Method for detecting multiple alkyl imidazole ionic liquids in serum based on liquid chromatography-mass spectrometry

Abstract

The application discloses a method for detecting a plurality of alkyl imidazole ionic liquids in serum based on liquid chromatography-mass spectrometry, and comprises the following steps: preparing a matrix standard solution, and drawing a matrix standard curve; carrying out pretreatment and solid phase extraction on a human serum sample to be detected; and finally, performing quantitative analysis by using LC-MS / MS. The method can quickly and accurately detect the alkyl imidazole ionic liquids in the human serum sample, is simple, efficient and high in sensitivity, can realize the detection of 10 target alkyl imidazole ionic liquids in human exposure, is good in separation effect, can provide an effective technical means for the biological monitoring research work of ionic liquid pollutants, and in the theoretical aspect, provides a certain scientific basis for the human exposure risk and preventive measures of ionic liquids.

Description

Technical Field

[0001] This invention relates to the field of detection and analysis technology, specifically to a method for detecting multiple alkylimidazolium ionic liquids in serum based on liquid chromatography-mass spectrometry (LC-MS). Background Technology

[0002] Ionic liquids (ILs), also known as room-temperature ionic liquids, are a class of molten salts composed of organic cations and inorganic or organic anions that are liquid at room temperature. Ionic liquids are hailed as a new type of green solvent for the 21st century. As compounds that are liquid at room temperature, they possess many excellent properties, such as low vapor pressure, non-flammability, low volatility, good stability, and strong conductivity, showing broad application prospects in numerous fields including chemical engineering, electronics, pharmaceuticals, biotechnology, and food. 1-alkyl-3-methyl-imidazolium cations (Cn[MIM], where the alkyl group Cn is C2-C12) are among the most studied ILs in ecological and toxicological research. Most notably, they exhibit extremely high stability in water. Furthermore, their low vapor pressure, low volatility, easy recovery, and lack of harmful gas generation during industrial use have led to their widespread use. This promising application prospect has resulted in a gradual increase in the demand for IL production, making them a substitute for traditional organic solvents.

[0003] The development and research of ionic liquids have enhanced human awareness of environmental and health concerns. In recent years, widely used ionic liquids have been proven to be less environmentally friendly and biofriendly. Currently, there are few reports on the environmental and biological hazards of ionic liquids. The rapid development of ionic liquids is not matched by the lagging understanding of their environmental and human health risks. Increasing human usage and wastewater discharge will inevitably lead to the environmental presence of ionic liquids and the risk of human exposure. Due to their high thermal and chemical stability, traditional physicochemical methods are insufficient for their elimination, and they are difficult to biodegrade, potentially becoming new persistent pollutants. The internationally renowned hepatology journal *Journal of Hepatology* reported that a research team discovered a high concentration of a novel exogenous compound in soil samples near a municipal landfill, which may be an environmental trigger for primary biliary cholangitis (PBC). This substance was identified as a 1-octyl-3-methylimidazolium ionic liquid by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HMS), indicating the presence of ILs in the environment and their potential threat to human health. This foreshadows the multiple risks associated with the release of such ILs into the environment. Therefore, accelerating the development of highly sensitive detection technologies for ILs and conducting monitoring of environmental pollution and human exposure levels to provide a scientific basis for environmental risk management are important issues facing environmental science today.

[0004] Early analytical methods for ionic liquids were primarily used for quality control after laboratory synthesis, mainly including liquid chromatography, ion chromatography, ion-pair chromatography, and capillary electrophoresis. These methods suffer from poor accuracy and insufficient sensitivity when dealing with trace-level analysis of complex biological matrices in environmental or human samples. Therefore, developing a highly sensitive detection method is crucial. Summary of the Invention

[0005] The main objective of this invention is to propose a method for detecting multiple alkylimidazolium ionic liquids in serum based on liquid chromatography-mass spectrometry (LC-MS), aiming to provide a rapid and accurate method for detecting ionic liquid contaminants in human serum samples.

[0006] To achieve the above objectives, this invention proposes a method for detecting multiple alkylimidazolium ionic liquids in serum based on liquid chromatography-mass spectrometry (LC-MS), comprising the following steps:

[0007] Multiple imidazole ionic liquid compounds were respectively prepared into multiple ionic liquid standards with a concentration of 100 mg / mL as stock solutions. The stock solutions were then mixed to prepare mixed standard solutions, and diluted with a mixed solution of acetonitrile and solution a as solvent to prepare mixed standard working solutions with different concentration gradients. Solution a includes any one of ammonium formate aqueous solution, ammonium acetate aqueous solution, and formic acid aqueous solution, and the volume ratio of acetonitrile to solution a is 2:8.

