Method for determining elements in whole blood based on matrix matching and collision reaction cell technology icp-ms / ms

Abstract

The application discloses a method for determining elements in whole blood based on a substrate matching and collision reaction cell technology ICP-MS / MS, and belongs to the technical field of clinical examination medicine, element analysis and biological sample detection. The method adopts a direct sampling scheme of an alkaline diluent, only needs a trace amount of whole blood, and does not need a complex pretreatment, thereby providing a key technical guarantee for realizing clinical batch detection. On this basis, the ICP-MS / MS collision reaction cell technology is used, a matching gas mode combination is adopted, and ClinChek Whole Blood Control Level I, II and III whole blood quality control samples are used to establish a standard curve for substrate matching, so that the physical and chemical interference of the complex substrate of the whole blood is eliminated. The method solves technical bottlenecks such as a long process, weak anti-interference ability, low detection flux, low quantitative precision and large sample consumption of a traditional detection method, realizes rapid and accurate analysis of elements in blood, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the fields of clinical laboratory medicine, elemental analysis and biological sample detection technology, and in particular to a method for determining whole blood elements based on matrix matching and collision reaction cell technology ICP-MS / MS. Background Technology

[0002] Metals and metalloids play crucial roles in maintaining life activities, and their levels in the human body are closely related to disease, metabolic disorders, environmental exposure, and nutritional status. Blood, as a bodily fluid obtained from a patient, effectively reflects the body's current physiological state. Therefore, quantitative analysis of elements in whole blood samples provides a comprehensive "elemental spectrum" of multiple essential trace elements (such as zinc, selenium, and manganese) and toxic heavy metals (such as lead, cadmium, and arsenic) in a single analysis. This is of great significance for accurately assessing an individual's nutritional status, diagnosing heavy metal poisoning, and assisting in the diagnosis of certain metabolic diseases. Inductively coupled plasma mass spectrometry (ICP-MS), with its advantages of high sensitivity, high throughput, and multi-element analysis, has become the mainstream technology for blood elemental analysis, making it an indispensable bridge connecting the microscopic world of elements with macroscopic life and health, and a key technological pillar for promoting the development of modern precision medicine and preventive medicine.

[0003] Blood has a complex matrix, containing a large amount of organic matter (such as proteins, glucose, hormones, red blood cells, and white blood cells) and inorganic salts (such as Na, K, Cl, Ca, and P). This complex composition and matrix effects can interfere with the accuracy of elemental measurements. Conventional digestion methods, such as microwave digestion and water bath digestion, while effectively removing organic interference, still have many potential risks, such as elemental loss, exogenous contamination, and cumbersome and time-consuming procedures, making them unsuitable for large-scale sample analysis. The latest direct dilution method simplifies the pretreatment process by directly treating blood samples with a diluent, reducing potential contamination and loss from excessive steps, and aligning better with the principles of green analytical chemistry. Typically, the dilution method uses a mixture of nitric acid solution and Triton X-100 to dilute the sample 10 to 100 times before testing. This method introduces less reagent blank, is easy to operate, and is suitable for testing large batches of clinical samples. For example, the research article (He Siqing, Nie Manqing, Zhang Jing, et al. Study on pretreatment methods for determining 21 elements in serum [J]. Journal of Preventive Medicine Information, 2024, 40(10):1318-1323.) optimized the conditions and used a mixed solvent dilution method combined with ICP-MS to complete the detection and analysis of 21 metal elements in serum, which is suitable for assessing the level of human element exposure. However, since the organic matter in the sample is not destroyed, these components will generate a large amount of polyatomic ion interference and double-charged ion interference in ICP, and there is still a large matrix effect, which affects the accuracy of the detection of target elements, especially key toxic metals and essential trace elements. Therefore, the direct dilution method also faces core technical problems such as how to select a suitable diluent and how to eliminate the interference caused by complex matrices.

[0004] Traditional single-bar ICP-MS uses only a single-stage mass analyzer, typically paired with a collision cell to eliminate some polyatomic ion interference through kinetic discrimination. However, whole blood matrix has a complex composition, containing high concentrations of carbon, hydrogen, oxygen, nitrogen, chlorine, sodium, calcium, and other components, which form a large number of polyatomic ions and isotopes in plasma, severely interfering with the accurate quantification of trace elements. Single-bar ICP-MS cannot completely separate isotopes and high-abundance polyatomic interference at the mass level, resulting in high background noise and limited detection limits, making it difficult to meet the detection requirements in scenarios such as low-dose exposure. To improve anti-interference capabilities, tandem quadrupole inductively coupled plasma mass spectrometry (ICP-MS / MS) is gradually being applied in the detection field. Compared to single-bar ICP-MS, the core of triple quadrupole inductively coupled plasma mass spectrometry lies in its Q1-Q2-Q3 triple quadrupole tandem design. For example, Chinese invention patent CN117470945A discloses an ICP-MS method for determining the content of inorganic elements in honey. It uses inductively coupled plasma mass spectrometry (ICP-MS / MS) to determine the content of 19 inorganic elements in honey. It establishes a faster, more accurate and convenient detection method through mass transfer. It utilizes the dynamic reaction mode of the collision / reaction cell to form high-abundance, interference-free cluster ions under ammonia conditions, thereby effectively eliminating mass spectrometric interference of each element.

