Determination of 26 elements in bone marrow supernatant by ICP-MS

Abstract

This invention belongs to the field of biological sample detection and discloses an ICP-MS detection method for 26 elements in bone marrow supernatant. The detection method includes: (1) preparation of sample diluent and matrix-matched diluent; (2) preparation of mixed internal standard solution; (3) preparation of first mixed standard curve solution, second mixed standard curve solution and third mixed standard curve solution respectively; (4) plotting standard curves for 26 elements; (5) diluting the bone marrow supernatant sample with sample diluent, and then vortexing and centrifuging sequentially to obtain the sample solution to be tested; (6) determining the content of 26 elements in the sample solution to be tested. The dual diluent strategy of this invention realizes the matching of standard, internal standard and sample matrix, and the three-segment standard curve reduces the memory effect. Only one injection is needed to simultaneously and accurately detect 26 elements. The spiked recovery rate is 70%~130%, and the internal standard recovery rate is 80%~120%, which is suitable for rapid clinical detection.

Description

Technical Field

[0001] This invention belongs to the field of biological sample detection, specifically relating to an ICP-MS method for detecting 26 elements in bone marrow supernatant. Background Technology

[0002] Chemical elements are fundamental to maintaining normal physiological functions in the human body. Imbalances in the metabolism of essential chemical elements or exposure to harmful elements can disrupt homeostasis and are closely related to human health. Bone marrow supernatant, as an important component of the bone marrow microenvironment, directly reflects its physiological and pathological state. Rapid and accurate determination of its elemental content is of great significance for the diagnosis and treatment of hematological diseases, bone marrow metastases, and other diseases.

[0003] Currently, the main methods for elemental detection in biological samples include atomic absorption spectrometry (AAS), atomic fluorescence spectrometry (AFS), atomic emission spectrometry (AES), and inductively coupled plasma mass spectrometry (ICP-MS). While AAS and AFS have lower detection costs, they can only analyze one element at a time, and AFS can only detect elements that easily form hydrides, such as As, Hg, and Se, resulting in lower detection efficiency and versatility. AES commonly uses inductively coupled plasma as the excitation source, enabling simultaneous multi-dimensional elemental analysis. However, in biological sample detection, matrix interference is more pronounced than in AAS, and its sensitivity is lower than ICP-MS, making it difficult to guarantee the accuracy of trace element detection. In contrast, ICP-MS offers advantages such as rapid and simultaneous multi-element detection, a wide linear range, good accuracy, and high sensitivity, making it an effective method for analyzing biological samples.

[0004] However, bone marrow supernatant contains high concentrations of proteins, salts, and lipids, resulting in a complex matrix, and samples are difficult to obtain and limited in quantity. Therefore, direct dilution is the only viable pretreatment method before instrumental analysis. Existing ICP-MS methods for plasma or serum typically use diluents containing HNO3 and Triton X-100, or add organic substances such as n-butanol and isopropanol. However, these methods do not consider the matrix mismatch between the sample and the standard curve and internal standard solution. Directly applying these to bone marrow supernatant leads to significant fluctuations in spiked and internal standard recoveries, compromising accuracy. Furthermore, while existing methods utilize multiple standard curve designs to cover a wide dynamic range, the preparation process is cumbersome, often involving stepwise dilution with multi-element stock solutions. The presence of easily adsorbed elements (such as Tl and Hg) in high-concentration stock solutions, along with numerous other elements, exacerbates instrument memory effects and cross-contamination, making them unsuitable for rapid detection of small sample volumes in bone marrow supernatant. Summary of the Invention

[0005] The purpose of this invention is to provide an ICP-MS method for detecting 26 elements in bone marrow supernatant. This method can simultaneously detect the content of 26 elements, namely Mg, Al, Ca, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, As, Se, Rb, Sr, Mo, Cd, Sn, Sb, Ba, Pt, Hg, Tl, and Pb, in a single instrumentation. This solves the technical problem of difficulty in simultaneous multi-element detection caused by the complex matrix of bone marrow supernatant samples, small sample volume, large range of element concentrations, and strong adsorption of some elements.

