A method for the separation and detection of multiple chiral drugs

Abstract

This invention discloses a method for the separation and detection of various chiral drugs, relating to the field of environmental pollutant detection and analysis. The method provided by this invention significantly improves experimental efficiency and reduces experimental costs while ensuring experimental accuracy, sensitivity, and low detection limits by optimizing sample pretreatment methods and parameters of the liquid chromatography-mass spectrometry (LC-MS) instrument. This method is applicable to aquatic and plant environments, including domestic sewage, medical wastewater, wastewater treatment plant effluent and effluent, surface water, and drinking water. It can be used for the separation of six chiral drugs and their transformation product enantiomers, and can also accurately quantify the content of these substances. This invention effectively solves the problems of traditional chiral detection methods requiring different pretreatment steps for different substances and multiple injection needles in the instrument.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of environmental pollutant detection and analysis, and in particular to a method for resolving and detecting multiple chiral drugs. BACKGROUND

[0002] In recent years, the production and consumption of drugs have shown a growing trend. Due to the incomplete metabolism of the human body and the incomplete removal of sewage treatment plants, drug pollutants have been widely detected in different types of water bodies, including domestic sewage, medical waste, sewage treatment plant influent and effluent, surface water and drinking water, etc., with concentrations ranging from ng / L to pg / L. Plants in contact with water bodies, including wetland plants or crops, can also absorb and transfer drug pollutants through cell membrane diffusion and transpiration. At present, at least 50% of the drugs on the market are chiral. Chiral drugs are prone to enantioselective metabolic transformation in the body, leading to changes in the enantiomeric fraction in the environment, but only a small number of studies have reported on the enantiomers of chiral drugs in the environment. Chiral drugs often cause abnormalities in reproduction, behavior, metabolism and stress response of non-target organisms, especially aquatic species, and their ecological toxicity is enantioselective, for example, the toxicity of S-fluoxetine to blackhead minnow is 9.4 times that of R-fluoxetine in terms of effective concentration for inhibiting 10% growth; the toxicity of R-venlafaxine and its metabolite (R-O-desmethylvenlafaxine) to freshwater algae Spiny Scenedesmus is greater than that of the corresponding S-enantiomer. Therefore, in the comprehensive ecological toxicity evaluation and risk assessment, different enantiomers of chiral drugs should be regarded as separate environmental pollutants.

[0003] Effective monitoring of chiral drug enantiomers is of great significance in exploring their environmental behavior and elimination mechanisms, and can also provide a more comprehensive and accurate basis for the risk of drug enantiomer residues in environmental media. However, the concentration of chiral drugs in environmental media is very low, and there is usually complex matrix interference in the sample, which affects the detection results. In addition, there are many types of chiral drugs with great differences in properties, and traditional detection methods need to design different pretreatment steps for different substances, and multiple needle injections are required in the instrument due to the difference in mobile phase conditions. This series of problems limits the popularization and application of chiral drug detection methods. Therefore, it is necessary to develop a detection method for chiral drugs in different environmental media at the enantiomer level, so as to achieve the purpose of resolving and quantifying more chiral drugs. SUMMARY

[0004] In view of the above problems in the prior art, the present application provides a method for resolving and detecting multiple chiral drugs, which improves the experimental efficiency, reduces the experimental cost, and effectively solves the problem that traditional chiral detection methods need to design different pretreatment steps for different substances and multiple needle injections in the instrument.

[0005] To achieve the above object, the technical scheme adopted by the present application to solve its technical problems is to provide a method for detecting resolution of multiple chiral drugs, comprising the following steps:

[0006] S1, adding working solvent and four isotopic internal standard standards to six chiral drug racemate standards to obtain a racemic drug mixed working solution;

[0007] S2, using liquid chromatography tandem mass spectrometry to analyze the racemic drug mixed working solution and the test sample liquid obtained in step S1, and resolving the racemic drug in positive ion mode;

[0008] S3, using liquid chromatography-triple quadrupole tandem mass spectrometry to analyze the racemic drug mixed working solution and the test sample liquid obtained in step S1, and determining the residual amount of the chiral enantiomer of the intermediate drug in multiple reaction monitoring positive ion mode.

[0009] Further, in step S1, the chiral drugs are salbutamol, atenolol, fluoxetine, citalopram, venlafaxine and O-desmethylvenlafaxine.

[0010] Further, the mass ratio of salbutamol, atenolol, fluoxetine, citalopram, venlafaxine and O-desmethylvenlafaxine is 1:1:1:1:1:1.

[0011] Further, the isotopic internal standard is salbutamol-D3, atenolol-D7, fluoxetine-D5 and venlafaxine-D6.

[0012] Further, the mass ratio of salbutamol-D3, atenolol-D7, fluoxetine-D5 and venlafaxine-D6 is 1:1:1:1.

[0013] Further, the gradient concentration of the racemate standard of the chiral drug in the racemic drug mixed working solution is 1, 5, 10, 25, 50, 100 and 500 μg / L.

[0014] Further, the concentration of the isotopic internal standard in the racemic drug mixed working solution is 10 μg / L.

[0015] Further, in step S1, when the test sample liquid is a water sample liquid, the working solvent is methanol.

[0016] Further, in step S1, when the test sample solution is a plant sample solution, the working solvent is prepared by the following method: freeze-dry the plant without detectable chiral drugs, add acetonitrile to the obtained freeze-dried powder, vortex, sonicate in an ice bath, freeze-centrifuge, and then take the supernatant and repeat twice. Collect the supernatant and concentrate it with nitrogen until it is just dried. After redissolving the residue with methanol, add ultrapure water and mix. Adjust the pH value, and pass the resulting mixture through an activated Oasis HLB solid-phase extraction column. After the sample has completely passed through the packing, add ultrapure water to rinse. After drying the packing under negative pressure, add methanol to elute, and filter the eluent to obtain the working solvent.

