Kit and detection method for detecting bisphenol compounds in dried blood spots

Abstract

The invention relates to the field of environmental chemistry and biological sample analysis, in particular to a kit for detecting bisphenol compounds in dried blood spots and a detection method. The kit comprises a mixed standard stock solution and a mixed internal standard stock solution, and can be used for simultaneously detecting bisphenol compounds at least comprising BPA, BPF, BPS, BPAF, BPAP, BPB, BPC, 2, 4-BPS, BPSIP, BPS-MAE, TGSA, DPS, DDS, TDP, D-90 and DD-70 in dried blood spots. The kit and the detection method disclosed by the invention have the advantages of high absolute recovery rate, low matrix effect, high sensitivity, high accuracy, high stability and the like, and are used as a first report for simultaneously detecting various bisphenol compounds in dried blood spots; the method has wide practical application value and significance for evaluating the health effect of the bisphenol compound and the health condition of a human body.

Description

Technical Field

[0001] This invention relates to the fields of environmental chemistry and biological sample analysis, specifically to a kit and detection method for detecting bisphenol compounds in dried blood spots. Background Technology

[0002] With the acceleration of industrialization and modernization, bisphenol compounds, one of the endocrine disruptors, are widely used in daily life and industrial production. Bisphenol compounds include bisphenol A (BPA), bisphenol F (BPF), and bisphenol S (BPS), which are important additives in products such as plastics, epoxy resins, and thermal paper. Their extensive use not only pollutes the environment but also enters the human body through the food chain, drinking water, and skin contact, posing a potential threat to human health. Existing research indicates that bisphenol compounds have endocrine disrupting properties and may be associated with various health problems, including reproductive system abnormalities, metabolic diseases, cardiovascular diseases, and an increased risk of cancer.

[0003] With the emergence of alternatives to bisphenol compounds, such as 2,4-dihydroxydiphenyl sulfone (2,4-BPS) and 4-hydroxy-4'-isopropoxydiphenyl sulfone (BPSIP), the detection of a single compound is no longer sufficient to comprehensively assess human exposure. These alternatives differ from the original compounds in chemical structure, physical properties, and biological activity, and their potential health risks to humans require further investigation. Biomonitoring, as an effective means of assessing the extent of human exposure to chemicals, can directly reflect the actual exposure level of an individual or group by detecting specific biomarkers in biological samples (such as blood and urine). However, traditional biomonitoring methods (such as venous blood collection) have limitations such as high invasiveness, complex operation, and high cost, especially limiting their application in sensitive populations such as children, pregnant women, and the elderly. Dried blood spots (DBS), as a minimally invasive, easily stored, and transportable biological sample collection method, has shown great potential in the field of biomonitoring in recent years. DBS technology involves collecting blood from the fingertip or heel, then applying a small amount of blood to a special filter paper to create a dried blood spot. This not only simplifies the sampling process but also reduces costs, making large-scale population monitoring possible.

[0004] Despite the numerous advantages of deep blood spot (DBS) technology in biomonitoring, its application in the detection of bisphenol compounds still faces significant bottlenecks. The complex matrix of dried blood spots contains a large amount of interfering substances such as proteins and lipids, placing higher demands on the recovery rate and purification efficiency of trace bisphenol compounds. Traditional pretreatment methods, such as protein precipitation and liquid-liquid extraction, often suffer from low recovery rates and poor impurity removal when processing DBS samples, making it difficult to meet the needs of simultaneously determining multiple bisphenol compounds. Furthermore, the stability, extraction efficiency, and detection sensitivity of bisphenol compounds in DBS are also key factors limiting their accurate determination.

[0005] Currently, there are no reported analytical methods for detecting bisphenol compounds in dried blood spots. These methods are significantly inadequate for newly emerging or poorly studied substances, especially novel bisphenol compounds such as BPSIP, BPS-MAE, and DBSP. Studies of unreported substances have revealed that existing methods suffer from low absolute recoveries during sample processing and detection, affecting the accuracy of the final analytical results. Therefore, there is an urgent need to develop a kit and detection method capable of simultaneously determining multiple bisphenol compounds in dried blood spots, providing a powerful tool for revealing the health effects of bisphenol compounds and assessing human health status. Summary of the Invention

[0006] The purpose of this invention is to overcome the problems existing in the prior art and provide a kit and detection method for detecting bisphenol compounds in dried blood spots. This kit and detection method can simultaneously perform quantitative analysis of multiple bisphenol compounds in dried blood spots, and has advantages such as high absolute recovery rate, low matrix effect, high sensitivity, high accuracy, and high stability.

