Method for detecting various forms of arsenide in whole blood based on dry blood spot sampling

Abstract

The invention relates to a method for detecting various forms of arsenide in whole blood based on dry blood spot sampling, and belongs to the technical field of blood arsenide clinical detection. In order to solve the problem that a dry blood spot sampling technology is difficult to accurately quantify in detection of various forms of arsenide in whole blood, the invention provides a method for detecting various forms of arsenide in whole blood based on dry blood spot (DBS) sampling, and the method comprises the following steps: preparing a DBS series mixed standard sample, performing pretreatment, then performing HPLC-HG-AFS detection, and drawing a standard curve equation of AsIII, MMA and DMA; a to-be-detected DBS sample of a subject is subjected to pretreatment and HPLC-HG-AFS detection, peak areas are substituted into the corresponding standard curve equations, and the concentrations of the three arsenide compounds in the to-be-detected DBS sample are measured. According to the method, efficient arsenide extraction in the DBS, precise retention of the original valence state, clear separation of polymorphic baselines and collaborative standard reaching of detection precision improvement are realized, and reliable data support can be provided for clinic.

Description

Technical Field

[0001] This invention belongs to the field of clinical detection technology of blood arsenic compounds, and particularly relates to a method for detecting multiple forms of arsenic compounds in whole blood based on dried blood spot sampling. Background Technology

[0002] Arsenic trioxide (ATO) is a first-line treatment for acute promyelocytic leukemia (APL). Its active ingredient, trivalent inorganic arsenic (As), is... Ⅲ Arsenic compounds require methylation metabolism to produce monomethylarsonicacid (MMA) and dimethylarsinicacid (DMA). This metabolic process serves both detoxification and activity prolongation functions, and the in vivo exposure levels of the three arsenic forms are directly related to therapeutic efficacy and toxicity risk. Ⅲ MMA and DMA are the main pharmacologically active components. They can enhance anti-APL effects by inducing apoptosis and inhibiting proliferation, while excessive accumulation can cause toxicity to multiple organs such as the liver, heart, and kidneys, as well as a series of adverse reactions. Therefore, accurate quantitative detection of different forms of arsenic compounds in the blood is a key prerequisite for optimizing treatment effects and predicting toxic reactions, and has important clinical value for guiding individualized medication.

[0003] Current blood arsenic testing mainly relies on venous blood collection to obtain samples. Blood samples need to be stored at low temperatures and transported in a cold chain throughout the process. This not only increases the cost and difficulty of sample preservation, but also makes it difficult to control the transportation conditions when sending samples for testing. Temperature fluctuations can affect the stability of the samples, thereby interfering with the accuracy of the test results. Furthermore, it cannot meet the monitoring needs of children and patients receiving home treatment, thus limiting its flexible application in clinical personalized medication monitoring.

[0004] Dry blood spot (DBS) sampling technology has been widely used in drug metabolism monitoring and genetic disease screening due to its advantages such as minimal blood collection, low invasiveness, sample stability at room temperature, and ease of storage and transportation. However, there are still technical challenges to be solved in applying DBS technology to the detection of multiple forms of arsenic compounds in whole blood. Existing conventional DBS pretreatment methods cannot simultaneously achieve extraction efficiency, valence stability, and chromatographic separation compatibility, making it difficult to accurately quantify different forms of arsenic compounds in blood samples, which restricts the application and transformation of this technology in clinical ATO monitoring. Summary of the Invention

[0005] To address the challenge of accurately quantifying various forms of arsenic compounds in whole blood using dried blood spot sampling technology, this invention provides a method for detecting various forms of arsenic compounds in whole blood based on dried blood spot sampling.

[0006] The technical solution of the present invention:

[0007] A method for detecting multiple forms of arsenic compounds in whole blood based on dried blood spot sampling includes the following steps:

[0008] Step 1: Prepare As III -MMA-DMA mixed stock solution was diluted with ultrapure water to form a series of mixed standard solutions with the same concentration of three arsenides. Human blank whole blood was added to each series of mixed standard solutions to obtain a series of mixed standard samples of blank whole blood.

[0009] Step 2: Prepare DBS series mixed standard samples from the blank whole blood series mixed standard samples; prepare DBS test samples from the subject's whole blood;

[0010] Step 3: DBS Sample Pretreatment

[0011] Take blood spots from DBS test samples or DBS series mixed standard samples, add formic acid, vortex and sonicate, incubate at 90℃ for 30 min, centrifuge and take the supernatant and mix it with an equal volume of pure water to prepare test loading solution and series mixed standard loading solution.

