Mixed adsorbent for detecting antoin in blood and determination method

Abstract

The invention discloses a mixed adsorbent for detecting antoin in blood and a determination method, and belongs to the technical field of blood concentration determination, the mixed adsorbent for detecting antoin in blood comprises zirconium dioxide, a PSA adsorbent, an EMR-lipid adsorbent and a PLS adsorbent; the mass ratio of the zirconium dioxide to the PSA adsorbent to the EMR-lipid adsorbent to the PLS adsorbent in the mixed adsorbent is 1: 2: 1: 1. The method for rapidly determining the antoin in the blood comprises the following steps: adding a blood sample into a 96-hole extraction plate filled with the mixed adsorbent, and pre-treating the sample; and determining the antoin concentration in the blood sample by adopting a liquid chromatography-tandem mass spectrometry combined with a standard curve method. According to the method, the detection accuracy and sensitivity are greatly improved, and rapid, accurate and sensitive detection of the 96-well plate extraction-liquid chromatography-tandem mass spectrometry of the blood is realized.

Description

Technical Field

[0001] This invention belongs to the field of blood drug concentration determination technology, and particularly relates to a mixed adsorbent and determination method for detecting benzodiazepines in blood. Background Technology

[0002] Antoxin, chemically known as 1-naphthiourea, has the chemical formula C0.05. 11 H 10 N2S, chemically stable and resistant to deterioration, is a thiourea-based acute rodenticide with high selectivity, primarily used to control brown and yellow-haired rats. Currently, rapid and sensitive detection of N2S in blood mainly utilizes liquid chromatography-tandem mass spectrometry (LC-MS / MS). Sample pretreatment and purification primarily employ methods such as dispersion solid-phase extraction, protein precipitation, and liquid-liquid extraction. While these pretreatment methods can be automated, they are relatively expensive, consume large amounts of organic reagents, and are environmentally unfriendly. Finding suitable sample processing and purification methods is a major challenge for quality control between laboratories. 96-well plate extraction and purification are widely used in blood sample extraction and purification; however, due to the differences in the chemical properties of the analytes, targeted optimization of the packing material is necessary to obtain analytical methods with good inter-laboratory quality control. The current literature reports on the determination of antacid content in blood by liquid chromatography-tandem mass spectrometry (LC-MS / MS) (Cai Xinxin et al., Simultaneous and rapid determination of 11 rodenticides in plasma and urine by ultra-high performance liquid chromatography triple quadrupole mass spectrometry, Analytical Chemistry, 2010, 38(10): 1411-1416) which uses protein precipitation for sample processing. This method has disadvantages such as low sensitivity, large sample volume, long analysis time, and difficulty in batch operation, making it difficult to meet the requirements of clinical and judicial authorities for rapid, accurate, and sensitive detection of antacid content in blood. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention proposes a mixed adsorbent and a determination method for detecting betamethasone in blood. A specific mixed adsorbent is used to extract purified blood samples in a 96-well plate, providing an accurate, efficient, rapid, and sensitive method for detecting betamethasone in blood.

[0004] To achieve the above objectives, the present invention provides a mixed adsorbent for detecting benzodiazepines in blood, comprising zirconium dioxide, PSA adsorbent, EMR-lipid adsorbent, and PLS adsorbent; wherein the mass ratio of zirconium dioxide: PSA adsorbent: EMR-lipid adsorbent: PLS adsorbent in the mixed adsorbent is 1:2:1:1.

[0005] This invention also provides a rapid method for determining benzodiazepines in blood, comprising the following steps:

[0006] Blood samples were added to a 96-well extraction plate filled with the mixed adsorbent for sample pretreatment; the concentration of benzodiazepine in the blood samples was determined by liquid chromatography-tandem mass spectrometry combined with a standard curve method.

[0007] Preferably, the blood sample is serum or plasma; the ratio of the blood sample to the mixed adsorbent is 100 μL: 25 mg.

