A sample preparation system and method for c-14 analysis in biological samples

Abstract

The present application relates to a kind of sample preparation system and method for C-14 analysis in biological sample, connect with the gas discharge of element analyzer or oxidizing combustion instrument through first interface, then sequentially connect CO2 purification and collection unit, measurement unit through pipeline, after the CO2 gas generated when the sample C analysis of element analyzer is collected, purified, transfer into measurement tube, measure the amount of CO2 collected, again transfer to reduction tube, for preparing graphite target sample to be measured accelerator mass spectrometry.The system provided by the present application is simple in structure, and strong in operability, and the target sample in carbon-14 accelerator mass spectrometry can be prepared by combining the method provided by the present application.The system provided by the present application is fixed on stainless steel structure, and can be assembled into modular mobile device.Based on the system and method provided by the present application, the application of oxidizing combustion furnace or element analyzer can be expanded.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical analysis technology, specifically relating to a sample preparation system and method for C-14 analysis in biological samples. Background Technology

[0002] Radiolabeled drug metabolism studies play a crucial role in new drug development. They provide information on drug absorption (A), metabolism (D), tissue distribution (M), and excretion (E), and are irreplaceable in the evaluation of metabolite safety and mass balance studies of new drugs. Low-energy... 14 C and 3 H is a commonly used radionuclide in drug research, with over 90% of drugs utilizing it. 14 C is used for labeling. After subjects or experimental animals are given a certain dose of the radiolabeled drug, plasma, whole blood, urine, and fecal samples are collected within a certain experimental period, and then the biological samples are analyzed. 14 C content analysis, based on urine and fecal excretion results, yielded... 14 C recovery rate, pharmacokinetic curves were obtained based on plasma and whole blood results.

[0003] Currently commonly used biological samples 14 The analytical method for C is liquid scintillation counting (LSC). For radioactive, clear liquid samples such as plasma, urine, and bile, the sample can be directly mixed with the scintillation fluid for detection. However, for biological samples such as whole blood and feces, an oxidative combustion analyzer is needed to convert the carbon components in the sample into CO2 gas, which is then collected and tested. When the measurement time is 1 hour, this method is effective for... 14 The detection limit for C is 0.17 mBq. When using this method for ADME studies, the radioactive dose in the drug administered to subjects or experimental animals can reach several hundred μCi.

[0004] Accelerator mass spectrometry (AMS) is an ultrasensitive nuclear analysis technique that can also be used for biological samples. 14 Analysis of C content, especially trace amounts 14 The analysis of C has a detection limit 300 to 1000 times lower than that of LSC technology, resulting in a radioactive dose of <1 μCi in the drug administered to subjects or experimental animals, significantly reducing the radiation dose received by subjects or experimental animals. Accelerator mass spectrometry is used for... 14 When performing carbon analysis, the carbon components in the sample need to be converted into a form that can be measured by AMS. The common method is to first convert the carbon components in the sample into CO2 gas through high-temperature oxidation, and then convert the CO2 gas into graphite through high-temperature reduction.

[0005] Currently, commonly used AMS sample preparation methods include two steps: high-temperature oxidation of CuO and high-temperature reduction based on Zn-Fe, Zn-TiH2-Fe, or H2-Fe. All reactions must be carried out under vacuum conditions. After the high-temperature oxidation step is completed, the generated gas must be purified by a secondary cold trap before the high-temperature reduction reaction is carried out.

[0006] Domestically, Northwest University's invention patent, "Organic Molecular Carbon-14 Dating Sample Preparation System and Method," employs a hydrogen synthesis method for graphite. Primarily designed for trace organic samples, the system comprises three independent test benches for carbon dioxide preparation, purification, and graphite synthesis, with laser heating used for the latter. The Institute of Geology and Geophysics, Chinese Academy of Sciences, has an invention patent, "A Graphite Sample Preparation System and Method for Accelerator Mass Spectrometry," which divides the system into a CO2 preparation unit and a CO2 purification and graphite preparation unit, equipped with an automated control unit. Utilizing a hydrogen synthesis method, it achieves fully automated control of the entire graphite preparation process.

[0007] Internationally, the Swiss company Ionplus has developed a dedicated device, model AGE3, which uses iron powder catalyst for hydrogen synthesis. It integrates an elemental analyzer with a graphite synthesis unit, enabling sample combustion, collection, purification, and reduction. The system costs approximately 3 million RMB. The graphitization system from the American company NEC also uses hydrogen synthesis, but adds a measurement step after carbon dioxide preparation. Its maximum reaction temperature is approximately 700°C, and the system costs approximately 2.8 million RMB. The graphitization synthesis system developed by the Japan Atomic Energy Agency uses zinc reduction, with a maximum reaction temperature of approximately 850°C.

