CN113866255A - Inductively coupled plasma mass spectrometry detection of 10 elements in peripheral blood - Google Patents

Abstract

The invention provides an ICPMS detection method of 10 elements in peripheral blood, which comprises the following steps: sample collection, sample preparation, sample storage and on-machine detection; wherein, a contact pressure type peripheral blood collector is adopted when a sample is collected, and the blood collection amount (40 muL, 60 muL, 80 muL and 100 muL) is verified through a contrast experiment; the sample is prepared by a dilution method, and the dilution liquid contains nitric acid, triton and isopropanol; the storage time of the diluted sample (0 hour, 24 hours, 48 hours and 72 hours) is verified by a comparative experiment; 10 elements of magnesium, calcium, manganese, iron, copper, zinc, arsenic, selenium, cadmium and lead are detected by an inductively coupled plasma mass spectrometry method. The mass spectrometry detection method for the inductively coupled plasma of 10 elements in peripheral blood has the advantages of small blood collection amount, long storage time, simple and reliable sample preparation method, high repeatability, low instrument detection limit and high precision, is suitable for ICPMS instruments of different brands, and is suitable for batch detection of trace elements in whole blood.

Description

Inductively coupled plasma mass spectrometry detection of 10 elements in peripheral blood

Technical Field

The invention relates to the field of inorganic trace element detection, and discloses a brand new detection method for inductively coupled plasma mass spectrometry (ICP-MS) detection of various elements in human peripheral blood.

Background

The trace elements are contained in a human body in a very small amount, generally less than 0.01% of the total weight of the human body, and the daily requirement of each person is less than 100 mg. Medical research shows that although the content of elements in human bodies or animals is not large, the elements have important effects on human health. The research of molecular biology reveals that elements are combined with proteins and other organic groups to form biomacromolecules such as enzymes, hormones, vitamins and the like, and play important physiological and biochemical functions. The trace elements have duality in human bodies, and can cause corresponding diseases when being ingested insufficiently; when the intake amount is excessive, the damage to the human body is also caused. With the increasingly clear relationship between trace elements and diseases and the close attention of people to health problems, the method for accurately, quickly and conveniently measuring the content of trace elements in human bodies becomes a problem to be solved in the field of trace element detection. In addition, the analysis of trace elements in blood is also often used as a biological monitoring means for food production, environmental pollution, agricultural production and occupational health research, and plays a monitoring role in related fields.

If the content of beneficial elements such as calcium, magnesium, iron, zinc, selenium and the like in human serum is lower than a standard range, the human serum is prompted to be supplemented correspondingly, or the correlation between the human serum and corresponding diseases such as iron deficiency anemia, growth and development retardation of children, osteoporosis and keshan disease is prompted. If the content of harmful elements such as lead, cadmium, mercury, arsenic and thallium in the serum exceeds the limit, poisoning is prompted, and corresponding detoxification measures must be taken. For some trace elements associated with specific diseases, the change of the content thereof indicates the development stage and prognosis of the disease.

The biological samples involved in the analysis of human body elements include blood, urine, hair, bone, nail, etc., among which blood is the most commonly used. Research shows that the total amount of Fe, Cu and Zn in the body is reduced, so that the immunity can be weakened, bacterial infection is facilitated, the death rate after infection is high, and the like; when some trace elements (heavy metal elements) exceed a certain concentration, acute poisoning can be caused or long-term poisoning effect can be generated in human body. In particular, with the development of the industry today, metal contamination involves various areas of human activity that can cause poisoning when the concentration of each metal element exceeds certain biological contact limits. Research shows that the content measurement of human toxicants or toxicant metabolites is the most key objective basis for clinically confirming toxicant poisoning, and the increase of toxic substances in biological materials can indicate the excessive absorption of the toxicants in the body.

In addition, in the process of detoxification, the detection of trace elements of various novel drugs has become the key point in the work of detoxification, and in the process of judicial practice, cases such as exposure to inorganic element poisons and suicide often occur, for example, in forensic identification, the cause and the degree of poisoning need to be accurately and quickly judged, but the uncertainty of the target object of the inorganic element and the universality of the type of poisons cause element detection to have certain complexity, and similar symptoms may appear in acute poisoning of different elements. In addition, in the detection work of the in vivo stimulant, due to the characteristics of rapid metabolism, low residue and difficult detection of various novel stimulants, the detection of the trace components of the stimulant after overtime metabolism is always a research hotspot of the anti-stimulant work in the sports community. Therefore, in addition to the health monitoring work such as disease diagnosis and environmental and occupational diseases, the method can be used for accurately and quickly analyzing and detecting inorganic elements in judicial and law enforcement works and other fields (such as anti-stimulant works), and has important significance for understanding the true situation.

The contents of trace elements such as magnesium, calcium, manganese, iron, copper, zinc, arsenic, selenium and lead in the whole blood can reflect the concentration of the elements in a human body, and can be used as a reliable basis for health care, auxiliary diagnosis, poisoning symptoms and observation of curative effects, so that accurate and rapid trace element analysis and medical evaluation of detection results provide powerful basis for human disease diagnosis and biological monitoring of environmental and occupational health research and toxin clearance and inhibition.

The current detection methods for elements are as follows: atomic Absorption Spectroscopy (AAS), Atomic Emission Spectroscopy (AES), Atomic Fluorescence Spectroscopy (AFS), ultraviolet-visible spectrophotometry (UVS), inductively coupled plasma mass Spectrometry (ICP-MS).

The measurement of trace elements in blood is often carried out by Atomic Absorption Spectroscopy (AAS), which can be classified into graphite furnace atomic absorption spectroscopy (GF-AAS), flow injection-cold vapor generation atomic absorption spectroscopy (FI-VGAAS), and Flame Atomic Absorption Spectroscopy (FAAS) depending on the atomization method. The GF-AAS has the advantages of small sampling amount, simple pretreatment, high sensitivity, capability of directly analyzing solid and high-viscosity liquid samples and the like, is suitable for analyzing biological samples, is widely applied to actual element inspection work, has the main defect of serious matrix interference, and can detect only one element at a time, thereby consuming a long time. FAAS is a classical analysis technology, has the advantages of stable signal, relatively small interference, high analysis speed, simple operation and the like, and is mostly adopted in trace element detection. The main disadvantage of this method is that it is not suitable for the determination of refractory elements, alkaline earth elements and elements with resonance absorption lines in the far ultraviolet range, which are not completely decomposed. The FI-VGAAS can be used for detecting elements formed by hydrides or cold steam, such as arsenic, selenium, bismuth, mercury and the like.

Yan Cheng et al [ Yan Cheng, Li Weixin, Deyuan, atomic absorption spectrometry and ICP-MS methods for thallium measurement, [ J ], Guangdong chemical industry, 2016,9: 232-. Compared with the two methods, the atomic absorption spectrometry has the defects that the operation process is complicated, the detection limit and the linear range are not as good as those of ICP-MS, the detection limit of AAS is 5ug/L, and the detection limit of ICP-MS is 0.024 ug/L. The linear range of the atomic absorption spectrometry is 0-10 mg/L, the linear range of the ICP-MS method is 0-100 ug/L, and the correlation coefficient r is 0.9998. The anti-interference capability is stronger than that of ICP-MS. The ICP-MS has the advantages of capability of realizing simultaneous measurement of multiple elements, more convenient and faster operation and suitability for mass samples.

Inductively Coupled plasma atomic emission spectrometry (ICP-AES) is an atomic emission spectrometry analysis and detection method using Inductively Coupled plasma as an excitation light source and an atomization device. The ICP-AES has the following advantages: 1. can simultaneously detect a plurality of elements in a sample, and has the capability of simultaneously detecting a plurality of elements; 2. the analysis speed is high, and dozens of elements can be quantitatively analyzed in a short time; 3. the detection limit is low, and is generally 1-10 ug/L; 4. the method has high analysis accuracy and precision, is one of various analysis methods with less interference, generally has a relative standard deviation of less than or equal to 10 percent, and has a relative standard deviation of less than or equal to 1 percent when the concentration of the analyte exceeds 100 times of the detection limit.

ICP-AES is applied more widely in the actual element analysis and detection work, for example, the Shen Guo et al [ Shen Guo, Xuxin Yun, Zhang Shun Xiang, Zhang ren Li, He Yaqing, ICP-AES and ICP-MS method for measuring trace elements [ J ] in blood, modern preventive medicine, 2003,30 (5): 644-645], detecting the content of arsenic, lead, selenium, barium, antimony and germanium in the blood of the student with ICP-MS at 0.004-1.08mg/kg, and detecting the content of aluminum, calcium, iron, potassium, magnesium, manganese, copper and zinc in the blood of the student with ICP-AES at 0.03-1409 mg/kg. Xujun et al [ Xujun et al, ICP-MS/ICP-AES method for analyzing 18 element content reference values in adult male serum, Guangdong microelement science, 2006,13(4):17-23], wherein the detection limit of K, Mg, Ca, Na and Fe is 0.02-0.60ug/mL by adopting ICP-AES, and the detection limit of As, Cd, Co and Cr is 0.005-0.1ng/mL by adopting ICP-MS. For the macroelements, ICP-AES can be used for detection, and for the trace elements, the detection limit does not meet the requirement of the trace elements, and ICP-MS is used for detection.

