A method for determining single conodont phosphate oxygen isotope based on ion chromatography-electrostatic field orbitrap mass spectrometry

Abstract

This invention discloses a method for determining the phosphate oxygen isotopes of a single dentomorph based on ion chromatography-electrostatic orbital trap mass spectrometry, belonging to the field of isotope geochemical analysis technology. The method includes: acid digestion step; lyophilization step; reconstitution step; ion chromatography separation and fraction collection step; mass spectrometry analysis step: acquiring the mass spectrometric signals of the target fragment ions; isotope ratio calculation step: calculating the oxygen isotope shift of the sample relative to the reference solution; and calibration step: establishing a calibration curve using multiple known phosphate reference materials to obtain the phosphate oxygen isotope composition of the sample. This invention simultaneously solves three technical bottlenecks in existing thermal conversion elemental analysis-isotope ratio mass spectrometry methods: high sample consumption and cumbersome procedures prone to fractionation; secondary ion mass spectrometry methods: mixed signals and inability to distinguish different oxygen-containing components; and pure solution electrostatic orbital trap mass spectrometry methods: lack of solid sample pretreatment support.

Description

Technical Field

[0001] This invention relates to the field of isotope geochemical analysis technology, and in particular to a method for determining the phosphate oxygen isotopes of a single dentin-like particle based on ion chromatography-electrostatic field orbital trap mass spectrometry. Background Technology

[0002] Conodonts, widely distributed phosphate microfossils in Paleozoic to Triassic marine strata, are crucial for reconstructing deep-sea paleoocean temperatures through their apatite lattice oxygen isotope composition. However, high-precision phosphate oxygen isotope analysis of individual conodonts remains a significant challenge due to limitations in current analytical techniques. While conventional thermal conversion elemental analysis-isotope ratio mass spectrometry offers high accuracy, it requires milligram-level samples (corresponding to dozens of conodonts), failing to meet the demands for high-resolution single-particle analysis. Secondary ion mass spectrometry, though enabling in-situ analysis of micro-areas, cannot distinguish the oxygen signals from phosphate groups and other oxygen-containing groups such as carbonates and hydroxyl groups in apatite. Measuring isotope values ​​yields mixed signals from multiple components, making it difficult to accurately reflect the true composition of oxygen in the phosphate lattice. While the latest electrospray ionization-electrostatic field orbital trap mass spectrometry method for pure solution analysis achieves nanomolar-level high sensitivity at the instrument level, it is only applicable to pure solution matrices and lacks a complete pretreatment solution for solid geological samples, including acid digestion, impurity separation, fraction collection, concentration, and solvent exchange. Therefore, it cannot be directly applied to complex solid samples such as denticles. Consequently, there is an urgent need in this field for a complete analytical method that combines nanomolar-level high sensitivity, high selectivity for phosphate oxygen only, low sample consumption down to the level of a single denticle, and controllable fractionation throughout the entire process.

[0003] However, current common solutions have many drawbacks, including: existing methods mainly rely on three types of techniques: thermal conversion elemental analysis-isotope ratio mass spectrometry, secondary ion mass spectrometry, and electrospray ionization-electrostatic field orbital trap mass spectrometry pure solution analysis. However, each of these three methods has its own inherent defects that are difficult to overcome: thermal conversion elemental analysis-isotope ratio mass spectrometry, although with high analytical precision, requires milligram-level samples, and the pretreatment process is cumbersome and lengthy, easily introducing non-equilibrium oxygen isotope fractionation, and cannot achieve independent high-resolution analysis of single tooth-shaped spikes; secondary ion mass spectrometry, although capable of in-situ analysis of micro-areas, cannot distinguish phosphorus ash. The oxygen signals of phosphate and other oxygen-containing groups such as carbonate and hydroxyl groups in phosphate rock are measured as a weighted average of multiple components, which is difficult to accurately reflect the true composition of oxygen in the phosphate lattice. At the same time, the equipment is extremely expensive and difficult to widely apply. Although the electrospray ionization-electrostatic field orbital trap mass spectrometry pure solution analysis method has achieved nanomolar-level high sensitivity detection at the instrument end, it is only applicable to pure solution matrix. It lacks a complete pretreatment solution for solid conodonts, including acid digestion, impurity separation, fraction collection, concentration and solvent exchange. Moreover, the recovery rate and fractionation controllability of the whole process have not been verified, so it cannot be directly applied to actual geological samples. Summary of the Invention

[0004] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0005] In view of the problems existing in the current method for determining the phosphate oxygen isotopes of a single dentin-like morphology based on ion chromatography-electrostatic field orbital trap mass spectrometry, the present invention is proposed.

