A method for online separation and determination of free thyroxine in serum

Abstract

The present invention discloses a method for online separation and absolute determination of free thyroid hormone in serum, comprising the following steps: (1) setting up a 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 by isotope dilution mass spectrometry; and (6) cleaning of the two-dimensional liquid chromatography system. The method of the present invention achieves online separation and absolute determination of free thyroid hormone in serum. Since the first-dimensional chromatographic mobile phase is a mild ammonium salt buffer solution that does not contain organic solvents, it can ensure that the thyroid hormone bound to proteins in the serum remains in a bound state, and the target substance entering the mass spectrometry analysis is the free thyroid hormone; and the determination result can be traced back to the International System of Units through national certified reference materials. Compared with traditional serum offline processing methods such as solid phase extraction and equilibrium dialysis, the method is efficient, reliable, and easy to operate.

Description

Technical Field

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

[0002] Accurate measurement of thyroid hormones is crucial for the diagnosis of thyroid disease, especially free hormones such as free triiodo-L-thyronine (FT3) and free L-thyroxine (FT4). These hormones, unaffected by iodine, more accurately reflect the functional status of the thyroid gland. FT3 is the first test to increase in the early stages of hyperthyroidism or during a relapse and is currently the preferred test for diagnosing hyperthyroidism. However, due to the low levels of free hormones and the lack of accurate and reliable reference materials, establishing reference measurement methods for FT3 and FT4 in serum remains challenging. In particular, there is no globally recognized solution to the problem of maintaining an endogenous balance between bound and free forms during the hormone separation process.

[0003] Currently, most researchers consider equilibrium dialysis, ultrafiltration, and solid-phase extraction to be the primary methods for separating free hormones. However, these methods also have significant limitations. For example, equilibrium dialysis is time-consuming, labor-intensive, and costly, and the target compound may adsorb onto the biofilm. Ultrafiltration can cause protein leakage and requires strict temperature and pH controls. Solid-phase extraction is subject to significant human influence and is difficult to achieve uniformity.

[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 solutions adopted by the present invention are as follows:

[0007] A method for online separation and determination of free thyroid hormones in serum, wherein the free thyroid hormones are FT3 and FT4. The method specifically comprises the following steps: (1) setting up a 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 of the free thyroid hormones by isotope dilution mass spectrometry; and (6) cleaning of the two-dimensional liquid chromatography system.

[0008] Specifically, step (1) is composed of two chromatographic systems, namely the first-dimensional chromatographic system and the second-dimensional chromatographic system, which realize the series and parallel connection of chromatographic flow paths by switching the six-way valve.

[0009] The first-dimension chromatography column is based on a hydrophobic ultramacroporous polymer resin (4 µm) and coated with a hydrophobic DVB (divinylbenzene) polymer, responsible for removing serum macromolecules such as fatty acids, peptides, and proteins. First-dimension liquid chromatography conditions include: A. mobile phase: 500 mM ammonium formate buffer (pH 7.4); B. flow rate: 0.2 mL / min; C. UV detection wavelength: 215 nm; and D. column temperature: 37°C.

[0010] The second-dimensional chromatography column was an octadecyl bonded silica gel (C18) column, responsible for the separation and analysis of FT3 and FT4. The second-dimensional liquid chromatography conditions included: A. 0.1% formic acid in water (v / v) as the aqueous mobile phase, 90% methanol in water (v / v) as the organic mobile phase, and isocratic elution at 60% (v / v) of the organic phase; B. Flow rate, 0.3 mL / min; C. UV detection wavelength, 215 nm; and D. Column temperature, 37°C.

[0011] Specifically, step (2) is as follows: before the formal analysis, the six-way valve is adjusted so that the two chromatograms are in a parallel state, and the serum to be analyzed is injected into the first-dimensional chromatogram under the action of the injector. The chromatographic conditions are conducive to the rapid outflow of large molecular 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) have strong retention, thereby achieving the purpose of online separation of target components in serum.

[0012] 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.

[0013] Specifically, step (4) comprises the following steps: when the two chromatographic columns 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 using the multiple reaction monitoring mode to collect mass spectrometer signals.

