Online separation and valuing method for free thyroxine in serum

Abstract

The invention discloses an on-line separation and valuing method for free thyroxine in serum. The method comprises the following steps: (1) setting a two-dimensional liquid chromatogram; (2) on-line separation and removal of macromolecular interferents in the serum; (3) retaining free thyroid hormone; (4) on-line separation of free thyroid hormone; (5) on-line valuing by isotope dilution mass spectrometry; and (6) cleaning the two-dimensional liquid chromatography system. The method provided by the invention realizes on-line separation of free thyroid hormone in serum and absolute constant value. A first-dimensional chromatographic mobile phase is a mild ammonium salt buffer solution and does not contain an organic solvent, so that thyroid hormones combined with protein in serum can be kept in a combined state, and a target object entering mass spectrometry is free thyroid hormones; and the valuing result can be traced to the international unit system through the national certified standard substance. Compared with traditional serum off-line treatment modes such as solid-phase extraction and equilibrium dialysis, the method has the characteristics of high efficiency, reliability and easiness in operation.

Description

Technical Field

[0001] The invention relates to the technical field of biochemical analysis, and in particular to an on-line separation and determination method of free thyroxine in serum. Background Art

[0002] Accurate measurement of thyroid hormones is crucial for the diagnosis of thyroid diseases, especially free hormones, such as free triiodo-L-thyronine (FT3) and free L-thyroxine (FT4). Since they are not affected by iodine, they can more accurately reflect the functional status of the thyroid gland. FT3 is the first to increase in the early stages of hyperthyroidism or in the early stages of relapse, and is currently the preferred test item for diagnosing hyperthyroidism. Due to the low content of free hormones and the lack of accurate and reliable standard substances, it is still difficult to establish a reference measurement method for FT3 and FT4 in serum, especially how to ensure the endogenous balance of bound and free states during the hormone separation process. There is no globally recognized solution.

[0003] At present, most researchers believe that equilibrium dialysis, ultrafiltration, and solid phase extraction are the main methods for separating free hormones. However, their limitations are also obvious, for example: equilibrium dialysis is time-consuming, labor-intensive, and costly, and the target may be adsorbed on the membrane; ultrafiltration may cause protein leakage, and requires strict temperature and pH limits; solid phase extraction is greatly affected by human factors and is difficult to achieve consistency.

[0004] Based on this, the field of laboratory medicine urgently needs to establish an online, repeatable, highly reliable, and traceable method to develop corresponding standard substances or establish reference measurement procedures for the standardization of free thyroxine testing. Summary of the invention

[0005] The purpose of the present invention is to provide an on-line separation and determination method for free thyroxine in serum, wherein the determination result has metrological traceability.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows: A method for online separation and determination of free thyroid hormones in serum, wherein the free thyroid hormones are FT3 and FT4. Specifically comprising the following steps: (1) setting up two-dimensional liquid chromatography; (2) online separation and removal of macromolecular interferences in serum; (3) retention of free thyroid hormones (FT3, FT4); (4) online separation of free thyroid hormones (FT3, FT4); (5) online determination by isotope dilution mass spectrometry; (6) cleaning of the two-dimensional liquid chromatography system; Specifically, step (1) comprises two groups of chromatographic systems, namely, a first-dimensional chromatogram and a second-dimensional chromatogram, which are connected in series and in parallel by switching of a six-way valve.

[0007] Among them, the chromatographic column in the first dimension chromatography is based on a hydrophobic ultra-macroporous polymer resin (4 µm) and coated with a hydrophobic DVB (divinylbenzene) polymer, which is responsible for removing serum macromolecular components such as fatty acids, peptides, and proteins; the first dimension liquid chromatography conditions include: A. The mobile phase is 500 mM ammonium formate buffer solution (pH 7.4); B. The flow rate is 0.2 mL / min; C. The ultraviolet detection wavelength is 215 nm; D. The column temperature is 37°C.

[0008] Among them, the chromatographic column in the second-dimensional chromatography is an octadecyl bonded silica gel (C18) chromatographic column, which is responsible for the separation and analysis of FT3 and FT4; the second-dimensional liquid chromatography conditions include: A. The aqueous mobile phase is 0.1% formic acid water (v / v), the organic mobile phase is 90% methanol aqueous solution (v / v), and the isocratic elution is performed at 60% (v / v) of the organic phase; B. The flow rate is 0.3 mL / min; C. The UV detection wavelength is 215 nm; D. The column temperature is 37°C.

