A method for rapid detection of lincomycin, clindamycin, erythromycin, clarithromycin and roxithromycin in water

Abstract

The present invention relates to a method for rapidly detecting lincomycin, clindamycin, erythromycin, clarithromycin and roxithromycin in water, belonging to the technical field of analytical detection. By means of specific fully automated on-line solid-phase extraction conditions in combination with specific high performance liquid chromatography-tandem mass spectrometry conditions, the present invention realizes sample enrichment, purification, separation and determination, and fully automatically analyzes lincomycin, clindamycin, erythromycin, clarithromycin and roxithromycin in water. The present invention realizes fully automated operation, and the total determination time only needs 11 min. The detection limit reaches the ng / L level. By washing the solid-phase extraction column with an ammonium bicarbonate aqueous solution, the problem of matrix effect commonly encountered in the analysis of complex matrix samples is overcome, meeting the requirements for ultra-trace detection of lincomycin, clindamycin, erythromycin, clarithromycin and roxithromycin in water.

Description

Technical Field

[0001] The present invention relates to a method for rapidly detecting lincomycin, clindamycin, erythromycin, clarithromycin and roxithromycin in water, and belongs to the technical field of analytical detection. Background Art

[0002] Antibiotics are drugs widely used in human medicine and animal husbandry. A large amount of antibiotics ultimately enter the environment and become one of the new important pollutants in the environment, posing a huge threat to the environment and human body.

[0003] For the pretreatment of antibiotic samples in water environment, solid-phase extraction method, solid-phase microextraction and liquid-liquid extraction method are usually adopted to purify and concentrate the analytes, so as to obtain more sensitive and reproducible results. However, the above pretreatment methods all require manual extraction experiments, with a large workload and long pretreatment and analysis testing time. The fully automatic on-line extraction ultra-high performance liquid chromatography-tandem mass spectrometry method is convenient, fast and has a low sample consumption, and is gradually applied to the detection of antibiotics in water environment. However, the current instrument analysis conditions are mainly applicable to antibiotics extracted under acidic and neutral conditions, and the detection and analysis of lincomycin, clindamycin, erythromycin, clarithromycin and roxithromycin extracted under alkaline conditions are still blank. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a method for rapidly detecting lincomycin, clindamycin, erythromycin, clarithromycin and roxithromycin in water, which can detect conveniently, efficiently and rapidly.

[0005] The fully automatic on-line extraction high performance liquid chromatography-tandem mass spectrometry method not only needs to consider sample enrichment, activation, purification and cleaning of the solid-phase extraction column, but also needs to consider the influence of the elution conditions of the liquid chromatography mobile phase on the elution efficiency of the enrichment column. When developing the method, it is necessary to optimize the conditions of the three devices of fully automatic on-line extraction, liquid chromatography and tandem mass spectrometer at the same time. The method development process is more complex than ordinary extraction methods and liquid chromatography methods. At present, there is no report at home and abroad on the determination of lincomycin, clindamycin, erythromycin, clarithromycin and roxithromycin antibiotics extracted under alkaline conditions by fully automatic on-line solid-phase extraction high performance liquid chromatography-tandem mass spectrometry, especially for samples with more complex matrices.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is: a method for rapidly detecting lincomycin, clindamycin, erythromycin, clarithromycin and roxithromycin in water, the method comprising the following steps:

[0007] (1) Sample pretreatment;

[0008] (2) Add the sample to an online solid-phase extraction column for solid-phase extraction and enrichment. The mobile phase conditions for the solid-phase extraction and enrichment are as follows: the initial proportion of mobile phase A is 100%; from 0 min to 0.5 min, it is 100% mobile phase A; from 0.5 min to 4.5 min, it is 98% mobile phase A and 2% mobile phase B; from 4.5 min to 7.5 min, it is 100% mobile phase C; from 7.5 min to 11 min, it is 100% mobile phase A.

[0009] Among them, mobile phase A is an aqueous solution of 50 mM ammonium bicarbonate, mobile phase B is a methanol solution, and mobile phase C is a mixed solution of methanol, acetone, and n-hexane.

