratio of the 60min and 750rpm group (the third group) to the 90min and 750rpm group (the fifth group) is more than 0.5, and the difference has no statistical significance. For the same extraction time, the peak area of the 750rpm group is higher than that of the 950rpm group, which may cause the overflow or volatilization of the extraction liquid due to the high rotation speed, thereby reducing the extraction efficiency. According to the invention, through vibration heating extraction, the optimal extraction time is 60min, the optimal rotation speed is 750rpm, the optimal extraction efficiency is reached in a short time, and the high efficiency and reliability of the experiment are ensured.
Finally, it should be noted that the above embodiments are intended to illustrate the technical solutions of the present invention and not to limit the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. A method for detecting thyroxine and triiodothyroxine in dried blood spots is characterized by comprising the following steps:
(1) adding the internal standard extraction liquid into the dry blood spot sample, mixing and oscillating, centrifugally filtering, blowing nitrogen, and redissolving to obtain a sample to be detected;
(2) and detecting the sample to be detected by adopting a liquid chromatography-mass spectrometry system.
2. The detection method according to claim 1, wherein in the step (1), the internal standard extract contains 13 C 6 -triiodothyroxine and 13 C 6 -thyroxine; in the internal standard extraction liquid 13 C 6 -triiodothyroxine and 13 C 6 thyroid glandThe mass concentration of each element is 1-10 ng/mL.
3. The detecting method according to claim 1, wherein in the step (1), the temperature of the mixing oscillation is 35-45 ℃, the rotation speed of the mixing oscillation is 700-950rpm, and the time of the mixing oscillation is 45-90 minutes.
4. The detection method according to claim 1, wherein in the step (1), the internal standard extraction liquid is obtained by diluting an internal standard stock solution with an extraction liquid;
the internal standard stock solution contains 13 C 6 -triiodothyroxine and 13 C 6 -thyroxine; in the internal standard extraction liquid 13 C 6 -triiodothyroxine and 13 C 6 -the mass concentration of thyroxine is 100ng/mL each;
the extraction liquid comprises any one of methanol, normal hexane, ethyl acetate and a mixed solution of methanol and acetonitrile; the volume ratio of the methanol to the acetonitrile in the mixed solution of the methanol and the acetonitrile is 3: 2.
5. The detection method according to claim 1, wherein in the step (1), after the mixing and shaking are finished, the supernatant is extracted and subjected to centrifugal filtration.
6. The detection method as claimed in claim 1, wherein in the step (1), the rotation speed of the centrifugation is 1400-1600rpm/min, and the time of the centrifugation is 2.5-3.5 minutes.
7. The detection method according to claim 1, wherein in the step (1), the solution used for re-dissolution is an aqueous methanol solution, and the volume percentage of methanol in the aqueous methanol solution is 30-45%.
8. The detection method according to claim 1, wherein in the step (2), the liquid chromatography conditions in the detection are as follows:
a chromatographic column: watts ACQUITY UPLC HSS T3 Column, 1.8 μm × 2.1mm × 50 mm;
column temperature: 37-45 ℃;
flow rate: 0.3-0.4 mL/min;
sample introduction amount: 10 mu L of the solution;
mobile phase A: 0.1-0.5% formic acid, 1-5 mmol/L ammonium acetate water solution;
mobile phase B: 0.1-0.5% formic acid, 1-5 mmol/L ammonium acetate methanol solution.
Gradient elution procedure: 0-0.01 min, the volume percentage of the mobile phase A is 80-70%, and the volume percentage of the mobile phase B is 20-30%;
reducing the volume percentage of the mobile phase A from 80-70% to 8-2% and increasing the volume percentage of the mobile phase B from 20-30% to 92-98% in 0.01-3 min;
3.01-4 min, 8-2% of mobile phase A by volume and 92-98% of mobile phase B by volume;
4.01-6 min, the volume percentage of the mobile phase A is 80-70%, and the volume percentage of the mobile phase B is 20-30%.
9. The detection method according to claim 1, wherein in the step (2), the mass spectrum parameters in the detection are as follows:
an ion source: an electrospray ionization ion source;
ion mode: a multiple reaction monitoring mode positive ion mode;
ion source temperature: 350 ℃;
ion source voltage: 4500V;
ion transfer tube temperature: 350 ℃;
sheath gas: 35 mL;
auxiliary gas: 10 mL.
10. A detection kit for simultaneously determining thyroxine and triiodothyroxine in dry blood spots is characterized by comprising a standard working solution, an internal standard stock solution, an extraction liquid and a liquid chromatogram mobile phase; the standard working solution is a thyroid solution and a triiodothyronine solution with standard mass concentration; the liquid chromatogram mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is 0.1-0.5% of formic acid and 1-5 mmol/L of ammonium acetate aqueous solution, and the mobile phase B is 0.1-0.5% of formic acid and 1-5 mmol/L of ammonium acetate methanol solution.
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