[0008] Each of the mother solutions was prepared into a single standard working solution with a concentration of 10 ng / mL. First, the mother ion was scanned by LC-MS / MS to determine the monitoring ion and quantitative ion pair of each ionic liquid. Then, the retention time of each ionic liquid was determined by LC-MS / MS detection.

[0009] A blank matrix serum sample without ionic liquid was pretreated, and then reconstituted with the mixed standard working solution to prepare mixed matrix standard working solutions with different concentration gradients. LC-MS / MS detection was then performed, and linear regression was performed on the peak area and mass concentration of the target ionic liquid quantitative ion pair to obtain the linear regression equation and standard curve for each ionic liquid.

[0010] The human serum samples to be tested were pretreated, then reconstituted with a mixture of acetonitrile and solution a, centrifuged to obtain the supernatant, and the supernatant was quantitatively analyzed by LC-MS / MS.

[0011] The preprocessing steps include:

[0012] Add phosphoric acid-acetonitrile solution to the sample to be treated, let stand overnight, centrifuge and take the supernatant. Add 5% ammonia solution to the supernatant to adjust the pH of the solution to 6.8, and then use ultrapure water to make up to 1 mL to obtain the extract.

[0013] The extractant is placed in a solid-phase extraction column and activated, rinsed, and eluted sequentially. The eluent is then purged with nitrogen at 35-40°C. The solid-phase extraction column is activated sequentially with acetonitrile and ultrapure water.

[0014] Optionally, the concentration gradients of the mixed standard working solution are 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL and 100 ng / mL, respectively.

[0015] The concentration gradients of the mixed matrix standard working solutions are 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL and 100 ng / mL.

[0016] Optionally, solution a is an aqueous solution of ammonium formate with a concentration of 10 mmol / L.

[0017] Optionally, in the step of pretreating the human serum sample to be tested, then reconstituted with a mixture of acetonitrile and solution a, and centrifuging to obtain the supernatant:

[0018] The centrifugation conditions were 0℃, 15000rpm, and 15min.

[0019] Optionally, in the preprocessing step:

[0020] The overnight settling temperature is 4°C; and / or,

[0021] The centrifugation conditions were 15000 r / min for 15 min.

[0022] Optionally, the solid-phase extraction column is a Waters Oasis WCX 3mL extraction column.

[0023] Optionally, in the preprocessing step:

[0024] The solvent used for activation is acetonitrile and ultrapure water; and / or,

[0025] The rinsing agent used in the rinsing is ammonia and acetonitrile; and / or,

[0026] The eluent used for elution is a mixed solution of formic acid and acetonitrile; and / or,

[0027] When the solid-phase extraction column is activated sequentially with acetonitrile and ultrapure water, the solution is allowed to drip naturally until it is level with the substance in the solid-phase extraction column. Then, the sample can be loaded. When loading the sample, the extractable is added to the activated solid-phase extraction column and the eluent is discarded.

[0028] Optionally, the detection parameter conditions for analysis using LC-MS / MS are as follows:

[0029] Column: ACQUITY TM Premier BEH C18 column, 100mm × 2.1mm, 1.7μm;

[0030] Column temperature: 35℃, sample chamber temperature: 20℃;

[0031] Injection volume: 5 μL;

[0032] Mobile phase A: Ammonium formate aqueous solution, ammonium acetate aqueous solution, or formic acid aqueous solution;

[0033] Mobile phase B: Acetonitrile;

[0034] Flow rate: 0.3 mL / min;

[0035] Gradient elution, the procedure for which is as follows:

[0036] 0min, 0.3mL / min, A 80%, B 20%;

[0037] 1.0min, 0.3mL / min, A 80%, B 20%;

[0038] 1.5min, 0.3mL / min, A 60%, B 40%;

[0039] 3min, 0.3mL / min, A 60%, B 40%;

[0040] 8min, 0.3mL / min, A 5%, B 95%;

[0041] 10min, 0.3mL / min, A 5%, B 95%;

[0042] 10.1min, 0.3mL / min, A 80%, B 20%;

[0043] 11min, 0.3mL / min, A 80%, B 20%;

[0044] Mass spectrometry conditions: ion source was electrospray ionization source in positive ion mode, scanning mode was multiple reaction monitoring mode, ionization voltage was 5500V, temperature was 500℃, spray gas was 40psi, auxiliary heating gas was 40psi, and curtain gas was 40psi.

[0045] Optionally, the mobile phase A is an aqueous solution of ammonium formate, and the concentration of the aqueous solution of ammonium formate is 10 mmol / L.

