A multi-target determination method for quaternary ammonium salt compounds in blood based on ultra-high performance liquid chromatography-tandem mass spectrometry

Abstract

The application discloses a method for determining multi-target quaternary ammonium salt compounds in blood based on ultra-high performance liquid chromatography-tandem mass spectrometry, and relates to the technical field of drug and poison analysis and detection, and comprises the following steps: preparing a mixed standard working solution, wherein the mixed standard working solution contains paraquat, diquat, chlormequat, mepiquat, pipradrol, tubocurarine, didecyldimethylammonium chloride (DDAC), neostigmine, succinylcholine chloride, vecuronium bromide and succinylcholine chloride; and determining the multi-target quaternary ammonium salt compounds in blood based on ultra-high performance liquid chromatography-tandem mass spectrometry, wherein the contents of paraquat, diquat, chlormequat, mepiquat, pipradrol, tubocurarine, didecyldimethylammonium chloride (DDAC), neostigmine, succinylcholine chloride, vecuronium bromide and succinylcholine chloride are determined simultaneously. The method is simple in operation, sensitive in detection and reliable in result, and is suitable for poisoning diagnosis and clinical toxicology research.

Description

Technical Field

[0001] This invention relates to the field of drug and toxicology analysis and detection technology, specifically to a method for multi-target determination of quaternary ammonium compounds in blood based on ultra-high performance liquid chromatography-tandem mass spectrometry. Background Technology

[0002] Quaternary ammonium compounds (QACs) are a class of organic compounds containing a permanent positive charge. Their molecular structure typically contains one or more tetravalent nitrogen atoms, exhibiting strong surface and biological activity. These compounds have wide applications in agriculture, public health, medicine, and daily chemicals. They include herbicides such as paraquat, diquat, wild glufosinate, and chlormequat chloride; disinfectants and fungicides such as dialcyldimethylammonium chloride (DDAC); and pharmaceutical or pharmaceutical-like substances such as neostigmine methylsulfate, succinylcholine chloride, vecuronium bromide, tubocurarine, and mepiperidine.

[0003] Although existing technologies have developed various detection methods, such as liquid chromatography (LC), gas chromatography (GC), and their coupled techniques, for analyzing quaternary ammonium compounds in environmental samples, urine, or surface swabs, significant challenges remain in the field of blood sample analysis. Existing methods are mostly focused on the detection of single or a few quaternary ammonium compounds, making it difficult to simultaneously determine multiple targets in blood, including pesticides such as wild wheat bran, diquat, and DDAC, as well as various neuromuscular blocking quaternary ammonium drugs. Furthermore, blood samples are complex, containing a large amount of proteins, lipids, and endogenous interfering substances. Quaternary ammonium compounds are typically highly polar, highly hydrophilic, easily adsorbed, and have weak chromatographic retention capabilities, resulting in significant deficiencies in existing detection methods in terms of sample pretreatment, separation and identification selectivity, sensitivity, and quantitative accuracy. Therefore, improvements are needed. Summary of the Invention

[0004] The purpose of this invention is to provide a multi-target determination method for quaternary ammonium compounds in blood based on ultra-high performance liquid chromatography-tandem mass spectrometry, in order to solve the problem that the existing technology lacks a standardized detection process that can simultaneously process multiple quaternary ammonium compounds in blood samples and achieve efficient separation and accurate quantification, resulting in limited detection range, insufficient sensitivity and quantitative accuracy.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for multi-target determination of quaternary ammonium compounds in blood based on ultra-high performance liquid chromatography-tandem mass spectrometry, comprising the following steps:

[0006] S1. Prepare a mixed standard working solution, wherein the mixed standard working solution contains paraquat, diquat, wild wheat bran, chlormequat chloride, mepiquat chloride, tubocurarine, didecyl dimethyl ammonium chloride, neostigmine, succinic acid monocholine chloride, vecuronium bromide and succinic acid monocholine chloride;

[0007] S2. Prepare an internal standard working solution, wherein the internal standard working solution contains D8-paraquat;

[0008] S3. Prepare a blood quality control solution, wherein the blood quality control solution contains paraquat, diquat, wild wheat bran, chlormequat chloride, mepiquat chloride, tubocurarine, didecyl dimethyl ammonium chloride, neostigmine, succinic acid monocholine chloride, vecuronium bromide and succinic acid monocholine chloride;

[0009] S4. Add the internal standard working solution to the blood sample to be tested and the blood quality control solution respectively, and dilute with an ammonia aqueous solution with a concentration of 0.1% to 0.5%. After vortexing and centrifugation, take the supernatant and extract it through a solid phase extraction column to obtain the blood test solution and the quality control sample solution respectively.

[0010] S5. Perform liquid chromatography-mass spectrometry analysis on the mixed standard working solution and the internal standard working solution to obtain the retention time, qualitative ion and quantitative ion information of each target analyte and internal standard;

[0011] S6. Prepare blood matrix spiked standard working curves at least five concentration points. After processing the blood matrix spiked standard working solutions of a series of concentrations according to step S4, perform liquid chromatography-mass spectrometry analysis according to the conditions of step S5. Plot the standard working curve with the ratio of the quantitative ion peak area of ​​each target analyte to the quantitative ion peak area of ​​the internal standard as the ordinate and the concentration of each target analyte as the abscissa.