[0006] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows:

[0007] An ICP-MS method for detecting 26 elements in bone marrow supernatant includes the following steps: (1) Prepare a mixed aqueous solution of HNO3 and Triton X-100 as a sample diluent; prepare a mixed aqueous solution of HNO3, Triton X-100, BSA and NaCl as a matrix matching diluent; (2) Select Sc, Y and Lu as internal standards and dilute them with the matrix-matched diluent to obtain a mixed internal standard solution; (3) Prepare a first mixed standard curve solution, a second mixed standard curve solution, and a third mixed standard curve solution respectively. The first mixed standard curve solution is a mixed standard curve solution of 26 elements including Mg, Al, Ca, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, As, Se, Rb, Sr, Mo, Cd, Sn, Sb, Ba, Pt, Hg, Tl, and Pb, with a concentration range of 0~20 μg / L. The second mixed standard curve solution is a mixed standard curve solution of 8 elements including Mg, Ca, Fe, Cu, Zn, Al, Rb, and Se, with a concentration range of 50~500 μg / L. The third mixed standard curve solution is a mixed standard curve solution of 3 elements including Mg, Ca, and Fe, with a concentration range of 1000~8000 μg / L. The first mixed standard curve solution, the second mixed standard curve solution, and the third mixed standard curve solution are all prepared using the matrix matching diluent. (4) The first mixed standard curve solution, the second mixed standard curve solution and the third mixed standard curve solution from step (3) are injected into the inductively coupled plasma mass spectrometer in sequence, and the mixed internal standard solution from step (2) is introduced online at the same time to plot the standard curves of 26 elements; (5) Using the direct dilution method, take the bone marrow supernatant sample to be tested, dilute it with the sample diluent, and vortex and centrifuge in sequence to obtain the sample solution to be tested; (6) Inject the sample solution to be tested described in step (5) into the inductively coupled plasma mass spectrometer, and simultaneously introduce the mixed internal standard solution described in step (2) online for determination. Using the standard curve and internal standard correction drawn in step (4), the content of 26 elements in the sample solution to be tested is calculated.

[0008] Preferably, in step (1), the final concentration of HNO3 in the sample diluent is 0.4%~0.6%vol, the final concentration of Triton X-100 is 0.04%~0.06%vol, and the final concentration of BSA in the matrix matching diluent is 0.35~0.55 g / L.

[0009] More preferably, in step (1), the final concentration of HNO3 in the sample diluent is 0.5%vol, the final concentration of Triton X-100 is 0.05%vol; and the final concentration of HNO3, Triton X-100, BSA, and NaCl in the matrix matching diluent is 0.45 g / L.

[0010] The design principle of the sample diluent and matrix-matched diluent is as follows: The sample diluent is free of BSA and NaCl and is used for 20-fold direct dilution of the sample, which can rapidly destroy protein structure and release elements; the matrix-matched diluent contains 3.5 g / L BSA and 0.45 g / L NaCl, and its concentration is designed based on the fact that after 20-fold dilution of bone marrow supernatant samples, the residual concentration of endogenous proteins and inorganic salts is equivalent to this concentration. Through the dual dilution strategy, the consistency of the matrix environment of the standard curve, internal standard solution and test sample during instrument detection is ensured without increasing sample consumption.

[0011] Preferably, in step (2), the concentrations of Sc, Y and Lu in the mixed internal standard solution are each 20~100μg / L; in step (5), the volume ratio of the bone marrow supernatant sample to be tested to the sample diluent is 1:15~30.

[0012] More preferably, in step (2), the concentrations of Sc, Y and Lu in the mixed internal standard solution are all 50 μg / L; in step (5), the volume ratio of the bone marrow supernatant sample to be tested to the sample diluent is 1:19.

[0013] Preferably, in step (3), the standard point concentrations of the first mixed standard curve solution are 0 μg / L, 0.02 μg / L, 0.05 μg / L, 0.1 μg / L, 0.2 μg / L, 0.5 μg / L, 1 μg / L, 2 μg / L, 5 μg / L, 10 μg / L, and 20 μg / L, respectively. The preparation of the first mixed standard curve solution is as follows: take a 33-element mixed standard solution containing 26 elements (Mg, Al, Ca, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, As, Se, Rb, Sr, Mo, Cd, Sn, Sb, Ba, Pt, Hg, Tl, and Pb) as the mother liquor, and dilute the mixed standard curve mother liquor with a matrix-matching diluent to obtain mixed standard curve solutions with different concentration gradients.

[0014] Preferably, in step (3), the standard point concentrations of the second mixed standard curve solution are 50 μg / L, 100 μg / L, and 500 μg / L, respectively; the preparation of the second mixed standard curve solution is as follows: take single-element standard solutions of Mg, Ca, Fe, Cu, Zn, Al, Rb, and Se, first dilute them with matrix matching diluent to obtain mixed standard curve stock solution, and then dilute the mixed standard curve stock solution with matrix matching diluent to obtain mixed standard curve solutions with different concentration gradients.

