A method for detecting multiple elements in dried blood spots

Abstract

The present invention discloses a method for detecting multiple elements in dried blood spots, and belongs to the technical field of element analysis. The detection method of the present invention comprises the following steps: using a puncher to take blood spot pieces of the same size of dried blood spot samples, dried blood spot calibrators and dried blood spot quality control products respectively in different injection tubes, adding diluent working solution, vortexing and mixing, and ultrasonicating for 50 to 70 minutes to obtain the sample solution to be tested, the calibration solution and the quality control solution; the sample solution to be tested, the calibration solution and the quality control solution are detected by inductively coupled plasma mass spectrometry; wherein the diluent is a mixed aqueous solution of nitric acid, disodium ethylenediaminetetraacetic acid, cysteine ​​and methanol; the dried blood spot calibrators and dried blood spot quality control products contain bovine whole blood matrix. The sample pretreatment process of the present invention is simple and fast, and is suitable for the detection of large batches of clinical samples; at the same time, the detection method of the present invention has high sensitivity, high specificity, high anti-interference, high precision, high repeatability, and good application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of element analysis, and in particular to a method for detecting multiple elements in dried blood spots. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance the understanding of the overall background of the invention and should not be necessarily regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Both deficiency and excess of trace elements can cause disease and even death. Arsenic, molybdenum, palladium, cadmium, indium, antimony, gadolinium, platinum, mercury, thallium, lead, and bismuth are toxic heavy metals with no known benefits in the human body. Their high levels can lead to an increase in reactive oxygen species, damaging proteins, lipids, and DNA. Lithium, vanadium, cobalt, and molybdenum are essential trace elements related to life. Although they are present in very small amounts in the human body, they play a vital role in metabolism. The quantification of these elements is crucial for nutritional research and disease intervention.

[0004] However, there are logistical and ethical limitations in the collection and analysis of biological samples, especially whole blood. Although it can provide various nutritional status indicators, blood collection by venipuncture requires professional personnel and a cold chain for sample storage and transportation; for remote or resource-limited areas, the cost of equipment, storage and transportation is relatively high. In addition, blood sampling is invasive and ethically challenging for vulnerable groups such as newborns, children and the elderly.

[0005] Dried blood spot (DBS) is a method of collecting capillary blood on a special filter paper by pricking an individual's finger or heel, which provides an alternative method for collecting biological samples to venipuncture. It has a relatively small sample volume, reduces the sampling burden on patients, and is conducive to blood sampling for vulnerable groups such as infants, children and the elderly who have difficulty in venipuncture. However, elemental analysis of dried blood spots is challenging because it requires low detection limits and low sample volumes.

[0006] Patent publication number CN110687190A discloses a method for detecting multiple elements in dried blood spots, which uses nitric acid, cysteine, and Triton solution as digestion solutions to digest the sample blood spots and then uses inductively coupled plasma mass spectrometry (ICP-MS) for detection. Although the invention has achieved a low detection limit, the pretreatment of the invention requires vortexing and standing for 1 hour, followed by ultrasound for 10 minutes, and filtering with a syringe filter after centrifugation at 8000g. The pretreatment is relatively complicated and time-consuming, and is not suitable for screening large quantities of samples.

[0007] Therefore, how to provide a method and a kit for detecting multiple elements in dry blood spots that has a simple pretreatment method, high accuracy, and can be used for large-scale screening is an urgent problem to be solved. Summary of the invention

[0008] In view of this, the present invention provides a method for detecting multiple elements in dry blood spots, which solves the problem that the pre-treatment of the detection of multiple elements in dry blood spots in the prior art is complicated and not suitable for large-scale screening.

[0009] The present invention provides a method for detecting multiple elements in a dried blood spot, comprising the following steps:

[0010] Using a puncher to take blood spot pieces of the same size of the dried blood spot sample, the dried blood spot calibrator and the dried blood spot quality control product into different injection tubes, adding the diluent working solution, vortexing and mixing, and then ultrasonicating for 50 to 70 minutes to obtain a sample solution, a calibration solution and a quality control solution; the sample solution, the calibration solution and the quality control solution are detected by inductively coupled plasma mass spectrometry;

[0011] The dilution working solution is obtained by mixing a diluent and an internal standard working solution in a set ratio, and the diluent is a mixed aqueous solution of nitric acid, disodium ethylenediaminetetraacetate, cysteine ​​and methanol; the dried blood spot calibrator and dried blood spot quality control product contain bovine whole blood matrix.