[0014] Specifically, step (5) comprises selecting T3 (triiodinated L-thyronine, national first-class purity standard substance, number GBW09326), T3*, and T4 (L-thyroxine, national first-class 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 liquors and working solutions with known mass concentrations; wherein, the mass fraction of the working solution after dilution of T3 and T3* is ≈5 ng / mg, and the mass fraction of the working solution after dilution of T4 and T4* 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. Based on the preliminary measurement results of FT3 and FT4, a certain mass of T3* and T4* working solutions is 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 on low-standard, equal-standard, and high-standard solutions, as well as T3, T3*, T4, and T4* in the serum sample under positive ion mode using an electrospray ionization source (ESI). The mass spectrometer parameters are shown in the following table:

[0015] 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

[0016] 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 spectrometry peak area ratio of T3 and T3* in low-standard, equal-standard, and high-standard solutions as the vertical axis; a standard curve for T4 and T4* was established in the same way; then, based on the mass spectrometry peak area ratios of T3, T3* and T4, T4* obtained by two-dimensional chromatography analysis of serum samples, the mass values ​​of T3, T3* and T4, T4* in serum samples could be calculated using the standard curve; 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 by calculation, and the content could be determined by dividing this mass number by the serum mass.

[0017] 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, completing one online analysis while preparing for the next analysis.

[0018] Compared with the prior art, the outstanding effects of the present invention are:

[0019] The online separation and determination method of this invention effectively separates bound and free thyroid hormones, eliminating membrane adsorption issues during the separation process and reducing errors introduced by operator proficiency. Compared with other separation methods such as equilibrium dialysis, ultrafiltration, and solid-phase extraction, it offers low loss, high efficiency, and a high degree of automation. Incorporating isotope dilution mass spectrometry and the use of certified reference materials, the method's determination results possess metrological traceability, demonstrating its potential as a reference measurement method.

[0020] The online separation and determination method of free thyroxine in serum of the present invention will be further described below with reference to the accompanying drawings and specific examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the chromatographic flow path, where A is in parallel and B is in series.

[0022] Figure 2 UV spectra of T3 and T4 standard solutions detected by one-dimensional liquid chromatography. A. Blank; B. UV spectra of T4 standard solution detected using a TurboFlowCyclone MAX column (1 × 50 mm, 4 μm); C. UV spectra of T4 standard solution detected using a TurboFlow Cyclone column (1 × 50 mm, 4 μm); D. UV spectra of T4 standard solution detected using a MAbPac™ Ph column (50 × 3 mm, 4 μm); E. UV spectra of T3 standard solution detected using a MAbPac™ Ph column (50 × 3 mm, 4 μm).

[0023] Figure 3 The recovery results of HSA protein eluted with ammonium formate buffer solutions of different concentration gradients and pH value gradients.

[0024] Figure 4 SDS-PAGE gel electrophoresis of the lyophilized and reconstituted flow-through of the protein mixture collected for the first-dimension chromatography column. 1. Marker, 2. HSA, 3. Myo, 4. Cyt-c, 5. PAB, 6. Protein mixture before loading, 7. Protein mixture after loading, lyophilized and reconstituted, 8. Blank, 9. Marker.

[0025] Figure 5 This is the recovery rate result of Hela cell extract detected by the first-dimension chromatographic column.

[0026] Figure 6 SDS-PAGE gel electrophoresis of HeLa cell extract collected from the lyophilized and reconstituted flow-through for the first-dimension chromatography column. 1. Marker, 2. Blank, 3. HeLa cell extract, 4. HeLa cell extract after lyophilization and reconstitution of the flow-through, 5. HSA, 6. Cyt-c.

[0027] Figure 7 The MRM diagrams of serum FT3 and FT4 are shown. DETAILED DESCRIPTION

[0028] A method for online separation and determination of free thyroxine in serum, comprising the following steps:

[0029] (1) Flow path setup for online separation and analysis of free thyroxine by two-dimensional liquid chromatography

[0030] 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 flow paths by switching the six-way valve.