[0015] Preferably, in step (3), the standard point concentrations of the third mixed standard curve solution are 1000 μg / L, 4000 μg / L, and 8000 μg / L, respectively; the preparation of the third mixed standard curve solution specifically involves taking single-element standard solutions of Mg, Ca, and Fe, diluting them first with matrix-matched diluent to obtain a mixed standard curve stock solution, and then diluting the mixed standard curve stock solution with matrix-matched diluent to obtain mixed standard curve solutions with different concentration gradients.

[0016] The design principle of the three-segment standard curve is as follows: The concentration range of the 26 elements in bone marrow supernatant spans five orders of magnitude, and heavy metals such as Tl and Hg have strong adsorption properties. If a single ultra-wide range standard curve and a high-concentration multi-element mixed mother liquor are used, the above-mentioned easily adsorbed elements will produce a severe memory effect in the instrument, interfering with subsequent detection. This invention divides the 26 elements into three groups according to the expected concentration: The first group covers the low concentration end of all 26 elements (0~20 μg / L), ensuring that the highest standard point concentration of easily adsorbed elements such as Tl and Hg is controlled within 20 μg / L, significantly reducing the memory effect; the second group (50~500 μg / L) and the third group (1000~8000 μg / L) are for medium and high concentration elements, respectively, and are prepared using single-element standard solutions. This design ensures wide dynamic range coverage while reducing element memory effect, and is simple to prepare, making it more suitable for rapid detection of bone marrow supernatant with small sample volumes.

[0017] Preferably, in step (5), the bone marrow supernatant sample to be tested is derived from human bone marrow, excluding bone marrow from other animals, and is obtained by centrifuging at 3000 rpm for 10 min and then taking the supernatant.

[0018] Preferably, in step (5), the volume ratio of the bone marrow supernatant sample to the sample diluent is 1:19; the vortexing time is 10-20 s, the centrifugation speed is 4000-6000 rpm, and the centrifugation time is 5-15 min. More preferably, the vortexing time is 10 s, the centrifugation speed is 5000 rpm, and the centrifugation time is 10 min.

[0019] Preferably, in steps (4) and (6), the inductively coupled plasma mass spectrometer uses collision mode, with a He gas flow rate of 4.5~5.5 mL / min and online internal standard calibration. More preferably, the helium gas flow rate is 5 mL / min.

[0020] More preferably, in steps (4) and (6), the inductively coupled plasma mass spectrometer is a Thermo Scientific iCAP MTX triple quadrupole inductively coupled plasma mass spectrometer.

[0021] Internal standard calibration is a routine method in ICP-MS analysis to eliminate signal drift and matrix effects. The selection of internal standard elements follows generally accepted principles in the field: elements with similar mass numbers and ionization energies to the analyte, and which are absent or negligible in the sample. Based on these principles, the mass numbers of the 26 elements used in this method and their corresponding internal standard elements are as follows: Low mass number element Mg... 24 Al 27 Ca 44 Ti 49 V 51 Cr 52 Mn 55 Fe 56 Co 59 Ni 60 Cu 63 Zn 66 Ga 69 As 75 Se 78 Select 45 Sc is the internal index element, and Rb is the medium mass number element. 85 、Sr 88 Mo 95 Cd 111 Sn 118 Sb 121 Ba 138 Select 89Y is the internal index element, and Pt is the high-quality number element. 195 Hg 202 、Tl 205 Pb 208 Select 175 Lu is an internal index element.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Dual-dilution matrix matching system: This invention innovatively adopts a dual-dilution strategy of "sample diluent (without BSA / NaCl) + matrix matching diluent (containing BSA / NaCl)" to ensure that the matrix environment of the standard curve, internal standard solution and bone marrow supernatant sample diluted 20 times is consistent. This design differs from the single diluent or the addition of organic substances such as isopropanol commonly used in existing biological sample detection, and significantly reduces the matrix effect caused by the complex matrix of high protein and high salt in bone marrow supernatant. The spiked recovery rate of all elements is stable at 70%~130%, and the internal standard recovery rate is stable at 80%~120%.

[0023] (2) Three-segment standard curve design: Considering the large range of element concentrations in bone marrow supernatant and the strong adsorption of elements such as Tl and Hg, the standard curve was optimized into three groups, with the second and third groups prepared using single-element standard solutions. This design ensures accurate quantification of low, medium, and high concentration elements within their respective optimal linear ranges while effectively controlling memory effects and cross-contamination. It is also simple to prepare and more suitable for rapid clinical testing with small sample sizes.

