Splitting detection method for multiple chiral drugs

Abstract

The invention discloses a resolution detection method for various chiral drugs, and relates to the field of environmental pollutant detection and analysis. According to the method provided by the invention, the experiment efficiency is greatly improved and the experiment cost is reduced under the conditions of ensuring the experiment accuracy, sensitivity and low detection limit by optimizing the sample pretreatment method and the parameters of the liquid chromatograph and mass spectrometer. The method is suitable for water bodies and plants, the water bodies comprise domestic sewage, medical wastewater, sewage treatment plant inlet and outlet water, surface water, drinking water and the like, the method can be used for splitting six chiral drugs and conversion product enantiomers, and the content of the substances can be accurately quantified. The problems that different pretreatment steps need to be designed for different substances and multi-needle sample injection is needed in an instrument in a traditional chiral detection method are effectively solved.

Description

Technical Field

[0001] The present invention relates to the field of environmental pollutant detection and analysis, and in particular to a method for separating and detecting multiple chiral drugs. Background Art

[0002] In recent years, the production and consumption of drugs have been on the rise. Since they cannot be completely metabolized by the human body and cannot be completely removed by sewage treatment plants, drug pollutants have been widely detected in different types of water bodies, including domestic sewage, medical wastewater, sewage treatment plant inlet and outlet water, surface water and drinking water, with concentrations ranging from ng / L to μg / 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 commercially available drugs are chiral. Chiral drugs are prone to enantioselective metabolic transformation in organisms, resulting in changes in the enantiomer fraction in the environment, but only a few studies have reported on the enantiomers of chiral drugs in the environment. Chiral drugs often cause abnormal reproduction, behavior, metabolism and stress response of non-target organisms (especially aquatic species), and their ecotoxicity is enantioselective. For example, in terms of the effective concentration of inhibiting 10% growth, the toxicity of S-fluoxetine to blackhead fish is 9.4 times that of R-fluoxetine; R-venlafaxine and its metabolite (R-desmethylvenlafaxine) are more toxic to the freshwater alga Spiny Scenedesmus than the corresponding S-enantiomer. Therefore, in the comprehensive ecotoxicity evaluation and risk assessment, the different enantiomers of chiral drugs should be regarded as separate environmental pollutants.

[0003] Effective monitoring of chiral drug enantiomer monomers is of great significance in exploring their environmental behavior and elimination mechanism, and can also provide a basis for a more comprehensive and accurate investigation of the residual risk of drug enantiomers in environmental media. However, the concentration of chiral drugs in environmental media is very low, and there are usually complex matrix effects in the samples that interfere with the detection results. In addition, there are many types of chiral drugs with large differences in properties. Traditional detection methods require different pretreatment steps to be designed for different substances, and multi-needle injections are required in the instrument due to different mobile phase conditions. This series of problems limits the promotion and application of chiral drug detection methods. Therefore, it is necessary to develop detection methods for chiral drugs in different environmental media at the enantiomeric level, so as to achieve the purpose of simultaneously separating and quantifying more chiral drugs. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a method for splitting and detecting a plurality of chiral drugs, which improves experimental efficiency, reduces experimental costs, and effectively solves the problem that the traditional chiral detection method requires different pretreatment steps to be designed for different substances and requires multiple needle injections in the instrument.

[0005] To achieve the above object, the technical solution adopted by the present invention to solve the technical problem is: to provide a method for separating and detecting multiple chiral drugs, comprising the following steps:

[0006] S1. Adding a working solvent and four isotope internal standard standards to six racemic standard products of chiral drugs to obtain a racemic drug mixed working solution;

[0007] S2, the racemic drug mixed working solution obtained in step S1 and the test sample solution are analyzed by liquid chromatography tandem mass spectrometry to separate the racemic drugs in positive ion mode;

[0008] S3. The racemic drug mixed working solution obtained in step S1 and the test sample solution are analyzed by liquid chromatography-triple quadrupole tandem mass spectrometry to determine the residual amount of chiral enantiomers of the drug in the multiple reaction monitoring positive ion mode.

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

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

[0011] Furthermore, the isotopic internal standards were salbutamol-D3, atenolol-D7, fluoxetine-D5, and venlafaxine-D6.

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

[0013] Furthermore, the gradient concentrations of the racemic standard of the chiral drug in the racemic drug mixed working solution are 1, 5, 10, 25, 50, 100 and 500 μg / L.

[0014] Furthermore, the concentration of the isotope internal standard in the racemic drug mixed working solution was 10 μg / L.