[0006] Therefore, the purpose of this invention is to provide a method for determining the phosphate oxygen isotope of a single dentin-like spur based on ion chromatography-electrostatic field orbital trap mass spectrometry. This method is suitable for solving the problem that existing technologies cannot simultaneously meet the comprehensive technical requirements of nanomolar-level high sensitivity, high selectivity for phosphate oxygen only, sample consumption at the level of a single dentin spur, and controllable fractionation throughout the entire process.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution: In a first aspect, embodiments of the present invention provide a method for determining the phosphate oxygen isotope of a single dentin skeletal specimen based on ion chromatography-electrostatic field orbital trap mass spectrometry, comprising: an acid digestion step: digesting the dentin skeletal specimen or apatite standard with hydrochloric acid solution to obtain an acid digest; a lyophilization step: diluting and filtering the acid digest, followed by lyophilization to remove excess hydrochloric acid and water, to obtain a lyophilized residue; a reconstitution step: adding ultrapure water to the lyophilized residue to dissolve the residue, obtaining a sample loading solution; an ion chromatography separation and fraction collection step: injecting the sample loading solution into an ion chromatography system, using KOH solution as the eluent for gradient elution to separate phosphate ions from other anions, and collecting the phosphate-containing fraction; a concentration and solvent exchange step: concentrating the collected phosphate fraction by vacuum centrifugation to remove water, then adding ultrapure water and an organic solvent to prepare a phosphate solution suitable for electrospray ionization; and a mass spectrometry analysis step: using the phosphate solution as a sample solution, and performing a mass spectrometry analysis with a known δ¹⁸O₂ value. 18 Phosphate reference solutions with an O value were alternately introduced into an electrospray ionization-electrostatic field orbital trap mass spectrometer for mass spectrometry analysis in negative ion mode to collect target fragment ions P. 16 O3 - and P 18 O 16 O2 - Mass spectrometry signal; isotope ratio calculation steps: based on the collected P 18 O 16 O2 - With P 16 O3- The ionic strength ratio is used to calculate the oxygen isotope shift of the sample relative to the reference solution; correction steps: using various known δ... 18 A calibration curve was established using phosphate reference material with O value, and the oxygen isotope offset value was corrected to the VSMOW scale to obtain the phosphate oxygen isotope composition of the sample.

[0008] As a preferred embodiment of the method for determining the phosphate oxygen isotope of a single dentin based on ion chromatography-electrostatic field orbital trap mass spectrometry according to the present invention, wherein: in the acid digestion step, the concentration of the hydrochloric acid solution is 0.1 mol / L; for apatite powder standard, the digestion time is 24 hours; for dentin fossil samples, the digestion time is 48 hours.

[0009] As a preferred embodiment of the method for determining the phosphate oxygen isotope of a single dentomorph based on ion chromatography-electrostatic field orbital trap mass spectrometry according to the present invention, wherein: in the freeze-drying step, the freeze-drying conditions are: cold trap temperature -50°C, vacuum degree less than 10 Pa, and freeze-drying time of 12 hours.

[0010] As a preferred embodiment of the method for determining the phosphate oxygen isotope of a single dentin-like organism based on ion chromatography-electrostatic field orbital trap mass spectrometry according to the present invention, wherein: in the ion chromatography separation and fraction collection steps, the ion chromatography system uses an anion exchange analytical column and a hydroxide system eluent, and the gradient elution program is as follows: 0-18 minutes, KOH concentration is 12 mM; 18-19 minutes, KOH concentration linearly increases from 12 mM to 46 mM; 19-25 minutes, KOH concentration linearly decreases from 46 mM to 12 mM.

[0011] As a preferred embodiment of the method for determining the phosphate oxygen isotope of a single dentomorph based on ion chromatography-electrostatic field orbital trap mass spectrometry according to the present invention, wherein: in the ion chromatography separation and fraction collection steps, the ion chromatography system has a switchable injection mode, including a fraction collection mode and a concentration determination mode; in the fraction collection mode, the injection loop is 1 mL and the fraction collection window is 10.5-12.5 minutes; in the concentration determination mode, the injection loop is 25 μL, isocratic elution is used, and the KOH concentration is 30 mM.