[0007] To achieve the above objectives, the present invention provides a kit for detecting bisphenol compounds in dried blood spots, which can simultaneously detect bisphenol compounds including at least BPA, BPF, BPS, BPAF, BPAP, BPB, BPC, 2,4-BPS, BPSIP, BPS-MAE, TGSA, DPS, DDS, TDP, D-90 and DD-70. The kit includes a mixed standard stock solution and a mixed internal standard stock solution; The mixed standard stock solution contains at least bisphenol compounds including BPA, BPF, BPS, BPAF, BPAP, BPB, BPC, 2,4-BPS, BPSIP, BPS-MAE, TGSA, DPS, DDS, TDP, D-90 and DD-70. The mixed internal standard stock solution contains at least BPA- 13 C 12 BPF-D 10 BPS- 13 C 12 BPAF- 13 C 12 BPAP-D5, BPB- 13 C 12 2,4-BPS- 13 C 12 DPS-D 10 Internal standard compounds including DDS-D4 and BPSIP-D7.

[0008] A second aspect of the present invention provides a method for detecting bisphenol compounds in dried blood spots, the method using the above-mentioned kit.

[0009] A third aspect of the present invention provides an application of the above-described reagent kit and detection method in the assessment of human health status.

[0010] This invention develops a kit and detection method for the simultaneous determination of multiple bisphenol compounds in dried blood spots. By using specific reagents in the kit to perform specific pretreatment on the dried blood spot samples, the samples are purified and enriched, thereby significantly reducing matrix effects and improving the absolute recovery rate of the analytes. The detection method employed in this kit is simple and efficient, possessing advantages such as high sensitivity, high accuracy, and high stability. Furthermore, as the first report of the simultaneous detection of multiple bisphenol compounds in dried blood spots, it has broad practical application value and significance for assessing the health effects of bisphenol compounds and human health status. In addition, this invention also solves the problem of difficult venous blood collection for sensitive populations such as infants and the elderly through a home-based fingertip blood spot detection solution. Detailed Implementation

[0011] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0012] The present invention provides a kit for detecting bisphenol compounds in dried blood spots, which can simultaneously detect bisphenol compounds including at least BPA, BPF, BPS, BPAF, BPAP, BPB, BPC, 2,4-BPS, BPSIP, BPS-MAE, TGSA, DPS, DDS, TDP, D-90 and DD-70. The kit includes a mixed standard stock solution and a mixed internal standard stock solution; The mixed standard stock solution contains at least bisphenol compounds including BPA, BPF, BPS, BPAF, BPAP, BPB, BPC, 2,4-BPS, BPSIP, BPS-MAE, TGSA, DPS, DDS, TDP, D-90 and DD-70. The mixed internal standard stock solution contains at least BPA- 13 C 12 BPF-D 10 BPS- 13 C 12 BPAF-13 C 12 BPAP-D5, BPB- 13 C 12 2,4-BPS- 13 C 12 DPS-D 10 Internal standard compounds including DDS-D4 and BPSIP-D7.

[0013] According to the present invention, the bisphenol compounds contained in the above-mentioned mixed standard stock solution are: BPA is 2,2-bis(p-hydroxyphenyl)propane; BPF is 4,4'-dihydroxydiphenylmethane; BPS is 4,4'-dihydroxydiphenyl sulfone; BPAF is 4,4'-(hexafluoroisopropylidene)bisphenol; BPAP is 4,4'-(1-phenylethyl)bisphenol; BPB is bisphenol butane; BPC is 4,4'-(1-methylethylidene)bis(2-methylphenol); and 2,4-BPS is 2,4-dihydroxydiphenylmethane. Sulfone; BPSIP is 4-hydroxy-4'-isopropoxydiphenyl sulfone; BPS-MAE is 4-allyloxy-4'-hydroxydiphenyl sulfone; TGSA is 4,4'-sulfonylbis[2-(2-propenyl)]phenol; DPS is diphenyl sulfone; DDS is 4,4'-diaminodiphenyl sulfone; TDP is 4,4'-dihydroxydiphenyl sulfide; D-90 is bis(2-chloroethyl) ether-4,4'-dihydroxydiphenyl sulfone copolymer; DD-70 is [2-(4-hydroxyphenylthio)ethoxy]methane.

[0014] According to the present invention, the internal standard compound contained in the above-mentioned mixed internal standard stock solution includes: BPA- 13 C 12 2,2-bis(p-hydroxyphenyl)propane- 13 C 12 BPF-D 10 It is 4,4'-dihydroxydiphenylmethane-D 10 BPS- 13 C 12 It is 4,4'-dihydroxydiphenyl sulfone- 13 C 12 BPAF- 13 C 12 4,4'-(hexafluoroisopropylidene)bisphenol- 13 C 12 BPAP-D5 is 4,4'-(1-phenylethyl)bisphenol-D5; BPB- 13 C 12 Bisphenol Butane- 13 C 12 ;2,4-BPS- 13 C 12 2,4-Dihydroxydiphenyl sulfone 13 C 12DPS-D 10 diphenyl sulfone-D 10 DDS-D4 is 4,4'-diaminodiphenyl sulfone-D4; BPSIP-D7 is 4-hydroxy-4'-isopropoxydiphenyl sulfone-D7.