[0017] Further, in step S1, when the test sample solution is a plant sample solution, the working solvent is prepared by the following method: Plant samples for which the target compound was not detected are placed into a 50 mL centrifuge tube, and then freeze-dried at 20 Pa for 48 h using a vacuum freeze dryer. During freeze-drying, the centrifuge tube opening is covered with a layer of plastic wrap, and a small hole is punctured with a needle to facilitate moisture evaporation. 0.5-0.6 g of freeze-dried plant powder is weighed, and 10-11 mL of acetonitrile is added. The mixture is vortexed at 2500-2600 rpm for 3-4 min, and then... Sonicate in an ice bath for 10-11 min, then centrifuge at 4000-4500 rpm for 10-11 min. Collect the supernatant, repeating twice. Concentrate the supernatant under nitrogen until just dry. Redissolve the residue in 1-2 mL of methanol, add 9-10 mL of ultrapure water, and adjust the pH to 3.5-4.5 with 0.9-1 mmol / L hydrochloric acid or sodium hydroxide solution. Pass the resulting mixture through activated Oasis solution at a rate of 2-2.1 mL / min. HLB solid-phase extraction column (activated sequentially by passing through 5-6 mL of methanol, 5-6 mL of ultrapure water at pH 7, and 5-6 mL of ultrapure water at pH 3.5-4.5). After the sample has completely passed through the packing material, 5-6 mL of ultrapure water at pH 3.5-4.5 is added for rinsing. The packing material is then dried under negative pressure for 10-11 min, followed by elution with 5-6 mL of methanol. The eluent is then filtered through a 0.22-0.23 μm PTFE membrane to obtain the working solvent.

[0018] Further, in step S1, when the test sample solution is a plant sample solution, the working solvent is prepared by the following method: Plant samples from which the target compound was not detected are placed into a 50 mL centrifuge tube, then freeze-dried at 20 Pa for 48 h using a vacuum freeze dryer. During freeze-drying, the centrifuge tube opening is covered with a layer of plastic wrap and a small hole is punctured with a needle to facilitate water evaporation. 0.5 g of freeze-dried plant powder is weighed, 10 mL of acetonitrile is added, and the mixture is vortexed at 2500 rpm for 3 min, followed by sonication in an ice bath for 10 min, and then freeze-centrifuged at 4000 rpm for 10 min. The supernatant is collected, and this process is repeated twice. The supernatant is collected and concentrated with nitrogen until just dry. The residue is redissolved in 1 mL of methanol, mixed with 9 mL of ultrapure water, and the pH is adjusted to 4 with 1 mmol / L hydrochloric acid or sodium hydroxide solution. The resulting mixture is passed at a rate of 2 mL / min through an activated Oasis HLB solid-phase extraction column (passed sequentially through 5 mL of methanol, 5 mL of ultrapure water at pH 7, and 5 mL of sodium hydroxide solution). (Activation was performed using ultrapure water at pH 4). After the sample had completely passed through the packing material, 5 mL of ultrapure water at pH 4 was added for rinsing. The packing material was then dried under negative pressure for 10 min, followed by the addition of 5 mL of methanol for elution. The eluent was then filtered through a 0.22 μm PTFE membrane to obtain the working solvent.

[0019] Furthermore, in steps S2 and S3, the test sample solution is either an aqueous sample solution or a plant sample solution.

[0020] Furthermore, the method for preparing the water sample solution specifically includes the following steps: filtering the water sample, adding an isotope internal standard, adjusting the pH value, and then passing the sample through an activated Oasis HLB solid-phase extraction column. After the sample has completely passed through the packing material, ultrapure water is added for rinsing. The packing material is dried under negative pressure, and methanol is added to elute the target substance. The eluent is concentrated by nitrogen blowing until it is just dried. The volume is adjusted with methanol, vortexed, and then filtered to obtain the water sample solution.

[0021] Furthermore, filtration is performed using a 0.45-0.5μm filter membrane.

[0022] Furthermore, filtration is performed using a 0.45μm filter membrane.

[0023] Furthermore, the volume-to-mass ratio of the water sample solution to the isotope internal standard was 200-210 mL: 10-11 ng.

[0024] Furthermore, the volume-to-mass ratio of the water sample solution to the isotope internal standard was 200 mL: 10 ng.

[0025] Further, the pH value was adjusted to 4 with a 0.9-1 mmol / L hydrochloric acid or sodium hydroxide solution, and the sample was passed through an activated Oasis HLB solid-phase extraction column at a rate of 2-2.1 mL / min. After the sample had completely passed through the packing material, 5-6 mL of ultrapure water with a pH of 4 was added for rinsing. The packing material was dried under negative pressure for 10-11 min, and then 5-6 mL of methanol was added to elute the target analyte. The eluent was concentrated by nitrogen blowing until it was just dried, and then brought to a final volume of 1 mL with methanol. After vortexing, the solution was filtered through a 0.22-0.23 μm PTFE membrane to obtain the aqueous sample solution.

[0026] Further, the pH was adjusted to 4 with a 1 mmol / L hydrochloric acid or sodium hydroxide solution, and the sample was passed through an activated Oasis HLB solid-phase extraction column at a rate of 2 mL / min. After the sample had completely passed through the packing material, 5 mL of ultrapure water at pH 4 was added for rinsing. The packing material was dried under negative pressure for 10 min, and then 5 mL of methanol was added to elute the target analyte. The eluent was concentrated by nitrogen blowing until it was just dried, and then brought to a final volume of 1 mL with methanol. After vortexing, the solution was filtered through a 0.22 μm PTFE membrane to obtain the aqueous sample solution.

[0027] Further, the cells were activated sequentially with 5 mL of methanol, 5 mL of ultrapure water with pH 7, and 5 mL of ultrapure water with pH 4.

[0028] Furthermore, the preparation method of the plant sample solution specifically includes the following steps: freeze-drying the plant, adding an isotope internal standard to the obtained freeze-dried powder, adding acetonitrile, vortexing, sonicating in an ice bath, centrifuging at freeze, and collecting the supernatant. This process is repeated twice, and the supernatant is collected and concentrated with nitrogen until just dry. The residue is redissolved in methanol, mixed with ultrapure water, and the pH value is adjusted. The resulting mixture is passed through an activated Oasis HLB solid-phase extraction column. After the sample has completely passed through the packing material, ultrapure water is added for rinsing. The packing material is dried under negative pressure, and methanol is added to elute the target analyte. The eluent is concentrated with nitrogen until just dry, brought to a final volume with methanol, vortexed, and filtered to obtain the plant sample solution.

[0029] Further, the plant was transferred to a 50mL centrifuge tube and then freeze-dried in a vacuum freeze dryer at a pressure of 20-25Pa for 48-50 hours. During freeze-drying, the centrifuge tube opening was covered with a layer of plastic wrap and small holes were poked with a needle to facilitate moisture evaporation.

[0030] Further, the plant was transferred to a 50mL centrifuge tube and then freeze-dried in a vacuum freeze dryer at a pressure of 20Pa for 48 hours. During freeze-drying, the opening of the centrifuge tube was covered with a layer of plastic wrap and small holes were poked with a needle to facilitate the evaporation of moisture.