[0031] Figure 1 In Figure A, when the six-port valve is in position 1, the flow path is marked in green, and the two chromatographic paths are connected in parallel, used for serum sample injection and macromolecule removal. The left pump of the dual-phase pump is connected to the autosampler, which is connected to port 1 of the six-port valve. Port 1 is connected to port 6, which is connected to the first-dimension chromatographic column. The first-dimension chromatographic column is connected to port 3 of the six-port valve, which is connected to port 2. The right pump of the dual-phase pump is connected to port 4, which is connected to port 5. Port 5 is connected to the second-dimension analytical column, which is connected to the detector.

[0032] Figure 1 In Figure B, when the six-port valve is in position 2, the flow path is marked in yellow, and the two chromatographic paths are connected in series, eluting FT3, FT4, T3*, and T4*. Thyroxine is reverse-eluted from the first-dimension column and enters the second-dimension analytical column. The left pump of the dual-phase pump is connected to the autosampler, which is connected to port 1 of the six-port valve, which is connected to port 2. The right pump of the dual-phase pump is connected to port 4, which is connected to port 3. Port 3 is connected to the first-dimension column, which is connected to port 6 of the six-port valve, which is connected to port 5. Port 5 is connected to the second-dimension analytical column, which is connected to the detector.

[0033] (2) Performance evaluation and optimization of chromatographic columns in two-dimensional liquid phase systems

[0034] Because macromolecules in serum, including fatty acids, peptides, and proteins, are the biggest obstacle to the analysis of low-abundance small molecules, achieving online separation of target compounds requires the use of a stationary phase that is less prone to clogging and has poor retention for macromolecules. For this reason, the three columns examined in the first-dimensional chromatography all used large-particle, large-pore stationary phases, which are not conducive to the retention of macromolecules. Specific information is shown in Table 1:

[0035] Table 1 Information on candidate columns for first-dimension chromatography

[0036] name size filler TurboFlow Cyclone 1×50 mm, 4 μm Phenyl based polymers TurboFlow Cyclone MAX 1×50 mm, 4 μm Mixed anion exchange column MAbPac™ ph 50×3 mm, 4 μm DVB (divinylbenzene) polymer

[0037] The three chromatographic columns in Table 1 were connected to the liquid chromatography system respectively, and the T4 standard solution was detected. The UV detector was used in the condition investigation and optimization process. The maximum absorption wavelength of thyroxine was between 200-220 nm. Figure 2 The following is a chromatogram of a thyroxine standard solution at 215 nm. While no T4 eluted when using the TurboFlow Cyclone or TurboFlow Cyclone MAX columns, the MAbPac™ Ph column detected T4 at 18.3 minutes. Subsequently, using the same chromatographic conditions, T3 was detected at 16.7 minutes. Ultimately, the MAbPac™ Ph column (50 × 3 mm, 4 μm) was selected as the first-dimension separation column in the 2D-LC system.

[0038] Subsequently, the first-dimension liquid chromatography mobile phase was investigated. Figure 3 As shown in the figure, 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.

[0039] A commercially available Waters BEH C18 ultra-high-performance reversed-phase column (50 × 2.1 mm, 1.7 μm) was used in the second dimension for the separation and analysis of FT3 and FT4. These chromatographic conditions were optimized during the development of the national primary reference material for thyroxine and have been thoroughly optimized and validated. The aqueous phase consisted of 0.1% formic acid in water (v / v), and the organic phase consisted of 90% methanol in water (v / v). Elution was isocratic with a 60% organic phase, at a flow rate of 0.3 mL / min, UV detection at 215 nm, and the column temperature at 37°C.

[0040] (3) Online separation and removal of macromolecular interfering substances in serum

[0041] 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 2As can be seen, both T3 and T4 elute after 16 minutes, so the valve switching time can be set at 15 minutes. At this time, the sample enters the preparative column from the autosampler through ports 1 and 6. FT3 and FT4 are retained in the preparative column. The large molecular weight proteins and some impurities in the sample flow out of the preparative column and pass through ports 3 and 2. Port 2 is connected to the waste liquid tank. The mobile phase and impurities ultimately flow through port 2 to the waste liquid tank.