[0015] According to the present invention, among the above-mentioned bisphenol compounds and internal standard compounds: the internal standard compound corresponding to BPA is BPA- 13 C 12 The internal standard compound corresponding to BPF is BPF-D. 10 The internal standard compound corresponding to BPS is BPS- 13 C 12 The internal standard compound corresponding to BPAF is BPAF- 13 C 12 The internal standard compound corresponding to BPAP is BPAP-D5; the internal standard compound corresponding to BPB is BPB- 13 C 12 The internal standard compound corresponding to BPC is BPAP-D5; the internal standard compound corresponding to 2,4-BPS is 2,4-BPS- 13 C 12 The internal standard compound corresponding to BPSIP is BPSIP-D7; the internal standard compound corresponding to BPS-MAE is BPS- 13 C 12 The internal standard compound corresponding to TGSA is BPAF- 13 C 12 The internal standard compound corresponding to DPS is DPS-D. 10 The internal standard compound corresponding to DDS is DDS-D4; the internal standard compound corresponding to TDP is BPS- 13 C 12 The internal standard compound corresponding to D-90 is BPAF- 13 C 12 The internal standard compound corresponding to DD-70 is BPA- 13 C 12 .

[0016] According to the present invention, in order to better perform quantitative analysis, the concentrations of the above-mentioned mixed standard stock solution and mixed internal standard stock solution can be set. Preferably, the concentrations of each bisphenol compound in the mixed standard stock solution are simultaneously 1-20 μg / mL, more preferably 5-15 μg / mL, for example, values ​​such as 5 μg / mL, 8 μg / mL, 10 μg / mL and 15 μg / mL and any range between these values.

[0017] Preferably, the concentration of each internal standard compound in the mixed internal standard stock solution is simultaneously 3-8 μg / mL, more preferably 4-6 μg / mL, for example, it can be 4 μg / mL, 4.5 μg / mL, 5 μg / mL and 6 μg / mL and any range between these values.

[0018] According to the present invention, the solvents of the mixed standard stock solution and the mixed internal standard stock solution can be selected from a wide range. In order to obtain better analytical results, the solvents of the mixed standard stock solution and the mixed internal standard stock solution are preferably methanol.

[0019] According to the present invention, the mixed standard stock solution and the mixed internal standard stock solution can be obtained by conventional preparation methods in the art, for example, by the following methods: Mixed standard stock solution: Weigh each bisphenol compound into a single volumetric flask, add solvent to make up to volume, and repeat this process to obtain single standard stock solutions for each bisphenol compound. Transfer equal volumes of the single standard stock solutions for each bisphenol compound into volumetric flasks, add solvent to make up to volume, and obtain the mixed standard stock solution.

[0020] Mixed internal standard stock solution: Weigh each internal standard compound into a separate volumetric flask, add solvent to make up to volume, and repeat this process to obtain individual standard stock solutions for each internal standard compound. Transfer equal volumes of the individual standard stock solutions for each internal standard compound into volumetric flasks, add solvent to make up to volume, and obtain the mixed internal standard stock solution.

[0021] According to the present invention, for better detection using the kit, preferably, the kit further includes a diluent, wherein the diluent is methanol. The diluent is used to dilute the mixed standard stock solution and the mixed internal standard stock solution.

[0022] According to the present invention, in order to perform good pretreatment of dried blood spot samples to reduce their matrix effect and improve the absolute recovery rate of the analyte, thereby obtaining more accurate and reliable detection results, the kit preferably further includes a first extraction solution, wherein the first extraction solution is water.

[0023] Preferably, the kit further includes a second extraction solution, which is acetonitrile.

[0024] Preferably, the kit further includes a solid-phase extraction plate, wherein the solid-phase extraction plate is an HMR 96-well plate.

[0025] According to the present invention, after pretreatment of dried blood spot samples, the samples need to be reconstituted to obtain a sample of appropriate volume and concentration for subsequent analysis and detection. In order to better dissolve the sample and improve the accuracy of detection, the kit preferably further includes a reconstituter, which is a methanol aqueous solution with a volume fraction of 10-30% (the volume fraction can be, for example, 10%, 15%, 20% and 30% and any range between these values).

[0026] According to the present invention, in order to obtain more accurate and stable analytical results, it is necessary to combine specific UHPLC-MS / MS detection conditions to complete the quantitative analysis. Preferably, the UHPLC-MS / MS detection conditions applicable to the kit include: using WatersAcquity BEH C 18 Chromatographic column; mobile phase A: water; mobile phase B: methanol; total injection time: at least 15 min; elution gradient: 0-2 min, 80% A, 20% B; 2-4 min, 80% A - 70% A, 20% B - 30% B; 4-7 min, 70% A, 30% B; 7-10 min, 70% A - 50% A, 30% B - 50% B; 10-12 min, 50% A - 20% A, 50% B - 80% B; 12-13 min, 20% A, 80% B; 13-13.01 min, 20% A - 80% A, 80% B - 20% B; 13.01 min - end of elution, 80% A, 20% B.

[0027] According to the present invention, in order to obtain faster detection speed and improve detection stability and accuracy, preferably, the UHPLC-MS / MS detection conditions applicable to the kit further include: injection volume 3-7 μL; column temperature 35-50℃; injection temperature 5-12℃; mobile phase flow rate 0.1-0.4 mL / min; ion source ESI; scanning mode positive and negative ion scanning; monitoring mode MRM; carrier gas nitrogen and collision gas helium; capillary voltage 2.3-2.6 kV; ion source temperature 120-180℃; and desolventizing temperature 400-600℃.