[0012] S7. Perform liquid chromatography-mass spectrometry analysis on the blood test solution and the quality control sample solution under the same conditions as in step S5 to obtain the quantitative ion peak areas of each target analyte and internal standard.

[0013] S8. Substitute the ratio of the quantitative ion peak area of ​​each target analyte to the internal standard obtained in step S7 into the standard working curve drawn in step S6, and calculate the content of each quaternary ammonium salt compound in the blood sample to be tested.

[0014] Further, in step S1, the preparation method of the mixed standard working solution is as follows: First, a mixed standard stock solution is prepared, containing paraquat, tubocurarine, vecuronium bromide, and succinylcholine chloride at a concentration of 40 μg / mL, succinylcholine chloride at a concentration of 20 μg / mL, chlormequat chloride and dialc-dimethylammonium chloride at a concentration of 10 μg / mL, and wild wheat bran, mepiquat chloride, diquat, and neostigmine at a concentration of 4 μg / mL; then, the mixed standard stock solution is taken and diluted with acetonitrile to obtain a mixed standard working solution, containing paraquat, tubocurarine, vecuronium bromide, and succinylcholine chloride at a concentration of 4 μg / mL, succinylcholine chloride at a concentration of 2 μg / mL, chlormequat chloride and dialc-dimethylammonium chloride at a concentration of 1 μg / mL, and wild wheat bran, mepiquat chloride, diquat, and neostigmine at a concentration of 0.4 μg / mL.

[0015] Further, in step S2, the internal standard working solution is a D8-paraquat solution with a concentration of 2 μg / mL.

[0016] Further, in step S3, the concentration of paraquat, tubocurarine, vecuronium bromide, and succinylcholine chloride in the blood quality control solution is 4.0 ng / mL, the concentration of chlormequat chloride and dialcyldimethylammonium chloride is 1.0 ng / mL, and the concentration of wild wheat bran, mepiquat chloride, diquat, and neostigmine is 0.4 ng / mL.

[0017] Further, in step S4, after dilution with ammonia aqueous solution, the mixture is vortexed for 1 minute, and then centrifuged at a speed of 9000-15000 rpm for 3-5 minutes; the solid phase extraction column is a WCX weak cation exchange solid phase extraction column.

[0018] Furthermore, in steps S5 and S7, the conditions for liquid chromatography-mass spectrometry analysis include:

[0019] Chromatographic column: HILIC column as stationary phase, column size 2.1×100mm, packing particle size 1.6~1.7μm;

[0020] Mobile phase: Phase A is a high-purity aqueous solution containing 50-200 mM ammonium formate, and Phase B is acetonitrile;

[0021] Flow rate: 0.35–0.45 mL / min;

[0022] Column temperature: 30~40℃;

[0023] Elution method: gradient elution;

[0024] Mass spectrometry conditions: positive ion scanning mode, multiple reaction monitoring.

[0025] Furthermore, the gradient elution procedure is as follows:

[0026] At 0.0 minutes, mobile phase A is 15% and mobile phase B is 85%.

[0027] At 2.0 minutes, mobile phase A was 20% and mobile phase B was 80%.

[0028] At 4.0 minutes, mobile phase A was 80% and mobile phase B was 20%.

[0029] At 6.0 minutes, mobile phase A was 80% and mobile phase B was 20%.

[0030] At 6.01 minutes, mobile phase A was 15% and mobile phase B was 85%.

[0031] At 8.0 minutes, mobile phase A was 15% and mobile phase B was 85%.

[0032] Furthermore, in steps S5 and S7, the retention times of each target substance are as follows: paraquat 4.91 minutes, diquat 2.93 minutes, chlormequat 2.44 minutes, mepiquat chloride 3.36 minutes, wild wheat bran 1.19 minutes, tubocurarine 3.43 minutes, dialc-decyl dimethyl ammonium chloride 0.87 minutes, neostigmine 2.29 minutes, succinylcholine chloride 3.69 minutes, vecuronium bromide 3.75 minutes, and succinylcholine chloride 4.62 minutes.

[0033] Further, in step S6, a standard working curve of blood matrix spiked at 5 concentration points is prepared, and the linear correlation coefficient R of the standard working curve is ≥0.99.

[0034] Furthermore, in step S8, when the deviation between the measured content of each quaternary ammonium salt compound in the quality control sample solution and the actual added amount is greater than 15%, the blood sample solution to be tested in the same batch needs to be retested.