[0046] Optionally, the plurality of imidazole ionic liquid compounds include 1-ethyl-3-methylimidazolium, 1-propyl-3-methylimidazolium, 1-butyl-3-methylimidazolium, 1-pentyl-3-methylimidazolium, 1-hexyl-3-methylimidazolium, 1-heptyl-3-methylimidazolium, 1-octyl-3-methylimidazolium, 1-nonyl-3-methylimidazolium, 1-decyl-3-methylimidazolium, and 1-dodecyl-3-methylimidazolium.

[0047] The method provided by this invention can rapidly and accurately detect alkylimidazolium ionic liquids in human serum samples. It is simple, efficient, and highly sensitive, and can detect multiple target alkylimidazolium ionic liquids in human exposure. It can provide an effective technical means for biomonitoring of ionic liquid pollutants and research on exposure health risks. In terms of theory, it also provides a certain scientific basis for human exposure risks and preventive measures of ionic liquids. Attached Figure Description

[0048] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0049] Figure 1 This is a schematic flowchart of an embodiment of the method for detecting multiple alkylimidazolium ionic liquids in serum based on liquid chromatography-mass spectrometry provided by the present invention.

[0050] Figure 2 The chromatograms are of 10 imidazole ionic liquid standards in Example 1 of this invention;

[0051] Figure 3a and Figure 3b The above are single-channel chromatograms of 10 imidazole ionic liquids in Example 1 of this invention;

[0052] Figure 4 The graph shows the results of the spiked recovery of 10 imidazole ionic liquid compounds in human serum samples in Example 1 of this invention.

[0053] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0054] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, or solution B, or a solution where both A and B are satisfied simultaneously. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0055] To address the current lack of methods for detecting the body's load of ionic liquid contaminants, this invention proposes a method based on liquid chromatography-mass spectrometry (LC-MS) for detecting multiple alkylimidazolium ionic liquids in serum. Figure 1 The image shows an embodiment of the method provided by the present invention for detecting multiple alkylimidazolium ionic liquids in serum based on liquid chromatography-mass spectrometry (LC-MS). See also... Figure 1 As shown, in this embodiment, the method for detecting multiple alkylimidazolium ionic liquids in serum based on liquid chromatography-mass spectrometry includes the following steps:

[0056] Step S10: Prepare various imidazole ionic liquid compounds into various ionic liquid standards with a concentration of 100 mg / mL as mother solutions. Then, mix the mother solutions to prepare a mixed standard solution. Dilute the mixed standard working solution with acetonitrile and solution a as a solvent to prepare a mixed standard working solution with different concentration gradients. The solution a includes any one of ammonium formate aqueous solution, ammonium acetate aqueous solution, and formic acid aqueous solution, and the volume ratio of acetonitrile to solution a is 2:8.

[0057] Step S20: Take each of the mother liquors and prepare a single standard working solution with a concentration of 10 ng / mL. First, perform a mother ion scan by LC-MS / MS to determine the monitoring ion and quantitative ion pair of each ionic liquid. Then, use an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometer (LC-MS / MS) to detect and determine the retention time of each ionic liquid.

[0058] Step S30: Take a blank matrix serum sample without ionic liquid for pretreatment, then reconstitute it with the mixed standard working solution to prepare mixed matrix standard working solutions with different concentration gradients, and then perform LC-MS / MS detection. Linear regression is performed on the peak area and mass concentration of the target ionic liquid quantitative ion pair to obtain the linear regression equation and standard curve of each ionic liquid.

[0059] Step S40: Pre-treat the human serum sample to be tested, then reconstitute it with a mixed solution of acetonitrile and solution a, centrifuge to collect the supernatant, and perform quantitative analysis on the supernatant using LC-MS / MS;

[0060] The preprocessing steps include:

[0061] Step a: Add phosphoric acid-acetonitrile solution to the sample to be treated, let stand overnight, centrifuge and take the supernatant, add 5% ammonia water to the supernatant to adjust the pH of the solution to 6.8, and then use ultrapure water to make up to 1 mL to obtain the extract.

[0062] Step b: The extractant is placed in a solid-phase extraction column and activated, rinsed, and eluted sequentially. The eluent is then purged with nitrogen at 35-40°C. The solid-phase extraction column is activated sequentially with acetonitrile and ultrapure water.

[0063] The method provided by this invention can rapidly and accurately detect alkylimidazolium ionic liquids in human serum samples. It is simple, efficient, and highly sensitive, and can detect multiple target alkylimidazolium ionic liquids in human exposure. It can provide an effective technical means for biomonitoring research on ionic liquid pollutants. In terms of theory, it also provides a certain scientific basis for the human exposure risk and prevention measures of ionic liquids.

