Mass spectrum rapid determination kit for strychnine and strychnine

Abstract

The invention discloses a rapid mass spectrum determination kit for strychnine and strychnine, and relates to the technical field of biological detection. The rapid mass spectrum determination kit comprises an MCX mixed type cationic adsorbent and a pyrrolidone-styrene polymer adsorbent. The rapid mass spectrum determination kit effectively improves the accuracy and sensitivity of detection, and through a porous plate extraction-liquid chromatography-tandem mass spectrum method, the detection sensitivity is greatly improved on a silica gel matrix C18 reversed-phase chromatographic column under the conditions of specific mobile phase composition and flow velocity. Separation of strychnine and strychnine from interference impurities in blood on a silica gel matrix C18 reversed-phase chromatographic column and multi-reaction monitoring (MRM) mass spectrometry detection under an electrospray ionization source positive ion mode are realized, and finally rapid, accurate and high-sensitivity detection of strychnine and strychnine in blood is realized.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, in particular to a mass spectrometry rapid determination kit for strychnine and strychnine. Background Art

[0002] Brucine (CAS: 357-57-3) and Stychnine (CAS: 57-24-9) (structural formulas are shown in Figure 1 Strychnine (A) and Strychnine (B) are the two most important alkaloids in the traditional Chinese medicine Strychnine. They are a class of nitrogen-containing alkaline organic compounds with significant biological activity that exist in plant organisms. They have strong physiological effects on humans and animals and have been widely used in the field of medicine. However, excessive or improper consumption will cause poisoning and even death. In recent years, poisoning cases caused by poisoning, suicide or improper consumption of strychnine and strychnine have occurred from time to time. In order to treat poisoned patients in a timely and efficient manner, it is very necessary to establish a rapid, sensitive and reliable qualitative and quantitative analysis method for strychnine and strychnine in biological samples.

[0003] At present, the main methods for determining strychnine and strychnine in biological samples include gas chromatography, gas chromatography-mass spectrometry, high performance liquid chromatography, high performance liquid chromatography-tandem mass spectrometry, etc. Since strychnine and strychnine are not easy to vaporize, the analytical sensitivity of gas chromatography and gas chromatography-mass spectrometry is low, and the accurate identification ability of high performance liquid chromatography is limited. High performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) has been widely used for the analysis of drug concentrations in blood, and is an ideal accurate and sensitive method for determining drug concentrations in blood. The sample pretreatment for LC-MS / MS of strychnine and strychnine in blood reported in the literature mainly adopts solid phase extraction (Wang Chaohong et al., LC-MS / MS method for determination of strychnine and strychnine in whole blood, Chinese Journal of Forensic Medicine, 2008, 23(4):226-227; Zhang Wangrong et al., CE-TOF / MS method for determination of strychnine and strychnine in human plasma and urine, ZL201210167511.3). However, when processing batch samples for detection, solid phase extraction method consumes a large amount of organic reagents, is costly, and is not environmentally friendly.

[0004] Multi-well plate extraction and purification has been widely used in the extraction and purification of blood samples. However, due to the differences in the chemical properties of the target objects to be tested, the optimization of the multi-well plate filler and the optimization of the elution and washing methods must be carried out according to the chemical properties of the specific target objects in order to obtain an analytical method with good inter-laboratory quality control. The present invention intends to use multi-well plate extraction and purification to provide an accurate, efficient, rapid and sensitive mass spectrometry rapid detection kit and detection method for strychnine and strychnine in blood, so as to prevent the damage caused by public emergencies to the greatest extent, protect the lives and property of the public, and maintain national security and social stability. Summary of the invention

[0005] The purpose of the present invention is to provide a mass spectrometry rapid determination kit for strychnine and strychnine to solve the problems existing in the above-mentioned prior art. The kit can be used to achieve rapid, accurate and highly sensitive detection of strychnine and strychnine.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides a mass spectrometry rapid determination kit for strychnine and strychnine, which comprises an MCX mixed cationic adsorbent and a pyrrolidone-styrene polymer adsorbent.

[0008] Furthermore, the mass ratio of the MCX mixed cationic adsorbent to the pyrrolidone-styrene polymer adsorbent is 4:1.

[0009] Furthermore, the mass spectrometry rapid determination kit also includes a multi-well plate.