[0009] Specifically, the step (2) comprises the following steps: before the formal analysis, the six-way valve is adjusted so that the two chromatograms are in parallel state, and the serum to be analyzed is injected into the first dimension chromatogram under the action of the injector. The chromatographic conditions are conducive to the rapid outflow of macromolecular components such as fatty acids, peptides, and proteins, and small organic molecules such as free triiodo-L-thyronine (Free T3, FT3) and free L-thyroxine (Free T4, FT4) are strongly retained, thereby achieving the purpose of online separation of target components in serum.

[0010] Specifically, step (3) is to set 15 min as the switching time of the six-way valve, that is, to adjust the six-way valve so that the two chromatograms are in series, so as to ensure that the macromolecular components enter the waste liquid pool through the first-dimensional chromatography, while retaining FT3 and FT4 in the chromatography system.

[0011] Specifically, step (4) comprises the following steps: when the two chromatographic paths are in series connection, under the action of the second-dimensional mobile phase, FT3 and FT4 flow out of the first-dimensional chromatographic column in reverse direction, then enter the second-dimensional chromatographic column, and enter the mass spectrometer detector one by one after separation; and the FT3 and FT4 and their isotope-labeled T3 (T3*) and T4 (T4*) entering the mass spectrometer detector are detected in a multiple reaction monitoring mode to collect mass spectrometer signals.

[0012] Specifically, step (5) comprises selecting T3 (triiodo-L-thyronine national first-grade purity standard substance, number GBW09326), T3*, and T4 (L-thyroxine national first-grade purity standard substance, number GBW09325), and T4* as materials, accurately weighing them using a precision balance, and using 0.5% v / v ammonia solution as a solvent to form four groups of mother solutions and working solutions with known mass concentrations; wherein the mass fraction of the working solutions of T3 and T3* after dilution is ≈5 ng / mg, and the mass fraction of the working solutions of T4 and T4* after dilution is ≈100 ng / mg. By weighing and mixing working solutions of different masses, three groups of standard mixed solutions, namely, low standard, equal standard and high standard, are formed; that is, the first group of low standard solutions contains T3:T3*≈0.8:1 (m / m), T4:T4*≈0.8:1 (m / m), the second group of equal standard solutions contains T3:T3*≈1.0:1 (m / m), T4:T4*≈1.0:1 (m / m), and the third group of high standard solutions contains T3:T3*≈1.2:1 (m / m), T4:T4*≈1.2:1 (m / m); similarly, a precision balance is used to weigh the serum to be tested, and according to the preliminary measurement results of FT3 and FT4, a certain mass of T3* and T4* working solutions are added to make the mass ratio of the TT3 and TT4 to be tested in the serum to the added T3* and T4* close to 1:1. In actual measurement, this ratio can be between 0.8-1.2 (m / m). A triple quadrupole mass spectrometer was used as the detector, and multiple reaction monitoring (MRM) was performed one by one on low-standard, equal-standard, high-standard solutions, and T3, T3*, T4, and T4* in the serum sample to be tested in the positive ion mode of the electrospray ion source (ESI). The mass spectrometer parameters are shown in the following table: name Chemical formula Precursor ion m / z Product ion m / z Fragmentation Energy T3 <![CDATA[C 15 H 12 I3NO4]]> 652.2 606.0 20V Isotope-labeled T3 (T3*) <![CDATA[ 13 C6C9H 12 I3NO4]]> 658.0 611.9 20V T4 <![CDATA[C 15 H 11 I4NO4]]> 777.7 731.7 24V Isotope-labeled T4 (T4*) <![CDATA[ 13 C6C9H 11 I4NO4]]> 783.7 737.7 24V A standard curve was established with the mass ratio of T3 and T3* in low-standard, equal-standard and high-standard solutions as the horizontal axis and the mass spectrum peak area ratio of T3 and T3* in low-standard, equal-standard and high-standard solutions as the vertical axis; a standard curve of T4 and T4* was established in the same way; then, the mass values ​​of T3, T3* and T4, T4* in serum samples were calculated through the standard curve based on the mass spectrum peak area ratios of T3, T3* and T4, T4* obtained by analyzing the serum samples through the two-dimensional chromatography system; since the mass numbers of T3* and T4* added to the serum samples were known, the mass numbers of endogenous TT3 and TT4 could be quickly obtained through calculation, and the content could be determined by dividing the mass numbers by the serum mass.