[0064] First, it is necessary to prepare standard solutions, matrix standard working solutions, and plot matrix standard curves. The specific steps include:

[0065] Accurately weigh a certain amount of imidazole ionic liquid standard into a brown glass bottle, add ultrapure water to dissolve it, and prepare an ionic liquid standard with a concentration of 100 mg / mL as the stock solution. For multiple imidazole ionic liquids, prepare corresponding synthetic ionic liquid standards, all of which are stock solutions. It should be noted that, due to the high viscosity of the imidazole ionic liquid standard, vortex mixing is preferred to ensure its complete dissolution in ultrapure water when preparing the standard solution. Then, take equal volumes of each ionic liquid standard and mix them to prepare a mixed standard solution. Using a mixture of acetonitrile and solution a (solution a includes any one of ammonium formate aqueous solution, ammonium acetate aqueous solution, and formic acid aqueous solution, with a volume ratio of acetonitrile to solution a of 2:8) as the solvent, prepare mixed standard working solutions with different concentration gradients for later use.

[0066] Each of the mother liquors was prepared into a single standard working solution with a concentration of 10 ng / mL. The mother ion was first scanned by LC-MS / MS to determine the monitoring ion and quantitative ion pair of each ionic liquid. Then, the retention time of each ionic liquid was determined by ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS / MS).

[0067] A blank matrix serum sample without ionic liquids was pretreated using the same method as the pretreatment of the subsequent human serum samples to be tested. Specifically, a phosphate-acetonitrile solution (phosphate concentration of 4% by volume) was added to the blank matrix serum sample, and after standing overnight, the supernatant was collected by centrifugation. Ammonia solution with a volume concentration of 5% was added to the supernatant to adjust the pH to 6.8, and then the volume was adjusted to 1 mL with ultrapure water to obtain the extract. The extract was placed in a solid-phase extraction column activated sequentially with acetonitrile and ultrapure water, and activated, eluted, and eluted sequentially. The eluent was then subjected to nitrogen blowing at 35-40°C. Next, the nitrogen-blown product was reconstituted using the mixed standard working solution to prepare mixed matrix standard working solutions with different concentration gradients. LC-MS / MS detection was then performed, and linear regression was conducted using the peak area and mass concentration of the target ionic liquid quantitative ion pairs to obtain the linear regression equations for each ionic liquid. The curves corresponding to each linear regression equation are the standard curves for the corresponding ionic liquids.

[0068] In some embodiments of the present invention, the structural formulas of the various imidazole ionic liquid compounds are as follows:

[0069]

[0070] In the above structural formulas, R represents ethyl, propyl, butyl...dodecyl, and the corresponding imidazole ionic liquid compounds are 1-ethyl-3-methylimidazolium (C2MIM), 1-propyl-3-methylimidazolium (C3MIM), 1-butyl-3-methylimidazolium (C4MIM), 1-pentyl-3-methylimidazolium (C5MIM), 1-hexyl-3-methylimidazolium (C6MIM), 1-heptyl-3-methylimidazolium (C7MIM), 1-octyl-3-methylimidazolium (C8MIM), 1-nonyl-3-methylimidazolium (C9MIM), 1-decyl-3-methylimidazolium (C9MIM), and 1-decyl-3-methylimidazolium (C9MIM). 10 MIM), 1-dodecyl-3-methylimidazole (C 12 MIM).

[0071] Accordingly, step S10 requires the preparation of stock solutions, mixed standard solutions, and mixed standard working solutions for the aforementioned 10 imidazole ionic liquid compounds. The detection method provided by this invention can effectively detect at least the aforementioned 10 imidazole ionic liquids.

[0072] In some embodiments of the present invention, the concentration range of the mixed standard working solution is 0.5~100 ng / mL, with specific concentration gradients of 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, and 100 ng / mL; the concentration range of the mixed matrix standard working solution is 0.5~100 ng / mL, with specific concentration gradients of 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, and 100 ng / mL. The method provided by the present invention can effectively reduce matrix effects by plotting a standard working curve matched to the matrix, enabling quantitative detection that is scientifically accurate and can achieve efficient detection within the range of 0.5~100 ng / mL. The linear correlation coefficient R of the regression equation involving the standard curves of the above 10 imidazole ionic liquid target matrix compounds is also relevant. 2 All values ​​were greater than 0.99. The recoveries of 10 imidazole ionic liquid targets in serum at spiking concentrations of 10 ng / mL, 50 ng / mL, and 100 ng / mL ranged from 79% to 118%. This method can accurately quantify ionic liquid targets in serum and has the advantages of high sensitivity, simple operation, speed, accuracy, and wide applicability. It can be used for the detection and analysis of large batches of samples and can provide methodological support for the exposure and risk assessment of pollutants to the population.