[0008] In studies of radiolabeled drug metabolism, especially in human clinical trials, it is necessary to process nearly 800 biological samples within a week. The sample processing throughput of existing graphite sample preparation systems is severely insufficient. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the present invention aims to provide a sample preparation system and method for C-14 analysis in biological samples. The CO2 purification unit is configured as an independent device, organically integrated with elemental analyzers, oxidative combustion analyzers, etc., through a variety of interface configurations. CO2 gas generated by the elemental analyzer or oxidative combustion analyzer is collected by freezing and transferred to reduction tubes. These reduction tubes can be batch-placed in a muffle furnace for high-temperature reduction reactions, thereby achieving… 14 Rapid preparation of C-AMS measurement samples significantly improves sample processing throughput. The method described in this invention can be used for biological samples in biopharmaceutical research. 14 C content analysis: The system described can be promoted to domestic systems with the capability to perform C content analysis. 14 A laboratory with C analytical capabilities.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows: a sample preparation system for C-14 analysis in biological samples, the system comprising: connected sequentially via pipes;

[0011] The first interface, the first end of the first interface is connected to the gas exhaust port of the elemental analyzer or the oxidation combustion instrument via a pipe;

[0012] The CO2 purification and collection unit includes, in sequence, a first tee port, a first cold trap, a second tee port, a second cold trap, and a third cold trap, all connected by pipes. Specifically, the first end of the first tee port is connected to the second end of the first port via a pipe; the second end of the first tee port is connected to the first end of the first cold trap via a pipe equipped with a second valve; and the third end of the first tee port is connected to the atmosphere via a pipe equipped with a first valve. The second end of the first cold trap is connected to the first end of the second tee port via a pipe equipped with a third valve; the second end of the second tee port is connected to the first end of the second cold trap via a pipe equipped with a fifth valve; and the third end of the second tee port is connected to the atmosphere via a pipe equipped with a fourth valve. The second end of the second cold trap is connected to the first end of the third cold trap via a pipe equipped with a sixth valve.

[0013] The measuring unit includes a measuring tube, a four-way connector, a first vacuum gauge, a third three-way connector, and a second vacuum gauge. The second end of the third cold trap is connected to the first end of the four-way connector via a pipe equipped with a seventh valve. The second end of the four-way connector is connected to the first end of the third three-way connector via a pipe equipped with an eighth valve. The third end of the four-way connector is connected to the first vacuum gauge, and the fourth end of the four-way connector is connected to the measuring tube. The second end of the third three-way connector is connected to a mechanical pump via a pipe sequentially equipped with a second vacuum gauge and a tenth valve. The third end of the third three-way connector is connected to the first end of the second connector via a pipe equipped with a ninth valve. The second end of the second connector is connected to a reduction tube.

[0014] Furthermore, the system also includes a liquid nitrogen cup for placing below the first cold trap, the third cold trap, the measuring tube, or the reduction tube to collect CO2.

[0015] Furthermore, the system also includes a dry ice-alcohol cup, which is placed below the second cold trap to remove impurity gases.

[0016] Furthermore, the first cold trap consists of multiple sets of first cold traps connected in parallel to the pipeline to increase the sample processing throughput.

[0017] The present invention also provides a sample preparation method for C-14 analysis in biological samples, the method being implemented based on the sample preparation system for C-14 analysis in biological samples, comprising the following steps:

[0018] S1. Prepare the reduction tube: The reduction tube includes a large quartz test tube and a small quartz test tube nested inside the large quartz test tube;

[0019] S2. Connect the system and check its airtightness: Connect the elemental analyzer to the CO2 purification and collection unit through the first interface, and connect the CO2 purification and collection unit to the reduction tube through the second interface. Close the second valve and the fourth valve, and open the other valves in the system. Ensure that the gas exhaust port of the elemental analyzer is connected to the first interface, the first valve is connected to the atmosphere, and the pipelines and components after the second valve are connected to the mechanical pump. Then, turn on the mechanical pump to check the airtightness of the system.

[0020] S3. CO2 Collection: When the elemental analyzer starts analyzing sample C, close the first and fifth valves, open the second, third, and fourth valves, and immerse the first cold trap in the liquid nitrogen cup to start collecting CO2 gas; after the carbon content analysis is completed, open the first valve, close the second and fourth valves, and stop collecting.

[0021] S4. Purify CO2 gas: Open the fifth valve and evacuate the pipeline and components between the second and fifth valves to keep the first cold trap completely immersed in the liquid nitrogen cup. CO2 remains in a solid state. When the vacuum degree of the second vacuum gauge is less than 0, the carrier gas He gas in the system is removed to achieve preliminary purification of CO2 gas. Then close the eighth valve.

[0022] The second and third cold traps are placed in a dry ice-alcohol cup and a liquid nitrogen cup, respectively. The liquid nitrogen cup at the first cold trap is removed. At room temperature, the solid CO2 in the first cold trap is converted into gaseous CO2 and diffuses towards the direction of lower pressure, passing through the second and third cold traps in sequence. The second cold trap is used to remove easily liquefiable impurities, and the third cold trap is used to collect the CO2 gas after the removal of easily liquefiable impurities. After the CO2 gas is transferred to the third cold trap, when the reading of the first vacuum gauge rises to a stable level, the eighth valve is opened. The second and third cold traps are still placed in the dry ice-alcohol cup and the liquid nitrogen cup, respectively. A mechanical pump is connected to evacuate the vacuum to remove low-boiling-point impurities. When the vacuum degree of the first vacuum gauge is less than 0, the eighth valve is closed.

