Method for simultaneous separation and quantitative analysis of NaYF4: Yb < 3 + >, Tm < 3 + > and degradation or metabolite thereof in complex system based on SE-HPLC-ICP-MS

Abstract

The invention relates to a method for simultaneous separation and quantitative analysis of NaYF4: Yb < 3 + >, Tm < 3 + > and degradation or metabolite of NaYF4: Yb < 3 + > and Tm < 3 + > in a complex system based on SE-HPLC-ICP-MS. The method for simultaneous separation and quantitative analysis of NaYF4: Yb < 3 + >, Tm < 3 + > and degradation or metabolite of NaYF4: Yb < 3 + > and Tm < 3 + > in the complex system based on SE-HPLC-ICP-MS comprises the following steps: mixing aqueous phase NaYF4: Yb < 3 + >, Tm < 3 + >, an additive and protein, and detecting by using a volume exclusion high performance liquid chromatography-inductively coupled plasma mass spectrometry technology. According to the method, NaYF4: Yb < 3 + >, Tm < 3 + > and degradation or metabolite of NaYF4: Yb < 3 + > and Tm < 3 + > can be separated at the same time, and the degradation or metabolite of NaYF4: Yb < 3 + > and Tm < 3 + > can be quantitatively analyzed.

Description

Technical Field

[0001] The present invention belongs to the technical field of rare earth ion analysis and research, and specifically relates to a method for analyzing NaYF in a complex system based on SE-HPLC-ICP-MS. 4 :Yb 3+ , Tm 3+ and simultaneous separation and quantitative analysis methods of its degradation or metabolites. Background Art

[0002] Rare-earth-elements-doped upconversion nanoparticles (REEs-UCNPs) are a new type of luminescent material that can absorb long-wavelength near-infrared light and emit short-wavelength ultraviolet and visible light. REEs-UCNPs have many advantages, such as stable chemical properties, narrow emission band, good biocompatibility, low background fluorescence, anti-bleaching and multi-color tunable spectrum. Due to their unique upconversion luminescence properties, they show broad application prospects in the fields of bioimaging, drug delivery, photodynamic therapy, etc. With the widespread application of REEs-UCNPs in many fields, their exposure to the environment and the population is increasing, and their biological toxicity and impact on the environment and human health have become important research topics.

[0003] In practical applications, REEs-UCNPs often exist in different forms, such as dispersed nanoparticles, degraded nanoparticles / rare earth ions, and nanomaterial complexes that may be combined with other substances. These different forms of REEs-UCNPs may have different toxic effects in vivo. Studies have shown that LaF 3 :Yb 3+ , Er 3+ La released in zebrafish 3+ Ions can bind to DNA, causing DNA damage and affecting the normal growth and development of organisms. 3+ Ions may also enter the cell nucleus, interfere with gene expression, and further aggravate biological toxicity. Studies have found that lanthanide rare earth ions released by rare earth oxide nanomaterials in an acidic environment are prone to interact with phosphorus-containing biological molecules in the body, thereby inducing cell autophagy and apoptosis. Therefore, the biological toxicity of REEs-UCNPs is closely related to its properties and its degradation or metabolites.

[0004] At present, the research on the toxicity of nanomaterials is still in its initial stage, and the understanding of their toxicity mechanism, the impact of degradation products and ecological safety is not deep enough. At present, the commonly used research methods for the morphological analysis of nanomaterials mainly include fluorescence spectroscopy (MFS), infrared spectroscopy (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM) and inductively coupled plasma mass spectrometry (ICP-MS). Among them, MFS has a low signal-to-noise ratio, is greatly interfered, and is easily affected by light and temperature; TEM and SEM have large human interference factors, high technical requirements for operators, and are time-consuming and labor-intensive; XRD testing time is long, signal-to-noise ratio is low, and working conditions are strict; ICP-MS can generally only perform total element analysis, and different morphological analysis requires more complex sample pretreatment steps, which leads to large errors in the results; the above analysis methods cannot achieve simultaneous and accurate quantitative analysis of REEs-UCNPs and their degradation or metabolites. Therefore, it is urgent to develop an analytical method with high sensitivity, low detection limit, high accuracy, good stability, fast analysis speed, and the ability to simultaneously analyze different morphological components such as REEs-UCNPs and their degradation or metabolites, so as to accurately understand the metabolic and distribution characteristics of REEs-UCNPs, grasp the degradation and change laws of REEs-UCNPs, provide technical support for in-depth research on their toxicity and safety evaluation, and realize the practical application of REEs-UCNPs separation and analysis methods, which is of great guiding significance for evaluating the environmental risks of REEs-UCNPs, formulating safe use standards, and application development. The main technical difficulties of the analysis method of different morphological components such as REEs-UCNPs and their degradation or metabolites are as follows: ① The properties of nanomaterials are different from those of general compounds or metal ions. REEs-UCNPs are generally charged and interact with the liquid chromatography stationary phase, so that REEs-UCNPs are adsorbed by the stationary phase and cannot leave the chromatographic column and enter the detector, resulting in no signal or low sensitivity. ② REEs-UCNPs are generally negatively charged and will bind to their degradation or metabolites (rare earth ions, etc.), so it is difficult to separate REEs-UCNPs from the rare earth ions produced by their degradation or metabolism. ③ When separating the rare earth ions produced by the degradation or metabolism of REEs-UCNPs, if gel chromatography is used, the separation is difficult due to the small difference in particle size between different rare earth ions. If ion chromatography is used, the simultaneous separation of nanomaterials and rare earth ions cannot be solved, and it may also cause the chromatographic column to be blocked, no peaks, and no signals. ④ In complex systems, such as biological systems, REEs-UCNPs and their degradation or metabolites will bind to various substances, resulting in complex separation and analysis. Summary of the invention

[0005] The purpose of the present invention is to provide a method for analyzing NaYF in complex systems based on SE-HPLC-ICP-MS. 4 :Yb 3+ , Tm 3+ The simultaneous separation and quantitative analysis method of their degradation or metabolites is used to solve the technical problem that REEs-UCNPs and rare earth ions are difficult to separate.