[0035] Compared with existing technologies, this invention provides a multi-target method for the determination of quaternary ammonium compounds in blood based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS). This method can simultaneously determine the content of paraquat, diquat, chlormequat chloride, mepiquat chloride, wild wheat bran, tubocurarine, dialc-2-methylammonium chloride (DDAC), neostigmine, succinylcholine chloride, vecuronium bromide, and succinylcholine chloride. This method is simple to operate, highly sensitive, and provides reliable results, making it suitable for poisoning diagnosis and clinical toxicology research. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0037] Figure 1 MRM chromatograms of 11 quaternary ammonium salt compounds provided in the embodiments of the present invention;

[0038] Figure 2 MRM chromatogram of wild wheatgrass is provided for embodiments of the present invention;

[0039] Figure 3 MRM chromatograms of chlormequat chloride are provided for embodiments of the present invention;

[0040] Figure 4 MRM chromatograms of mepiperidine are provided for embodiments of the present invention;

[0041] Figure 5 MRM chromatogram of diquat is provided for embodiments of the present invention;

[0042] Figure 6 MRM chromatogram of paraquat is provided for embodiments of the present invention;

[0043] Figure 7 MRM chromatograms of tubocurarine chloride are provided for embodiments of the present invention;

[0044] Figure 8 MRM chromatograms of DDAC are provided for embodiments of the present invention;

[0045] Figure 9 MRM chromatograms of neostigmine are provided for embodiments of the present invention;

[0046] Figure 10 MRM chromatogram of succinylcholine chloride is provided for embodiments of the present invention;

[0047] Figure 11 MRM chromatograms of vecuronium bromide are provided for embodiments of the present invention;

[0048] Figure 12 The MRM chromatogram of succinylcholine chloride is provided for embodiments of the present invention. Detailed Implementation

[0049] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0050] As attached Figure 1 To be continued Figure 12 As shown:

[0051] Example 1:

[0052] This invention provides a method for the multi-target determination of quaternary ammonium compounds in blood based on ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) and equipped with an electrospray ionization (ESI) source.

[0053] Chromatographic conditions:

[0054] Chromatographic column: UPLC HILIC column, 2.1×100mm, packing particle size 1.6~1.7μm;

[0055] Mobile phase: Phase A is high-purity water + 50-200mM ammonium formate, and Phase B is acetonitrile;

[0056] Flow rate: 0.35–0.45 mL / min;

[0057] Column temperature: 30~40℃;

[0058] Sample chamber temperature: 10℃;

[0059] Gradient elution program: 0.0 min (15% A, 85% B), 2.0 min (20% A, 80% B), 4.0 min (80% A, 20% B), 6.0 min (80% A, 20% B), 6.01 min (15% A, 85% B), 8.0 min (15% A, 85% B).

[0060] Mass spectrometry conditions:

[0061] Scanning method: positive ion scan;

[0062] Detection method: Multiple reaction monitoring (MRM);

[0063] Capillary voltage: 3.0kV;

[0064] Ion source temperature: 150℃;

[0065] Desolvent gas removal temperature: 550℃;

[0066] Atomizing airflow rate: 900L / hr;

[0067] Conical orifice airflow velocity: 150 L / hr;

[0068] Collision gas: Argon, flow rate 0.15 mL / min.

[0069] Solution preparation:

[0070] Preparation of mixed standard working solutions:

[0071] Accurately weigh the standards of paraquat, diquat, chlormequat chloride, mepiquat chloride, wild wheat bran, tubocurarine, DDAC, neostigmine, succinicotinamide chloride, vecuronium bromide, and succinicotinamide chloride, respectively, and dissolve and dilute them with acetonitrile water at a ratio of 1:1 (V / V) to prepare standard stock solutions of paraquat, diquat, chlormequat chloride, mepiquat chloride, wild wheat bran, tubocurarine, DDAC, neostigmine, succinicotinamide chloride, vecuronium bromide, and succinicotinamide chloride with a concentration of 2.0 mg / mL.

[0072] Take appropriate amounts of the standard stock solutions and dilute them with acetonitrile to prepare mixed standard stock solutions with concentrations of 40 μg / mL for paraquat, tubocurarine, vecuronium bromide, and succinylcholine chloride; 20 μg / mL for succinylcholine chloride; 10 μg / mL for chlormequat chloride and DDAC; and 4 μg / mL for wild wheatgrass, mepiquat chloride, diquat, and neostigmine.

[0073] Take appropriate amounts of the mixed standard stock solution and add acetonitrile to prepare mixed standard working solutions with the following concentrations: paraquat, tubocurarine, vecuronium bromide, succinylcholine chloride (4 μg / mL), succinylcholine chloride (2 μg / mL), chlormequat chloride, DDAC (1 μg / mL), and wild wheatgrass, mepiquat chloride, diquat, neostigmine (0.4 μg / mL).

[0074] All the above-mentioned prepared solutions should be stored at -20°C and brought to room temperature before use.

[0075] Preparation of internal standard working solution:

[0076] Accurately pipette an appropriate amount of D8-paraquat internal standard stock solution, add acetonitrile as a diluent, and prepare a 2 μg / mL D8-paraquat internal standard working solution. Store the above solution at -20℃ and allow it to reach room temperature before use.

[0077] Preparation of blood quality control solution:

[0078] Take 1 mL of blank blood and add 20 μL of C5 mixed standard working solution to prepare blood quality control solutions with a concentration of 4 ng / mL for paraquat, tubocurarine, vecuronium bromide, and succinylcholine chloride; a concentration of 2 ng / mL for succinylcholine chloride; a concentration of 1 ng / mL for chlormequat chloride and DDAC; and a concentration of 0.4 ng / mL for wild wheatgrass, mepiquat chloride, diquat, and neostigmine.