[0023] S5. Transfer CO2 gas into the measuring tube: Remove the liquid nitrogen cup at the third cold trap, immerse the lower end of the measuring tube in the liquid nitrogen cup, and the solid CO2 at the third cold trap at room temperature will turn into gaseous CO2. After diffusing into the measuring tube, it will turn into solid CO2 again. The liquid nitrogen cup will rise slowly and stop at the top of the measuring tube. When the reading of the first vacuum gauge is stable, open the eighth valve and observe the reading of the second vacuum gauge. When the vacuum degree of the second vacuum gauge is less than 0, close the seventh and eighth valves and remove the liquid nitrogen cup below the measuring tube.

[0024] S6. Transfer CO2 gas to the reduction tube: Close the tenth valve and open the eighth valve. The CO2 gas diffuses towards the direction of lower pressure, and the reading of the second vacuum gauge rises. Immerse the lower end of the reduction tube in the liquid nitrogen cup and slowly raise it to 1 / 3 of the way from the top of the reduction tube. The CO2 turns into a solid in the reduction tube. When the vacuum reading of the second vacuum gauge stabilizes, close the ninth valve and keep 1 / 2 of the reduction tube immersed in the liquid nitrogen cup. Use a flame gun to melt and seal the reduction tube.

[0025] S7. Preparation of graphite target for accelerator mass spectrometry: After sealing the reduction tube, it is placed in a muffle furnace for high-temperature reduction reaction to generate graphite target for accelerator mass spectrometry.

[0026] Furthermore, in step S1, the bottom of the large quartz test tube contains 30 mg of zinc granules and 12 mg of titanium hydride powder, while the small quartz test tube contains 3.5 mg of 325 mesh iron powder.

[0027] Furthermore, in step S2, the carrier gas He gas is discharged from the fourth valve.

[0028] Furthermore, in step S3, the elemental analyzer oxidation combustion process includes sample injection, N analysis, C analysis, and S analysis, with prompts provided for each step of the analysis process.

[0029] Furthermore, in step S4, the easily liquefiable impurity gas includes water vapor and sulfur dioxide; the low-boiling-point impurity gas includes nitrogen, oxygen, and helium.

[0030] Furthermore, in step S5, after the CO2 in the measuring tube is converted into gas, the reading of the first vacuum gauge is read.

[0031] Furthermore, the method also includes the following steps:

[0032] S8. Replace the reduction tube and prepare the next sample: Close the second and fourth valves, confirm that the ninth valve has been closed in step S6, replace the reduction tube, and open all valves in the system except for the second and fourth valves; evacuate the system again and prepare the next sample.

[0033] The beneficial effects of the present invention are as follows: The sample preparation system and method for C-14 analysis in biological samples provided by the present invention are connected to a CO2 purification and collection unit and a measurement unit in sequence through a first interface connected to the gas emission port of an elemental analyzer or an oxidizing combustion instrument. The CO2 gas generated during the C analysis of the sample by the elemental analyzer is collected, purified, and then transferred into a measurement tube to measure the amount of collected CO2. The CO2 is then transferred to a reduction tube for the preparation of graphite accelerator mass spectrometry target samples.

[0034] Compared with existing technologies, the main advantages of this invention are that the system provided by this invention has a simple structure and is highly operable. Combined with the method provided by this invention, it can be used to prepare target samples for carbon-14 accelerator mass spectrometry analysis. The system provided by this invention can be fixed to a stainless steel structure and assembled into a modular mobile device. Based on the system and method provided by this invention, applications in oxidation combustion furnaces or elemental analyzers can be expanded. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the sample preparation system for C-14 analysis in biological samples as described in this invention;

[0036] Figure 2 This is a schematic diagram of a sample preparation method for C-14 analysis in biological samples provided by the present invention;

[0037] In the diagram, 101—first interface, 102—second interface, 201—first valve, 202—second valve, 203—third valve, 204—fourth valve, 205—fifth valve, 206—sixth valve, 207—seventh valve, 208—eighth valve, 209—ninth valve, 210—tenth valve, 301—first cold trap, 302—second cold trap, 303—third cold trap, 401—first vacuum gauge, 402—second vacuum gauge. Detailed Implementation

[0038] The technical solutions in the embodiments of the present invention will be further clearly and completely described below with reference to the accompanying drawings and examples. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0039] It should be noted that in the description of the embodiments of the present invention, the terms "upper," "lower," "front," "rear," "front," "back," "left," "right," "top," "bottom," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the present invention. In addition, the terms "first," "second," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0040] Among existing high-temperature reduction methods using CO2 gas (Zn-Fe method, Zn-TiH2-Fe method, H2-Fe method), the Zn-Fe method is highly sensitive to temperature changes and requires two furnaces with different temperatures; the H2-Fe method requires high-purity H2, which is a flammable and explosive gas, posing high safety requirements for the laboratory; in comparison, the experimental conditions required for the Zn-TiH2-Fe method are more suitable for biological samples. 14 C-AMS sample preparation.