[0012] As a preferred embodiment of the method for determining the phosphate oxygen isotope of a single dentomorph based on ion chromatography-electrostatic field orbital trap mass spectrometry according to the present invention, wherein: in the concentration and solvent exchange steps, the vacuum centrifugation concentration temperature is 45°C, the vacuum degree is less than 100 Pa, and the running time is 3 hours; the organic solvent is methanol; the final concentration of the phosphate solution is 50 μmol / L, and the volume ratio of water to methanol is 1:4.

[0013] As a preferred embodiment of the method for determining the phosphate oxygen isotope of a single dentin-like organism based on ion chromatography-electrostatic field orbital trap mass spectrometry according to the present invention, wherein: in the mass spectrometry analysis step, the parameters of the electrospray ionization are: negative ion mode, sheath gas flow rate 3.5 Arb, auxiliary gas flow rate 1 Arb, purge gas flow rate 1 Arb, spray voltage 2.6 kV, ion transmission tube temperature 325°C, and nebulizer gas temperature 70°C; the parameters of the electrostatic field orbital trap mass spectrometer are: full scan mode, scan range m / z 78-84, mass resolution 30,000 (at m / z 200), in-source fragmentation enabled, and in-source fragmentation energy 40 V.

[0014] Secondly, to further solve the above-mentioned technical problems, the present invention provides an analytical system for determining the phosphate oxygen isotope of a single tooth-shaped acorn phosphate group based on ion chromatography-electrostatic field orbital trap mass spectrometry, comprising: an ion chromatography separation and fraction collection module, which includes a first infusion pump, an online eluent generator, an injector, a quantitative injection loop, a guard column, an analytical column, a suppressor, a conductivity detector, and an automatic fraction collector; the outlet of the first infusion pump is connected to the inlet of the online eluent generator, the outlet of the online eluent generator is connected to the inlet of the injector, the injector is provided with a quantitative injection loop, the outlet of the injector is sequentially connected to the inlets of the guard column and the analytical column, the outlet of the analytical column is connected to the inlet of the suppressor, and the outlet of the suppressor is connected to the conductivity detector. The inlet of the conductivity detector is connected to the inlet of the automatic fraction collector; the mass spectrometry detection module includes an electrospray ion source, an ion transmission tube, an S-Lens, a quadrupole mass filter, an electrostatic orbital trap mass analyzer, a detector, a dual-channel syringe pump, and a six-way switching valve; the first and second channels of the dual-channel syringe pump are respectively connected to the first and second inlets of the six-way switching valve, the outlet of the six-way switching valve is connected to the inlet of the electrospray ion source, and the outlet of the electrospray ion source is connected in series with the ion transmission tube, S-Lens, quadrupole mass filter, electrostatic orbital trap mass analyzer, and detector; the ion chromatography separation and fraction collection module and the mass spectrometry detection module are connected via an offline manual transfer method.

[0015] The beneficial effects of this invention are as follows: This invention, through the combined use of ion chromatography and electrostatic orbital trap mass spectrometry, simultaneously solves three technical bottlenecks in existing thermal conversion elemental analysis-isotope ratio mass spectrometry (TCMS), namely, high sample consumption, cumbersome procedures prone to fractionation, mixed signals and inability to distinguish different oxygen-containing components in secondary ion mass spectrometry, and the lack of solid sample pretreatment support in pure solution electrostatic orbital trap mass spectrometry. It reduces sample consumption from milligrams to nanomoles, achieving independent high-resolution analysis of individual dentin spikes. Simultaneously, the complete separation through ion chromatography ensures high selectivity for phosphate oxygen determination, eliminates isotopic interference from non-target oxygen-containing components, and guarantees the accuracy and reliability of the analytical results. This provides a novel analytical tool for high-resolution paleotemperature reconstruction and sedimentary phosphorus cycle research. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein: Figure 1 This is a flowchart of the method for determining the phosphate oxygen isotope of a single tooth-shaped spur according to Example 1.

[0017] Figure 2 This is a schematic diagram of the electrospray ionization-electrostatic field orbital trap mass spectrometry stable isotope testing structure of the present invention in Example 1.