[0013] Specifically, step (6) is to adjust the six-way valve at 25 minutes so that the two chromatographic columns are restored to a parallel state, and the two chromatographic columns are fully cleaned and balanced under the action of the first-dimensional and second-dimensional mobile phases, respectively, to complete one online analysis while preparing for the next analysis.

[0014] Compared with the prior art, the outstanding effects of the present invention are: The online separation and determination method of the present invention effectively separates bound and free thyroid hormones, solves the problem of membrane adsorption during the separation process, reduces the result error introduced by different operating levels of personnel, and has the characteristics of low loss, high efficiency, and high automation compared with separation methods such as equilibrium dialysis, ultrafiltration, and solid phase extraction. Combined with isotope dilution mass spectrometry technology and the use of certified reference materials, the determination results of this method have metrological traceability and have the potential to become a reference measurement method.

[0015] The online separation and determination method of free thyroxine in serum of the present invention will be further described below in conjunction with the accompanying drawings and specific examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the chromatographic flow path, where A is in parallel and B is in series.

[0017] Figure 2 The UV images of T3 and T4 standard solutions detected by one-dimensional liquid chromatography. A. Blank; B. UV image of T4 standard solution detected by TurboFlowCyclone MAX column (1×50 mm, 4 μm); C. UV image of T4 standard solution detected by TurboFlow Cyclone column (1×50 mm, 4 μm); D. UV image of T4 standard solution detected by MAbPac™ Ph column (50×3 mm, 4 μm); E. UV image of T3 standard solution detected by MAbPac™ Ph column (50×3 mm, 4 μm).

[0018] Figure 3 The recovery results of HSA protein eluted with ammonium formate buffer solutions of different concentration gradients and pH gradients.

[0019] Figure 4 The SDS-PAGE gel electrophoresis diagram of the lyophilized and reconstituted effluent collected for the first-dimension chromatographic column detection of the protein mixed solution. Among them, 1. Marker, 2. HSA, 3. Myo, 4. Cyt-c, 5. PAB, 6. Protein mixed solution before loading, 7. Protein mixed solution lyophilized and reconstituted after loading, 8. Blank, 9. Marker.

[0020] Figure 5 This is the recovery result of Hela cell extract detected by the first dimension chromatographic column.

[0021] Figure 6The SDS-PAGE gel electrophoresis diagram of the lyophilized and reconstituted effluent collected for the first-dimension chromatography column detection of Hela cell extract. Among them, 1. Marker, 2. Blank, 3. Hela cell extract, 4. Hela cell extract after lyophilization and reconstitution of the effluent, 5. HSA, 6. Cyt-c.

[0022] Figure 7 The MRM diagrams of serum FT3 and FT4 are shown in Figure 2. DETAILED DESCRIPTION

[0023] An online separation and determination method for free thyroxine in serum, comprising the following specific steps: (1) Flow path setting for online separation and analysis of free thyroxine by two-dimensional liquid chromatography

[0024] The analytical system consists of the first-dimension chromatography and the second-dimension chromatography. Figure 1 As shown, the two can realize series and parallel connection of flow paths by switching the six-way valve.

[0025] Figure 1 In A, when the six-way valve is in position 1, the flow path is marked in green, and the two chromatograms are in parallel, which is used for the injection and removal of macromolecules of serum samples. The left pump of the dual-liquid phase pump is connected to the autosampler, the autosampler is connected to the interface 1 of the six-way valve, the interface 1 is connected to the interface 6, the interface 6 is connected to the first-dimension chromatographic column, the first-dimension chromatographic column is connected to the interface 3 of the six-way valve, and the interface 3 is connected to the interface 2; the right pump of the dual-liquid phase pump is connected to the interface 4, the interface 4 is connected to the interface 5, the interface 5 is connected to the second-dimension analytical column, and the second-dimension analytical column is connected to the detector.