[0006] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0007] A novel method for analyzing NaYF in complex systems based on SE-HPLC-ICP-MS 4 :Yb 3+ , Tm 3+ and a method for simultaneous separation and quantitative analysis of degradation or metabolites thereof, comprising the following steps: 4 :Yb 3+ , Tm 3+ After mixing the additives and proteins, the samples were detected using size exclusion high performance liquid chromatography-inductively coupled plasma mass spectrometry.

[0008] Further, the following steps are included:

[0009] S1: Preparation of different concentrations of Y 3+ , Yb 3+ 、Tm 3+ Standard solution of standard ions;

[0010] S2: Prepare buffer solution system: Add different concentrations of Y 3+ , Yb 3+ 、Tm 3+ The standard solutions of the standard ions were mixed with aqueous NaYF 4 :Yb 3+ , Tm 3+ , and the additives are mixed; adding protein into the buffer solution system;

[0011] S3: using size exclusion high performance liquid chromatography-inductively coupled plasma mass spectrometry to detect the buffer solution system to which the protein is added;

[0012] S4: Y 3+ , Yb 3+ 、Tm 3+ The standard ion concentration is the horizontal axis, Y 3+ , Yb 3+ 、Tm 3+ The chromatographic peak area of ​​the standard ion is plotted as the ordinate. 3+ , Yb 3+ 、Tm 3+Quantitative analysis curve of standard ions, calculate Y 3+ , Yb 3+ 、Tm 3+ Linear regression equation for standard ions.

[0013] Furthermore, the additive is disodium ethylenediaminetetraacetate, the protein is fetal bovine serum, the final volume concentration of the fetal bovine serum is 12.5-50%, and the disodium ethylenediaminetetraacetate and the aqueous phase NaYF 4 :Yb 3+ , Tm 3+ The molar ratio of NaYF is (20-25):8; the aqueous phase NaYF 4 :Yb 3+ , Tm 3+ The concentration is 3.0×10 -10 ~1.6×10 -9 mol / mL.

[0014] Further, the parameters of the size exclusion high performance liquid chromatography in the size exclusion high performance liquid chromatography-inductively coupled plasma mass spectrometry technology are: using a gel chromatography column, the mobile phase is a sodium hydrogen phosphate solution with a concentration of 25 to 50 mmol / L and a pH of 8.0 to 9.0, the flow rate of the mobile phase is 1.0 to 1.5 mL / min, and the injection volume is 10 μL; the operating parameters of the inductively coupled plasma mass spectrometer in the size exclusion high performance liquid chromatography-inductively coupled plasma mass spectrometry technology are: radio frequency power: 1550 W; argon gas flow rate: 14 to 16 L / min; auxiliary gas, carrier gas, compensation / dilution gas flow rate: 0.7 to 0.9 L / min; argon pressure range: 650 to 750 KPa; peristaltic pump flow rate: 0.10 rps; signal acquisition mode: three-level converter; circulating water pressure value: 230 to 400 KPa; air volume: 10 to 15 m / s; measured elements: 89 Y. 172 Yb and 169 Tm.

[0015] Furthermore, the Y 3+ The linear regression equation of the standard ion is Y = 111.543X + 132.979, r 2 =0.995, the detection linear range is 0.1-50 μg / L, the Yb 3+ The linear regression equation of the standard ion is Y = 19.573X + 47.698, r 2 =0.990, the detection linear range is 0.1-50 μg / L, the Tm 3+ The linear regression equation of the standard ion is Y = 10.805X + 25.433, r 2=0.990, and the detection linear range is 0.1~20μg / L.

[0016] Furthermore, the aqueous phase NaYF 4 :Yb 3+ , Tm 3+ Oil phase NaYF 4 :Yb 3+ , Tm 3+ After ultrasonic degreasing, the oil phase NaYF 4 :Yb 3+ , Tm 3+ It was prepared by solvothermal method.

[0017] Furthermore, the aqueous phase NaYF 4 :Yb 3+ , Tm 3+ The preparation method is as follows: after mixing an aqueous solution of yttrium chloride, an aqueous solution of ytterbium chloride and an aqueous solution of thulium chloride, the mixture is heated to 100-120°C and reacted for 15-25 minutes to obtain a solid, the solid is cooled to room temperature, and then 6-9 mL of oleic acid and 10-15 mL of 1-octadecene are added to obtain a mixture, the mixture is heated to 120-150°C and maintained for 30-40 minutes to obtain a solution 1, after the solution 1 is cooled to 55-70°C, 4.5-5.5 mL of a methanol mixed solution containing 0.1-0.15 g of sodium hydroxide solid and 0.1-0.15 g of ammonium fluoride solid is added, the mixture is heated to 90-100°C and reacted for 20-30 minutes, argon is introduced and maintained for 2-3 minutes, the mixture is heated to 280-310°C and reacted for 60-90 minutes to obtain a solution 2, after the solution 2 is cooled to room temperature, ethanol is added, and centrifugation is performed to obtain a precipitate 1, and the precipitate 1 is washed and freeze-dried to obtain the oil phase NaYF 4 :Yb 3+ , Tm 3+ , the oil phase NaYF 4 :Yb 3+ , Tm 3+ The solution was dispersed in cyclohexane to obtain solution 3, and the solution was subjected to ultrasound and centrifugation to obtain precipitate 2, and the precipitate 2 was freeze-dried to obtain aqueous NaYF 4 :Yb 3+ , Tm 3+ .

[0018] Furthermore, the concentration of the yttrium chloride aqueous solution is 1.0-1.5 mol / L, the concentration of the ytterbium chloride aqueous solution is 0.1-0.2 mol / L, the concentration of the thulium chloride aqueous solution is 0.03-0.05 mol / L, and the volume fraction of the oleic acid is 20-25%.