[0008] Preferably, the sample pretreatment specifically includes the following steps:

[0009] Add acetonitrile-water solution to a 96-well extraction plate and vortex.

[0010] Preferably, the volume ratio of the blood sample to the acetonitrile-water solution is 1:4; and the volume ratio of acetonitrile to water in the acetonitrile-water solution is 5:95.

[0011] Preferably, the determination of benzodiazepine in blood samples using liquid chromatography-tandem mass spectrometry combined with a standard curve method specifically includes the following steps:

[0012] The blood sample in the 96-well extraction plate was transferred to the 96-well plate injection plate to obtain the sample 96-well plate injection plate;

[0013] Prepare a series of Antonine standard working solutions, add them to a 96-well extraction plate filled with mixed adsorbent, then add acetonitrile-water solution, and transfer the Antonine standard working solution sample from the 96-well extraction plate to the 96-well plate injection plate to obtain the Antonine sample 96-well plate injection plate;

[0014] The sample 96-well plate injection plate and the Antu sample 96-well plate injection plate were injected into a liquid chromatography-tandem mass spectrometer. The liquid chromatography used acetonitrile-0.1% formic acid aqueous solution as the mobile phase, and the mass spectrometry used negative ion electrospray ionization multi-ion reaction and LC-MS / MS detection.

[0015] Using the peak area of ​​a series of concentrations of Antu standard working solutions as the ordinate and the concentration of Antu standard working solutions as the abscissa, a linear regression equation was obtained to calculate the concentration of Antu in blood samples.

[0016] Preferably, the conditions for the liquid chromatography are as follows: mobile phase A: acetonitrile-0.1% formic acid aqueous solution with a volume ratio of 25:75; chromatographic column: Shim-pack XR-ODSⅢ 75mm×2.0mm id, 1.6μm; column temperature: 40℃; flow rate: 300μL / min; injection volume: 5μL.

[0017] Preferably, the mass spectrometry conditions are as follows: using a multi-reaction monitoring mode with negative ion electrospray ionization; quantitative detection method: multi-reaction monitoring mode; curtain gas pressure: 20 psi; collision gas pressure: 7 psi; nebulizer gas pressure: 50 psi; auxiliary gas pressure: 50 psi; declustering voltage: -60 V; inlet voltage: -15 V; collision energy: -20 eV; collision chamber outlet voltage: -15 V; ion spray voltage: -5000 V; ion source temperature: 650 °C; and the quantitative ion pair is m / z 203.2 → m / z 144.0, and the qualitative ion pair is m / z 203.2 → m / z 186.0.

[0018] The present invention also provides a kit for determining antacid in blood using the aforementioned assay method, comprising antacid standard working solutions of different concentrations, acetonitrile-water solution at a volume ratio of 5:95, acetonitrile-0.1% formic acid water solution at a volume ratio of 25:75, a 96-well plate extraction plate filled with the aforementioned mixed adsorbent, a 96-well plate injection plate, and quality control materials.

[0019] The quality control product is prepared by adding Antu standard substance to blank plasma. The quality control product contains three concentration levels of quality control plasma: low, medium, and high. The concentrations of these plasmas are consistent with the first, fourth, and seventh gradient concentrations in the eight gradients of the standard solution, respectively. The target value is determined by detection.

[0020] The present invention also provides the application of the aforementioned assay method or the aforementioned reagent kit for determining the content of antacid in blood in the detection of antacid content.

[0021] Compared with the prior art, the present invention has the following advantages and technical effects:

[0022] (1) This invention uses an acetonitrile-water solution with a volume ratio of 5:95 to transfer blood samples into a 96-well extraction plate. The 96-well plate is filled with a mixed adsorbent of a specific composition for extraction and purification. Compared with the dispersion solid phase extraction method, it can effectively reduce non-specific adsorption, greatly improve the recovery rate of the test sample, reduce the matrix effect, and is of great significance for improving the accuracy of the test results.