[0041] The Swiss company Ionplus invented an accelerator mass spectrometry gas introduction system that, when coupled with an elemental analyzer and an AMS, can achieve the extraction of CO2 gas samples. 14 The system analyzes carbon content, enabling rapid, high-throughput measurement of biological samples; however, it costs approximately 3.4 million RMB and requires professional operation.

[0042] Some domestic pharmacokinetics laboratories or environmental monitoring laboratories have this capability. 14 The ability to perform C liquid scintillation counting analysis is also commonly found in systems equipped with oxidative combustion analyzers or elemental analyzers. However, in current research on the metabolism of radiolabeled drugs, especially in human clinical trials, it is necessary to process nearly 800 biological samples within a week, and the sample processing throughput of existing graphite sample preparation systems is severely insufficient. To address this issue, this invention sets up a CO2 purification unit as an independent device, organically integrating it with elemental analyzers, oxidative combustion analyzers, etc., through a rich array of interface configurations. CO2 gas generated by the elemental analyzer or oxidative combustion analyzer is collected by freezing and transferred to reduction tubes. These reduction tubes can then be placed in batches in a muffle furnace for high-temperature reduction reactions, achieving… 14 Rapid preparation of samples for C-AMS measurement; this method can be used for biological samples in biopharmaceutical research. 14 C content analysis: This device can be promoted to domestic manufacturers with the capability. 14 A laboratory with C analytical capabilities.

[0043] like Figure 1As shown, an embodiment of the present invention provides a sample preparation system for C-14 analysis in biological samples, the system comprising, from left to right, connected by pipes:

[0044] The first interface 101, the first end of the first interface 101 is connected to the gas exhaust port of the elemental analyzer or the oxidation combustion instrument through a pipe;

[0045] The CO2 purification and collection unit, from left to right, includes a first tee port, a first cold trap 301, a second tee port, a second cold trap 302, and a third cold trap 303 connected by pipes. The first end of the first tee port is connected to the second end of the first port 101 via a pipe; the second end of the first tee port is connected to the first end of the first cold trap 301 via a pipe equipped with a second valve 202; and the third end of the first tee port is connected to the atmosphere via a pipe equipped with a first valve 201. The second end of the first cold trap 301 is connected to the first end of the second tee port via a pipe equipped with a third valve 203; the second end of the second tee port is connected to the first end of the second cold trap 302 via a pipe equipped with a fifth valve 205; and the third end of the second tee port is connected to the atmosphere via a pipe equipped with a fourth valve 204. The second end of the second cold trap 302 is connected to the first end of the third cold trap 303 via a pipe equipped with a sixth valve 206.

[0046] The measuring unit includes a measuring tube, a four-way connector, a first vacuum gauge 401, a third three-way connector, and a second vacuum gauge 402. The second end of the third cold trap 303 is connected to the first end of the four-way connector via a pipe equipped with a seventh valve 207. The second end of the four-way connector is connected to the first end of the third three-way connector via a pipe equipped with an eighth valve 208. The third end of the four-way connector is connected to the first vacuum gauge 401, and the fourth end of the four-way connector is connected to the measuring tube. The second end of the third three-way connector is connected to a mechanical pump via a pipe sequentially equipped with the second vacuum gauge 402 and a tenth valve 210. The third end of the third three-way connector is connected to the first end of the second connector 102 via a pipe equipped with a ninth valve 209. The second end of the second connector 102 is connected to a reduction tube.

[0047] In one specific embodiment, the first cold trap 301 uses multiple sets of first cold traps 301 connected in parallel to the pipeline, thereby increasing the sample processing throughput.

[0048] Specifically, the system also includes a liquid nitrogen cup, which is placed below the first cold trap 301, the third cold trap 303, the measuring tube, or the reduction tube to collect CO2. When the generated CO2 gas begins to be discharged from the gas outlet of the elemental analyzer along with the carrier gas He gas, the first valve 201 and the fifth valve 205 are closed, and the second valve 202, the third valve 203, and the fourth valve 204 are opened. When the first cold trap 301 is immersed in the liquid nitrogen cup, gas collection begins. Since the temperature of liquid nitrogen is -196℃, the CO2 gas (boiling point -78.5℃) turns into a solid as it flows through this point, and the carrier gas He gas is discharged from the fourth valve 204. After the carbon content analysis is completed, the first valve 201 is opened, and the second valve 202 and the fourth valve 204 are closed to stop the collection.