[0028] More preferably, the conditions for UHPLC-MS / MS detection applicable to the kit further include: injection volume of 4-6 μL (e.g., 4 μL, 5 μL, and 6 μL, or any range thereof); column temperature of 40-45℃ (e.g., 40℃, 42℃, 43℃, and 45℃, or any range thereof); injection temperature of 8-10℃ (e.g., 8℃, 9℃, and 10℃, or any range thereof); and mobile phase flow rate of 0.2-0.3 mL / min (e.g., 0.2 mL / min, 0.25 mL / min, and 0.3 mL / min, or any range thereof). (The range of any values); Ion source: ESI; Scan mode: positive and negative ion scanning; Monitoring mode: MRM; Carrier gas: nitrogen; Collision gas: helium; Capillary voltage: 2.4-2.5kV (e.g., 2.4kV, 2.45kV, and 2.5kV, etc., and any range between these values); Ion source temperature: 140-160℃ (e.g., 140℃, 145℃, 150℃, and 160℃, etc., and any range between these values); Desolventizing temperature: 450-550℃ (e.g., 450℃, 480℃, 500℃, and 550℃, etc., and any range between these values).

[0029] According to the present invention, under the above-mentioned UHPLC-MS / MS detection conditions, the retention time of BPA is 12.29 min; the retention time of BPF is 10.97 min; the retention time of BPS is 5.85 min; the retention time of BPAF is 12.91 min; the retention time of BPAP is 12.95 min; the retention time of BPB is 12.73 min; the retention time of BPC is 13.00 min; the retention time of 2,4-BPS is 7.81 min; the retention time of BPSIP is 12.34 min; the retention time of BPS-MAE is 12.14 min; the retention time of TGSA is 12.57 min; the retention time of DPS is 11.27 min; the retention time of DDS is 2.37 min; the retention time of TDP is 11.86 min; the retention time of D-90 (n=1) is 12.15 min; and the retention time of BPA- 13 C 12 The retention time was 12.29 min; BPF-D 10 The retention time was 10.82 min; BPS- 13 C 12 The retention time was 5.84 min; BPAF- 13 C 12 The retention time was 12.91 min; the retention time of BPAP-D5 was 8.63 min; the retention time of BPB- 13 C 12The retention time was 12.73 min; 2,4-BPS- 13 C 12 The retention time was 7.81 min; DPS-D 10 The retention time was 11.15 min; the retention time of DDS-D4 was 2.34 min; and the retention time of BPSIP-D7 was 12.31 min.

[0030] A second aspect of the present invention provides a method for detecting bisphenol compounds in dried blood spots, the method using the above-mentioned kit.

[0031] According to the present invention, the method for detecting bisphenol compounds in dried blood spots includes: mixing dried blood spot sample, mixed internal standard working solution and first extraction solution and performing a first ultrasonic treatment; adding second extraction solution and performing a second ultrasonic treatment; taking the supernatant and filtering it under positive pressure using the solid phase extraction plate; repeating the above operation once; combining the organic phases obtained from the two operations; drying them and resolving them with the redissolving solvent to obtain the sample to be tested; performing UHPLC-MS / MS detection on the sample to be tested; and quantifying the bisphenol compounds in the sample to be tested by establishing a standard curve.

[0032] According to the present invention, in order to perform quantitative analysis, it is necessary to set the amount of each component. Preferably, the dried blood spot sample is obtained by drying 30-80 μL of whole blood, and more preferably by drying 40-60 μL of whole blood (for example, values ​​such as 40 μL, 50 μL, 55 μL and 60 μL and any range between these values).

[0033] Preferably, the mixed internal standard working solution is obtained by diluting the mixed internal standard stock solution with the diluent, and the concentration of each internal standard compound in the mixed internal standard working solution is simultaneously 0.3-0.8 μg / mL, preferably 0.4-0.6 μg / mL, for example, it can be 0.4 μg / mL, 0.5 μg / mL, 0.55 μg / mL and 0.6 μg / mL and any range between these values.

[0034] Preferably, the volume of the mixed internal standard working solution is 5-30 μL, more preferably 10-20 μL, for example, it can be 10 μL, 15 μL, 18 μL and 20 μL and any range between these values.

[0035] According to the present invention, in order to better remove impurities and reduce matrix effect, preferably, the amount of the first extract is 50-150 μL, more preferably 80-120 μL, for example, it can be 80 μL, 100 μL, 110 μL and 120 μL and any range between these values.

[0036] Preferably, the duration of the first ultrasonic treatment is 5-50 min, more preferably 10-30 min, for example, it can be 10 min, 15 min, 20 min and 30 min and any range between these values.

[0037] According to the present invention, in order to better recover the analyte and improve the absolute recovery rate, preferably, the amount of the second extract is 150-250 μL, more preferably 180-220 μL, for example, it can be 180 μL, 200 μL, 210 μL and 220 μL and any range between these values.