[0073] In some embodiments of the present invention, solution a is an aqueous solution of ammonium formate with a concentration of 10 mmol / L, and a mixed solution of ammonium formate and acetonitrile is used as a solvent, resulting in a high response.

[0074] Then, the human serum sample to be tested is pretreated. The specific pretreatment method includes: adding phosphate-acetonitrile solution (phosphate concentration of 4% by volume) to the serum sample to be tested and letting it stand overnight to precipitate proteins, which can effectively remove protein substances in the serum and prevent them from clogging the chromatographic column. Then, centrifuge and take the supernatant. Add 5% ammonia water by volume to the supernatant, vortex to mix, and use pH test paper to test and adjust the pH of the solution to 6.8. Then, use ultrapure water to make up to 1 mL to obtain the extract. The extract is placed in a solid phase extraction column and activated, rinsed and eluted in sequence. Then, the eluent is purged with nitrogen at 35~40℃. The solid phase extraction column is activated with 3 mL of acetonitrile and 3 mL of ultrapure water in sequence. During activation, the solution is allowed to drip naturally. When the solution is level with the substance in the solid phase extraction column, the sample can be loaded. When loading the sample, the extract is added to the activated solid phase extraction column and the eluent is discarded.

[0075] In some embodiments of the present invention, the pretreatment step involves adding a phosphoric acid-acetonitrile solution and allowing it to stand overnight at a temperature of 4°C. The centrifugation conditions for the step of centrifuging to obtain the supernatant in the pretreatment step are 15000 r / min for 15 min.

[0076] Next, the extracted sample was analyzed and detected, specifically including: reconstituted the nitrogen-blown product with a mixed solution of acetonitrile and solution a, centrifuged to obtain the supernatant, and then passed the supernatant through a 0.22 mm organic filter membrane for analysis by LC-MS / MS. A standard working curve was established based on the peak area and concentration of the target ion liquid quantitative ion pair. The corresponding sample concentration was obtained by measuring the target substance in the human serum sample, thereby obtaining the content of the target substance in the serum sample.

[0077] This invention employs solid-phase extraction purification to process serum samples, which is rapid and simple, requires no other extraction equipment, is time-efficient, and requires little solvent.

[0078] In some embodiments of the present invention, the solid phase extraction column is a mixed cation exchange column, specifically a 3 mL (waters Oasis WCX) extraction column.

[0079] In some embodiments of the present invention, in the pretreatment step: the solvent used for activation is acetonitrile and ultrapure water.

[0080] In some embodiments of the present invention, in the pretreatment step, the rinsing agent used is ammonia and acetonitrile. Preferably, after rinsing, the rinsing solution is dried by vacuum extraction before adding eluent for elution.

[0081] In this invention, the pretreatment for solid-phase extraction was optimized. The extraction efficiency of different eluents for ILs was investigated by comparing different eluent types, and the optimal eluent was selected. In some embodiments of this invention, the pretreatment step involves using a mixed solution of formic acid and acetonitrile as the eluent.

[0082] In some embodiments of the present invention, in step S40: the centrifugation conditions are 0°C, 15000 rpm, and 15 min.

[0083] Furthermore, in some embodiments of the present invention, the detection parameter conditions for analysis using LC-MS / MS are as follows:

[0084] Column: ACQUITY TM Premier BEH C18 column, 100mm × 2.1mm, 1.7μm;

[0085] Column temperature: 35℃, sample chamber temperature: 20℃;

[0086] Injection volume: 5 μL;

[0087] Mobile phase A: Ammonium formate aqueous solution, ammonium acetate aqueous solution, or formic acid aqueous solution;

[0088] Mobile phase B: Acetonitrile;

[0089] Flow rate: 0.3 mL / min;

[0090] Gradient elution, the procedure of which is shown in Table 1 below:

[0091] Table 1 Gradient elution program

[0092]

[0093] Mass spectrometry conditions: ion source was electrospray ionization source in positive ion mode, scanning mode was multiple reaction monitoring mode, ionization voltage was 5500V, temperature was 500℃, spray gas was 40psi, auxiliary heating gas was 40psi, and curtain gas was 40psi.

[0094] Furthermore, in some embodiments of the present invention, the mobile phase A is preferably an aqueous solution of ammonium formate with a concentration of 10 mmol / L, resulting in a better peak pattern and higher response. Correspondingly, the solvent used for resolution in step S40 is a mixed solution of ammonium formate and acetonitrile, wherein the concentration of the ammonium formate aqueous solution is 10 mmol / L, and the volume ratio of ammonium formate aqueous solution to acetonitrile is 8:2.