[0010] (3) Elute and separate the sample after solid-phase extraction and enrichment by high-performance liquid chromatography, and detect the eluate with a mass spectrometer.

[0011] As a preferred embodiment of the method of the present invention, in the step (1), the specific method for sample pretreatment is: filter the sample through a filter membrane, and adjust the pH to 9.5 - 10.5 with ammonia water.

[0012] As a preferred embodiment of the method of the present invention, the filter membrane is a polytetrafluoroethylene filter membrane with a pore size of 0.22 μm.

[0013] As a preferred embodiment of the method of the present invention, in the step (2), the volume ratio of methanol, acetone, and n-hexane in mobile phase C is 1:1:1.

[0014] As a preferred embodiment of the method of the present invention, in the step (2), the flow rate of the mobile phase for the solid-phase extraction and enrichment is set as follows: from 0 min to 3.5 min, the flow rate is 2.0 mL / min; from 3.5 min to 4.5 min, the flow rate is 0.01 mL / min; from 4.5 min to 11.0 min, the flow rate is 2.0 mL / min; the online solid-phase extraction column is an Oasis HLB column.

[0015] As a preferred embodiment of the method of the present invention, in the step (2), the sample loading volume for the online solid-phase extraction column to perform solid-phase extraction and enrichment is 0.1 - 5 mL.

[0016] As a preferred embodiment of the method of the present invention, in step (3), the mobile phase conditions for elution separation by high performance liquid chromatography are as follows: Mobile phase A1 and mobile phase B1 are mixed in proportion. Initially, it is 90% of mobile phase A1 and 10% of mobile phase B1; from 4.5 min to 8.5 min, the proportion of mobile phase B1 increases from 10% to 95%, while the proportion of mobile phase A1 decreases from 90% to 5%; from 8.5 min to 9.0 min, it is 95% of mobile phase B1 and 5% of mobile phase A1; from 9.0 min to 10.5 min, the proportion of mobile phase A1 increases from 5% to 90%, while the proportion of mobile phase B1 decreases from 95% to 10%; from 10.5 min to 11.0 min, it is 90% of mobile phase A1 and 10% of mobile phase B1;

[0017] Among them, mobile phase A1 is an aqueous solution containing acetic acid and ammonia water. The volume concentration of acetic acid in the aqueous solution is 0.2%, and the volume concentration of ammonia water is 0.1%. Mobile phase B1 is an acetonitrile solution.

[0018] As a preferred embodiment of the method of the present invention, in step (3), the flow rate for elution separation by high performance liquid chromatography is as follows: from 0 min to 4.5 min, the flow rate is 0.1 mL / min; from 4.5 min to 11.0 min, the flow rate is 0.4 mL / min.

[0019] As a preferred embodiment of the method of the present invention, in step (3), the chromatographic column for elution separation by high performance liquid chromatography is an Acquity BEH130 (100 mm × 2.1 mm i.d., 1.7 μm) liquid chromatography column, and the column temperature is 40 °C.

[0020] As a preferred embodiment of the method of the present invention, in step (3), the detection conditions of the mass spectrometry are as follows: The multiple reaction monitoring (MRM) mode is adopted, and the ESI+ source is used for analysis. The spray voltage is 5500 V, the curtain gas is 20 Psi, the nebulizing gas is 55 Psi, the auxiliary heating gas is 60 Psi, and the needle temperature is 450 °C.

[0021] The detection method of the present invention can also be used for quantitative detection of lincomycin, clindamycin, erythromycin, clarithromycin, and roxithromycin in water. By using the method of the present invention, the peak area values of a series of concentration standard solutions are measured, and a standard curve is constructed by taking the peak area and the concentration of the corresponding substance standard solution, thereby obtaining a quantitative detection model.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] (1) The present invention provides a detection method for conveniently, efficiently and rapidly determining lincomycin, clindamycin, erythromycin, clarithromycin and roxithromycin in water. The sample enrichment, elution, washing and analysis are completed in one step, without manual pretreatment, with a small sample requirement and a fast analysis time.