[0038] Preferably, the duration of the second ultrasonic treatment is 5-50 min, more preferably 10-30 min, for example, it can be 10 min, 20 min, 25 min and 30 min and any range between these values.

[0039] According to the present invention, in order to facilitate subsequent UHPLC-MS / MS detection, the amount of the reconstitution solvent is preferably 50-150 μL, more preferably 80-120 μL, for example, it can be 80 μL, 90 μL, 100 μL and 120 μL and any range between these values.

[0040] According to the present invention, preferably, the gas used for drying is a non-reactive gas, such as nitrogen and / or argon.

[0041] According to the present invention, preferably, the drying is performed at 30-50°C, more preferably at 35-45°C, for example, values ​​such as 35°C, 40°C and 45°C and any range between these values.

[0042] According to the present invention, when the sample to be tested is used for UHPLC-MS / MS detection, the conditions for UHPLC-MS / MS detection are as described in the first aspect above.

[0043] According to the present invention, the quantitative analysis is performed using the internal standard method, which can minimize errors caused by pretreatment and instrument instability, thereby improving the accuracy of the analytical results. The standard curve used in the internal standard method can be established by existing known methods. Preferably, the method for establishing the standard curve includes: diluting the mixed standard stock solution with the diluent to form a series of mixed standard working solutions with gradient mass concentrations (e.g., more than three concentrations, such as at least three concentration values ​​among 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 250 ng / mL, and 500 ng / mL); mixing each of the mixed standard working solutions of different concentrations with the mixed internal standard working solution; adjusting the volume to a certain concentration with the diluent; and then performing UHPLC-MS / MS detection. The standard curve is obtained by using the ratio of the peak area of ​​each bisphenol compound to the peak area of ​​the corresponding internal standard compound in the detection results as the ordinate and the detection concentration as the abscissa.

[0044] More preferably, the method for establishing the standard curve includes: diluting the mixed standard stock solution with the diluent to prepare a series of mixed standard working solutions with mass concentrations of 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 250 ng / mL and 500 ng / mL respectively; taking 50 μL of each mixed standard working solution and adding 50 μL of the mixed internal standard working solution; then adjusting the volume to 500 μL with the diluent; and performing UHPLC-MS / MS detection respectively. The standard curve is obtained by using the ratio of the peak area of ​​each bisphenol compound to the peak area of ​​the corresponding internal standard compound in the detection results as the ordinate and the detection concentrations of 0.1 ng / mL, 0.25 ng / mL, 0.5 ng / mL, 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL and 50 ng / mL as the abscissa.

[0045] A third aspect of the present invention provides an application of the above-described reagent kit and detection method in the assessment of human health status.

[0046] The kit and detection method of this invention enable the simultaneous determination of multiple bisphenol compounds in dried blood spots, exhibiting advantages such as high sensitivity, high accuracy, and high stability. As the first report of simultaneous detection of multiple bisphenol compounds in dried blood spots, it has broad practical application value and significance for assessing the health effects of bisphenol compounds and human health status. The limits of detection for bisphenol compounds are 0.04-1.17 ng / mL, the limits of quantitation are 0.15-1.48 ng / mL, the spiked recovery rate is 70-128%, the precision is <20%, and the matrix effect is 76-124%, achieving excellent analytical detection results.

[0047] The present invention will be described in detail below through embodiments.

[0048] Unless otherwise specified, the apparatus used in the following examples are all conventional in the art, the operations performed are all conventional in the art, and the raw materials and reagents used are all commercially available. The instrument used for UHPLC-MS / MS detection was an IClass plus / XEVOTQXS. All bisphenol compounds and their corresponding internal standards were purchased from Toronto Research Chemicals (TRC), Cambridge Isotope Laboratories (CIL), Dr. Ehrenstorfer, Tokyo Chemical Industry Co., Ltd. (TCI), and Tianjin Altascientific Co., Ltd., respectively. HMR 96-well plates were purchased from Beijing Anavo Technologies Co., Ltd.

[0049] Example 1 This embodiment provides a kit and detection method for detecting bisphenol compounds in dried blood spots. The kit includes: a mixed standard stock solution; a mixed internal standard stock solution; a diluent: methanol; a first extraction solution: water; a second extraction solution: acetonitrile; a solid-phase extraction plate: an HMR 96-well plate; and a reconstitution solvent: a 20% (v / v) methanol-water solution.

[0050] The preparation methods for the mixed standard stock solution and the mixed internal standard stock solution are as follows: Single-standard stock solutions of BPA, BPF, BPS, BPAF, BPAP, BPB, BPC, 2,4-BPS, BPSIP, BPS-MAE, TGSA, DPS, DDS, TDP, D-90, and DD-70 with a concentration of 100 μg / L were prepared with methanol. Then, equal volumes of each of the above 16 single-standard stock solutions were transferred to the same volumetric flask and diluted with methanol to prepare mixed standard stock solutions with a concentration of 10 μg / mL.

