A rapid mass spectrometry determination kit for brucine and strychnine

Abstract

The present invention discloses a kit for rapid determination of brucine and strychnine by mass spectrometry, which relates to the technical field of biological detection. The kit for rapid determination of mass spectrometry includes an MCX mixed cationic adsorbent and a pyrrolidone-styrene polymer adsorbent. The kit for rapid determination of mass spectrometry effectively improves the accuracy and sensitivity of detection. Through the method of extraction by multi-well plate - liquid chromatography - tandem mass spectrometry, on a silica-based C18 reversed-phase chromatography column, under specific mobile phase composition and flow rate conditions, the separation of brucine and strychnine from interfering impurities in blood on the silica-based C18 reversed-phase chromatography column and the multi-reaction monitoring (MRM) mass spectrometry detection in the positive ion mode of the electrospray ionization source are realized, and finally the rapid, accurate and highly sensitive detection of brucine and strychnine in blood is achieved.

Description

Technical Field

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

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

[0003] At present, the methods for determining brucine and strychnine in biological samples mainly include gas chromatography, gas chromatography-mass spectrometry, high performance liquid chromatography, high performance liquid chromatography-tandem mass spectrometry, etc. Since brucine and strychnine are not easily vaporized, the analysis sensitivity of gas chromatography and gas chromatography-mass spectrometry is relatively low, and the qualitative ability of high performance liquid chromatography is limited. High performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) has been widely used in the analysis of drug concentrations in blood and is an accurate and sensitive method for determining drug concentrations in blood. At present, the sample pretreatment of LC-MS / MS for the determination of brucine and strychnine contents in blood reported in the literature mainly adopts solid phase extraction method (Wang Zhaohong et al., Determination of brucine and strychnine contents in whole blood by LC-MS / MS, Chinese Journal of Forensic Medicine, 2008, 23(4): 226-227; Zhang Wangrong et al., Method for determining brucine and strychnine contents in human plasma and urine by CE-TOF / MS, ZL201210167511.3). However, when the solid phase extraction method is used for the detection of batch samples, the consumption of organic reagents is large, the cost is high, and it is not conducive to environmental protection.

[0004] The extraction and purification using porous plates have been widely applied in the extraction and purification of blood samples. However, due to the differences in the chemical properties of the analytes to be measured, it is necessary to optimize the porous plate packing materials and the elution and washing methods specifically according to the chemical properties of the specific analytes in order to obtain an analytical method with good inter-laboratory quality control. The present invention intends to use porous plate extraction and purification to provide a rapid mass spectrometry detection kit and detection method for strychnine and brucine in blood, so as to maximize the prevention of damage caused by sudden public events, safeguard the lives and property of the public, and maintain national security and social stability. Summary of the Invention

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

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

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

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

[0009] Further, the rapid mass spectrometry determination kit further includes a porous plate.

[0010] Further, the rapid mass spectrometry determination kit further includes an acetonitrile-aqueous solution, a methanol-ammonia aqueous solution, and an acetonitrile-0.1% formic acid aqueous solution;

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

[0012] The methanol-ammonia aqueous 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] Further, the rapid mass spectrometry determination kit further includes a quality control reagent package; the quality control reagent package includes a mixed standard series solution of strychnine and brucine and a quality control sample;

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

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

[0017] Add the sample to be tested into a porous plate filled with the MCX mixed cationic adsorbent and the pyrrolidone-styrene polymer adsorbent, then add the acetonitrile-aqueous solution. After mixing evenly, elute with the methanol-ammonia aqueous solution to obtain an eluate. The eluate is then detected by liquid chromatography-tandem mass spectrometry to obtain the peak area of brucine or strychnine. Finally, calculate the concentration of brucine or strychnine in the sample to be tested through the standard curve of brucine or strychnine;

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

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

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

[0021] Furthermore, the C18 reversed-phase chromatographic column is a Waters BEH C18 column.

[0022] The present invention also provides a method for extracting and purifying brucine and strychnine, including the step of extracting and purifying brucine and strychnine in the 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 extraction and purification method include:

[0026] Load the mixed filler into a porous plate, add the sample to be tested, then add the acetonitrile-aqueous solution. After mixing evenly, elute with the methanol-ammonia aqueous solution to obtain an eluate;

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

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

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

[0030] (1) The present invention uses a porous plate filled with highly efficient mixed fillers for the extraction and purification of brucine and strychnine in blood samples. Compared with the purification method of solid-phase extraction in the prior art, it effectively reduces the loss of target substances caused by non-specific adsorption, greatly improves the detection recovery rate of samples, effectively reduces the matrix effect, and well improves the accuracy of detection results.