[0079] Preparation of matrix spiking standard working curve:

[0080] Take 5 centrifuge tubes, add 1 mL of blank serum to each tube, and add different volumes of mixed standard working solution to each tube to make the concentration of each target substance in the blood as shown in Table 1 (five concentration points C1 to C5). Then add 10 μL of internal standard working solution and 3 mL of 0.1% to 0.5% ammonia solution to each tube. Centrifuge at 9000 to 15000 r / min for 5 minutes. Take the supernatant and extract it into a WCX weak cation exchange solid phase extraction column to obtain a series of matrix-spiked mixed standard working solutions.

[0081] The above-mentioned matrix-spiked series of mixed standard working solutions were injected sequentially and analyzed according to the liquid chromatography-mass spectrometry conditions in step 1. The standard working curve was plotted with the ratio of the peak area of ​​the quantitative ion peak of each target analyte to the peak area of ​​the corresponding internal standard analyte as the ordinate and the corresponding target analyte concentration as the abscissa.

[0082] The linear regression equation for paraquat is Y=3.69489X+5.76794, with a linear correlation coefficient R>0.99.

[0083] Preparation and processing of test sample and quality control solution:

[0084] Take 1 mL of the blood sample to be tested into a centrifuge tube, add 2.5 μL of internal standard working solution, and dilute with 3 mL of 0.1% ammonia water solution. Vortex for 1 min, centrifuge at 9000-15000 r / min for 5 min, and take the supernatant to extract it into a WCX solid phase extraction column to obtain the blood test solution.

[0085] Take 1 mL of blood quality control solution and perform the same procedure to obtain the quality control sample solution.

[0086] Sample determination:

[0087] The obtained blood test solution and quality control sample solution were analyzed under liquid chromatography-mass spectrometry conditions to obtain the retention time, qualitative ion and quantitative ion information of each compound.

[0088] Using the above liquid chromatography-mass spectrometry method, the retention time of paraquat was determined to be 4.91 min.

[0089] The peak area of ​​the quantitative ion peak of paraquat in the blood test solution was measured to be 628612.88, and the peak area of ​​the quantitative ion peak of the internal standard was 31119.03.

[0090] The peak area of ​​the quantitative ion peak of paraquat in the quality control sample solution was measured to be 295937.03, and the peak area of ​​the quantitative ion peak of the internal standard was 35730.29.

[0091] Content calculation:

[0092] Substituting the ratio of the quantitative ion peak area of ​​paraquat to that of the internal standard in the blood test solution into the standard working curve Y=3.69489X+5.76794, the paraquat content in the blood sample was calculated to be 101 ng / mL.

[0093] The ratio of the quantitative ion peak area of ​​paraquat to that of the internal standard in the quality control sample solution was substituted into the standard working curve to calculate the paraquat content in the quality control sample. The result was compared with the actual addition amount of 4.0 ng / mL, and the deviation was 2.5%, which meets the requirement of deviation ≤15%.

[0094] Analysis using the above liquid chromatography-mass spectrometry method showed that the abundance ratios of the qualitative ion pairs (186.0 to 171.0, 186.0 to 85.0) of paraquat were consistent with those of the standard, confirming the detection of paraquat in the blood sample. The MRM chromatogram of this sample is shown below. Figure 6 As shown.

[0095] Example 2:

[0096] Examining linear relationships:

[0097] Take 5 centrifuge tubes, add 1 mL of blank serum to each tube, and add different volumes of mixed standard working solution to each tube to achieve the concentrations of each target analyte in the blood (C1, C2, C3, C4, and C5). Then add 10 μL of internal standard working solution and 3 mL of 0.1% ammonia solution to each tube. Centrifuge at 9500 r / min for 15 minutes. Take the supernatant and extract it onto a WCX weak cation exchange solid phase extraction column to obtain a series of matrix-spiked mixed standard working solutions.

[0098] Table 1. Five concentration points (concentration unit ng / mL) of the standard working curve are shown below:

[0099]

[0100] The above-mentioned matrix-spiked series of mixed standard working solutions were sequentially injected and analyzed according to the liquid chromatography-mass spectrometry conditions of Example 1. The standard working curve was plotted with the ratio of the peak area of ​​the quantitative ion peak of each target analyte to the peak area of ​​the corresponding internal standard quantitative ion peak as the ordinate and the corresponding target analyte concentration as the abscissa.

[0101] The linear regression equations for each target are as follows:

[0102] Paraquat: Y = 3.69489X + 5.76794;

[0103] Dichlorvos: Y = 3.43086X + 0.0444636;

[0104] Wild wheat withering: Y = 13.2818X + 0.620124;

[0105] Chlormequat chloride: Y = 2.24568X + 0.0515763;

[0106] Mepiperidine: Y = 1.35479X + 0.0281676;

[0107] Tubocurarine: Y = 0.551152X - 0.830955;

[0108] DDAC: Y = 0.551152X + 1.32183;

[0109] Neostigmine: Y = 26.2694X - 0.19138;

[0110] Succinylcholine chloride: Y = 0.566027X - 0.570214;

[0111] Vecuronium bromide: Y = 0.367767X - 0.723901;

[0112] Succinylcholine chloride: Y = 1.15566X - 27.1643.