[0095] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0096] Example 1

[0097] (1) Preparation of mixed matrix standard solutions and determination of retention time:

[0098] Accurately weigh 0.01 g of 10 imidazole ionic liquid standards into a 15 mL brown glass bottle, dissolve in 10 mL of ultrapure water to prepare an ionic liquid standard with a concentration of 100 mg / mL, which will be used as the stock solution. Take the same volume of the 10 ionic liquid standards, mix them to prepare a mixed standard solution, and then use a mixed solution of acetonitrile and ammonium formate aqueous solution (the concentration of ammonium formate aqueous solution is 10 mmol / L, and the volume ratio of acetonitrile to ammonium formate aqueous solution is 2:8) as the solvent to dilute the mixed standard solution into mixed standard working solutions with concentration gradients of 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, and 100 ng / mL.

[0099] Prepare single standard working solutions with a concentration of 10 ng / mL using the above-mentioned mother solutions. First, perform a mother ion scan by LC-MS / MS to determine the monitor ion and quantitative ion pair of each ionic liquid. Then, determine the retention time of each ionic liquid by LC-MS / MS detection.

[0100] Eight blank matrix serum samples without ionic liquids were pretreated as follows: Phosphoric acid-acetonitrile solution (4% phosphate concentration) was added to the blank matrix serum samples, and the mixture was allowed to stand overnight. The supernatant was then centrifuged, and 5% ammonia solution was added to the supernatant to adjust the pH to 6.8. The solution was then diluted to 1 mL with ultrapure water to obtain the extract. The extract was placed in a solid-phase extraction column activated sequentially with acetonitrile and ultrapure water, and activated, eluted, and eluted sequentially. The eluent was then subjected to nitrogen blowing at 35–40 °C. Next, the nitrogen-blown product was reconstituted using the prepared mixed standard working solution to prepare eight mixed matrix standard working solutions with concentration gradients of 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, and 100 ng / mL.

[0101] (2) Plot the matrix standard curve:

[0102] A specific set of parent ions and daughter ions were selected as quantitative ions, and a specific set of parent ions and daughter ions were selected as qualitative ions. The retention time, parent ion, daughter ion, fragmentation voltage, collision energy and other parameters of 10 imidazole ionic liquid compounds are shown in Table 2 below (where * indicates the quantitative ions selected for quantitative ion analysis).

[0103] Table 2 Ion Pair Information Table

[0104]

[0105] A standard working curve was established using the peak area of ​​the target ionic liquid quantitative ion pair and the mass concentration of the target ionic liquid. The regression equation and correlation coefficient of the ionic liquid standard curve were obtained. The limit of detection was set at the mass concentration corresponding to a signal-to-noise ratio (SNR) of 3 for the chromatographic peak of the analyte ionic liquid compound in the spiked sample, and the limit of quantitation was set at the mass concentration corresponding to a SNR of 10. The linear range and correlation coefficient (R²) of the ionic liquid standard curve were then determined. 2 The detection range is 0.5–100 ng / mL, the limit of detection is 0.001 ng / mL, and the limit of quantitation is 0.355 ng / mL. The matrix standard curves for the 10 target analytes are shown in Table 3 below.

[0106] Table 3. Matrix standard curves for 310 target substances

[0107]

[0108] As can be seen from Table 3, the method provided by this invention has a wide linear range, good correlation, and low detection and quantitation limits for detecting imidazole ionic liquids, indicating that the method has high sensitivity and stability.

[0109] Figure 2 Figure 3 shows the chromatograms of 10 imidazole ionic liquid standards.

[0110] (3) Serum sample pretreatment:

[0111] Transfer 200 μL of human serum sample (sampled from Hubei CDC) into a 1.5 mL EP tube. Add 10 μL of mixed standard working solution with concentrations of 10 ng / mL, 50 ng / mL, and 100 ng / mL to the serum sample, respectively. Add phosphate-acetonitrile solution (phosphate concentration of 4%) and let stand overnight at 4℃. Then centrifuge at 15000 r / min for 15 min. Take the supernatant, add 5% ammonia water (v / v), vortex to mix, and use pH test paper to determine that the pH of the supernatant is 6.8. Then add ultrapure water to make up to 1 mL, vortex, and obtain the extract to be extracted.

[0112] (4) Solid-phase extraction and instrumental testing:

[0113] Extraction was performed using a 3 mL Waters Oasis PRIME WCX extraction column. The extraction procedure was as follows:

[0114] Activation: 3 mL acetonitrile, 3 mL ultrapure water, sample loading;

[0115] Rinsing: 3 mL 5% ammonia solution, 3 mL acetonitrile;

[0116] Elution: Elute with 6 mL of 4% formic acid-acetonitrile solution; evaporate the eluent under nitrogen at 40°C until nearly dry.