[0012] Step 4: Plot the standard curve:

[0013] The series of mixed standard sample solutions were analyzed by HPLC-HG-AFS, with As III Linear regression was performed with the concentrations of three arsenides—MMA, DMA, and arsenic trioxide (A, MMA, and DMA)—on the x-axis and the corresponding peak areas on the y-axis to obtain the As... III Standard curve equations for MMA and DMA;

[0014] Step 5: Testing and Data Analysis

[0015] The sample solution to be tested was analyzed under the same HPLC-HG-AFS conditions. The obtained peak area was substituted into the corresponding standard curve equation, and quantification was performed using the external standard method to obtain the As content in the DBS sample. III The concentrations of three arsenides: MMA and DMA.

[0016] Furthermore, as described in step one... III -MMA-DMA mixed stock solution containing As III The concentrations of the three arsenides, MMA and DMA, were all 2000 ng / mL.

[0017] Furthermore, the gradient concentrations of the three arsenides in the series of mixed standard solutions described in step one are 200 ng / mL, 500 ng / mL, 800 ng / mL, 1000 ng / mL, 1500 ng / mL, 1800 ng / mL and 2000 ng / mL, respectively.

[0018] Furthermore, the blank whole blood series mixed standard sample obtained in step one was prepared by mixing 20 μL of series mixed standard solution with 180 μL of human blank whole blood.

[0019] Furthermore, the gradient concentrations of the three arsenic compounds in the blank whole blood series mixed standard samples were 20 ng / mL, 50 ng / mL, 80 ng / mL, 100 ng / mL, 150 ng / mL, 180 ng / mL and 200 ng / mL, respectively.

[0020] Furthermore, in step two, the DBS series mixed standard sample is prepared by quantitatively aspirating 40 μL of blank whole blood series mixed standard sample using a microcapillary tube and spotting it inside the circle on a Whatman 903# filter paper. After the filter paper has completely absorbed the blood, the microcapillary tube is removed, and the filter paper is dried and sealed for later use.

[0021] The DBS test sample is prepared by quantitatively aspirating 40 μL of whole blood from the subject using a microcapillary tube, spotting it inside the circle on a Whatman903# filter paper, removing the microcapillary tube after the filter paper has completely absorbed the blood, drying the filter paper, and sealing it for later use.

[0022] Furthermore, in step three, the blood spots are obtained by taking samples from the DBS test sample or the DBS series mixed standard sample using a 3mm diameter punch. Four blood spots are taken from each sample and 100μL of 2M formic acid is added. The vortexing time is 1min, the ultrasonic power is 120w for 10min, and the centrifugation is performed at 4℃ and 18311 centrifugal force for 15min. 70μL of the centrifugal supernatant is then mixed with 70μL of pure water.

[0023] Furthermore, the HPLC conditions for HPLC-HG-AFS detection described in step four are as follows:

[0024] Anion exchange column: Hamilton PRP-X100, 250 mm × 4.1 mm, 10 μm; mobile phase: a combination solution of 13 mmol / L anhydrous sodium acetate, 3 mmol / L anhydrous sodium dihydrogen phosphate, 4 mmol / L potassium nitrate and 0.2 mmol / L disodium ethylenediaminetetraacetate; flow rate: 1.0 mL / min; injection volume: 100 μL.

[0025] The HG-AFS analysis conditions were as follows: main current: 80mA; auxiliary current: 40mA; shielding gas flow rate: 900mL / min; carrier gas flow rate: 300mL / min; negative high voltage: 300V; pump speed: 60r / min; reducing solution: containing 3% potassium borohydride and 0.5% potassium hydroxide; carrier solution: 5% hydrochloric acid.

[0026] Furthermore, the As obtained in step four IIIThe standard curve equation is: Y = 1.178 × 10⁻⁶ 5 +2.184×10 5 X, r 2 =0.996, linear range is 20~200ng / mL;

[0027] The standard curve equation for MMA is Y = 4.527 × 10⁻⁶. 4 +2.287×10 5 X, r 2 =0.995, linear range is 20~200ng / mL;

[0028] The standard curve equation for DMA is Y = 3.714 × 10⁻⁶. 3 +1.778×10 5 X, r 2 =0.996, with a linear range of 20~200 ng / mL.