[0023] (2) The present invention utilizes a 96-well extraction plate for extraction and purification, which can reduce the steps of liquid-liquid extraction, enrichment and purification in the early stage. Furthermore, the operation of the 96-well extraction plate is designed for batch quantitative processing and can be automatically processed by automated equipment, which can greatly shorten the pretreatment time, effectively improve the sample analysis throughput, greatly accelerate the analysis speed of batch samples, and further reduce the detection cost.

[0024] (3) The present invention uses an acetonitrile-0.1% formic acid aqueous solution system with a volume ratio of 25:75 as the mobile phase and a triple quadrupole mass spectrometer ESI source for ionization and multiple reaction monitoring (MRM) detection, which greatly improves the accuracy and sensitivity of detection and realizes rapid, accurate and sensitive detection of blood antagonist by 96-well plate extraction-liquid chromatography-tandem mass spectrometry. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The image shows the MRM chromatogram for the detection of benzodiazepines in a serum sample. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] The reagents and experimental instruments used in the embodiments of this invention were sourced from the following sources: acetonitrile (LC / MS grade, Thermo Fisher Scientific), methanol (LC / MS grade, Thermo Fisher Scientific), ammonium acetate (HPLC grade, Merck AG, Germany), ammonia (HPLC grade, Shanghai Aladdin Biochemical Technology Co., Ltd.), distilled water (Watsons Group Co., Ltd.), formic acid (HPLC grade, Thermo Fisher Scientific), PSA adsorbent (N-propylethylenediamine, particle size 40-50 μm, Yingruicheng Biochemical Technology Co., Ltd.), PLS adsorbent (pyrrolidone-styrene polymer, particle size 40-50 μm, Yingruicheng Biochemical Technology Co., Ltd.), and EMR-lipid adsorbent (Enhanced Matrix Removal-lipid, BondElut). EMR-Lipid enhanced lipid removal agent (Agilent Technologies (China) Co., Ltd.), sodium chloride (AR, Sinopharm Chemical Reagent Co., Ltd.), filter head (0.22μm, Shanghai Anpu Experimental Technology Co., Ltd.), disposable syringe (Jiangxi Hongda Medical Instrument Group Co., Ltd.), blank plasma (collected from a tertiary hospital in Zhejiang), 10mL polypropylene centrifuge tube (Zhejiang Gongdong Medical Technology Co., Ltd.), zirconium dioxide (AR, Sinopharm Chemical Reagent Co., Ltd.), Antu standard stock solution (1.0mg / L, Tanmo Quality Inspection Standard Material Center); UFLC ultra-fast liquid chromatograph (Shimadzu Corporation, Japan), AB6500 liquid chromatography-tandem mass spectrometer (AB Sciex Corporation, USA) with electrospray ionization source, Vortex-vortex mixer (Sigma-Aldrich, USA), XS205 0.0001 g electronic analytical balance (Mettler, Switzerland), 3-30K high-speed refrigerated centrifuge (Sigma-Aldrich, USA).

[0033] The preparation method of the Anton standard working solution used in the embodiments of the present invention:

[0034] Preparation of standard stock solution: Accurately pipette 100 μL of Antu standard stock solution (1.0 mg / L) into a 2 mL volumetric flask, and dilute to volume with acetonitrile to prepare a 50.0 μg / L standard stock solution.

[0035] Preparation of standard solution series: Accurately pipette 50.0 μg / L of Antonide standard stock solution to prepare a series of standard working solutions with concentrations of 0.05, 0.1, 0.2, 1.0, 5.0, 10.0, 50.0, and 100.0 μg / L.

[0036] Example 1

[0037] Preparation of standard curves: 100 μL of Antonide standard working solution (concentrations of 0.05, 0.1, 0.5, 1.0, 5.0, 10.0, 50.0, and 100.0 μg / L, respectively) were added sequentially to the wells of a 96-well extraction plate. Each well was filled with 25 mg of a mixed adsorbent (5 mg zirconium dioxide, 10 mg PSA adsorbent, 5 mg EMR-lipid adsorbent, and 5 mg PLS adsorbent). Then, 400 μL of an acetonitrile-water solution (5:95 v / v) was added. Using a 96-well plate positive pressure device, the sample from the 96-well extraction plate was transferred to the 96-well sample injection plate. 5 μL of the sample was accurately absorbed and injected into the liquid chromatography-tandem mass spectrometry (LC-MS / MS) instrument. Analysis was performed according to the following LC-MS / MS analytical conditions.