[0012] Preferably, the plurality of elements include Li, V, Co, As, Mo, Pd, Cd, In, Sb, Cs, Gd, Pt, Hg, Tl, Pb and Bi.

[0013] Preferably, the preparation method of the bovine whole blood matrix is ​​as follows: take heparin sodium bovine anticoagulated whole blood in a centrifuge tube, centrifuge to separate plasma and blood cells, and then mix them in a volume ratio of plasma: blood cells = (54-58): (42-46) to obtain the bovine whole blood matrix.

[0014] Preferably, the preparation method of the dried blood spot calibrator is as follows: take the multi-element calibrator stock solution and dilute it proportionally with 0.5wt% HNO3 solution to form a series of concentration solutions, then mix the series of concentration solutions with bovine whole blood matrix at a volume ratio of 10% to obtain a series of matrix-containing calibrator solutions, and then drop the series of matrix-containing calibrator solutions into blood collection cards respectively to prepare different blood spot slices, and dry them for at least 4 hours to prepare dried blood spot calibrators.

[0015] Preferably, the dry blood spot quality control product includes a high concentration quality control product and a low concentration quality control product, and the preparation method of the dry blood spot quality control product is as follows: single element standard solutions of multiple elements are prepared into high concentration quality control theoretical concentrations and low concentration quality control theoretical concentrations, and then added to bovine whole blood matrix while mixing at a volume ratio of 1% to obtain high concentration quality control solutions and low concentration quality control solutions; the quality control target values ​​and ranges of the high concentration quality control solutions and low concentration quality control solutions are as follows:

[0016]

[0017] The high-concentration quality control solution and the low-concentration quality control solution were respectively dripped into two blood collection cards and dried for at least 4 hours to prepare dried blood spot quality control products.

[0018] Preferably, in the internal standard working solution, the concentration of each element is: beryllium 800 mg / L, scandium 100 mg / L, germanium 200 mg / L, yttrium 100 mg / L, rhodium 10 mg / L, rhenium 10 mg / L, and uranium 0.5 mg / L.

[0019] Preferably, the diluent is a mixed aqueous solution of 0.1wt% nitric acid, 0.01wt% disodium ethylenediaminetetraacetate, 0.2wt% cysteine ​​and 2wt% methanol; the volume ratio of the diluent to the internal standard working solution is 5000:1.

[0020] Preferably, the determination conditions of the inductively coupled plasma mass spectrometry are as follows: using a Burgener ari nebulizer, KED mode to collect data, and the collision gas is helium; the instrument parameters are as follows: the radio frequency power is 1500-1600W; the nebulizing gas flow rate is 0.9-1.1L / min; the auxiliary gas flow rate is 0.7-1.0L / min; the cooling gas flow rate is 12-15L / min; the sampling depth is 5-6mm, and the quantitative loop specification is 0.4-0.6mL.

[0021] Preferably, a standard curve is drawn with the concentration of a series of matrix calibrator solutions as the abscissa and the signal ratio of the calibrator solution to the internal standard as the ordinate to obtain a linear equation y=bx+a, and the signal ratio of the sample solution to be tested to the internal standard is substituted into the equation to calculate the concentration of each element in the sample solution to be tested.

[0022] Preferably, the diameter of the blood spot is 9 to 11 mm; the volume of the diluted internal standard solution added is 2 to 3 mL.

[0023] Preferably, the sample solution, calibration solution and quality control solution are transferred to an empty tube and detected by inductively coupled plasma mass spectrometry.

[0024] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0025] (1) The present invention uses a dried blood spot calibrator with bovine whole blood matrix, which is consistent with the blood spot size of the dried blood spot sample to be tested, and the sample pretreatment method is consistent. On the one hand, the loss of blood spots after extraction is corrected to avoid the situation of unknown sample size. On the other hand, the interference of matrix effect and physical effect is corrected, effectively improving the accuracy.

[0026] (2) The present invention adds nitric acid, disodium ethylenediaminetetraacetate, cysteine ​​and methanol to the diluent, wherein the addition of methanol can reduce the surface tension of the solution, help to better introduce the sample into the plasma, improve the atomization efficiency, thereby improving the sensitivity of the detection and reducing the detection limit; at the same time, methanol can reduce the degradation of certain elements and organic compounds in the sample, especially when the sample needs to be stored for a long time before or during analysis. Cysteine ​​and disodium ethylenediaminetetraacetate can form stable complexes with metal ions in the sample, thereby more efficiently extracting the elements in the blood spot, and at the same time can improve the stability of the sample, thereby improving the sensitivity and accuracy of the analysis.