[0015] Furthermore, in step S1, when the test sample liquid is an aqueous 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-drying a plant from which no chiral drug is detected, adding acetonitrile to the obtained freeze-dried powder and vortexing, performing ice bath sonication, freezing centrifugation and then aspirating the upper supernatant and repeating the process twice, collecting the supernatant, and concentrating it by nitrogen blowing until it is just blown dry, re-dissolving the residue with methanol, adding ultrapure water to mix, adjusting the pH value, passing the obtained mixture through an activated Oasis HLB solid phase extraction column, adding ultrapure water to rinse the sample after it has completely passed through the filler, draining the filler under negative pressure, adding methanol to elute, and filtering the eluate 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: taking a plant without detected target compound into a 50 mL centrifuge tube, and then freeze-drying it at a pressure of 20 Pa in a vacuum freeze dryer for 48 hours, covering the mouth of the centrifuge tube with a layer of plastic wrap during freeze-drying, and poking a small hole with a needle to facilitate water evaporation, weighing 0.5-0.6 g of freeze-dried plant powder, adding 10-11 mL of acetonitrile, vortexing at 2500-2600 rpm for 3-4 minutes, and then Ultrasonicate in an ice bath for 10-11 minutes, centrifuge at 4000-4500 rpm for 10-11 minutes, draw the supernatant, repeat twice, collect the supernatant, and concentrate it with nitrogen until it is just dry. After the residue is redissolved with 1-2 mL of methanol, add 9-10 mL of ultrapure water and mix, 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 the activated Oasis at a rate of 2-2.1 mL / min. HLB solid phase extraction column (activated by 5-6 mL methanol, 5-6 mL ultrapure water with a pH of 7, and 5-6 mL ultrapure water with a pH of 3.5-4.5 in sequence). After the sample has completely passed through the filler, 5-6 mL ultrapure water with a pH of 3.5-4.5 is added for elution. After the filler is drained for 10-11 minutes under negative pressure, 5-6 mL methanol is added for elution, and the eluate is filtered through a 0.22-0.23 μm PTFE filter 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: taking a plant without detected target compound into a 50mL centrifuge tube, and then freeze-drying it at a pressure of 20Pa for 48h in a vacuum freeze dryer, covering the mouth of the centrifuge tube with a layer of plastic wrap during freeze-drying, and poking a small hole with a needle to facilitate water volatilization, weighing 0.5g of freeze-dried plant powder, adding 10mL of acetonitrile, vortexing at 2500rpm for 3min, then ultrasonicating in an ice bath for 10min, and refrigerating and centrifuging at 4000rpm for 10min, absorbing the upper clear liquid, repeating twice, collecting the supernatant, and concentrating it with nitrogen blowing until it is just blown dry, re-dissolving the residue with 1mL of methanol, adding 9mL of ultrapure water to mix, and adjusting the pH to 4 with 1mmol / L hydrochloric acid or sodium hydroxide solution, and passing the resulting mixture through an activated Oasis HLB solid phase extraction column at a rate of 2mL / min (passing through 5mL of methanol, 5mL of ultrapure water with a pH of 7, and 5mL in sequence). After the sample has completely passed through the filler, 5 mL of ultrapure water with a pH of 4 is added for elution. After the filler is drained for 10 min under negative pressure, 5 mL of methanol is added for elution. The eluate is filtered through a 0.22 μm PTFE filter membrane to obtain the working solvent.

[0019] Furthermore, in step S2 and step S3, the test sample liquid is a water sample liquid or a plant sample liquid.

[0020] Furthermore, the preparation method of the water sample liquid specifically includes the following steps: filtering the water sample, adding an isotope internal standard, adjusting the pH value, and then passing through an activated Oasis HLB solid phase extraction column. After the sample has completely passed through the filler, ultrapure water is added for elution, the filler is drained under negative pressure conditions, methanol is added to elute the target, and the eluted liquid nitrogen is concentrated until it is just dried, the volume is made up with methanol, and it is vortexed and filtered to obtain the water sample liquid.

[0021] Further, the solution was filtered through a 0.45-0.5 μm filter membrane.

[0022] Furthermore, the solution was filtered through a 0.45 μm filter membrane.

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

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

[0025] Furthermore, the pH value was adjusted to 4 with 0.9-1mmol / L hydrochloric acid or sodium hydroxide solution, and the sample was passed through the activated Oasis HLB solid phase extraction column at a rate of 2-2.1mL / min. After the sample completely passed through the filler, 5-6mL of ultrapure water with a pH value of 4 was added for elution. After the filler was drained for 10-11min under negative pressure, 5-6mL of methanol was added to elute the target substance, and the eluted liquid nitrogen was concentrated until just dry, and the volume was made up to 1mL with methanol. After vortexing, it was filtered through a 0.22-0.23μm PTFE filter membrane to obtain the water sample liquid.

[0026] Furthermore, the pH was adjusted to 4 with 1 mmol / L hydrochloric acid or sodium hydroxide solution, and the sample was passed through the activated Oasis HLB solid phase extraction column at a rate of 2 mL / min. After the sample had completely passed through the filler, 5 mL of ultrapure water with a pH of 4 was added for elution. After the filler was drained for 10 min under negative pressure, 5 mL of methanol was added to elute the target substance, and the eluted liquid nitrogen was concentrated until just dry, and the volume was made up to 1 mL with methanol. After vortexing, it was filtered through a 0.22 μm PTFE filter membrane to obtain the water sample solution.

[0027] Furthermore, activation was performed with 5 mL of methanol, 5 mL of ultrapure water with a pH of 7, and 5 mL of ultrapure water with a pH of 4 in sequence.

[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, vortexing after adding acetonitrile, ultrasonicating in an ice bath, and absorbing the upper clear liquid after frozen centrifugation, repeating twice, collecting the supernatant, and concentrating it by nitrogen blowing until it is just dry, re-dissolving the residue with methanol, adding ultrapure water to mix, adjusting the pH value, passing the obtained mixture through an activated Oasis HLB solid phase extraction column, and after the sample has completely passed through the filler, adding ultrapure water to rinse, draining the filler under negative pressure conditions, adding methanol to elute the target, and concentrating the eluted liquid with nitrogen blowing until it is just dry, making up to volume with methanol, vortexing and filtering to obtain the plant sample solution.