[0038] LC-MS / MS determination conditions:

[0039] The liquid chromatography conditions were as follows: mobile phase A: acetonitrile-0.1% formic acid aqueous solution (25:75, V / V); column: Shim-pack XR-ODSⅢ (75mm×2.0mm id, 1.6μm); flow rate: 300μL / min; injection volume: 5μL; column temperature: 40℃.

[0040] The mass spectrometry conditions were as follows: negative ion electrospray ionization was used; quantitative detection method: multiple reaction monitoring (MRM) mode; curtain gas pressure: 20 psi; collision gas pressure: 7 psi; nebulizer gas pressure: 50 psi; auxiliary gas pressure: 50 psi; declustering voltage: -60 V; inlet voltage: -15 V; collision energy: -20 eV; collision chamber outlet voltage: -15 V; ion spray voltage: -5000 V; ion source temperature: 600 ℃; the quantitative ion pair was m / z 203.2→m / z 144.0, and the qualitative ion pair was m / z 203.2→m / z 186.0.

[0041] Based on the peak area of ​​antacid at different mass concentrations, a linear regression was performed on the peak area (y) against the mass concentration (x, μg / L), yielding the regression equation y = 1.245 × 10⁻⁶. 5 x + 1.218 × 10 3 .

[0042] Blood sample processing and testing: Take 100 μL of serum sample and add it to the wells of a 96-well extraction plate. Each well is filled with 25 mg of a mixed adsorbent (5 mg zirconium dioxide, 10 mg PSA adsorbent, 5 mg EMR-lipid adsorbent, and 5 mg PLS adsorbent). Then add 400 μL of acetonitrile-water solution (5:95 v / v). Using a 96-well positive pressure device, transfer the sample from the 96-well extraction plate to the 96-well sample injection plate. Accurately absorb 5 μL and inject it into a liquid chromatography-tandem mass spectrometer. Perform LC-MS / MS analysis under the same conditions as the standard curve to obtain the peak area of ​​clopidogrel in the serum sample. Quantitatively calculate the mass concentration of clopidogrel in serum using the external standard method. A typical MRM chromatogram for the determination of clopidogrel in a serum sample is shown below. Figure 1 .

[0043] Comparative Example 1

[0044] The only difference from Example 1 is that the mixed adsorbent consists of 25 mg of zirconium dioxide.

[0045] Comparative Example 2

[0046] The only difference from Example 1 is that the mixed adsorbent consists of 25 mg of PSA adsorbent.

[0047] Comparative Example 3

[0048] The only difference from Example 1 is that the mixed adsorbent consists of 25 mg of EMR-lipid adsorbent.

[0049] Comparative Example 4

[0050] The only difference from Example 1 is that the mixed adsorbent consists of 25 mg of PLS ​​adsorbent.

[0051] Comparative Example 5

[0052] The only difference from Example 1 is that the mass ratio of zirconium dioxide: PSA adsorbent: EMR-lipid adsorbent: PLS adsorbent in the 25mg mixed adsorbent is 1:1:1:1.

[0053] Comparative Example 6

[0054] The only difference from Example 1 is that the mass ratio of zirconium dioxide: PSA adsorbent: EMR-lipid adsorbent: PLS adsorbent in the 25mg mixed adsorbent is 1:1:1:2.

[0055] Comparative Example 7

[0056] The only difference from Example 1 is that the mass ratio of zirconium dioxide: PSA adsorbent: EMR-lipid adsorbent: PLS adsorbent in the 25mg mixed adsorbent is 1:1:2:1.