[0024] (2) Through an online solid-phase extraction column, rapid enrichment of lincomycin, clindamycin, erythromycin, clarithromycin and roxithromycin in water is achieved. The entire enrichment time only requires 4 minutes, and the solid-phase extraction column is washed with an ammonium bicarbonate aqueous solution to remove matrix interference and improve the detection sensitivity.

[0025] (3) By optimizing the liquid chromatography conditions, lincomycin, clindamycin, erythromycin, clarithromycin and roxithromycin are separated to meet the requirements of analytical detection.

[0026] (4) Through the combination of an online solid-phase extraction column and ultra-high performance liquid chromatography tandem mass spectrometry, the sample only needs to be filtered and all analysis steps are automatically completed by the instrument, realizing fully automated operation. The total determination time only requires 11 minutes, and the detection limit reaches the ng / L level. Due to automated operation, the reproducibility and precision are better than those of traditional manual pretreatment methods, reducing the errors caused by manual operation.

[0027] (5) Through the pumping and filtration of the automatic station water pump, real-time online monitoring of lincomycin, clindamycin, erythromycin, clarithromycin and roxithromycin in water at the monitoring points of the automatic station can be realized. Description of the Drawings

[0028] Figure 1 It is the chromatogram of antibiotic reference substances. Among them, (a) is the chromatogram of lincomycin reference substance, (b) is the chromatogram of clindamycin reference substance, (c) is the chromatogram of erythromycin reference substance, (d) is the chromatogram of clarithromycin reference substance, (e) is the chromatogram of roxithromycin reference substance, and (f) is the chromatogram of roxithromycin-D7 reference substance.

[0029] Figure 2 It is the comparison chart of different mobile phases A in Comparative Example 1. Among them, (a) is the chromatogram with mobile phase A being pure aqueous solution, and (b) is the chromatogram with mobile phase A being 50 mM ammonium bicarbonate aqueous solution.

[0030] Figure 3 It is the comparison chart of different mobile phases A1 in Comparative Example 2. Among them, (a) is the chromatogram with mobile phase A1 being pure aqueous solution, and (b) is the chromatogram with mobile phase A1 being an aqueous solution containing 0.2% acetic acid and 0.1% ammonia water.

[0031] Figure 4 It is the graph of the sample spiked recovery rate at different storage times in Comparative Example 3. Detailed Embodiments

[0032] To better illustrate the objectives, technical solutions, and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0033] Example 1

[0034] A method for rapidly detecting lincomycin, clindamycin, erythromycin, clarithromycin, and roxithromycin in water, characterized in that the method comprises the following steps:

[0035] (1) Sample pretreatment: Take the water sample and filter it through a polytetrafluoroethylene filter membrane with a pore size of 0.22 μm. If there are many particulate matters in the water sample, it can be centrifuged at 6000 rmp for 10 min first, and then the supernatant is taken and filtered through the filter membrane to remove the particulate matters; Add ammonia water to adjust its pH value to 10, and put it into a sample bottle for further measurement;

[0036] (2) Add the sample to an online solid-phase extraction column for solid-phase extraction and enrichment. The online extraction pretreatment equipment consists of a Waters ACQUITY UPLC quaternary solvent manager (SPE pump) and a 2777 high-pressure sample manager. The mobile phase conditions for the solid-phase extraction and enrichment are as follows: The initial proportion of mobile phase A is 100%; from 0 min to 0.5 min, it is 100% mobile phase A, from 0.5 min to 4.5 min, it is 98% mobile phase A and 2% mobile phase B; from 4.5 min to 7.5 min, it is 100% mobile phase C; from 7.5 min to 11 min, it is 100% mobile phase A;

[0037] Among them, mobile phase A is a 50 mM ammonium bicarbonate aqueous solution, mobile phase B is a methanol solution, and mobile phase C is a mixed solution of methanol, acetone, and n-hexane. The volume ratio of methanol, acetone, and n-hexane in mobile phase C is 1:1:1;