[0026] Figure 1 In B, when the six-way valve is in position 2, the flow path is marked in yellow, and the two chromatograms are in series, which are used for the elution of FT3, FT4, T3*, and T4*. Thyroxine is reversely eluted from the first-dimensional chromatographic column and enters the second-dimensional analytical column. The left pump of the dual-phase pump is connected to the autosampler, which is connected to interface 1 of the six-way valve, and interface 1 is connected to interface 2; the right pump of the dual-phase pump is connected to interface 4, which is connected to interface 3; interface 3 is connected to the first-dimensional chromatographic column, which is connected to interface 6 of the six-way valve, and interface 6 is connected to interface 5; interface 5 is connected to the second-dimensional analytical column, and the second-dimensional analytical column is connected to the detector.

[0027] (2) Performance investigation and optimization of chromatographic columns in two-dimensional liquid phase systems

[0028] Since macromolecules in serum, including fatty acids, peptides, and proteins, are the biggest obstacle to the analysis of low-abundance small molecules, if the goal of online separation of the target is to be achieved, a stationary phase that is not easily clogged and has poor retention for macromolecules is needed to achieve this goal. Based on this, the three chromatographic columns examined in the first-dimensional chromatography are all large-particle, large-pore filler stationary phases, which are not conducive to the retention of macromolecules. The specific information is shown in Table 1: Table 1 Information on candidate columns in the first dimension chromatography name size filler TurboFlow Cyclone 1×50 mm, 4 μm Phenyl based polymer TurboFlow Cyclone MAX 1×50 mm, 4 μm Mixed anion exchange column MAbPac™ ph 50×3 mm, 4 μm DVB (divinylbenzene) polymer The three chromatographic columns in Table 1 were connected to the liquid chromatography system respectively, and the T4 standard solution was detected. The ultraviolet detector was used in the condition investigation and optimization process. The maximum absorption wavelength of thyroxine was between 200-220 nm. Figure 2 The chromatogram of the thyroxine standard solution at 215 nm. When TurboFlow Cyclone and TurboFlow Cyclone MAX columns were used, no T4 was eluted. However, the MAbPac™ Ph column detected T4 at 18.3 min. Subsequently, using the same chromatographic conditions, T3 was detected at 16.7 min. Finally, the MAbPac™ Ph column (50×3 mm, 4 μm) was selected as the first-dimension chromatographic separation column in the 2D-LC system.

[0029] Subsequently, the first-dimension liquid chromatography mobile phase was investigated. Figure 3 As shown, according to the recovery efficiency of thyroxine at different pH and different mobile phase concentrations, 500 mM ammonium formate buffer solution (pH 7.4) was selected as the mobile phase.

[0030] The second-dimension chromatographic column selected a commercial Waters BEH C18 ultra-high-efficiency reversed-phase column (50×2.1 mm, 1.7 μm) for the separation and analysis of FT3 and FT4. The chromatographic conditions were optimized during the development of the national first-level standard material for thyroxine and have been fully optimized and verified. The aqueous phase was 0.1% formic acid water (v / v), the organic phase was 90% methanol water solution (v / v), the organic phase was eluted isocratically at 60% organic phase, the flow rate was 0.3 mL / min, the UV detection wavelength was 215 nm, and the column temperature was 37°C.

[0031] (3) Online separation and removal of macromolecular interferences in serum

[0032] When the six-way valve is in position 1, the two columns are in parallel operation, and the flow rate of the first-dimension mobile phase is 0.3 mL / min. Figure 2It can be seen that both T3 and T4 elute after 16 minutes, so 15 minutes can be set as the valve switching time. At this time, the sample reaches the preparation column from the automatic sampler through the No. 1 and No. 6 ports, FT3 and FT4 are retained in the preparation column, and the large molecular proteins and some impurities in the sample flow out of the preparation column through the No. 3 and No. 2 ports. The No. 2 port is connected to the waste liquid barrel, and the mobile phase and the impurities finally flow to the waste liquid barrel through the No. 2 port.

[0033] Since organic solvents such as methanol and acetonitrile are powerful protein denaturants, they will destroy the binding of thyroid hormones to TBG proteins, resulting in increased levels of FT3 and FT4 in serum, and should therefore be avoided in serum sample analysis. At the same time, since a mass spectrometer detector needs to be connected for quantitative analysis later, non-volatile buffer salts should also be avoided. Based on this, ammonium formate buffer solution was selected as the mobile phase in the experiment.