[0010] Furthermore, the mass spectrometry rapid determination kit also includes acetonitrile-water solution, methanol-ammonia solution and acetonitrile-0.1% formic acid solution;

[0011] The acetonitrile-water solution is prepared by mixing acetonitrile and water in a volume ratio of 5:95;

[0012] The methanol-ammonia solution is prepared by mixing methanol and ammonia water in a volume ratio of 95:5;

[0013] The acetonitrile-0.1% formic acid aqueous solution is prepared by mixing acetonitrile and 0.1% formic acid aqueous solution in a volume ratio of 20:80.

[0014] Furthermore, the mass spectrometry rapid determination kit also includes a quality control reagent package; the quality control reagent package includes a mixed standard series solution of strychnine and strychnine and a quality control product;

[0015] The quality control product is prepared by adding a mixed standard solution of strychnine and strychnine into artificial plasma.

[0016] Furthermore, the detection method of the mass spectrometry rapid determination kit is as follows:

[0017] The sample to be tested is added to the porous plate filled with the MCX mixed cationic adsorbent and the pyrrolidone-styrene polymer adsorbent, and then the acetonitrile-water solution is added, and after being evenly mixed, it is eluted with the methanol-ammonia solution to obtain an eluate, and the eluate is then subjected to liquid chromatography-tandem mass spectrometry detection to obtain the peak area of ​​strychnine or strychnine, and finally the concentration of strychnine or strychnine in the sample to be tested is calculated by the standard curve of strychnine or strychnine;

[0018] In the liquid chromatography-tandem mass spectrometry detection, the mobile phase of the liquid chromatography detection is the acetonitrile-0.1% formic acid aqueous solution;

[0019] The standard curve of strychnine or strychnine is obtained by detecting mixed standard solutions of strychnine and strychnine with different concentrations.

[0020] Furthermore, the chromatographic column for liquid chromatography detection is a C18 reverse phase chromatographic column.

[0021] Furthermore, the C18 reverse phase chromatography column is a Waters BEH C18 column.

[0022] The present invention also provides a method for extracting and purifying strychnine and strychnine, comprising the steps of extracting and purifying strychnine and strychnine in a sample to be tested by using a mixed filler;

[0023] The mixed filler includes an MCX mixed cationic adsorbent and a pyrrolidone-styrene polymer adsorbent;

[0024] The mass ratio of the MCX mixed cationic adsorbent to the pyrrolidone-styrene polymer adsorbent is 4:1.

[0025] Furthermore, the specific operation steps of the purification method include:

[0026] The mixed filler is loaded into a porous plate, a sample to be tested is added, and then an acetonitrile-water solution is added, and after being mixed evenly, the mixture is eluted with a methanol-ammonia solution to obtain an eluent;

[0027] The acetonitrile-water solution is prepared by mixing acetonitrile and water in a volume ratio of 5:95;

[0028] The methanol-ammonia water solution is prepared by mixing methanol and ammonia water in a volume ratio of 95:5.

[0029] The present invention discloses the following technical effects:

[0030] (1) The present invention utilizes a porous plate filled with a high-efficiency mixed filler to extract and purify strychnine and strychnine in a blood sample. Compared with the solid phase extraction purification method in the prior art, the present invention effectively reduces the loss of target substances caused by nonspecific adsorption, greatly improves the detection recovery rate of the sample, effectively reduces the matrix effect, and greatly improves the accuracy of the detection result.

[0031] (2) The present invention utilizes a porous plate for extraction and purification, which greatly simplifies the tedious operation steps of manual solid phase extraction, improves the convenience of operation, and greatly shortens the pretreatment time, thereby effectively improving the sample analysis throughput and accelerating the analysis speed of batch samples.