[0029] Furthermore, a series of mixed quality control samples of blank whole blood were prepared by mixing 200 ng / mL of mixed standard solution with different volumes of human blank whole blood to obtain a series of mixed quality control samples of blank whole blood with gradient concentrations of three arsenic compounds of 25 ng / mL, 75 ng / mL, 125 ng / mL and 175 ng / mL, respectively. The mixed quality control samples of blank whole blood were prepared using the same method as the mixed standard samples of blank whole blood, including DBS series mixed quality control sample preparation, pretreatment and HPLC-HG-AFS detection. The accuracy and precision of the detection method were judged by the recovery rate and relative standard deviation of the mixed quality control samples of blank whole blood.

[0030] The beneficial effects of this invention are:

[0031] This invention innovatively employs a weak acid high-temperature pretreatment combination strategy, which achieves synergistic results in efficient extraction of arsenic compounds from DBS, accurate preservation of original valence states, clear separation of multiple arsenic forms, and improved detection accuracy through systematic regulation. This breaks through the technical barriers to the application of DBS technology in the detection of multiple arsenic forms in whole blood and achieves a comprehensive improvement in detection performance.

[0032] This invention relies on precise adaptation and optimization of weak acid and high temperature. On the one hand, it can efficiently separate and release arsenic compounds of different valence states from DBS filter paper and erythrocyte matrix, achieving simultaneous and efficient extraction of inorganic and organic arsenic, while strictly maintaining the stability of various arsenic compounds, especially As, throughout the process. ⅢThe original valence state and structural integrity are preserved; on the other hand, by scientifically matching the mild characteristics of weak acid with high temperature parameters, the damage to the valence state of arsenic compounds and the breaking of carbon-arsenic covalent bonds in organic arsenic are avoided by high temperature and acid treatment, thus avoiding interference from valence state changes and structural degradation on the detection results, and simultaneously improving extraction efficiency and target concentration, providing a guarantee for subsequent accurate detection.

[0033] This invention also precisely optimizes the compatibility of the extraction solution, ensuring that the treated sample fully meets the requirements for chromatographic separation, and can achieve As Ⅲ The baselines of the three target arsenic forms, MMA and DMA, were clearly separated without peak overlap interference, solving the separation problem of simultaneous analysis of multiple arsenic forms.

[0034] The recovery rate, relative standard deviation, and valence stability verification data provided by this invention demonstrate that the detection method of this invention has extremely high accuracy and precision. It can be accurately applied to the monitoring of arsenic speciation in APL patients after ATO treatment, providing reliable data support for clinical efficacy evaluation and toxicity prediction. Meanwhile, the DBS matrix possesses inherent advantages such as high sample stability, convenient room-temperature transportation, minimal invasiveness, and small blood volume, making it suitable for routine monitoring scenarios, especially for children and patients at home. Attached Figure Description

[0035] Figure 1 As of the DBS mixed standard sample measured in Example 1 III Total chromatograms of MMA and DMA;

[0036] Figure 2 As of the DBS test sample measured in Example 1 III Total chromatograms of MMA and DMA;

[0037] Figure 3 As measured in the methanol pretreatment DBS mixed standard sample of Comparative Example 1 III Total chromatograms of MMA and DMA;

[0038] Figure 4 As measured in the DBS mixed standard sample of Comparative Example 2 without high-temperature incubation before pretreatment III Total chromatograms of MMA and DMA;

[0039] Figure 5 As measured in Comparative Example 3, the pH of the sample solution was not adjusted before pretreatment. III Total chromatograms of MMA and DMA;

[0040] Figure 6 As measured in the standard whole blood mixed sample of Comparative Example 4 III Total chromatograms of MMA and DMA. Detailed Implementation

[0041] The technical solution of the present invention will be further described below with reference to embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention. In the following embodiments, the process equipment or apparatus not specifically specified are all conventional equipment or apparatus in the art. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available; unless otherwise specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0042] Example 1

[0043] This embodiment provides a method for detecting multiple forms of arsenic compounds in whole blood based on dried blood spot sampling, including the following steps:

[0044] Step 1: Prepare a series of blank whole blood mixed standard samples:

[0045] (a) Preparation of As III -MMA-DMA Mixed Stock Solution

[0046] Prepared with pure water, containing As III A mixed stock solution of arsenic compounds in three forms: MMA and DMA, with a concentration of 2000 ng / mL for each of the three arsenic compounds.

[0047] (ii) Preparation of a series of mixed standard solutions with the same concentration of three arsenic compounds

[0048] The mixed stock solution was diluted with pure water to obtain a series of mixed standard solutions with gradient concentrations of 200 ng / mL, 500 ng / mL, 800 ng / mL, 1000 ng / mL, 1500 ng / mL, 1800 ng / mL and 2000 ng / mL for the three arsenic compounds, respectively.