The inductively coupled plasma mass spectrometry (ICP-MS) method uses an ICP torch as an atomization and ionization device of an instrument, uses mass spectrometry as a detection means, can detect most elements in a periodic table, and can meet the requirement on the detection sensitivity. The method has the advantages of simple spectrogram, simple sample preparation and introduction technology, quick mass scanning, high analysis speed, short operation period, good selectivity, wide linear range and low detection limit, can be used for detecting various elements and measuring isotope ratio, is a trace (ultra-trace) inorganic multi-element analysis technology with wide prospect, is widely applied to the fields of metallurgy, environment, medicine, biology and the like at present, becomes the strongest element analysis technology, and is an analysis method suitable for detecting human body fluid samples. ICP-MS has ng/L detection limit for most metal elements, and enrichment treatment is not needed, so that pollution of the environment to a sample is reduced, and reliability and accuracy of an analysis method are improved.

Trace element analysis of blood is often used as a biological monitoring tool for disease diagnosis and environmental and occupational health research. However, because the content of trace elements is very low, most elements are in the ng/L-ug/L level, and the matrix of human blood is complex, a plurality of uncertain influence factors are brought to the detection process, so that the establishment of a set of method for accurately detecting the trace elements in the blood clinically has important significance. ICP-MS (inductively coupled plasma-mass spectrometry) is used for detecting trace elements in a human body, a sample needs to be pretreated firstly, the complex serum components contain proteins, inorganic salts, cholesterol and other components, the complex matrix brings interference to measurement, the pretreatment of the sample is beneficial to reducing matrix interference, and a proper sample pretreatment method can improve the measurement accuracy. The main pretreatment methods include ashing and melting, wet digestion, microwave digestion and direct dilution. The sample treatment is to prevent the loss of the trace component to be measured caused by volatilization, generation of insoluble compound or difficult elution of the trace component adsorbed on the wall of the vessel, and to prevent the pollution introduced in the pretreatment process, and to ensure that the finally obtained sample solution can meet the requirements of the measurement conditions, such as reducing or eliminating the matrix interference as much as possible. Therefore, the selection of an appropriate sample processing method is very important for the analysis work.

The melting technology is mostly used for samples with more silicon, and because the reagent blank adopted by the melting technology is higher, and the content of trace elements in serum is very low (ug/L-ng/L magnitude), the detection of the trace elements can be greatly influenced, the element volatilization can be caused in the melting process, the accuracy and reliability of the detection of the trace elements are influenced, and the ashing method does not adopt melting and ashing methods because large interference can be introduced in the treatment process of a vessel.

Wet digestion is a method that can be used in trace element analysis of biological samples, and although the volatile loss of trace elements in the digestion process is less than that of ashing, the trace elements in serum are low in content and interfere with the determination result of the trace elements, so the method is not suitable for serum trace element detection.

In recent years, microwave digestion is widely applied to biological sample digestion, and can break organic molecules in a sample matrix, convert the organic molecules into clear transparent liquid, convert macromolecules into inorganic elements and facilitate ICP-MS (inductively coupled plasma-mass spectrometry) detection. In the microwave digestion process, because a quartz tube or a Teflon test tube needs to be repeatedly used, pollution is easily introduced between two times of digestion, and the detection result of trace elements is higher. In addition, high-temperature heating is required in the digestion process, and the possibility of element loss exists in the serum sample. Meanwhile, the serum sample is treated by microwave digestion, the process is complicated, and the cooling time is longer after digestion is finished although the digestion time is short, so that the detection of a sample with a large sample amount is not facilitated.

The direct dilution method is a method of directly detecting a diluted sample by adding a diluent to the sample. The direct dilution method is used for preprocessing the sample, can well avoid interference introduced in the digestion process, adopts a high dilution multiple, can inhibit a matrix effect to a certain extent, is simple and convenient to operate, can reduce element loss caused by the preprocessing process as much as possible, is high in sample processing efficiency, and can preprocess a large amount of samples in a short time.

In chemical analysis, precision refers to the degree of consistency between measurements obtained from repeated analysis of a homogeneous sample under controlled conditions using a particular analytical procedure. Precision is determined by occasional errors (except gross errors) indicating reproducibility of the measurement. For example, measurement of intra-day precision and inter-day precision: sampling a control substance with a certain concentration repeatedly for 3 times for three consecutive days, and measuring the precision within and between days. The detection limit then represents the minimum concentration or amount of the substance to be detected that can be detected by the detection method at a given confidence level, where concentration is referred to as the relative detection limit and mass is referred to as the absolute detection limit. Therefore, the higher precision is better, and the value representing the detection limit is real and credible, so in the prior art of detecting trace elements, how to obtain higher precision RSD (namely relative standard deviation) and further finally obtain higher sensitive detection limit is called as an important index for measuring the accuracy of the detection method.

Chinese patent application 201610100493.5 entitled "method for detecting microelements in peripheral blood" discloses a method for detecting microelements in peripheral blood, wherein the used diluent contains HNO with equal volume concentration₃And triton, and is prepared by diluting 100 times of stock solution with volume concentration of 5-15%. Although the invention discloses detectable trace amountsThe elements of magnesium, iron, copper, zinc, lead and calcium, but the invention does not disclose the precise density values and detection limits of each element which can prove the detection effect, so the result is lack of credibility in the absence of test data, and finally, no authorization is given.

Chinese invention patent application 201910486480.X, the name of the invention, "a method for determining the content of lead and cadmium in human whole blood", discloses a method for determining the content of lead and cadmium in human whole blood. The method adopts an ICP-MS (inductively coupled plasma mass spectrometry) method to measure the content of lead and cadmium in human whole blood, and comprises the steps of pretreatment of a blood sample and analysis of the inductively coupled plasma mass spectrometry (ICP-MS). And quantifying the content of lead and cadmium in the blood sample to be detected by an internal standard method. Wherein 1% HNO was used₃Preparing standard working solution, internal standard working solution and tuning working solution from the solutions, and sequentially using 5% HNO₃、1％HNO₃The solution treats and dilutes the whole blood sample before the assay is completed. Although the method combines an internal standard method with an inductively coupled plasma mass spectrometry method, the accuracy of a quantitative result is improved to a certain extent, but the HNO under the concentration is₃The solution still has the problems that the red blood cells cannot be fully digested to fully release the trace elements, the obtained sample has poor homogeneity, a certain residue exists on the tube wall of the reagent, and the detection of all the trace elements is influenced, for example, the precision RSD (relative standard deviation) of the lead and cadmium to be detected is 3.10-4.07 percent and 3.78-4.52 percent, although the solution meets the requirement that the detection is consistent with the standard deviation<The requirement of 5% limits lead quantitation to 0.0625ug/L, but still further improvement possibilities are needed.

Chinese patent 201810107419.5 discloses a kit for detecting elements in whole blood by inductively coupled plasma mass spectrometry and application thereof, which are used for detecting 32 trace elements including 9 elements. The present invention surprisingly found that 0.1% of HNO is present₃And 0.1% triton, the detection result is not ideal and is not suitable for detecting a large amount of clinical samples (see example 2). At the same time, a lower concentration of 0.01% tranatone and 0.06% HNO was used₃When the combined diluent is used, the whole blood diluent can generate precipitates, the stability of the detection result is not ideal, and the combined diluent is not suitable for a large number of clinical samples(see example 3). However, at a specific low concentration of 0.016-0.033% HNO₃The RSD and detection limit of the diluent combined with 0.02-0.04% triton have better values. Based on this discovery, the invention is ultimately authorized.

In addition, as the closest prior art, chinese patent application 201610197111.5, entitled "method for measuring trace elements in human whole blood by inductively coupled plasma mass spectrometry" discloses a method for measuring 7 trace elements in human whole blood, in which a mixture of 0.1% nitric acid and triton is used as a diluent, and whole blood is diluted at a ratio of 1: 30. Wherein the diluent contains low-concentration nitric acid, which can decompose blood cells in blood and release elements contained in the blood cells into the diluent; meanwhile, the triton in the diluent can ensure the uniformity of the fully and uniformly mixed solution. In addition, the invention also prepares the internal standard stock solution into an internal standard working solution with the mass concentration of 100 mu g/L for later use by using a nitric acid solution with the volume concentration of 0.2%, and the internal standard elements are scandium, germanium, yttrium, rhodium, indium, terbium and bismuth. Although the invention obtains better internal precision (i.e. daily precision) and inter-batch precision and has high repeatability, the detection result has poor accuracy, and the detection limit is mostly 0.1% order of magnitude, wherein the detection limits of the iron element and the copper element are 2.491mg/L and 1.908 mug/L respectively, which are far higher than the detection limit value reported in the granted patent 201810107419.5, which further verifies that the accuracy of trace element detection can be seriously influenced by using 0.1% of mixed solution of nitric acid and triton as diluent.

In addition, the existing human whole blood detection means usually takes venous blood, and has the defects of large sampling amount, complex pretreatment method, high sample storage difficulty and great pain to patients. Meanwhile, in the detection of elements in a human body, the concentration of partial elements is very low, even reaches trace level, and the matrix is complex, so that the detection is interfered, and how to accurately, rapidly and efficiently perform multi-element simultaneous determination is a problem to be solved urgently.

Based on the above researches, there is a need for a detection technology for various trace elements (such as magnesium, calcium, manganese, iron, copper, zinc, arsenic, selenium, lead, cadmium, and more) capable of further improving precision and detection limit.