[0005] However, existing methods for applying ICP-MS / MS to whole blood element detection have not been optimized for instrument performance and still suffer from several technical shortcomings. For example, most mainstream methods employ microwave or wet digestion, requiring the addition of concentrated acid and high-temperature heating. Processing a single sample can take several hours, and the numerous steps introduce environmental contaminants. Volatile elements are easily lost during heating, leading to low recovery rates. Some methods using direct dilution suffer from insufficient cell lysis and easy precipitation of proteins and lipids, still requiring centrifugation and filtration, making rapid sample preparation difficult. Other methods use high-dilution of samples to mitigate matrix interference, which significantly lowers the method's detection limit, placing extremely high demands on instrument sensitivity. Secondly, the types of mass spectrometric interferences for different elements in whole blood vary significantly, resulting in different optimal collision and reaction modes. Current detection methods employ a single-mode, batch-injection approach, where some elements are detected under one reaction mode, and the remaining elements are detected after switching modes. This results in low throughput, long detection cycles, and high sample consumption, making it difficult to meet the high-efficiency testing needs of large batches of clinical samples. Furthermore, most methods use aqueous standard curves for quantification, and the signal inhibition or enhancement effects of whole blood matrices can lead to significant quantitative deviations. Some methods use matrix-matched calibration, which suffers from poor consistency between the calibration matrix and the whole blood matrix of the sampler, and a narrow concentration gradient coverage, making it difficult to guarantee accurate quantification across a wide range from normal physiological levels to abnormally elevated levels. This makes quantitative accuracy susceptible to matrix effects. Finally, current detection methods require large sample volumes, typically requiring hundreds of microliters to several milliliters of whole blood sample for digestion. For populations with difficult blood collection, such as infants and elderly patients, the large sample volume limits the applicability of these methods. Therefore, overcoming these technical obstacles is still necessary to achieve compatibility between ICP-MS / MS and whole blood element detection protocols.

[0006] In summary, developing a simple and rapid pretreatment method with strong anti-interference ability, high detection throughput, accurate quantification, and low sample volume for the detection of multiple elements in whole blood by ICP-MS / MS is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0007] In view of the above-mentioned deficiencies of the prior art, the purpose of the present invention is to provide a rapid, accurate, trace-level, and highly sensitive method for determining 11 elements (including but not limited to Cr, Mn, Co, Ni, As, Se, Sr, Cd, Hg, Tl, and Pb) in whole blood, so as to solve the problems of complex pretreatment, large sample volume, severe physical and chemical interference in complex matrices, and inability to guarantee the quantitative accuracy of low-concentration elements in the prior art.

[0008] To achieve the above objectives, the technical solution provided by the present invention is as follows: This invention provides a method for determining whole blood elements based on matrix matching and collision reaction cell technology using ICP-MS / MS, comprising the following steps: (1) Using water as a solvent, prepare a diluent solution containing tetramethylammonium hydroxide (TMAH), ammonium pyrrolidinedithiocarbamate (APDC) and Triton X-100. The diluent solution is used for the direct dilution of whole blood samples. (2) Take the whole blood sample to be tested and dilute it with the diluent solution according to a certain volume ratio, vortex shake and let it stand to obtain the test solution for direct injection; (3) Use certified whole blood standard substances ClinChek Whole BloodControl Level I, Level II and Level III with the same matrix as the sample to be tested for matrix matching calibration. Dilute the diluent solution with the three certified whole blood standard substances according to the dilution ratio in step (2) to prepare calibration standard solutions with low, medium and high concentration gradients. (4) The test solution from step (2) and the calibration standard solution from step (3) are respectively introduced into a series quadrupole inductively coupled plasma mass spectrometer (ICP-MS / MS). The gas mode combination is matched according to the mass spectrometry interference characteristics of the test element for determination. The sample is collected in time segments during a single injection to achieve simultaneous determination of multiple elements. (5) Quantitative calculations were performed based on the internal standard method to obtain the element concentrations in the original whole blood.

[0009] In this field, ClinChek Whole Blood Control Level I, Level II, and Level III are certified reference materials for trace elements in lyophilized human whole blood matrix from the ClinChek brand, a subsidiary of RECIPE GmbH in Germany. These matrix-matched standards are widely used in clinical, occupational toxicology, and environmental toxicology laboratories for whole blood element detection. They are used for internal quality assurance in clinical laboratories to monitor the accuracy and precision of specific analytical items in whole blood samples. Level I, Level II, and Level III represent low, medium, and high concentration levels to meet the needs of multi-point quality control in testing.

[0010] Preferably, in step (1), the final concentration of tetramethylammonium hydroxide in the diluent solution is 0.1 vol.%~1.0 vol.%, the final concentration of pyrrolidine dithiocarbamate is 0.01%~0.05% (w / v), and the final concentration of Triton X-100 is 0.01 vol.%~0.05 vol.%.

[0011] Preferably, in step (2), the sampling volume of the whole blood sample to be tested is 20~100 μL, and the dilution volume factor is 20~100 times.

[0012] Based on the design of this invention, the sample size in step (2) is small, and the pretreatment process does not require heating, digestion, centrifugation or filtration. The total sample processing time is ≤30 s / sample, which can greatly reduce the processing time and achieve the effect of rapid detection.