[0031] (2) The present invention uses a porous plate for extraction and purification, greatly simplifies the cumbersome operation steps of manual solid-phase extraction, improves the convenience of operation, 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. Through the method of porous plate extraction - liquid chromatography - tandem mass spectrometry, on a silica-based C18 reversed-phase chromatographic column, under specific mobile phase composition and flow rate conditions, the separation of brucine and strychnine from interfering impurities in blood on the silica-based C18 reversed-phase chromatographic column and the multi-reaction monitoring (MRM) mass spectrometry detection in the positive ion mode of the electrospray ionization source are realized, and finally the rapid, accurate and highly sensitive detection of brucine and strychnine in blood is achieved. Description of the Drawings

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0034] Figure 1 Structural formulas of brucine (A) and strychnine (B);

[0035] Figure 2 Chromatogram of the separation of brucine and strychnine using a Waters BEH C18 column (100 mm × 2.1 mm i.d., 1.7 μm) chromatographic column;

[0036] Figure 3 Chromatogram of the separation of brucine and strychnine using a Waters Premier BEH C18 (100 mm × 2.1 mm i.d., 1.7 μm) chromatographic column;

[0037] Figure 4 Chromatogram of the separation of brucine and strychnine using a Shim-pack XR-ODSⅡ column (75 mm × 2.0 mm i.d., 1.6 μm) chromatographic column;

[0038] Figure 5Chromatogram of separating brucine and strychnine using a Shim-pack XR-ODSⅢ column (75mm×2.0mm i.d., 1.6μm);

[0039] Figure 6 Chromatogram of separating brucine and strychnine using an Agela Technologies Venusil MP C18 column (150mm×2.1mm i.d., 3.0μm);

[0040] Figure 7 Chromatogram of separating brucine and strychnine using an Agilent HILIC Plus column (100mm×4.6mm i.d., 3.5μm);

[0041] Figure 8 MRM chromatogram of separating brucine and strychnine with an acetonitrile - water mobile phase;

[0042] Figure 9 MRM chromatogram of separating brucine and strychnine with an acetonitrile - 5mM ammonium acetate aqueous solution mobile phase;

[0043] Figure 10 MRM chromatogram of separating brucine and strychnine with an acetonitrile - 0.1% formic acid aqueous solution (20:80, V / V) mobile phase;

[0044] Figure 11 MRM chromatogram of separating brucine and strychnine with an acetonitrile - 0.1% formic acid aqueous solution (10:90, V / V) mobile phase;

[0045] Figure 12 MRM chromatogram of separating brucine and strychnine with an acetonitrile - 0.2% ammonia water mobile phase;

[0046] Figure 13 MRM chromatogram of separating brucine and strychnine with a methanol - water mobile phase;

[0047] Figure 14 MRM chromatogram of separating brucine and strychnine with a methanol - 0.1% formic acid aqueous solution mobile phase;

[0048] Figure 15 MRM chromatogram of strychnine in serum samples. Detailed implementation manners

[0049] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be regarded as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0050] It should be understood that the terms used in the present invention are only for describing particular embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0051] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice 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 related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0052] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0053] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all 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 KGaA, Germany), ammonia water (HPLC grade, Shanghai Aladdin Biochemical Technology Co., Ltd.), distilled water (Watsons Water Co., Ltd.), formic acid (HPLC grade, Thermo Fisher Scientific Inc.), PLS adsorbent (pyrrolidone-styrene polymer, Pyrolidonelink styrol beads, particle size 400 - 500 nm, Enrich Biochemical Technology Co., Ltd.), MCX mixed cationic adsorbent (particle size 40 - 50 μm, Welch Materials (Shanghai) Co., Ltd.), C18 adsorbent (particle size 10 μm, Beijing Dikma Technologies Inc.), PSA adsorbent (N-propylethylenediamine, particle size 400 - 500 nm, Welch Materials (Shanghai) Co., Ltd.), filter head (0.22 μm, Shanghai ANPEL Laboratory Technologies Inc.), disposable syringe (Jiangxi Hongda Medical Instrument Group Co., Ltd.), brucine and strychnine standard samples (content greater than 98%, National Institutes for Food and Drug Control), blank plasma (collected from a certain tertiary hospital), 10 mL polypropylene centrifuge tube (Zhejiang Gongdong Medical Technology Co., Ltd.).

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

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

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

[0061] 1.2 Experimental instruments

[0062] UFLC ultra-fast liquid chromatograph (Shimadzu Corporation, Japan), AB6500 liquid chromatography-tandem mass spectrometer (ABSciex Corporation, USA) with electrospray ionization source, Vortex - vortex mixer (Sigma Corporation, USA), XS205 type one in a hundred thousandth electronic analytical balance (Mettler Toledo AG, Switzerland), 3 - 30K type high-speed refrigerated centrifuge (Sigma Corporation, USA).