[0113] The linear correlation coefficients R of each target analyte within the corresponding concentration range were all ≥0.99, indicating a good linear relationship.

[0114] Accuracy verification:

[0115] Take 1 mL of blank blood, add an appropriate amount of mixed standard working solution, and prepare a blood quality control solution according to the method in Example 1. The concentrations of paraquat, tubocurarine, vecuronium bromide, and succinylcholine chloride are 4.0 ng / mL, the concentration of succinylcholine chloride is 2.0 ng / mL, the concentrations of chlormequat chloride and DDAC are 1.0 ng / mL, and the concentrations of wild wheat bran, mepiquat chloride, diquat, and neostigmine are 0.4 ng / mL.

[0116] The above quality control sample solution was analyzed according to the liquid chromatography-mass spectrometry conditions of Example 1 to determine the content of each target analyte and calculate the deviation between the measured value and the actual amount added.

[0117] The quality control sample test results showed that:

[0118] The measured value of paraquat was 4.1 ng / mL, which deviated from the actual added amount of 4.0 ng / mL by 2.5%.

[0119] The measured value of chlormequat chloride was 1.02 ng / mL, which deviated from the actual added amount of 1.0 ng / mL by 2.0%.

[0120] The measured value of wild wheat bran was 0.39 ng / mL, which deviated from the actual added amount of 0.4 ng / mL by -2.5%.

[0121] The measured value of mepiperidine was 0.41 ng / mL, which deviated from the actual added amount of 0.4 ng / mL by 2.5%.

[0122] The measured value of diquat was 0.39 ng / mL, which deviated from the actual added amount of 0.4 ng / mL by -2.5%.

[0123] The measured value of tubocurarine was 3.9 ng / mL, which deviated from the actual added amount of 4.0 ng / mL by -2.5%.

[0124] The DDAC measurement was 0.98 ng / mL, which deviated from the actual addition amount of 1.0 ng / mL by -2.0%.

[0125] The measured value of neostigmine was 0.41 ng / mL, which deviated from the actual added amount of 0.4 ng / mL by 2.5%.

[0126] The measured value of succinic acid monocholine chloride was 2.05 ng / mL, which deviated from the actual added amount of 2.0 ng / mL by 2.5%.

[0127] The measured value of vecuronium bromide was 3.9 ng / mL, which deviated from the actual added amount of 4.0 ng / mL by -2.5%.

[0128] The measured value of succinylcholine chloride was 4.1 ng / mL, which deviated from the actual added amount of 4.0 ng / mL by 2.5%.

[0129] The deviations between the measured values ​​and the actual amounts added for each target analyte were all ≤15%, which meets the accuracy requirements of the method.

[0130] Precision verification:

[0131] Take blank blood samples and add mixed standard solutions of low, medium and high concentrations respectively. After processing according to the method in Example 1, analyze the samples according to the liquid chromatography-mass spectrometry conditions in Example 1. Each concentration is measured in parallel 6 times.

[0132] The relative standard deviation (RSD) is calculated based on the measured values ​​of each target, and the intraday precision is examined.

[0133] The results show that the intraday precision RSD of each target at low, medium and high concentration levels is ≤8.5%, and the interday precision RSD is ≤10.2%, indicating that the method has good repeatability.

[0134] Sensitivity verification:

[0135] The lowest concentration point (C1) in the matrix-spiked series of mixed standard working solutions was analyzed according to the liquid chromatography-mass spectrometry conditions in step 1 of Example 1, and the detection limit of each target analyte was determined with a signal-to-noise ratio (S / N) ≥ 3.

[0136] The detection limits for each target analyte are as follows:

[0137] The detection limit for paraquat is 0.5 ng / mL;

[0138] The detection limit for diquat was 0.1 ng / mL;

[0139] The detection limit for wild wheat bran was 0.05 ng / mL;

[0140] The detection limit for chlormequat chloride was 0.2 ng / mL;

[0141] The detection limit for mepiperidine is 0.1 ng / mL;

[0142] The detection limit for tubocurarine is 1.0 ng / mL;

[0143] The detection limit for DDAC is 0.2 ng / mL;

[0144] The detection limit for neostigmine is 0.1 ng / mL;

[0145] The detection limit for succinic acid monocholine chloride is 0.5 ng / mL;

[0146] The detection limit for vecuronium bromide is 1.0 ng / mL;

[0147] The detection limit for succinylcholine chloride is 1.0 ng / mL.

[0148] The detection limits of each target analyte meet the requirements for detecting quaternary ammonium compounds in the blood of poisoned patients.

[0149] Example 3:

[0150] Sample source:

[0151] Blood samples were collected from 11 poisoning patients or poisoning victims, and were numbered No.1 to No.11.