[0049] (III) Preparation of blank whole blood series mixed standard samples

[0050] In this embodiment, blood samples were collected from 7 healthy volunteers using anticoagulant tubes, and all blood samples were mixed to obtain arsenic-free human blank whole blood.

[0051] Take 20 μL of the series mixed standard solution into a 1.5 mL EP tube, add 180 μL of human blank whole blood, and gently invert to mix to obtain the blank whole blood series mixed standard sample. The gradient concentrations of the three arsenic compounds in the blank whole blood series mixed standard sample are 20 ng / mL, 50 ng / mL, 80 ng / mL, 100 ng / mL, 150 ng / mL, 180 ng / mL and 200 ng / mL, respectively.

[0052] Step 2: Prepare DBS series mixed standard samples from blank whole blood series mixed standard samples; prepare DBS test samples from whole blood of subjects;

[0053] Using a microcapillary tube, 40 μL of blank whole blood series mixed standard sample was quantitatively aspirated and spotted into the circle on a Whatman 903# filter paper. After the filter paper had completely absorbed the blood, the microcapillary tube was removed, and the filter paper was placed face up on a drying rack and allowed to air dry at room temperature for at least 2 hours. After drying, it was placed in a sealed bag to obtain DBS series mixed standard samples with gradient concentrations of three arsenic compounds of 20 ng / mL, 50 ng / mL, 80 ng / mL, 100 ng / mL, 150 ng / mL, 180 ng / mL, and 200 ng / mL, respectively. These samples were stored at -20℃ for later use.

[0054] Quantitatively draw 40 μL of whole blood from the subject using a microcapillary tube and spot it inside the circle on a filter paper disc (whatman903#). After the filter paper disc has completely absorbed the blood, remove the microcapillary tube, allow the filter paper disc to dry, and obtain the DBS test sample. Seal and store for later use.

[0055] Step 3: DBS Sample Pretreatment

[0056] Blood spots were collected from the DBS test sample or the DBS series mixed standard sample using a 3mm diameter punch. Four blood spots were collected from each sample and placed in a 2mL flat-bottomed EP tube. 100μL of 2M formic acid was added, and the tube was vortexed for 1 min, followed by sonication at 120W for 10 min. The tube was then sealed and incubated at 90℃ for 30 min. After incubation, the tube was centrifuged at 18311 at 4℃ for 15 min. 70μL of the supernatant was collected in a new EP tube, and 70μL of pure water was added and mixed to obtain the test sample solution and the series mixed standard sample solution.

[0057] Step 4: Plot the standard curve:

[0058] The series of mixed standard sample solutions were sequentially analyzed by HPLC-HG-AFS. In this example, the HPLC conditions were as follows: anion exchange column: Hamilton PRP-X100, 250 mm × 4.1 mm, 10 μm; mobile phase: a combination solution consisting of 13 mmol / L anhydrous sodium acetate, 3 mmol / L anhydrous sodium dihydrogen phosphate, 4 mmol / L potassium nitrate and 0.2 mmol / L disodium ethylenediaminetetraacetate; flow rate: 1.0 mL / min; injection volume: 100 μL.

[0059] HG-AFS analysis conditions: Main current: 80mA; Auxiliary current: 40mA; Shielding gas flow rate: 900mL / min; Carrier gas flow rate: 300mL / min; Negative high voltage: 300V; Pump speed: 60r / min; Reducing solution: containing 3% potassium borohydride and 0.5% potassium hydroxide; Carrier solution: 5% hydrochloric acid.

[0060] The total chromatogram obtained by HPLC-HG-AFS detection of a DBS mixed standard sample with three arsenic compounds at a concentration of 100 ng / mL is shown below. Figure 1 As shown in the figure, the three peaks correspond to As in sequence. III The peaks of MMA and DMA were clear and non-overlapping, and the baseline separation between the peaks was good, proving that the anion exchange column (Hamilton PRP-X100) and mobile phase combination used in this embodiment can effectively distinguish these three arsenic forms.

[0061] As III Linear regression was performed using the concentrations of three arsenic compounds—MMA, DMA, and DMA—on the x-axis and the corresponding peak areas on the y-axis to obtain the As... III Standard curve equations for MMA and DMA:

[0062] As III The standard curve equation is Y = 1.178 × 10⁻⁶. 5 +2.184×10 5 X, r 2 =0.996, linear range is 20~200ng / mL;

[0063] The standard curve equation for MMA is Y = 4.527 × 10⁻⁶. 4 +2.287×10 5 X, r 2 =0.995, linear range is 20~200ng / mL;

[0064] The standard curve equation for DMA is Y = 3.714 × 10⁻⁶. 3 +1.778×10 5 X, r 2 =0.996, with a linear range of 20~200 ng / mL.