[0013] In step (3), a certified whole blood standard, Seronorm Trace Elements Whole Blood L2 lyophilized whole blood, can be used as an independent source for quality control. After dissolving it, take the same amount of Seronorm Trace Elements Whole Blood L2 quality control solution as the whole blood sample to be tested, and then dilute it with a diluent solution to the same ratio as the test solution for accuracy verification.

[0014] In this field, Seronorm Trace Elements Whole Blood L2 is a certified quality control standard for trace elements in lyophilized human whole blood matrix produced by SERO AB of Norway. It is a commonly used third-party whole blood element detection quality control material in clinical testing, occupational toxicology, and environmental exposure studies. It is an independent quality control sample used in this invention for method accuracy validation.

[0015] Preferably, in step (4), the element to be tested includes at least one of Cr, Mn, Co, Ni, As, Se, Sr, Cd, Sb, Pb, and I.

[0016] Preferably, in step (4), the gas mode combination includes ammonia reaction mode, oxygen reaction mode, oxygen transfer mode, and helium collision mode.

[0017] Further preferably, the determination of the elements to be tested, Cr, Mn, and Ni, adopts the ammonia reaction mode; the determination of the element to be tested, Cd, adopts the oxygen reaction mode; the determination of the elements to be tested, As and Se, adopts the oxygen transfer mode; and the determination of the elements to be tested, Sb, Pb, Sr, Co, and I, adopts the helium collision mode.

[0018] More preferably, in the ammonia reaction mode, the ammonia flow rate is 0.3~1.0 mL / min; and in the oxygen reaction mode, the oxygen flow rate is 0.1~0.5 mL / min.

[0019] Preferably, in step (5), the internal standard method includes the following steps: pre-adding an internal standard element to the diluent, using the ratio of the response values ​​of the element to be tested and the internal standard element in the calibration standard solution (response value / internal standard response value) as the response signal, and plotting a standard curve for the theoretical concentration; substituting the response ratio of the sample to be tested into the standard curve, calculating the concentration after dilution, and then multiplying by the dilution factor to obtain the original element concentration in whole blood.

[0020] More preferably, the internal standard element includes at least one of Sc, Ge, Rh, and Ir.

[0021] Based on the properties of the analyte, the internal standard element can be selected as an element with a mass number similar to that of the analyte and which is not present or is considered to be almost absent in whole blood.

[0022] More preferably, the linear correlation coefficient R of the standard curve 2 ≥0.991.

[0023] Preferably, when the method for determining whole blood elements based on matrix matching and collision reaction cell technology by ICP-MS / MS is used for the determination of blood samples from clinical patients, the spiked recovery rate is 80%~128%.

[0024] Based on the above solutions, the design concept and principle of this invention are as follows: This invention is based on triple quadrupole inductively coupled plasma mass spectrometry (ICP-MS / MS), with its core being a triple quadrupole series design of Q1-Q2-Q3. Q1 acts as the first mass filter, pre-selecting ions with the same mass number (m / z) as the target element to enter the reaction cell (Q2). In Q2, a carefully selected reactive gas (such as oxygen, ammonia, or hydrogen) is introduced using dynamic reaction cell technology to react chemically with the target ions. This technology eliminates isotope and polyatomic interference based on the ion-molecule reaction principle. By applying radio frequency and DC voltages to the quadrupoles in the reaction cell to form a bandpass filter, interfering substances are eliminated before entering the analyzer. Finally, Q3 detects the product ions after the reaction. This invention employs a "mass screening, chemical reaction, mass re-screening" model, which significantly improves the signal-to-noise ratio due to the effective elimination of background interference. This enables ICP-MS / MS to detect elements at extremely low concentrations (down to the ng / L or ppt level), thus meeting the needs of low-dose environmental exposure studies and trace element nutritional studies.

[0025] This invention employs a direct sample introduction scheme using an alkaline diluent, requiring only a small volume of whole blood (e.g., 40 µL) and eliminating complex pretreatment, thus providing crucial technical support for large-scale clinical testing. Based on this, it utilizes ICP-MS / MS collision reaction cell technology, combining He collision, O2 mass transfer, and NH3 or O2 in-situ reaction modes, and establishes standard curves using ClinChek Whole Blood Control Level I, II, and III whole blood quality control samples for matrix matching, effectively eliminating physicochemical interference from the complex matrix of whole blood. This method enables rapid and accurate analysis of 11 elements in blood, including Cr, Mn, Ni, Cd, As, Se, Sb, Co, Pb, Sr, and I. As presented in one or more embodiments of this invention, by comparing pretreatment methods such as acidic diluents, alkaline diluents, and microwave digestion, this scheme solves the technical bottlenecks of traditional detection methods, such as long workflows, weak anti-interference capabilities, low throughput, low quantitative accuracy, and large sample volumes.