[0024] (3) Simultaneous detection of trace samples in a single injection: Using the direct dilution method, only 80 μL of bone marrow supernatant sample is required. After dilution by 20 times, 26 elements can be detected simultaneously in a single injection, covering essential trace elements, heavy metals, alkali metals, and transition metals, meeting the clinical needs for bone marrow elemental spectrum analysis. At the same time, by optimizing parameters such as pump tube length, pump speed, and sample lift time, the injection volume is minimized while increasing the number of elements, solving the problem of extremely small bone marrow supernatant sample volume; (4) The pretreatment process is simple and fast: the bone marrow supernatant sample is processed by direct dilution method, which only requires three steps: dilution, vortexing and centrifugation. The operation is simple and reduces the possibility of sample element contamination or loss due to complex pretreatment. (5) Excellent linear relationship: The correlation coefficient r of the standard curves of the 26 elements is ≥0.999, which ensures the reliability and accuracy of the detection data.

[0025] (6) Good detection stability: Through online internal standard calibration combined with matrix-matched dilution design, the internal standard recovery rate is stable at 80%~120%, indicating that the matrix of the method is well matched and the detection process is stable and controllable.

[0026] (7) High accuracy: The spiked recovery rates of the 26 elements are basically between 70% and 130%, which meets the accuracy requirements for the detection of biological sample elements. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a standard curve diagram of elements Mg, Al, V, Cr, Co, Ni, Ga, and As in an embodiment of the present invention.

[0029] Figure 2 This is a standard curve diagram of elements Ca, Ti, Mn, Fe, Cu, Zn, Se, and Rb in an embodiment of the present invention.

[0030] Figure 3 This is a standard curve diagram of elements Sr, Mo, Sb, Ba, Tl, and Pb in an embodiment of the present invention.

[0031] Figure 4 This is a standard curve diagram of elements Cd, Sn, Pt, and Hg in an embodiment of the present invention.

[0032] Figure 5 This is a graph showing the recovery rate of the internal standard under dilution A conditions in this embodiment of the invention.

[0033] Figure 6 This is a graph showing the recovery rate of the internal standard under dilution B conditions in an embodiment of the present invention.

[0034] Figure 7 This is a graph showing the recovery rate of the internal standard under diluent C conditions in this embodiment of the invention.

[0035] Figure 8 These are daytime and intraday precision graphs in an embodiment of the present invention. Detailed Implementation

[0036] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0037] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0038] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0039] The bone marrow supernatant sample tested in this embodiment of the invention is derived from human bone marrow, excluding bone marrow from other animals. It is obtained by centrifuging at 3000 rpm for 10 min and then collecting the supernatant.

[0040] Example: An ICP-MS method for detecting 26 elements in bone marrow supernatant includes the following steps: 1. Major instruments, consumables, and reagents 1.1 Main Instruments Thermo Scientific iCAP MTX triple quadrupole inductively coupled plasma mass spectrometry (ICP-MS) from the USA, Milli-Q Advantage A10 ultrapure water system from the USA, Eppendorf Centrifuge 5430 high-speed centrifuge from Germany, and QL-902 vortex mixer from Haimen Qilin Bell Instrument Manufacturing Co., Ltd.

[0041] 1.2 Main Consumables Eppendorf pipettes (5mL; 1; 200μL; 100μL, 20μL), Corning centrifuge tubes (50mL, 15mL), Guangzhou Jet Biofiltration Co., Ltd. microcentrifuge tubes (2mL), Corning volumetric flasks (100mL, 200mL).

[0042] 1.3 Main Reagents Table 1 Main Reagents

[0043] 2. ICP-MS method for detecting the content of 26 elements in bone marrow supernatant samples 2.1 ICP-MS Instrument Detection Parameter Settings Table 2 Instrument Detection Parameter Settings

[0044] 2.2 Mass numbers of the 26 elements and the internal standard elements used Table 3. Mass numbers of 26 elements and their internal standard elements.

[0045] 2.3 Solution Preparation 2.3.1 Preparation of sample diluent: First, pour a certain amount of ultrapure water into a 200 mL volumetric flask, then accurately add 1.0 mL of 68% vol HNO3, followed by 100 μL of Triton X-100. Make up the volume with ultrapure water to 200 mL and sonicate to mix. The resulting solution is a mixed aqueous solution of 0.5% vol HNO3 and 0.05% vol Triton X-100, used for direct dilution of bone marrow supernatant samples.