[0038] The flow rate of the mobile phase for the solid-phase extraction and enrichment is set as follows: from 0 min to 3.5 min, the flow rate is 2.0 mL / min; from 3.5 min to 4.5 min, the flow rate is 0.01 mL / min, and from 4.5 min to 11.0 min, the flow rate is 2.0 mL / min; The online solid-phase extraction column is an Oasis HLB column (2.1×30 mm i.d., 20 μm); the injection volume is 5 mL;

[0039] (3) Elute and separate the sample after solid-phase extraction and enrichment by high-performance liquid chromatography, and detect the eluate with a mass spectrometer;

[0040] When performing elution separation by high performance liquid chromatography, the mobile phase conditions are as follows: Mobile phase A1 and mobile phase B1 are mixed in proportion. Initially, it is 90% of mobile phase A1 and 10% of mobile phase B1; from 4.5 min to 8.5 min, the proportion of mobile phase B1 increases from 10% to 95%, and at the same time, the proportion of mobile phase A1 decreases from 90% to 5%; from 8.5 min to 9.0 min, it is 95% of mobile phase B1 and 5% of mobile phase A1; from 9.0 min to 10.5 min, the proportion of mobile phase A1 increases from 5% to 90%, and at the same time, the proportion of mobile phase B1 decreases from 95% to 10%; from 10.5 min to 11.0 min, it is 90% of mobile phase A1 and 10% of mobile phase B1;

[0041] Among them, the mobile phase A1 is an aqueous solution containing acetic acid and ammonia water. The volume concentration of acetic acid in the aqueous solution is 0.2%, and the volume concentration of ammonia water is 0.1%. The mobile phase B1 is an acetonitrile solution;

[0042] The ultra-high performance liquid chromatography equipment includes Waters ACQUITY UPLC binary solvent manager (analytical pump) and ACQUITY UPLC column heater cooler. When performing elution separation by high performance liquid chromatography, the flow rate is set as follows: from 0 min to 4.5 min, the flow rate is 0.1 mL / min; from 4.5 min to 11.0 min, the flow rate is 0.4 mL / min; when performing elution separation by high performance liquid chromatography, the chromatographic column is Acquity BEH130 (100 mm × 2.1 mm i.d., 1.7 μm) liquid chromatography column, and the column temperature is 40 °C;

[0043] The detection conditions of the mass spectrometry are: the tandem mass spectrometry equipment is AB SCIEX API4000 + Tandem mass spectrometer, using the multiple reaction monitoring (MRM) mode, analyzed with the ESI+ source. The spray voltage is 5500 V, the curtain gas is 20 Psi, the nebulizing gas is 55 Psi, the auxiliary heating gas is 60 Psi, and the needle temperature is 450 °C. After optimizing factors such as the cone voltage and collision voltage for the characteristic ions, the mass spectrometry analysis parameters are determined, as shown in Table 1. Figure 1 It is the chromatogram of the antibiotic reference substance. Among them, (a) is the chromatogram of the lincomycin reference substance, (b) is the chromatogram of the clindamycin reference substance, (c) is the chromatogram of the erythromycin reference substance, (d) is the chromatogram of the clarithromycin reference substance, (e) is the chromatogram of the roxithromycin reference substance, and (f) is the chromatogram of the roxithromycin-D7 reference substance.

[0044] Table 1 Mass spectrometry analysis parameters of six compounds

[0045]

[0046] Example 2

[0047] Example 2 is different from Example 1 in that in step (1) sample pretreatment: the water sample is filtered through a polytetrafluoroethylene filter membrane with a pore size of 0.22 μm. If there are many particulate matters in the water sample, it can be centrifuged at 6000 rmp for 10 min first, and after taking the supernatant, the particulate matters are removed by filter membrane filtration; ammonia water is added to adjust its pH value to 9.5, and it is put into a sample bottle for measurement.

[0048] Other conditions are the same as those in Example 1.

[0049] Example 3

[0050] Example 3 is different from Example 1 in that in step (1) sample pretreatment: the water sample is filtered through a polytetrafluoroethylene filter membrane with a pore size of 0.22 μm. If there are many particulate matters in the water sample, it can be centrifuged at 6000 rmp for 10 min first, and after taking the supernatant, the particulate matters are removed by filter membrane filtration; ammonia water is added to adjust its pH value to 10.5, and it is put into a sample bottle for measurement.