[0051] BPA-1 was prepared with methanol at a concentration of 1 mg / mL. 13 C 12 BPF- 13 C 12 BPS- 13 C 12 BPAF- 13 C 12 BPAP-D5, BPB- 13 C 12 2,4-BPS- 13 C 12 DPS-D 10 The single-standard stock solutions of DDS-D4 and BPSIP-D7 were prepared, and then the same volume of each of the above 10 single-standard stock solutions was transferred to the same volumetric flask and diluted with methanol to prepare a mixed internal standard stock solution of 5 μg / mL.

[0052] The detection method used in this kit is as follows.

[0053] (a) Establishing standard curves for 16 bisphenol compounds The mixed standard stock solution was diluted with methanol to prepare a series of mixed standard working solutions with mass concentrations of 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 250 ng / mL, and 500 ng / mL. 50 μL of each mixed standard working solution was taken, and 50 μL of mixed internal standard working solution (obtained by diluting the mixed internal standard stock solution with methanol, concentration of 0.5 μg / mL) was added. The solution was then diluted with methanol to a final volume of 500 μL. UHPLC-MS / MS was performed for detection under the conditions shown in step (III) below. The standard curve was obtained by plotting the ratio of the peak area of ​​each bisphenol compound to the peak area of ​​the corresponding internal standard compound on the ordinate and the detection concentrations of 0.1 ng / mL, 0.25 ng / mL, 0.5 ng / mL, 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL, and 50 ng / mL on the abscissa. Calculate its linear range based on the standard curve, when the linear coefficient (R) 2 When the linearity is greater than 0.999, it indicates good linearity, and quantitative calculations can be performed accordingly. The linear correlation parameters of each bisphenol compound are shown in Table 1.

[0054] Table 1

[0055] Note: In the linear equation, Y represents the ratio of the standard response value to the internal standard response value, and X represents the mass concentration.

[0056] As shown in Table 1, the method detection limits for the 16 bisphenol compounds in this invention are 0.04-1.17 ng / mL, the method quantification limits are 0.15-1.48 ng / mL, and the linear coefficients (R0) are [not specified in the original text]. 2 All values ​​are above 0.999, indicating a good linear relationship.

[0057] (II) Pretreatment of dried blood spots A dried blood spot sample obtained by drying 50 μL of whole blood was added to a centrifuge tube, along with 10 μL of mixed internal standard working solution (obtained by diluting mixed internal standard stock solution with methanol, concentration 0.5 μg / mL) and 100 μL of water. The mixture was sonicated for 20 min. Then, 200 μL of acetonitrile was added, and the mixture was sonicated for another 20 min. The resulting supernatant was transferred to an HMR 96-well plate and filtered under positive pressure. The above operation was repeated once, and the organic phases obtained from the two tests were combined. After drying under nitrogen at 40 °C, the mixture was reconstituted with 100 μL of methanol-water solution (volume fraction 20%) to obtain the sample to be tested.

[0058] (III) UHPLC-MS / MS detection The sample to be tested was analyzed by UHPLC-MS / MS, and the bisphenol compounds in the sample were quantified according to the standard curve.

[0059] The conditions for UHPLC-MS / MS detection include: using Waters Acquity BEH C 18 Chromatographic column; mobile phase A: water; mobile phase B: methanol; total injection time: 15 min; elution gradient: 0-2 min, 80% A, 20% B; 2-4 min, 80% A-70% A, 20% B-30% B; 4-7 min, 70% A, 30% B; 7-10 min, 70% A-50% A, 30% B-50% B; 10-12 min, 50% A-20% A, 50% B-80% B; 12-13 min, 20% A 80%B; 13-13.01 min, 20%A-80%A, 80%B-20%B; 13.01 min - elution end, 80%A, 20%B; injection volume 5 μL; column temperature 40℃; injection temperature 8℃; mobile phase flow rate 0.3 mL / min; ion source: ESI; scanning mode: positive and negative ion scanning; monitoring mode: MRM; carrier gas: nitrogen, collision gas: helium; capillary voltage: 2.46 kV; ion source temperature: 150℃; desolvation temperature: 500℃.

[0060] The retention times, monitoring ions, cone voltages, and collision energies of 16 bisphenol compounds and their corresponding 10 internal standard compounds are shown in Table 2.

[0061] Table 2

[0062] (iv) Evaluation of spiked recovery, relative standard deviation and precision Dried blood spot samples from the same source were divided into four aliquots. Three of these aliquots were adjusted to spike concentrations of 0.5 μg / mL, 5 μg / mL, and 20 μg / mL with a certain amount of mixed standard stock solution, respectively. All samples were processed and analyzed according to steps (I) to (III), with each sample undergoing n parallel experiments. The spike recovery rate and relative standard deviation were calculated, where: Spike recovery rate = (Spiked sample value - Unspiked sample value) ÷ Spike amount × 100%; Relative standard deviation (RSD) = Deviation of spike recovery rate ÷ Average spike recovery rate × 100%; Precision = (Standard deviation (SD) ÷ Average) × 100%. The results are shown in Table 3.