[0046] 2.3.2 Preparation of matrix-matching diluent: First, pour a certain amount of ultrapure water into a 200 mL volumetric flask, then accurately add 1.0 mL of 68% vol HNO3, followed by 100 μL of Triton X-100, 0.70 g of BSA, and 0.09 g of NaCl. Make up the volume with ultrapure water to 200 mL and sonicate thoroughly. The resulting solution is a mixed aqueous solution with a final concentration of 0.5% vol HNO3, 0.05% vol Triton X-100, 3.5 g / L BSA, and 0.45 g / L NaCl. This solution is used to prepare the standard curve solution and internal standard solution to ensure matrix consistency with the diluted bone marrow supernatant sample.

[0047] The design of the BSA and NaCl concentrations in the matrix-matched diluent is based on the following: after the bone marrow supernatant sample is diluted 20-fold, the concentrations of its endogenous proteins and inorganic salts are comparable to the concentrations of 3.5 g / L BSA and 0.45 g / L NaCl in the matrix-matched diluent, thereby ensuring that the standard curve, internal standard solution and the matrix environment of the sample to be tested are consistent during the instrument detection.

[0048] 2.3.3 Preparation of mixed internal standard solution: First, pour a certain amount of the matrix-matched diluent into a 100 mL volumetric flask. Then, add 50 μL of an 8-element standard solution containing Sc and Lu, and 50 μL of a single-element standard solution containing Y. Make up to 100 mL with the matrix-matched diluent and mix thoroughly by inverting the flask. The concentrations of Sc, Y, and Lu in the mixed internal standard solution are all 50 μg / L.

[0049] 2.3.4 Preparation of mixed standard curve solution: (1) Preparation of the first mixed standard curve solution (0~20 μg / L): Take a 33-element standard solution containing 26 elements (Mg, Al, Ca, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, As, Se, Rb, Sr, Mo, Cd, Sn, Sb, Ba, Pt, Hg, Tl, and Pb), first dilute it with the matrix-matched diluent to obtain a mixed standard curve stock solution (each element concentration is 200 μg / L), and then dilute it with the matrix-matched diluent to obtain mixed standard curve solutions with different concentration gradients.

[0050] Table 4. Preparation method of the first mixed standard curve (STD0~STD10)

[0051] (2) Preparation of the second mixed standard curve solution (50~500 μg / L): Take single-element standard solutions of Mg, Ca, Fe, Cu, Zn, Al, Rb, and Se, and first dilute them with the matrix-matched diluent to obtain a mixed standard curve stock solution (each element concentration is 5 mg / L). Then dilute them with the matrix-matched diluent to obtain mixed standard curve solutions with different concentration gradients.

[0052] Table 5. Preparation method of the second mixed standard curve (STD11~STD13)

[0053] (3) Preparation of the third mixed standard curve solution (1000~8000 μg / L): Take single-element standard solutions of Mg, Ca, and Fe, and first dilute them with the matrix-matched diluent to obtain a mixed standard curve stock solution (each element concentration is 100 mg / L). Then dilute them with the matrix-matched diluent to obtain mixed standard curve solutions with different concentration gradients.

[0054] Table 6. Preparation method of the third mixed standard curve (STD14~STD16)

[0055] 2.4 Sample Pretreatment Take 80 μL of the bone marrow supernatant sample to be tested into a 2 mL microcentrifuge tube, add 1.52 mL of the above sample diluent, vortex mix for 10 s, centrifuge at 5000 rpm for 10 min, and take the supernatant as the sample solution to be tested.

[0056] 2.5 On-machine testing The first, second, and third mixed standard curve solutions were sequentially injected into the inductively coupled plasma mass spectrometer (ICP-MS), while a mixed internal standard solution was introduced online. After subtracting the detection value of STD0 from the detection values ​​of all 16 standard points (STD1-STD16), five consecutive standard points were selected according to the linear range of each element in Table 7 to plot standard curves for 26 elements. Then, the sample solution and the mixed internal standard solution were injected into the ICP-MS to determine the content of the 26 elements in the sample solution.

[0057] 2.6 Methodological Validation 2.6.1 Standard Curve Standard curves for 26 elements, as shown Figures 1 to 4 As shown in Table 8, the specific correlation coefficient values ​​r are all ≥0.999 for all 26 elements, ensuring the reliability and accuracy of the detection data.