[0042] Organic solvents such as methanol and acetonitrile are potent protein denaturants that can disrupt the binding of thyroid hormone to TBG proteins, leading to elevated serum FT3 and FT4 levels. Therefore, their use should be avoided in serum sample analysis. Furthermore, since subsequent quantitative analysis requires connection to a mass spectrometer, the use of non-volatile buffer salts should also be avoided. For this reason, ammonium formate buffer was selected as the mobile phase in this experiment.

[0043] 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.

[0044] Single protein system: 50 mM, 100 mM, 200 mM, 500 mM, 800 mM, and 1000 mM ammonium formate buffer 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 HSA protein solution was tested in these ammonium formate buffer solutions with different concentration gradients and different pH value gradients; the effluent was collected and vacuum-freezed, and then reconstituted and analyzed by electrophoresis. The results are as follows Figure 3 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.

[0045] Mixed protein system: Using the previously verified 500 mM, pH=7.4 ammonium formate buffer solution as the mobile phase, a mixed solution of four proteins was analyzed, 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 lyophilization and reconstitution. The results are as follows: Figure 4 As shown in the figure, the protein band width and staining intensity before loading (band 6) and after lyophilization and reconstitution (band 7) are basically consistent, indicating that the selected mobile phase conditions can effectively remove proteins in the mixed protein system and have a good protein removal effect.

[0046] Hela cell protein extract system: To more realistically simulate the serum environment 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 sample 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 As shown. The protein band width and staining intensity before loading (band 3) and after lyophilization 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.

[0047] (4) Retention of free thyroid hormones (FT3, FT4)

[0048] Thyroxine in serum binds to TBG protein for transport. In the first dimension of separation, bound T3 and T4 flow out along 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.

[0049] (5) Separation of free thyroid hormones (FT3, FT4)

[0050] After the first-dimension chromatography completes its work, the six-port valve is switched to position 2 starting at 15 minutes and held there for 10 minutes. FT3, FT4, and other retained organic small molecules in the serum are backflushed and carried to the second-dimension column by the new mobile phase, allowing separation to continue. This reduces co-elution of substances at the same time and improves analytical sensitivity and reliability. The second-dimension chromatography conditions were thoroughly investigated during the development of the reference material and did not require further optimization.

[0051] (6) Online determination of isotope dilution mass spectrometry

[0052] T3 (triiodinated L-thyronine, National Grade 1 Standard Purity Reference Material, No. GBW09326), T3*, and T4 (L-thyroxine, National Grade 1 Standard Purity Reference Material, No. GBW09325), T4*, were selected as materials. They were accurately weighed using a precision balance and prepared using 0.5% ammonia solution (v / v) to create four sets of stock solutions and working solutions of known concentrations. The working solutions of T3 and T3* after dilution had a concentration of approximately 5 ng / mg, and the working solutions of T4 and T4* had a concentration of approximately 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. Based on the preliminary measurement results of FT3 and FT4, a certain mass of T3* and T4* working solutions is 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 ionization 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.

[0053] Table 2 Mass spectrometry parameters

[0054] 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

[0055] A standard curve was constructed using the mass ratio of T3 to T3* in low-, equal-, and high-standard solutions as the horizontal axis and the mass spectrometry peak area ratio of T3 to T3* in low-, equal-, and high-standard solutions as the vertical axis. Standard curves for T4 and T4* were constructed in the same manner. Subsequently, the mass values ​​of T3, T3*, T4, and T4* in serum samples were calculated using the standard curve based on the mass spectrometry peak area ratios of T3, T3* and T4, T4* obtained by two-dimensional chromatography analysis of serum samples. Because the mass of T3* and T4* added to the serum samples is known, the mass of endogenous TT3 and TT4 can be quickly calculated and then divided by the serum mass to determine the content. Figure 7 The MRM ion current chromatograms of FT3 and FT4 are displayed. The peak area ratios on the vertical axis of the standard curve are calculated based on the peak area integrals of the ion current chromatograms.