[0018] Figure 3 This is a schematic diagram of the configuration of the ion chromatography system used for phosphate purification and fraction collection in Example 1. Detailed Implementation

[0019] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0020] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0021] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0022] Example 1 Reference Figures 1-3 This is the first embodiment of the present invention, which provides a method for determining the phosphate oxygen isotope of a single tooth-like protozoan based on ion chromatography-electrostatic orbital trap mass spectrometry, comprising the following steps: S1: Acid digestion step: The conodont sample or apatite standard is digested with hydrochloric acid solution to obtain acid digestion solution.

[0023] Preferably, in the acid digestion step, the concentration of the hydrochloric acid solution is 0.1 mol / L.

[0024] For apatite powder standard material, the digestion time is 24 hours.

[0025] For the conodont fossil samples, the digestion time was 48 hours.

[0026] Furthermore, the conodont samples were single conodont fossils, with a sample mass of 0.01–0.05 mg. Before acid digestion, the clay minerals and organic film adhering to the surface of the conodonts were carefully removed under a stereomicroscope using a needle. The samples were then ultrasonically cleaned three times with ultrapure water (5 minutes each time) and dried in a 60°C oven for 2 hours.

[0027] Furthermore, acid digestion was carried out in 1.5 mL centrifuge tubes with the caps tightly closed and allowed to stand at room temperature (20-25°C) for digestion. For conodont fossil samples, the centrifuge tubes were gently shaken every 12 hours to promote uniform reaction and avoid local acid depletion. During digestion, the conodont shells were visibly dissolved gradually, and the final solution was white and transparent.

[0028] S2: Freeze-drying step: After diluting and filtering the acid hydrolysate, freeze-dry it to remove excess hydrochloric acid and water, and obtain the freeze-dried residue.

[0029] Preferably, in the freeze-drying step, the freeze-drying conditions are: cold trap temperature -50°C, vacuum degree less than 10 Pa, and freeze-drying time of 12 hours.

[0030] Specifically, after the acid digestion is completed, 1.5 mL of ultrapure water is added to the centrifuge tube to dilute the acid digestion solution. Then, the solution is filtered through a 0.22 μm needle filter to remove insoluble residues. The filtrate is collected and transferred to a lyophilization bottle, which is then placed in a freeze dryer for lyophilization.

[0031] Furthermore, the residue inside the lyophilized tube is a white solid powder (mainly phosphates and trace salts). The lyophilized bottle should be sealed and stored to prevent moisture absorption.

[0032] S3: Reconstitution step: Add ultrapure water to the lyophilized residue to dissolve the residue and obtain the solution to be loaded.

[0033] Preferably, 1.3 mL of ultrapure water (18.2 MΩ·cm) is added to the freeze-dried residue, and the residue is shaken thoroughly to completely dissolve it.

[0034] Furthermore, the resistivity of ultrapure water is 18.2 MΩ·cm, and the total organic carbon (TOC) is ≤1.8 ppb.

[0035] Specifically, the solution to be loaded after redissolved is transferred to an ion chromatography autosampler vial and placed on the autosampler of the ion chromatography system for later use.

[0036] S4: Ion Chromatography Separation and Fraction Collection Steps: Inject the sample solution into the ion chromatography system and perform gradient elution with KOH solution as the eluent to separate phosphate ions from other anions, and collect the fraction containing phosphate.

[0037] Preferably, in the ion chromatography separation and fraction collection steps, the ion chromatography system uses an anion exchange analytical column and a hydroxide ion system eluent, with the gradient elution program as follows: The KOH concentration was 12 mM for 0-18 minutes.

[0038] Within 18–19 minutes, the KOH concentration increased linearly from 12 mM to 46 mM.

[0039] Within 19–25 minutes, the KOH concentration decreased linearly from 46 mM to 12 mM.

[0040] Specifically, in the ion chromatography separation and fraction collection steps, the ion chromatography system has switchable injection modes, including fraction collection mode and concentration determination mode.

[0041] In fraction collection mode, the sample injection loop is 1 mL, and the fraction collection window is 10.5–12.5 minutes.

[0042] In concentration determination mode, the injection loop is 25 μL, isocratic elution is used, and the KOH concentration is 30 mM.