[0057] Comparative Example 8

[0058] The only difference from Example 1 is that the mass ratio of zirconium dioxide: PSA adsorbent: EMR-lipid adsorbent: PLS adsorbent in the 25mg mixed adsorbent is 1:1:3:1.

[0059] Comparative Example 9

[0060] The only difference from Example 1 is that the mass ratio of zirconium dioxide: PSA adsorbent: EMR-lipid adsorbent: PLS adsorbent in the 25mg mixed adsorbent is 1:0.5:1:1.

[0061] Comparative Example 10

[0062] The only difference from Example 1 is that the mass ratio of zirconium dioxide: PSA adsorbent: EMR-lipid adsorbent: PLS adsorbent in the 25mg mixed adsorbent is 1:1:0.5:1.

[0063] Comparative Example 11

[0064] The only difference from Example 1 is that the mass ratio of zirconium dioxide: PSA adsorbent: EMR-lipid adsorbent: PLS adsorbent in the 25mg mixed adsorbent is 1:1:1:0.5.

[0065] Comparative Example 12

[0066] The only difference from Example 1 is that the mass ratio of zirconium dioxide: PSA adsorbent: EMR-lipid adsorbent: PLS adsorbent in the 25mg mixed adsorbent is 1:0.5:2:1.

[0067] Comparative Example 13

[0068] The only difference from Example 1 is that the mass ratio of zirconium dioxide: PSA adsorbent: EMR-lipid adsorbent: PLS adsorbent in the 25mg mixed adsorbent is 1:1:0.5:2.

[0069] Comparative Example 14

[0070] The only difference from Example 1 is that the mass ratio of zirconium dioxide: PSA adsorbent: EMR-lipid adsorbent: PLS adsorbent in the 25mg mixed adsorbent is 1:2:1:0.5.

[0071] Experimental Example 1

[0072] Different types of mixed adsorbents were packed into 96-well plates in Examples 1 and Comparative Examples 1-14. The effects on the adsorption and purification recovery rate and matrix effect of antacid in blood were determined, and the data are shown in Table 1.

[0073] Table 1 Effects of different types of mixed adsorbents

[0074]

[0075]

[0076] The results shown in Table 1 indicate that zirconium dioxide, PSA adsorbent, EMR-lipid adsorbent, and PLS adsorbent all exhibit some matrix removal activity, but significant matrix effects remain, resulting in low recovery rates. When using mixed adsorbents with different mass ratios, both recovery rates and matrix effects differ. Compared to using a single packing material, using mixed adsorbents with different mass ratios significantly improves both recovery rate and matrix effect. In Example 1, when a mixed adsorbent of zirconium dioxide, PSA adsorbent, EMR-lipid adsorbent, and PLS adsorbent (mass ratio 1:2:1:1) was used, the recovery rate and matrix effect evaluation data met the requirements for the established method.

[0077] Experiment Example 2

[0078] I. Linear range, limit of detection, and limit of quantitation

[0079] 100 μL of a series of concentrations of Antonex standard working solution (concentrations of 0.05, 0.1, 0.5, 1.0, 5.0, 10.0, 50.0, and 100.0 μg / L) were respectively added to the wells of a 96-well extraction plate (the wells were filled with a mixed adsorbent of 5 mg zirconium dioxide, 10 mg PSA adsorbent, 5 mg EMR-lipid adsorbent, and 5 mg PLS adsorbent). Then, 400 μL of acetonitrile-water solution with a volume ratio of 5:95 was added. Using a 96-well plate positive pressure device, the solution in the 96-well extraction plate was transferred to the 96-well plate injection plate. Then, 5 μL was accurately absorbed and injected into the liquid chromatography-tandem mass spectrometry instrument.

[0080] LC-MS / MS determination conditions:

[0081] The liquid chromatography conditions were as follows: mobile phase A: acetonitrile-0.1% formic acid aqueous solution (25:75, V / V); column: Shim-pack XR-ODSⅢ (75mm×2.0mm id, 1.6μm); flow rate: 300μL / min; injection volume: 5μL; column temperature: 40℃.