[0056] (7) Chromatographic system cleaning

[0057] To avoid potential carryover and ensure reliable subsequent analysis, the two-stage chromatography system must be cleaned after serum processing. At this point, the six-port valve is switched back to position 1 (6-1 connection). Equilibrate the column for 5 minutes using ammonium formate (500 mM, pH 7.4) as the aqueous phase and methanol (90%, 0.1% formic acid) as the organic phase at a flow rate of 0.3 mL / min in a 40:60 ratio (ammonium formate:methanol, v / v).

[0058] Finally, a complete analysis process requires 15 minutes of serum cleanup, 10 minutes of free thyroxine separation and analysis, and 5 minutes of chromatographic system cleaning and activation, for a total of 30 minutes.

[0059] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the 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 fall within the scope of protection determined by the claims of the present invention.

Claims

1. A method for online separation and determination of free thyroxine in serum, characterized by: in, The free thyroxine is FT3 and FT4; specifically comprising the following steps: (1) 2D-LC setup; It consists of two chromatographic systems, one for the first dimension and the other for the second dimension, which are connected in series and in parallel by switching a six-way valve. The first-dimension chromatography column is based on a hydrophobic ultramacroporous polymer resin and coated with a hydrophobic divinylbenzene polymer, which is responsible for removing serum macromolecular components. The first-dimension liquid chromatography conditions include: mobile phase: 500 mM ammonium formate buffer solution, pH 7.4; flow rate: 0.2 mL / min; UV detection wavelength: 215 nm; column temperature: 37°C; The second-dimension chromatography column was a C18 column, responsible for the separation and analysis of FT3 and FT4. The second-dimension liquid chromatography conditions included: 0.1% formic acid in water as the mobile phase, 90% methanol in water as the organic phase, and isocratic elution with 60% organic phase; flow rate 0.3 mL / min; UV detection wavelength 215 nm; column temperature 37°C. (2) Online separation and removal of macromolecular interferences in serum; Before the formal analysis, the six-way valve is adjusted to make the two chromatograms in parallel. The serum to be analyzed is injected into the first-dimension chromatogram under the action of the injector. The large molecular components are quickly discharged, and the small organic molecules are strongly retained, achieving the purpose of online separation of the target components in the serum. (3) retention of free thyroxine; Set the switching time of the six-way valve, that is, 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 chromatogram, while retaining FT3 and FT4 in the chromatographic system; (4) Online separation of free thyroxine; When the two chromatographic columns are in series, under the action of the second-dimensional mobile phase, FT3 and FT4 flow out of the first-dimensional column in reverse, then enter the second-dimensional column, and after separation, enter the mass spectrometer detector one by one. The multiple reaction monitoring mode is used to detect the FT3, FT4, and their isotope-labeled T3* and T4* entering the mass spectrometer detector, and the mass spectrometer signals are collected. (5) Online determination of isotope dilution mass spectrometry; (6) Cleaning of two-dimensional liquid chromatography system.

2. The method for online separation and determination of free thyroxine in serum according to claim 1, 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; 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 acquiring signals of the compounds T3, T3*, T4, and T4* in the multiple reaction monitoring (MRM) mode.

3. The method for online separation and determination of free thyroxine in serum according to claim 2, characterized in that: The mass spectrometry parameters are shown in the following table: 。 4. The method for online separation and determination of free thyroxine in serum according to claim 3, 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 spectrometry peak area ratio of T3 and T3* in low-standard, equal-standard, and high-standard solutions as the vertical axis; a standard curve for T4 and T4* was established in the same manner; based on the mass spectrometry peak area ratios of T3, T3* and T4, T4* obtained from serum samples analyzed by a two-dimensional chromatography system, the mass values ​​of T3, T3* and T4, T4* in serum samples were calculated using the standard curve; the mass numbers of T3* and T4* added to the serum samples were known, and the mass numbers of endogenous T3 and T4 were obtained by calculation. This mass number was divided by the serum mass to achieve the content determination.

5. The method for online separation and determination of free thyroxine in serum according to claim 4, 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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