[0117] Reconstitution: 200 μL of solvent (a mixed solution of acetonitrile and 10 mmol / L ammonium formate aqueous solution, with a volume ratio of acetonitrile to ammonium formate aqueous solution of 2:8).

[0118] The reconstituted solution was centrifuged at 15000 r / min for 15 min at 0℃. The supernatant was then passed through a 0.22 mm organic filter membrane and analyzed by LC-MS / MS under the following conditions:

[0119] Column: ACQUITY TM Premier BEH C18 column, 100mm × 2.1mm, 1.7μm;

[0120] Column temperature: 35℃, sample chamber temperature: 20℃;

[0121] Injection volume: 5 μL;

[0122] Mobile phase A: Ammonium formate aqueous solution;

[0123] Mobile phase B: Acetonitrile;

[0124] Flow rate: 0.3 mL / min;

[0125] Gradient elution, the gradient elution run time is 11 minutes, and the specific procedure is shown in Table 1 above:

[0126] Mass spectrometry conditions: Ion source was electrospray ionization source in positive ion mode, scanning mode was multiple reaction monitoring mode, ionization voltage was 5500V, temperature was 500℃, spray gas (GS1) was 40psi, auxiliary heating gas (GS2) was 40psi, and curtain gas (CUR) was 40psi.

[0127] The instrumental analysis was repeated three times, and the average recovery rate and relative standard deviation for each spiked level were calculated. The results are as follows: Figure 4 As shown. By Figure 4As can be seen, by adding mixed standard working solution to human serum samples, the average recovery rate of this method at the spiking level was 82-113%, and the RSD (relative standard deviation) was less than 10.44%. This indicates that the method provided by the present invention has good recovery effect, high sensitivity, can meet the quantitative detection requirements of ionic liquids in serum samples, has good separation effect on spiked serum samples, good response, and no interference from other impurities.

[0128] Table 4 shows the precision of the imidazole ionic liquid detection method of the present invention:

[0129] Table 4 Precision of the Imidazole Ionic Liquid Detection Method

[0130]

[0131] As shown in Table 5, the same sample was tested at six different time points within one day, and the monitoring was conducted continuously for six days. The RSD of the intraday and interday variations was calculated to assess the stability of the method. As shown in the table above, the intraday precision of the ionic liquid detection method was 6.99%, and the interday precision was 7.46%, indicating that the method provided by this invention has good stability and meets the requirements of quantitative analysis.

[0132] Example 2

[0133] Human serum samples from the Hubei Provincial Center for Disease Control and Prevention were selected as the analysis object, and the method provided in this invention was used for detection and analysis. The analysis results are as follows:

[0134] After testing actual human serum samples, the detection value of C8MIM was relatively high, at 1.7 ng / ml, while other values ​​were not detected.

[0135] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. A method for detecting multiple alkylimidazolium ionic liquids in serum based on liquid chromatography-mass spectrometry (LC-MS), characterized in that, Includes the following steps: Multiple imidazole ionic liquid compounds were respectively prepared into multiple ionic liquid standards with a concentration of 100 mg / mL as stock solutions. The stock solutions were then mixed to prepare mixed standard solutions, and diluted with a mixed solution of acetonitrile and solution a as solvent to prepare mixed standard working solutions with different concentration gradients. Solution a includes any one of ammonium formate aqueous solution, ammonium acetate aqueous solution, and formic acid aqueous solution, and the volume ratio of acetonitrile to solution a is 2:

8. Each of the mother solutions was prepared into a single standard working solution with a concentration of 10 ng / mL. First, the mother ion was scanned by LC-MS / MS to determine the monitoring ion and quantitative ion pair of each ionic liquid. Then, the retention time of each ionic liquid was determined by LC-MS / MS detection. A blank matrix serum sample without ionic liquid was pretreated, and then reconstituted with the mixed standard working solution to prepare mixed matrix standard working solutions with different concentration gradients. LC-MS / MS detection was then performed, and linear regression was performed on the peak area and mass concentration of the target ionic liquid quantitative ion pair to obtain the linear regression equation and standard curve for each ionic liquid. The human serum samples to be tested were pretreated, then reconstituted with a mixture of acetonitrile and solution a, centrifuged to obtain the supernatant, and the supernatant was quantitatively analyzed by LC-MS / MS. The preprocessing steps include: Add phosphoric acid-acetonitrile solution to the sample to be treated, let stand overnight, centrifuge and take the supernatant. Add 5% ammonia solution to the supernatant to adjust the pH of the solution to 6.8, and then use ultrapure water to make up to 1 mL to obtain the extract. The extractant is placed in a solid-phase extraction column and activated, rinsed, and eluted sequentially. The eluent is then purged with nitrogen at 35-40°C. The solid-phase extraction column is activated sequentially with acetonitrile and ultrapure water.