[0019] The additive of the present invention uses disodium ethylenediaminetetraacetic acid (EDTA-2Na), which can react with aqueous NaYF 4 :Yb 3+ , Tm 3+ Combined to generate NaYF 4 :Yb 3+ , Tm 3+ -EDTA complexes, avoid NaYF 4 :Yb 3+ , Tm 3+ The NaYF 4 :Yb 3+ , Tm 3+ Difficult to separate and no chromatographic peak signal can be detected.

[0020] The aqueous NaYF prepared by the present invention 4 :Yb 3+ , Tm 3+ Negatively charged, it will react with water NaYF 4 :Yb 3+ , Tm 3+ Degradation or metabolite Yb 3+ 、Tm 3+ , Y 3+ Combination leads to aqueous NaYF 4 :Yb 3+ , Tm 3+ With Yb 3+ 、Tm 3+ , Y 3+ The chromatographic peaks overlap. The present invention adds protein, which can be adsorbed on the surface of metal ions to form a complex, thus avoiding the aqueous phase NaYF 4 :Yb 3+ , Tm 3+ With Yb 3+ 、Tm 3+ , Y 3+ Combined, so that the aqueous phase NaYF 4 :Yb 3+ , Tm 3+ With Yb 3+ 、Tm 3+ , Y 3+ Baseline separation is difficult to achieve.

[0021] Y 3+ At a concentration of 0.1-50 μg / L, Y 3+ The concentration of Yb is proportional to the chromatographic peak area. 3+ At a concentration of 0.1-50 μg / L, Yb 3+ The concentration is proportional to the chromatographic peak area, Tm 3+At a concentration of 0.1-20 μg / L, Tm 3+ The concentration of NaYF is proportional to the chromatographic peak area. The present invention can quantitatively detect the aqueous phase NaYF 4 :Yb 3+ , Tm 3+ Degradation or metabolite Yb 3+ 、Tm 3+ , Y 3+ concentration. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The aqueous phase NaYF in Example 1 4 :Yb 3+ , Tm 3+ Characterization diagram, where a is the SEM diagram, b is the TEM diagram, c is the physical diagram, d is the XRD diagram, and e is the MFS diagram;

[0023] Figure 2 The aqueous phase NaYF in Example 1 4 :Yb 3+ , Tm 3+ Zeta potential diagram in water at different times;

[0024] Figure 3 The aqueous phase NaYF in Example 1 was separated and analyzed by EDTA-2Na. 4 :Yb 3+ , Tm 3+ SE-HPLC-ICP-MS diagram, where a is the aqueous phase NaYF 4 :Yb 3+ , Tm 3+ solution, b is EDTA-2Na solution, c is aqueous NaYF 4 :Yb 3+ , Tm 3++ and a mixed solution of EDTA-2Na;

[0025] Figure 4 The aqueous phase NaYF in the buffer solution system of Example 1 4 :Yb 3+ , Tm 3+ and Yb 3+ 、Tm 3+ , Y 3+ SE-HPLC-ICP-MS graph of standard ions, where a is Yb 3+ 、Tm 3+ , Y 3+ Standard ion, b is aqueous NaYF 4 :Yb 3+ , Tm 3+ and Yb 3+ Solution, c is Yb3+ 、Tm 3+ , Y 3+ Standard ions and aqueous NaYF 4 :Yb 3+ , Tm 3+ and Yb 3+ Solution;

[0026] Figure 5 The aqueous phase NaYF in FBS in Example 1 4 :Yb 3+ , Tm 3+ and Y 3+ SE-HPLC-ICP-MS diagram of standard ions, where a is aqueous NaYF 4 :Yb 3+ , Tm 3+ solution, b is aqueous NaYF 4 :Yb 3+ , Tm 3+ and Y 3+ Mixed solution of standard ions;

[0027] Figure 6 The aqueous phase NaYF in FBS in Example 1 4 :Yb 3+ , Tm 3+ and Yb 3+ SE-HPLC-ICP-MS diagram of standard ions, where a is phase NaYF 4 :Yb 3+ , Tm 3+ solution, b is aqueous NaYF 4 :Yb 3+ , Tm 3+ and Yb 3+ Mixed solution of standard ions;

[0028] Figure 7 The aqueous phase NaYF in FBS in Example 1 4 :Yb 3+ , Tm 3+ and Tm 3+ SE-HPLC-ICP-MS diagram of standard ions, where a is phase NaYF 4 :Yb 3+ , Tm 3+ solution, b is aqueous NaYF 4 :Yb 3+ , Tm 3+ and Tm 3+ Mixed solution of standard ions;

[0029] Figure 8 The aqueous phase NaYF in FBS in Example 14 :Yb 3+ , Tm 3+ and Y 3+ , Yb 3+ 、Tm 3+ SE-HPLC-ICP-MS graphs of standard ions, where the final volume concentration of FBS in a is 12.5%, the final volume concentration of FBS in b is 25%, the final volume concentration of FBS in c is 37.5%, and the final volume concentration of FBS in d is 50%;

[0030] Fig. 9 For different concentrations of Y 3+ , Yb 3+ 、Tm 3+ Standard ions and aqueous NaYF 4 :Yb 3+ , Tm 3+ SE-HPLC-ICP-MS diagram, where Y 3+ , Yb 3+ 、Tm 3+ The ion concentration of the standard products is 0.1 μg / L. 3+ , Yb 3+ 、Tm 3+ The ion concentration of the standard products is 1 μg / L. 3+ , Yb 3+ 、Tm 3+ The ion concentration of the standard products is 5 μg / L. 3+ , Yb 3+ 、Tm 3+ The ion concentration of the standard products is 20 μg / L. 3+ , Yb 3+ 、Tm 3+ The ion concentration of the standard products is 50 μg / L. 3+ , Yb 3+ 、Tm 3+ The ion concentration of the standard products was 100 μg / L;

[0031] Fig.10 Y 3+ , Yb 3+ 、Tm 3+ Standard curve of standard ions, where a is Y 3+ Standard ion, b is Yb 3+ Standard ion, c is Tm 3+ Standard ions. DETAILED DESCRIPTION

[0032] The present invention will be further described below in conjunction with the embodiments of the present invention and the accompanying drawings.