[0049] In one specific embodiment, after the first cold trap 301 has collected all the gas, considering that there is still carrier gas He gas remaining in the pipeline between the second valve 202 and the fifth valve 205, the fifth valve 205 is slowly opened to evacuate the pipeline between the second valve 202 and the fifth valve 205, keeping the first cold trap 301 completely immersed in the liquid nitrogen cup, and the CO2 remains in a solid state. When the vacuum degree of the second vacuum gauge 402 is less than 0, the carrier gas He gas is evacuated, and the eighth valve 208 is closed.

[0050] Optionally, the system further includes a dry ice-alcohol cup (a mixture of dry ice and alcohol, with a co-bath point of -60 to -70°C), placed below the second cold trap 302 to remove impurity gases. The second cold trap 302 and the third cold trap 303 are placed in the dry ice-alcohol cup and the liquid nitrogen cup, respectively. The liquid nitrogen cup at the first cold trap 301 is removed. At room temperature, the solid CO2 in the first cold trap 301 is converted into gaseous CO2 and diffuses towards the direction of lower pressure, passing sequentially through the second cold trap 302 and the third cold trap 303. The second cold trap 302 is used to remove any possible impurity gases such as water vapor, and the third cold trap 303 is used to collect the CO2 gas again. This operation achieves one CO2 gas purification. During operation, the reading of the first vacuum gauge 401 will rise. When the reading of the first vacuum gauge stabilizes, the eighth valve 208 is opened. The second cold trap 302 and the third cold trap 303 are still placed in the dry ice-alcohol cup and the liquid nitrogen cup, respectively. A mechanical pump is connected to evacuate the vacuum. When the vacuum degree of the first vacuum gauge 401 is less than 0, the eighth valve 208 is closed.

[0051] Remove the liquid nitrogen cup from the third cold trap 303 and immerse the lower end of the measuring tube in the liquid nitrogen cup. At room temperature, the solid CO2 in the third cold trap 303 transforms into gaseous CO2, diffuses into the measuring tube, and transforms back into solid CO2. The liquid nitrogen cup rises slowly, stopping at the top of the measuring tube. This operation controls the generation of solid CO2 from the bottom of the measuring tube. During this operation, the reading of the first vacuum gauge 401 will first rise and then fall. When the reading of the first vacuum gauge 401 stabilizes, open the eighth valve 208 and observe the reading of the second vacuum gauge 402. When the vacuum degree of the second vacuum gauge is less than 0, close the seventh valve 207 and the eighth valve 208, and remove the liquid nitrogen cup below the measuring tube. After the CO2 in the measuring tube transforms into gas, read the reading of the first vacuum gauge 401.

[0052] like Figure 1-2 As shown, this invention also provides a sample preparation method for C-14 analysis in biological samples, implemented using the aforementioned sample preparation system for C-14 analysis in biological samples. The method includes the following steps:

[0053] S1. Prepare the reduction tube: The reduction tube includes a large quartz test tube and a small quartz test tube nested inside the large quartz test tube;

[0054] Optionally, in step S1, the bottom of the large quartz test tube contains 30 mg of zinc granules and 12 mg of titanium hydride powder, and the small quartz test tube contains 3.5 mg of 325 mesh iron powder.

[0055] Specifically, in step S1, the large quartz test tube has a diameter of 10mm and a length of 30cm, while the small quartz test tube has a diameter of 5mm and a length of 3cm.

[0056] S2. Connect the system and check its airtightness: Connect the elemental analyzer to the CO2 purification and collection unit through the first interface 101, and connect the CO2 purification and collection unit to the reduction tube through the second interface 102. Close the second valve 202 and the fourth valve 204, and open the other valves in the system (first valve 201, third valve 203, fifth valve 205, sixth valve 206, seventh valve 207, eighth valve 208, ninth valve 209, and tenth valve 210). With the gas exhaust port of the elemental analyzer connected to the first interface 101, the first valve 201 connected to the atmosphere, and the pipelines and components after the second valve 202 connected to the mechanical pump, turn on the mechanical pump to check the airtightness of the system. The vacuum levels of the first vacuum gauge and the second vacuum gauge decrease to ensure the airtightness of the system.

[0057] S3. CO2 Collection: When the elemental analyzer starts analyzing the carbon content of the sample, the generated CO2 gas begins to be discharged from the gas outlet of the elemental analyzer along with the carrier gas He. At this time, the first valve 201 and the fifth valve 205 are closed, and the second valve 202, the third valve 203, and the fourth valve 204 are opened. The first cold trap 301 is then immersed in the liquid nitrogen cup to start collecting CO2 gas. Since the temperature of liquid nitrogen is -196℃, the CO2 gas (boiling point -78.5℃) turns into a solid as it flows through this point. The carrier gas He is discharged from the fourth valve 204. After the carbon content analysis is completed, the first valve 201 is opened, and the second valve 202 and the fourth valve 204 are closed to stop the collection.

[0058] Specifically, the elemental analyzer's oxidation-combustion process includes sample introduction, N analysis, C analysis, and S analysis, with prompts for each step of the analysis process.