[0051] Other conditions are the same as those in Example 1.

[0052] Comparative Example 1

[0053] It is the same as the detection method in Example 1, and the difference is only in the conditions of the on-line extraction pretreatment in step (2), and the others remain unchanged: the sample injection volume is 5 mL, the proportion of mobile phase A in the initial mobile phase is 100%; the proportion of mobile phase A in the mobile phase from 0 min to 4.5 min is 100%; the proportion of mobile phase C in the mobile phase from 4.5 min to 7.5 min is 100%; the proportion of mobile phase A in the mobile phase from 7.5 min to 11 min is 100%. Among them, mobile phase A is an aqueous solution, mobile phase B is a methanol solution, and mobile phase C is methanol / acetone / n-hexane (1:1:1). The chromatograms of different mobile phases in this comparative example are as Figure 2 shown.

[0054] It can be Figure 2 seen that when mobile phase A is an aqueous solution, the absolute response value of the analyte on the mass spectrum is significantly lower than that of a 50 mM ammonium bicarbonate aqueous solution. For example, the peak areas of lincomycin and clarithromycin are only one-third of those under the condition of a 50 mM ammonium bicarbonate aqueous solution. It can be seen that the enrichment efficiency of the analyte on the on-line solid phase extraction column is greatly reduced in a neutral mobile phase. In addition, continuing to increase the ammonium bicarbonate concentration does not increase the response of lincomycin and clarithromycin, but instead leads to a decrease in the response of roxithromycin and erythromycin. Therefore, 50 mM ammonium bicarbonate is the optimal concentration. Compared with adjusting the pH value of the mobile phase by adding ammonia water, the ammonium bicarbonate solution is more stable, the pH value is not easy to fluctuate, and it can meet the needs of a large number of samples and long-term analysis, ensuring the accuracy of the results.

[0055] When the proportion of mobile phase A is 100% from 0 min to 4.5 min, it has little impact on samples with a relatively clean matrix. However, for samples of wastewater or sewage with a more complex matrix, matrix effects are likely to occur, leading to deviation of results. When the proportion of mobile phase A is 98% and the proportion of mobile phase B is 2.0% from 0.5 min to 4.5 min, interfering substances in the sample can be washed away by elution, eliminating matrix effects.

[0056] Comparative Example 2

[0057] The detection method is the same as that in Example 1, except for the conditions of the high-performance liquid chromatography in step (3), with other conditions remaining unchanged: Mobile phase A1 and mobile phase B1 are mixed in proportion. Mobile phase conditions: Initially, it is 90% mobile phase A1 and 10% mobile phase B1; from 4.5 min to 8.5 min, the proportion of mobile phase B1 increases from 10% to 95%, while the proportion of mobile phase A1 decreases from 90% to 5%; from 8.5 min to 9.0 min, it is 95% mobile phase B1 and 5% mobile phase A1; from 9.0 min to 10.5 min, the proportion of mobile phase A1 increases from 5% to 90%, while the proportion of mobile phase B1 decreases from 95% to 10%; from 10.5 min to 11.0 min, it is 90% mobile phase A1 and 10% mobile phase B1. Among them, mobile phase A1 is a pure aqueous solution, and mobile phase B1 is an acetonitrile solution. The chromatograms of different mobile phases in this comparative example are as Figure 3 shown.

