阅读说明：本技术 吗氯贝胺的检测方法 (Detection method of moclobemide ) 是由 刘杏立 贾永娟 倪君君 于 2021-01-12 设计创作，主要内容包括：本发明提供了吗氯贝胺的检测方法,包括：制备具有吗氯贝胺和内标物的至少三种浓度的标准溶液,标准溶液中的内标物的量相同；利用液相色谱仪在检测条件下检测每一种标准溶液,获得标准溶液对应的第一检测结果；根据各个第一检测结果、标准溶液中吗氯贝胺的浓度和内标物的浓度,拟合吗氯贝胺的标准曲线方程；取待处理样品离心后的第一上清液；向第一上清液内加入内标物,涡旋混匀,顺次加入萃取剂,对第一上清液进行萃取,得到待测样品；利用液相色谱仪在检测条件下检测待测样品,获得待测样品的第二检测结果；基于标准曲线方程和第二检测结果,得到待测样品中吗氯贝胺的浓度。本方案能够缩短样品检测时间。(The invention provides a detection method of moclobemide, which comprises the following steps: preparing standard solutions having at least three concentrations of moclobemide and an internal standard, the internal standard being present in the standard solutions in the same amount; detecting each standard solution by using a liquid chromatograph under detection conditions to obtain a first detection result corresponding to the standard solution; fitting a standard curve equation of the moclobemide according to each first detection result, the concentration of the moclobemide in the standard solution and the concentration of the internal standard substance; taking a first supernatant after a sample to be treated is centrifuged; adding an internal standard substance into the first supernatant, uniformly mixing by vortex, sequentially adding an extracting agent, and extracting the first supernatant to obtain a sample to be detected; detecting the sample to be detected by using a liquid chromatograph under the detection condition to obtain a second detection result of the sample to be detected; and obtaining the concentration of the moclobemide in the sample to be detected based on the standard curve equation and the second detection result. The scheme can shorten the sample detection time.)

1. The detection method of moclobemide is characterized by comprising the following steps:

preparing at least three concentrations of standard solutions, wherein the standard solutions are solutions with moclobemide and internal standard substances, and the amount of the internal standard substances in the at least three concentrations of standard solutions is the same;

respectively detecting each standard solution by using a liquid chromatograph under a preset detection condition to obtain a first detection result corresponding to each standard solution;

fitting a standard curve equation of the moclobemide according to each first detection result, the concentration of the moclobemide in the standard solution and the concentration of an internal standard substance;

centrifuging a sample to be processed, and taking a centrifuged first supernatant;

adding an internal standard substance into the first supernatant, uniformly mixing by vortex, sequentially adding an extracting agent, and extracting the first supernatant to obtain a sample to be detected;

detecting the sample to be detected by using a liquid chromatograph under the detection condition to obtain a second detection result of the sample to be detected;

and obtaining the concentration of the moclobemide in the sample to be detected based on the standard curve equation and the second detection result.

2. The method of detecting moclobemide of claim 1,

the liquid phase condition among the detection conditions includes:

a reverse phase C18 chromatography column;

the aqueous phase in the elution mobile phase comprises: an aqueous solution containing sodium dihydrogen phosphate and disodium hydrogen phosphate;

eluting the organic phase in the mobile phase comprises: acetonitrile solution;

the column temperature is 20-50 ℃;

flow rates include 0.3-2.0 mL/min.

3. The method of detecting moclobemide of claim 1,

the liquid phase condition among the detection conditions includes:

the volume ratio of aqueous phase to organic phase in the elution mobile phase comprises: 72 percent, 28 percent to 82 percent and 18 percent.

4. The method of detecting moclobemide of claim 1,

the ultraviolet detection condition in the detection conditions comprises:

detection wavelength of ultraviolet detector: 230-250 nm.