[0059] In a specific embodiment, the operation method for sample analysis using an elemental analyzer is as follows: ① Biological samples need to be frozen at -80°C. Before sample preparation, they should be thawed in a refrigerator at 0~4°C to restore them to a liquid state; ② A 300μL tin cup is heated at 200°C for 3 hours; ③ The treated tin cup is placed on a balance, tare it, and 100μL of biological sample is accurately transferred using a pipette to the tin cup. The sample mass is then weighed and recorded; ④ Using forceps, the sample is transferred from the tin cup to the tin cup... Starting from the middle of the cup, clamp the upper half of the tin cup tightly. This operation can reduce the air inside the tin cup. Then roll the tin cup from top to bottom twice to ensure that the sample does not leak out. ⑤ Turn on the elemental analyzer, select CNS mode, set the combustion tube temperature to 1150℃, the reduction tube temperature to 850℃, the TCD temperature to 60℃, the oxygen flow rate to 75mL / min, and the oxygen flow time to 50s. ⑥ Place the prepared sample into the sample tray of the elemental analyzer, enter the sample information, start the oxidation combustion and perform carbon content analysis.

[0060] Combustion of biological samples generates gases such as carbon oxides, nitrogen oxides, sulfur oxides, and nitrogen. Meanwhile, the elemental analyzer uses oxygen and helium during operation. During the collection of CO2 in the first cold trap 301, CO2 is converted to a solid state, and most of the impurity gases are discharged through the fourth valve 204. However, a small amount of these impurity gases may still be trapped within the CO2 solid. Therefore, in the subsequent step S4, when purifying the CO2 gas, the second cold trap 302 is placed in a dry ice-alcohol cup at a temperature between -60 and -70°C to remove easily liquefiable impurity gases such as water vapor and sulfur dioxide. The third cold trap 303 is placed in a liquid nitrogen cup, which can again convert CO2 to a solid state. Low-boiling-point impurity gases such as nitrogen, oxygen, and helium require even lower condensation temperatures (oxygen and helium have lower boiling points than liquid nitrogen, while nitrogen requires temperatures below its boiling point (i.e., liquid nitrogen temperature) to condense into liquid nitrogen). These gases remain in gaseous form when passing through the third cold trap 303, so a second vacuum is applied to remove these low-boiling-point impurity gases.

[0061] S4. Purify CO2 gas: Open the fifth valve 205, evacuate the pipeline and components between the second valve 202 and the fifth valve 205, keep the first cold trap 301 completely immersed in the liquid nitrogen cup, keep CO2 in a solid state, when the vacuum degree of the second vacuum gauge 402 is less than 0, the carrier gas He gas in the system is pumped away, realizing the initial purification of CO2 gas, and close the eighth valve 208.

[0062] To remove potential impurities such as water vapor from the system, the second cold trap 302 and the third cold trap 303 are placed in a dry ice-alcohol cup (a mixture of dry ice and alcohol, with a temperature range of -60 to -70°C) and a liquid nitrogen cup, respectively. The liquid nitrogen cup at the first cold trap 301 is removed. At room temperature, the solid CO2 at the first cold trap 301 transforms into gaseous CO2 and diffuses towards lower pressure, passing sequentially through the second cold trap 302 and the third cold trap 303. The second cold trap 302 is used to remove potentially present easily liquefiable impurities such as water vapor and sulfur dioxide; the third cold trap 303 is used to remove... The CO2 gas, after the removal of easily liquefiable impurities, is collected again, and this operation step achieves another purification of CO2 gas. During the operation, after the CO2 gas is transferred to the third cold trap 303, the reading of the first vacuum gauge 401 will rise. When the reading of the first vacuum gauge 401 stabilizes, the eighth valve 208 is opened. The second cold trap 302 and the third cold trap 303 are still placed in the dry ice-alcohol cup and the liquid nitrogen cup, respectively. A mechanical pump is connected to evacuate the vacuum to remove low-boiling-point impurities such as nitrogen, oxygen, and helium. When the vacuum degree of the first vacuum gauge 401 is less than 0, the eighth valve 208 is closed.

[0063] S5. Transfer the purified CO2 gas into the measuring tube: Remove the liquid nitrogen cup from the third cold trap 303, immerse the lower end of the measuring tube in the liquid nitrogen cup. At room temperature, the solid CO2 in the third cold trap 303 transforms into gaseous CO2, diffuses into the measuring tube, and then transforms back into solid CO2. The liquid nitrogen cup rises slowly, at a rate <5 mm / s, stopping at the top of the measuring tube. This operation controls the generation of solid CO2 from the bottom of the measuring tube. During this operation, the reading of the first vacuum gauge 401 will first rise and then fall. When the reading of the first vacuum gauge 401 stabilizes, open the eighth valve 208 and observe the reading of the second vacuum gauge 402. When the vacuum degree of the second vacuum gauge is less than 0, close the seventh valve 207 and the eighth valve 208, and remove the liquid nitrogen cup below the measuring tube. After the CO2 in the measuring tube transforms into gas, read the reading of the first vacuum gauge 401, and confirm the relationship between the gas pressure at the measuring tube and the corresponding carbon content through experiments to monitor the efficiency of CO2 transfer in the experiment.