[0058] 2.6.2 Accuracy – Spike Recovery Experiment Measurement procedure: Take 2 mL of each of the four mixed bone marrow supernatant samples. One sample is used as the original sample, and the other three samples are spiked with low-concentration, medium-concentration, and high-concentration standard solutions, respectively. The low-concentration and medium-concentration standard solutions are 33-element standard solutions, and the high-concentration standard solution is a mixed solution prepared from single-element standard solutions of Mg, Ca, Fe, Cu, Zn, Al, Rb, and Se (Mg, Ca, and Fe concentrations are 1000 mg / L, and Cu, Zn, Al, and Rb concentrations are 100 mg / L). Then, process the samples according to the above sample pretreatment method, and perform instrumental analysis. Calculate the spike recovery rate of each element using the formula [Spiked recovery rate = (Spiked sample value - Original sample value) / Spiked concentration × 100%]. Based on the principle that the concentration of each element in the original sample is close to the spiked concentration, select one of the spiked samples (1, 2, and 3) to calculate the spike recovery rate of each element. Typically, a spiked recovery rate of 70% to 130% is preferred. The spiked recovery rate results of this method are shown in Table 8. The spiked recoveries of all 26 elements are between 70% and 130%, which meets the accuracy requirements for element detection in biological samples.

[0059] Table 7. Sample Spiking Method

[0060] 2.6.3 Internal standard recovery rate Internal standard recovery rate is mainly used to determine whether the sample matrix matches and is stable with the matrix of the standard curve. Generally, an internal standard recovery rate between 80% and 120% is considered optimal. The internal standard recovery rate in this method for detecting bone marrow supernatant samples is shown in [link to relevant documentation]. Figures 5-7It can be observed that the internal standard recoveries of the three internal standard elements, Sc, Y, and Lu, are relatively stable, fluctuating between 80% and 120%, indicating that this method can ensure matrix stability and that the standard curve matches the sample matrix well.

[0061] 2.6.4 Limit of Detection and Limit of Quantification The sample blank (FQBLK), i.e., the above sample dilution, was measured 11 times. The limit of detection (LOD) and limit of quantitation (LOQ) were calculated using the following formulas: LOD = 3 × standard deviation (S) of the signal response value of the analyte in FQBLK / slope of the working curve (k); LQ = 10S / k. The LOD and LQ of this method are shown in Table 8.

[0062] Table 8. Linear range, correlation coefficient, detection limit, and quantitation limit for 26 elements.

[0063] 2.6.5 Precision The intra-day precision is calculated by repeatedly testing the same spiked sample six times within a day and calculating the relative standard deviation (RSD). The inter-day precision is calculated by testing the same spiked sample three times daily over three days and calculating the RSD. A precision of ≤10% for both intra-day and inter-day precision is considered excellent. Figure 8 The results show that the intra-day and inter-day precision of this method are both ≤10%, indicating that the method has good repeatability.

[0064] 2.7 Optimization Experiment of Key Parameters Currently, the pretreatment dilution system for biological samples often uses a mixed aqueous solution containing 0.1%–1% HNO3 and 0.01%–0.1% Triton X-100. The role of dilute HNO3 is to denature proteins, releasing bound elements and maintaining solution stability; however, excessively high concentrations may lead to protein precipitation and loss of certain elements through co-precipitation. The surfactant Triton X-100 improves sample homogeneity, prevents protein aggregation after denaturation, and reduces instrument adsorption; however, excessively high concentrations can generate excessive foam, interfering with detection. In ICP-MS detection, introducing He gas can reduce signal overlap caused by polyatomic ions; however, excessively high He gas flow rates can significantly reduce the signal values ​​of the analytes, while excessively low flow rates are insufficient to eliminate interference. Therefore, to screen the optimal conditions for multi-element detection in bone marrow supernatant samples, L9(3) was used. 4A three-factor, three-level orthogonal experiment was designed using an orthogonal experimental design table. The factors and levels were set as follows: HNO3 concentration (factor A) was 0.1%, 0.5%, and 1.0%; Triton X-100 concentration (factor B) was 0.01%, 0.05%, and 0.10%; and He gas flow rate (factor C) was 3 mL / min, 4 mL / min, and 5 mL / min. Nine parallel experiments were conducted, using the overall concentration (C), signal-to-noise ratio (S / N), and relative standard deviation (RSD) of 26 elements in the bone marrow supernatant as comprehensive evaluation indicators. Range analysis was used to determine the order of influence of each factor on the detection performance and the optimal combination of levels. Combined with the results of analysis of variance, the optimal basic experimental conditions were determined.

[0065] Range analysis showed that the order of influence of each factor on the concentration index was Triton X-100 concentration > HNO3 concentration > He gas flow rate, with the optimal level combination being A3B2C3; the order of influence of each factor on the signal-to-noise ratio index was HNO3 concentration > He gas flow rate > Triton X-100 concentration, with the optimal level combination being A2B2C3; the order of influence of each factor on the relative standard deviation index was Triton X-100 concentration > Helium gas flow rate > HNO3 concentration, with the optimal level combination being A1B3C1. Analysis of variance showed that Triton X-100 concentration had a significant impact on the relative standard deviation, and the results are shown in Tables 10-12. Considering the three evaluation indicators, and the fact that 1% HNO3 causes precipitation in the sample, the S / N ratio is the most important evaluation indicator. Furthermore, 0.1% Triton X-100 easily generates a large number of bubbles during reagent preparation. Therefore, the basic optimal conditions were determined to be: 0.5% HNO3, 0.05% Triton X-100, and 5 mL / min He gas flow rate.