[0031] Further, weigh 0.5-0.6 g of lyophilized plant powder, add 10 ng each of isotope internal standards (salbutamol-D3, atenolol-D7, fluoxetine-D5, and venlafaxine-D6), add 10-11 mL of acetonitrile, vortex at 2500-2600 rpm for 3-4 min, then sonicate on ice for 10-11 min, and centrifuge at 4000-4500 rpm for 10-11 min. Collect the supernatant, repeat twice, and concentrate under nitrogen until just dry. Redissolve the residue in 1-2 mL of methanol, add 9-10 mL of ultrapure water, and adjust the pH to 3.5-4.5 with 0.9-1 mmol / L hydrochloric acid or sodium hydroxide solution. Pass the resulting mixture at a rate of 2-2.1 mL / min through an activated Oasis HLB solid-phase extraction column (passing sequentially through 5-6 mL of methanol, 5-6 mL of ultrapure water at pH 7, and 5-6 mL of...). (Activation with ultrapure water at pH 3.5-4.5) After the sample has completely passed through the packing material, add 5-6 mL of ultrapure water at pH 3.5-4.5 to rinse. Under negative pressure, dry the packing material for 10-11 min, then add 5-6 mL of methanol to elute the target analyte. Concentrate the eluent with nitrogen until it is just dry. Make up the volume of the residue to 1 mL with methanol. After vortexing, filter through a 0.22-0.23 μm PTFE membrane to obtain the plant sample solution.

[0032] Further, weigh 0.5 g of lyophilized plant powder, add 10 ng each of isotope internal standards (salbutamol-D3, atenolol-D7, fluoxetine-D5, and venlafaxine-D6), add 10 mL of acetonitrile, vortex at 2500 rpm for 3 min, then sonicate on ice for 10 min, and centrifuge at 4000 rpm for 10 min. Collect the supernatant, repeat twice, collect the supernatant, and concentrate it under nitrogen until just dry. Redissolve the residue in 1 mL of methanol, add 9 mL of ultrapure water, and adjust the pH to 4 with 1 mmol / L hydrochloric acid or sodium hydroxide solution. Pass the resulting mixture through an activated Oasis HLB solid-phase extraction column at a rate of 2 mL / min (activated sequentially by passing through 5 mL of methanol, 5 mL of ultrapure water at pH 7, and 5 mL of ultrapure water at pH 4). After the sample has completely passed through the packing material, add 5 mL of... The sample was rinsed with ultrapure water at pH 4, and the packing material was dried under negative pressure for 10 min. Then, 5 mL of methanol was added to elute the target analyte. The eluent was concentrated by nitrogen blowing until it was just dried. The residue was brought to a final volume of 1 mL with methanol, vortexed, and then filtered through a 0.22 μm PTFE membrane to obtain the plant sample solution.

[0033] Further, in steps S2 and S3, the liquid chromatography uses a CHIROBIOTIC V column and a CHIROBIOTIC V guard column. The column has dimensions of 250 × 2.1 mm and 5 μm, and the guard column has dimensions of 20 × 4 mm and 5 μm. Silica gel coated with vancomycin is used as the chiral stationary phase. Mobile phase A is ultrapure water containing 0.005-0.006 vt% formic acid and 4-5 mM ammonium formate, and mobile phase B is methanol containing 0.005-0.006 vt% formic acid. The volume ratio of mobile phase A is 49-51%, and the volume ratio of mobile phase B is 49-51%.

[0034] Further, in steps S2 and S3, the liquid chromatography uses a CHIROBIOTIC V column and a CHIROBIOTIC V guard column. The column has dimensions of 250 × 2.1 mm and 5 μm, and the guard column has dimensions of 20 × 4 mm and 5 μm. Silica gel coated with vancomycin is used as the chiral stationary phase. Mobile phase A is ultrapure water containing 0.005 vt% formic acid and 4 mM ammonium formate, and mobile phase B is methanol containing 0.005 vt% formic acid. The volume ratio of mobile phase A is 50%, and the volume ratio of mobile phase B is 50%.

[0035] Furthermore, the column temperature of the liquid chromatography was 25-26℃, the flow rate was 0.1-0.15mL / min, the injection volume was 20-21μL, and the elution method was isocratic elution.

[0036] Furthermore, the column temperature of the liquid chromatography was 25℃, the flow rate was 0.15mL / min, the injection volume was 20μL, and the elution method was isocratic elution.

[0037] Furthermore, in steps S2 and S3, the MS / MS detection mode used in the mass spectrometry analysis is multiple reaction monitoring (MRM); the electrospray ionization source is in positive ion mode with a voltage of 4500-5000V; the nebulizer gas flow rate is 3-4L / min; the heating gas flow rate is 10-11L / min; the drying gas flow rate is 10-11L / min; the interface temperature is 300-350℃; the DL temperature is 250-300℃; and the heating block temperature is 400-450℃.

[0038] Furthermore, in steps S2 and S3, the MS / MS detection mode used in the mass spectrometry analysis is multiple reaction monitoring mode (MRM); the electrospray ionization source is in positive ion mode with a voltage of 4500V; the nebulizer gas flow rate is 3L / min; the heating gas flow rate is 10L / min; the drying gas flow rate is 10L / min; the interface temperature is 300℃; the DL temperature is 250℃; and the heating block temperature is 400℃.

[0039] The above-mentioned methods for the separation and detection of various chiral drugs are applied to the quality detection of drug enantiomers.

[0040] The present invention has the following beneficial effects:

[0041] 1. This invention achieves the separation and quantification of enantiomeric substances of five common drug active substances and one metabolite from three major classes (anti-asthmatic drugs, β-blockers and antidepressants) with a single injection by optimizing sample pretreatment design and improving liquid chromatography conditions.

[0042] 2. Compared with common methods, this method has greatly improved the number of chiral target substances; at the same time, by optimizing the key steps of preparing racemic drug mixture working solutions and sample processing, and adjusting liquid chromatography conditions and parameters, the method has achieved the goal of reducing detection time and matrix effects in complex media.

[0043] 3. Compared with previous methods, this method can significantly reduce experimental operation costs, including labor costs, instrument usage costs, and reagent costs, while also saving laboratory management costs, including sampling, transportation, and waste disposal. Attached Figure Description

[0044] Figure 1 This is the enantiomeric separation chromatogram of the drug in Example 2.