[0065] Step 5: Testing and Data Analysis

[0066] The sample solution to be tested was analyzed under the same HPLC-HG-AFS conditions. The obtained peak area was substituted into the corresponding standard curve equation, and quantification was performed using the external standard method to obtain the As content in the DBS sample. III The concentrations of three arsenides: MMA and DMA.

[0067] The subject included in this embodiment was one APL patient who received ATO induction remission therapy on day 10. The subject's basic information is as follows: male, 32 years old, body surface area 1.72 m² 2 The ATO dosage was 0.16 mg / (kg·d), and no significant abnormalities were observed in liver and kidney function during the treatment period.

[0068] The DBS sample from this subject was analyzed by HPLC-HG-AFS, and the resulting chromatogram is shown below. Figure 2 As shown, three characteristic peaks appeared in the sample to be tested, and their retention times were related to... Figure 1 As of the DBS standard sample III The retention times of MMA and DMA were completely consistent (deviation < 0.1 min), with no interference from stray peaks, confirming that the detected target analyte was As. III MMA, DMA.

[0069] Based on the quantitative analysis using the standard curve, the patient's various indicators were within the normal concentration range during the ATO treatment induction period, indicating that the patient's arsenic metabolism was good.

[0070] This demonstrates that the method of this embodiment can effectively monitor the metabolic levels of different forms of arsenic compounds in the body during the treatment of APL patients. This embodiment uses HPLC-HG-AFS, which is less expensive and simpler to operate than ICP-MS, and its sensitivity meets the detection requirements for clinical ATO treatment.

[0071] Example 2

[0072] This embodiment verifies the accuracy and precision of the detection method for multiple forms of arsenic compounds in whole blood based on dried blood spot sampling provided in Example 1 as follows:

[0073] Three arsenic compound gradient concentrations of 25 ng / mL, 75 ng / mL, 125 ng / mL, and 175 ng / mL were prepared by mixing 200 ng / mL of mixed standard solution with different volumes of blank human whole blood. DBS series mixed control samples were prepared using the same method as the blank whole blood series mixed standard samples in Example 1, including sample pretreatment and HPLC-HG-AFS detection. Three batches were analyzed repeatedly over three consecutive days. The recoveries and intra-day and inter-day precision are shown in Table 1.

[0074] Table 1

[0075]

[0076] As can be seen from the data in Table 1, the method provided in Example 1, under different spiking levels, AsIII The spiked recoveries of MMA and DMA were all within ±15%; the intra-day precision and inter-day precision were both within ±15%, indicating that the detection method provided in Example 1 has good precision and repeatability and can be used for accurate quantitative analysis.

[0077] Example 3

[0078] This embodiment verifies the stability of the method for detecting multiple forms of arsenic compounds in whole blood based on dried blood spot sampling provided in Example 1 as follows:

[0079] Three groups of blank whole blood series mixed quality control samples with gradient concentrations of three arsenic compounds of 25 ng / mL, 75 ng / mL, 125 ng / mL and 175 ng / mL were prepared according to the method in Example 2. Group 1 was placed at room temperature (25℃) for 3 days; Group 2 was frozen at -4℃ for 7 days; and Group 3 was frozen at -80℃ for 30 days.

[0080] The test was performed according to the test method in Example 1. Groups 1, 2 and 3 were analyzed repeatedly for 3 batches. The room temperature stability, -4℃ stability and -80℃ stability (30 days) are shown in Table 2.

[0081] Table 2

[0082]

[0083] As shown in Table 2, in groups 1, 2, and 3, As... III The recoveries of MMA and DMA at different spiking levels were all within ±20%, demonstrating that the stability of the detection method provided in Example 1 meets the standard.

[0084] Example 4

[0085] This embodiment verifies the sample homogeneity of the detection method for multiple forms of arsenic compounds in whole blood based on dried blood spot sampling provided in Example 1 as follows:

[0086] Two groups of blank whole blood series mixed quality control samples with gradient concentrations of three arsenic compounds of 25 ng / mL, 75 ng / mL, 125 ng / mL, and 175 ng / mL were prepared according to the method in Example 2. Four blood spots were punched in the central region of the DBS blood spot in Group 1, and four blood spots were punched in the edge region of the DBS blood spot in Group 2. The determination was performed according to the method provided in Example 1. The analysis was repeated for 3 batches, and the recovery rates are shown in Table 3.