[0029] Further, the plant was placed in a 50 mL centrifuge tube, and then freeze-dried in a vacuum freeze dryer at a pressure of 20-25 Pa for 48-50 h. During freeze-drying, a layer of plastic wrap was used to cover the mouth of the centrifuge tube, and a small hole was poked with a needle to facilitate evaporation of water.

[0030] Furthermore, the plant was placed in a 50 mL centrifuge tube and then freeze-dried in a vacuum freeze dryer at a pressure of 20 Pa for 48 h. During freeze-drying, a layer of plastic wrap was used to cover the mouth of the centrifuge tube, and a small hole was poked with a needle to facilitate evaporation of water.

[0031] Further, 0.5-0.6 g of freeze-dried plant powder was weighed, 10 ng of each isotope internal standard (salbutamol-D3, atenolol-D7, fluoxetine-D5 and venlafaxine-D6) was added, 10-11 mL of acetonitrile was added, vortexed at 2500-2600 rpm for 3-4 min, then ultrasonicated in an ice bath for 10-11 min, and centrifuged at 4000-4500 rpm for 10-11 min, the supernatant was aspirated, and the process was repeated twice. The supernatant was collected and concentrated by nitrogen blowing until just dry. The residue was redissolved with 1-2 mL of methanol, and then 9-10 mL of ultrapure water was added and mixed, and the pH was adjusted to 3.5-4.5 with 0.9-1 mmol / L hydrochloric acid or sodium hydroxide solution. The resulting mixture was passed through an activated Oasis HLB solid phase extraction cartridge at a rate of 2-2.1 mL / min (5-6 mL of methanol, 5-6 mL of ultrapure water with a pH of 7, and 5-6 mL of HPLC in sequence). After the sample has completely passed through the filler, add 5-6mL of ultrapure water with a pH of 3.5-4.5 to elute. After the filler is drained for 10-11 minutes under negative pressure, add 5-6mL of methanol to elute the target. The eluted liquid nitrogen is concentrated until it is just dried. The residue is fixed to 1mL with methanol, vortexed, and filtered through a 0.22-0.23μm PTFE filter membrane to serve as the plant sample solution.

[0032] Further, 0.5 g of freeze-dried plant powder was weighed, 10 ng of each isotope internal standard (salbutamol-D3, atenolol-D7, fluoxetine-D5 and venlafaxine-D6) was added, 10 mL of acetonitrile was added, vortexed at 2500 rpm for 3 min, then ultrasonicated in an ice bath for 10 min, and centrifuged at 4000 rpm for 10 min, the supernatant was aspirated, and the process was repeated twice. The supernatant was collected and concentrated by nitrogen blowing until just dry. The residue was redissolved with 1 mL of methanol, and then 9 mL of ultrapure water was added and mixed. The pH was adjusted to 4 with 1 mmol / L hydrochloric acid or sodium hydroxide solution. The resulting mixture was passed through an activated Oasis HLB solid phase extraction column (activated by 5 mL of methanol, 5 mL of ultrapure water with a pH of 7, and 5 mL of ultrapure water with a pH of 4) at a rate of 2 mL / min. After the sample had completely passed through the filler, 5 mL The solution was eluted with ultrapure water at pH 4. The filler was drained for 10 min under negative pressure and 5 mL of methanol was added to elute the target substance. The eluted liquid nitrogen was concentrated until just dry. The residue was made up to 1 mL with methanol, vortexed and filtered through a 0.22 μm PTFE filter membrane as the plant sample solution.

[0033] Furthermore, in step S2 and step S3, a CHIROBIOTIC V chromatographic column and a CHIROBIOTIC V guard column are used in liquid chromatography, the specifications of the chromatographic column are 250×2.1 mm, 5 μm, the specifications of the guard column are 20×4 mm, 5 μm, and silica gel-coated vancomycin is used as the chiral stationary phase; the mobile phase A is ultrapure water containing 0.005-0.006 vt% formic acid and 4-5 mM ammonium formate, and the mobile phase B is methanol containing 0.005-0.006 vt% formic acid; the volume ratio of the mobile phase A is 49-51%, and the volume ratio of the mobile phase B is 49-51%.

[0034] Furthermore, in step S2 and step S3, a CHIROBIOTIC V chromatographic column and a CHIROBIOTIC V guard column are used in liquid chromatography, the specifications of the chromatographic column are 250×2.1 mm, 5 μm, the specifications of the guard column are 20×4 mm, 5 μm, and silica gel-coated 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 the mobile phase A is 50%, and the volume ratio of the mobile phase B is 50%.

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

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

[0037] Furthermore, in step S2 and step S3, in the mass spectrometry analysis, the MS / MS detection mode used is the multiple reaction monitoring mode (MRM); the electrospray ion source positive ion mode voltage is 4500-5000V; the nebulizing 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°C; the DL temperature is 250-300°C; and the heating block temperature is 400-450°C.

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

[0039] The above-mentioned multiple chiral drug separation and detection methods are applied in the quality detection of drug enantiomers.

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

[0041] 1. The present invention achieves the separation and quantification of five common active pharmaceutical ingredients and one metabolite enantiomer in three categories (anti-asthma drugs, beta-blockers and antidepressants) by one-shot 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 racemic drug mixed working solution preparation and sample treatment, and adjusting liquid chromatography conditions and parameters, the purpose of reducing detection time and matrix effects in complex media is achieved.