Disclosure of Invention

The invention aims to solve the problems that: the method is to detect 10 trace elements by collecting peripheral blood, so that the problems of complicated venous blood collection operation, large blood collection amount, poor compliance on children and the like are solved; the method overcomes the defects that the detection limit of the existing detection technology can not meet the requirement of detecting the trace peripheral blood, and the existing sample pretreatment method has complex process and is easy to introduce interference. Provides a mass spectrometry detection method of multiple (10 or more) inductively coupled plasmas in peripheral blood, which has the advantages of less blood sampling amount, patient pain alleviation, simple sample preparation, long sample storage time and high detection capability of the used instrument.

In order to solve the above problems, the present invention is improved from the following three inventive principles:

the first principle of the invention is that: nitric acid with low concentration in the known diluent in the field is beneficial to promoting blood cells to release trace elements, and under the condition that the triton ensures the uniformity of the fully-mixed solution, the specific concentration of the diluent is not searched, but non-mass spectrum interference influencing precision and detection limit is mainly eliminated. Therefore, the present invention is different from conventional pretreatment reagents in that: isopropanol with specific content is added into the pretreatment reagent to overcome the adverse effect of matrix effect and enhance the intensity of the detection signal of the trace element to be detected. Meanwhile, in the research, diluted nitric acid is not selected for diluting to perform standard curve constant volume, and element extracting solution with a specific proportion is selected for diluting the internal standard substance and the external standard substance, so that the interference of matrix effect and physical effect is corrected, and the specificity of the reaction is effectively improved.

The second principle of the invention is that: on the basis of the principle, the whole blood quality control product introduced with the standard element to be detected with a specific content range is a solid quality control product for measuring the inorganic element in the whole blood: the quality control product has three concentration levels of low, medium and high, and has the following functions: the measurement precision and measurement accuracy were evaluated or verified.

The third principle of the invention is that: on the basis of the above principle, peripheral blood collection and storage conditions were confirmed by designing peripheral blood sampling amount screening experiments (40. mu.L, 60. mu.L, 80. mu.L, 100. mu.L) and peripheral blood storage time evaluation experiments (24 hours, 48 hours, 72 hours). The peripheral blood and the venous blood are collected by recruiting 38 volunteers, the content of 10 elements is detected respectively, and the consistency of the peripheral blood detection of a plurality of (10) elements is fully verified by comparing the detection results of the peripheral blood and the venous blood of the same volunteer.

Therefore, the first object of the present invention is to provide a method for detecting multiple trace elements in peripheral blood by ICP-MS, which comprises the following steps:

(1) collecting samples: sterilizing the skin surface by using 75% ethanol or isopropanol, collecting blood by using a touch pressure type peripheral blood collector, collecting blood by using a disposable micro blood collection tube, and quantitatively transferring the collected peripheral blood by using a glass capillary latex suction head;

(2) preparing a sample solution to be tested: taking (80 mu L) peripheral blood sample according to the method in the step (1), mixing the peripheral blood sample with 1.9mL sample diluent in a 2.5mL centrifuge tube, and then placing the mixture on a vortex oscillation mixer for mixing for 30 seconds, wherein the sample diluent contains 0.1% (v/v) triton solution and 0.1% (v/v) HNO₃And 2% (v/v) isopropyl alcohol, the balance being purified water;

(3) preparing a blank sample: taking a 2.5mL centrifuge tube filled with 1.9mL of sample diluent, adding no solution, and then placing the centrifuge tube on a vortex oscillation mixer for mixing for 30 seconds;

(4) preparing a quality control detection solution: taking the redissolved whole blood inorganic element quality control product according to the method of (1) to mix with 1.9mL sample diluent in a centrifugal tube of 2.5mL, and then placing the mixture on a vortex oscillation mixer to mix for 30 seconds, wherein the whole blood inorganic element quality control product is a mixed solid quality control product of the whole blood inorganic element quality control product containing heparin anticoagulated bovine whole blood and national standard solution, and has high, medium and low concentrations, and the concentration of each element is shown below:

(5) and (3) storing the sample solution to be tested: storing the diluted sample in a refrigerator at 2-8 deg.C, and testing on a computer for storage time (less than or equal to 72 hr);

(6) preparing an external standard calibrator of an element calibration curve to be measured with corresponding concentration and an internal standard calibrator of calibration instrument drift, wherein the internal standard calibrator is a mixed solution containing Ge, Y, In and Tb elements and prepared by 2% nitric acid, and the concentration of each element is 40 mug/L;

(7) and (3) computer detection:

sequentially introducing a blank sample, 0-5 parts of an external standard calibrator, a quality control detection solution and a sample solution to be detected into an instrument for determination, and adding an internal standard calibrator on line, wherein the sample solution and the internal standard solution are injected in a ratio of 1: 1; taking the ratio of the element signal to be detected in the external standard calibrator to the element signal in the internal standard calibrator as the Y axis, taking the concentration of the element to be detected in the external standard calibrator as the X axis, drawing a calibration curve Y as ax + b, and when the correlation coefficient r of each element calibration curve is²>When the concentration is more than 0.999, the quality control detection liquid can be detected; when the error of the detection result of the quality control detection solution is within a 95% confidence interval, the sample solution to be detected can be continuously detected;

the external standard calibration product is a mixed 6-concentration gradient solution containing Mg, Ca, Mn, Fe, Cu, Zn, As, Se, Cd and Pb elements and prepared by 2% nitric acid, and the gradient concentration of each element is As follows:

(8) result processing

The software automatically calculates the mass concentration (mu g/L) of each element to be detected in the sample liquid to be detected according to the regression equation in the step (7), and the calculation of the concentration of the trace elements in the peripheral blood sample is as follows:

ρ_(X)＝(C_X-C₀)×K…………………………(1)

in the formula:

ρ_(X)-mass concentration of trace elements in peripheral blood samples in micrograms per liter (μ g/L);

C_Xthe mass concentration of the trace elements in the sample solution to be tested is determined by a standard curve, and the unit is microgram per liter (mu g/L);

C₀-mass concentration of trace elements in microgram per liter (. mu.g/L) in blank samples from a standard curve;

k is the whole blood dilution factor;

V_X-peripheral blood volume in milliliters (mL).

In one embodiment, both hands are washed with soapy water or a hand sanitizer before collection in the step (1), so that exogenous pollution is reduced as much as possible, and then the hands are disinfected by using 75% ethanol or isopropanol, so that the disinfection by using iodophor/povidone iodine is avoided, and the blood sample can be polluted and the detection result is influenced.

In another embodiment, the sample amount (40 μ L, 60 μ L, 80 μ L, 100 μ L) measured in step (2) is compared with the target value, and the number of samples in the sample amount comparison experiment is 30. In a preferred embodiment, the minimum blood collection amount of magnesium, calcium, copper, selenium, lead and cadmium in peripheral blood in the step (2) is 60 μ L, and the minimum blood collection amount of zinc, iron, manganese and arsenic is 80 μ L, so 80 μ L is the minimum blood collection amount.

It should be noted that due to the extremely low levels of cadmium and arsenic in the human body, quality control was used to evaluate the different blood sampling levels of cadmium and arsenic. Taking a quality control product index value or a 100 mu L result as a target value, and determining the total error of 5 elements of magnesium, manganese, copper, selenium and zinc according to 1/2 indoor evaluation (< 12.5%); the total error of iron was determined (< 10%) according to the 1/2 compartment test, the total error of calcium was determined (target value. + -. 0.125%) according to the 1/2 compartment test, and the total error of lead was determined (target value. + -. 10%) according to the 1/4 compartment test.

In another embodiment, in the step (5), the test results of the sample solutions to be tested stored for 24 hours, 48 hours and 72 hours are compared with the test results immediately after collection, and the number of samples in the peripheral blood storage time comparison test is 20. In a preferred embodiment, wherein the calcium, magnesium, manganese, iron, copper, zinc, selenium, arsenic, cadmium and lead samples in whole blood are stored in step (5) for up to 72 hours.

It should be noted that the cadmium and arsenic content is extremely low in humans. The quality control product is adopted for evaluation, the detection result of 0h of arsenic and cadmium elements is the standard value of the quality control product, the detection result of 0h is taken as the target value, the deviation between the detection result of 0h and the results of different storage times is calculated, 5 elements of standard magnesium, manganese, copper, selenium and zinc are judged according to 1/2 room quality evaluation allowable total error (less than 12.5%), iron elements are judged according to 1/2 room quality evaluation allowable total error (less than 10%), calcium elements are judged according to 1/2 room quality evaluation allowable total error (target value +/-0.125), lead elements are judged according to 1/4 room quality evaluation allowable total error (target value +/-10), and when the standard is met, the storage time can be taken as the longest storage time.

In another embodiment, the quality control product in the step (4) is a whole blood inorganic element quality control product containing a mixed solid quality control product of heparin anticoagulated bovine whole blood and a national standard solution, and has three concentrations of high, medium and low. In a preferred embodiment, wherein the calculation of the concentration of the 10 elements is obtained by (sample to be tested-blank sample) x dilution factor, wherein the calculation of the dilution factor is obtained by (sample amount + 1.9)/sample amount.