[0043] Furthermore, the ion chromatography system employed a Thermo Scientific Dionex ICS-6000 ion chromatograph, equipped with an EGC 500 KOH online eluent generator, an ADRS 600 anion dynamic suppressor, and an AS-AP autosampler; the flow rate of the first infusion pump was 1.5 mL / min; the suppressor current was 146 mA in gradient elution mode and 112 mA in isocratic elution mode; the conductivity detector was activated, and the detection signal output range was set to 0–100 μS.

[0044] Furthermore, the start and end times of the collection window should be fine-tuned according to the position of the phosphate peak in the actual chromatogram. The adjustment principle is as follows: start collecting 0.2 minutes before the phosphate peak rises and end collecting 0.2 minutes after the phosphate peak has completely fallen back to the baseline to ensure quantitative collection of all phosphate components; the outflow volume in the collection window is approximately 3-4.5 mL.

[0045] S5: Concentration and Solvent Exchange Steps: The collected phosphate fraction is concentrated by vacuum centrifugation to remove water, and then ultrapure water and organic solvent are added to prepare a phosphate solution suitable for electrospray ionization.

[0046] Specifically, in the concentration and solvent exchange steps, the vacuum centrifugation concentration temperature is 45°C, the vacuum degree is less than 100Pa, and the running time is 3 hours. The organic solvent is methanol; The final concentration of the phosphate solution was 50 μmol / L, and the volume ratio of water to methanol was 1:4.

[0047] Furthermore, before vacuum centrifugation concentration, the total amount of phosphate in the collected fraction is calculated based on the phosphate concentration measured in the concentration measurement mode; the corresponding volume of 1 mol / L sodium hydroxide solution is added to the collection tube to adjust the pH of the solution to 7.0, so that the phosphate is completely converted into sodium dihydrogen phosphate; then the fraction containing an integer multiple of 25 nmol of phosphate is transferred from the collected fraction to a new 15 mL centrifuge tube for concentration.

[0048] Further, after drying, 100 μL of ultrapure water was added to the white solid residue in the centrifuge tube, and the residue was gently shaken to completely dissolve it; then LC-MS grade methanol (purity ≥99.9%) was added to dilute the solution to a final phosphate concentration of 50 μmol / L.

[0049] S6: Mass spectrometry analysis procedure: Use phosphate solution as the sample solution, and analyze it with a known δ... 18 Phosphate reference solutions with an O value were alternately introduced into an electrospray ionization-electrostatic field orbital trap mass spectrometer for mass spectrometry analysis in negative ion mode to collect target fragment ions P. 16 O3 - and P 18 O 16 O2 - The mass spectrometry signal.

[0050] Specifically, in the mass spectrometry analysis procedure, the parameters for electrospray ionization are: negative ion mode, sheath gas flow rate 3.5 Arb, auxiliary gas flow rate 1 Arb, purge gas flow rate 1 Arb, spray voltage 2.6 kV, ion transfer tube temperature 325°C, and nebulizer gas temperature 70°C.

[0051] The parameters of the electrostatic field orbital trap mass spectrometer are as follows: full scan mode, scan range m / z 78-84, mass resolution 30,000 (at m / z 200), source fragmentation enabled, source fragmentation energy 40 V.

[0052] Furthermore, the phosphate-methanol mixed solution prepared in step S5 is transferred to a mass spectrometry injection vial as the sample solution and placed in the first channel of a dual-channel syringe pump; three known δ-type solutions are then prepared. 18 The phosphate reference solutions with an O value (ICIER-PO1, USGS 80, B2207) were prepared using the same process steps S1 to S5, and finally a 50 μmol / L water-methanol mixed solution was prepared as a reference solution and placed in the second channel of the dual-channel syringe pump.

[0053] Furthermore, the sample solution and reference solution are alternately introduced via a six-way switching valve according to the following timing sequence: 0-10 minutes, the valve is in the first working position, connecting the sample channel; 10-20 minutes, the valve switches to the second working position (transitional flushing stage), and the reference solution enters the ion source; 20-30 minutes, the valve remains in the second working position, and mass spectrometry data of the reference signal are acquired; 30-40 minutes, the valve switches back to the first working position (transitional flushing stage); 40-50 minutes, the valve remains in the first working position, and mass spectrometry data of the sample signal are acquired; 50-60 minutes, the valve switches back to the first working position (transitional flushing stage); 60-70 minutes, the valve remains in the first working position, and mass spectrometry data of the sample signal are acquired. At least three data blocks are acquired for each sample and reference solution.