[0027] (3) The sample pretreatment process of the present invention only requires ultrasonic extraction of the blood spot slices, and then detection by inductively coupled plasma mass spectrometry. The pretreatment process is simpler and faster, and is more suitable for the detection of large quantities of clinical samples.

[0028] (4) The present invention establishes a detection method suitable for multi-element dried blood spots with high sensitivity, high specificity, high anti-interference, high precision and high repeatability. The method can be used for heavy metal screening and post-healing monitoring, such as detecting trace toxin pollution in the environment, detecting the concentration of therapeutic drugs or post-poisoning recovery monitoring, and has good application prospects. DETAILED DESCRIPTION

[0029] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0030] The present invention provides a method for detecting multiple elements in a dried blood spot, comprising the following steps:

[0031] Using a puncher to take blood spot pieces of the same size of the dried blood spot sample, the dried blood spot calibrator and the dried blood spot quality control product into different injection tubes, adding the diluent working solution, vortexing and mixing, and then ultrasonicating for 50 to 70 minutes to obtain a sample solution, a calibration solution and a quality control solution; the sample solution, the calibration solution and the quality control solution are detected by inductively coupled plasma mass spectrometry;

[0032] The dilution working solution is obtained by mixing a diluent and an internal standard working solution in a set ratio, and the diluent is a mixed aqueous solution of nitric acid, disodium ethylenediaminetetraacetate, cysteine ​​and methanol; the dried blood spot calibrator and dried blood spot quality control product contain bovine whole blood matrix.

[0033] The present invention does not impose any special restrictions on the vortex mixing and ultrasonic operation steps, and the vortex mixing and ultrasonic operation steps commonly used in the art can be used. The present invention extracts elements from dried blood spot samples, dried blood spot calibrators and dried blood spot quality control products through the above steps.

[0034] In the present invention, the multiple elements include Li, V, Co, As, Mo, Pd, Cd, In, Sb, Cs, Gd, Pt, Hg, Tl, Pb and Bi. Among them, arsenic, molybdenum, palladium, cadmium, indium, antimony, gadolinium, platinum, mercury, thallium, lead and bismuth are toxic heavy metals with no known benefits in the human body. Their high content will lead to an increase in reactive oxygen species and damage proteins, lipids and DNA. Lithium, vanadium, cobalt and molybdenum are essential trace elements related to life. Although their content in the human body is extremely small, they play a vital role in metabolism. The quantification of these elements is crucial to nutritional research and disease intervention.

[0035] In the present invention, the preparation method of the bovine whole blood matrix is ​​as follows: take heparin sodium anticoagulated bovine whole blood in a centrifuge tube, centrifuge to separate plasma and blood cells, and then mix them in a volume ratio of plasma: blood cells = (54-58): (42-46) after stratification to obtain the bovine whole blood matrix. Due to the batch difference of heparin sodium anticoagulated bovine whole blood, the dilution and viscosity between batches are inconsistent, so there will be differences in the diffusion degree of filter paper that affect the results. The present invention adopts a centrifugal re-proportioning method to avoid the difference between batches, while keeping the dilution and viscosity consistent with human blood, thereby improving the accuracy of detecting real samples.

[0036] In the present invention, the preparation method of the dried blood spot calibrator is as follows: take the multi-element calibrator stock solution and dilute it into a series of concentration solutions in proportion using 0.5wt% HNO3 solution, then mix the series of concentration solutions with bovine whole blood matrix at a volume ratio of 10% to obtain a series of matrix-containing calibrator solutions, then drip the series of matrix-containing calibrator solutions into blood collection cards to prepare different blood spot pieces, and dry them for at least 4 hours to prepare dried blood spot calibrators. The present invention mixes the multi-element calibrator stock solution dilution with the bovine whole blood matrix to avoid inaccurate results caused by inconsistency between the calibrator and the sample matrix, and on the other hand, it can also correct the loss caused by blood spot extraction.

[0037] In the present invention, the dry blood spot quality control product includes a high-concentration quality control product and a low-concentration quality control product. The preparation method of the dry blood spot quality control product is as follows: single-element standard solutions of multiple elements are prepared into high-concentration quality control theoretical concentrations and low-concentration quality control theoretical concentrations, and then added to bovine whole blood matrix while mixing at a volume ratio of 1% to obtain high-concentration quality control solutions and low-concentration quality control solutions; the quality control target values ​​and ranges of the high-concentration quality control solutions and the low-concentration quality control solutions are as follows:

[0038]

[0039] The high concentration quality control solution and the low concentration quality control solution are respectively dripped into two blood collection cards, and dried for at least 4 hours to prepare a dry blood spot quality control product. The target value of the quality control product used in the present invention is determined by a reagent kit that has been verified under liquid conditions, and then a dry blood spot quality control is prepared from a blood collection card to examine the accuracy and effectiveness of the method and determine the accuracy of the test results.