[0082] The mass spectrometry conditions were as follows: negative ion electrospray ionization was used; quantitative detection method: multiple reaction monitoring (MRM) mode; curtain gas pressure: 20 psi; collision gas pressure: 7 psi; nebulizer gas pressure: 50 psi; auxiliary gas pressure: 50 psi; declustering voltage: -60 V; inlet voltage: -15 V; collision energy: -20 eV; collision chamber outlet voltage: -15 V; ion spray voltage: -5000 V; ion source temperature: 600 ℃; the quantitative ion pair was m / z 203.2→m / z 144.0, and the qualitative ion pair was m / z 203.2→m / z 186.0.

[0083] The peak areas of antacid at different mass concentrations were obtained by analyzing the chromatographic and mass spectrometric conditions. Then, a linear regression was performed on the peak area (y) against the mass concentration (x, μg / L), and the regression equation was y = 1.245 × 10⁻⁶. 5 x + 1.218 × 10 3 The method exhibits good linearity with a correlation coefficient (r) of 0.9999. The detection limit is 0.01 μg / L, calculated using the concentration at a signal-to-noise ratio of 3:1, and the quantitation limit is 0.03 μg / L, calculated using the concentration at a signal-to-noise ratio of 10:1.

[0084] II. Recovery Rate and Precision

[0085] Quality control plasma samples at three concentration levels (0.05, 1.0, and 50.0 μg / L) of Antu working solution were collected. Six 100 μL aliquots of the same concentration were taken from each well and added to the wells of a 96-well extraction plate. 400 μL of acetonitrile-water solution (5:95 v / v) was added and mixed thoroughly on a 96-well plate vortex mixer. Using a 96-well positive pressure device, the sample was transferred from the extraction plate to the injection plate. 5 μL was accurately absorbed and injected into a liquid chromatography-tandem mass spectrometer (LC-MS / MS). Analysis was performed under the same chromatographic and mass spectrometric conditions to obtain the peak areas of Antu at different concentrations. Quantification was performed using the external standard method based on the standard curve. The recoveries for the low, medium, and high concentrations were 93.3%, 96.6%, and 95.7%, respectively, with precisions of 4.3%, 3.5%, and 3.1%, respectively.

[0086] III. Compared with existing measurement methods

[0087] Quality control plasmas containing low, medium, and high concentrations of Antu working solution (0.05, 1.0, and 50.0 μg / L) were used. Blood samples were pretreated and purified using the determination method described in Example 1, as well as protein precipitation, dispersion solid-phase extraction, and liquid-liquid extraction methods. The Antu content in the blood was measured six times repeatedly, and the results are shown in Table 2.

[0088] Table 2. Relative deviations and precision results of different measurement methods.

[0089]

[0090] Note: [1] Cai Xinxin et al., Simultaneous and rapid determination of 11 rodenticides in plasma and urine by ultra-high performance liquid chromatography triple quadrupole mass spectrometry, Analytical Chemistry, 2010, 38(10): 1411-1416;

[0091] [2] Jin Micong et al., A method, test kit and application for determining phenobarbital in blood, ZL202010091510.X;

[0092] [3] Guan Fuyu et al. Determination of 7 rodenticides in plasma by high performance liquid chromatography. Analytical Chemistry, 1995, 23(2): 159-162.

[0093] As shown in Table 2, using the method described in Example 1 of this invention, the relative deviation between the measured values ​​of the low, medium, and high concentration quality control samples and the theoretical quality control values ​​is the smallest, and the measurement precision is also the best.

[0094] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A mixed adsorbent for detecting beta-carotene in blood, characterized in that: It includes zirconium dioxide, PSA adsorbent, EMR-lipid adsorbent and PLS adsorbent; the mass ratio of zirconium dioxide: PSA adsorbent: EMR-lipid adsorbent: PLS adsorbent in the mixed adsorbent is 1:2:1:

1.