[0066] Table 9. Results of Orthogonal Experiments

[0067] Table 10. Results of ANOVA for Concentration C

[0068] Table 11. Results of S / N variance analysis for signal-to-noise ratio (S / N)

[0069] Table 12. Results of Analysis of Variance for Relative Standard Deviation (RSD)

[0070] 2.8 Verification experiment on the selection of diluent in pretreatment conditions To verify the limitations of using traditional diluents (aqueous solutions containing HNO3 and Triton X-100) suitable for biological samples such as blood and urine, and common modified diluents with added organic substances such as isopropanol and n-butanol, directly for bone marrow supernatant sample detection, and to demonstrate the irreplaceable nature of the matrix-matched diluent of this invention for the complex matrix of bone marrow supernatant, parallel comparative experiments were conducted using the same bone marrow supernatant sample with the following three diluents: Diluent A (conventional diluent): a mixed aqueous solution of 0.5% HNO3 and 0.05% Triton X-100, used for sample pretreatment, preparation of standard curves and internal standard solutions; Diluent B (common modified diluent): a mixed aqueous solution of 0.5% HNO3, 0.05% Triton X-100 and 2% isopropanol, used for sample pretreatment, preparation of standard curves and internal standard solutions; Diluent C (matrix-matched diluent of this invention): a mixed aqueous solution of 0.5% HNO3, 0.05% Triton X-100, 3.5 g / L BSA and 0.45 g / L NaCl, used for preparation of standard curves and internal standard solutions. Diluent A is used for sample pretreatment to achieve the purpose of matching the standard solution with the sample matrix.

[0071] Following the experimental steps described above, reagents were prepared, samples were pretreated, and the samples were tested. The internal standard recovery rate and spiked recovery rate were compared under different dilution conditions.

[0072] The results showed that under dilution A, the spiked recovery rate of one element (Zn) exceeded 70-130%; under dilution B, the spiked recoveries of four elements (Ca, Mn, Ba, and Pb) exceeded 70-130%; and under dilution C, the spiked recoveries of no element exceeded 70-130%. The concentrations and spiked recoveries of 26 elements in the pretreated bone marrow supernatant samples under the three dilution conditions are shown in Table 13. Under dilution A, the internal standard recoveries of Y and Lu exceeded 80-120%; under dilution B, the internal standard recovery of Lu exceeded 80-120%; and under dilution C, the internal standard recoveries of no element exceeded 80-120%. Specific results are as follows: Figures 5-7 As shown. In summary, diluent C, namely a mixed aqueous solution of 0.5% HNO3, 0.05% Triton X-100, 3.5 g / L BSA, and 0.45 g / L NaCl (the above matrix-matched diluent), was selected as the diluent for the preparation of the standard curve and internal standard solution; a mixed aqueous solution of 0.5% HNO3 and 0.05% Triton X-100 (the above sample diluent) was used for sample pretreatment, which can significantly eliminate the interference of complex matrices on the simultaneous detection of multiple elements.

[0073] Table 13. Pretreatment bone marrow supernatant concentration and spiked recovery rate results for three dilutions (A, B, and C).

[0074] In summary, the detection method of this invention can simultaneously detect the content of 26 elements (Mg, Al, Ca, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, As, Se, Rb, Sr, Mo, Cd, Sn, Sb, Ba, Pt, Hg, Tl, and Pb) in bone marrow supernatant in a single run. Furthermore, through matrix matching design, it significantly reduces the matrix effect caused by the complex matrix of high protein and high salt content in bone marrow supernatant. The method also requires a small sample size, has a rapid and simple pretreatment process, provides an excellent standard curve, high accuracy, and good sample-matrix matching.