[0033] The instruments used in the present invention are shown in Table 1, and the reagents used are shown in Table 2.

[0034] Table 1 NaYF 4 :Yb 3+ , Tm 3+ Main instruments for separation and analysis

[0035]

[0036] Table 2NaYF 4 :Yb 3+ , Tm 3+ Main reagents for separation and analysis

[0037]

[0038]

[0039] Example 1

[0040] In this example, the SE-HPLC-ICP-MS was used to analyze the NaYF 4 :Yb 3+ , Tm 3+ The method for simultaneous separation and quantitative analysis of the degradation or metabolites thereof comprises the following steps:

[0041] 1. Aqueous NaYF 4 :Yb 3+ , Tm 3+ Preparation

[0042] YCl 3 6H 2 O, YbCl 3 6H 2 O、TmCl 3 6H 2 O was fully dissolved in deionized water and prepared into 1 mol / L YCl 3 Aqueous solution, 0.1 mol / L YbCl 3 Aqueous solution, 0.03 mol / L TmCl 3 Aqueous solution, after ultrasonic treatment to obtain clear YCl 3 Aqueous solution, YbCl 3 Aqueous solution, TmCl 3 Aqueous solution. Take 0.747mL of YCl 3 Aqueous solution, 0.25 mL of YbCl 3 Aqueous solution and 0.1 mL of TmCl 3The aqueous solution was transferred to a three-necked round-bottom flask, stirred rapidly in a magnetic stirrer, heated to 110°C, maintained for 20 minutes, and the water was evaporated to obtain a white solid. After the white solid was cooled to room temperature, 6 mL of oleic acid and 15 mL of 1-octadecene were added to the three-necked round-bottom flask to obtain a mixture, and the mixture was heated to 150°C and maintained for 30 minutes to obtain a clear and uniform solution 1. After solution 1 was cooled to 60°C, 5 mL of a methanol mixed solution containing 0.1 g of sodium hydroxide solid and 0.148 g of ammonium fluoride solid was added, the temperature was raised to 110°C, maintained for 20 minutes, methanol and residual water were evaporated, and then high-purity argon was introduced for 2 minutes, the condenser was connected, the temperature was raised to 300°C, and the temperature was maintained for 60 minutes to obtain solution 2. Solution 2 was cooled to room temperature and transferred to a centrifuge tube, anhydrous ethanol was added, and precipitate 1 was obtained after centrifugation. After washing precipitate 1 twice, the oil phase NaYF 4 :Yb 3 + , Tm 3+ , the oil phase NaYF 4 :Yb 3+ , Tm 3+ The powder is freeze-dried and stored for future use. The volume fraction of oleic acid is 20.6%.

[0043] Take the freeze-dried powdered oil phase NaYF 4 :Yb 3+ , Tm 3+ Evenly dispersed in cyclohexane, prepared into 3 mg / mL solution three, ultrasonicated solution three for 20 min and centrifuged for 5 min, repeated the "ultrasound-centrifugation" step 3 times, discarded the supernatant to obtain precipitation two, lyophilized precipitation two into powder and stored for later use, and then the aqueous phase NaYF 4 :Yb 3+ , Tm 3+ .

[0044] 2. Basic working conditions of SE-HPLC-ICP-MS

[0045] 1. SE-HPLC basic working conditions

[0046] Power supply: 220V, 50HZ; Binary high pressure pump type: four-pressure type; Binary high pressure pump: digitally controlled linear transmission chromatographic double pump; Vacuum degassing: online automatic degasser; Maximum working pressure: no more than 400bar; Injection volume: 10μL.

[0047] Gel chromatography column: Agilent Bio SEC-5 gel chromatography column, the filling material of the gel chromatography column is porous silica coated with hydrophilic polymer, the particle size is 5μm, the pore structure Outer diameter 4.6mm, length 300mm, pH range: 2.0~8.5; maximum working pressure: no more than 240bar.

[0048] 2. Basic working conditions of ICP-MS

[0049] RF power: 1550W; Argon gas flow rate: 15L / min; Auxiliary gas (Ar), carrier gas, compensation / dilution gas flow rate: 0.8L / min; Argon pressure range: 650-750KPa; Peristaltic pump flow rate: 0.10rps; Signal acquisition method: three-level converter; Circulating water (distilled water) pressure value: 230-400KPa; Air volume: 10-15m / s; Determined elements: 89 Y. 172 Yb and 169 Tm.

[0050] 3. Results and Discussion

[0051] 3.1 Aqueous NaYF 4 :Yb 3+ , Tm 3+ Synthesis and characterization of nanomaterials

[0052] Figure 1 For aqueous NaYF 4 :Yb 3+ , Tm 3+ Characterization diagram, the optimal rare earth element doping ratio is Y 3+ :Yb 3+ :Tm 3+ =74.7%:25%:0.3%, reaction time is 60min, reaction temperature is 300℃, oleic acid volume fraction is 20.6%, from Figure 1 It can be seen that under the optimal conditions, the aqueous NaYF 4 :Yb 3+ , Tm 3 It is a hexagonal crystal structure with uniform size and good dispersion. The average particle size between the two planes of the hexagon is about 50nm. Under the excitation of 980nm near-infrared light, the fluorescence intensity is the highest at 452nm and 478nm, and it emits bright blue fluorescence. 4 :Yb 3+ , Tm 3+ Zeta potential graphs were measured after being placed in water at room temperature for 0.5h, 1h, 5h, 12h, and 24h. Figure 2 It can be seen that as time increases, the aqueous NaYF 4 :Yb 3+ , Tm 3+ The Zeta value of the aqueous phase NaYF 4 :Yb 3+, Tm 3+ The surface has a negative charge, and the absolute value is greater than 15mV, indicating that the aqueous NaYF 4 :Yb 3 + , Tm 3+ There is no coagulation phenomenon and the dispersion in water is good.