5. The method of detecting moclobemide of claim 1,

the two variables of the standard curve equation are respectively: the ratio of the chromatographic peak area of the moclobemide in the standard solution to the chromatographic peak area of the internal standard substance, and the ratio of the concentration of the moclobemide in the standard solution to the concentration of the internal standard substance.

6. The method of detecting moclobemide of claim 1,

the extraction agent comprises: n-hexane containing 0% -40% methyl tert-butyl ether.

7. The method of detecting moclobemide of claim 1,

the volume ratio of the first supernatant to the extracting agent is any one of 1:5-1: 15.

8. The method of detecting moclobemide of claim 1,

adding an internal standard substance into the first supernatant, uniformly mixing by vortex, sequentially adding an extracting agent, and extracting the first supernatant to obtain a sample to be detected, wherein the method comprises the following steps:

adding an internal standard substance into the first supernatant, and carrying out vortex mixing for 0.5-1min at the rotating speed of 1500-2500 rpm;

adding the extracting agent in sequence, mixing for 8-12min at the rotation speed of 1500-;

blowing the second supernatant fluid by using nitrogen, sequentially adding the complex solution, carrying out vortex mixing for 0.5-1.5min at the rotating speed of 1500-charge 2500rpm, centrifuging for 4-6min at the rotating speed of 10000-charge 14000rpm, and taking the centrifuged third supernatant fluid as a sample to be detected.

9. The method of detecting moclobemide of claim 8,

the compound solution comprises: methanol solution containing 0% -70% water.

10. The method of detecting moclobemide according to any one of claims 1 to 9,

the internal standard comprises pirfenidone.

Technical Field

The invention relates to the technical field of biological detection, in particular to a detection method of moclobemide.

Background

Moclobemide, namely 4-chloro-N-2- (4-morpholinyl) ethylbenzamide, is white or off-white crystal or crystalline powder; no smell, slightly bitter taste. Is soluble in methanol, ethanol or chloroform, soluble in acetone, and slightly soluble in water.

At present, the method generally adopted for detecting the content of moclobemide in a sample is high performance liquid chromatography. The existing detection method usually carries out more complex pretreatment on a sample to be detected, and the complex operation comprises adding an alkaline solution, drying by high-temperature nitrogen and the like, so that the consumed time is more, and the sample detection time is longer.

Disclosure of Invention

The invention provides a detection method of moclobemide, which can shorten the detection time of a sample.

In order to solve the above problem, an embodiment of the present invention provides a detection method of moclobemide, including:

preparing at least three concentrations of standard solutions, wherein the standard solutions are solutions with moclobemide and internal standard substances, and the amount of the internal standard substances in the at least three concentrations of standard solutions is the same;

respectively detecting each standard solution by using a liquid chromatograph under a preset detection condition to obtain a first detection result corresponding to each standard solution;

fitting a standard curve equation of the moclobemide according to each first detection result, the concentration of the moclobemide in the standard solution and the concentration of an internal standard substance;

centrifuging a sample to be processed, and taking a centrifuged first supernatant;

adding an internal standard substance into the first supernatant, uniformly mixing by vortex, sequentially adding an extracting agent, and extracting the first supernatant to obtain a sample to be detected;

detecting the sample to be detected by using a liquid chromatograph under the detection condition to obtain a second detection result of the sample to be detected;

and obtaining the concentration of the moclobemide in the sample to be detected based on the standard curve equation and the second detection result.

Preferably, in order to more accurately detect the concentration of moclobemide in the sample to be detected, the internal standard substance in the standard solution and the internal standard substance in the sample to be detected are both pirfenidone.

It is noted that the first supernatant comprises serum or plasma, and the upper organic phase is selected after extraction.

Specifically, the standard solutions of the series of concentrations were prepared as follows:

(1) preparation of standard stock solution

Accurately weighing the moclobemide standard substance, placing the moclobemide standard substance in a volumetric flask, dissolving the moclobemide standard substance in a methanol solution containing 0-70% of water, and fixing the volume to the marked line of the volumetric flask to obtain a standard stock solution, and storing the standard stock solution at-80 ℃.