[0063] 2. Methods and results

[0064] 2.1 Selection of the type and ratio of adsorbents in 96-well plates

[0065] Different adsorbents (C18, MCX, PSA, and PLS) were used and packed into 96-well plates in different weight ratios (see Table 1), with the total packing amount controlled at 25 mg, and then used for the adsorption and purification of brucine and strychnine in blood, and evaluated using the recovery rate and matrix effect indicators.

[0066] The calculation formula for the matrix effect is as follows: Matrix effect = [(slope of the sample curve - slope of the standard curve) / slope of the standard curve] × 100%. The calculation formula for the spiked recovery rate is as follows: Spiked recovery rate = (measured value of the spiked sample - measured value of the sample) / 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 on reducing the matrix effect, but the matrix effect is still relatively obvious, and the recovery rate of the target substances is also low. When using mixed fillers with different weight ratios, the recovery rate and matrix effect are also different. Compared with using a single filler, the mixed filler makes the recovery rate and matrix effect improved to a certain extent. When using the MCX:PLS (weight ratio 4:1) mixed adsorbent, the evaluation data of the recovery rate and matrix effect both meet the requirements for method establishment. Considering the comprehensive situation, the MCX:PLS (weight ratio 4:1) mixed adsorbent, that is, a mixed adsorbent of 20 mg of MCX and 5 mg of PLS, was selected as the filler for the 96-well plate.

[0068] Table 1

[0069]

[0070] 2.2 Selection of the chromatographic column

[0071] In this invention, 6 C18 reversed-phase chromatographic columns produced by different companies were tested, namely Waters BEH C18 column (100 mm × 2.1 mm i.d., 1.7 μm), Waters Premier BEH C18 (100 mm × 2.1 mm i.d., 1.7 μm), Shim-pack XR-ODSⅡ column (75 mm × 2.0 mm i.d., 1.6 μm), Shim-pack XR-ODSⅢ column (75 mm × 2.0 mm i.d., 1.6 μm), Agela Technologies Venusil MP C18 column (150 mm × 2.1 mm i.d., 3.0 μm), and Agilent HILIC Plus column (100 mm × 4.6 mm i.d., 3.5 μm) for their influence on the separation of brucine and strychnine drugs. The chromatograms of brucine and strychnine in the 6 chromatographic columns are asFigure 2-7 As shown, the results indicate that the Waters PremierBEH C18 column (100 mm × 2.1 mm i.d., 1.7 μm) has the highest mass spectrometry response value, but relatively lower resolution; the Waters BEH C18 column (100 mm × 2.1 mm i.d., 1.7 μm) has the second highest response value and better resolution; for other chromatographic columns, either the resolution is poor or the chromatographic peak shape is not ideal. Considering the overall situation, the Waters BEH C18 column (100 mm × 2.1 mm i.d., 1.7 μm) was selected.

[0072] 2.3 Selection of mobile phase

[0073] In this invention, different types of mobile phases were used on the Waters BEH C18 column (100 mm × 2.1 mm i.d., 1.7 μm) to study their effects on the chromatographic separation and mass spectrometry response of brucine and strychnine. A total of 7 mobile phases were mainly used for the experiments: 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 this 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, and the most appropriate overall elution time, with better effects. Considering the overall situation, the acetonitrile - 0.1% formic acid aqueous solution (20:80, V / V) was selected.

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

[0076] Respectively pipette 100 μL of the standard S1a - S8a in reagent pack B (a series of brucine 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) and add them successively into the wells of a 96 - well extraction and purification plate in reagent pack C (each well contains a mixed adsorbent of 20 mg of MCX and 5 mg of PLS). Then add 400 μL of acetonitrile - water (5:95, V / V) solution, vortex and mix well. Successively wash with 1.0 mL of 0.1 M hydrochloric acid and 1.0 mL of methanol, elute with methanol - ammonia (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 introduction plate. Then accurately pipette 5 μL and inject it into a liquid chromatography - tandem mass spectrometer, and analyze according to the described chromatographic and mass spectrometric conditions to obtain the peak areas of brucine and strychnine at different mass concentrations. Then perform a linear regression with the peak area (y) against the mass concentration (x, μg / L). The regression equation for brucine is y = 3.211×10 5 x + 1.518×10 3 , and the regression equation for strychnine is y = 3.745×10 5 x + 2.281×10 3 . This method has good linearity, and the correlation coefficient (r) is 0.9999. The corresponding concentration at a signal - to - noise ratio of 3:1 is taken as the detection limit, and the detection limit is 0.01 μg / L for both. The corresponding concentration at a signal - to - noise ratio of 10:1 is taken as the quantitative detection limit, and the quantitative detection limit is 0.03 μg / L for both.