[0054] S7: Isotope ratio calculation steps: Based on the collected P... 18 O 16 O2 - With P 16 O3 - The ionic strength ratio is used to calculate the oxygen isotope shift of the sample relative to the reference solution.

[0055] Specifically, the raw mass spectrometry data (.RAW file) acquired during the mass spectrometry analysis step is imported into data extraction software (Xcalibur software) to identify and extract the target ion P. 16 O3 - (m / z≈78.9) and P 18 O 16 O2 -The chromatographic peak area integral value (m / z≈80.9) was obtained; the extracted data was imported into IsoX software, and after peak alignment, background subtraction, and smoothing, the net ion count of each target ion was output; the data file output by IsoX software was uploaded to the Iso-Orbi Web data analysis platform (version 0.53), and the platform automatically calculated the P of each data block. 18 O 16 O2 - / P 16 O3 - Ion intensity ratio (R): Calculate the average ratio of the sample data block and the adjacent reference data block, and substitute it into the following formula to calculate the original oxygen isotope shift value: δ 18 O PO3, raw = (R sample / R reference ) - 1 Furthermore, P 16 O3 - The precise mass-to-charge ratio is approximately 78.9, P 18 O 16 O2 - The accurate mass-to-charge ratio is approximately 80.9, and baseline separation is achieved between the two in the Orbitrap mass analyzer.

[0056] S8: Correction steps: Utilizing multiple known δ 18 A calibration curve was established using phosphate reference material with O value, and the oxygen isotope offset value was corrected to the VSMOW scale to obtain the phosphate oxygen isotope composition of the sample.

[0057] Specifically, within the same analytical sequence, for three known δ... 18 Phosphate reference solutions (ICIER-PO1, USGS 80, B2207) with O values ​​were measured according to steps S6 and S7, respectively, to obtain their original δ values. 18 O PO3,raw Value; the known VSMOW scaling values ​​(δ) of the three reference materials. 18 O VSMOW,known A three-point correction equation was established by performing linear regression fitting between the measured original values ​​and the actual values. δ 18 O VSMOW = a × δ 18 O PO3,raw + b Where a is the regression slope and b is the regression intercept, both obtained through least squares linear regression; the original δ of the unknown sample... 18 O PO3,raw Substituting the value into the above correction equation, the VSMOW scaling calibration value (δ) of the sample is calculated. 18 OVSMOW,calibrated The final phosphate oxygen isotope composition of the sample is denoted as .

[0058] Furthermore, for the determination of different types of samples, select the values ​​that correspond to the expected δ values ​​of the sample. 18 The reference solution with the closest O value was used for matching: ICIER-PO1 was used as the reference solution when determining Qinghu apatite; USGS 80 was used as the reference solution when determining Durango apatite and dog tooth samples; and B2207 was used as the reference solution when determining shark tooth, NBS 120C apatite, and tooth-shaped spike samples.

[0059] Furthermore, each analytical sequence must include the following quality control measures: (A) Recovery control: When transferring integer multiples of 25 nmol phosphate, simultaneously record the total amount of phosphate collected and the actual amount of phosphate transferred, ensuring that the transferred amount does not exceed the total collected amount; (B) Isotope fractionation verification: For each analytical sequence, at least one apatite standard material with the same matrix as the sample should be analyzed in parallel throughout the entire process, and its measured value should be compared with the known reference value, with a deviation not exceeding 0.5‰; (C) Repeatability verification: Each sample should be analyzed at least 2-3 times independently throughout the entire process, and the average value and standard deviation (1SD) of the multiple repeated measurements should be reported, with the standard deviation ≤ 0.5‰.

[0060] In summary, this invention, through ion chromatography-electrostatic orbital trap mass spectrometry (ECOSOL), simultaneously addresses three technical bottlenecks in existing thermal conversion elemental analysis-isotope ratio mass spectrometry (TCMS), namely, high sample consumption, cumbersome procedures prone to fractionation, mixed signals and inability to distinguish different oxygen-containing components in secondary ion mass spectrometry, and the lack of solid sample pretreatment support in pure solution EMS. It reduces sample consumption from milligrams to nanomoles, enabling independent high-resolution analysis of individual dentin spikes. Furthermore, the complete separation achieved by ion chromatography ensures high selectivity for phosphate oxygen determination, eliminates isotopic interference from non-target oxygen-containing components, and guarantees the accuracy and reliability of the analytical results. This provides a novel analytical tool for high-resolution paleotemperature reconstruction and sedimentary phosphorus cycle research.