[0053] 3.2 Effect of EDTA-2Na Additive on NaYF 4 :Yb 3+ , Tm 3+ Impact of separation

[0054] Based on the basic working conditions of SE-HPLC-ICP-MS, 25 mmol / L sodium hydrogen phosphate solution with pH = 8.0 was used as the mobile phase, and the flow rate of the mobile phase was 1.0 mL / min; NaYF 4 :Yb 3+ , Tm 3+ Concentration: 1.6×10 -9 mol / mL; explore the concentration of additive EDTA-2Na to separate and analyze NaYF 4 :Yb 3+ , Tm 3+ The SE-HPLC-ICP-MS images are shown in Figure 3 As shown, from Figure 3 aIt can be seen that NaYF 4 :Yb 3+ , Tm 3+ No signal was detected without adding EDTA-2Na, which may be due to the NaYF 4 :Yb 3+ , Tm 3+ Adsorbed by the stationary phase and does not flow out of the chromatographic column; Figure 3 b It can be seen that the detection of EDTA-2Na solution alone also failed to produce a signal; Figure 3 c It can be seen that when in NaYF 4 :Yb 3+ , Tm 3+ After adding EDTA-2Na to the solution, NaYF 4 :Yb 3+ , Tm 3+ The chromatographic peak has a smooth elution curve and a high signal intensity. This phenomenon may be caused by the interaction between the additive EDTA-2Na and NaYF 4 :Yb 3 + , Tm 3+ Combined to form NaYF 4 :Yb 3+ , Tm 3+-EDTA complex is not adsorbed by the stationary phase in the chromatographic column, so it can flow out of the chromatographic column smoothly, and the chromatographic peak signal is detected. After the experimental conditions are optimized, the nanomaterial NaYF 4 :Yb 3+ , Tm 3+ The optimal molar ratio of EDTA-2Na to the additive is 8:25. The concentration of EDTA-2Na is 5.0×10 -9 mol / mL.

[0055] 3.3 NaYF in buffer solution system 4 :Yb 3+ , Tm 3+ and Y 3+ , Yb 3+ 、Tm 3+ Optimization of SE-HPLC-ICP-MS separation conditions

[0056] Buffer solution system: NaYF 4 :Yb 3+ , Tm 3+ , Additives EDTA-2Na, Y 3+ , Yb 3+ 、Tm 3+ The standard ions are mixed and obtained; NaYF 4 :Yb 3+ , Tm 3+ The concentration is 3.0×10 -10 mol / mL, the concentration of EDTA-2Na is 9.375×10 -10 mol / mL, i.e. NaYF 4 :Yb 3+ , Tm 3+ The molar ratio of Y to EDTA-2Na is 8:25; 3+ , Yb 3+ 、Tm 3+ The concentration of standard ions is 187.5 μg / L.

[0057] Study on NaYF in buffer solution system 4 :Yb 3+ , Tm 3+ and Y 3+ , Yb 3+ 、Tm 3+ The SE-HPLC-ICP-MS chart shows Figure 4 As shown, from Figure 4 a It can be seen that the three standard ions Y 3+ , Yb 3+ 、Tm 3+ The retention time of Figure 4 As can be seen in b, NaYF 4 :Yb 3+ , Tm 3+ The retention time of Figure 4 As can be seen from c, NaYF 4 :Yb 3+ , Tm 3+ and Y 3+ , Yb 3+ 、Tm 3+ The retention time of the standard ion mixed solution is 3.897min, which is similar to that of NaYF 4 :Yb 3+ , Tm 3+ The retention time of the NaYF 4 :Yb 3+ , Tm 3+ negatively charged, and Y 3 + , Yb 3+ 、Tm 3+ The standard ions are all positively charged. Due to the interaction between positive and negative charges, Y 3+ , Yb 3+ 、Tm 3+ The standard ions are adsorbed onto NaYF 4 :Yb 3+ , Tm 3+ The surface causes the two chromatographic peaks to overlap.

[0058] 3.4 Effect of Fetal Bovine Serum (FBS) on NaYF 4 :Yb 3+ , Tm 3+ and Y 3+ Effect of Ion Separation of Standards

[0059] When the sample solution (NaYF 4 :Yb 3+ , Tm 3+ and Y 3+ When 1 mL of 60% FBS was added to a mixed solution of standard ions (the final volume concentration was 37.5%), Figure 5 b It can be seen that two types of Y appear in the chromatogram 3+ Ion form peak, probably Y 3+ The products formed by the binding of standard ions with different proteins in FBS are expressed as Y 3+ [2] and Y 3+ [3] indicates that when only NaYF 4 :Yb 3+ , Tm 3+When testing, the results are as follows Figure 5 As shown in a, Y in the chromatogram 3+ and Figure 5 Y in b 3+ [1] The peak signal size (peak area) is consistent, which indicates that NaYF 4 :Yb 3+ , Tm 3+ and Y 3+ [2]+Y 3+ [3] Achieve complete baseline separation, Y 3+ [1] NaYF 4 :Yb 3+ , Tm 3+ Middle Y 3+ The mechanism for achieving baseline separation is that a certain protein in FBS binds to Y 3+ The interaction forms a stable complex, which changes the Y 3+ surface charge, and the size of the complex is larger than that of NaYF 4 :Yb 3+ , Tm 3+ Large, thus achieving the purpose of separation.

[0060] 3.5 FBS vs NaYF 4 :Yb 3+ , Tm 3+ and Yb 3+ Effect of Ion Separation of Standards

[0061] When the sample solution (NaYF 4 :Yb 3+ , Tm 3+ and Yb 3+ When 1 mL of 60% FBS was added to the mixed solution of standard ions (the final volume concentration was 37.5%), Figure 6 b, a Yb appeared in the chromatogram 3+ Ion form peak, probably Yb 3+ The product formed by the binding of the standard ions to the proteins in FBS is Yb 3+ [2] indicates that when only NaYF 4 :Yb 3+ , Tm 3+ When testing, the results are as follows Figure 6 a, Yb in the chromatogram 3+ and Figure 6 Yb in b 3+ [1] The peak signal size (peak area) is consistent, which indicates that NaYF 4 :Yb 3+ , Tm 3+ and Yb 3+[2] Achieve complete baseline separation, Yb 3+ [1] NaYF 4 :Yb 3+ , Tm 3+ Medium 3+ The mechanism for achieving baseline separation is that a certain protein in FBS interacts with Yb 3+ The interaction forms a stable complex, which changes the Yb 3+ surface charge, and the size of the complex is larger than that of NaYF 4 :Yb 3+ , Tm 3+ Large, thus achieving the purpose of separation.