[0034] In order to investigate the effect of corresponding chromatographic conditions on the removal of macromolecular components in serum, a single protein system, a mixed protein system, and Hela cell extracts were selected as objects for optimization of the process, and analytical systems from simple to complex were simulated respectively.

[0035] Single protein system: 50 mM, 100 mM, 200 mM, 500 mM, 800 mM and 1000 mM ammonium formate buffer saline solutions were set, and three different pH values ​​were set at each concentration: acidic (5.6), neutral (7.4) and alkaline (9.2). The protein removal effect of these ammonium formate buffer saline solutions with different concentration gradients and different pH value gradients on HSA protein solution was investigated; the effluent was collected and vacuum-freezed, and then reconstituted and analyzed by electrophoresis. The results are as follows: Figure 3 As shown in the figure, it can be seen that the best protein removal effect is achieved when the concentration of ammonium formate buffer solution is 500 mM and the pH value is 7.4.

[0036] Mixed protein system: The previously verified 500 mM, pH=7.4 ammonium formate buffer solution was used as the mobile phase to analyze the mixed solution of four proteins, including serum albumin (HSA), myoglobin (Myo), cytochrome c (Cyt-c) and human serum prealbumin (PAB). After the mixed protein solution was loaded, the effluent was collected and vacuum-freezed, and then reconstituted and analyzed by SDS-PAGE gel electrophoresis. The mixed protein solution before loading was compared with the effluent after freeze-drying and reconstitution. The results are as follows: Figure 4 As shown in the figure, it can be seen that the width and staining intensity of the protein bands before loading (band 6) and after freeze-drying and reconstitution (band 7) are basically the same, indicating that in the mixed protein system, the selected mobile phase conditions can effectively remove proteins and have a good protein removal effect.

[0037] Hela cell protein extract system: In order to simulate the serum environment more realistically and verify the protein removal effect of the mobile phase, Hela cell protein extract was used as the experimental object. Its composition is similar to that of serum and can better reflect the complexity of actual serum sample processing. The results showed that the effluent collected before and after loading was freeze-dried and re-dissolved, and the protein concentration was measured using Nanodrop. The calculated recovery rates were 85.02% (enzymatic hydrolysis) and 88% (non-enzymatic hydrolysis), respectively. The specific results are shown in Figure 5 In addition, the samples before and after loading were analyzed by SDS-PAGE gel electrophoresis. Figure 6 The protein band width and staining intensity before loading (band 3) and after freeze-drying and reconstitution (band 4) are basically the same. Figure 5 and Figure 6 The results showed that in the Hela cell system, 500 mM ammonium formate buffer solution, pH 7.4, as the mobile phase had a significant protein removal effect, further verifying the applicability of this condition as a preparative column mobile phase.

[0038] (4) Retention of free thyroid hormones (FT3, FT4)

[0039] Thyroxine in serum will bind to TBG protein for transport. In the first dimension of separation, bound T3 and T4 flow out with macromolecular substances and also leave the chromatographic system. At the same time, FT3 and FT4 that are not bound to TBG interact with and are retained by the MAbPac™ Ph column when the six-way valve is in position 1. Figure 2 The results show that 15 min can be selected as the valve switching time.

[0040] (5) Separation of free thyroid hormones (FT3, FT4)

[0041] After the first-dimension chromatography is completed, the six-way valve is switched to position 2 from 15 minutes and maintained for 10 minutes. At this time, FT3, FT4, and other organic small molecules with retention properties in the serum retained in the chromatography system will be backwashed and brought into the second-dimension chromatography column under the action of the new mobile phase, so that separation will continue, thereby reducing the co-elution of substances at the same time and improving the sensitivity and reliability of analysis. The second-dimension chromatography conditions have been deeply investigated during the development of standard substances and do not need further optimization.