[0061] Example 2, an embodiment of the present invention, provides a system for determining the phosphate oxygen isotope of a single tooth-shaped acorn phosphate molecule based on ion chromatography-electrostatic field orbital trap mass spectrometry. The system includes an ion chromatography separation and fraction collection module, comprising a first infusion pump, an online eluent generator, an injector, a quantitative injection loop, a guard column, an analytical column, a suppressor, a conductivity detector, and an automatic fraction collector. The outlet of the first infusion pump is connected to the inlet of the online eluent generator, and the outlet of the online eluent generator is connected to the inlet of the injector. The injector contains a quantitative injection loop. The outlet of the injector is sequentially connected to the inlets of the guard column and the analytical column, the outlet of the analytical column is connected to the inlet of the suppressor, and the outlet of the suppressor is connected to the inlet of the conductivity detector. The conductivity detector outlet is connected to the inlet of the automatic fraction collector; the mass spectrometry detection module includes an electrospray ion source, an ion transmission tube, an S-Lens, a quadrupole mass filter, an electrostatic orbital trap mass analyzer, a detector, a dual-channel syringe pump, and a six-way switching valve; the first and second channels of the dual-channel syringe pump are respectively connected to the first and second inlets of the six-way switching valve, and the outlet of the six-way switching valve is connected to the inlet of the electrospray ion source; the outlet of the electrospray ion source is connected in series with the ion transmission tube, S-Lens, quadrupole mass filter, electrostatic orbital trap mass analyzer, and detector; the ion chromatography separation and fraction collection module is connected to the mass spectrometry detection module via offline manual transfer.

[0062] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for determining the phosphate oxygen isotope of a single tooth-like acorn based on ion chromatography-electrostatic field orbital trap mass spectrometry, characterized in that: include: Acid digestion step: The conodont sample or apatite standard is digested with hydrochloric acid solution to obtain acid digestion solution; Freeze-drying step: The acid hydrolysate is diluted and filtered, and then freeze-dried to remove excess hydrochloric acid and water, resulting in freeze-dried residue; Reconstitution step: Add ultrapure water to the lyophilized residue to dissolve the residue and obtain the solution to be loaded. Ion chromatography separation and fraction collection steps: The sample solution is injected into the ion chromatography system, and gradient elution is performed using KOH solution as the eluent to separate phosphate ions from other anions, and the fraction containing phosphate is collected. Concentration and solvent exchange steps: The collected phosphate fraction is concentrated by vacuum centrifugation to remove water, and then ultrapure water and organic solvent are added to prepare a phosphate solution suitable for electrospray ionization. Mass spectrometry analysis steps: Use the phosphate solution as the sample solution, and react it with a known δ... 18 Phosphate reference solutions with an O value were alternately introduced into an electrospray ionization-electrostatic field orbital trap mass spectrometer for mass spectrometry analysis in negative ion mode to collect target fragment ions P. 16 O3 - and P 18 O 16 O2 - The mass spectrometry signal; Steps for calculating isotope ratios: Based on the collected P... 18 O 16 O2 - With P 16 O3 - The ratio of ionic strength is used to calculate the oxygen isotope shift of the sample relative to the reference solution. Correction steps: Utilizing multiple known δ 18 A calibration curve was established using phosphate reference material with O value, and the oxygen isotope offset value was corrected to the VSMOW scale to obtain the phosphate oxygen isotope composition of the sample.

2. The method for determining the phosphate oxygen isotope of a single tooth-like acorn based on ion chromatography-electrostatic field orbital trap mass spectrometry as described in claim 1, characterized in that: In the acid hydrolysis digestion step, the concentration of the hydrochloric acid solution is 0.1 mol / L; For apatite powder standard material, the digestion time is 24 hours; For the conodont fossil samples, the digestion time was 48 hours.