[0045] Figure 2 Example 3 shows the recovery rate of enantiomeric extraction of drugs from plants. Detailed Implementation

[0046] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0047] Example 1

[0048] A method for the separation and detection of multiple chiral drugs, comprising the following steps:

[0049] S1. Take 20 mg of each of the racemic standard compounds of six chiral drugs: salbutamol, atenolol, fluoxetine, citalopram, venlafaxine, and O-norvenlafaxine. Dilute with methanol, and then add 10 ng of each of the four isotope internal standard compounds: salbutamol-D3, atenolol-D7, fluoxetine-D5, and venlafaxine-D6. This will give you a mixed working solution of racemic drugs. The gradient concentrations of the racemic standard compounds of chiral drugs in the mixed working solution of racemic drugs are 1, 5, 10, 25, 50, 100, and 500 μg / L. The mass concentration of the isotope internal standard in the mixed working solution of racemic drugs is 10 μg / L.

[0050] S2. The racemic drug mixture working solution and the aqueous sample solution obtained in step S1 were analyzed by liquid chromatography-tandem mass spectrometry. The racemic drugs were separated in positive ion mode. The preparation method of the above-mentioned aqueous sample solution is as follows: Take 200 mL of water sample, filter it through a 0.45 μm filter membrane, add 10 ng each of isotope internal standards (salbutamol-D3, atenolol-D7, fluoxetine-D5, and venlafaxine-D), adjust the pH to 4 with 1 mmol / L hydrochloric acid, activate the Oasis HLB solid-phase extraction column (specification 6 mL / 200 mg) sequentially with 5 mL of methanol, 5 mL of ultrapure water at pH 7, and 5 mL of ultrapure water at pH 4, and pass the resulting mixture through the activated solid-phase extraction column at a rate of 2 mL / min. Discard the eluent and add 5 mL of... The sample was rinsed with ultrapure water at pH 4, then the packing material was dried under negative pressure for 10 min. 5 mL of methanol was added to elute the target analyte, and the eluent was concentrated by nitrogen blowing until just dry. The residue was brought to a final volume of 1 mL with methanol, vortexed, and filtered through a 0.22 μm PTFE membrane to obtain the aqueous sample solution. The above liquid chromatography conditions were as follows: a CHIROBIOTIC V column and a CHIROBIOTIC V guard column were used. The column dimensions were 250 mm × 2.1 mm, 5 μm, and the guard column dimensions were 20 × 4 mm, 5 μm. Silica gel coated with vancomycin was used as the chiral stationary phase. Mobile phase A consisted of ultrapure water containing 0.005% formic acid and 4 mM ammonium formate, and mobile phase B consisted of methanol containing 0.005 wt% formic acid. The volume ratio of mobile phase A to mobile phase B was 50%, the flow rate was 0.1 mL / min, and the column temperature was 25 °C. The injection volume was 20 μL. The mass spectrometry conditions were as follows: ion source: electrospray ionization (ESI), positive ion mode voltage 4500 V, detection mode: multiple reaction monitoring (MRM), nebulizer gas flow rate: 3 L / min, heating gas flow rate: 10 L / min, drying gas flow rate: 10 L / min, interface temperature: 300 °C, DL temperature: 250 °C, heating block temperature: 400 °C. The MRM mass spectrometry parameters of the chiral drug and internal standard are shown in Table 1.

[0051] S3, the racemic drug mixture working solution, and the aqueous sample solution were analyzed by liquid chromatography-triple quadrupole tandem mass spectrometry in multiple reaction monitoring positive ion mode to determine the chiral enantiomer residues of the drug. The above liquid chromatography conditions were: CHIROBIOTIC V column and CHIROBIOTIC... The chromatographic column was 250 mm × 2.1 mm, 5 μm, and the guard column was 20 × 4 mm, 5 μm. Silica gel coated with vancomycin was used as the chiral stationary phase. Mobile phase A consisted of ultrapure water containing 0.005 wt% formic acid and 4 mM ammonium formate, and mobile phase B consisted of methanol containing 0.005 wt% formic acid. The volume ratio of mobile phase A to mobile phase B was 50%. The flow rate was 0.1 mL / min, the column temperature was 25 °C, and the injection volume was 20 μL. The mass spectrometry conditions were as follows: ion source: electrospray ionization (ESI), positive ion mode voltage 4500 V, detection mode: multiple reaction monitoring (MRM), nebulizer gas flow rate: 3 L / min, heating gas flow rate: 10 L / min, drying gas flow rate: 10 L / min, interface temperature: 300 °C, DL temperature: 250 °C, and heating block temperature: 400 °C.

[0052] Table 1. MRM mass spectrometry parameters of chiral drugs and internal standards

[0053]

[0054]

[0055] *: Quantitative ions

[0056] Example 2

[0057] A method for the separation and detection of multiple chiral drugs, comprising the following steps:

[0058] S1. Take 20 mg of each of the racemic standard compounds of six chiral drugs: salbutamol, atenolol, fluoxetine, citalopram, venlafaxine, and O-norvenlafaxine. Dilute with methanol, and then add 10 ng of each of the four isotope internal standard compounds: salbutamol-D3, atenolol-D7, fluoxetine-D5, and venlafaxine-D6. This will give you a mixed working solution of racemic drugs. The gradient concentrations of the racemic standard compounds of chiral drugs in the mixed working solution of racemic drugs are 1, 5, 10, 25, 50, 100, and 500 μg / L. The mass concentration of the isotope internal standard in the mixed working solution of racemic drugs is 10 μg / L.