[0062] 3.6 FBS vs NaYF 4 :Yb 3+ , Tm 3+ and Tm 3+ Effect of Ion Separation of Standards

[0063] When the sample solution (NaYF 4 :Yb 3+ , Tm 3+ and Tm 3+ When 1 mL of 60% FBS was added to the mixed solution of standard ions (the final volume concentration was 37.5%), Figure 7 , a Tm 3+ Ion form peak, probably Tm 3+ The product formed by the binding of the standard ions to the proteins in FBS is expressed as Tm 3+ [2] indicates that when only NaYF 4 :Yb 3+ , Tm 3+ When testing, the results are as follows Figure 7 a, Tm in the chromatogram 3+ and Figure 7 Tm in b 3+ [1] The peak signal size (peak area) is consistent, which indicates that NaYF 4 :Yb 3+ , Tm 3+ and Tm 3+ [2] Achieve complete baseline separation, Tm 3+ [1] NaYF 4 :Yb 3+ , Tm 3+ Medium Tm 3+ The mechanism for achieving baseline separation is that a certain protein in FBS binds to the Tm 3+ Interact to form a stable complex, changing the Tm 3+ surface charge, and the size of the complex is larger than that of NaYF4 :Yb 3+ , Tm 3+ Large, thus achieving the purpose of separation.

[0064] 3.7 FBS vs NaYF 4 :Yb 3+ , Tm 3+ and Y 3+ , Yb 3+ 、Tm 3+ Effect of Ion Separation of Standards

[0065] When the sample solution (NaYF 4 :Yb 3+ , Tm 3+ and Y 3+ , Yb 3+ 、Tm 3+ When 1 mL of 20% FBS was added to a mixed solution of standard ions (the final volume concentration was 12.5%), Figure 8 As can be seen from a, NaYF 4 :Yb 3+ , Tm 3+ and Y 3+ , Yb 3+ 、Tm 3+ The standard ions seemed to be baseline separated, but under these conditions, two Y 3+ , Yb 3+ 、Tm 3+ Ion form peak, probably Y 3+ , Yb 3+ 、Tm 3+ The products formed by the standard ions binding to different proteins in FBS are expressed as Y 3+ [2] and Y 3+ [3] Yb 3+ [2] and Yb 3+ [3] Tm 3+ [2] and Tm 3+ [3] indicates that part Y 3+ [2] and Y 3+ [3] Yb 3+ [2] and Yb 3+ [3] Tm 3+ [2] and Tm 3+ [3] The chromatographic peak of the ion is similar to that of NaYF 4 :Yb 3+ , Tm 3+ overlap; when 1 mL of 40% FBS was added to the sample solution (final volume concentration was 25%), Figure 8 b It can be seen that NaYF4 :Yb 3 + , Tm 3+ and Y 3+ , Yb 3+ 、Tm 3+ The separation of the standard ions is further increased, and Y 3+ [1]+Y 3+ [2]+Y 3+ [3] Yb 3+ [1]+Yb 3+ [2]+Yb 3+ [3] Tm 3+ [1]+Tm 3+ [2]+Tm 3+ [3] The ion signal intensity decreases, Y 3+ [2]+Y 3+ [3] Yb 3+ [2]+Yb 3+ [3] Tm 3+ [2]+Tm 3+ [3] The signal intensity increased, but complete baseline separation was not achieved. When 1 mL of FBS with a volume fraction of 60% was added to the sample solution (the final volume concentration was 37.5%), Figure 8 cIt can be seen that NaYF 4 :Yb 3+ , Tm 3+ and Y 3+ , Yb 3+ 、Tm 3+ The resolution of the standard ions was further increased, and NaYF 4 :Yb 3+ , Tm 3+ There is also a small amount of Y 3+ , Yb 3+ 、Tm 3+ Standard ions; when 1 mL of 80% FBS was added to the sample solution (the final volume concentration was 50.0%), Figure 8 As can be seen from d, NaYF 4 :Yb 3+ , Tm 3+ There is still a small amount of Y 3+ , Yb 3+ 、Tm 3+ In addition, with the increase of FBS volume concentration, the Y 3+ [2] Yb 3+ [2] Tm 3+ The content of [2] gradually increased, and the peak time was 2.148min. 3+ [3] Yb3+ [3] Tm 3+ The content of [3] gradually decreases, from which it can be inferred that Y 3+ , Yb 3+ 、Tm 3+ It preferentially binds to the corresponding protein at 2.648 min, and secondarily binds to the corresponding protein at 2.148 min.

[0066] 3.8 Performance Evaluation

[0067] Under optimal conditions, NaYF 4 :Yb 3+ , Tm 3+ The concentration is 3.0×10 -10 mol / mL, the concentration of additive EDTA-2Na is 9.375×10 -10 mol / mL, FBS volume concentration: 37.5%, for NaYF in the buffer solution system 4 :Yb 3+ , Tm 3+ and Y 3 + , Yb 3+ 、Tm 3+ The standard series of standard ions (the concentration gradients were 0.1 μg / L, 1 μg / L, 5 μg / L, 20 μg / L, 50 μg / L, and 100 μg / L) were mixed and analyzed by SE-HPLC-ICP-MS. The chromatogram is shown in Fig. 9 As shown, Y 3+ , Yb 3+ 、Tm 3+ The standard ion concentration is the horizontal axis, Y 3+ , Yb 3+ 、Tm 3+ The chromatographic peak area of ​​the standard ion concentration was used as the ordinate to draw the quantitative analysis curve and determine its linear range. The detection limit was calculated according to the formula D = 3σ / A (where D is the detection limit, σ is the standard deviation, and A is the slope of the standard curve). In addition, under the optimal experimental conditions, NaYF 4 :Yb 3+ , Tm 3+ The aqueous solution was tested for Y in the buffer solution system at 1 μg / L, 5 μg / L and 20 μg / L. 3+ , Yb 3+ 、Tm 3+ The spike recovery experiment was performed, and each sample was measured 3 times to calculate the relative standard deviation (RSD) and spike recovery rate. All performance parameters of the SE-HPLC-ICP-MS method are shown in Table 3.