2. The method for detecting multiple alkylimidazolium ionic liquids in serum based on liquid chromatography-mass spectrometry as described in claim 1, characterized in that, The concentration gradients of the mixed standard working solutions are 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL and 100 ng / mL, respectively. The concentration gradients of the mixed matrix standard working solutions are 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL and 100 ng / mL.

3. The method for detecting multiple alkylimidazolium ionic liquids in serum based on liquid chromatography-mass spectrometry as described in claim 1, characterized in that, Solution a is an aqueous solution of ammonium formate with a concentration of 10 mmol / L.

4. The method for detecting multiple alkylimidazolium ionic liquids in serum based on liquid chromatography-mass spectrometry as described in claim 1, characterized in that, In the process of pretreating the human serum sample to be tested, then reconstituted it with a mixture of acetonitrile and solution a, and centrifuging to obtain the supernatant: The centrifugation conditions were 0℃, 15000rpm, and 15min.

5. The method for detecting multiple alkylimidazolium ionic liquids in serum based on liquid chromatography-mass spectrometry as described in claim 1, characterized in that, In the preprocessing steps: The overnight settling temperature is 4°C; and / or, The centrifugation conditions were 15000 r / min for 15 min.

6. The method for detecting multiple alkylimidazolium ionic liquids in serum based on liquid chromatography-mass spectrometry as described in claim 1, characterized in that, The solid-phase extraction column used was a Waters Oasis WCX 3mL extraction column.

7. The method for detecting multiple alkylimidazolium ionic liquids in serum based on liquid chromatography-mass spectrometry as described in claim 1, characterized in that, In the preprocessing steps: The solvent used for activation is acetonitrile and ultrapure water; and / or, The rinsing agent used in the rinsing is ammonia and acetonitrile; and / or, The eluent used for elution is a mixed solution of formic acid and acetonitrile; and / or, When the solid-phase extraction column is activated sequentially with acetonitrile and ultrapure water, the solution is allowed to drip naturally until it is level with the substance in the solid-phase extraction column. Then, the sample can be loaded. When loading the sample, the extractable is added to the activated solid-phase extraction column and the eluent is discarded.

8. The method for detecting multiple alkylimidazolium ionic liquids in serum based on liquid chromatography-mass spectrometry as described in claim 1, characterized in that, The detection parameter conditions for analysis using LC-MS / MS are as follows: Column: ACQUITY TM Premier BEH C18 column, 100mm × 2.1mm, 1.7μm; Column temperature: 35℃, sample chamber temperature: 20℃; Injection volume: 5 μL; Mobile phase A: Ammonium formate aqueous solution, ammonium acetate aqueous solution, or formic acid aqueous solution; Mobile phase B: Acetonitrile; Flow rate: 0.3 mL / min; Gradient elution, the procedure for which is as follows: 0min, 0.3mL / min, A 80%, B 20%; 1.0min, 0.3mL / min, A 80%, B 20%; 1.5min, 0.3mL / min, A 60%, B 40%; 3min, 0.3mL / min, A 60%, B 40%; 8min, 0.3mL / min, A 5%, B 95%; 10min, 0.3mL / min, A 5%, B 95%; 10.1min, 0.3mL / min, A 80%, B 20%; 11min, 0.3mL / min, A 80%, B 20%; Mass spectrometry conditions: ion source was electrospray ionization source in positive ion mode, scanning mode was multiple reaction monitoring mode, ionization voltage was 5500V, temperature was 500℃, spray gas was 40psi, auxiliary heating gas was 40psi, and curtain gas was 40psi.

9. The method for detecting multiple alkylimidazolium ionic liquids in serum based on liquid chromatography-mass spectrometry as described in claim 8, characterized in that, The mobile phase A is an aqueous solution of ammonium formate, and the concentration of the aqueous solution of ammonium formate is 10 mmol / L.

10. The method for detecting multiple alkylimidazolium ionic liquids in serum based on liquid chromatography-mass spectrometry as described in claim 1, characterized in that, The various imidazole ionic liquid compounds include 1-ethyl-3-methylimidazolium, 1-propyl-3-methylimidazolium, 1-butyl-3-methylimidazolium, 1-pentyl-3-methylimidazolium, 1-hexyl-3-methylimidazolium, 1-heptyl-3-methylimidazolium, 1-octyl-3-methylimidazolium, 1-nonyl-3-methylimidazolium, 1-decyl-3-methylimidazolium, and 1-dodecyl-3-methylimidazolium.

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