[0040] In the internal standard working solution of the present invention, the concentration of each element is: beryllium 800mg / L, scandium 100mg / L, germanium 200mg / L, yttrium 100mg / L, rhodium 10mg / L, rhenium 10mg / L, uranium 0.5mg / L. The present invention does not impose any special restrictions on the preparation method of the internal standard working solution, and the preparation method commonly used by those skilled in the art can be adopted.

[0041] In the present invention, the diluent is preferably a mixed aqueous solution of 0.1wt% nitric acid, 0.01wt% disodium ethylenediaminetetraacetate, 0.2wt% cysteine ​​and 2wt% methanol. The present invention does not impose any special restrictions on the preparation method of the diluent, and the preparation method commonly used by those skilled in the art can be used. The water used in the present invention is preferably ultrapure water. In the present invention, the volume ratio of the diluent to the internal standard working solution is 5000:1.

[0042] In the present invention, the determination conditions of the inductively coupled plasma mass spectrometry are as follows: using a Burgener ari nebulizer, KED mode data collection, collision gas is helium; instrument parameters are as follows: radio frequency power is 1500-1600W; nebulizing gas flow rate is 0.9-1.1L / min; auxiliary gas flow rate is 0.7-1.0L / min; cooling gas flow rate is 12-15L / min; sampling depth is 5-6mm, and the quantitative loop specification is 0.4-0.6mL. Inductively coupled plasma mass spectrometry (ICP-MS) detection is based on the mass-to-charge ratio of elements, so the present invention has strong specificity for the detection of trace elements in peripheral blood and accurate quantification.

[0043] In the present invention, a standard curve is drawn with the concentration of a series of matrix calibrator solutions as the horizontal coordinate and the signal ratio of the calibrator solution to the internal standard as the vertical coordinate to obtain a linear equation y=bx+a, and the signal ratio of the sample solution to be tested to the internal standard is substituted into the equation to calculate the concentration of each element in the sample solution to be tested. The present invention adopts this method to correct the loss caused by extraction and avoid the difference in the extraction rate of each element. The obtained detection sample concentration is the real human blood sample concentration.

[0044] In the present invention, the diameter of the blood spot piece is 9 to 11 mm; the volume of the diluted internal standard solution added is 2 to 3 mL. The present invention uses blood spot pieces of the same size to ensure the same sampling volume. The present invention uses a neonatal blood collection card, whose pre-printed circle is a circle with a diameter of 12 mm. The production of the dry blood spot calibrator and the dry blood spot quality control product of the present invention uses 40 μL of blood to fill the pre-printed circle, and the clinical dry blood spot sample only needs to pierce the fingertips or heels to fill the pre-printed circle with blood drops. There is no need for accurate quantitative blood collection, and it can be dried, folded and stored. This method simplifies the blood collection process, and also eliminates the problem of unknown sample size that has always existed in dry blood spots. Taking a circular blood spot piece with a diameter of 9 to 11 mm can also maximize its sensitivity and reduce the quantitative limit.

[0045] In the present invention, the sample solution, calibration solution and quality control solution are transferred to an empty tube and detected by inductively coupled plasma mass spectrometry. The purpose of transferring to the empty tube is to remove the extracted blood spot pieces and the impurities precipitated at the bottom of the injection tube. If the detection is completed within 1 hour, the transfer is not required, and the blood spot pieces are shaken to sink to the bottom of the tube, and the injection needle is raised so that it does not touch the blood spot pieces. At the same time, the injection volume meets the injection requirements and can be directly tested on the machine.

[0046] The technical solution of the present invention is further described below in conjunction with specific embodiments.

[0047] Example

[0048] 1. Instrument:

[0049] The inductively coupled plasma mass spectrometer was YS EXT 8600MD (Shandong Yingsheng Biotechnology Co., Ltd.), the nebulizer was Burgener ari (Burgener Research Inc), the high-speed desktop refrigerated centrifuge was (Xiangyi), the vortex mixer was (Scientific industries), the ultrasonic cleaner was (240 W, Kangshijie Ultrasonic), and the hole puncher was (10 mm).