In another embodiment, the method further comprises the steps of: and (3) debugging and correcting the ICP-MS instrument by using a tuning liquid before detecting by using an inductively coupled plasma mass spectrometry (ICP-MS) in the step (7). In a preferred embodiment, wherein said modulator fluid is formulated as: diluting a stock solution used for tuning an inductively coupled plasma mass spectrometer by using a nitric acid solution with the mass concentration of 2%, preparing the stock solution into a tuning solution containing lithium element, cobalt element, indium element, uranium element, cerium element and barium element with the mass concentration of 10 mu g/L for later use, wherein each element in the tuning solution is selected from the following elements: the national standard substance research center comprises lithium with code number of GSB 04-1734-type 2004, cobalt with code number of GSB 04-1722-type 2004, indium with code number of GSB 04-1731-type 2004, uranium with code number of GBW (E)080173, cerium with code number of GSB 04-1775-type 2004 and barium with code number of GSB 04-1717-type 2004.

In any of the above embodiments, the steps (2) to (5) are a whole blood sample pretreatment method for ICP-MS detection of trace elements.

In any of the above embodiments, the blood sample comprises blood samples from healthy adults, children, and pregnant women, as well as blood samples from people suspected of being poisoned.

In other embodiments, the detection methods are used for environmental trace toxin contamination, trace stimulant composition, forensic toxicant detection or identification of human or cadavers.

The second purpose of the invention is to provide a kit for detecting a plurality of trace elements in peripheral blood by the ICP-MS method, wherein the kit comprises:

(1) sample diluent: the sample diluent contains 0.1% (v/v) triton solution and 0.1% (v/v) HNO₃And 2% (v/v) isopropyl alcohol, the balance being purified water;

(2) an internal standard calibrator: the internal standard calibration substance is a mixed solution containing Ge, Y, In and Tb elements and prepared by 2% nitric acid, the concentration of each element is 40 mug/L,

(3) external standard calibrator: the external standard calibration product is a mixed 6-concentration gradient solution containing Mg, Ca, Mn, Fe, Cu, Zn, As, Se, Cd and Pb, which is prepared by 2% nitric acid, and the gradient concentration of each element is As follows:

(4) whole blood inorganic element quality control product: the whole blood inorganic element quality control product is a mixed solid quality control product containing heparin anticoagulated bovine whole blood and a national standard solution, and has three concentrations of high, medium and low, wherein the concentrations of the elements are shown as follows:

in one embodiment, the kit further comprises a disinfectant solution and a blood sampling device, wherein the disinfectant solution is 75% ethanol or isopropanol, and the blood sampling device comprises a touch-press type peripheral blood sampler, a disposable micro blood sampling tube (100 μ L), and a glass capillary latex suction head.

In one embodiment, the internal standard elements of the internal standard calibrator are selected from the group consisting of: the code number of the national standard substance research center is germanium element with GSB 04-1728-2004, the code number of the national standard substance research center is yttrium element with GSW 08657, the code number of the national standard substance research center is indium element with GSB 04-1731-2004 and the code number of the national standard substance research center is terbium element with GSB 04-1781-2004.

In another embodiment, the external standard calibrator STD0 is a calibration blank with each element at a concentration of 0 μ g/L, and the external standard calibrator STD1-5 is diluted (32, 16, 8, 4, 2 fold) from a mixed standard solution. .

In still another embodiment, the standard mixed solution used in step (3) for preparing the gradient concentration solution of the external standard calibrator by dilution is composed of 16mg/L magnesium, 32mg/L calcium, 32. mu.g/L manganese, 160mg/L iron, 480. mu.g/L copper, 2400. mu.g/L zinc, 32. mu.g/L arsenic, 64. mu.g/L selenium, 32. mu.g/L cadmium and 64. mu.g/L lead. In a preferred embodiment, the external standard elements in the external standard calibrator are selected from the group consisting of: magnesium element with code number GNM-SMG-001-.

In any embodiment, the solid quality control materials (high, medium and low) used in the whole blood inorganic element quality control material need to be accurately dissolved by 5mL of ultrapure water according to the specification before use, and severe vibration is avoided in the dissolving process, so that bubbles are generated; the dissolved liquid quality control product needs to be shaken up and effectively stored for 30 days under the refrigeration condition. In a preferred embodiment, at room temperature, at least two bottles of mixed solid quality control products containing heparin anticoagulated bovine whole blood and national standard solution with different concentrations are taken, 5mL of ultrapure water is accurately added into each bottle, the mixed solution is uniformly mixed for 30min until the mixed solution is completely dissolved, 80 μ L of the redissolved quality control product is added into 1.9mL of sample diluent, and the sample diluent is placed on a vortex oscillation mixer and uniformly mixed for 30 seconds, and is marked as a quality control sample.

The third purpose of the invention is to provide the application of the kit for detecting the trace elements in the whole blood.

In one embodiment, the use is in detection of environmental trace toxin contamination, detection of trace stimulant composition, forensic toxicant detection or identification of human or cadavers, and human trace element content assessment.

Technical effects

1. In the inductively coupled plasma mass spectrometry detection method for multiple (10 or more) elements in peripheral blood, provided by the invention, a detection method which is suitable for multiple elements in peripheral blood, has high sensitivity, high specificity, high anti-interference performance, high precision and high repeatability is established, and the method can be used for detection purposes of non-disease diagnosis, such as detection of environmental trace toxin pollution, detection of trace stimulant components, detection or identification of forensic toxicants of human bodies or corpses, and evaluation of the content of trace elements in human bodies.

2. The inductively coupled plasma mass spectrometry detection method for multiple (10 or more) elements in peripheral blood provided by the invention has good detection precision, wherein the internal precision is less than 10%, and the inter-batch precision is less than 15%.

3. In the inductively coupled plasma mass spectrometry detection method for multiple (10 or more) elements in peripheral blood provided by the invention, the detection limit on an ICP-MS detection platform is generally lower than or equal to that of the prior art, and the detection limit of each element is as follows: 0.001. mu.g/L magnesium, 0.0122. mu.g/L calcium, 0.0443. mu.g/L manganese, 0.0015. mu.g/L iron, 0.0335. mu.g/L copper, 0.3368. mu.g/L zinc, 0.059. mu.g/L arsenic, 0.0244. mu.g/L selenium, 0.0009. mu.g/L cadmium and 0.0034. mu.g/L lead.

4. The inductively coupled plasma mass spectrometry detection method for multiple (10 or more) elements in peripheral blood provided by the invention has strong specificity of the detected elements: the ICP-MS detection is based on the mass-to-charge ratio of elements, so that the invention has strong detection specificity and accurate quantification on trace elements in peripheral blood.

5. In the inductively coupled plasma mass spectrometry detection method for multiple (10 or more) elements in peripheral blood, the peripheral blood is adopted, so that compared with venous blood collection, the method has the advantages of better experience of patients, less sample amount, simpler and more convenient pretreatment method, and suitability for collection and detection of a large number of samples.

6. In the inductively coupled plasma mass spectrometry detection method for multiple (10 or more) elements in peripheral blood, a dilution method is adopted for sample preparation, the dilution method is convenient to operate and low in cost, interference is not easily introduced, and the method is suitable for rapid treatment of a large number of samples.

7. In the inductively coupled plasma mass spectrometry detection method for multiple (10 or more) elements in peripheral blood, the minimum blood collection amount of magnesium, calcium, copper, selenium, lead and cadmium in whole blood is 60 muL, and the minimum blood collection amount of zinc, iron, manganese and arsenic is 80 muL, so that the final blood collection amount is 80 muL.

8. In the inductively coupled plasma mass spectrometry detection method for multiple (10 or more) elements in peripheral blood, the storage time (0 hour, 24 hours, 48 hours and 72 hours) of a diluted sample can reach 72 hours at most through comparison experiment verification, so that the method is very suitable for medical institutions which cannot provide detection instruments on site, has sufficient sample storage and transport time, and simultaneously has reliable guarantee on the quality of the stored sample.

9. In the inductively coupled plasma mass spectrometry detection method for multiple (10 or more) elements in peripheral blood, 10 elements including magnesium, calcium, manganese, iron, copper, zinc, arsenic, selenium, cadmium and lead in venous blood and peripheral blood are detected by inductively coupled plasma mass spectrometry, and the result shows that the result of trace elements in peripheral blood has comparability with venous blood.

Principles and definitions

1. The detection principle of the invention is that an inductively coupled plasma source of a mass spectrum system is excited by high-energy radio frequency energy to form plasma, and an external standard calibrator containing an element to be detected enters a high-vacuum quadrupole fast scanning mass spectrum detection part through a cone interface and an ion transmission system in sequence through the processes of evaporation, dissociation, atomization, ionization and the like under the action of high-temperature plasma to form univalent positive ions. And (3) drawing a standard curve in software according to the ratio of the to-be-detected element signal and the internal standard signal in the mixed element external standard calibrator and the concentration of the to-be-detected element in the mixed element external standard calibrator, so as to calibrate the instrument.

2. The term "element" refers to the collective term for a class of atoms having the same number of nuclear charges (i.e., the number of protons in the nucleus). In the human body, the number of protons is different for the same element, for example, C12 and C14. For the selection of the detection element isotope in the present invention, the following 2 criteria are referenced: the isotope with higher relative abundance is selected, and the isotope with smaller relative interference (mass spectrum interference such as oxide, polyatomic and the like) is selected. In the case of Se element, isotopes 77, 78, 80 and 82 exist in Se, the isotopes 78 and 80 are in high relative abundance, and the 80 isotope has interference of Ar diatom, which can seriously affect the detection result, so 78 isotope is selected as the detection object.