[0043] 3. Compared with previous methods, this method can save a lot of experimental operation costs including labor costs, instrument usage costs, reagent costs, etc., and can also save laboratory management costs including sampling, transportation, waste disposal, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The enantiomer separation chromatogram of the drug in Example 2.

[0045] Figure 2 This is the recovery rate of drug enantiomer extraction in plants in Example 3. DETAILED DESCRIPTION

[0046] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0047] Example 1

[0048] A method for separating and detecting multiple chiral drugs comprises the following steps:

[0049] S1. Take 6 kinds of racemic standard products of chiral drugs, 20 mg of each of salbutamol, atenolol, fluoxetine, citalopram, venlafaxine and O-desmethylvenlafaxine, add methanol to dilute, then add 4 kinds of isotope internal standard products, 10 ng of each of salbutamol-D3, atenolol-D7, fluoxetine-D5 and venlafaxine-D6, to obtain a racemic drug mixed working solution, the gradient concentrations of the racemic standard products of chiral drugs in the racemic drug mixed working solution are 1, 5, 10, 25, 50, 100 and 500 μg / L, and the mass concentration of the isotope internal standard products in the racemic drug mixed working solution is 10 μg / L;

[0050] S2. The racemic drug mixed working solution obtained in step S1 and the water sample solution are analyzed by liquid chromatography tandem mass spectrometry to separate the racemic drugs in positive ion mode. The preparation method of the above water sample solution is as follows: take 200 mL of water sample, filter it through a 0.45 μm filter membrane, add 10 ng 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) with 5 mL of methanol, 5 mL of ultrapure water with a pH of 7 and 5 mL of ultrapure water with a pH of 4 in sequence, pass the obtained mixture through the activated solid phase extraction column at a speed of 2 mL / min, discard the effluent, add 5 mL The sample was washed with ultrapure water at pH 4, and then the filler was drained for 10 minutes under negative pressure. 5 mL of methanol was added to elute the target, and the eluted liquid nitrogen was blown and concentrated until it was just blown dry. The residue was fixed to 1 mL with methanol, vortexed, and filtered through a 0.22 μm PTFE filter membrane to obtain an aqueous sample liquid. The above liquid chromatography conditions: a CHIROBIOTIC V chromatographic column and a CHIROBIOTIC V guard column were used. The specifications of the chromatographic column were 250 mm × 2.1 mm, 5 μm, and the specifications of the guard column were 20 × 4 mm, 5 μm. Silica gel-coated vancomycin was used as the chiral stationary phase. Mobile phase A was ultrapure water containing 0.005% formic acid and 4 mM ammonium formate, and mobile phase B was methanol containing 0.005 vt% formic acid. The volume ratio of mobile phase A was 50%, the volume ratio of 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, and the mass spectrometry conditions were as follows: ion source: electrospray ionization (ESI), positive ion mode voltage 4500 V, detection mode: multiple reaction monitoring mode (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, and the MRM mass spectrometry parameters of chiral drugs and internal standards are shown in Table 1 ;

[0051] S3. The racemic drug mixed working solution and aqueous sample solution were analyzed by liquid chromatography-triple quadrupole tandem mass spectrometry, and the residual chiral enantiomers of the drugs were determined in the multiple reaction monitoring positive ion mode. The above liquid chromatography conditions: CHIROBIOTIC V column and CHIROBIOTIC V guard column, the specifications of the chromatographic column are 250mm×2.1mm, 5μm, the specifications of the guard column are 20×4mm, 5μm, silica gel coated vancomycin is used as the chiral stationary phase, mobile phase A is ultrapure water containing 0.005vt% formic acid and 4mM ammonium formate, mobile phase B is methanol containing 0.005vt% formic acid, the volume ratio of mobile phase A is 50%, the volume ratio of mobile phase B is 50%, the flow rate is 0.1mL / min, the column temperature is 25℃, the injection volume is 20μL, and 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℃.

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

[0053]

[0054]

[0055] *: Quantitative ion

[0056] Example 2

[0057] A method for separating and detecting multiple chiral drugs comprises the following steps:

[0058] S1. Take 6 kinds of racemic standard products of chiral drugs, 20 mg of each of salbutamol, atenolol, fluoxetine, citalopram, venlafaxine and O-desmethylvenlafaxine, add methanol to dilute, then add 4 kinds of isotope internal standard products, 10 ng of each of salbutamol-D3, atenolol-D7, fluoxetine-D5 and venlafaxine-D6, to obtain a racemic drug mixed working solution, the gradient concentrations of the racemic standard products of chiral drugs in the racemic drug mixed working solution are 1, 5, 10, 25, 50, 100 and 500 μg / L, and the mass concentration of the isotope internal standard products in the racemic drug mixed working solution is 10 μg / L;