3. The method for determining the phosphate oxygen isotope of a single tooth-like acorn based on ion chromatography-electrostatic field orbital trap mass spectrometry as described in claim 1, characterized in that: In the freeze-drying step, the freeze-drying conditions are: cold trap temperature -50°C, vacuum degree less than 10 Pa, and freeze-drying time of 12 hours.

4. The method for determining the phosphate oxygen isotope of a single tooth-like acorn based on ion chromatography-electrostatic field orbital trap mass spectrometry as described in claim 1, characterized in that: In the ion chromatography separation and fraction collection steps, the ion chromatography system uses an anion exchange analytical column and a hydroxide ion system eluent, with the gradient elution program as follows: 0–18 minutes, KOH concentration is 12 mM; Within 18–19 minutes, the KOH concentration increased linearly from 12 mM to 46 mM; Within 19–25 minutes, the KOH concentration decreased linearly from 46 mM to 12 mM.

5. The method for determining the phosphate oxygen isotope of a single tooth-like acorn based on ion chromatography-electrostatic field orbital trap mass spectrometry as described in claim 1, characterized in that: In the ion chromatography separation and fraction collection steps, the ion chromatography system has switchable injection modes, including a fraction collection mode and a concentration determination mode. In the fraction collection mode, the sample injection loop is 1 mL, and the fraction collection window is 10.5–12.5 minutes. In the concentration determination mode, the injection loop is 25 μL, isocratic elution is used, and the KOH concentration is 30 mM.

6. The method for determining the phosphate oxygen isotope of a single tooth-like acorn based on ion chromatography-electrostatic field orbital trap mass spectrometry as described in claim 1, characterized in that: In the concentration and solvent exchange steps, the vacuum centrifugation concentration is carried out at a temperature of 45°C, a vacuum degree of less than 100 Pa, and a running time of 3 hours. The organic solvent is methanol; The final concentration of the phosphate solution is 50 μmol / L, and the volume ratio of water to methanol is 1:

4.

7. The method for determining the phosphate oxygen isotope of a single tooth-like acorn based on ion chromatography-electrostatic field orbital trap mass spectrometry as described in claim 1, characterized in that: In the mass spectrometry analysis step, the parameters of the electrospray ionization are: negative ion mode, sheath gas flow rate 3.5 Arb, auxiliary gas flow rate 1 Arb, purge gas flow rate 1 Arb, spray voltage 2.6 kV, ion transmission tube temperature 325°C, and nebulizing gas temperature 70°C. The parameters of the electrostatic field orbital trap mass spectrometer are as follows: full scan mode, scan range m / z 78-84, mass resolution 30,000 (at m / z 200), source fragmentation enabled, source fragmentation energy 40 V.

8. A system for determining phosphate oxygen isotopes in single dentin-like cells based on ion chromatography-electrostatic orbital trap mass spectrometry, and a method for determining phosphate oxygen isotopes in single dentin-like cells based on ion chromatography-electrostatic orbital trap mass spectrometry as described in any one of claims 1 to 7, characterized in that: include, An ion chromatography separation and fraction collection module includes a first infusion pump, an online eluent generator, an injector, a quantitative injection loop, a guard column, an analytical column, a suppressor, a conductivity detector, and an automatic fraction collector. The outlet of the first infusion pump is connected to the inlet of the online eluent generator, the outlet of the online eluent generator is connected to the inlet of the injector, the injector is equipped with a quantitative injection loop, the outlet of the injector is sequentially connected to the inlets of the guard column and the analytical column, the outlet of the analytical column is connected to the inlet of the suppressor, the outlet of the suppressor is connected to the inlet of the conductivity detector, and the outlet of the conductivity detector is connected to the inlet of the automatic fraction collector. The mass spectrometry detection module includes an electrospray ionization source, an ion transmission tube, an S-Lens, a quadrupole mass filter, an electrostatic orbital trap mass analyzer, a detector, a dual-channel syringe pump, and a six-way switching valve. The first and second channels of the dual-channel syringe pump are respectively connected to the first and second inlets of the six-way switching valve. The outlet of the six-way switching valve is connected to the inlet of the electrospray ionization source. The outlet of the electrospray ionization source is connected in series with the ion transmission tube, the S-Lens, the quadrupole mass filter, the electrostatic orbital trap mass analyzer, and the detector. The ion chromatography separation and fraction collection module and the mass spectrometry detection module are connected via an offline manual transfer method.

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