[0152] Sample pretreatment:

[0153] Take 1 mL of each of the above 11 blood samples into a centrifuge tube, add 2.5 μL of internal standard working solution to each, and dilute with 3 mL of 0.1% ammonia aqueous solution. Vortex for 1 min, centrifuge at 9000-15000 r / min for 3-5 minutes, and extract the supernatant into a WCX weak cation exchange solid phase extraction column to obtain the blood test solutions of the 11 samples.

[0154] Instrumental analysis conditions:

[0155] Analysis was performed according to the liquid chromatography-mass spectrometry conditions of Example 1, including:

[0156] Chromatographic column: UPLCHILIC column (2.1×100mm, 1.6~1.7μm);

[0157] Mobile phase: Phase A is high-purity water + 50-200mM ammonium formate, and Phase B is acetonitrile;

[0158] Flow rate: 0.35–0.45 mL / min; Column temperature: 30–40 °C;

[0159] Gradient elution program: 0.0 min (15% A, 85% B), 2.0 min (20% A, 80% B), 4.0 min (80% A, 20% B), 6.0 min (80% A, 20% B), 6.01 min (15% A, 85% B), 8.0 min (15% A, 85% B);

[0160] Mass spectrometry conditions: positive ion scan, multiple reaction monitoring (MRM) mode.

[0161] Standard operating curve:

[0162] Quantitative analysis was performed using the standard working curves for each target analyte established in Example 2:

[0163] Paraquat: Y = 3.69489X + 5.76794;

[0164] Dichlorvos: Y = 3.43086X + 0.0444636;

[0165] Wild wheat withering: Y = 13.2818X + 0.620124;

[0166] Chlormequat chloride: Y = 2.24568X + 0.0515763;

[0167] Mepiperidine: Y = 1.35479X + 0.0281676;

[0168] Tubocurarine: Y = 0.551152X - 0.830955;

[0169] DDAC: Y = 0.551152X + 1.32183;

[0170] Neostigmine: Y = 26.2694X - 0.19138;

[0171] Succinylcholine chloride: Y = 0.566027X - 0.570214;

[0172] Vecuronium bromide: Y = 0.367767X - 0.723901;

[0173] Succinylcholine chloride: Y = 1.15566X - 27.1643.

[0174] The linear correlation coefficient R of each standard working curve is ≥0.99.

[0175] Test results

[0176] Eleven blood sample solutions were analyzed by liquid chromatography-mass spectrometry under the above conditions to obtain the retention time, qualitative ion and quantitative ion information of each target analyte. The peak area of ​​the quantitative ion of each target analyte and the peak area of ​​the quantitative ion of the internal standard were measured. The peak area ratio was substituted into the corresponding standard working curve to calculate the content of quaternary ammonium salt compounds in each blood sample.

[0177] The test results are as follows:

[0178] Sample No. 1: Wild wheatgrass was detected, with a retention time of 1.19 min. The peak area of ​​the quantitative ion of wild wheatgrass was 1084599.13, and the peak area of ​​the quantitative ion of the internal standard was 37913.88. Substituting these values ​​into the wild wheatgrass standard curve Y = 13.2818X + 0.620124, the calculated content of wild wheatgrass in the blood was 10.72 ng / mL. The MRM chromatogram of this sample is shown below. Figure 2 As shown.

[0179] Sample No. 2: Chlormequat was detected, with a retention time of 2.44 min. The peak area of ​​the quantitative ion for chlormequat was 295937.03, and the peak area of ​​the quantitative ion for the internal standard was 35730.29. Substituting these values ​​into the chlormequat standard curve Y = 2.24568X + 0.0515763, the calculated chlormequat concentration in the blood was 18.41 ng / mL. The MRM chromatogram of this sample is shown below. Figure 3 As shown.

[0180] Sample No. 3: Mepiride was detected, with a retention time of 3.37 min. The quantitative ion peak area of ​​mepiride was 86104.62, and the quantitative ion peak area of ​​the internal standard was 49340.52. Substituting these values ​​into the mepiride standard curve Y = 1.35479X + 0.0281676, the calculated mepiride concentration in the blood was 6.42 ng / mL. The MRM chromatogram of this sample is shown below. Figure 4 As shown.

[0181] Sample No. 4: Diquat was detected, with a retention time of 2.93 min. The quantitative ion peak area of ​​diquat was 164631.97, and the quantitative ion peak area of ​​the internal standard was 43967.4. Substituting these values ​​into the diquat standard curve Y = 3.43086X + 0.0444636, the calculated diquat content in the blood was 5.43 ng / mL. The MRM chromatogram of this sample is shown below. Figure 5 As shown.

[0182] Sample No. 5: Paraquat was detected, with a retention time of 4.93 min. The peak area of ​​the quantitative ion for paraquat was 628612.88, and the peak area of ​​the quantitative ion for the internal standard was 31119.03. Substituting these values ​​into the paraquat standard curve Y = 3.69489X + 5.76794, the calculated paraquat concentration in the blood was 101 ng / mL. The MRM chromatogram of this sample is shown below. Figure 6 As shown.