[0064] S6. Transfer CO2 gas to the reduction tube: Close the tenth valve 210 and open the eighth valve 208. The CO2 gas diffuses towards the direction of lower pressure, and the reading of the second vacuum gauge 402 rises. Immerse the lower end of the reduction tube in the liquid nitrogen cup and slowly raise it to 1 / 3 of the distance from the top of the reduction tube. The rising speed is <5 mm / s. CO2 is converted into solid in the reduction tube, thus achieving the purpose of transferring CO2 gas to the reduction tube. When the vacuum reading of the second vacuum gauge 402 stabilizes, close the ninth valve 209 and keep 1 / 2 of the reduction tube immersed in the liquid nitrogen cup. Use a flame gun to perform a melting and sealing operation on the reduction tube.

[0065] S7. Preparation of graphite target for accelerator mass spectrometry: After sealing the reduction tube, it is placed in a muffle furnace for high-temperature reduction reaction to generate graphite target for accelerator mass spectrometry.

[0066] S8. Replace the reduction tube and prepare the next sample: Close the second valve 202 and the fourth valve 204, confirm that the ninth valve 209 has been closed in step S6, replace the reduction tube, and open all valves in the system except for the second and fourth valves (first valve 201, third valve 203, fifth valve 205, sixth valve 206, seventh valve 207, eighth valve 208, ninth valve 209, tenth valve 210); evacuate the system again and prepare the next sample.

[0067] Other embodiments of the invention will readily occur to those skilled in the art upon consideration of the specification and practice of the invention disclosed herein. This application is intended to cover any variations, uses, or adaptations of the invention that follow the general principles of the invention and include common knowledge or customary techniques in the art not disclosed herein. Thus, the invention also intends to include such variations and adaptations if they fall within the scope of the claims and their equivalents.

[0068] The above embodiments are merely illustrative examples of the present invention. The present invention may also be implemented in other specific ways or forms without departing from its spirit or essential characteristics. Therefore, the described embodiments should be considered illustrative rather than limiting in any respect. The scope of protection of the present invention should be defined by the claims, and any variations equivalent to the intent and scope of the claims should also be included within the scope of the present invention.

Claims

1. A sample preparation system for C-14 analysis in biological samples, characterized in that, The system comprises the following components connected sequentially by pipes: The first interface, the first end of the first interface is connected to the gas exhaust port of the elemental analyzer or the oxidation combustion instrument via a pipe; The CO2 purification and collection unit includes, in sequence, a first tee port, a first cold trap, a second tee port, a second cold trap, and a third cold trap, all connected by pipes. Specifically, the first end of the first tee port is connected to the second end of the first port via a pipe; the second end of the first tee port is connected to the first end of the first cold trap via a pipe equipped with a second valve; and the third end of the first tee port is connected to the atmosphere via a pipe equipped with a first valve. The second end of the first cold trap is connected to the first end of the second tee port via a pipe equipped with a third valve; the second end of the second tee port is connected to the first end of the second cold trap via a pipe equipped with a fifth valve; and the third end of the second tee port is connected to the atmosphere via a pipe equipped with a fourth valve. The second end of the second cold trap is connected to the first end of the third cold trap via a pipe equipped with a sixth valve. The measuring unit includes a measuring tube, a four-way connector, a first vacuum gauge, a third three-way connector, and a second vacuum gauge. The second end of the third cold trap is connected to the first end of the four-way connector via a pipe equipped with a seventh valve. The second end of the four-way connector is connected to the first end of the third three-way connector via a pipe equipped with an eighth valve. The third end of the four-way connector is connected to the first vacuum gauge, and the fourth end of the four-way connector is connected to the measuring tube. The second end of the third three-way connector is connected to a mechanical pump via a pipe sequentially equipped with a second vacuum gauge and a tenth valve. The third end of the third three-way connector is connected to the first end of the second connector via a pipe equipped with a ninth valve. The second end of the second connector is connected to a reduction tube.

2. The sample preparation system for C-14 analysis in biological samples according to claim 1, characterized in that, The system also includes a liquid nitrogen cup, which is placed below the first cold trap, the third cold trap, the measuring tube, or the reduction tube to collect CO2.

3. The sample preparation system for C-14 analysis in biological samples according to claim 2, characterized in that, The system also includes a dry ice-alcohol cup, which is placed below the second cold trap to remove impurity gases.

4. The sample preparation system for C-14 analysis in biological samples according to claim 1, characterized in that, The first cold trap consists of multiple sets of first cold traps connected in parallel to the pipeline to increase the sample processing throughput.