[0058] From Figure 3It can be seen that when the mobile phase A1 is a pure aqueous solution, the peak shapes of erythromycin, clarithromycin, and roxithromycin are broadened, and the detector response is low. When the mobile phase A1 is an aqueous solution containing 0.2% acetic acid and 0.1% ammonia water, the peak shapes of erythromycin, clarithromycin, and roxithromycin are significantly improved, and the detector response value is significantly increased. In the present invention, on the one hand, the mobile phases A1 and B1 serve as the mobile phases of ultra-high performance liquid chromatography, responsible for the gradient elution and separation of the analytes on the liquid chromatography column; on the other hand, the mobile phases A1 and B1 also play a role in eluting the analytes from the online solid-phase extraction column. Therefore, (1) 0.1% acetic acid and 0.1% ammonia water in the mobile phase A1 react to form ammonium acetate, thereby producing a salting-out effect, improving the peak shapes of erythromycin, clarithromycin, and roxithromycin during the separation process on the liquid chromatography column, and increasing the response of these substances on the detector; (2) the generated ammonium acetate and the excess acetic acid after the reaction form an acetic acid-ammonium acetate system, which can be used as a mobile phase regulator to lower the pH of the mobile phase to about 4-5.5, improving the elution ability of the mobile phase on the online solid-phase extraction column. Compared with the formic acid-formic acid ammonium system (pH = 3-4.5), the pH value of the acetic acid-ammonium acetate system is more suitable for the elution of the above-mentioned substances; (3) the acetic acid-ammonium acetate system can adjust the ionic strength, enhance the ionization degree of the sample molecules, making them more easily detected by the mass spectrometer and improving the detection sensitivity.

[0059] Comparative Example 3

[0060] The detection method is the same as that in Example 1, except for the conditions of online solid-phase extraction in step (2), and the others remain unchanged: the sample loading volume is 5 mL, and the initial proportion of the mobile phase A is 100%; from 0 min to 0.5 min, it is 100% of the mobile phase A, from 0.5 min to 4.5 min, it is 98% of the mobile phase A and 2% of the mobile phase B; from 4.5 min to 7.5 min, it is 100% of the mobile phase C; from 7.5 min to 11 min, it is 100% of the mobile phase A;. Among them, the mobile phase A is a 2% ammonia water solution, the mobile phase B is a methanol solution, and the mobile phase C is methanol / acetone / n-hexane (1:1:1). The results of the spike recovery rates of samples with different storage times are as Figure 4 shown.

[0061] As Figure 4As can be seen, the spiked recovery rate of the samples gradually decreases with the increase or decrease of the storage time of the 2% ammonia water solution. This is mainly because more than 95% of the ammonia water in the dilute ammonia water solution exists in the form of NH3·H2O, and the decomposition of ammonia monohydrate will occur in the aqueous solution: NH3·H2O = NH3↑ + H2O. Therefore, the ammonia water in the solution will decompose and have strong volatility, and gradually volatilize with the increase of time, resulting in a gradual decrease in the proportion of ammonia water in the mixed solution, thereby changing the composition of the mobile phase, causing the pH of the mixed solution to change, and the spiked recovery rate of the samples to decrease accordingly. However, the 50 mM ammonium bicarbonate aqueous solution does not have the above-mentioned volatility problem.

[0062] Test Example 1 Standard Curve and Detection Limit

[0063] Prepare a standard solution of 1 ng / ~100 ng / L with ultrapure water, detect it under the above instrument conditions, record the peak area, and draw a peak area-concentration standard curve (n = 7). The results are shown in Table 2.

[0064] Table 2 Standard Curves, Correlation Coefficients and Detection Limits of Five Compounds (n = 7)

[0065]

[0066] As can be seen from Table 2, the linear correlation coefficient of the standard curve is greater than 0.9950, and the detection limit is 0.2~0.4 ng / L, which can meet the requirements of relevant determinations.

[0067] Test Example 2 Precision and Accuracy

[0068] Under these conditions, the actual samples were extracted in this invention for precision and accuracy experiments (n = 7), and the results are shown in Table 3.

[0069] The calculation formula for the recovery rate is: K(%) = (A - B) / C × 100%; where: A: the measured amount of the sample added with the standard substance, unit ng / L, B: the measured amount of this substance in the sample, unit ng / L, C: the added amount of the standard substance, unit ng / L.