[0063] Table 3

[0064] As shown in Table 3, the average recoveries of the 16 bisphenol compounds in this invention ranged from 80.5% to 112%, which is relatively high; however, the relative standard deviations were <19.1% and the precision was <20%, which is relatively low. Therefore, the analytical method of this invention exhibits excellent accuracy and reliability.

[0065] (v) Assessment of matrix effect Seven dried blood spot samples were prepared from bovine whole blood anticoagulated with EDTA (ethylenediaminetetraacetic acid). The processing followed steps (I) to (III), with the difference that the organic phase obtained in step (II) was used as the matrix to prepare standard curves for 16 bisphenol compounds at concentrations of 0.1 ng / mL, 0.25 ng / mL, 0.5 ng / mL, 1 ng / mL, 2.5 ng / mL, 5 ng / mL, and 10 ng / mL. Simultaneously, a methanol matrix standard curve of the same concentration was prepared. The matrix effect (ME) was calculated as: ME = (slope of matrix calibration curve / slope of solvent calibration curve) × 100%. The matrix effect was evaluated by the ratio of the slopes of the dried blood spot matrix standard curve and the methanol matrix standard curve. When ME is 80%-120%, it is judged as a weak matrix effect; when ME is 50%-80% or 120%-150%, it is judged as a moderate matrix effect; when ME is <50% or >150%, it is judged as a strong matrix effect.

[0066] The matrix effects of different dried blood spot samples are shown in Table 4.

[0067] Table 4

[0068] As can be seen from Table 4, after internal standard correction, the matrix effect of the 16 bisphenol compounds in this invention is 76-124%, which shows a weak matrix effect.

[0069] (vi) Actual sample determination Thirty-three dried blood spot samples from different sources were collected and processed and analyzed using a kit according to steps (I) to (III). The test results are shown in Table 5.

[0070] Table 5

[0071] As shown in Table 5, except for six bisphenol compounds (BPC, 2,4-BPS, BPSIP, BPS-MAE, TGSA, and TDP) which were not detected in all samples, the overall detection rate of the remaining 10 bisphenol compounds ranged from 3% to 64%, with concentrations below the detection limit to 3.01 ng / mL. BPF had the highest detection rate (64%). In terms of concentration distribution, the median and 75th percentile values ​​of most bisphenol compounds were below the method limit of quantitation or detection limit. However, some bisphenol compounds showed higher concentrations at the 95th percentile, particularly BPA (0.97 ng / mL) and DPS (2.19 ng / mL), with maximum values ​​of 2.03 ng / mL and 3.01 ng / mL, respectively. These data indicate that although the concentrations of bisphenol compounds were low in most samples, significant exposure was observed in some samples, with particular attention needed to be paid to the high detection rate of BPF and the high concentration exposure of BPA and DPS.

[0072] Example 2 The method is the same as that in step (i) to step (iii) of Example 1, except that in step (ii), the mixed internal standard working solution, water and acetonitrile are added into the centrifuge tube at the same time and sonicated for only 20 minutes.

[0073] According to the method in step (iv) of Example 1, the absolute recoveries of 16 bisphenol compounds were 27-52%, with standard deviations of 2-12%.

[0074] It is evident that the reliability of this method is lower than that of Example 1.

[0075] Example 3 According to Example 1, the difference is that the kit does not contain the first extract, i.e., it does not contain water.

[0076] The method is the same as that in step (i) to step (iii) of Example 1, except that in step (ii), water is not used, but the mixed internal standard working solution and acetonitrile are added to the centrifuge tube and sonicated once for 20 minutes.

[0077] According to the method in step (iv) of Example 1, except for BPSIP and DPS, whose absolute recoveries are all above 75%, the absolute recoveries of other target compounds are not ideal. Specifically, the absolute recoveries of BPAP, BPB, DD-70 and TDP are all 0%, the absolute recoveries of BPC and BPF are all below 5%, the absolute recoveries of BPAF and TGSA are all below 10%, and the absolute recoveries of other bisphenol compounds are 11-45%, with relative standard deviations of 10-79% and precision of 10-79%.

[0078] It is evident that the reliability of this method is lower than that of Example 1.

[0079] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A kit for detecting bisphenol compounds in dried blood spots, characterized in that, This kit can simultaneously detect bisphenol compounds including at least BPA, BPF, BPS, BPAF, BPAP, BPB, BPC, 2,4-BPS, BPSIP, BPS-MAE, TGSA, DPS, DDS, TDP, D-90 and DD-70. The kit includes a mixed standard stock solution and a mixed internal standard stock solution; The mixed standard stock solution contains at least bisphenol compounds including BPA, BPF, BPS, BPAF, BPAP, BPB, BPC, 2,4-BPS, BPSIP, BPS-MAE, TGSA, DPS, DDS, TDP, D-90 and DD-70. The mixed internal standard stock solution contains at least BPA- 13 C 12 BPF-D 10 BPS- 13 C 12 BPAF- 13 C 12 BPAP-D5, BPB- 13 C 12 2,4-BPS- 13 C 12 DPS-D 10 Internal standard compounds including DDS-D4 and BPSIP-D7.