[0183] Sample No. 6: Tubacurarine chloride was detected, with a retention time of 3.43 min. The peak area of ​​the quantitative ion of tubocurarine was 159045.75, and the peak area of ​​the quantitative ion of the internal standard was 107682.7. Substituting these values ​​into the tubocurarine standard curve Y = 0.551152X - 0.830955, the calculated concentration of tubocurarine in the blood was 14.91 ng / mL. The MRM chromatogram of this sample is shown below. Figure 7 As shown.

[0184] Sample No. 7: DDAC was detected, with a retention time of 0.87 min. The peak area of ​​the quantitative ion for DDAC was 75963.68, and the peak area of ​​the quantitative ion for the internal standard was 89579.94. Substituting these values ​​into the DDAC standard curve Y = 0.551152X + 1.32183, the calculated DDAC content in the blood was 1.34 ng / mL. The MRM chromatogram of this sample is shown below. Figure 8 As shown.

[0185] Sample No. 8: Neostigmine was detected, with a retention time of 2.30 min. The peak area of ​​the quantitative ion of neostigmine was 2167760.75, and the peak area of ​​the quantitative ion of the internal standard was 81365.1. Substituting these values ​​into the neostigmine standard curve Y = 26.2694X - 0.19138, the calculated neostigmine content in the blood was 5.08 ng / mL. The MRM chromatogram of this sample is shown below. Figure 9 As shown.

[0186] Sample No. 9: Succinylcholine chloride was detected, with a retention time of 3.69 min. The peak area of ​​the quantitative ion of succinylcholine chloride was 244633.75, and the peak area of ​​the quantitative ion of the internal standard was 83467.84. Substituting these values ​​into the succinylcholine chloride standard curve Y = 0.566027X - 0.570214, the calculated concentration of succinylcholine chloride in the blood was 26.90 ng / mL. The MRM chromatogram of this sample is shown below. Figure 10 As shown.

[0187] Sample No. 10: Vecuronium bromide was detected, with a retention time of 3.75 min. The quantitative ion peak area of ​​vecuronium bromide was 568818.19, and the quantitative ion peak area of ​​the internal standard was 84310.65. Substituting these values ​​into the vecuronium bromide standard curve Y = 0.367767X - 0.723901, the calculated vecuronium bromide concentration in the blood was 93.69 ng / mL. The MRM chromatogram of this sample is shown below. Figure 11 As shown.

[0188] Sample No. 11: Succinylcholine chloride was detected, with a retention time of 4.62 min. The peak area of ​​the quantitative ion of succinylcholine chloride was 1534792.75, and the peak area of ​​the quantitative ion of the internal standard was 78548.21. Substituting these values ​​into the succinylcholine chloride standard curve Y = 1.15566X - 27.1643, the calculated concentration of succinylcholine chloride in the blood was 64.68 ng / mL. The MRM chromatogram of this sample is shown below. Figure 12 As shown.

[0189] Results Summary: The results of the detection of quaternary ammonium compounds in the blood of 11 poisoned / deceased individuals are summarized in the table below.

[0190] Results of quaternary ammonium compound content tests in the blood of 11 poisoned / deceased individuals:

[0191]

[0192] Results confirmed: MRM chromatograms for each case ( Figures 2 to 12 The results showed that the chromatographic peaks of each target compound were well-shaped, the retention times were consistent with those of the standards, the abundance ratio of qualitative ion pairs was consistent with that of the standards, and there was no matrix interference, confirming the detection of the corresponding quaternary ammonium salt compounds in each sample.

[0193] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A method for multi-target determination of quaternary ammonium compounds in blood based on ultra-high performance liquid chromatography-tandem mass spectrometry, characterized in that, Includes the following steps: S1. Prepare a mixed standard working solution, wherein the mixed standard working solution contains paraquat, diquat, wild wheat bran, chlormequat chloride, mepiquat chloride, tubocurarine, didecyl dimethyl ammonium chloride, neostigmine, succinic acid monocholine chloride, vecuronium bromide and succinic acid monocholine chloride; S2. Prepare an internal standard working solution, wherein the internal standard working solution contains D8-paraquat; S3. Prepare a blood quality control solution, wherein the blood quality control solution contains paraquat, diquat, wild wheat bran, chlormequat chloride, mepiquat chloride, tubocurarine, didecyl dimethyl ammonium chloride, neostigmine, succinic acid monocholine chloride, vecuronium bromide and succinic acid monocholine chloride; S4. Add the internal standard working solution to the blood sample to be tested and the blood quality control solution respectively, and dilute with an ammonia aqueous solution with a concentration of 0.1% to 0.5%. After vortexing and centrifugation, take the supernatant and extract it through a solid phase extraction column to obtain the blood test solution and the quality control sample solution respectively. S5. Perform liquid chromatography-mass spectrometry analysis on the mixed standard working solution and the internal standard working solution to obtain the retention time, qualitative ion and quantitative ion information of each target analyte and internal standard; S6. Prepare blood matrix spiked standard working curves at least five concentration points. After processing the blood matrix spiked standard working solutions of a series of concentrations according to step S4, perform liquid chromatography-mass spectrometry analysis according to the conditions of step S5. Plot the standard working curve with the ratio of the quantitative ion peak area of ​​each target analyte to the quantitative ion peak area of ​​the internal standard as the ordinate and the concentration of each target analyte as the abscissa. S7. Perform liquid chromatography-mass spectrometry analysis on the blood test solution and the quality control sample solution under the same conditions as in step S5 to obtain the quantitative ion peak areas of each target analyte and internal standard. S8. Substitute the ratio of the quantitative ion peak area of ​​each target analyte to the internal standard obtained in step S7 into the standard working curve drawn in step S6, and calculate the content of each quaternary ammonium salt compound in the blood sample to be tested.