[0050] 2. Reagents:

[0051] Nitric acid (CNW, concentration ≥ 65%, trace metal analysis); methanol (mass spectrometry grade, Merck); disodium ethylenediaminetetraacetate (EDTA-2Na) (analytical grade, Macklin); L-cysteine ​​(purity 99%, Macklin); sodium heparin bovine anticoagulated whole blood (sodium heparin anticoagulation, Borsi); neonatal blood collection card (Best).

[0052] 3. Preparation of relevant solutions

[0053] (1) Preparation of diluent:

[0054] Weigh nitric acid, L-cysteine, methanol, and EDTA-2Na solution into a HPDE bottle, then add ultrapure water to make the concentration of each component (mass fraction of solute) as follows: 0.1% nitric acid, 0.2% cysteine, 2% methanol, 0.01% EDTA-2Na. After ultrasonic mixing for 10 minutes, use it as a diluent for later use.

[0055] (2) Preparation of cleaning solution:

[0056] Weigh nitric acid and EDTA-2Na solution into a HPDE bottle, add ultrapure water to make the concentration of each component (mass fraction of solute) as follows: nitric acid: 0.5%, EDTA-2Na: 0.002%, mix by ultrasonic for 10 minutes, and use as cleaning solution for later use.

[0057] (3) Preparation of internal standard working solution:

[0058] Take the internal standard stock solutions of beryllium, scandium, germanium, yttrium, rhodium, rhenium, and uranium respectively, take 400 μL beryllium element standard solution (concentration: 2000 mg / L) + 100 μL scandium element standard solution (concentration: 1000 mg / L) + 200 μL germanium element standard solution (concentration: 1000 mg / L) + 100 μL yttrium element standard solution (concentration: 1000 mg / L) + 10 μL rhodium element standard solution (concentration: 1000 mg / L) + 10 μL rhenium element standard solution (concentration: 1000 mg / L) + 5 μL uranium element standard solution (concentration: 100 mg / L) + 175 μL ultrapure water, mix well and prepare the internal standard working solution. The concentrations of each element are shown in Table 1.

[0059] Table 1 Concentration of each element in the internal standard working solution (unit: mg / L)

[0060] Beryllium Scandium Sc GermaniumGe Yttrium Rhodium Rhenium UraniumU 800 100 200 100 10 10 0.5

[0061] (4) Bovine whole blood matrix: Sodium heparinized bovine anticoagulated whole blood was placed in a centrifuge tube and centrifuged at 4000 rpm for 10 min to separate the plasma and blood cells. The blood cells were then mixed at a volume ratio of plasma:blood cells = 4:3 to obtain the bovine whole blood matrix.

[0062] (5) Multi-element calibrator stock solution: Prepare multi-element calibrator stock solution according to the concentrations shown in Table 2.

[0063] Table 2 Concentration of each element in the multi-element calibrant stock solution

[0064] element Mass number Concentration (μg / mL) element Mass number Concentration (μg / mL) Li 7 350 Sb 121 20 V 51 10 Cs 133 40 Co 59 10 G 157 10 As 75 40 Pt 195 10 Mo 95 50 Hg 202 20 Pd 105 10 Tl 205 10 Cd 111 20 Pb 208 100 In 115 10 Bi 209 6.4

[0065] 4. Sample collection and preparation

[0066] (1) Dried blood spot sample:

[0067] Use a disposable blood collection needle to prick the inside or outside of the heel to a depth of less than 3 mm, wipe off the first drop of blood with a dry cotton ball, and take the second drop of blood; touch the filter paper to the blood drop, and do not touch the heel skin, so that the blood can naturally penetrate to the back of the filter paper, and collect at least three blood spots; place the blood spot in clean air, avoid direct sunlight, and dry naturally to a dark brown color to obtain a dry blood spot sample, and store it in a sealed bag.

[0068] (2) Dried blood spot calibrator:

[0069] The multi-element calibrant stock solution was diluted 10 times with 0.5% HNO3 to obtain the high-concentration calibrant intermediate solution. The high-concentration calibrant intermediate solution was diluted with 0.5% HNO3 in a 1:1 ratio, with a total of 7 concentration gradients (including the high-concentration calibrant intermediate solution), from high concentration to low concentration, 7, 6, 5, 4, 3, 2, 1, respectively. The 7, 6, 5, 4, 3, 2, 1 solution and 0.5% HNO3 100μL to 900μL (volume ratio of 10%) bovine whole blood matrix were taken to obtain S7, S6, S5, S4, S3, S2, S1, Blank, a total of 8 matrix calibrant solutions. The theoretical concentrations in each solution are shown in Table 3.