[0070] Table 3 Precision and Accuracy (n = 7)

[0071]

[0072] As can be seen from Table 3, the recovery rates of lincomycin, clindamycin, erythromycin, clarithromycin and roxithromycin in the samples are 87.8~112%, and the deviations are 3.4~9.5%, with good precision and accuracy.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for rapid detection of lincomycin, clindamycin, erythromycin, clarithromycin and roxithromycin in water, characterized in that, The method includes the following steps: (1) Sample pretreatment; (2) Adding the sample to an online solid-phase extraction column for solid-phase extraction enrichment. The mobile phase conditions for the solid-phase extraction enrichment are as follows: the initial proportion of mobile phase A is 100%; from 0 min to 0.5 min, it is 100% mobile phase A; from 0.5 min to 4.5 min, it is 98% mobile phase A and 2% mobile phase B; from 4.5 min to 7.5 min, it is 100% mobile phase C; from 7.5 min to 11 min, it is 100% mobile phase A; wherein, mobile phase A is an aqueous solution of 50 mM ammonium bicarbonate, mobile phase B is a methanol solution, and mobile phase C is a mixed solution of methanol, acetone, and n-hexane; (3) Eluting and separating the sample after solid-phase extraction enrichment by high-performance liquid chromatography, and detecting the eluate with a mass spectrometer; In the step (3), the mobile phase conditions for eluting and separating by high-performance liquid chromatography are as follows: mobile phase A1 and mobile phase B1 are mixed in proportion. Initially, it is 90% mobile phase A1 and 10% mobile phase B1; from 4.5 min to 8.5 min, the proportion of mobile phase B1 increases from 10% to 95%, and at the same time, the proportion of mobile phase A1 decreases from 90% to 5%; from 8.5 min to 9.0 min, it is 95% mobile phase B1 and 5% mobile phase A1; from 9.0 min to 10.5 min, the proportion of mobile phase A1 increases from 5% to 90%, and at the same time, the proportion of mobile phase B1 decreases from 95% to 10%; from 10.5 min to 11.0 min, it is 90% mobile phase A1 and 10% mobile phase B1; wherein, mobile phase A1 is an aqueous solution containing acetic acid and ammonia. The volume concentration of acetic acid in the aqueous solution is 0.2%, and the volume concentration of ammonia is 0.1%. Mobile phase B1 is an acetonitrile solution; The flow rate for eluting and separating by high-performance liquid chromatography is as follows: from 0 min to 4.5 min, the flow rate is 0.1 mL / min; from 4.5 min to 11.0 min, the flow rate is 0.4 mL / min; The chromatographic column for eluting and separating by high-performance liquid chromatography is an Acquity BEH130 liquid chromatography column, with a specification of 100 mm × 2.1 mm i.d., 1.7 μm.

2. The method according to claim 1, wherein In the step (1), the specific method for sample pretreatment is: filtering the sample through a filter membrane and adjusting the pH to 9.5 - 10.5 with ammonia water.

3. The method according to claim 2, characterized in that The filter membrane is a polytetrafluoroethylene filter membrane with a pore size of 0.22 μm.

4. The method according to claim 1, characterized in that, In the step (2), the volume ratio of methanol, acetone, and n-hexane in mobile phase C is 1:1:

1.

5. The method according to claim 1, wherein In the step (2), the flow rate setting for the solid-phase extraction enrichment is as follows: from 0 min to 3.5 min, the flow rate is 2.0 mL / min; from 3.5 min to 4.5 min, the flow rate is 0.01 mL / min; from 4.5 min to 11.0 min, the flow rate is 2.0 mL / min. The online solid-phase extraction column is an Oasis HLB column.

6. The method according to claim 1, characterized in that, In the step (2), the sample loading volume for the online solid-phase extraction column to perform solid-phase extraction enrichment is 0.1 - 5 mL.

7. The method according to claim 1, wherein In the step (3), the column temperature during the elution separation by high performance liquid chromatography is 40°C.

8. The method according to claim 1, characterized in that, In the step (3), the detection conditions of the mass spectrometry are as follows: the multiple reaction monitoring mode is adopted, the ESI+ source is used for analysis, the spray voltage is 5500 V, the curtain gas is 20 Psi, the nebulizing gas is 55 Psi, the auxiliary heating gas is 60 Psi, and the needle temperature is 450°C.

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