[0059] S2. The racemic drug mixed working solution obtained in step S1 and the water sample solution are analyzed by liquid chromatography tandem mass spectrometry to separate the racemic drugs in positive ion mode. The preparation method of the above water sample solution is as follows: take 200 mL of water sample, filter it through a 0.45 μm filter membrane, add 10 ng 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) with 5 mL of methanol, 5 mL of ultrapure water with a pH of 7 and 5 mL of ultrapure water with a pH of 4 in sequence, pass the obtained mixture through the activated solid phase extraction column at a speed of 2 mL / min, discard the effluent, add 5 mL The sample was washed with ultrapure water at pH 4, and then the filler was drained for 10 minutes under negative pressure. 5 mL of methanol was added to elute the target, and the eluted liquid nitrogen was concentrated until it was just dried. The residue was fixed to 1 mL with methanol, vortexed, and filtered through a 0.22 μm PTFE filter membrane to obtain an aqueous sample liquid. The above liquid chromatography conditions: a CHIROBIOTIC V chromatographic column and a CHIROBIOTIC V guard column were used. The specifications of the chromatographic column were 250 mm × 2.1 mm, 5 μm, and the specifications of the guard column were 20 × 4 mm, 5 μm. Silica gel-coated vancomycin was used as the chiral stationary phase. Mobile phase A was ultrapure water containing 0.005% formic acid and 4 mM ammonium formate, and mobile phase B was methanol containing 0.005 vt% formic acid. The volume ratio of mobile phase A was 50%, the volume ratio of mobile phase B was 50%, the flow rate was 0.15 mL / min, and the column temperature was 25 °C , injection volume was 20 μL, the above mass spectrometry conditions were: ion source: electrospray ionization (ESI), positive ion mode voltage 4500 V, detection mode: multiple reaction monitoring mode (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, and the MRM mass spectrometry parameters of chiral drugs and internal standards are shown in Table 1;

[0060] S3. The racemic drug mixed working solution and aqueous sample solution were analyzed by liquid chromatography-triple quadrupole tandem mass spectrometry, and the residual chiral enantiomers of the drugs were determined in the multiple reaction monitoring positive ion mode. The above liquid chromatography conditions: CHIROBIOTIC V column and CHIROBIOTIC V guard column, the specifications of the chromatographic column are 250mm×2.1mm, 5μm, the specifications of the guard column are 20×4mm, 5μm, silica gel coated with vancomycin is used as the chiral stationary phase, mobile phase A is ultrapure water containing 0.005vt% formic acid and 4mM ammonium formate, mobile phase B is methanol containing 0.005vt% 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, and 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℃.

[0061] Example 3

[0062] A method for separating and detecting multiple chiral drugs comprises the following steps:

[0063] S1. Take 6 kinds of racemic standard products of chiral drugs, 20 mg each of salbutamol, atenolol, fluoxetine, citalopram, venlafaxine and O-desmethylvenlafaxine, add working solvent to dilute, then add 4 kinds of isotope internal standard products, 10 ng each of salbutamol-D3, atenolol-D7, fluoxetine-D5 and venlafaxine-D6, to obtain a racemic drug mixed working solution, the gradient concentrations of the racemic standard products of chiral drugs in the racemic drug mixed working solution are 1, 5, 10, 25, 50, 100 and 500 μg / L, the mass concentration of the isotope internal standard products in the racemic drug mixed working solution is 10 μg / L, and the above working solvent is prepared by the following method: take the plant without the target compound detected into a 50 mL centrifuge tube, and then cool it with a vacuum condenser. The tube was freeze-dried in a freeze dryer at a pressure of 20 Pa for 48 hours. During freeze-drying, a layer of plastic wrap was used to cover the mouth of the centrifuge tube, and a small hole was pierced with a needle to facilitate the evaporation of water. 0.5 g of freeze-dried plant powder was weighed, and 10 ng of each isotope internal standard (salbutamol-D3, atenolol-D7, fluoxetine-D5 and venlafaxine-D6) was added. 10 mL of acetonitrile was added, and the tube was vortexed at 2500 rpm for 3 minutes, followed by ice bath ultrasound for 10 minutes, and refrigerated centrifugation at 4000 rpm for 10 minutes. The upper clear liquid was aspirated, and the mixture was repeated twice. The supernatant was collected and concentrated by nitrogen blowing until it was just blown dry. The residue was redissolved with 1 mL of methanol, and 9 mL of ultrapure water was added to mix. The pH value was adjusted to 4 with 1 mmol / L of hydrochloric acid. The resulting mixture was passed through the activated Oasis at a rate of 2 mL / min. HLB solid phase extraction cartridge (activated by 5 mL of methanol, 5 mL of ultrapure water with a pH of 7, and 5 mL of ultrapure water with a pH of 4 in sequence), after the sample has completely passed through the filler, 5 mL of ultrapure water with a pH of 4 is added for elution, the filler is drained under negative pressure for 10 min, and then 5 mL of methanol is added for elution, and the eluate is filtered through a 0.22 μm PTFE filter membrane to obtain the working solvent;