[0059] S2. The racemic drug mixture working solution and the aqueous sample solution obtained in step S1 were analyzed by liquid chromatography-tandem mass spectrometry. The racemic drugs were separated in positive ion mode. The preparation method of the above-mentioned aqueous sample solution is as follows: Take 200 mL of water sample, filter it through a 0.45 μm filter membrane, add 10 ng each of isotope internal standards (salbutamol-D3, atenolol-D7, fluoxetine-D5, and venlafaxine-D), adjust the pH to 4 with 1 mmol / L hydrochloric acid, activate the Oasis HLB solid-phase extraction column (specification 6 mL / 200 mg) sequentially with 5 mL of methanol, 5 mL of ultrapure water at pH 7, and 5 mL of ultrapure water at pH 4, and pass the resulting mixture through the activated solid-phase extraction column at a rate of 2 mL / min. Discard the eluent and add 5 mL of... The sample was rinsed with ultrapure water at pH 4, then the packing material was dried under negative pressure for 10 min. 5 mL of methanol was added to elute the target analyte, and the eluent was concentrated by nitrogen blowing until just dry. The residue was brought to a final volume of 1 mL with methanol, vortexed, and filtered through a 0.22 μm PTFE membrane to obtain the aqueous sample solution. The above liquid chromatography conditions were as follows: a CHIROBIOTIC V column and a CHIROBIOTIC V guard column were used. The column dimensions were 250 mm × 2.1 mm, 5 μm, and the guard column dimensions were 20 × 4 mm, 5 μm. Vancomycin-coated silica gel was used as the chiral stationary phase. Mobile phase A consisted of ultrapure water containing 0.005% formic acid and 4 mM ammonium formate, and mobile phase B consisted of methanol containing 0.005 wt% formic acid. The volume ratio of mobile phase A to mobile phase B was 50%, the flow rate was 0.15 mL / min, and the column temperature was 25 °C. The injection volume was 20 μL. The mass spectrometry conditions were as follows: ion source: electrospray ionization (ESI), positive ion mode voltage 4500 V, detection mode: multiple reaction monitoring (MRM), nebulizer gas flow rate: 3 L / min, heating gas flow rate: 10 L / min, drying gas flow rate: 10 L / min, interface temperature: 300 °C, DL temperature: 250 °C, heating block temperature: 400 °C. The MRM mass spectrometry parameters of the chiral drug and internal standard are shown in Table 1.

[0060] S3, the racemic drug mixture working solution, and the aqueous sample solution were analyzed by liquid chromatography-triple quadrupole tandem mass spectrometry in multiple reaction monitoring positive ion mode to determine the chiral enantiomer residues of the drug. The above liquid chromatography conditions were: CHIROBIOTIC V column and CHIROBIOTIC... The chromatographic column was 250 mm × 2.1 mm, 5 μm, and the guard column was 20 × 4 mm, 5 μm. Silica gel coated with vancomycin was used as the chiral stationary phase. Mobile phase A consisted of ultrapure water containing 0.005 wt% formic acid and 4 mM ammonium formate, and mobile phase B consisted of methanol containing 0.005 wt% formic acid. The volume ratio of mobile phase A to mobile phase B was 50%. The flow rate was 0.15 mL / min, the column temperature was 25 °C, and the injection volume was 20 μL. The mass spectrometry conditions were as follows: ion source: electrospray ionization (ESI), positive ion mode voltage 4500 V, detection mode: multiple reaction monitoring (MRM), nebulizer gas flow rate: 3 L / min, heating gas flow rate: 10 L / min, drying gas flow rate: 10 L / min, interface temperature: 300 °C, DL temperature: 250 °C, and heating block temperature: 400 °C.

[0061] Example 3

[0062] A method for the separation and detection of multiple chiral drugs, comprising the following steps:

[0063] S1. Take 20 mg of each of the racemic standards of six chiral drugs: salbutamol, atenolol, fluoxetine, citalopram, venlafaxine, and O-norvenlafaxine. Dilute with the working solvent, then add 10 ng of each of the four isotope internal standard standards: salbutamol-D3, atenolol-D7, fluoxetine-D5, and venlafaxine-D6. This yields a mixed working solution of racemic drugs. The concentration gradients of the racemic standards of chiral drugs in the mixed working solution are 1, 5, 10, 25, 50, 100, and 500 μg / L. The mass concentration of the isotope internal standard in the mixed working solution is 10 μg / L. The working solvent is prepared by the following method: Place the plant sample containing the undetected target compound into a 50 mL centrifuge tube, then refrigerate under vacuum. The freeze-dried plant powder was freeze-dried at 20 Pa for 48 h. During freeze-drying, the centrifuge tube opening was covered with a layer of plastic wrap and small holes were punctured with a needle to facilitate moisture evaporation. 0.5 g of freeze-dried plant powder was weighed, and 10 ng each of isotope internal standards (salbutamol-D3, atenolol-D7, fluoxetine-D5, and venlafaxine-D6) were added. 10 mL of acetonitrile was added, and the mixture was vortexed at 2500 rpm for 3 min, followed by sonication in an ice bath for 10 min, and then centrifuged at 4000 rpm for 10 min. The supernatant was collected, and the process was repeated twice. The supernatant was collected and concentrated with nitrogen until just dry. The residue was redissolved in 1 mL of methanol, mixed with 9 mL of ultrapure water, and the pH was adjusted to 4 with 1 mmol / L hydrochloric acid. The resulting mixture was then passed through activated Oasis at a rate of 2 mL / min. HLB solid-phase extraction column (activated sequentially by passing through 5 mL of methanol, 5 mL of ultrapure water at pH 7, and 5 mL of ultrapure water at pH 4). After the sample has completely passed through the packing material, 5 mL of ultrapure water at pH 4 is added for rinsing. The packing material is dried under negative pressure for 10 min, and then 5 mL of methanol is added for elution. The eluent is then filtered through a 0.22 μm PTFE filter membrane to obtain the working solvent.

[0064] S2. The racemic drug mixture working solution and plant sample solution obtained in step S1 were analyzed by liquid chromatography-tandem mass spectrometry. The racemic drug was separated in positive ion mode. The preparation method of the above plant sample solution is as follows: the plant was placed into a 50 mL centrifuge tube, and then freeze-dried in a vacuum freeze dryer at 20 Pa pressure for 48 h. During freeze-drying, the centrifuge tube opening was covered with a layer of plastic wrap and a small hole was poked with a needle to facilitate water evaporation. 0.5 g of freeze-dried plant powder was accurately weighed, 10 ng of the target isotope internal standard was added, 10 mL of acetonitrile was added, and the mixture was vortexed at 2500 rpm for 3 minutes. Then, it was sonicated in an ice bath for 10 min, and then frozen and centrifuged at 4000 rpm for 10 min. The supernatant was collected, and the above steps were repeated twice. The supernatants were combined and concentrated with nitrogen until just dry. The residue was redissolved in 1 mL of methanol and mixed with 9 mL of ultrapure water. The pH of the plant sample was adjusted to 4 using 1 mmol / L NaOH. The Oasis HLB solid-phase extraction column (6 mL / 200 mg) was activated sequentially with 5 mL of methanol, 5 mL of ultrapure water at pH 7, and 5 mL of ultrapure water at pH 4. The mixture from the previous step was passed through the activated solid-phase extraction column at a rate of 2 mL / min. The eluent was discarded, and 5 mL of ultrapure water at pH 4 was added for rinsing. The packing material was then dried under negative pressure for 10 min. 5 mL of methanol was added to elute the target analyte, and the eluent was concentrated by nitrogen blowing until just dry. The residue was brought to a final volume of 1 mL with methanol. After vortexing, the solution was filtered through a 0.22 μm PTFE membrane to obtain the plant sample solution. The above liquid chromatography conditions were: CHIROBIOTIC V column and CHIROBIOTIC... V guard column, chromatographic column specifications are 250mm×2.1mm, 5μm, guard column specifications are 20×4mm, 5μm, using silica gel coated with vancomycin as chiral stationary phase, mobile phase A is ultrapure water containing 0.005% formic acid and 4mM ammonium formate, mobile phase B is methanol containing 0.005% formic acid, the volume ratio of mobile phase A is 50%, the volume ratio of mobile phase B is 50%, the flow rate is 0.15mL / min, the column temperature is 25℃, the injection volume is 20μL, the above mass spectrometry conditions are: ion source: electrospray ionization (ESI), positive ion mode voltage 4500V, detection mode: multiple reaction monitoring mode (MRM), nebulizer gas flow rate: 3L / min, heating gas flow rate: 10L / min, drying gas flow rate: 10L / min, interface temperature: 300℃, DL temperature: 250℃, heating block temperature: 400℃;