[0087] Table 3

[0088]

[0089] As shown in Table 3, in Group 1 and Group 2, As IIIThe recovery rates of MMA and DMA samples obtained by punching samples in different regions of DBS were all within ±20%, proving that the sample homogeneity of the detection method provided in Example 1 meets the standard.

[0090] Comparative Example 1

[0091] This comparative example demonstrates a method for detecting multiple forms of arsenic compounds in whole blood using existing dried blood spot pretreatment methods.

[0092] The only difference between this comparative example and Example 1 is the DBS sample pretreatment method. The pretreatment method for this comparative example is as follows:

[0093] Using a DBS mixed standard sample with a concentration of 100 ng / mL for each of the three arsenic compounds as the test sample, four blood spots were taken from the DBS mixed standard sample using a 3 mm diameter punch and placed in a 2 mL flat-bottomed EP tube. 200 μL of methanol was added to the EP tube, and the mixture was vortexed for 1 min, then sonicated at 120 W for 10 min. The mixture was then centrifuged at 18311 at 4 °C for 15 min, and 100 μL of the supernatant was extracted into a new EP tube as the sample loading solution.

[0094] HPLC-HG-AFS detection was performed using the same method as in Example 1, and the results were as follows: Figure 3 The total chromatogram shown does not contain As. III The characteristic peaks corresponding to MMA and DMA only exhibit baseline noise. This indicates that the commonly used methanol pretreatment method for DBS is not suitable for the extraction of arsenic speciation from this DBS sample.

[0095] Comparative Example 2

[0096] The only difference between this comparative example and Example 1 is that high-temperature incubation was not used during the DBS sample pretreatment. The pretreatment method for this comparative example is as follows:

[0097] Using a DBS mixed standard sample with three arsenic compounds at a concentration of 100 ng / mL as the test sample, four blood spots were taken from the DBS mixed standard sample using a 3 mm diameter punch and placed in a 2 mL flat-bottomed EP tube. 100 μL of 2M formic acid was added, and the sample was vortexed for 1 min, then sonicated at 120 W for 10 min. The sample was then centrifuged at 18311 at 4 °C for 15 min. 70 μL of the supernatant was taken into a new EP tube, and 70 μL of pure water was added and mixed to obtain the mixed standard loading solution.

[0098] HPLC-HG-AFS detection was performed using the same method as in Example 1, and the results were as follows: Figure 4 The total chromatogram shown is consistent with... Figure 1 compared to, Figure 4 As IIIThe characteristic peak areas corresponding to MMA and DMA were significantly reduced. This indicates that without high-temperature incubation, the weak acid dissolution of formic acid combined with ultrasonic action alone is insufficient to completely extract arsenic compounds from DBS, resulting in a low arsenic compound concentration in the extract, ultimately manifested as a significant reduction in chromatographic peak area. This result verifies that weak acid combined with high-temperature incubation is the key step in improving the arsenic compound extraction efficiency of DBS samples in this invention. This invention overcomes the technical bias that "high temperature easily causes changes in the valence state of arsenic compounds." Through the synergistic control of weak acid and heating temperature, the binding of arsenic compounds to the DBS filter paper matrix and hemoglobin is disrupted by high temperature, achieving full release of the target compound; simultaneously, the mild properties of weak acid inhibit the arsenic compounds from being extracted under high-temperature conditions. Ⅲ The oxidation reaction also avoids the breakage of the carbon-arsenic covalent bond in organic arsenic, ensuring extraction efficiency while fully preserving the original form of each arsenic compound, thus solving the technical contradiction of difficulty in balancing extraction efficiency and morphological stability in traditional DBS arsenic speciation detection.

[0099] Comparative Example 3

[0100] The only difference between this comparative example and Example 1 is that the compatibility of the loading solution was not optimized. The pretreatment method for this comparative example is as follows:

[0101] Using a DBS mixed standard sample with three arsenic compounds at a concentration of 100 ng / mL as the test sample, blood spots were collected from the DBS mixed standard sample using a 3 mm diameter punch. Four blood spots from each sample were placed in a 2 mL flat-bottomed EP tube, and 100 μL of 2M formic acid was added. After vortexing for 1 min, the tube was sonicated at 120 W for 10 min, followed by sealing and incubation at 90 °C for 30 min. After incubation, the tube was centrifuged at 18311 rpm for 15 min at 4 °C, and the supernatant was collected into a new EP tube to obtain the mixed standard loading solution.