2. The method for multi-target determination of quaternary ammonium compounds in blood based on ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, In step S1, the preparation method of the mixed standard working solution is as follows: First, a mixed standard stock solution is prepared, which contains paraquat, tubocurarine, vecuronium bromide, and succinylcholine chloride at a concentration of 40 μg / mL, succinylcholine chloride at a concentration of 20 μg / mL, chlormequat chloride and dialc-dimethylammonium chloride at a concentration of 10 μg / mL, and wild wheat bran, mepiquat chloride, diquat, and neostigmine at a concentration of 4 μg / mL; then, the mixed standard stock solution is taken and diluted with acetonitrile to obtain a mixed standard working solution, which contains paraquat, tubocurarine, vecuronium bromide, and succinylcholine chloride at a concentration of 4 μg / mL, succinylcholine chloride at a concentration of 2 μg / mL, chlormequat chloride and dialc-dimethylammonium chloride at a concentration of 1 μg / mL, and wild wheat bran, mepiquat chloride, diquat, and neostigmine at a concentration of 0.4 μg / mL.

3. The method for multi-target determination of quaternary ammonium compounds in blood based on ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, In step S2, the internal standard working solution is a D8-paraquat solution with a concentration of 2 μg / mL.

4. The method for multi-target determination of quaternary ammonium compounds in blood based on ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, In step S3, the blood quality control solution contains paraquat, tubocurarine, vecuronium bromide, and succinylcholine chloride at a concentration of 4.0 ng / mL, chlormequat chloride and dialcyldimethylammonium chloride at a concentration of 1.0 ng / mL, and wild wheatgrass, mepiquat chloride, diquat, and neostigmine at a concentration of 0.4 ng / mL.

5. The method for multi-target determination of quaternary ammonium compounds in blood based on ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, In step S4, after dilution with ammonia water, the mixture is vortexed for 1 minute and then centrifuged at 9000-15000 rpm for 3-5 minutes; the solid phase extraction column is a WCX (weak cation exchange) solid phase extraction column.

6. The method for multi-target determination of quaternary ammonium compounds in blood based on ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, In steps S5 and S7, the conditions for liquid chromatography-mass spectrometry analysis include: Chromatographic column: HILIC column as stationary phase, column size 2.1×100mm, packing particle size 1.6~1.7μm; Mobile phase: Phase A is a high-purity aqueous solution containing 50-200 mM ammonium formate, and Phase B is acetonitrile; Flow rate: 0.35–0.45 mL / min; Column temperature: 30~40℃; Elution method: gradient elution; Mass spectrometry conditions: positive ion scanning mode, multiple reaction monitoring.

7. The method for multi-target determination of quaternary ammonium compounds in blood based on ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 6, characterized in that, The gradient elution procedure is as follows: At 0.0 minutes, mobile phase A is 15% and mobile phase B is 85%. At 2.0 minutes, mobile phase A was 20% and mobile phase B was 80%. At 4.0 minutes, mobile phase A was 80% and mobile phase B was 20%. At 6.0 minutes, mobile phase A was 80% and mobile phase B was 20%. At 6.01 minutes, mobile phase A was 15% and mobile phase B was 85%. At 8.0 minutes, mobile phase A was 15% and mobile phase B was 85%.

8. The method for multi-target determination of quaternary ammonium compounds in blood based on ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, In steps S5 and S7, the retention times of each target substance are as follows: paraquat 4.91 minutes, diquat 2.93 minutes, chlormequat 2.44 minutes, mepiquat chloride 3.36 minutes, wild wheat bran 1.19 minutes, tubocurarine 3.43 minutes, decyl dimethyl ammonium chloride 0.87 minutes, neostigmine 2.29 minutes, succinylcholine chloride 3.69 minutes, vecuronium bromide 3.75 minutes, and succinylcholine chloride 4.62 minutes.

9. The method for multi-target determination of quaternary ammonium compounds in blood based on ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, In step S6, a standard working curve of blood matrix spiked at 5 concentration points is prepared, and the linear correlation coefficient R of the standard working curve is ≥0.

99.

10. The method for multi-target determination of quaternary ammonium compounds in blood based on ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, In step S8, if the deviation between the measured content of each quaternary ammonium salt compound in the quality control sample solution and the actual added amount is greater than 15%, the blood sample solution to be tested in the same batch needs to be retested.

Images