[0065] S3, the racemic drug mixed standard working solution and the test sample solution were analyzed by liquid chromatography-triple quadrupole tandem mass spectrometry in multiple reaction monitoring positive ion mode to determine the chiral enantiomer residues of the drug. The above liquid chromatography conditions were: CHIROBIOTIC V column and CHIROBIOTIC... V guard column, chromatographic column specifications are 250mm×2.1mm, 5μm, guard column specifications are 20×4mm, 5μm, using silica gel coated with vancomycin as chiral stationary phase, mobile phase A is ultrapure water containing 0.005% formic acid and 4mM ammonium formate, mobile phase B is methanol containing 0.005% formic acid, the volume ratio of mobile phase A is 50%, the volume ratio of mobile phase B is 50%, the flow rate is 0.15mL / min, the column temperature is 25℃, the injection volume is 20μL, the above mass spectrometry conditions are: ion source: electrospray ionization (ESI), positive ion mode voltage 4500V, detection mode: multiple reaction monitoring mode (MRM), nebulizer gas flow rate: 3L / min, heating gas flow rate: 10L / min, drying gas flow rate: 10L / min, interface temperature: 300℃, DL temperature: 250℃, heating block temperature: 400℃.

[0066] Example 4

[0067] A method for the separation and detection of multiple chiral drugs, comprising the following steps:

[0068] S1. Take 20 mg of each of the racemic standard compounds of six chiral drugs: salbutamol, atenolol, fluoxetine, citalopram, venlafaxine, and O-norvenlafaxine. Dilute with methanol, and then add 10 ng of each of the four isotope internal standard compounds: salbutamol-D3, atenolol-D7, fluoxetine-D5, and venlafaxine-D6. This will give you a mixed working solution of racemic drugs. The gradient concentrations of the racemic standard compounds of chiral drugs in the mixed working solution of racemic drugs are 1, 5, 10, 25, 50, 100, and 500 μg / L. The mass concentration of the isotope internal standard in the mixed working solution of racemic drugs is 10 μg / L.

[0069] S2. The racemic drug mixture working solution and the aqueous sample solution obtained in step S1 were analyzed by liquid chromatography-tandem mass spectrometry. The racemic drug was separated in positive ion mode. The preparation method of the above-mentioned aqueous sample solution is as follows: Take 210 mL of water sample, filter it through a 0.5 μm filter membrane, and add 10 ng each of isotope internal standards (salbutamol-D3, atenolol-D7, fluoxetine-D5, and venlafaxine-D6). Adjust the pH to 4 with 0.9 mmol / L hydrochloric acid. Activate the Oasis HLB solid-phase extraction column (6 mL / 200 mg) sequentially with 5 mL of methanol, 5 mL of ultrapure water at pH 7, and 5 mL of ultrapure water at pH 4. Pass the resulting mixture through the activated solid-phase extraction column at a rate of 2.1 mL / min. Discard the eluent and add 5 mL of... The sample was rinsed with ultrapure water at pH 4, then the packing material was dried under negative pressure for 11 min. 5 mL of methanol was added to elute the target analyte, and the eluent was concentrated by nitrogen blowing until just dry. The residue was brought to a final volume of 1 mL with methanol, vortexed, and then filtered through a 0.23 μm PTFE membrane to obtain the aqueous sample solution. The above liquid chromatography conditions were as follows: a CHIROBIOTIC V column and a CHIROBIOTIC... The chromatographic column was 250 mm × 2.1 mm, 5 μm, and the guard column was 20 × 4 mm, 5 μm. Silica gel coated with vancomycin was used as the chiral stationary phase. Mobile phase A was ultrapure water containing 0.005 wt% formic acid and 4 mM ammonium formate, and mobile phase B was methanol containing 0.005 wt% formic acid. The volume ratio of mobile phase A was 49%, and the volume ratio of mobile phase B was 51%. The flow rate was 0.15 mL / min, the column temperature was 26 °C, and the injection volume was 21 μL. The mass spectrometry conditions were as follows: ion source: electrospray ionization (ESI), positive ion mode voltage 5000 V, detection mode: multiple reaction monitoring (MRM), nebulizer gas flow rate: 4 L / min, heating gas flow rate: 11 L / min, drying gas flow rate: 11 L / min, interface temperature: 350 °C, DL temperature: 300 °C, heating block temperature: 450 °C.

[0070] S3, the racemic drug mixture working solution and the test sample solution were analyzed by liquid chromatography-triple quadrupole tandem mass spectrometry in multiple reaction monitoring positive ion mode to determine the chiral enantiomer residues of the drug. The above liquid chromatography conditions were: CHIROBIOTIC V column and CHIROBIOTIC... The chromatographic column was 250 mm × 2.1 mm, 5 μm, and the guard column was 20 × 4 mm, 5 μm. Silica gel coated with vancomycin was used as the chiral stationary phase. Mobile phase A consisted of ultrapure water containing 0.005% formic acid and 4 mM ammonium formate, and mobile phase B consisted of methanol containing 0.005% formic acid. The volume ratio of mobile phase A was 49%, and the volume ratio of mobile phase B was 51%. The flow rate was 0.15 mL / min, the column temperature was 26 °C, and the injection volume was 21 μL. The mass spectrometry conditions were as follows: ion source: electrospray ionization (ESI), positive ion mode voltage 5000 V, detection mode: multiple reaction monitoring (MRM), nebulizer gas flow rate: 4 L / min, heating gas flow rate: 11 L / min, drying gas flow rate: 11 L / min, interface temperature: 350 °C, DL temperature: 300 °C, and heating block temperature: 450 °C.