[0026] The core innovation of this invention lies in the synergistic effect of matrix-matched standard curves and multi-mode interference elimination technology. Specifically, the reaction cell technology eliminates interference to the maximum extent at the mass spectrometry level, while matrix matching compensates for residual matrix effects at the chemometric level. The combined approach achieves highly sensitive (ppt-level), highly accurate (recovery rate 80%~128%), and highly reliable (deviation from certified reference materials <8%) determination of 11 elements in whole blood. This method requires small sample volumes, simplifies pretreatment, significantly reduces contamination and losses introduced by steps, and significantly improves the efficiency of batch clinical sample testing. Simultaneously, the standard curve concentration range closely approximates the true level, and the quantification accuracy for low-concentration elements is high, providing a reliable methodological reference for whole blood element analysis in large-scale population health screenings.

[0027] Compared with the prior art, the present invention has the following advantages and beneficial effects: This invention provides a method for determining whole blood elements using ICP-MS / MS based on matrix matching and collision reaction cell technology. The method features simple, rapid, and efficient pretreatment, employing diluent solutions such as TMAH, APDC, and Triton X-100. Hemolysis, protein dissolution, and matrix dispersion are completed in one step via direct dilution, eliminating the need for heating, digestion, centrifugation, or filtration. The pretreatment time for a single sample is less than 30 seconds, making it suitable for large-scale clinical screening. Furthermore, this method requires only a small sample volume and has wide applicability, requiring only 40 µL of whole blood, significantly less than traditional methods (200–500 µL), making it suitable for newborns, infants, the elderly, and critically ill patients. Regarding interference elimination and sensitivity, this invention utilizes a matrix matching standard curve and multi-mode interference elimination technology to achieve a synergistic effect. The reaction cell technology eliminates interference to the maximum extent from the mass spectrometry end, while matrix matching compensates for residual matrix effects from the chemometric end. The combination of these two techniques achieves high sensitivity (ppt level), high accuracy (recovery rate 80%–128%), and high reliability (deviation from certified reference materials <8%) in the determination of elements in whole blood. Furthermore, this method exhibits good linearity and high accuracy. It utilizes certified whole blood reference materials to prepare a series of standard curves, with concentration ranges closely approximating true levels. This effectively improves the quantitative accuracy of elements at both high and low concentrations. The 11 elements show good linear correlation coefficients (R0) within the linear range. 2 With a value ≥0.991, the actual clinical sample spike recovery rate is 80.3%~127.8%, which meets the accuracy requirements of clinical testing and has good application prospects. Detailed Implementation

[0028] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0029] The instruments and reagents involved in the embodiments of this invention are prepared as follows: Instruments and equipment: Triple quadrupole inductively coupled plasma mass spectrometer: NexION 5000G triple quadrupole inductively coupled plasma mass spectrometer (Perkin Elmer, USA), equipped with a standard platinum sampling cone and a cutoff cone.

[0030] Ultrapure water system: RODL-220AIS continuous desalination ultrapure water system (Xiamen Ruisijie Scientific Instruments Co., Ltd.) Microwave Digestion System: M6 Microwave Digestion System (Shanghai Yiyao Technology) Vortex oscillator: VM-500Pro vortex oscillator (Qun'an Experimental Instruments).

[0031] Micropipettes: 1 µL to 1000 µL (Eppendorf, Germany).

[0032] Sample tubes: 5 mL centrifuge tubes (polypropylene, metal content tested and qualified).

[0033] Reagents and Standards Tetramethylammonium hydroxide (TMAH): 25% aqueous solution, analytical grade (Sigma-Aldrich, USA).

[0034] Ammonium pyrrolidine dithiocarbamate (APDC): Analytical grade (≥99%) (Sigma-Aldrich, USA).

[0035] Triton X-100: Laboratory grade (Sigma-Aldrich, USA).

[0036] Concentrated nitric acid: 67%~70%, electronic grade (Suzhou Jingrui Chemical Co., Ltd.).

[0037] Internal standard mixed solution: containing 10 mg / L each of Sc, Ge, Rh and Ir (Perkin Elmer, USA).

[0038] Certified whole blood standard substances: ClinCheck Level 1 and Level 2 (Recipe, Germany).

[0039] Quality control whole blood samples: Seronorm Trace Elements Whole Blood L-1, L-2 (Sero, Norway).

[0040] Elemental standard stock solutions: National Nonferrous Metals and Electronic Materials Analysis and Testing Center standard solution GNM-M2810566-2013 Al, As, B, Ba, Be, Bi, Cd, Co, Cr, Cs, Cu, Fe, Ga, Ge, Li, Mg, Mn, Mo, Ni, Pb, Rb, Sb, Se, Sn, Sr, Ti, Zn multi-element standard solution and GNM-SI-002-2013 I single-element standard solution 100 mg / L.

[0041] Preparation of diluent solution: Weigh / measure the following components: Tetramethylammonium hydroxide (TMAH): 10.0 mL Ammonium pyrrolidine dithiocarbamate (APDC): 0.05 g Triton X-100: 0.25 mL Add the above components sequentially to approximately 300 mL of ultrapure water, sonicate for 10 min to completely dissolve APDC, then bring the volume to 500 mL with ultrapure water and mix well. The resulting diluted solution consists of: 0.5% (v / v) TMAH, 0.01% (w / v) APDC, and 0.05% (v / v) Triton X-100. Store in a sealed container at 4 °C for later use.

[0042] Clinical whole blood sample collection and preservation: Whole blood samples were collected from clinical patients in heparin sodium anticoagulant tubes (after passing background testing) and transported to the laboratory at 4°C within 2 hours of collection. Samples can be stored at 4°C for 7 days or at -20°C for 6 months. Before testing, samples must be brought to room temperature and thoroughly mixed.