Claims

1. An ICP-MS method for detecting 26 elements in bone marrow supernatant, characterized in that, Includes the following steps: (1) Prepare a mixed aqueous solution of HNO3 and Triton X-100 as a sample diluent; prepare a mixed aqueous solution of HNO3, Triton X-100, BSA and NaCl as a matrix matching diluent; (2) Select Sc, Y and Lu as internal standards and dilute them with the matrix-matched diluent to obtain a mixed internal standard solution; (3) Prepare a first mixed standard curve solution, a second mixed standard curve solution, and a third mixed standard curve solution respectively. The first mixed standard curve solution is a mixed standard curve solution of 26 elements including Mg, Al, Ca, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, As, Se, Rb, Sr, Mo, Cd, Sn, Sb, Ba, Pt, Hg, Tl, and Pb, with a concentration range of 0~20 μg / L. The second mixed standard curve solution is a mixed standard curve solution of 8 elements including Mg, Ca, Fe, Cu, Zn, Al, Rb, and Se, with a concentration range of 50~500 μg / L. The third mixed standard curve solution is a mixed standard curve solution of 3 elements including Mg, Ca, and Fe, with a concentration range of 1000~8000 μg / L. The first mixed standard curve solution, the second mixed standard curve solution, and the third mixed standard curve solution are all prepared using the matrix matching diluent. (4) The first mixed standard curve solution, the second mixed standard curve solution and the third mixed standard curve solution from step (3) are injected into the inductively coupled plasma mass spectrometer in sequence, and the mixed internal standard solution from step (2) is introduced online at the same time to plot the standard curves of 26 elements; (5) Using the direct dilution method, take the bone marrow supernatant sample to be tested, dilute it with the sample diluent, and vortex and centrifuge in sequence to obtain the sample solution to be tested; (6) Inject the sample solution to be tested described in step (5) into the inductively coupled plasma mass spectrometer, and simultaneously introduce the mixed internal standard solution described in step (2) online for determination. Using the standard curve and internal standard correction drawn in step (4), the content of 26 elements in the sample solution to be tested is calculated.

2. The ICP-MS detection method according to claim 1, characterized in that, In step (1), the final concentration of HNO3 in the sample diluent is 0.4%~0.6%vol, the final concentration of Triton X-100 is 0.04%~0.06%vol, and the final concentration of BSA in the matrix matching diluent is 0.35~0.55 g / L.

3. The ICP-MS detection method according to claim 2, characterized in that, In step (1), the final concentration of HNO3 in the sample diluent is 0.5%vol, and the final concentration of Triton X-100 is 0.05%vol; the final concentration of HNO3 in the matrix matching diluent is 0.5%vol, the final concentration of Triton X-100 is 0.05%vol, the final concentration of BSA is 3.5 g / L, and the final concentration of NaCl is 0.45 g / L.

4. The ICP-MS detection method according to claim 1, characterized in that, In step (2), the concentrations of Sc, Y and Lu in the mixed internal standard solution are each 20~100 μg / L; in step (5), the volume ratio of the bone marrow supernatant sample to be tested to the sample diluent is 1:15~30.

5. The ICP-MS detection method according to claim 4, characterized in that, In step (2), the concentrations of Sc, Y and Lu in the mixed internal standard solution are all 50 μg / L; in step (5), the volume ratio of the bone marrow supernatant sample to be tested to the sample diluent is 1:

19.

6. The ICP-MS detection method according to claim 1, characterized in that, In step (3), the standard point concentrations of the first mixed standard curve solution are 0 μg / L, 0.02 μg / L, 0.05 μg / L, 0.1 μg / L, 0.2 μg / L, 0.5 μg / L, 1 μg / L, 2 μg / L, 5 μg / L, 10 μg / L, and 20 μg / L, respectively; the standard point concentrations of the second mixed standard curve solution are 50 μg / L, 100 μg / L, and 500 μg / L, respectively; and the standard point concentrations of the third mixed standard curve solution are 1000 μg / L, 4000 μg / L, and 8000 μg / L, respectively.

7. The ICP-MS detection method according to claim 1, characterized in that, In step (5), the bone marrow supernatant sample to be tested is derived from human bone marrow, excluding bone marrow from other animals. It is obtained by centrifuging at 3000 rpm for 10 min and then taking the supernatant.

8. The ICP-MS detection method according to claim 1, characterized in that, In step (5), the volume ratio of the bone marrow supernatant sample to the sample diluent is 1:19; the vortexing time is 10~20 s, the centrifugation speed is 4000~6000 rpm, and the centrifugation time is 5~15 min.

9. The ICP-MS detection method according to claim 1, characterized in that, In steps (4) and (6), the inductively coupled plasma mass spectrometer uses collision mode for analysis, with a He gas flow rate of 4.5~5.5 mL / min and online internal standard calibration.

10. The ICP-MS detection method according to claim 9, characterized in that, In steps (4) and (6), the inductively coupled plasma mass spectrometer is a Thermo Scientific iCAP MTX triple quadrupole inductively coupled plasma mass spectrometer.

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