3. The term "standard substance", i.e. a standard substance/standard sample, is a material or substance, one or more characteristic values of which are sufficiently uniform and stable and well defined for calibrating a measuring system, evaluating a measuring procedure or assigning a value to a material.

4. The term "calibrator" means: calibrators are standard substances whose values are used as arguments in a calibration function for calibrating a measurement system or assigning values to a material; the correct property control substance is a standard substance for evaluating the measurement deviation of a measurement system.

5. The standard substance has two main functions of calibrating and evaluating a measuring system, and has traceability to national standard substances. A standard substance can be used as both a calibration substance and a correct property control substance in one measurement procedure or measurement system, but not as both a calibration substance and a correct property control substance. In general, reference substances are higher-order reference substances, most of which are certified reference substances. Certified standard substances are certified standard substances, one or more characteristic values of which are determined by a traceability-establishing procedure so as to be traceable to an accurately reproduced measurement slip representing the characteristic value, each determined characteristic value having an uncertainty of a given confidence level. It can be seen that the difference between the certified standard substance and the standard substance is that the former has clear traceability and uncertainty requirements. The standard substance standard sample producer capacity approval criteria which are officially released and implemented in China at 8.1.2007 put higher requirements on the organization production management of standard substance producers, standard substance assignment and statistical methods, uncertainty evaluation, performance indexes which the standard substances need to reach, and the like.

6. The term "test product" refers to any conventional product for detecting elements in human serum, including: detection reagents, detection chips (such as solid chips, liquid chips and the like), detection carriers, detection kits and the like.

7. Non-mass spectrometric interference: because the blood sample contains a large amount of protein mixture, carbohydrate, inorganic salt and the like and has complex components, the matrix effects of signal inhibition or sensitization and the like of the element to be detected are caused; the physical effect is that the background of the salt concentration in the blood solution is high, and the cone hole is blocked due to the deposition of matrix oxide powder along with the increase of the sample introduction time, and the ion current signal to be detected is degraded in stability and drifts.

Drawings

Fig. 1-10 show standard curves established for 10 elements, α: measurement of the curve. According to the experimental design, it is expected that each curve should achieve:

(1) the smaller the absolute value of the error, the closer the detection point is to the fitted curve, indicating the better the linear fit.

(2) A more strict judgment index is selected, a linear fitting coefficient R is specified by 5.5 of national standard (GB/T26124-. And R of the present invention²>0.9996-0.9997-0.9998。

(3) The standard concentration is accurately configured, so that high fitting is brought; the overall description is far beyond the accuracy specified by the national standard. The internal standard element selected by the element is also shown to be proper and accurate.

(4) The data points of the curve are distributed more uniformly, which shows that the curve is reasonable in extension.

Fig. 1 is a standard curve of elemental magnesium, showing the achievement of the above expected effect, and the interruption of the test results in this curve for an unknown sample (example of actual patient test), proving the range of the curve to be reasonable.

Fig. 2 is a standard curve of elemental calcium, showing that the above expected effect is achieved, and that the test results of an unknown sample (example of actual test patient) are interrupted in the curve, proving that the range of the curve is reasonable.

Fig. 3 is a standard curve of elemental manganese, showing the achievement of the above expected effect, and the interruption of the test results in this curve for an unknown sample (example of actual patient test), proving the range of the curve to be reasonable.

Fig. 4 is a standard curve of elemental iron, showing that the above expected effect is achieved, and that the test results of an unknown sample (example of actually testing a patient) are interrupted in the curve, proving that the range of the curve is reasonable.

Fig. 5 is a standard curve of elemental copper, showing the achievement of the above expected effect, and the interruption of the test results in this curve for an unknown sample (example of an actual patient test), justifying the range of the curve.

Fig. 6 is a standard curve of elemental zinc, showing that the above expected effect is achieved, and that the test results of an unknown sample (example of actual test patient) are interrupted in the curve, proving that the range of the curve is reasonable.

FIG. 7 is a standard curve of arsenic, which shows that the expected effect is achieved, and the element is a toxic element, the detection result of the unknown sample is in the lower section of the curve, the upper detection limit of the element is 15.9 mug/L, and the range of the curve is proved to be reasonable.

Fig. 8 is a standard curve of elemental selenium, showing that the above expected effect is achieved, and that the test results of an unknown sample (example of an actual patient test) are interrupted in the curve, proving that the range of the curve is reasonable.

FIG. 9 is a standard curve of cadmium, which shows that the above expected effect is achieved, and the element is a toxic element, the detection result of the unknown sample is in the lower section of the curve, the upper detection limit of the element is 15.8 μ g/L, and the range of the curve is proved to be reasonable.

FIG. 10 is a standard curve of lead element, and the result shows that the expected effect is achieved, and the element is a toxic element, the detection result of the unknown sample is in the lower section of the curve, and the upper detection limit of the element is 32.1 mug/L, which proves that the range of the curve is reasonable.

Detailed Description

The invention is further described below with reference to specific preferred embodiments, without thereby limiting the scope of protection of the invention.

Example 1 Instrument operating parameters

The trace element analysis was performed using a trace element analyzer (Clin-ICP-QMS-I) whose basic operating parameters are shown in the following table:

TABLE 1 Instrument parameter settings

Example 2 screening experiments with different dilutions

2.1 sample treatment

2.1.1 reconstitution

Redissolving 1 bottle of freeze-dried whole blood quality control product with 5mL of purified water, slightly covering a bottle cap, slowly and continuously rolling for about 30 minutes to ensure that the powder is completely dissolved, and cannot be shaken and uniformly mixed to avoid generating a large amount of bubbles to form the reconstituted whole blood quality control product solution.

2.1.2 dilution

And (3) taking 100uL of the whole blood quality control product compound solution, dissolving in 1.9mL of sample diluent, fully and uniformly mixing to form a quality control solution to be detected, and waiting for detection on a computer.

2.1.3 sample dilutions

Sample diluent a: 0.1% nitric acid (v/v) + 0.1% Triton (v/v) + 0.1% isopropanol (v/v)

Sample diluent B: 0.1% nitric acid (v/v) + 0.1% Triton (v/v) + 0.5% isopropanol (v/v)

Sample diluent C: 0.1% nitric acid (v/v) + 0.1% Triton (v/v) + 1% isopropanol (v/v)

Sample diluent D: 0.1% nitric acid (v/v) + 0.1% Triton (v/v) + 2% isopropanol (v/v)

Sample diluent E: 0.1% nitric acid (v/v) + 0.1% Triton (v/v) + 5% isopropanol (v/v)

Sample diluent F: 0.1% nitric acid (v/v) + 0.1% triton (v/v) + 2% 1, 4-butanediol (v/v)

Sample diluent G: 0.1% nitric acid (v/v) + 0.1% Triton (v/v) + 2% ethanol (v/v)

Sample diluent H: 0.1% nitric acid (v/v) + 0.1% triton (v/v) + 2% acetone (v/v)

Sample diluent I: 0.1% nitric acid (v/v) + 0.1% Triton (v/v) + 2% methanol (v/v)

Sample diluent J: 0.1% nitric acid (v/v) + 0.1% triton (v/v) + 2% n-butanol (v/v)

2.1.4 external standard calibrator

The concentration of 10 elements is prepared by 6 gradient concentrations of 2% nitric acid, and the concentration of 10 elements is shown in the following table:

TABLE 2 concentrations of 10 elements of external standard calibrator

Table 3 information on 10 element raw materials in external standard calibrators

2.1.5 internal standard calibrator

Scandium Se, germanium Ge, rhodium Rh, yttrium Y, indium In, lutetium Lu and bismuth Bi elements are prepared by using 2% nitric acid, and the concentration is 40 mu g/L.

Table 4 information on 4-element raw materials in internal standard calibrators

2.1.6 quality control

The whole blood inorganic element quality control product is a mixed solid quality control product containing heparin anticoagulation bovine whole blood and national standard solution, and has three concentrations of high, medium and low, wherein the concentrations are shown in the following table.

TABLE 5 Whole blood inorganic element quality control index value

2.2 sample assay

The signal of the instrument is adjusted to Be strongest by using tuning liquid (10 mu g/L of lithium Li, cobalt Co, indium In, uranium U, cerium Ce and barium Be), and the interference of the instrument is adjusted to Be minimum by using He mode. The tuning liquid comprises elements selected from: the national standard substance research center comprises lithium with code number of GSB 04-1734-type 2004, cobalt with code number of GSB 04-1722-type 2004, indium with code number of GSB 04-1731-type 2004, uranium with code number of GBW (E)080173, cerium with code number of GSB 04-1775-type 2004 and barium with code number of GSB 04-1717-type 2004.

The external standard calibrator and the internal standard calibrator are simultaneously injected, and the instrument can automatically establish a calibration curve (of each element in the external standard calibrator) according to the concentration of 6 points in the external standard calibratorConcentration is in the abscissa and the ratio of the signals of the elements of the external and internal calibration standards is in the ordinate). The calibration curve needs to satisfy R²Not less than 0.999. And then sequentially carrying out 9-element detection on the sample diluent and the quality control solution to be detected. Each sample was tested 3 times in succession and the relative deviation of the diluted quality control samples of each sample dilution was calculated.