[0102] HPLC-HG-AFS detection was performed using the same method as in Example 1, and the results were as follows: Figure 5 The total chromatogram shown indicates that As Ⅲ The characteristic peaks of MMA and DMA were not separated, showing only a single mixed peak. This indicates that the high concentration of acid matrix in the formic acid extract has compatibility issues with the HPLC mobile phase, interfering with the column's retention and separation of different arsenides, ultimately preventing baseline separation of the three target compounds.

[0103] This invention uses pure water to dilute and adjust the pH instead of the traditional strong acid / base / buffer solution adjustment. While ensuring separation in a compatible chromatographic system, it avoids the introduction of impurities and changes in speciation, ultimately achieving a simultaneous improvement in separation efficiency, speciation stability, and detection sensitivity in arsenic speciation detection.

[0104] Comparative Example 4

[0105] This comparative example provides a method for detecting arsenic compounds in whole blood without using dried blood spot sampling. The steps are as follows:

[0106] The blank whole blood series mixed standard samples were prepared according to the method of Example 1. The blank whole blood mixed standard sample with the concentration of each of the three arsenic compounds being 100 ng / mL was used as the test sample. 50 μL of the blank whole blood mixed standard sample was taken into a 2 mL flat-bottomed EP tube, 200 μL of 30% hydrogen peroxide was added, and the mixture was vortexed. The resulting sample was left to stand at room temperature overnight. The next day, the sample was centrifuged at low speed for 5 min, 150 μL of the supernatant was taken, and 50 μL of 20% perchloric acid was mixed. The mixture was centrifuged at 18311 at 4°C for 15 min. The supernatant was extracted into a new EP tube to obtain the test sample.

[0107] HPLC-HG-AFS detection was performed using the same method as in Example 1, and the results were as follows: Figure 6 The total chromatogram shown has three characteristic peaks, which, combined with the retention times, correspond to DMA, MMA, and As. Ⅴ However, As did not appear. Ⅲ The characteristic peaks indicate that As in the blank whole blood mixed standard sample Ⅲ It has been oxidized to As Ⅴ .

[0108] The comparative analysis results show that the existing hydrogen peroxide + perchloric acid pretreatment method for arsenic compound detection causes trivalent arsenic to be oxidized to pentavalent arsenic, affecting the accuracy of arsenic compound analysis. The pretreatment method provided by this invention avoids the use of strong oxidizing agents and effectively maintains the original valence state of arsenic compounds during sample processing, thereby significantly improving the accuracy and reliability of arsenic speciation detection.

Claims

1. A method for detecting multiple forms of arsenic compounds in whole blood based on dried blood spot sampling, characterized in that, Includes the following steps: Step 1: Prepare As III -MMA-DMA mixed stock solution was diluted with ultrapure water to form a series of mixed standard solutions with the same concentration of three arsenides. Human blank whole blood was added to each series of mixed standard solutions to obtain a series of mixed standard samples of blank whole blood. Step 2: Prepare DBS series mixed standard samples from the blank whole blood series mixed standard samples; prepare DBS test samples from the subject's whole blood; Step 3: DBS Sample Pretreatment Take blood spots from DBS test samples or DBS series mixed standard samples, add formic acid, vortex and sonicate, incubate at 90℃ for 30 min, centrifuge and take the supernatant and mix it with an equal volume of pure water to prepare test loading solution and series mixed standard loading solution. Step 4: Plot the standard curve: The series of mixed standard sample solutions were analyzed by HPLC-HG-AFS, with As III Linear regression was performed with the concentrations of three arsenides—MMA, DMA, and arsenic trioxide (A, MMA, and DMA)—on the x-axis and the corresponding peak areas on the y-axis to obtain the As... III Standard curve equations for MMA and DMA; Step 5: Testing and Data Analysis The sample solution to be tested was analyzed under the same HPLC-HG-AFS conditions. The obtained peak area was substituted into the corresponding standard curve equation, and quantification was performed using the external standard method to obtain the As content in the DBS sample. III The concentrations of three arsenides: MMA and DMA.

2. The method for detecting multiple forms of arsenic compounds in whole blood based on dried blood spot sampling according to claim 1, characterized in that, As described in step one III -MMA-DMA mixed stock solution containing As III The concentrations of the three arsenides, MMA and DMA, were all 2000 ng / mL.

3. The method for detecting multiple forms of arsenic compounds in whole blood based on dried blood spot sampling according to claim 1 or 2, characterized in that, The gradient concentrations of the three arsenides in the series of mixed standard solutions described in step one are 200 ng / mL, 500 ng / mL, 800 ng / mL, 1000 ng / mL, 1500 ng / mL, 1800 ng / mL and 2000 ng / mL, respectively.