[0042] (6) Online determination of isotope dilution mass spectrometry

[0043] T3 (triiodo-L-thyronine national first-level purity standard material, number GBW09326), T3*, T4 (L-thyroxine national first-level purity standard material, number GBW09325), and T4* were selected as materials, accurately weighed using a precision balance, and 0.5% ammonia solution (v / v) was used as the solvent to form 4 groups of mother solutions and working solutions with known mass concentrations. Among them, the mass fraction of the working solution of T3 and T3* after dilution is ≈5 ng / mg, and the mass fraction of the working solution of T4 and T4* after dilution is ≈100 ng / mg. By weighing and mixing working solutions of different masses, three groups of standard mixed solutions, namely, low standard, equal standard and high standard, are formed; that is, the first group of low standard solutions contains T3:T3*≈0.8:1 (m / m), T4:T4*≈0.8:1 (m / m), the second group of equal standard solutions contains T3:T3*≈1.0:1 (m / m), T4:T4*≈1.0:1 (m / m), and the third group of high standard solutions contains T3:T3*≈1.2:1 (m / m), T4:T4*≈1.2:1 (m / m); similarly, a precision balance is used to weigh the serum to be tested, and according to the preliminary measurement results of FT3 and FT4, a certain mass of T3* and T4* working solutions are added to make the mass ratio of the TT3 and TT4 to be tested in the serum to the added T3* and T4* close to 1:1. In actual measurement, this ratio can be between 0.8-1.2 (m / m). In the positive ion mode of the electrospray ion source (ESI), multiple reaction monitoring (MRM) was performed one by one on T3, T3*, T4, and T4* in the low-standard, equal-standard, and high-standard solutions, as well as the serum samples to be tested. The corresponding mass spectrometry parameters are shown in Table 2 below.

[0044] Table 2 Mass spectrometry parameters name Chemical formula Precursor ion m / z Product ion m / z Fragmentation Energy T3 <![CDATA[C 15 H 12 I3NO4]]> 652.2 606.0 20V Isotope-labeled T3 (T3*) <![CDATA[ 13 C6C9H 12 I3NO4]]> 658.0 611.9 20V T4 <![CDATA[C 15 H 11 I4NO4]]> 777.7 731.7 24V Isotope-labeled T4 (T4*) <![CDATA[ 13 C6C9H 11 I4NO4]]> 783.7 737.7 24V The standard curve was established with the mass ratio of T3 and T3* in low-standard, equal-standard and high-standard solutions as the horizontal axis and the mass peak area ratio of T3 and T3* in low-standard, equal-standard and high-standard solutions as the vertical axis; the standard curve of T4 and T4* was established in the same way. Subsequently, the mass values ​​of T3, T3* and T4, T4* in serum samples can be calculated through the standard curve according to the mass peak area ratio of T3, T3* and T4, T4* obtained by analyzing the serum samples by the two-dimensional chromatography system; since the mass numbers of T3* and T4* added to the serum samples are known, the mass numbers of endogenous TT3 and TT4 can be quickly obtained by calculation, and the content can be determined by dividing the mass number by the serum mass. Figure 7 The MRM ion flow chromatograms of FT3 and FT4 are shown. The peak area ratio on the ordinate of the standard curve is calculated based on the peak area integral of the ion flow chromatogram.

[0045] (7) Chromatographic system cleaning

[0046] To avoid potential carryover and conduct reliable subsequent analysis, the two-position chromatography system needs to be cleaned after serum treatment. At this point, the six-port valve is switched to position 1 (6-1 connection) again, and ammonium formate (500 mM, pH 7.4) is used as the aqueous phase and methanol (90%, 0.1% formic acid) is used as the organic phase. The column is equilibrated at a flow rate of 0.3 mL / min at a ratio of 40:60 (ammonium formate: methanol, v / v) for 5 min.

[0047] Finally, a complete analysis process requires 15 min of serum cleanup, 10 min of free thyroxine separation and analysis, and 5 min of chromatographic system cleaning and activation, a total of 30 min.

[0048] The embodiments described above are merely descriptions of preferred implementation modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for online separation and determination of free thyroxine in serum, characterized in that: The method comprises the following steps: (1) setting up two-dimensional liquid chromatography; (2) online separation and removal of macromolecular interferences in serum; (3) retention of free thyroid hormone; (4) online separation of free thyroid hormone; (5) online determination of isotope dilution mass spectrometry; (6) cleaning of the two-dimensional liquid chromatography system; Wherein, the free thyroid hormones are FT3 and FT4.