[0070] Table 3 Theoretical concentrations of elements in a series of matrix calibrant solutions (unit: μg / L)

[0071]

[0072]

[0073] Take 40 μL of the above 8 solutions respectively and drop them into two blood collection cards (each containing 4 pre-printed circles), dry them for at least 4 hours to make dried blood spot calibrators, and fold and seal them for storage.

[0074] (3) Dried blood spot quality control products:

[0075] Single element standard solutions of 16 elements were respectively prepared into two theoretical quality control concentrations, which were added into bovine whole blood matrix while being mixed at a volume ratio of 1%. 40 μL of the two solutions were respectively dropped into two blood collection cards, and dried for at least 4 hours to prepare dried blood spot quality control products, which were folded and sealed for storage. The theoretical quality control concentrations are shown in Table 4.

[0076] Table 4 Quality Control Theoretical Concentration

[0077]

[0078]

[0079] 5. Sample pretreatment

[0080] (1) Preparation of dilution working solution

[0081] Prepare the diluted working solution by mixing the diluent and the internal standard working solution at a volume ratio of 5000:1. Prepare it before use and calculate the amount of the diluted working solution based on the number of samples to be tested.

[0082] (2) Sample preparation

[0083] ① Sample solution to be tested: Use a hole puncher to take a dry blood spot sample with a diameter of 10 mm and put it into an injection tube. Add 2 mL of dilution working solution, vortex to mix, put it into an ultrasonic machine for 60 minutes, and transfer the extracted solution to an empty tube to obtain the sample solution to be tested.

[0084] ② Calibrator solution: Use a hole puncher to take a 10mm diameter dry blood spot calibrator into an injection tube, add 2mL of dilution working solution, vortex to mix, put it into an ultrasonic machine for 60min, transfer the extracted solution to an empty tube to obtain the calibrator solution, and use it for testing.

[0085] ③ Quality control solution: Use a hole puncher to take a 10mm diameter dry blood spot quality control product into the injection tube, add 2mL of dilution working solution, vortex to mix, put it into the ultrasonic machine for 60min, transfer the extracted solution to an empty tube to obtain the quality control solution, and use it for testing.

[0086] 6. Detection method

[0087] Place the sample tube on the sample rack, start the application software (Qtegra software for YS EXT 8600MD), and create a test method and sample list.

[0088] The data were collected using a Burgener ari nebulizer (Burgener Research Inc) in KED mode, with helium as the collision gas, and an inductively coupled plasma mass spectrometer for detection. Instrument parameters: RF power (Plasma power) 1550W; Nebulizer flow (Nebulizer flow) 0.99L / min; Auxiliary flow (Auxiliary flow) 0.8L / min; Cool flow (Cool flow) 14L / min; Sampling depth (Sampling depth) 5.5mm, quantitative loop specification: 0.5mL.

[0089] 7. Performance Verification

[0090] (1) Standard curves of 16 elements

[0091] Open Qtegra software and select the corresponding data for result analysis. First, use the concentration of a series of matrix calibrant solutions as the horizontal axis (Table 3) and the ratio of the calibrant solution to the internal standard signal as the vertical axis to draw a standard curve to obtain the linear equation y=bx+a. Substitute the signal ratio of the sample to the internal standard into the equation to calculate the concentration of each element in the sample, the linear concentration range of each element, the standard curve, and the correlation coefficient (R 2 ) as shown in Table 5.

[0092] Table 5 Linear results of 16 elements

[0093]

[0094] (2) Limit of detection and limit of quantification

[0095] According to the reference range of each element in human whole blood and the minimum concentration that the instrument can detect, 16 element calibration mixtures were diluted with diluent (the concentration of each element is shown in Table 3), and 20 copies of each concentration were prepared in parallel; the coefficient of variation (CV) of the measurement results of all analytes was ≤20.0%, and the deviation between the measured value and the specified value was ≤20.0% as the quantitative limit, and 1 / 2 times the quantitative limit was used as the detection limit of 27 analytes. The detection limit and quantitative limit results are shown in Table 6.

[0096] Table 6 Results of detection limits and quantification limits of 16 elements

[0097]

[0098] Results: The coefficient of variation (CV) of the quantitative limits of 16 elements was between 3.34% and 13.60%, and the deviations between the measured values ​​and the specified values ​​were between -11.89% and 14.72%. The coefficient of variation (CV) of all analytes was ≤20.0%, and the deviations between the measured values ​​and the specified values ​​were ≤20.0%.