[0043] This embodiment provides a method for determining whole blood elements using ICP-MS / MS based on matrix matching and collision reaction cell technology. The specific steps are as follows: (1) Diluent solution: A diluent solution for direct dilution of whole blood samples was prepared using tetramethylammonium hydroxide (TMAH), pyrrolidine dithiocarbamate (APDC), and Triton X-100, prepared with ultrapure water, with a final concentration of 0.5% (v / v) TMAH, 0.01% (w / v) APDC, and 0.05% (v / v) Triton X-100. (2) Sample pretreatment: Take 40 µL of whole blood sample from the person to be tested, add it to a 5 mL centrifuge tube, and then accurately add 1960 µL of the diluent solution prepared in step (1) to make the dilution factor 50 (1:49, v / v). Mix using a vortex mixer and let stand at room temperature to obtain the solution to be directly injected for testing. The pretreatment process requires no heating, digestion, centrifugation or filtration, and the total processing time is ≤30 s / piece; Blank sample preparation: Use 40 µL of ultrapure water instead of whole blood, and perform the remaining operations as above; (3) Preparation of standard curve and quality control samples: Matrix matching calibration was performed using certified whole blood standards, ClinChek Whole Blood Control Level I, Level II, and Level III, which use the same matrix as the test samples. Using the diluent described in step (1), the whole blood standard substance was diluted with the same dilution factor (1:49, v / v) as the sample according to ClinChek Whole Blood Control Level Ⅰ, Ⅱ, Ⅲ to prepare calibration standard solutions with three different concentration gradients: low, medium, and high. Simultaneously, a certified whole blood standard, Seronorm Trace Elements Whole Blood L2, a lyophilized whole blood sample from an independent source, was added to 3 mL of ultrapure water according to the instructions and vortexed for 30 min to ensure complete dissolution. 40 µL of the dissolved Seronorm Trace Elements Whole Blood L2 control solution was then placed in a 2 mL polypropylene centrifuge tube and diluted 50 times with diluent solution for accuracy verification. (4) ICP-MS / MS instrument determination: The solutions obtained in steps (2) and (3) were introduced into a series quadrupole inductively coupled plasma mass spectrometer for measurement. The instrument conditions were set as shown in Table 1. Table 1: Basic Instrument Parameters

[0044] Based on the different mass spectrometry interference characteristics of the 11 analytes (Cr, Mn, Co, Ni, As, Se, Sr, Cd, Hg, Tl, Pb), multiple mode combinations as shown in Table 2 were used for determination. The four modes were switched and acquired in time segments in a single injection sequence to achieve simultaneous determination of multiple elements. Table 2: Mass number and gas mode settings for target elements tested by ICP-MS / MS

[0045] (5) Quantitative calculation: Quantitative calculations were performed using the internal standard method: Internal standard element: Select an element with a mass number similar to that of the analyte and which is almost absent in whole blood (such as Sc, Ge, Rh, Ir), and pre-add it to the diluent at a concentration of 1~10 µg / L; Calibration curve: A standard curve is plotted against the theoretical concentration using the response signals (response value / internal standard response value) of each analyte in the calibration standard solution. The linear correlation coefficient R0 is used. 2 ≥0.991.

[0046] Sample concentration calculation: Substitute the response ratio of the sample to be tested into the standard curve, calculate the concentration after dilution, and then multiply by the dilution factor (50 times) to obtain the original element concentration in whole blood.

[0047] In the above operation, the internal standard is configured as follows: the internal standard solution is introduced online through the three-way valve, and the mixed internal standard solution is pre-added to the diluent so that the Rh concentration in the final solution is 10 µg / L.

[0048] All prepared samples were analyzed on the instrument within 4 hours.

[0049] In step (3) above, a standard curve is established for whole blood quality control samples to perform matrix matching, and a series of standard solutions (S1, S2, S3) with three different concentration gradients (low, medium, and high) are prepared as shown in Table 3.

[0050] Table 3: Series of standard curves for 11 elements established from whole blood quality control samples