2. The method for online separation and determination of free thyroxine in serum according to claim 1, characterized in that: Specifically, the step (1) comprises two groups of chromatographic systems, namely, a first-dimension chromatogram and a second-dimension chromatogram, which are connected in series and in parallel by switching of a six-way valve; The chromatographic column of the first dimension chromatography is based on a hydrophobic super-macroporous polymer resin and coated with a hydrophobic divinylbenzene polymer, which is responsible for removing serum macromolecular components. The first dimension liquid chromatography conditions include: the mobile phase is 500 mM ammonium formate buffer solution, pH 7.4; the flow rate is 0.2 mL / min; the ultraviolet detection wavelength is 215 nm; the column temperature is 37°C; The chromatographic column of the second-dimensional chromatography was a C18 column, which was responsible for the separation and analysis of FT3 and FT4; the second-dimensional liquid chromatography conditions included: the aqueous mobile phase was 0.1% formic acid water, the organic mobile phase was 90% methanol water solution, and isocratic elution was performed at 60% of the organic phase; the flow rate was 0.3 mL / min; the UV detection wavelength was 215 nm; and the column temperature was 37°C.

3. The method for online separation and determination of free thyroxine in serum according to claim 2, characterized in that: The step (2) specifically includes, before the formal analysis, adjusting the six-way valve so that the two chromatograms are in parallel state, and the serum to be analyzed is injected into the first dimension chromatogram under the action of the injector, and the chromatographic conditions are conducive to the rapid outflow of large molecular components and strong retention of small organic molecules, thereby achieving the purpose of online separation of target components in serum.

4. The method for online separation and determination of free thyroxine in serum according to claim 3, characterized in that: The step (3) specifically includes setting the switching time of the six-way valve, that is, adjusting the six-way valve so that the two chromatograms are in series connection, so as to ensure that the macromolecular components enter the waste liquid pool through the first-dimensional chromatography, while retaining FT3 and FT4 in the chromatography system.

5. The method for online separation and determination of free thyroxine in serum according to claim 4, characterized in that: The step (4) specifically comprises the following steps: when the two chromatographic paths are in series, under the action of the second-dimensional mobile phase, FT3 and FT4 flow out of the first-dimensional chromatographic column in reverse direction, then enter the second-dimensional chromatographic column, and enter the mass spectrometer detector one by one after separation; and the FT3 and FT4 and their isotope-labeled T3 (T3*) and T4 (T4*) entering the mass spectrometer detector are detected in a multiple reaction monitoring mode to collect mass spectrometer signals.

6. The method for online separation and determination of free thyroxine in serum according to claim 5, characterized in that: The step (5) specifically comprises the following steps: selecting T3, T3*, and T4, T4* as materials, accurately weighing them using a precision balance, and using 0.5% ammonia solution as a solvent to form four groups of mother solutions and working solutions with known mass concentrations; weighing and mixing working solutions of different masses to form three groups of standard mixed solutions of low standard, equal standard, and high standard; similarly, weighing the serum to be tested, and adding a certain mass of T3* and T4* working solutions respectively according to the preliminary measured serum FT3 and FT4 concentrations, so that the mass ratio of the FT3 and FT4 to be tested in the serum and the added T3* and T4* is close to 1:1; selecting a triple quadrupole mass spectrometer as a detector, and collecting signals of T3, T3*, T4, and T4* compounds in the multiple reaction monitoring (MRM) mode.

7. The method for online separation and determination of free thyroxine in serum according to claim 6, characterized in that: The mass spectrometry parameters are shown in the following table: 。 8. The method for online separation and determination of free thyroxine in serum according to claim 7, characterized in that: A standard curve was established with the mass ratio of T3 and T3* in low-standard, equal-standard and high-standard solutions as the horizontal axis and the mass spectrum peak area ratio of T3 and T3* in low-standard, equal-standard and high-standard solutions as the vertical axis; a standard curve of T4 and T4* was established in the same way; Subsequently, based on the mass spectrum peak area ratios of T3, T3* and T4, T4* obtained by analyzing the serum samples through the two-dimensional chromatography system, the mass values ​​of T3, T3* and T4, T4* in the serum samples can be calculated through the standard curve; since the mass numbers of T3* and T4* added to the serum samples are known, the mass numbers of endogenous TT3 and TT4 can be quickly obtained by calculation, and this mass number can be divided by the serum mass to achieve content determination.

9. The method for online separation and determination of free thyroxine in serum according to claim 8, characterized in that: The step (6) specifically includes adjusting the six-way valve so that the two chromatographic columns are restored to a parallel state, and the two chromatographic columns are fully cleaned and balanced under the action of the first-dimensional and second-dimensional mobile phases, respectively, completing one online analysis while preparing for the next analysis.

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