[0099] (3) Recovery rate

[0100] Clinical samples were selected and divided into three parts; different concentrations of the same volume of the standard solution of the analyte were added to two of the samples, and 40 μL of the sample with the standard solution was dripped onto the blood collection card to prepare the recovered dry blood spot sample, and the volume of the standard solution added did not exceed 10% of the volume of the clinical sample; the same volume of blank reagent without the analyte was added to the other sample as the basic dry blood spot sample. According to the instructions, the basic dry blood spot sample was measured in parallel 3 times, and the low and high concentration recovery dry blood spot samples were measured in parallel 10 times each. The mean was calculated respectively, and the recovery rate was calculated according to the formula. The recovery rate deviations of the 16 analytes were all within the range of ±15%, and the coefficient of variation (CV) was less than 15%. The results are shown in Tables 7 and 8.

[0101] Table 7 Results of experimental determination of the recovery rates of 16 analytes

[0102]

[0103] Table 8 Theoretical values ​​and deviations of recoveries of 16 analytes

[0104]

[0105]

[0106] Results: The recovery rate deviations of 16 elements were all within the range of -13.11% to 13.19%, and the coefficient of variation (CV) was within the range of 0.65% to 9.82%. The recovery rate deviations of all analytes were within the range of ±15%, and the coefficient of variation (CV) was less than 15%, indicating the accuracy of the method.

[0107] (4) Accuracy

[0108] To verify the accuracy of the method, the whole blood 29-element kit of Shandong Yingsheng Biotechnology Co., Ltd. was used to determine the prepared quality control products and obtain the target values. The quality control products were then prepared into dried blood spot quality control for accuracy and precision verification, as shown in Tables 9 to 11.

[0109] As can be seen from Table 9, the coefficient of variation (CV) of the measurement results of the 16 analytes was ≤10.0%, the measured values ​​were all within the given target value range, and the deviations from the target values ​​were all within the range of ±15%, demonstrating good accuracy.

[0110] Table 9 Accuracy results of 16 analytes (unit: μg / L, except CV)

[0111]

[0112]

[0113] Results: The coefficients of variation (CV) of the measurement results of 16 elements were all in the range of 1.14%-5.44%, and the deviations from the target values ​​were all in the range of -12.39%-16.84%. All analyte results were within the given target value range, indicating the reliability of the method.

[0114] Table 10 Intra-day precision results of 16 analytes (unit: μg / L, except CV)

[0115]

[0116]

[0117] Table 11 Inter-day precision results of 16 analytes (unit: μg / L, except CV)

[0118]

[0119]

[0120] Results: The coefficients of variation (CV) of intra-day and inter-day measurement results of 16 elements were all in the range of 3.57%-11.08%, indicating the reliability of the method.

[0121] Comparative Example 1

[0122] In order to verify the effect of adding bovine whole blood matrix to the calibrator on the detection of 16 elements in dried blood spots, this comparison was verified by recovery experiments.

[0123] (1) Recovery rate experiment

[0124] The specific experimental process is the same as the recovery rate test in the embodiment, but the bovine whole blood matrix is ​​not added to the multi-element calibrator. The multi-element calibrator stock solution is diluted 100 times with 0.5% HNO3 solution to obtain a high-concentration calibrator intermediate solution. Take 100 μL of the high-concentration calibrator intermediate solution and dilute it with 0.5% HNO3 solution in a 1:1 ratio, with a total of 7 concentration gradients (including the high-concentration calibrator intermediate solution), from high concentration to low concentration, 7, 6, 5, 4, 3, 2, 1, dilute with diluent, the concentration of each element is shown in Table 12, and the recovery results are shown in Table 13.

[0125] Table 12 Theoretical concentration of each compound at different concentration levels (unit: μg / L)

[0126]

[0127]

[0128] Table 13 Recovery rate deviation experimental results (unit: %)

[0129]

[0130] Comparative Example 2

[0131] In order to verify the influence of each component in the diluent on the extraction of 16 elements from blood spots, this comparative example was verified by a recovery rate experiment.

[0132] The specific experimental process is the same as the recovery rate test in the performance verification experiment, but the dilution working solution does not contain EDTA-2Na and cysteine, respectively. The results are shown in Table 14.

[0133] Table 14 Recovery rate deviation experimental results (unit: %)

[0134]

[0135]

[0136] Results: According to the results in Table 14, EDTA-2Na in the diluent mainly acts as a metal complexing agent, which stably complexes with elements such as cobalt, cadmium, indium, lead, and bismuth in the blood spots, ensuring that the elements in the blood spots are extracted into the diluent and ensuring their stability in the solution. Methanol in the diluent can compensate for the carbon matrix, thereby improving the sensitivity of elements such as arsenic and molybdenum. At the same time, cysteine ​​in the diluent can complex with mercury, increase the extraction of mercury in the blood spots, and increase the stability of mercury in the solution and in subsequent detection.