[0071] Comparative Example 1

[0072] The difference from Example 1 is that in Comparative Example 1, the volume ratio of mobile phase A is 40% and the volume ratio of mobile phase B is 60%.

[0073] Comparative Example 2

[0074] The difference from Example 1 is that in Comparative Example 2, the volume ratio of mobile phase A is 3% and the volume ratio of mobile phase B is 97%.

[0075] Comparative Example 3

[0076] The difference from Example 1 is that in Comparative Example 3, the flow rate was 0.2 mL / min.

[0077] Comparative Example 4

[0078] The difference from Example 2 is that in Comparative Example 4, the volume ratio of mobile phase A is 65% and the volume ratio of mobile phase B is 35%.

[0079] Comparative Example 5

[0080] The difference from Example 2 is that in Comparative Example 5, the pH was adjusted to 7, and the Oasis HLB solid-phase extraction column (6 mL / 200 mg) was activated sequentially with 5 mL of methanol, 5 mL of ultrapure water at pH 7, and 5 mL of ultrapure water at pH 7. The resulting mixture was then passed through the activated solid-phase extraction column at a rate of 2 mL / min. The eluent was discarded, and 5 mL of ultrapure water at pH 7 was added for rinsing.

[0081] Comparative Example 6

[0082] The difference from Example 2 is that in Comparative Example 6, the pH was adjusted to 9, and the Oasis HLB solid-phase extraction column (6 mL / 200 mg) was activated sequentially with 5 mL of methanol, 5 mL of ultrapure water with pH 7, and 5 mL of ultrapure water with pH 9. The resulting mixture was passed through the activated solid-phase extraction column at a rate of 2 mL / min, the eluent was discarded, and 5 mL of ultrapure water with pH 9 was added for rinsing.

[0083] Experimental Example 1

[0084] By optimizing the effects of factors such as the ratio and flow rate of the mobile phase on chromatographic behavior, the resolution was used to examine the chiral separation effect of enantiomers.

[0085] The enantiomer resolution (Rs) is calculated according to the formula shown below.

[0086]

[0087] In the formula, Rs represents the resolution; T1 and T2 represent the retention times of the first and second eluted enantiomers; W1 and W2 represent the peak widths of the first and second eluted enantiomers.

[0088] (1) Separation effect of drug enantiomers under different mobile phase ratios

[0089] Table 2 shows the chiral separation effect of drug enantiomers under different mobile phase ratios in Examples 1, Comparative Examples 1 and 2.

[0090] Table 2. Separation effect of drug enantiomers under different mobile phase ratios.

[0091]

[0092]

[0093] Table 2 shows that mobile phase A is ultrapure water containing 0.005% formic acid and 4 mM ammonium formate, and mobile phase B is methanol containing 0.005% formic acid. With a volume ratio of 50%:50% for mobile phases A and B, under isocratic elution conditions, the resolution of chiral enantiomers of various drugs ranged from 1.58 to 6.31, achieving baseline separation, and exhibiting the highest enantiomer chromatographic peak response values. With a volume ratio of 40%:60% for mobile phases A and B, under isocratic elution conditions, the resolution ranged from 1.16 to 3.84, with baseline separation slightly lower than in Example 1. With a volume ratio of 3%:97% for mobile phases A and B, under isocratic elution conditions, the resolution ranged from 0.04 to 0.85, failing to achieve complete baseline separation of drug enantiomers. Furthermore, within the range of enantiomeric concentrations of 0.5, 2.5, 5, 12.5, 25, 50, and 250 μg / L, the linear relationships between the peak area ratio and the mass concentration ratio of various drug enantiomeric and internal standard enantiomeric chromatographic peaks were good, with correlation coefficients R>0.998.

[0094] (2) Separation effect of drug enantiomers under different flow rates

[0095] Table 3 shows the chiral separation effect of drug enantiomers under different flow rates and isocratic elution conditions in Examples 1, 2, and 3.

[0096] Table 3. Separation effect of drug enantiomers under different flow rates

[0097]

[0098]

[0099] As shown in Table 3, the chiral separation of the drug enantiomers in Example 1 was significantly better than that in Example 2 and Comparative Example 3, but the separation time was too long (72.46-143.34 min). In Comparative Example 3, the resolution of salbutamol was 0.19, which could not achieve complete baseline separation of the enantiomers. In Example 2, the resolution of the chiral drug was 1.14-5.28, which could shorten the separation time while ensuring good baseline separation. Therefore, 0.15 mL / min is the preferred flow rate.

[0100] (3) The effect of different mobile phase ratios on drug enantiomer separation under optimized flow rate conditions

[0101] Table 4 shows the separation effect of different mobile phase ratios on drug enantiomers under preferred flow rate conditions in Examples 2 and Comparative Example 4.

[0102] Table 4. Effects of different mobile phase ratios on drug enantiomer separation under optimal flow rates.

[0103]

[0104]

[0105] As shown in Table 4, under the preferred flow rate conditions, the chiral separation effect of various drug enantiomers in Example 2 is better than that in Comparative Example 4. Therefore, a 50%:50% volume ratio of mobile phase A to B is the preferred mobile phase ratio.

[0106] Experimental Example 2

[0107] The detection method for multiple drug enantiomers constructed in this invention was applied to the separation and residual analysis of multiple drug enantiomers in the mixed influent of a domestic sewage treatment plant in Chongqing over a period of 24 hours.

[0108] Six racemic target drugs were added at 50 ng / L to the influent matrix of a wastewater treatment plant, and three parallel determinations were performed. A blank control experiment was also conducted. Following the steps described in Example 2 and Comparative Examples 5-6, the pH of the solution to be treated was adjusted using hydrochloric acid and sodium hydroxide. The retention and recovery rates of drug enantiomers under different acid and alkaline conditions were investigated. The results are shown in Table 5.

[0109] Table 5. Spike recoveries and relative standard deviations of various drug enantiomers in water samples (n=3)

[0110]

[0111] As shown in Table 5, when the pH of the treatment solution in Example 2 was 4, the retention and recovery rates of the six drug enantiomers were all better than those in Comparative Example 6, which met the experimental requirements. When the pH of the treatment solution in Comparative Example 5 was 7, the retention and recovery rates of fluoxetine and O-desvenlafaxine were both less than 30%, indicating that the neutral treatment solution was not suitable for their retention and adsorption on the column. Therefore, the treatment solution with pH 4 in Example 2 was selected as the optimal loading solution.