[0051] In step (4) above, during multi-element determination by ICP-MS / MS, the main interferences include multi-atom / multi-ion interference generated by plasma and interface region, isotope interference, double charge interference, and multi-atom ion interference generated by side reactions in the reaction cell. To address the above-mentioned mass spectrometry interferences, mass screening Q1 can be used to pre-separate ions outside the target mass range, creating a cleaner reaction environment for the subsequent reaction cell and significantly reducing the possibility of new interferences forming in the reaction cell. Through dynamic reaction cell Q2, carefully selected reactive gases are introduced into the reaction cell to cause the target ions to undergo chemical reactions, while interfering ions do not react or undergo different reactions. Finally, through mass screening Q3, only specific product ions after the reaction are detected. Even if new byproducts are generated in the reaction cell, they will be excluded as long as their mass number is different from the set detection mass, thus ensuring the purity of the results. Blood is directly diluted and injected for analysis, which contains a large amount of organic matter and inorganic salts. On the one hand, matrix effects can be reduced using aerosol mass spectrometer (AMS) technology; on the other hand, the interference of argon, carbon, and chlorides on Cr and V mass spectra can be eliminated through optimization using ICP-MS / MS multi-mode methods. In the reaction mode, NH3 is an effective cell gas capable of eliminating... 35 Cl 16 O + right 51 V + , 40 Ar 12 C + right 52 Cr + Interference, etc. 75 As and 80 Se is susceptible 40 Ar 35 Cl and 40 Ar 40 Ar interference is present, while O2 reacts with As and Se to form clusters, allowing for mass transfer determination of these elements based on the mass number of oxide ions in the absence of mass spectrometry interference. In whole blood samples, Fe is abundant in erythrocytes, and Fe-Mn peak overlap generally leads to overestimation of Mn quantification. Applying the triple quadrupole technique can significantly improve abundance sensitivity, resolving the two peaks to baseline and eliminating the interference from high concentrations of Fe at mass numbers 54 and 56.55 Interference from Mn. As shown in Table 1, by optimizing the instrument conditions, ICP-MS / MS was used to analyze 11 representative elements in blood samples, including Cr, Mn, Ni, Cd, As, Se, Sb, Co, Pb, Sr, and I. Based on the different elemental characteristics, the corresponding modes were selected for determination.

[0052] This implementation method completes the methodological verification based on the above process in order to study its practical application effect.

[0053] Linear range and detection limit: A series of standard solutions were prepared as described above. ClinChek Whole Blood Control Level I, II, and III whole blood standards with low, medium, and high elemental concentrations were diluted 50-fold using an alkaline diluent to construct a standard curve. This ensures perfect matching with the blood sample matrix, thereby reducing matrix effects. A standard curve was plotted with the response ratio (elemental signal / internal standard signal) against concentration on the ordinate, and the corresponding linear correlation coefficient (R²) was calculated. 2 The calculation results are shown in Table 4. Simultaneously, 11 blank solutions were measured in parallel. The limit of detection (LOD) of each element was calculated as the ratio of the standard deviation of 3 times the blank response value to the slope of the standard curve.

[0054] Table 4: Linear range, regression equation, correlation coefficient, and method detection limit of 11 elements in whole blood directly injected into the diluent solution.

[0055] Accuracy: Take an independently sourced whole blood quality control sample, Seronorm Trace Elements Whole Blood L2 lyophilized certified whole blood standard, add 3 mL of ultrapure water according to the instructions, and vortex for 30 min to ensure complete dissolution. Take 40 µL of the dissolved Seronorm Trace Elements Whole Blood L2 quality control sample solution into a 5 mL centrifuge tube, add 1960 µL of diluent solution, dilute 50 times, vortex, and analyze. Compare the measured value with the certified value.

[0056] Analysis of actual blood sample test results and spiked recovery rate: Using a three-way in-line system to introduce the internal standard solution, and employing the aforementioned diluent solution, three different concentration levels of ClinChek Whole Blood Control Level I, II, and III blood standard samples were diluted 50-fold. An 11-element standard curve was prepared as an external standard, and the external standard method was used to analyze the patient blood samples. Blood samples were collected by Zhongnan Hospital of Wuhan University. After removing the patient blood samples from the refrigerator, they were placed at room temperature, thoroughly shaken, and then 40 µL of the patient blood sample was taken and diluted 50-fold using alkaline diluents of 0.5% (v / v) TMAH, 0.01% (w / v) APDC, and 0.05% (v / v) Triton X-100 before analysis. To ensure consistency between the sample matrix and the standard solution matrix, the amount of blood added and the dilution dose must be controlled consistently. Therefore, 20 µL of ClinChek standard sample and 20 µL of patient blood sample were mixed and diluted 50-fold with an alkaline diluent of 0.5% (v / v) TMAH, 0.01% (w / v) APDC, and 0.05% (v / v) Triton X-100. The results were analyzed by ICP-MS / MS, and the spiked recovery rate was calculated. The results are listed in Table 5.

[0057] Table 5: Analysis of Test Results and Spike Recovery Rate of Actual Blood Samples

[0058] In comparison with the method of the present invention, this embodiment provides the following three comparison groups: Comparison Group 1 Sample pretreatment was performed using the direct dilution method with acidic diluents: all sample preparation steps were performed at room temperature. Each sample was thawed only once before analysis and homogenized using a vortex mixer for 30 min. 40 µL of the Ronorm TraceElements Whole Blood L2 quality control sample was placed in a 2 mL polypropylene centrifuge tube and diluted 50-fold with acidic diluent (0.5% HNO3 + 0.05% Triton X). Simultaneously, the corresponding blood standard samples (Whole Blood Control Level I, II, and III) were diluted 50-fold using the corresponding diluents to prepare a blood sample analysis standard curve. The samples were then tested, and the measured values ​​were compared with the certified values.

[0059] Comparative Example 2: Sample pretreatment was performed using microwave digestion: 500 µL of Seronorm Trace Elements WholeBlood L2 quality control sample was placed in a PTFE digestion tube, 7 mL of concentrated HNO3 was added, and the temperature was programmed to reach 190 °C and held for 30 min. The sample was then heated to 160 °C for 90 min to remove acid, and finally diluted to 10 mL with 1% nitric acid. A series of standard curves were prepared using multi-element standard solutions, and the results were analyzed using the microwave digestion method. The measured values ​​were compared with the certified values.