TABLE 60.1% nitric acid + 0.1% Triton + 0.1% Isopropanol relative deviations of the quality control samples after dilution

Element to be measured

Mg

Ca

Mn

Fe

Cu

Zn

As

Se

Pb

Relative deviation (%)

31.9％

2.4％

7.6％

6.7％

10.2％

5.6％

21.8％

1.2％

6.0％

TABLE relative deviation of quality control substances after dilution with 70.1% nitric acid, 0.1% triton and 0.5% isopropanol

Element to be measured

Mg

Ca

Mn

Fe

Cu

Zn

As

Se

Pb

Relative deviation (%)

24.9％

0.8％

9.0％

6.7％

12.0％

4.6％

3.3％

3.8％

7.2％

TABLE 80.1% nitric acid + 0.1% Triton + 1% Isopropanol relative deviations of the quality control samples after dilution

Element to be measured

Mg

Ca

Mn

Fe

Cu

Zn

As

Se

Pb

Relative deviation (%)

19.1％

3.3％

6.4％

6.2％

13.6％

5.0％

4.0％

2.2％

8.4％

TABLE relative deviation of quality control after dilution of 90.1% nitric acid + 0.1% Triton + 2% isopropanol

Element to be measured

Mg

Ca

Mn

Fe

Cu

Zn

As

Se

Pb

Relative deviation (%)

2.3％

4.5％

4.3％

8.4％

7.3％

2.0％

0.2％

7.2％

6.5％

TABLE 100.1% nitric acid + 0.1% Triton + 5% Isopropanol relative deviations of the quality control samples after dilution

TABLE 110.1% nitric acid + 0.1% Triton + 2% 1, 4-butanediol relative deviation of the quality control samples after dilution

Element to be measured

Mg

Ca

Mn

Fe

Cu

Zn

As

Se

Pb

Relative deviation (%)

39.0％

5.3％

6.8％

15.1％

7.9％

0.6％

4.7％

4.9％

5.0％

TABLE 120.1% nitric acid + 0.1% Triton + 2% ethanol diluted quality control relative deviation

Element to be measured

Mg

Ca

Mn

Fe

Cu

Zn

As

Se

Pb

Relative deviation (%)

21.5％

4.8％

5.3％

10.5％

9.4％

0.7％

5.7％

0.5％

7.0％

TABLE 130.1 relative deviations of the quality control samples diluted with nitric acid + 0.1% Triton + 2% propanol

Element to be measured

Mg

Ca

Mn

Fe

Cu

Zn

As

Se

Pb

Relative deviation (%)

42.3％

3.2％

1.0％

3.0％

11.5％

4.3％

35.1％

44.6％

5.8％

TABLE 140.1 relative deviations of the quality control diluted with nitric acid, 0.1% Triton and 2% methanol

Element to be measured

Mg

Ca

Mn

Fe

Cu

Zn

As

Se

Pb

Relative deviation (%)

34.9％

3.9％

3.8％

14.2％

8.2％

1.0％

9.6％

9.1％

4.9％

TABLE 150.1% nitric acid + 0.1% Triton + 2% n-butanol relative deviation of the quality control samples after dilution

Element to be measured

Mg

Ca

Mn

Fe

Cu

Zn

As

Se

Pb

Relative deviation (%)

8.8％

3.6％

9.4％

11.1％

11.5％

1.9％

5.5％

6.8％

8.4％

2.3 conclusion

The quality control product diluted by using the sample diluent (0.1 percent nitric acid, 0.1 percent triton and 2 percent isopropanol) has the best effect when the relative deviation of the 9 elements is less than or equal to 10 percent. Therefore, with the use of dilutions of the components and ratios, it is expected that the best relative deviation will be found for the 10 elements.

Example 3 differential experiment of internal and external standard calibrators prepared with 2% nitric acid and sample diluent (0.1% nitric acid + 0.1% Triton + 2% isopropanol)

The operation method and the instrument parameters are the same as those described above, and the internal standard and the external standard calibration sample prepared by 2 percent nitric acid are sequentially injected to make calibrationCurve (R)²>0.999), the quality control product diluted with the sample diluent (0.1% nitric acid + 0.1% triton + 2% isopropanol) is injected 3 times, then the internal standard and the external standard calibration products prepared with the sample diluent (0.1% nitric acid + 0.1% triton + 2% isopropanol) are injected, and the calibration curve (R) is formed²>0.999), the quality control diluted with a sample diluent (0.1% nitric acid + 0.1% triton + 2% isopropyl alcohol) was injected 3 times. The relative deviations of the two methods are shown in the table below.

Table 162% nitric acid formulated internal and external standard calibrators

Element to be measured

Mg

Ca

Mn

Fe

Cu

Zn

As

Se

Pb

Relative deviation (%)

2.3％

4.5％

4.3％

8.4％

7.3％

2.0％

0.2％

7.2％

6.5％

TABLE internal and external Standard calibrators made with 170.1% nitric acid + 0.1% Triton + 2% Isopropanol

Element to be measured

Mg

Ca

Mn

Fe

Cu

Zn

As

Se

Pb

Relative deviation (%)

2.1％

4.4％

2.5％

1.0％

0.8％

0.4％

3.8％

1.5％

0.8％

And (4) conclusion: when the internal standard calibrator, the external standard calibrator and the sample are used in the same diluent, the relative deviation of the detection results is smaller.

Example 4 determination of blood sampling volume and relative deviation

4.1 quality control product preliminary determination of blood sampling volume

The signal of the instrument is adjusted to Be strongest by using tuning liquid (10 mu g/L of Li, Co, In, uranium mu m, Ce and Ba Be), and the interference of the instrument is adjusted to Be minimum by using He mode.

And (3) simultaneously injecting the external standard calibrator and the internal standard calibrator, and automatically establishing a calibration curve by the instrument according to the concentration of 6 points in the external standard calibrator (taking the concentration of each element in the external standard calibrator as an abscissa and the ratio of signals of each element in the external standard calibrator and the internal standard calibrator as an ordinate). The calibration curve needs to satisfy R²Not less than 0.999. And then sequentially carrying out 10-element detection on the blank sample and the quality control solution to be detected. The test is carried out 10 times in succession for each sample, and the relative deviation (%) and precision RSD (%) of the diluted quality control product from the standard value are calculated (the results are shown in the following table).

Relative deviation calculation formula:

wherein:

x-detection result

T-mark value of quality control article

Precision calculation formula:

wherein:

in the formula:

-average of n sets of measurement data;

x_i-ith set of measurements (average of 3 scans);

s-standard deviation;

n-number of measurement sets.

TABLE 18 relative deviation of different blood sampling volume of whole blood quality control product

4.2 clinical samples determination of blood sampling volume

The results of the above 4.1 quality control samples from different blood sampling amounts showed (see table above), and when the sampling amount was 40. mu.L, the relative deviation was large, so that only 60. mu.L and 80. mu.L sampling amounts were evaluated in the clinical sample quantitative determination experiments. 30 samples of heparin anticoagulant venous blood are randomly collected, samples meeting the requirements are accurately transferred by using a sample adding gun, 60 mu L, 80 mu L and 100 mu L of venous blood are simultaneously sucked by each sample, the samples are placed in a sample diluent containing 1.9mL, and the sample diluent is fully and uniformly mixed to mark the sample to be detected. The mean value was calculated by repeating the test 3 times for each sample. Using 100. mu.L of venous blood results as the target, the deviation of the test results from 60. mu.L and 80. mu.L were compared, respectively (results are shown in the following Table). Because the content of cadmium and arsenic in human bodies is low, different blood sampling amounts of cadmium and arsenic are evaluated by adopting quality control product indicating values. 5 elements of magnesium, manganese, copper, selenium and zinc are determined according to 1/2 indoor evaluation allowed total error (< 12.5%); iron was determined as the total error allowed under the 1/2 chamber test (< 10%), calcium was determined as the total error allowed under the 1/2 chamber test (< 0.125 target), and lead was determined as the total error allowed under the 1/4 chamber test (< 10 target), with the sample size being considered the minimum sample size when the above criteria were met (data not shown).

TABLE 19 mean relative/absolute deviation of different blood sampling of clinical specimens (n. 30 cases)¹

¹Note: since the arsenic and cadmium contents in human bodies are very low, the blood sampling amount is not calculated.

²Note: due to space limitation, "mean absolute/relative bias" is the mean bias of 30 clinical specimens, and the specific bias data of ten different blood sampling amounts of each sample are not shown.

4.3 conclusion

Evaluating the blood collection quantity of cadmium and arsenic by using a quality control product, wherein the result shows that the minimum blood collection quantity of cadmium is 40uL, and the minimum blood collection quantity of arsenic is 80 muL; the other elements are evaluated by adopting clinical samples, and the result shows that the minimum blood collection quantity of 5 elements of magnesium, calcium, copper, selenium and lead is 60 ul; the minimum blood collection amount of 3 elements of zinc, iron and manganese was 80. mu.L (detailed data not shown), and thus 80. mu.L was the minimum blood collection amount.

Example 5: determining maximum storage time and relative deviation

5.1 pretreatment

5.1.1 sample treatment

20 cases of heparin anticoagulated venous blood are randomly collected, 80 mu L of heparin anticoagulated venous blood is accurately transferred by a sample adding gun and placed in 1.9mL of sample diluent, and the sample is fully and uniformly mixed to be marked for detection. The average value of each sample is calculated by repeating the test for 3 times, and the deviation of the test results of 24h, 48h and 72h is compared respectively by taking the result of 0h as a reference (the results are shown in the table 20)

5.1.2 external standard calibrator

The external standard calibrator with 10 elements and 6 gradient concentrations is prepared by using 2% nitric acid, and the concentrations and raw material information of the 10 elements are the same as those of example 2.