[0112] Experimental Example 3

[0113] The method provided in Example 3 was applied to the separation and residue analysis of multiple drug enantiomers in *Gnaphalium affine* planted in an artificial wetland in Chongqing. The results are as follows: Figure 2 As shown.

[0114] Depend on Figure 2 It can be seen that the sample pretreatment purification effect is good, the method has strong applicability, high accuracy, and good reproducibility. Six racemic target drugs were added at 10 ng / g (dry weight) to the plant sample matrix, and three parallel determinations were performed, with a blank control experiment also conducted. The spiked recoveries ranged from 70% to 130%, making it suitable for the analysis of enantiomer residues of multiple drugs in plant media.

[0115] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting resolution of a plurality of chiral drugs, characterized in that, The method comprises the following steps: S1, adding working solvent and four isotopic internal standard substances to six racemic standard substances of chiral drugs to obtain a mixed working solution of racemic drugs; the chiral drugs are salbutamol, atenolol, fluoxetine, citalopram, venlafaxine and O-desmethylvenlafaxine; the isotopic internal standards are salbutamol-D3, atenolol-D7, fluoxetine-D5 and venlafaxine-D6; S2, using liquid chromatography tandem mass spectrometry to analyze the mixed working solution of racemic drugs and the test sample liquid obtained in step S1 to separate the racemic drugs in a positive ion mode; the liquid chromatography uses a CHIROBIOTIC V chromatographic column and a CHIROBIOTIC V guard column, the specification of the chromatographic column is 250*2.1 mm, 5 μm, the specification of the guard column is 20*4 mm, 5 μm, and silica gel coated with vancomycin is used as a chiral stationary phase; mobile phase A is ultrapure water containing 0.005-0.006 vt% formic acid and 4-5 mM ammonium formate, and mobile phase B is methanol containing 0.005-0.006 vt% formic acid; the volume ratio of the mobile phase A is 49-51%, the volume ratio of the mobile phase B is 49-51%, the column temperature of the liquid chromatography is 25-26 ℃, the flow rate is 0.1-0.15 mL / min, the injection volume is 20 μL, and the elution mode is isocratic elution; the mass spectrometry analysis uses a MS / MS detection mode of multiple reaction monitoring mode (MRM); the voltage of the electrospray ion source in a positive ion mode is 4500-5000 V; the flow rate of the atomization gas is 3-4 L / min; the flow rate of the heating gas is 10-11 L / min; the flow rate of the dry gas is 10-11 L / min; the interface temperature is 300-350 ℃; the DL temperature is 250-300 ℃; and the heating block temperature is 400-450 ℃. S3, the racemic drug mixed working solution and the water sample liquid are analyzed by liquid chromatography-triple quadrupole tandem mass spectrometry, and the chiral enantiomer residual amount of the traditional Chinese medicine is determined in a positive ion mode of multiple reaction monitoring. The above liquid chromatography conditions are as follows: a CHIROBIOTIC V chromatographic column and a CHIROBIOTIC V guard column are used, the size of the chromatographic column is 250 mm x 2.1 mm, 5 μm, the size of the guard column is 20 x 4 mm, 5 μm, vancomycin coated with silica gel is used as a chiral stationary phase, mobile phase A is ultrapure water containing 0.005 vt% formic acid and 4 mM ammonium formate, mobile phase B is methanol containing 0.005 vt% formic acid, the volume ratio of mobile phase A is 50%, the volume ratio of mobile phase B is 50%, the flow rate is 0.1 mL / min, the column temperature is 25℃, and the injection amount is 20 μL. The above mass spectrometry conditions are as follows: ion source: electrospray ionization (ESI), positive ion mode voltage 4500 V, detection mode: multiple reaction monitoring mode (MRM), atomization gas flow rate: 3 L / min, heating gas flow rate: 10 L / min, dry gas flow rate: 10 L / min, interface temperature: 300℃, DL temperature: 250℃, heating block temperature: 400℃.

2. The method for separating and detecting multiple chiral drugs as described in claim 1, characterized in that, In step S1, when the test sample liquid is a water sample liquid, the working solvent is methanol. When the test sample liquid is a plant sample liquid, the working solvent is prepared by the following method: the plant is freeze-dried without detecting chiral drugs, acetonitrile is added to the obtained freeze-dried powder, and then vortexed, ice-bath ultrasonic, freeze centrifugation, and then the upper clear liquid is repeatedly absorbed twice, the supernatant is collected, and then concentrated to just dry by nitrogen blowing, the residue is redissolved with methanol, then mixed with ultrapure water, adjust the pH value, and then the obtained mixture is passed through an activated Oasis HLB solid phase extraction cartridge, the sample is completely filled with the filler, then ultrapure water is added for washing, the filler is dried under negative pressure, then methanol is added for elution, and then the eluent is filtered to obtain the working solvent.

3. The method for separating and detecting multiple chiral drugs as described in claim 1, characterized in that, In steps S2 and S3, the test sample liquid is a water sample liquid or a plant sample liquid.

4. The method for separating and detecting multiple chiral drugs as described in claim 3, characterized in that, The preparation method of the water sample liquid specifically comprises the following steps: filtering the water sample, adding an isotopic internal standard, adjusting the pH value, then passing through an activated Oasis HLB solid phase extraction cartridge, the sample is completely filled with the filler, then ultrapure water is added for washing, the filler is dried under negative pressure, then methanol is added for eluting the target, and then the eluent is concentrated to just dry by nitrogen blowing, then methanol is used for constant volume, vortexed, then filtered to obtain the water sample liquid.

5. The method for separating and detecting multiple chiral drugs as described in claim 3, characterized in that, The preparation method of the plant sample liquid specifically comprises the following steps: freeze-drying the plant, adding an isotopic internal standard to the obtained freeze-dried powder, vortexing after adding acetonitrile, ice-bath ultrasonic, freezing and centrifuging, and then sucking the supernatant, repeating twice, collecting the supernatant, and nitrogen-blowing to concentrate to just dry, re-dissolving the residue in methanol, mixing with ultrapure water, adjusting the pH value, passing the obtained mixture through an activated Oasis HLB solid-phase extraction column, adding ultrapure water to elute the column, drying the column under negative pressure, adding methanol to elute the target, and nitrogen-blowing to concentrate the eluent to just dry, using methanol to make up the volume, vortexing, and then filtering to obtain the plant sample liquid.

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