5. A sample preparation method for C-14 analysis in biological samples, characterized in that, Based on the sample preparation system for C-14 analysis in biological samples as described in claim 3, the method includes the following steps: S1. Prepare the reduction tube: The reduction tube includes a large quartz test tube and a small quartz test tube nested inside the large quartz test tube; S2. Connect the system and check its airtightness: Connect the elemental analyzer to the CO2 purification and collection unit through the first interface, and connect the CO2 purification and collection unit to the reduction tube through the second interface. Close the second valve and the fourth valve, and open the other valves in the system. Ensure that the gas exhaust port of the elemental analyzer is connected to the first interface, the first valve is connected to the atmosphere, and the pipelines and components after the second valve are connected to the mechanical pump. Then, turn on the mechanical pump to check the airtightness of the system. S3. CO2 Collection: When the elemental analyzer starts analyzing sample C, close the first and fifth valves, open the second, third, and fourth valves, and immerse the first cold trap in the liquid nitrogen cup to start collecting CO2 gas; after the carbon content analysis is completed, open the first valve, close the second and fourth valves, and stop collecting. S4. Purify CO2 gas: Open the fifth valve and evacuate the pipeline and components between the second and fifth valves to keep the first cold trap completely immersed in the liquid nitrogen cup. CO2 remains in a solid state. When the vacuum degree of the second vacuum gauge is less than 0, the carrier gas He gas in the system is removed to achieve preliminary purification of CO2 gas. Then close the eighth valve. The second and third cold traps are placed in a dry ice-alcohol cup and a liquid nitrogen cup, respectively. The liquid nitrogen cup at the first cold trap is removed. At room temperature, the solid CO2 in the first cold trap is converted into gaseous CO2 and diffuses towards the direction of lower pressure, passing through the second and third cold traps in sequence. The second cold trap is used to remove easily liquefiable impurities, and the third cold trap is used to collect the CO2 gas after the removal of easily liquefiable impurities. After the CO2 gas is transferred to the third cold trap, when the reading of the first vacuum gauge rises to a stable level, the eighth valve is opened. The second and third cold traps are still placed in the dry ice-alcohol cup and the liquid nitrogen cup, respectively. A mechanical pump is connected to evacuate the vacuum to remove low-boiling-point impurities. When the vacuum degree of the first vacuum gauge is less than 0, the eighth valve is closed. S5. Transfer CO2 gas into the measuring tube: Remove the liquid nitrogen cup at the third cold trap, immerse the lower end of the measuring tube in the liquid nitrogen cup, and the solid CO2 at the third cold trap at room temperature will turn into gaseous CO2. After diffusing into the measuring tube, it will turn into solid CO2 again. The liquid nitrogen cup will rise slowly and stop at the top of the measuring tube. When the reading of the first vacuum gauge is stable, open the eighth valve and observe the reading of the second vacuum gauge. When the vacuum degree of the second vacuum gauge is less than 0, close the seventh and eighth valves and remove the liquid nitrogen cup below the measuring tube. S6. Transfer CO2 gas to the reduction tube: Close the tenth valve and open the eighth valve. The CO2 gas diffuses towards the direction of lower pressure, and the reading of the second vacuum gauge rises. Immerse the lower end of the reduction tube in the liquid nitrogen cup and slowly raise it to 1 / 3 of the way from the top of the reduction tube. The CO2 turns into a solid in the reduction tube. When the vacuum reading of the second vacuum gauge stabilizes, close the ninth valve and keep 1 / 2 of the reduction tube immersed in the liquid nitrogen cup. Use a flame gun to melt and seal the reduction tube. S7. Preparation of graphite target for accelerator mass spectrometry: After sealing the reduction tube, it is placed in a muffle furnace for high-temperature reduction reaction to generate graphite target for accelerator mass spectrometry.

6. A sample preparation method for C-14 analysis in biological samples according to claim 5, characterized in that, In step S1, the bottom of the large quartz test tube contains 30 mg of zinc granules and 12 mg of titanium hydride powder, while the small quartz test tube contains 3.5 mg of 325 mesh iron powder.

7. A sample preparation method for C-14 analysis in biological samples according to claim 5, characterized in that, In step S2, the carrier gas He gas is discharged from the fourth valve.

8. A sample preparation method for C-14 analysis in biological samples according to claim 5, characterized in that, In step S3, the elemental analyzer oxidation combustion process includes sample injection, N analysis, C analysis, and S analysis, with prompts for each step of the analysis process.

9. A sample preparation method for C-14 analysis in biological samples according to claim 5, characterized in that, In step S4, the easily liquefiable impurity gas includes water vapor and sulfur dioxide; the low-boiling-point impurity gas includes nitrogen, oxygen, and helium.

10. A sample preparation method for C-14 analysis in biological samples according to claim 5, characterized in that, In step S5, after the CO2 in the measuring tube is converted into gas, the reading of the first vacuum gauge is read.

11. A sample preparation method for C-14 analysis in biological samples according to claim 5, characterized in that, The method further includes the following steps: S8. Replace the reduction tube and prepare the next sample: Close the second and fourth valves, confirm that the ninth valve has been closed in step S6, replace the reduction tube, and open all valves in the system except for the second and fourth valves; evacuate the system again and prepare the next sample.

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