[0068] Table 3 Performance parameters of SE-HPLC-ICP-MS method

[0069]

[0070] As can be seen from Table 3, the method of the present invention has a significant effect on Y 3+ , Yb 3+ The linear range of detection is 0.1-50 μg / L, and the detection limits are 1.3×10 -2 μg / L, 3.0×10 -2 μg / L, the spiked recoveries were 96.6% to 102.5% and 92.6% to 100.6% respectively; this method has a good 3+ The linear range of detection is 0.1-20 μg / L, and the detection limit is 4.3×10 -2 μg / L, and the spike recovery rate was 96.6% to 104.7%.

[0071] 3.9NaYF 4 :Yb 3+ , Tm 3+ and its metabolites or degradation products Y 3+ , Yb 3+ 、Tm 3+ SE-HPLC-ICP-MS quantitative analysis

[0072] Under the optimal experimental conditions, NaYF 4 :Yb 3+ , Tm 3+ and Y 3+ , Yb 3+ 、Tm 3+ Baseline separation was achieved. According to Table 3, Fig.10 It can be seen that Y 3+ , Yb 3+ 、Tm 3+ Within a certain concentration range, the peak area (A) is proportional to the concentration (C), that is, A = K·C. Therefore, the NaYF 4 :Yb 3+ , Tm 3+ and its degradation or metabolite Y 3+ , Yb 3+ 、Tm 3+ The specific quantitative method can be Fig.10 Carry out. 4 :Yb 3+ , Tm 3+ Middle Y 3+ (with NaYF 4 :Yb 3+ , Tm 3+ -Y 3+The content of 3+ The standard curve of NaYF was used for direct quantification. 4 :Yb 3+ , Tm 3+ Medium 3+ (with NaYF 4 :Yb 3+ , Tm 3+ -Yb 3+ The content of Yb 3+ The standard curve of NaYF was used for direct quantification. 4 :Yb 3+ , Tm 3+ Medium Tm 3+ (with NaYF 4 :Yb 3+ , Tm 3+ -Tm 3+ The content of 3+ The standard curve was used for direct quantification.

[0073] 3.10 NaYF in rat serum 4 :Yb 3+ , Tm 3+ Changes in content

[0074] SPF healthy male Wistar rats (4 weeks old) were purchased from Beijing Spike Biotechnology Co., Ltd. (animal production license number SCXK (Beijing) 2019-0010). There were 10 rats weighing 50-70 g. The male Wistar rats were randomly divided into two groups (control group and NaYF 4 :Yb 3+ ,Tm 3+ The NaYF synthesized by the present invention was injected into the 4 :Yb 3+ ,Tm 3+ Dissolve in saline and immediately inject via tail vein. 4 :Yb 3+ ,Tm 3+ The poisoning dose of the poisoning group was 20 mg / kg, and the control group was injected with an equal volume of physiological saline. After the poisoning was completed, about 0.4 mL of blood was collected from the rat tail vein every 30 minutes, 1 hour, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and 7 days in a 0.5 mL centrifuge tube. After standing for 4 hours, the blood was centrifuged at 3000 rpm for 20 minutes at 4°C to prepare serum. The serum samples were stored in a -80°C refrigerator or immediately analyzed by the SE-HPLC-ICP-MS developed by the present invention for the NaYF poisoning. 4 :Yb 3+ , Tm3+ NaYF in rat serum at different time points 4 :Yb 3+ , Tm 3+ and its metabolite Y 3 + , Yb 3+ 、Tm 3+ The results are shown in Table 4. It can be seen from Table 4 that this method successfully detected NaYF in serum 4 :Yb 3+ , Tm 3 + -Y 3+ ,NaYF 4 :Yb 3+ , Tm 3+ -Yb 3+ and the degraded Y 3+ , Yb 3+ ,NaYF 4 :Yb 3+ , Tm 3+ -Tm 3+ and Tm 3+ Not detected may be related to the fact that the dose of the poison is too small and below the detection limit of the instrument. ND stands for Not Detected.

[0075] Table 4 NaYF in serum of rats exposed at different time 4 :Yb 3+ , Tm 3+ Changes in concentrations of chloramphenicol and its metabolites (n=5)

[0076]

Claims

1. A method for the analysis of NaYF4:Yb in complex systems based on SE-HPLC-ICP-MS 3+ , Tm 3+ and a method for simultaneous separation and quantitative analysis of its degradation or metabolites, characterized in that: The following steps are involved: The aqueous phase NaYF4:Yb 3+ , Tm 3+ After mixing the additives and proteins, the samples were detected using size exclusion high performance liquid chromatography-inductively coupled plasma mass spectrometry.