4.1.3 internal standard calibrator

Preparing an internal standard calibrator by using 2% nitric acid, wherein the internal standard calibrator contains 4 elements such as germanium Ge, yttrium Y, indium In, terbium Tb and the like, and the concentration of the internal standard calibrator is 40 mu g/L. Internal standard calibrator raw material information was the same as in example 2.

4.2 measurement of quality control and venous blood sample storage time

The tuning liquid (10 ug/L Li, Co, In, u m, Ce and Be) was used to adjust the signal of the instrument to the strongest level and He mode was used to adjust the interference of the instrument to the lowest level.

And (3) simultaneously injecting the external standard calibrator and the internal standard calibrator, and automatically establishing a calibration curve by the instrument according to the concentration of 6 points in the external standard calibrator (taking the concentration of each element in the external standard calibrator as an abscissa and the ratio of signals of each element in the external standard calibrator and the internal standard calibrator as an ordinate). The calibration curve needs to satisfy R²Not less than 0.999. And then sequentially carrying out 10-element detection on the blank sample, the quality control solution to be detected and the sample. Comparing the detection results of the quality control products for 24h, 48h and 72h with the marking values; comparing the detection results of the clinical samples 24h, 48h and 72h with the results of 0h, and calculating the deviation percent. And (3) judging standard: 5 elements of magnesium, manganese, copper, selenium and zinc are judged according to 1/2 room evaluation allowable total error (< 12.5%), iron is judged according to 1/2 room evaluation allowable total error (< 10%), calcium is judged according to 1/2 room evaluation allowable total error (target value +/-0.125), lead is judged according to 1/4 room evaluation allowable total error (target value +/-10), and when the above standard is met, the storage time is determined to be the longest storage time. (the results of the measurements are shown in the following table).

TABLE 20 quality control product preservation for 24h, 48h, 72h detection result relative deviation

TABLE 21 clinical specimen preservation 24h, 48h, 72h average relative/absolute deviation of assay results (n 20 cases)¹

¹Note: the storage time was not calculated because the arsenic and cadmium content in humans was very low.

²Note: due to space limitations, "mean absolute/relative variance" is the mean variance of 20 clinical specimens, and the specific variance data of ten elements in each sample with different retention times is not shown.

5.3 conclusion

Evaluating the longest storage time of cadmium and arsenic by adopting a quality control product, wherein the result shows that the longest storage time of cadmium and arsenic is 72 h; the remaining elements were evaluated using clinical samples and the results showed that the longest storage time for the eight elements magnesium, calcium, copper, selenium, lead, zinc, iron and manganese was 72h (data not shown), and thus 72h was the longest storage time.

Example 6: instrument detection limit

6.1 Instrument detection limits

Mixing the solution: 2% nitric acid is used for preparing elements of magnesium Mg, calcium Ca, manganese Mn, iron Fe, copper Cu, zinc Zn, arsenic As, selenium Se, cadmium Cd and lead Pb, and the concentration is 10 mug/L.

According to the GBT-34826-. The instrument detection limit is 3 SD/S.

6.2 method detection limits

Diluting the low-concentration quality control product by multiple times according to GB/T-27415-2013 analytical method detection limit and quantitative limit evaluation, detecting 10 elements by a sample detection method until the detection result is lower than the instrument detection limit, and taking the detection result of the last dilution multiple as the method detection limit (see the following table).

TABLE 22 detection limit results

Example 7: recovery rate of added standard

7.1 preparation of the spiking solution

The mixed solution of Mg, Ca, Mn, Fe, Cu, Zn, As, Se, Cd and Pb elements prepared by 2% nitric acid is taken As the concentration (320Mg/L, 640Mg/L, 1280 mug/L, 3200Mg/L, 9600 mug/L, 48000 mug/L, 1280 mug/L and 1280 mug/L).

7.2 preparation of spiked samples

The formulation methods for the samples and the spiked samples are shown in the following table.

Preparation method of table 23 sample and standard sample

7.3 detection on computer

The operating method and instrument parameters were as above, and a calibration curve (R) was generated by injecting internal and external standard calibrators prepared from a sample diluent (0.1% nitric acid + 0.1% triton + 2% isopropanol)²>0.999), the sample 1 and the sample 2 are detected 3 times each, and the normalized recovery rate is calculated as shown in the following table.

TABLE 24 recovery of each element normalized

And (4) conclusion: the standard recovery rates of the ten elements all meet the requirement of 80-120%.

Example 8: precision degree

8.1 pretreatment

8.1.1 reconstitution

Taking 1 bottle of each of the high, medium and low concentration quality control products, redissolving the product by 5mL of ultrapure water, slightly covering a bottle cap, and slowly and continuously rolling for about 30 minutes to ensure that the powder is completely dissolved to form a whole blood quality control product redissolution. Attention is paid to that the mixture cannot be violently shaken during mixing, so that a large amount of bubbles are avoided.

8.1.2 dilution

Sample 1: and (3) taking 80 mu L of high-concentration whole blood quality control product compound solution, dissolving in 1.9mL of sample diluent, fully and uniformly mixing to form a quality control solution to be detected, and waiting for detection on a computer.

Sample 2: and (3) taking 80 mu L of the medium-concentration whole blood quality control product compound solution, dissolving in 1.9mL of sample diluent, fully and uniformly mixing to form a quality control solution to be detected, and waiting for detection on a computer.

Sample 3: and (3) taking 80 mu L of low-concentration whole blood quality control product redissolved solution, dissolving in 1.9mL of sample diluent, fully and uniformly mixing to form quality control solution to be detected, and waiting for detection on a computer.

8.1.4 external standard calibrator

The external standard calibrator with 10 elements and 6 gradient concentrations is prepared by using 2% nitric acid, and the concentrations and raw material information of the 10 elements are the same as those of example 2.

8.1.5 internal standard calibrator

An internal standard calibrator is prepared by using 2% nitric acid, and the internal standard calibrator contains 4 elements such as germanium Ge, yttrium Y, indium In, terbium Tb and the like, and the concentration of the internal standard calibrator is 40 mu g/L. Internal standard calibrator raw material information was the same as in example 2.

8.2 sample assay

The signal of the instrument is adjusted to Be strongest by using tuning liquid (10 mu g/L of lithium Li, cobalt Co, indium In, uranium U, cerium Ce and barium Be), and the interference of the instrument is adjusted to Be minimum by using He mode.

And (3) simultaneously injecting the external standard calibrator and the internal standard calibrator, and automatically establishing a calibration curve by the instrument according to the concentration of 6 points in the external standard calibrator (taking the concentration of each element in the external standard calibrator as an abscissa and the ratio of signals of each element in the external standard calibrator and the internal standard calibrator as an ordinate). The calibration curve needs to satisfy R²Not less than 0.999. And then sequentially carrying out 10-element detection on the sample diluent and the quality control solution to be detected. And (3) evaluating the precision in the batch: each sample was examined seriallyMeasuring for 10 times; and (3) evaluating batch precision: each sample is measured for 3 groups of data respectively, the detection is carried out for 2 times every day and for 5 days continuously, and the precision RSD (see the following table) of quality control products with different concentrations is calculated.

8.3 peripheral hemospermia Density measurement

According to the standard blood collection process in (1), peripheral blood is collected from 10 subjects, 80 μ L of each sample is collected, the sample is placed in 1.9mL of sample diluent, 10 samples are mixed, and the sample is marked to be tested. The precision RSD of the different elements was calculated (see table below).

TABLE 25 Low and high concentration of quality control Intra and Inter-batch precision RSD (%)

TABLE 26 peripheral sample precision RSD¹(％)

¹Note: since the arsenic and cadmium contents in the human body are very low, the precision thereof is not calculated.

²Note: the lower precision indicates a more reproducible measurement, indicating a more stable result for the instrument to detect the sample.

8.4 inter-lot precision evaluation of quality control products

According to the above operation process, performing quality control detection with each batch of samples every day, accumulating for 30 days, and calculating average value

And standard deviation, RSD% (see table below) between the evaluation batches was < 15%.

Table 2730 days of accumulated daytime precision of quality control product

Conclusion 8.5: the method detects the internal precision of 10 elements in the high-concentration and low-concentration quality control products which are all less than 10 percent. The precision in the peripheral blood batches is less than 10 percent, and the precision between the quality control product batches is less than 15 percent.

Example 9: comparison of venous blood with peripheral blood

9.1 sample Collection

(1) A blood collecting device: heparin anticoagulant blood collection tubes and venous blood collection needles; a disposable touch-press type peripheral blood hemostix, a disposable peripheral blood collection tube, a micro blood collection sucker and a latex sucker;

(2) subject: 38 healthy volunteers;

(3) the acquisition method comprises the following steps: venous (2mL) and peripheral (80. mu.L) blood were collected from the same volunteer at the same time and labeled as end-1 and quiet-1, respectively.

9.2 on-machine detection

By utilizing an ICP-MS detection method, an internal standard calibrator is added on line, and the sample injection is carried out according to the following sequence, wherein the external standard calibrator is 0-5, a blank sample, a quality control sample, venous blood (quiet-1-quiet 38) and peripheral blood (quiet-1-quiet-38).