[0064] S2. The racemic drug mixed working solution obtained in step S1 and the plant sample solution are analyzed by liquid chromatography-tandem mass spectrometry to separate the racemic drugs in positive ion mode. The preparation method of the above plant sample solution is as follows: put the plant into a 50mL centrifuge tube, and then freeze-dry it in a vacuum freeze dryer at a pressure of 20Pa for 48 hours. During freeze-drying, cover the mouth of the centrifuge tube with a layer of plastic wrap, and poke a small hole with a needle to facilitate water evaporation. Accurately weigh 0.5g of freeze-dried plant powder, add 10ng of the isotope internal standard of the target substance, add 10mL of acetonitrile, vortex at 2500rpm for 3 minutes, then ultrasonicate in an ice bath for 10min, and then freeze-centrifuge at 4000rpm for 10min, draw the upper clear liquid, repeat the above steps twice, combine the supernatants, and concentrate them with nitrogen blow until they are just dry. The residue was redissolved with 1 mL of methanol, and 9 mL of ultrapure water was added to mix. The pH of the plant sample was adjusted to 4 with 1 mmol / L NaOH. The Oasis HLB solid phase extraction column (specification: 6 mL / 200 mg) was activated with 5 mL of methanol, 5 mL of ultrapure water with a pH of 7, and 5 mL of ultrapure water with a pH of 4. The mixture from the previous step was passed through the activated solid phase extraction column at a rate of 2 mL / min. The effluent was discarded, and 5 mL of ultrapure water with a pH of 4 was added for elution. The filler was then drained for 10 min under negative pressure conditions, 5 mL of methanol was added to elute the target, and the eluted liquid nitrogen was blown and concentrated until it was just blown dry. The residue was made up to 1 mL with methanol, vortexed, and filtered through a 0.22 μm PTFE filter membrane to obtain the plant sample solution. The above liquid chromatography conditions: using a CHIROBIOTIC V column and CHIROBIOTIC V guard column, the specifications of the chromatographic column are 250 mm × 2.1 mm, 5 μm, the specifications of the guard column are 20 × 4 mm, 5 μm, and silica gel coated vancomycin is used as the chiral stationary phase. The mobile phase A is ultrapure water containing 0.005% formic acid and 4 mM ammonium formate, and the mobile phase B is methanol containing 0.005% formic acid. The volume ratio of mobile phase A is 50%, and the volume ratio of mobile phase B is 50%. The flow rate is 0.15 mL / min, the column temperature is 25 ° C, and the injection volume is 20 μL. The above mass spectrometry conditions are: ion source: electrospray ionization (ESI), positive ion mode voltage 4500 V, detection mode: multiple reaction monitoring mode (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;

[0065] S3. The racemic drug mixed standard working solution and the test sample solution were analyzed by liquid chromatography-triple quadrupole tandem mass spectrometry, and the residual chiral enantiomers of the drugs were determined in the multiple reaction monitoring positive ion mode. The above liquid chromatography conditions: CHIROBIOTIC V column and CHIROBIOTIC V guard column, the specifications of the chromatographic column are 250mm×2.1mm, 5μm, the specifications of the guard column are 20×4mm, 5μm, silica gel coated vancomycin is used as the 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, and 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 separating and detecting multiple chiral drugs comprises the following steps:

[0068] S1. Take 6 kinds of racemic standard products of chiral drugs, 20 mg of each of salbutamol, atenolol, fluoxetine, citalopram, venlafaxine and O-desmethylvenlafaxine, add methanol to dilute, then add 4 kinds of isotope internal standard products, 10 ng of each of salbutamol-D3, atenolol-D7, fluoxetine-D5 and venlafaxine-D6, to obtain a racemic drug mixed working solution, the gradient concentrations of the racemic standard products of chiral drugs in the racemic drug mixed working solution are 1, 5, 10, 25, 50, 100 and 500 μg / L, and the mass concentration of the isotope internal standard products in the racemic drug mixed working solution is 10 μg / L;

[0069] S2. The racemic drug mixed working solution obtained in step S1 and the water sample solution are analyzed by liquid chromatography tandem mass spectrometry to separate the racemic drugs in positive ion mode. The preparation method of the above water sample solution is as follows: 210 mL of water sample is taken, filtered through a 0.5 μm filter membrane, and 10 ng of each isotopic internal standard (salbutamol-D3, atenolol-D7, fluoxetine-D5 and venlafaxine-D6) is added, the pH is adjusted to 4 with 0.9 mmol / L hydrochloric acid, and an Oasis HLB solid phase extraction column (specification 6 mL / 200 mg) is activated with 5 mL of methanol, 5 mL of ultrapure water with a pH of 7, and 5 mL of ultrapure water with a pH of 4 in sequence, and the obtained mixture is completely passed through the activated solid phase extraction column at a speed of 2.1 mL / min, the effluent is discarded, and 5 mL The ultrapure water with pH 4 was used for elution, and then the filler was drained for 11 minutes under negative pressure. 5 mL of methanol was added to elute the target, and the eluted liquid nitrogen was blown and concentrated until it was just blown dry. The residue was made up to 1 mL with methanol, vortexed, and filtered through a 0.23 μm PTFE filter membrane to obtain the water sample liquid. The above liquid chromatography conditions: using CHIROBIOTIC V chromatographic column and CHIROBIOTIC V guard column, the specifications of the chromatographic column are 250mm×2.1mm, 5μm, the specifications of the guard column are 20×4mm, 5μm, and silica gel coated vancomycin is used as the chiral stationary phase. The mobile phase A is ultrapure water containing 0.005vt% formic acid and 4mM ammonium formate, and the mobile phase B is methanol containing 0.005vt% formic acid. The volume ratio of mobile phase A is 49%, and the volume ratio of mobile phase B is 51%. The flow rate is 0.15mL / min, the column temperature is 26°C, and the injection volume is 21μL. The above mass spectrometry conditions are: ion source: electrospray ionization (ESI), positive ion mode voltage 5000V, detection mode: multiple reaction monitoring mode (MRM), nebulizer gas flow rate: 4L / min, heating gas flow rate: 11L / min, drying gas flow rate: 11L / min, interface temperature: 350°C, DL temperature: 300°C, and heating block temperature: 450°C;