[0060] Comparative Example 3: Internal standard solutions were introduced online using a three-way valve. The standard solutions from the National Center for Analysis and Testing of Nonferrous Metals and Electronic Materials (GNM-M2810566-2013) for Al, As, B, Ba, Be, Bi, Cd, Co, Cr, Cs, Cu, Fe, Ga, Ge, Li, Mg, Mn, Mo, Ni, Pb, Rb, Sb, Se, Sn, Sr, Ti, and Zn, and the single-element iodine standard solution (GNM-SI-002-2013) were used as external standards. Standard curves for 11 elements were prepared according to the concentration gradients of each element in Table 6. 40 µL of the dissolved Seronorm Trace Elements Whole Blood L2 control sample solution was placed in a 2 mL polypropylene centrifuge tube and diluted 50-fold with acidic diluent (0.5% HNO3 + 0.05% Triton X). The Seronorm Trace Elements Whole Blood L2 control sample was analyzed using collision mode.

[0061] Table 6: Standard Curves for Each Element

[0062] To more intuitively evaluate the impact of matrix effects on the accuracy of test results during pretreatment methods and standard curve preparation, Seronorm Trace Elements Whole Blood L2 whole blood quality control samples were used as samples for analysis and testing, and the results were compared with the certified values.

[0063] To evaluate the impact of blood sample pretreatment methods on test results, under the premise of ensuring the establishment of a standard curve and matrix matching for whole blood quality control samples, three methods were used to process Seronorm Trace Elements Whole Blood L2 whole blood quality control samples: direct injection with acidic diluent, direct injection with alkaline diluent, and microwave digestion. The content of each target element was measured, and the test results are shown in Table 7 (test values ​​A, B, and C).

[0064] Table 7: Seronorm standard product certification values, certification ranges, and measurement values ​​under three different treatment conditions (Group A is the method of this invention, Group B is control group 1, Group C is control group 2, and Group D is control group 3).

[0065] The above experimental results show that the test results obtained by the direct injection of alkaline diluent have a good agreement with the reference values ​​of each element given in the certificate, indicating that this method can meet the requirements for accurate quantification of elements in blood samples. A comparison of the test results obtained by the direct injection of acidic diluent with the reference values ​​shows that acidic diluent can also meet the analysis requirements of most elements in blood samples. The test results of some elements, such as Ni, Mn, and Pb, are near the limit of the certificate reference value, but the test results of I show a significant deviation. A comparison of the test results obtained by microwave digestion with the certificate reference values ​​reveals that the I test result is significantly lower, which is due to the instability of I in an acidic environment. The test results of elements such as Cr, Mn, and Ni are much higher than the upper limit of the certificate reference value range, presumably because microwave digestion poses a higher risk of contamination for common elements, leading to higher measured values.

[0066] In summary, alkaline diluents are more compatible with the chemical environment of blood systems. Due to the complex matrix of whole blood, its high salt content, and abundant organic matter, alkaline diluents can better maintain the stability of the sample system, preserve the colloidal solubility of proteins, and prevent analyte precipitation or loss. Therefore, this invention preferably uses direct injection of alkaline diluents for blood sample pretreatment.

[0067] To further evaluate the impact of matrix effects on detection accuracy during standard curve preparation, this invention compares existing technical approaches. Currently published patents (such as CN119224103A and CN1133960153A) primarily use nitric acid aqueous solution as the medium and conventional standard solutions to prepare standard curves in plasma and serum analyses. However, whole blood matrices are more complex than plasma and serum matrices, characterized by high salt content, high carbon content, and high levels of easily ionized elements. In ICP-MS detection, this easily leads to significant ionization inhibition, polyatomic ion interference, and differences in nebulization transmission. Without matrix matching, difficult-to-correct systematic errors will be introduced, resulting in severely distorted detection results. Currently, there are no reports on using whole blood quality control samples with different concentration gradients to establish standard curves for matrix matching. Therefore, this invention compares the impact of two standard curve preparation methods on the test results of Seronorm Trace Elements Whole Blood L2 quality control samples: (1) A standard curve (test value B) was prepared using certified whole blood standard substances ClinChek Whole Blood Control Level I, II, and III, which are consistent with the matrix of the sample to be tested. (2) Using dilute nitric acid / pure water as the medium, a standard curve (test value D) was prepared using conventional standard solutions.

[0068] The comparative results show that iodine cannot exist stably in an acidic system, hence both test values ​​B and D deviate from the certified range for iodine. Furthermore, in test value D obtained using a standard curve prepared with conventional dilute nitric acid / pure water, the test results for elements such as As, Se, Sb, and Pb all exceed the upper limit of the certified range, indicating significant distortion. However, establishing a standard curve using whole blood quality control samples matched to the sample matrix ensures consistency between the standard system and the sample system in terms of ionization efficiency, nebulization transmission efficiency, and mass spectrometry interference levels. This eliminates matrix effects at their source and significantly improves the recovery rate, accuracy, precision, and linear reliability of the detection method.