[0032] (3) The kit provided by the present invention effectively improves the accuracy and sensitivity of detection. By using a multi-well plate extraction-liquid chromatography-tandem mass spectrometry method, on a silica-based C18 reverse-phase chromatographic column, under specific mobile phase composition and flow rate conditions, the separation of strychnine and strychnine from interfering impurities in the blood is achieved on a silica-based C18 reverse-phase chromatographic column and multiple reaction monitoring (MRM) mass spectrometry detection in the positive ion mode of an electrospray ionization source is achieved, ultimately achieving rapid, accurate and highly sensitive detection of strychnine and strychnine in the blood. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0034] Figure 1 are the structural formulas of strychnine (A) and strychnine (B);

[0035] Figure 2 The chromatogram is for the separation of strychnine and strychnine using a Waters BEH C18 column (100 mm × 2.1 mm id, 1.7 μm);

[0036] Figure 3 The chromatogram is for the separation of strychnine and strychnine using a Waters Premier BEH C18 (100 mm × 2.1 mm id, 1.7 μm) column;

[0037] Figure 4 The chromatogram is the separation of strychnine and strychnine using a Shim-pack XR-ODSⅡ column (75mm×2.0mm id, 1.6μm);

[0038] Figure 5The chromatogram is the separation of strychnine and strychnine using a Shim-pack XR-ODSⅢ column (75mm×2.0mm id, 1.6μm);

[0039] Figure 6 The chromatogram is for the separation of strychnine and strychnine using an Agela Technologies Venusil MP C18 column (150 mm × 2.1 mm id, 3.0 μm);

[0040] Figure 7 The chromatogram is for the separation of strychnine and strychnine using an Agilent HILIC Plus column (100 mm × 4.6 mm id, 3.5 μm);

[0041] Figure 8 The MRM chromatogram of acetonitrile-water mobile phase for separation of strychnine and strychnine;

[0042] Fig. 9 The MRM chromatogram of acetonitrile-5 mM ammonium acetate aqueous solution for separation of strychnine and strychnine;

[0043] Fig.10 The MRM chromatogram of acetonitrile-0.1% formic acid aqueous solution (20:80, V / V) mobile phase for separation of strychnine and strychnine;

[0044] Fig.11 The MRM chromatogram of acetonitrile-0.1% formic acid aqueous solution (10:90, V / V) mobile phase for separation of strychnine and strychnine;

[0045] Fig.12 The MRM chromatogram of acetonitrile-0.2% ammonia water mobile phase for separation of strychnine and strychnine;

[0046] Fig.13 The MRM chromatogram of separation of strychnine and strychnine with methanol-water mobile phase;

[0047] Fig.14 The MRM chromatogram of separation of strychnine and strychnine using methanol-0.1% formic acid aqueous solution as mobile phase;

[0048] Fig.15 The MRM chromatogram of strychnine in serum samples. DETAILED DESCRIPTION

[0049] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0050] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0051] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0052] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0053] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0054] Example 1

[0055] 1. Experimental Materials

[0056] 1.1 Reagents

[0057] Acetonitrile, methanol (LC / MS grade, Thermo Fisher Scientific Inc.), ammonium acetate (HPLC grade, Merck, Germany), ammonia (HPLC grade, Shanghai Aladdin Biochemical Technology Co., Ltd.), distilled water (Watson Group Co., Ltd.), formic acid (HPLC grade, Thermo Fisher Scientific Inc.), PLS adsorbent (pyrrolidone-styrene polymer, Pyrolidonelink styrol beads, particle size 400-500nm, Yingruicheng Biochemical Technology Co., Ltd.), MCX mixed cationic adsorbent (particle size 40-50μm, Yuexu Technology (Shanghai) Co., Ltd.), C18 adsorbent (particle size 10μm, Beijing Dicoma Technology Co., Ltd.), PSA adsorbent (N-propylethylenediamine, particle size 400-500nm, Yuexu Technology (Shanghai) Co., Ltd.), filter head (0.22μm, Shanghai Anpu Experiment Technology Co., Ltd.), disposable syringe (Jiangxi Hongda Medical Equipment Group Co., Ltd.), strychnine and strychnine standard samples (content greater than 98%, China Food and Drug Biological Products Control Institute), blank plasma (obtained from a tertiary hospital), 10mL polypropylene centrifuge tube (Zhejiang Gongdong Medical Technology Co., Ltd.).

[0058] Preparation of strychnine and strychnine standard stock solutions (100.0 mg / L): Accurately weigh 10.0 mg of strychnine standard and 10.0 mg of strychnine standard into a 100 mL volumetric flask, dissolve them in a small amount of acetonitrile and make up to the mark with acetonitrile.

[0059] Preparation of standard application solution: Accurately pipette 50 μL of strychnine and strychnine standard stock solution (100.0 mg / L) into a 10 mL volumetric flask, dilute to volume with acetonitrile to prepare a 500.0 μg / L standard stock solution.