2. The kit according to claim 1, wherein, The concentration of each bisphenol compound in the mixed standard stock solution is simultaneously 1-20 μg / mL, preferably 5-15 μg / mL; And / or, the concentration of each internal standard compound in the mixed internal standard stock solution is simultaneously 3-8 μg / mL, preferably 4-6 μg / mL; And / or, the solvent for the mixed standard stock solution and the mixed internal standard stock solution is methanol; And / or, the kit further includes a diluent, wherein the diluent is methanol; And / or, the kit further includes a first extraction solution, wherein the first extraction solution is water; And / or, the kit further includes a second extraction solution, wherein the second extraction solution is acetonitrile; And / or, the kit further includes a solid-phase extraction plate, wherein the solid-phase extraction plate is an HMR 96-well plate; And / or, the kit further includes a reconstitution solvent, which is a methanol aqueous solution with a volume fraction of 10-30%.

3. The kit according to claim 1 or 2, wherein, The applicable UHPLC-MS / MS detection conditions for the kit include: using Waters Acquity BEH C 18 Chromatographic column; mobile phase A: water; mobile phase B: methanol; total injection time: at least 15 min; elution gradient: 0-2 min, 80% A, 20% B; 2-4 min, 80% A - 70% A, 20% B - 30% B; 4-7 min, 70% A, 30% B; 7-10 min, 70% A - 50% A, 30% B - 50% B; 10-12 min, 50% A - 20% A, 50% B - 80% B; 12-13 min, 20% A, 80% B; 13-13.01 min, 20% A - 80% A, 80% B - 20% B; 13.01 min - end of elution, 80% A, 20% B.

4. The kit according to claim 3, wherein, The applicable UHPLC-MS / MS detection conditions for the kit also include: injection volume 3-7 μL; column temperature 35-50℃; injection temperature 5-12℃; mobile phase flow rate 0.1-0.4 mL / min; ion source ESI; scanning mode positive and negative ion scanning; monitoring mode MRM; carrier gas nitrogen and collision gas helium; capillary voltage 2.3-2.6 kV; ion source temperature 120-180℃; and desolvation temperature 400-600℃.

5. The kit according to claim 4, wherein, The applicable UHPLC-MS / MS detection conditions for the kit also include: injection volume 4-6 μL; column temperature 40-45℃; injection temperature 8-10℃; mobile phase flow rate 0.2-0.3 mL / min; ion source ESI; scanning mode positive and negative ion scanning; monitoring mode MRM; carrier gas nitrogen and collision gas helium; capillary voltage 2.4-2.5 kV; ion source temperature 140-160℃; and desolvation temperature 450-550℃.

6. A method for detecting bisphenol compounds in dried blood spots, characterized in that, The method uses the kit described in any one of claims 1-5.

7. The method according to claim 6, wherein, The method includes: mixing a dried blood spot sample, a mixed internal standard working solution, and the first extraction solution and performing a first ultrasonic treatment; adding the second extraction solution and performing a second ultrasonic treatment; taking the supernatant and filtering it under positive pressure using the solid phase extraction plate; repeating the above operation once; combining the organic phases obtained from the two operations; drying them and resolving them with the redissolving solvent to obtain the sample to be tested; performing UHPLC-MS / MS detection on the sample to be tested; and quantifying the bisphenol compounds in the sample to be tested by establishing a standard curve.

8. The method according to claim 7, wherein, The dried blood spot sample is obtained by drying 30-80 μL of whole blood, preferably by drying 40-60 μL of whole blood; And / or, the mixed internal standard working solution is obtained by diluting the mixed internal standard stock solution with the diluent, and the concentration of each internal standard compound in the mixed internal standard working solution is simultaneously 0.3-0.8 μg / mL, preferably 0.4-0.6 μg / mL; And / or, the volume of the mixed internal standard working solution is 5-30 μL, preferably 10-20 μL; And / or, the amount of the first extract is 50-150 μL, preferably 80-120 μL; And / or, the duration of the first ultrasonic treatment is 5-50 min, preferably 10-30 min; And / or, the amount of the second extract is 150-250 μL, preferably 180-220 μL; And / or, the duration of the second ultrasonic treatment is 5-50 min, preferably 10-30 min; And / or, the amount of the resolvent used is 50-150 μL, preferably 80-120 μL.

9. The method according to claim 7 or 8, wherein, The method for establishing the standard curve includes: diluting the mixed standard stock solution into a series of mixed standard working solutions with gradient mass concentrations using the diluent; mixing each mixed standard working solution of different concentrations with the mixed internal standard working solution; adjusting the volume to a certain concentration using the diluent; and then performing UHPLC-MS / MS detection. The standard curve is obtained by using the ratio of the peak area of ​​each bisphenol compound to the peak area of ​​the corresponding internal standard compound in the detection results as the ordinate and the detection concentration as the abscissa.

10. The application of the kit according to any one of claims 1-5 and the detection method according to any one of claims 6-9 in the assessment of human health status.

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