[0069] In summary, this invention, compared with various pretreatment methods, utilizes a limited dilution buffer containing 0.5% tetramethylammonium hydroxide, 0.01% pyrrolidine dithiocarbamate, and 0.05% Triton X-100 for direct dilution and injection of whole blood, requiring only 40 µL of a small volume of whole blood and eliminating the need for complex pretreatment. Employing ICP-MS / MS collision cell technology, it combines multiple modes including He collision, O2 mass transfer, and NH3 / O2 in-situ reaction, and establishes standard curves using ClinChek Whole Blood Control Level I, II, and III whole blood quality control samples for matrix matching. This effectively eliminates physicochemical interference from the complex matrix of whole blood, enabling the simultaneous and accurate determination of 11 elements: Cr, Mn, Ni, Cd, As, Se, Sb, Co, Pb, Sr, and I. This invention, through the synergistic effect of matrix-matched standard curves and multi-mode interference elimination technology, eliminates interference from both the mass spectrometry and chemometric perspectives, offering advantages of high sensitivity, high accuracy, and high reliability. This method requires small sample volumes, has simple pretreatment, and high detection efficiency, making it suitable for rapid batch analysis of whole blood elements in large-scale population health screenings.

[0070] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A method for determining whole blood elements based on matrix matching and collision reaction cell technology by ICP-MS / MS, characterized in that, Includes the following steps: (1) Using water as a solvent, prepare a diluent solution containing tetramethylammonium hydroxide, pyrrolidine dithiocarbamate and Triton X-100. The diluent solution is used for the direct dilution of whole blood samples. (2) Take the whole blood sample to be tested and dilute it with the diluent solution according to a certain volume ratio, vortex shake and let it stand to obtain the test solution for direct injection; (3) Use certified whole blood standard substances ClinChek Whole Blood Control Level I, Level II and Level III with the same matrix as the sample to be tested for matrix matching calibration. Dilute the diluent solution with the three certified whole blood standard substances according to the dilution ratio in step (2) to prepare calibration standard solutions with three concentration gradients of low, medium and high. (4) The test solution from step (2) and the calibration standard solution from step (3) are respectively introduced into a series quadrupole inductively coupled plasma mass spectrometer. The gas mode combination is matched according to the mass spectrometry interference characteristics of the test element for measurement. The sample is collected in time segments during a single injection to achieve simultaneous multi-element measurement. (5) Quantitative calculations were performed based on the internal standard method to obtain the element concentrations in the original whole blood.

2. The method for determining whole blood elements based on matrix matching and collision reaction cell technology ICP-MS / MS according to claim 1, characterized in that: In step (1), the final concentration of tetramethylammonium hydroxide in the diluent solution is 0.1 vol.%~1.0 vol.%, the final concentration of pyrrolidine dithiocarbamate is 0.01%~0.05% (w / v), and the final concentration of Triton X-100 is 0.01 vol.%~0.5 vol.%.

3. The method for determining whole blood elements based on matrix matching and collision reaction cell technology ICP-MS / MS according to claim 1, characterized in that: In step (2), the sample volume of the whole blood sample to be tested is 20~100 μL, and the dilution volume factor is 20~100 times.

4. The method for determining whole blood elements based on matrix matching and collision reaction cell technology ICP-MS / MS according to claim 1, characterized in that: In step (4), the element to be tested includes at least one of Cr, Mn, Co, Ni, As, Se, Sr, Cd, Sb, Pb, and I.

5. The method for determining whole blood elements based on matrix matching and collision reaction cell technology ICP-MS / MS according to claim 1, characterized in that: In step (4), the gas mode combination includes ammonia reaction mode, oxygen reaction mode, oxygen transfer mode, and helium collision mode.

6. The method for determining whole blood elements based on matrix matching and collision reaction cell technology ICP-MS / MS according to claim 5, characterized in that: The determination of the elements Cr, Mn, and Ni was performed using the ammonia reaction mode; the determination of the element Cd was performed using the oxygen reaction mode; the determination of the elements As and Se was performed using the oxygen transfer mode; and the determination of the elements Sb, Pb, Sr, Co, and I was performed using the helium collision mode.

7. The method for determining whole blood elements based on matrix matching and collision reaction cell technology ICP-MS / MS according to claim 5, characterized in that: In the ammonia reaction mode, the ammonia flow rate is 0.3~1.0 mL / min; in the oxygen reaction mode, the oxygen flow rate is 0.1~0.5 mL / min.

8. The method for determining whole blood elements based on matrix matching and collision reaction cell technology ICP-MS / MS according to claim 1, characterized in that: In step (5), the internal standard method includes the following steps: pre-adding an internal standard element to the diluent, using the ratio of the response values ​​of the analyte and the internal standard element in the calibration standard solution (response value / internal standard response value) as the response signal, and plotting a standard curve for the theoretical concentration; Substitute the response ratio of the sample to be tested into the standard curve, calculate the concentration after dilution, and then multiply by the dilution factor to obtain the elemental concentration in the original whole blood.

9. The method for determining whole blood elements based on matrix matching and collision reaction cell technology ICP-MS / MS according to claim 8, characterized in that: The internal standard element includes at least one of Sc, Ge, Rh, and Ir; the linear correlation coefficient R of the standard curve 2 ≥0.

991.

10. The method for determining whole blood elements based on matrix matching and collision reaction cell technology ICP-MS / MS according to claim 1, characterized in that: When used for testing blood samples from clinical patients, the spiked recovery rate is 80%–128%.

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