[0060] Preparation of standard solution series: Accurately pipette a certain amount of strychnine and strychnine standard application solution (500.0 μg / L) to prepare a series of standard solutions with concentrations of 0.05, 0.1, 0.2, 1.0, 5.0, 10.0, 50.0, and 100.0 μg / L, respectively.

[0061] 1.2 Experimental instruments

[0062] UFLC ultra-fast liquid chromatograph (Shimadzu Corporation, Japan), AB6500 liquid chromatograph tandem mass spectrometer (ABSciex Corporation, USA) with electrospray ionization source, Vortex-vortex mixer (Sigma Corporation, USA), XS205 1 / 100,000 electronic analytical balance (Mettler-Toledo Corporation, Switzerland), 3-30K high-speed refrigerated centrifuge (Sigma Corporation, USA).

[0063] 2. Methods and Results

[0064] 2.196-well plate adsorbent type and ratio selection

[0065] Different adsorbents (C18, MCX, PSA and PLS) were used to fill 96-well plates in different weight proportions (see Table 1). The total filling amount was controlled to 25 mg, and then used for the adsorption and purification of strychnine and strychnine in blood. The recovery rate and matrix effect indicators were used for evaluation.

[0066] The calculation formula of matrix effect is as follows: Matrix effect = [(slope of sample curve - slope of standard curve) / slope of standard curve] × 100%. The calculation formula of spiked recovery is as follows: Spiked recovery = (spike sample measurement value - sample measurement value) / spiked amount × 100%.

[0067] The results are shown in Table 1. The test results show that C18, MCX, PSA and PLS all have a certain effect in reducing the matrix effect, but the matrix effect is still relatively obvious, and the recovery rate of the target is also low. When mixed fillers with different weight ratios are used, the recovery rate and matrix effect are also different. Compared with the use of a single filler, the mixed filler improves the recovery rate and matrix effect to a certain extent. When MCX:PLS (weight ratio 4:1) mixed adsorbent is used, the recovery rate and matrix effect evaluation data meet the requirements of the method establishment. Combined with the comprehensive situation, MCX:PLS (weight ratio 4:1) mixed adsorbent, that is, MCX 20mg and PLS 5mg mixed adsorbent is selected as the filler for the 96-well plate.

[0068] Table 1

[0069]

[0070] 2.2 Selection of chromatographic columns

[0071] The present invention tests the effects of 6 C18 reverse phase chromatographic columns produced by different companies, namely Waters BEH C18 column (100mm×2.1mm id, 1.7μm), Waters Premier BEH C18 (100mm×2.1mm id, 1.7μm), Shim-pack XR-ODSⅡ column (75mm×2.0mm id, 1.6μm), Shim-pack XR-ODSⅢ column (75mm×2.0mmi.d., 1.6μm), Agela Technologies Venusil MP C18 column (150mm×2.1mm id, 3.0μm), and Agilent HILIC Plus column (100mm×4.6mm id, 3.5μm) on the separation of strychnine and strychnine drugs. The chromatograms of strychnine and strychnine in the 6 chromatographic columns are shown in FIG. Figure 2-7 As shown in the figure, the results show that the mass spectrometer response value of Waters PremierBEH C18 (100mm×2.1mm id, 1.7μm) is the highest, but the separation is relatively low; the response value of Waters BEH C18 column (100mm×2.1mm id, 1.7μm) is the second highest, but the separation is good; other chromatographic columns either have poor separation or unsatisfactory chromatographic peak shape. Based on the comprehensive situation, Waters BEH C18 column (100mm×2.1mm id, 1.7μm) was selected.

[0072] 2.3 Selection of mobile phase

[0073] The present invention uses different types of mobile phases on a Waters BEH C18 column (100 mm×2.1 mm id, 1.7 μm) to study the effects of different types of mobile phases on the chromatographic separation and mass spectrometric response of strychnine and strychnine. Seven mobile phases are mainly used for the experiment: acetonitrile-water, acetonitrile-5 mM ammonium acetate aqueous solution, acetonitrile-0.1% formic acid aqueous solution (20:80, V / V), acetonitrile-0.1% formic acid aqueous solution (10:90, V / V), acetonitrile-5 mM ammonium acetate-0.1% formic acid aqueous solution, acetonitrile-0.2% ammonia water, methanol-water and methanol-0.1% formic acid aqueous solution). In the present invention, the percentages involved in the mobile phase are all volume fractions.