4. The method for detecting multiple forms of arsenic compounds in whole blood based on dried blood spot sampling according to claim 3, characterized in that, The blank whole blood series mixed standard sample obtained in step one was prepared by mixing 20 μL of series mixed standard solution with 180 μL of human blank whole blood.

5. The method for detecting multiple forms of arsenic compounds in whole blood based on dried blood spot sampling according to claim 4, characterized in that, The gradient concentrations of the three arsenic compounds in the blank whole blood series mixed standard samples were 20 ng / mL, 50 ng / mL, 80 ng / mL, 100 ng / mL, 150 ng / mL, 180 ng / mL and 200 ng / mL, respectively.

6. The method for detecting multiple forms of arsenic compounds in whole blood based on dried blood spot sampling according to claim 5, characterized in that, Step 2 describes the DBS series mixed standard sample, which involves quantitatively aspirating 40 μL of blank whole blood mixed standard sample using a microcapillary tube and spotting it inside the circle on a Whatman 903# filter paper. After the filter paper has completely absorbed the blood, the microcapillary tube is removed, and the filter paper is dried and sealed for later use. The DBS test sample is prepared by quantitatively aspirating 40 μL of whole blood from the subject using a microcapillary tube, spotting it inside the circle on a Whatman903# filter paper, removing the microcapillary tube after the filter paper has completely absorbed the blood, drying the filter paper, and sealing it for later use.

7. The method for detecting multiple forms of arsenic compounds in whole blood based on dried blood spot sampling according to claim 6, characterized in that, The blood spots described in step three are obtained by taking samples from the DBS test sample or the DBS series mixed standard sample using a 3mm diameter punch. Four blood spots are taken from each sample and 100μL of 2M formic acid is added. The vortexing time is 1min, the ultrasonic power is 120w and the time is 10min, and the centrifugation is carried out at 4℃ and 18311 centrifugal force for 15min. 70μL of the centrifugation supernatant is taken and mixed with 70μL of pure water.

8. The method for detecting multiple forms of arsenic compounds in whole blood based on dried blood spot sampling according to claim 7, characterized in that, The HPLC conditions for HPLC-HG-AFS detection described in step four are as follows: Anion exchange column: Hamilton PRP-X100, 250 mm × 4.1 mm, 10 μm; mobile phase: a combination solution of 13 mmol / L anhydrous sodium acetate, 3 mmol / L anhydrous sodium dihydrogen phosphate, 4 mmol / L potassium nitrate and 0.2 mmol / L disodium ethylenediaminetetraacetate; flow rate: 1.0 mL / min; injection volume: 100 μL. The HG-AFS analysis conditions were: main current: 80mA; auxiliary current: 40mA; shielding gas flow rate: 900mL / min; carrier gas flow rate: 300mL / min. Negative high voltage: 300V; Pump speed: 60r / min; Reducing solution: contains 3% potassium borohydride and 0.5% potassium hydroxide by mass; Carrier fluid: 5% hydrochloric acid (by volume).

9. The method for detecting multiple forms of arsenic compounds in whole blood based on dried blood spot sampling according to claim 8, characterized in that, As obtained in step four III The standard curve equation is: Y = 1.178 × 10⁻⁶ 5 +2.184×10 5 X, r 2 =0.996, linear range is 20~200ng / mL; The standard curve equation for MMA is Y = 4.527 × 10⁻⁶. 4 +2.287×10 5 X, r 2 =0.995, linear range is 20~200ng / mL; The standard curve equation for DMA is Y = 3.714 × 10⁻⁶. 3 +1.778×10 5 X, r 2 =0.996, with a linear range of 20~200 ng / mL.

10. The method for detecting multiple forms of arsenic compounds in whole blood based on dried blood spot sampling according to claim 9, characterized in that, A series of mixed quality control samples of blank whole blood were prepared by mixing 200 ng / mL of mixed standard solution with different volumes of human blank whole blood to obtain a series of mixed quality control samples of blank whole blood with gradient concentrations of three arsenic compounds of 25 ng / mL, 75 ng / mL, 125 ng / mL and 175 ng / mL respectively. The mixed quality control samples of blank whole blood were prepared using the same method as the mixed standard samples of blank whole blood, including DBS series mixed quality control sample preparation, pretreatment and HPLC-HG-AFS detection. The accuracy and precision of the detection method were judged by the recovery rate and relative standard deviation of the mixed quality control samples of blank whole blood.

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