[0077] 2.5 Recovery and precision of the method

[0078] Take the quality control plasma at low, medium, and high concentration levels (0.05, 1.0, 50.0 μg / L) in reagent pack B. For each concentration level, take 6 aliquots of 100 μL of the quality control plasma samples of the same concentration and add them into the wells of the 96-well extraction plate in reagent pack C. Add 400 μL of acetonitrile-water (5:95, V / V) solution in reagent pack A as the extractant. Mix well on a 96-well plate vortex oscillator, then wash successively 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 positive pressure device to transfer the samples in the 96-well extraction plate to the 96-well plate sample injection plate. Then accurately pipette 5 μL and inject it into the liquid chromatography-tandem mass spectrometer, and analyze according to the described chromatographic and mass spectrometric conditions to obtain the peak areas of brucine and strychnine at different mass concentrations. Quantify by the external standard method of the standard curve. The recoveries of brucine at low, medium, and high concentration levels are 93.4%, 95.6%, and 91.7% respectively, and the precisions of 6 determinations are 4.4%, 3.8%, and 3.1% respectively. The recoveries of strychnine at low, medium, and high concentration levels are 89.4%, 91.6%, and 95.7% respectively, and the precisions of 6 determinations are 5.4%, 3.3%, and 3.0% respectively.

[0079] Example 2

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

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

[0082] Reagent pack B includes: (1) a series of mixed standard solutions of brucine and strychnine; a series of mixed standard solutions of brucine 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, which are dispensed in 2 mL brown bottles; (2) three quality control products at low, medium, and high levels (the low, medium, and high quality controls are all prepared by adding standard solutions of brucine and strychnine to artificial plasma, and their concentrations are the same as the first, fourth, and seventh gradient concentrations in the eight gradients of the standard solutions, that is, three plasma quality control products of brucine and strychnine at low, medium, and high levels with concentrations of 0.05, 1.0, and 50.0 μg / L respectively).

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

[0084] Packing of the 96-well extraction and purification plate: Take a 96-well solid phase extraction plate with a specification of 8×12 wells, and successively pack 20 mg of MCX and 5 mg of PLS into each well.

[0085] 2. Determination of the contents of brucine and strychnine in serum samples:

[0086] (1) Preparation of the standard curve: Respectively pipette 100 μL of the standard solutions in Reagent Pack B (a series of brucine 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) and add them successively into the wells of a 96-well extraction and purification plate in Reagent Pack C (the wells are filled with a mixed adsorbent of 20 mg of MCX and 5 mg of PLS). Then add 400 μL of acetonitrile-water (5:95, V / V) solution, vortex and mix evenly. Subsequently, wash with 1.0 mL of 0.1 M hydrochloric acid and 1.0 mL of methanol successively, 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 the 96-well sample introduction plate. Then accurately pipette 5 μL and inject it into the liquid chromatography-tandem mass spectrometer.

[0087] LC-MS / MS determination conditions:

[0088] The liquid chromatography conditions are 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 i.d., 1.7 μm); flow rate: 300 μL / min; column temperature: 40 °C; injection volume: 5 μL;

[0089] The mass spectrometry conditions are as follows: Positive ion electrospray ionization is adopted; quantitative detection mode: multiple reaction monitoring (MRM) mode; curtain gas pressure: 20 psi; collision gas pressure: 7 psi; nebulizing gas pressure: 50 psi; auxiliary gas pressure: 50 psi; declustering voltage: 80 V; entrance voltage: 15 V; collision energy: 60 eV; collision cell outlet voltage: 15 V; ion spray voltage: 5000 V; ion source temperature: 550 °C. The quantitative ion pair of brucine is m / z 395.2→244.1, and the qualitative ion pair is m / z 395.2→213.1. The quantitative ion pair of strychnine is m / z 335.1→184.0, and the qualitative ion pair is m / z 335.1→156.1.

[0090] Obtain the peak areas of brucine and strychnine at different mass concentrations, and then perform linear regression with the peak area (y) against the mass concentration (x, μg / L). The regression equation of brucine is y = 3.211×10 5 x + 1.518×10 3 and the regression equation of strychnine is y = 3.745×10 5 x + 2.281×10 3 .