[0074] The separation chromatograms under different mobile phase conditions are shown in Figure 8-14 The results show that the acetonitrile-0.1% formic acid aqueous solution (20:80, V / V) system has the highest response value, better peak shape, the most appropriate overall peak time, and better effect. Based on the comprehensive situation, acetonitrile-0.1% formic acid aqueous solution (20:80, V / V) is selected.

[0075] 2.4 Linear range, detection limit and quantification limit

[0076] 100 μL of standard S1a-S8a (a series of strychnine and strychnine standard solutions with concentrations of 0.05, 0.1, 0.5, 1.0, 5.0, 10.0, 50.0 and 100.0 μg / L, respectively) in reagent pack B were added to the wells of the 96-well extraction cleanup plate in reagent pack C (the wells contained MCX 20 mg and PLS 5 mg of mixed adsorbent), then add 400 μL of acetonitrile-water (5:95, V / V) solution, vortex mix, successively elute with 1.0 mL of 0.1 M hydrochloric acid and 1.0 mL of methanol, and elute with methanol-ammonia water (95:5, V / V). Use a 96-well plate positive pressure device to transfer the solution in the 96-well extraction and purification plate to a 96-well sample injection plate, then accurately draw 5 μL, inject into a liquid chromatography-tandem mass spectrometer, analyze according to the chromatography and mass spectrometry conditions, obtain the peak areas of strychnine and strychnine at different mass concentrations, and then use the peak area (y) to perform linear regression on the mass concentration (x, μg / L). The regression equation of strychnine is y=3.211×10 5 x+1.518×10 3 , the regression equation of strychnine is y = 3.745 × 10 5 x+2.281×10 3 The method had good linearity, with a correlation coefficient (r) of 0.9999. The detection limit was 0.01 μg / L when the signal-to-noise ratio was 3:1, and the quantitative detection limit was 0.03 μg / L when the signal-to-noise ratio was 10:1.

[0077] 2.5 Recovery and precision of the method

[0078] Take the quality control plasma of low, medium and high concentration levels (0.05, 1.0, 50.0 μg / L) in reagent pack B, take 6 100 μL samples of the same concentration of quality control plasma samples, add them to the 96-well extraction plate wells in reagent pack C, add 400 μL of acetonitrile-water (5:95, V / V) solution in reagent pack A as the extraction agent, mix well on a 96-well plate vortex oscillator, elute successively with 1.0 mL of 0.1 M hydrochloric acid and 1.0 mL of methanol, and use methanol-ammonia (95:5, V / V) Elution, using a 96-well positive pressure device, transfer the sample in the 96-well extraction plate to a 96-well plate injection plate, then accurately absorb 5 μL, inject into a liquid chromatography-tandem mass spectrometer, analyze according to the chromatography and mass spectrometry conditions, obtain the peak areas of strychnine and strychnine at different mass concentrations, quantify according to the standard curve external standard method, and obtain the recovery rates of low, medium and high concentrations of strychnine are 93.4%, 95.6% and 91.7%, respectively, and the precisions of 6 measurements are 4.4%, 3.8% and 3.1%, respectively. The recovery rates of low, medium and high concentrations of strychnine are 89.4%, 91.6% and 95.7%, respectively, and the precisions of 6 measurements are 5.4%, 3.3% and 3.0%, respectively.

[0079] Example 2

[0080] 1. A kit for detecting strychnine and strychnine in blood, comprising reagent pack A, reagent pack B and reagent pack C:

[0081] Reagent package A includes acetonitrile-water solution (5:95, V / V), acetonitrile-0.1% formic acid aqueous solution (20:80, V / V) and methanol-ammonia aqueous solution (95:5, V / V).