2. A rapid method for determining benzodiazepines in blood, characterized in that: Includes the following steps: Blood samples were added to a 96-well extraction plate packed with the mixed adsorbent described in claim 1 for sample pretreatment; the concentration of benzodiazepine in the blood samples was determined by liquid chromatography-tandem mass spectrometry combined with a standard curve method.

3. The determination method according to claim 2, characterized in that: The blood sample is serum or plasma; the ratio of the blood sample to the mixed adsorbent is 100 μL: 25 mg.

4. The determination method according to claim 2, characterized in that: The sample pretreatment specifically includes the following steps: Add acetonitrile-water solution to a 96-well extraction plate and vortex.

5. The determination method according to claim 4, characterized in that: The volume ratio of the blood sample to the acetonitrile-water solution is 1:4; the volume ratio of acetonitrile to water in the acetonitrile-water solution is 5:

95.

6. The determination method according to claim 2, characterized in that: The determination of benzodiazepine in blood samples using liquid chromatography-tandem mass spectrometry combined with a standard curve method specifically includes the following steps: The blood sample in the 96-well extraction plate was transferred to the 96-well plate injection plate to obtain the sample 96-well plate injection plate; Prepare a series of Antonine standard working solutions, add them to a 96-well extraction plate filled with mixed adsorbent, then add acetonitrile-water solution, and transfer the Antonine standard working solution sample from the 96-well extraction plate to the 96-well plate injection plate to obtain the Antonine sample 96-well plate injection plate; The sample 96-well plate injection plate and the Antu sample 96-well plate injection plate were injected into a liquid chromatography-tandem mass spectrometer. The liquid chromatography used acetonitrile-0.1% formic acid aqueous solution as the mobile phase, and the mass spectrometry used negative ion electrospray ionization multi-ion reaction and LC-MS / MS detection. Using the peak area of ​​a series of concentrations of Antu standard working solutions as the ordinate and the concentration of Antu standard working solutions as the abscissa, a linear regression equation was obtained to calculate the concentration of Antu in blood samples.

7. The determination method according to claim 6, characterized in that: The conditions for the liquid chromatography were as follows: mobile phase A: acetonitrile-0.1% formic acid aqueous solution with a volume ratio of 25:75; column: Shim-pack XR-ODSⅢ 75mm×2.0mm id, 1.6μm; column temperature: 40℃; flow rate: 300μL / min; injection volume: 5μL.

8. The determination method according to claim 6, characterized in that: The mass spectrometry conditions were as follows: multi-reaction monitoring mode with negative ion electrospray ionization; quantitative detection method: multi-reaction monitoring mode; curtain gas pressure: 20 psi. Impact air pressure: 7 psi; Nebulizer gas pressure: 50 psi; Auxiliary gas pressure: 50 psi; declustering voltage: -60 V; inlet voltage: -15 V; collision energy: -20 eV; collision chamber outlet voltage: -15 V; ion spray voltage: -5000 V; ion source temperature: 650 ℃; Anto quantitative ion pair: m / z 203.2→m / z 144.0, qualitative ion pair: m / z 203.2→m / z 186.

0.

9. A kit for determining benzodiazepines in blood using the method according to any one of claims 2 to 8, characterized in that: The mixture includes standard working solutions of different concentrations of antacid, an acetonitrile-water solution with a volume ratio of 5:95, an acetonitrile-0.1% formic acid-water solution with a volume ratio of 25:75, a 96-well plate extraction plate filled with the mixed adsorbent described in claim 1, a 96-well plate injection plate, and quality control materials. The quality control product is prepared by adding Antu standard substance to blank plasma. The quality control product contains three concentration levels of quality control plasma: low, medium, and high. The concentrations of these plasmas are consistent with the first, fourth, and seventh gradient concentrations in the eight gradients of the standard solution, respectively. The target value is determined by detection.

10. The application of the determination method according to any one of claims 2 to 8 or the kit for determining the concentration of anti-antagon in blood according to claim 9 in the detection of anti-antagon content.

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