9.3 results calculation: dilution factor ═ (amount of sample + 1.9)/amount of sample; concentration ═ concentration (assay result-blank) × dilution factor. Taking a venous blood detection result as a target value, judging 5 elements of magnesium, manganese, copper, selenium and zinc according to 3/4 (18.75) of the total allowable error of the compartment evaluation, judging iron element according to 3/4 (15) of the total allowable error of the compartment evaluation, judging calcium element according to 3/4 of the total allowable error of the compartment evaluation (target value +/-0.188), and judging lead element according to 1/4 of the total allowable error of the compartment evaluation (target value +/-10). (see the following Table)

Conclusion 9.4: the elements of magnesium, calcium, iron, selenium, copper, zinc, lead and cadmium 8 are comparable (the results are shown in the following table).

TABLE 28 comparison of venous blood and peripheral blood (38 cases)

¹Admission to ChamberTotal error + -25%, 3/4TEA + -18.75%, 1/2 Total error + -12.5%

²The total tolerance for the indoor scores was. + -. 20%, 3/4 TEA. + -. 15%, 1/2. + -. 10%

³Total error tolerance of. + -. 0.25 for the indoor scoring, 0.188 for 3/4TEA, 0.125 for 1/2

⁴Indoor scoring tolerance of 40, 1/4 tolerance of 10

Example 10: practical test of 20 patient samples

Blood samples were taken from 20 patients clinically and tested according to the method of the above example. The data results are as follows.

TABLE 29 actual examination of 20 patient samples

Reference ranges of Table 3010 Trace elements

In conclusion, the method established by the invention is combined with the test data of the embodiment, and the positive sample and the negative sample can be accurately distinguished and identified.

Claims (10)

1. An ICP-MS method for detecting multiple trace elements in peripheral blood, the method comprises the following steps:

(1) collecting samples: sterilizing the skin surface by using 75% ethanol or isopropanol, collecting blood by using a touch pressure type peripheral blood collector, collecting blood by using a disposable micro blood collection tube, and quantitatively transferring the collected peripheral blood by using a glass capillary latex suction head;

(2) sample (A)The preparation of the solution to be tested: taking (80 mu L) peripheral blood sample according to the method in the step (1), mixing the peripheral blood sample with 1.9mL sample diluent in a 2.5mL centrifuge tube, and then placing the mixture on a vortex oscillation mixer for mixing for 30 seconds, wherein the sample diluent contains 0.1% (v/v) triton solution and 0.1% (v/v) HNO₃And 2% (v/v) isopropyl alcohol, the balance being purified water;

(3) preparing a blank sample: taking a 2.5mL centrifuge tube filled with 1.9mL of sample diluent, adding no solution, and then placing the centrifuge tube on a vortex oscillation mixer for mixing for 30 seconds;

(4) preparing a quality control detection solution: taking the redissolved whole blood inorganic element quality control product according to the method of (1) to mix with 1.9mL sample diluent in a centrifugal tube of 2.5mL, and then placing the mixture on a vortex oscillation mixer to mix for 30 seconds, wherein the whole blood inorganic element quality control product is a mixed solid quality control product of the whole blood inorganic element quality control product containing heparin anticoagulated bovine whole blood and national standard solution, and has high, medium and low concentrations, and the concentration of each element is shown below:

(5) and (3) storing the sample solution to be tested: storing the diluted sample in a refrigerator at 2-8 deg.C, and testing on a computer for storage time (less than or equal to 72 hr);

(6) preparing an external standard calibrator of an element calibration curve to be measured with corresponding concentration and an internal standard calibrator of calibration instrument drift, wherein the internal standard calibrator is a mixed solution containing Ge, Y, In and Tb elements and prepared by 2% nitric acid, and the concentration of each element is 40 mug/L;

(7) and (3) computer detection:

sequentially introducing a blank sample, 0-5 parts of an external standard calibrator, a quality control detection solution and a sample solution to be detected into an instrument for determination, and adding an internal standard calibrator on line, wherein the sample solution and the internal standard solution are injected in a ratio of 1: 1; taking the ratio of the element signal to be detected in the external standard calibrator to the element signal in the internal standard calibrator as the Y axis, taking the concentration of the element to be detected in the external standard calibrator as the X axis, drawing a calibration curve Y as ax + b, and when each element is detectedCorrelation coefficient r of element calibration curve²>When the concentration is more than 0.999, the quality control detection liquid can be detected; when the error of the detection result of the quality control detection solution is within a 95% confidence interval, the sample solution to be detected can be continuously detected;

the external standard calibration product is a mixed 6-concentration gradient solution containing Mg, Ca, Mn, Fe, Cu, Zn, As, Se, Cd and Pb elements and prepared by 2% nitric acid, and the gradient concentration of each element is As follows:

(8) result processing

The software automatically calculates the mass concentration (mu g/L) of each element to be detected in the sample liquid to be detected according to the regression equation in the step (7), and the calculation of the concentration of the trace elements in the peripheral blood sample is as follows:

ρ_(X)＝(C_X-C₀)×K…………………………(1)

in the formula:

ρ_(X)-mass concentration of trace elements in peripheral blood samples in micrograms per liter (μ g/L);

C_Xthe mass concentration of the trace elements in the sample solution to be tested is determined by a standard curve, and the unit is microgram per liter (mu g/L);

C₀-mass concentration of trace elements in microgram per liter (. mu.g/L) in blank samples from a standard curve;

k is the whole blood dilution factor;

V_X-peripheral blood volume in milliliters (mL).

2. The method according to claim 1, wherein the results of the sample amount (40 μ L, 60 μ L, 80 μ L, 100 μ L) in step (2) are compared with the target value, and the number of samples in the sample amount comparison experiment is 30, preferably the minimum blood collection amount of magnesium, calcium, copper, selenium, lead, and cadmium in peripheral blood in step (2) is 60 μ L, the minimum blood collection amount of zinc, iron, manganese, and arsenic in step (2) is 80 μ L, and 80 μ L is the minimum blood collection amount.

3. The method of claim 1 or 2, wherein the step (2) is performed with a target value of a quality control index value or a 100 μ L result, and 5 elements of magnesium, manganese, copper, selenium and zinc are determined to allow a total error of less than 12.5% according to 1/2 interstitial evaluation; the total error of iron was determined (< 10%) according to the 1/2 compartment test, the total error of calcium was determined (target value. + -. 0.125%) according to the 1/2 compartment test, and the total error of lead was determined (target value. + -. 10%) according to the 1/4 compartment test.

4. The method according to claim 1 or 2, wherein in the step (5), the results of the test of the sample to be tested are stored for 24 hours, 48 hours and 72 hours and the results of the test immediately after collection are compared, and the number of samples in the peripheral blood storage time comparison test is 20, and preferably, in the step (5), the samples of calcium, magnesium, manganese, iron, copper, zinc, selenium, arsenic, cadmium and lead in the whole blood are stored for 72 hours at most.

5. The method as set forth in claim 5, wherein in the step (5), the evaluation is performed by using the quality control material, the 0h detection result of arsenic and cadmium is a standard value of the quality control material, the 0h detection result is a target value, the deviation between the 0h detection result and the results of different storage times is calculated, 5 elements of magnesium, manganese, copper, selenium and zinc are judged according to 1/2 compartment evaluation allowable total error (< 12.5%), iron is judged according to 1/2 compartment evaluation allowable total error (< 10%), calcium is judged according to 1/2 compartment evaluation allowable total error (target value ± 0.125%), lead is judged according to 1/4 compartment evaluation allowable total error (target value ± 10), and the storage time is determined as the longest storage time when the above standard is satisfied.

6. The method of any one of claims 1 to 5, wherein the calculation of the concentration of 10 elements of the quality control material in the step (4) is obtained by (sample solution to be tested-blank sample) x dilution factor, wherein the calculation of the dilution factor is obtained by (amount of sample + 1.9)/amount of sample.

7. The method of claim 7, wherein the ICP-MS instrument is debugged and calibrated with a tuning liquid prior to detection by inductively coupled plasma mass spectrometry (ICP-MS) in step (7), wherein said tuning liquid is formulated: diluting a stock solution used for tuning an inductively coupled plasma mass spectrometer by using a nitric acid solution with the mass concentration of 2%, preparing the stock solution into a tuning solution containing lithium element, cobalt element, indium element, uranium element, cerium element and barium element with the mass concentration of 10 mu g/L for later use, wherein each element in the tuning solution is selected from the following elements: the national standard substance research center comprises lithium with code number of GSB 04-1734-type 2004, cobalt with code number of GSB 04-1722-type 2004, indium with code number of GSB 04-1731-type 2004, uranium with code number of GBW (E)080173, cerium with code number of GSB 04-1775-type 2004 and barium with code number of GSB 04-1717-type 2004.

8. The method according to any one of claims 1 to 7, wherein the steps (2) to (5) are a whole blood sample pretreatment method for ICP-MS detection of trace elements.

9. The method of claim 8, wherein the blood samples comprise blood samples of healthy adults, children, and pregnant women, and blood samples of persons suspected of being poisoned.

10. The method of claim 9, wherein the detection method is used for detecting environmental trace toxin contamination, detecting trace stimulant composition, forensic toxicant detection or identification of human or cadavers, and human trace element content assessment.

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