[0091] (2) Treatment and testing of serum samples: Take 100 μL of serum samples and add them into the wells of a 96-well extraction plate in reagent kit C (the wells are filled with a mixed adsorbent of 20 mg of MCX and 5 mg of PLS). Then add 400 μL of acetonitrile-water (5:95, V / V) solution, vortex and mix evenly. Successively wash with 1.0 mL of 0.1 M hydrochloric acid and 1.0 mL of methanol, and elute with methanol-ammonia (95:5, V / V). Use a positive pressure device for 96-well plates to transfer the solution in the 96-well extraction and purification plate to the 96-well sample introduction plate. Then accurately pipette 5 μL and inject it into a liquid chromatography-tandem mass spectrometer.

[0092] Perform tests under the same LC-MS / MS analysis conditions as those for preparing the standard curve to obtain the peak areas of brucine and strychnine in the serum samples, and perform quantitative calculations by the external standard method. The MRM chromatogram for the determination of strychnine in a typical serum sample is shown in Figure 15 . The measured blood drug concentration of strychnine in the serum is 22.8 μg / L, and the blood drug concentration of brucine is not detected.

[0093] Effect verification example 1

[0094] Use quality control plasma of brucine and strychnine at three concentration levels of low, medium, and high (0.05, 1.0, 50.0 μg / L), and perform pretreatment and purification respectively by the method of the present invention (i.e., the detection method of Example 2) and the MCX small column solid-phase extraction method (reference: "Wang Zhaohong et al., Determination of brucine and strychnine in whole blood by LC-MS / MS method, Chinese Journal of Forensic Medicine, 2008, 23(4): 226-227"). Perform 6 repeated detections of the contents of brucine and strychnine by LC-MS / MS method, and the results are shown in Table 2. It can be seen from Table 2 that by using the method of the present invention, the relative deviations of the measured values of the quality control products at the three concentration levels of low, medium, and high from the quality control theoretical values are relatively small, and the measurement precision is good.

[0095] Table 2

[0096]

[0097] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A rapid mass spectrometry determination kit for strychnine and brucine based on extraction and purification with a porous plate, characterized in that, It 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; The rapid mass spectrometry determination kit further includes a multi-well plate; The rapid mass spectrometry determination kit further includes an acetonitrile-aqueous solution, a methanol-ammonia aqueous solution, and an acetonitrile-0.1% formic acid aqueous solution; The acetonitrile-aqueous solution is prepared from acetonitrile and water in a volume ratio of 5:95; The methanol-ammonia aqueous solution is prepared from methanol and ammonia water in a volume ratio of 95:5; The acetonitrile-0.1% formic acid aqueous solution is prepared from acetonitrile and 0.1% formic acid aqueous solution in a volume ratio of 20:80; The detection method of the rapid mass spectrometry determination kit is as follows: Add the sample to be tested into the multi-well plate filled with the MCX mixed cationic adsorbent and the pyrrolidone-styrene polymer adsorbent, then add the acetonitrile-aqueous solution, mix evenly and elute with the methanol-ammonia aqueous solution to obtain an eluate. The eluate is then detected by liquid chromatography-tandem mass spectrometry to obtain the peak area of brucine or strychnine, and finally the concentration of brucine or strychnine in the sample to be tested is calculated through the standard curve of brucine or strychnine; In the liquid chromatography-tandem mass spectrometry detection, the mobile phase for liquid chromatography detection is the acetonitrile-0.1% formic acid aqueous solution; The standard curve of brucine or strychnine is obtained by detecting a mixed standard solution of brucine and strychnine at different concentrations; The chromatographic column for liquid chromatography detection is a C18 reversed-phase chromatographic column.

2. The rapid mass spectrometry determination kit according to claim 1, characterized in that The rapid mass spectrometry determination kit further includes a quality control reagent package; the quality control reagent package includes a mixed standard series solution of brucine and strychnine and a quality control sample; The quality control sample is prepared by adding a mixed standard solution of brucine and strychnine to artificial plasma.

3. The rapid mass spectrometry determination kit according to claim 1, characterized in that, The C18 reversed-phase chromatographic column is a Waters BEH C18 column.

4. A method for extracting and purifying brucine and strychnine, characterized in that, It includes the step of performing multi-well plate extraction and purification on brucine and strychnine in the sample to be tested by using the rapid mass spectrometry determination kit according to any one of claims 1-3.

5. The extraction and purification method according to claim 4, characterized in that, The specific operation steps of the extraction and purification method include: Load the MCX mixed cationic adsorbent and the pyrrolidone-styrene polymer adsorbent into a multi-well plate, add the sample to be tested, then add the acetonitrile-aqueous solution, mix evenly and elute with the methanol-ammonia aqueous solution to obtain an eluate.

Images