[0082] Reagent package B includes: (1) a series of mixed standard solutions of strychnine and strychnine; a series of mixed standard solutions of strychnine and strychnine with concentrations of 0.05, 0.1, 0.5, 1.0, 5.0, 10.0, 50.0 and 100.0 μg / L, respectively, packaged in 2 mL brown bottles; (2) three quality controls of low, medium and high quality controls (the low, medium and high quality controls are all prepared by adding standard solutions of strychnine and strychnine to artificial plasma, and their concentrations are respectively consistent with the first, fourth and seventh gradient concentrations of the eight gradients of the standard solution, that is, the low, medium and high quality controls of plasma strychnine and strychnine with concentrations of 0.05, 1.0 and 50.0 μg / L, respectively).

[0083] Reagent Pack C includes a 96-well extraction and purification plate and a 96-well injection plate.

[0084] Filling of 96-well extraction and purification plate: Take a 96-well solid phase extraction plate with a specification of 8×12 wells, and fill each well with 20 mg of MCX and 5 mg of PLS ​​fillers respectively.

[0085] 2. Determination of strychnine and strychnine content in serum samples:

[0086] (1) Preparation of standard curve: 100 μL of the standard solution in reagent pack B (a series of strychnine and strychnine standard solutions with concentrations of 0.05, 0.1, 0.5, 1.0, 5.0, 10.0, 50.0 and 100.0 μg / L, respectively) was added sequentially to the wells of the 96-well extraction cleanup plate in reagent pack C (the wells contained a mixed adsorbent of MCX 20 mg and PLS 5 mg), and then 400 μL of acetonitrile-water (5:95, V / V) solution was added and vortexed to mix. The wells were then washed with 1.0 mL of 0.1 M hydrochloric acid and 1.0 mL of methanol, and eluted with methanol-ammonia water (95:5, V / V). The solution in the 96-well extraction cleanup plate was transferred to the 96-well sample injection plate using a 96-well plate positive pressure device, and then 5 μL was accurately aspirated and injected into the liquid chromatography-tandem mass spectrometer.

[0087] LC-MS / MS conditions:

[0088] Liquid chromatography conditions were as follows: mobile phase A: acetonitrile-0.1% formic acid aqueous solution (20:80, V / V); chromatographic column: Waters BEH C18 column (100 mm×2.1 mm id, 1.7 μm); flow rate: 300 μL / min; column temperature: 40°C; injection volume: 5 μL;

[0089] The mass spectrometry conditions were as follows: positive ion electrospray ionization; quantitative detection mode: 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: 80 V; inlet voltage: 15 V; collision energy: 60 eV; collision chamber outlet voltage: 15 V; ion spray voltage: 5000 V; ion source temperature: 550 ° C. The quantitative ion pair of strychnine was m / z 395.2→244.1, and the qualitative ion pair was m / z 395.2→213.1. The quantitative ion pair of strychnine was m / z 335.1→184.0, and the qualitative ion pair was m / z 335.1→156.1.

[0090] The peak areas of strychnine and strychnine at different mass concentrations were obtained, and then a linear regression was performed on the mass concentration (x, μg / L) using the peak area (y). The regression equation of strychnine was y = 3.211 × 10 5 x+1.518×10 3 , the regression equation of strychnine is y = 3.745 × 10 5 x+2.281×10 3 .

[0091] (2) Treatment and testing of serum samples: 100 μL of serum sample was taken and added to the wells of the 96-well extraction plate in reagent package C (the wells contained a mixed adsorbent of MCX 20 mg and PLS 5 mg), and then 400 μL of acetonitrile-water (5:95, V / V) solution was added, vortexed and mixed, and then rinsed with 1.0 mL of 0.1 M hydrochloric acid and 1.0 mL of methanol, and eluted with methanol-ammonia water (95:5, V / V). Using a 96-well plate positive pressure device, the solution in the 96-well extraction cleanup plate was transferred to the 96-well injection plate, and then 5 μL was accurately aspirated and injected into the liquid chromatography-tandem mass spectrometer.

[0092] The same LC-MS / MS analysis conditions were prepared according to the standard curve to obtain the peak areas of strychnine and strychnine in the serum samples, and quantitative calculation was performed according to the external standard method. The typical MRM chromatogram of strychnine determination in serum samples is shown in Fig.15 The blood concentration of strychnine in serum was measured to be 22.8 μg / L, and the blood concentration of strychnine was not detected.