2. The method according to claim 1 for analyzing NaYF4:Yb in complex system based on SE-HPLC-ICP-MS 3+ , Tm 3+ and a method for simultaneous separation and quantitative analysis of its degradation or metabolites, characterized in that: The following steps are involved: S1: Preparation of different concentrations of Y 3+ , Yb 3+ 、Tm 3+ Standard solution of standard ions; S2: Prepare buffer solution system: Add different concentrations of Y 3+ , Yb 3+ 、Tm 3+ The standard solutions of the standard ions were mixed with aqueous NaYF4:Yb 3+ , Tm 3+ , and the additives are mixed; adding protein into the buffer solution system; S3: using size exclusion high performance liquid chromatography-inductively coupled plasma mass spectrometry to detect the buffer solution system to which the protein is added; S4: Y 3+ , Yb 3+ 、Tm 3+ The standard ion concentration is the horizontal axis, Y 3+ , Yb 3+ 、Tm 3+ The chromatographic peak area of ​​the standard ion is plotted as the ordinate. 3+ , Yb 3+ 、Tm 3+ Quantitative analysis curve of standard ions, calculate Y 3+ , Yb 3+ 、Tm 3+ Linear regression equation for standard ions.

3. The method according to claim 2 for analyzing NaYF4:Yb in complex system based on SE-HPLC-ICP-MS 3+ , Tm 3+ and a method for simultaneous separation and quantitative analysis of its degradation or metabolites, characterized in that: The additive is disodium ethylenediaminetetraacetate, the protein is fetal bovine serum, the final volume concentration of the fetal bovine serum is 12.5-50%, the disodium ethylenediaminetetraacetate and the aqueous phase NaYF4:Yb 3+ , Tm 3+ The molar ratio of NaYF4:Yb 3+ , Tm 3+ The concentration is 3.0×10 -10 ~1.6×10 -9 mol / mL.

4. The method according to claim 2 for analyzing NaYF4:Yb in complex system based on SE-HPLC-ICP-MS 3+ , Tm 3+ and a method for simultaneous separation and quantitative analysis of its degradation or metabolites, characterized in that: The parameters of the size exclusion high performance liquid chromatography in the size exclusion high performance liquid chromatography-inductively coupled plasma mass spectrometry technology are as follows: a gel chromatography column is used, the mobile phase is a sodium hydrogen phosphate solution with a concentration of 25 to 50 mmol / L and a pH of 8.0 to 9.0, and the flow rate of the mobile phase is 1.0 to 1.5 mL / min; the operating parameters of the inductively coupled plasma mass spectrometer in the size exclusion high performance liquid chromatography-inductively coupled plasma mass spectrometry technology are as follows: argon gas flow rate: 14 to 16 L / min; auxiliary gas, carrier gas, compensation / dilution gas flow rate: 0.7 to 0.9 L / min; argon pressure range: 650 to 750 KPa; signal acquisition mode: three-level converter; circulating water pressure value: 230 to 400 KPa; air volume: 10 to 15 m / s; measured elements: 89 Y. 172 Yb and 169 Tm.

5. The method according to claim 2 for analyzing NaYF4:Yb in complex system based on SE-HPLC-ICP-MS 3+ , Tm 3+ and a method for simultaneous separation and quantitative analysis of its degradation or metabolites, characterized in that: The Y 3+ The linear regression equation of the standard ion is Y = 111.543X + 132.979, r 2 =0.995, the detection linear range is 0.1-50 μg / L, the Yb 3+ The linear regression equation of the standard ion is Y = 19.573X + 47.698, r 2 =0.990, the detection linear range is 0.1-50 μg / L, the Tm 3+ The linear regression equation of the standard ion is Y = 10.805X + 25.433, r 2 =0.990, and the detection linear range is 0.1~20μg / L.

6. The method according to claim 2 for analyzing NaYF4:Yb in complex system based on SE-HPLC-ICP-MS 3+ , Tm 3+ and a method for simultaneous separation and quantitative analysis of its degradation or metabolites, characterized in that: The aqueous phase NaYF4:Yb 3+ , Tm 3+ Oil phase NaYF4:Yb 3+ , Tm 3+ After ultrasonic degreasing, the oil phase NaYF4:Yb 3+ , Tm 3+ It was prepared by solvothermal method.

7. The method according to claim 6 for analyzing NaYF4:Yb in a complex system based on SE-HPLC-ICP-MS 3+ , Tm 3+ and a method for simultaneous separation and quantitative analysis of its degradation or metabolites, characterized in that: The aqueous phase NaYF4:Yb 3+ , Tm 3+ The preparation method is as follows: after mixing an aqueous solution of yttrium chloride, an aqueous solution of ytterbium chloride and an aqueous solution of thulium chloride, the mixture is heated to 100-120°C and reacted for 15-25 minutes to obtain a solid, the solid is cooled to room temperature, and then 6-9 mL of oleic acid and 10-15 mL of 1-octadecene are added to obtain a mixture, the mixture is heated to 120-150°C and maintained for 30-40 minutes to obtain a solution 1, after the solution 1 is cooled to 55-70°C, 4.5-5.5 mL of a methanol mixed solution containing 0.1-0.15 g of sodium hydroxide solid and 0.1-0.15 g of ammonium fluoride solid is added, the mixture is heated to 90-100°C and reacted for 20-30 minutes, argon is introduced and maintained for 2-3 minutes, the mixture is heated to 280-310°C and reacted for 60-90 minutes to obtain a solution 2, after the solution 2 is cooled to room temperature, ethanol is added, and centrifugation is performed to obtain a precipitate 1, and the precipitate 1 is washed and freeze-dried to obtain the oil phase NaYF4:Yb 3+ , Tm 3+ , the oil phase NaYF4:Yb 3+ , Tm 3+ The solution was dispersed in cyclohexane to obtain solution 3, and the solution was subjected to ultrasound and centrifugation to obtain precipitate 2, and the precipitate 2 was freeze-dried to obtain aqueous phase NaYF4:Yb 3+ , Tm 3+ .

8. The method according to claim 7 for analyzing NaYF4:Yb in a complex system based on SE-HPLC-ICP-MS 3+ , Tm 3+ and a method for simultaneous separation and quantitative analysis of its degradation or metabolites, characterized in that: The concentration of the yttrium chloride aqueous solution is 1.0-1.5 mol / L, the concentration of the ytterbium chloride aqueous solution is 0.1-0.2 mol / L, the concentration of the thulium chloride aqueous solution is 0.03-0.05 mol / L, and the volume fraction of the oleic acid is 20-25%.

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