[0070] S3. The racemic drug mixed working solution and the test sample solution were analyzed by liquid chromatography-triple quadrupole tandem mass spectrometry, and the residual chiral enantiomers of the drugs were determined in the multiple reaction monitoring positive ion mode. The above liquid chromatography conditions: CHIROBIOTIC V column and CHIROBIOTIC V guard column, the specifications of the chromatographic column are 250mm×2.1mm, 5μm, the specifications of the guard column are 20×4mm, 5μm, silica gel coated with vancomycin is used as the 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 49%, the volume ratio of mobile phase B is 51%, the flow rate is 0.15mL / min, the column temperature is 26°C, the injection volume is 21μL, and the above mass spectrometry conditions are: ion source: electrospray ionization (ESI), positive ion mode voltage 5000V, detection mode: multiple reaction monitoring mode (MRM), nebulizer gas flow rate: 4L / min, heating gas flow rate: 11L / min, drying gas flow rate: 11L / min, interface temperature: 350°C, DL temperature: 300°C, 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 is 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 is adjusted to 7, the Oasis HLB solid phase extraction column (specification: 6 mL / 200 mg) is activated with 5 mL of methanol, 5 mL of ultrapure water at pH 7, and 5 mL of ultrapure water at pH 7, respectively, the resulting mixture is passed through the activated solid phase extraction column at a speed of 2 mL / min, the effluent is discarded, and 5 mL of ultrapure water at pH 7 is added for elution.

[0081] Comparative Example 6

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

[0083] Test Example 1

[0084] By optimizing the influence of factors such as the ratio and flow rate of the mobile phase on the chromatographic behavior, the chiral separation effect of enantiomers was investigated by the resolution.

[0085] The resolution of enantiomers (Rs) was calculated according to the following formula.

[0086]

[0087] Wherein, Rs represents the separation degree; T1 and T2 represent the retention time of the first and second eluting enantiomers; W1 and W2 represent the peak width of the first and second eluting enantiomer chromatographic peaks.

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

[0089] In Example 1, Comparative Example 1 and Comparative Example 2, the chiral separation effects of drug enantiomers under different mobile phase ratios are shown in Table 2.

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

[0091]

[0092]

[0093] As can be seen from Table 2, 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, and the volume ratio of mobile phase A to B is 50%:50%. Under isocratic elution conditions, the separation degree of multiple drug chiral enantiomers is 1.58-6.31, achieving a baseline separation effect, and the enantiomer chromatographic peak response value is the highest. The volume ratio of mobile phase A to B is 40%:60%, and under isocratic elution conditions, the separation degree is 1.16-3.84, and the baseline separation effect is slightly lower than that of Example 1. The volume ratio of mobile phase A to B is 3%:97%, and under isocratic elution conditions, the separation degree is between 0.04-0.85, and complete baseline separation of drug enantiomers cannot be achieved. In addition, when the enantiomer mass concentration was in the range of 0.5, 2.5, 5, 12.5, 25, 50 and 250 μg / L, the linear relationship between the chromatographic peak area ratios and mass concentration ratios of multiple drug enantiomers and internal standard enantiomers was good, and the correlation coefficient R>0.998.

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

[0095] In Example 1, Example 2 and Comparative Example 3, the chiral separation effects of drug enantiomers under isocratic elution conditions at different flow rates are shown in Table 3.

[0096] Table 3 Separation effect of drug enantiomers under different flow rate conditions

[0097]

[0098]

[0099] As shown in Table 3, the chiral separation effect of drug enantiomer in Example 1 is obviously better than that in Example 2 and Comparative Example 3, but the separation time is too long (72.46-143.34min). The separation degree of salbutamol in Comparative Example 3 is 0.19, and the complete baseline separation of enantiomer cannot be achieved. The separation degree of chiral drug in Example 2 is 1.14-5.28, which can shorten the separation time while ensuring a good baseline separation effect. Therefore, 0.15mL / min is the preferred flow rate condition.

[0100] (3) Separation effect of different mobile phase ratios on drug enantiomers under optimal flow rate conditions

[0101] In Example 2 and Comparative Example 4, under the preferred flow rate conditions, the separation effects of different mobile phase ratios on drug enantiomers are shown in Table 4.

[0102] Table 4 Separation effect of different mobile phase ratios on drug enantiomers under optimal flow rate conditions

[0103]

[0104]

[0105] It can be seen from Table 4 that under the preferred flow rate conditions, the chiral separation effect of multiple drug enantiomers in Example 2 is better than that in Comparative Example 4. Therefore, the 50%:50% volume ratio of mobile phase A to B is the preferred mobile phase ratio condition.

[0106] Test Example 2

[0107] The detection method of multiple drug enantiomers constructed by the present invention is applied to the separation and residual analysis of multiple drug enantiomers in the 24-hour mixed influent of a domestic sewage treatment plant in Chongqing.

[0108] 50 ng / L of 6 racemic target drugs were added to the influent matrix of the sewage treatment plant, and 3 parallel determinations were performed. At the same time, a blank control test was performed. According to the steps described in Example 2 and Comparative Examples 5-6, hydrochloric acid and sodium hydroxide were used to adjust the pH of the treated liquid, and the retention recovery rate of the drug enantiomers under different acid-base conditions was investigated. The results are shown in Table 5.

[0109] Table 5 Recovery and relative standard deviation of spiked water samples of various drug enantiomers (n=3)

[0110]

[0111] As shown in Table 5, when the pH of the treated solution of Example 2 is 4, the retention recovery rates of the six drug enantiomers are better than those of Comparative Example 6, which can meet the experimental requirements. When the pH of the treated solution of Comparative Example 5 is 7, the retention recovery rates of fluoxetine and O-desmethylvenlafaxine are both lower than 30%, indicating that the neutral treated solution is not suitable for its retention and adsorption on the small column, so the treated solution with a pH of 4 in Example 2 is selected as the optimal column loading solution.