[0093] Effect verification example 1

[0094] The strychnine and strychnine quality control plasmas at three concentration levels (0.05, 1.0, 50.0 μg / L) were used for pretreatment and purification by the method of the present invention (i.e., the detection method of Example 2) and the MCX column solid phase extraction method (reference "Wang Chaohong et al., LC-MS / MS method for determination of strychnine and strychnine content in whole blood, Chinese Journal of Forensic Medicine, 2008, 23(4): 226-227"), and the LC-MS / MS method was used for 6 repeated detections of strychnine and strychnine content. The results are shown in Table 2. As can be seen from Table 2, by using the method of the present invention, the relative deviations between the measured values ​​of the three concentrations of the quality control products at low, medium and high concentrations and the theoretical values ​​of the quality control are small, and the determination precision is good.

[0095] Table 2

[0096]

[0097] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A mass spectrometry rapid determination kit for strychnine and strychnine, characterized in that: Including MCX mixed cationic adsorbent and pyrrolidone-styrene polymer adsorbent.

2. The mass spectrometry rapid determination kit according to claim 1, characterized in that: The mass ratio of the MCX mixed cationic adsorbent to the pyrrolidone-styrene polymer adsorbent is 4:

1.

3. The mass spectrometry rapid determination kit according to claim 2, characterized in that: The mass spectrometry rapid assay kit also includes a multi-well plate.

4. The mass spectrometry rapid determination kit according to claim 3, characterized in that: The mass spectrometry rapid determination kit also includes acetonitrile-water solution, methanol-ammonia solution and acetonitrile-0.1% formic acid solution; The acetonitrile-water solution is prepared by mixing acetonitrile and water in a volume ratio of 5:95; The methanol-ammonia solution is prepared by mixing methanol and ammonia water in a volume ratio of 95:5; The acetonitrile-0.1% formic acid aqueous solution is prepared by mixing acetonitrile and 0.1% formic acid aqueous solution in a volume ratio of 20:

80.

5. The mass spectrometry rapid determination kit according to claim 3, characterized in that: The mass spectrometry rapid determination kit also includes a quality control reagent package; the quality control reagent package includes a mixed standard series solution of strychnine and strychnine and a quality control product; The quality control product is prepared by adding a mixed standard solution of strychnine and strychnine into artificial plasma.

6. The mass spectrometry rapid determination kit according to claim 4, characterized in that: The detection method of the mass spectrometry rapid determination kit is as follows: The sample to be tested is added to the porous plate filled with the MCX mixed cationic adsorbent and the pyrrolidone-styrene polymer adsorbent, and then the acetonitrile-water solution is added, and after being mixed evenly, it is eluted with the methanol-ammonia water solution to obtain an eluate, and the eluate is then subjected to liquid chromatography-tandem mass spectrometry detection to obtain the peak area of ​​strychnine or strychnine, and finally the concentration of strychnine or strychnine in the sample to be tested is calculated by the standard curve of strychnine or strychnine; In the liquid chromatography-tandem mass spectrometry detection, the mobile phase of the liquid chromatography detection is the acetonitrile-0.1% formic acid aqueous solution; The standard curve of strychnine or strychnine is obtained by detecting mixed standard solutions of strychnine and strychnine with different concentrations.

7. The mass spectrometry rapid determination kit according to claim 6, characterized in that: The chromatographic column for the liquid chromatography detection is a C18 reverse phase chromatographic column.

8. The mass spectrometry rapid determination kit according to claim 6, characterized in that: The C18 reverse phase chromatographic column is a Waters BEH C18 column.

9. A method for extracting and purifying strychnine and strychnine, characterized in that: The method comprises the steps of extracting and purifying strychnine and strychnine in the sample to be tested by using a mixed filler; The mixed filler includes an MCX mixed cationic adsorbent and a pyrrolidone-styrene polymer adsorbent; The mass ratio of the MCX mixed cationic adsorbent to the pyrrolidone-styrene polymer adsorbent is 4:

1.

10. The purification method according to claim 9, characterized in that: The specific operation steps of the purification method include: The mixed filler is loaded into a porous plate, a sample to be tested is added, and then an acetonitrile-water solution is added, and after being mixed evenly, the mixture is eluted with a methanol-ammonia solution to obtain an eluent; The acetonitrile-water solution is prepared by mixing acetonitrile and water in a volume ratio of 5:95; The methanol-ammonia water solution is prepared by mixing methanol and ammonia water in a volume ratio of 95:5.

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