[0137] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting multiple elements in dried blood spots, characterized in that: The steps include: Using a puncher to take blood spot pieces of the same size of the dried blood spot sample, the dried blood spot calibrator and the dried blood spot quality control product into different injection tubes, adding the diluent working solution, vortexing and mixing, and then ultrasonicating for 50 to 70 minutes to obtain a sample solution, a calibration solution and a quality control solution; the sample solution, the calibration solution and the quality control solution are detected by inductively coupled plasma mass spectrometry; The dilution working solution is obtained by mixing a diluent and an internal standard working solution in a set ratio, and the diluent is a mixed aqueous solution of nitric acid, disodium ethylenediaminetetraacetate, cysteine ​​and methanol; the dried blood spot calibrator and dried blood spot quality control product contain bovine whole blood matrix; The plurality of elements include Li, V, Co, As, Mo, Pd, Cd, In, Sb, Cs, Gd, Pt, Hg, Tl, Pb and Bi; The diluent is a mixed aqueous solution of 0.1wt% nitric acid, 0.01wt% disodium ethylenediaminetetraacetate, 0.2wt% cysteine ​​and 2wt% methanol; the volume ratio of the diluent to the internal standard working solution is 5000:1; The determination conditions of the inductively coupled plasma mass spectrometry are as follows: a Burgener ari nebulizer is used, data is collected in KED mode, and the collision gas is helium; the instrument parameters are as follows: the radio frequency power is 1550 W; the nebulizing gas flow rate is 0.99 L / min; the auxiliary gas flow rate is 0.8 L / min; the cooling gas flow rate is 14 L / min; the sampling depth is 5.5 mm, and the quantitative loop specification is 0.5 mL.

2. The detection method according to claim 1, characterized in that The preparation method of the bovine whole blood matrix is ​​as follows: taking heparin sodium bovine anticoagulated whole blood in a centrifuge tube, centrifuging to separate plasma and blood cells, and then mixing them in a volume ratio of plasma: blood cells = (54-58): (42-46) to obtain the bovine whole blood matrix.

3. The detection method according to claim 1, characterized in that The preparation method of the dried blood spot calibrator is as follows: take the multi-element calibrator stock solution and dilute it into a series of concentration solutions in proportion using 0.5wt% HNO3 solution, then mix the series of concentration solutions with bovine whole blood matrix at a volume ratio of 10% to obtain a series of matrix-containing calibrator solutions, and then drop the series of matrix-containing calibrator solutions into blood collection cards to prepare different blood spot pieces, and dry them for at least 4 hours to prepare dried blood spot calibrators.

4. The detection method according to claim 1, characterized in that The dry blood spot quality control product includes a high-concentration quality control product and a low-concentration quality control product. The preparation method of the dry blood spot quality control product is as follows: single-element standard solutions of multiple elements are prepared into high-concentration quality control theoretical concentrations and low-concentration quality control theoretical concentrations, and then added to bovine whole blood matrix while mixing at a volume ratio of 1% to obtain high-concentration quality control solutions and low-concentration quality control solutions; the quality control target values ​​and ranges of the high-concentration quality control solutions and the low-concentration quality control solutions are as follows: The high-concentration quality control solution and the low-concentration quality control solution were respectively dripped into two blood collection cards and dried for at least 4 hours to prepare dried blood spot quality control products.

5. The detection method according to claim 1, characterized in that The concentration of each element in the internal standard working solution is: beryllium 800 mg / L, scandium 100 mg / L, germanium 200 mg / L, yttrium 100 mg / L, rhodium 10 mg / L, rhenium 10 mg / L, and uranium 0.5 mg / L.

6. The detection method according to claim 3, characterized in that: A standard curve is drawn with the concentration of a series of matrix calibrant solutions as the horizontal axis and the signal ratio of the calibrant solution to the internal standard as the vertical axis to obtain the linear equation y=bx+a. The signal ratio of the sample solution to be tested and the internal standard is substituted into the equation to calculate the concentration of each element in the sample solution to be tested.

7. The detection method according to claim 1, characterized in that The diameter of the blood spot is 9-11 mm; the volume of the diluted internal standard solution added is 2-3 mL; the sample solution, calibration solution and quality control solution are transferred to an empty tube, and the inductively coupled plasma mass spectrometry is used for detection.

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