[0112] Test Example 3

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

[0114] Depend on Figure 2 It can be seen that the sample pretreatment purification effect is good, the method is highly applicable, accurate and reproducible. Six racemic target drugs were added to the plant sample matrix at 10 ng / g (dry weight), and three parallel determinations were performed. At the same time, a blank control test was performed. The spike recovery rate was in the range of 70-130%, which is suitable for the analysis of the residual amount of multiple drug enantiomers 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for separation and detection of multiple chiral drugs, characterized in that: The following steps are involved: S1. Adding a working solvent and four isotope internal standard standards to six racemic standard products of chiral drugs to obtain a racemic drug mixed working solution; S2, the racemic drug mixed working solution obtained in step S1 and the test sample solution are analyzed by liquid chromatography tandem mass spectrometry to separate the racemic drugs in positive ion mode; S3. The racemic drug mixed working solution obtained in step S1 and the test sample solution are analyzed by liquid chromatography-triple quadrupole tandem mass spectrometry to determine the residual amount of the chiral enantiomer of the drug in the multiple reaction monitoring positive ion mode.

2. The method for separation and detection of multiple chiral drugs according to claim 1, characterized in that: In step S1, the chiral drug is salbutamol, atenolol, fluoxetine, citalopram, venlafaxine and O-desmethylvenlafaxine; The isotope internal standards are salbutamol-D3, atenolol-D7, fluoxetine-D5 and venlafaxine-D6.

3. The method for separation and detection of multiple chiral drugs according to claim 1, characterized in that: In step S1, when the test sample liquid is an aqueous sample liquid, the working solvent is methanol; When the test sample solution is a plant sample solution, the working solvent is prepared by the following method: freeze-drying a plant in which no chiral drug is detected, adding acetonitrile to the obtained freeze-dried powder and vortexing, performing ice bath sonication, and refrigerated centrifugation and then aspirating the upper supernatant, repeating the process twice, collecting the supernatant, and concentrating it by nitrogen blowing until it is just dry, re-dissolving the residue with methanol, adding ultrapure water to mix, adjusting the pH value, and passing the obtained mixture through an activated Oasis HLB solid phase extraction column. After the sample has completely passed through the filler, ultrapure water is added for elution, the filler is drained under negative pressure, and methanol is added for elution, and the eluate is filtered to obtain the working solvent.

4. The method for separation and detection of multiple chiral drugs according to claim 1, characterized in that: In step S2 and step S3, the test sample solution is a water sample solution or a plant sample solution.

5. The method for separation and detection of multiple chiral drugs according to claim 4, characterized in that: The preparation method of the water sample liquid specifically includes the following steps: filtering the water sample, adding an isotope internal standard, adjusting the pH value, and then passing through an activated Oasis HLB solid phase extraction column. After the sample has completely passed through the filler, ultrapure water is added for elution, the filler is drained under negative pressure conditions, methanol is added to elute the target, and the eluted liquid nitrogen is blown and concentrated until it is just blown dry, methanol is used to make up the volume, and the sample is filtered after vortexing to obtain the water sample liquid.

6. The method for separation and detection of multiple chiral drugs according to claim 4, characterized in that: The method for preparing the plant sample solution specifically comprises the following steps: freeze-drying the plant, adding an isotope internal standard to the obtained freeze-dried powder, vortexing after adding acetonitrile, ultrasonicating in an ice bath, and absorbing the upper clear liquid after freezing centrifugation, repeating twice, collecting the supernatant, and concentrating it by nitrogen blowing until it is just blown dry, re-dissolving the residue with methanol, adding ultrapure water to mix, adjusting the pH value, passing the obtained mixture through an activated Oasis HLB solid phase extraction column, adding ultrapure water to rinse after the sample has completely passed through the filler, draining the filler under negative pressure conditions, adding methanol to elute the target, and concentrating the eluted liquid with nitrogen blowing until it is just blown dry, making up to volume with methanol, vortexing and filtering to obtain the plant sample solution.

7. The method for separation and detection of multiple chiral drugs according to claim 1, characterized in that: The liquid chromatography described in step S2 and step S3 uses a CHIROBIOTIC V chromatographic column and a CHIROBIOTIC V guard column. The specifications of the chromatographic column are 250×2.1 mm, 5 μm, and the specifications of the guard column are 20×4 mm, 5 μm. Silica gel-coated 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 the mobile phase A is 49-51%, and the volume ratio of the mobile phase B is 49-51%.

8. The method for separation and detection of multiple chiral drugs according to claim 7, characterized in that: The column temperature of the liquid chromatography is 25-26° C., the flow rate is 0.1-0.15 mL / min, the injection volume is 20 μL, and the elution mode is isocratic elution.

9. The method for separation and detection of multiple chiral drugs according to claim 1, characterized in that: For the mass spectrometry analysis described in step S2 and step S3, the MS / MS detection mode used was the multiple reaction monitoring mode (MRM); the electrospray ion source positive ion mode voltage was 4500-5000 V; the nebulizing gas flow rate was 3-4 L / min; the heating gas flow rate was 10-11 L / min; the drying gas flow rate was 10-11 L / min; the interface temperature was 300-350°C; the DL temperature was 250-300°C; and the heating block temperature was 400-450°C.

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