(2) Preparation of standard working solution

Taking a proper amount of the standard stock solution in the step (1), diluting and mixing the standard stock solution by using a methanol solution containing 0-70% of water as a diluent to obtain a standard working solution containing 1000-30000ng/mL moclobemide, and storing the standard working solution at-80 ℃.

(3) Preparation of internal standard stock solution

Accurately weighing an internal standard substance pirfenidone standard substance, placing the internal standard substance into a volumetric flask, dissolving the internal standard substance by using methanol, and fixing the volume to the marked line of the volumetric flask to obtain an internal standard stock solution, and storing the internal standard stock solution at the temperature of minus 80 ℃.

(4) Preparation of internal standard working solution

Taking a proper amount of the internal standard stock solution obtained in the step (3), diluting the internal standard stock solution by using a methanol solution containing 0-70% of water as a diluent to obtain an internal standard working solution containing pirfenidone, and storing the internal standard working solution at-80 ℃.

(5) Calibration of standard solutions

And (3) respectively transferring the standard working solution with different concentrations in the step (2) and the internal standard working solution in the step (4) into centrifuge tubes, respectively adding methanol solution containing 0-70% of water into each centrifuge tube, mixing to prepare at least three mixed solutions with different concentrations, and uniformly mixing the mixed solutions in a vortex manner at the rotation speed of 1500-2500rpm for 0.5-1min to obtain the standard solutions.

In order to ensure that the moclobemide is fully dissolved, and simultaneously, in order to reduce the volatilization of the standard stock solution and the standard working solution in the repeated use process and ensure the stability of the standard working solution, a methanol solution containing 0-70% of water is selected for dissolving and diluting.

In order to improve the solubility of pirfenidone, methanol is selected for dissolving, and meanwhile, in order to reduce volatilization of the internal standard working solution in the repeated use process and ensure the stability of the internal standard working solution, the diluent of pirfenidone is a methanol solution containing 0-70% of water.

Preferably, the liquid phase condition among the detection conditions includes: a reverse phase C18 chromatography column;

the aqueous phase in the elution mobile phase comprises: an aqueous solution containing sodium dihydrogen phosphate and disodium hydrogen phosphate;

eluting the organic phase in the mobile phase comprises: acetonitrile solution;

the column temperature is 20-50 ℃; flow rates include 0.3-2.0 mL/min.

Specifically, the chromatographic column comprises Phenomenex-a C18 column, a column having a length of 100mm, an internal diameter of 4.6mm and a packing particle size of 5 μm; an Agilent Eclipse Plus C18 column having a length of 100mm, an internal diameter of 4.6mm and a packing particle size of 3.5 μm; waters Xbridge C18, column with a length of 150mm, an internal diameter of 4.6mm and a packing particle size of 5 μm.

Specifically, the pH range of the aqueous solution containing sodium dihydrogen phosphate and disodium hydrogen phosphate in the aqueous phase includes pH6.5 to pH8, for example, pH6.5, pH6.7, pH7, pH7.3, pH7.5, pH7.7 and pH8, wherein when the elution mobile phase is at pH6.7, an aqueous solution containing 10mM of sodium dihydrogen phosphate and 5mM of disodium hydrogen phosphate can be used, so that the chromatographic peak shape of the target substance can be ensured to be good.

For the column temperature, 20-50 ℃ means any temperature value in the range of 20 ℃ to 50 ℃, for example, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃ and 50 ℃.

With respect to flow rates, 0.3-2.0mL/min refers to any flow rate in the range of 0.3mL/min to 2.0mL/min, such as 0.3mL/min, 0.5mL/min, 0.8mL/min, 1.0mL/min, 1.5mL/min, and 2.0 mL/min.

Preferably, the liquid phase condition among the detection conditions includes:

the volume ratio of aqueous phase to organic phase in the elution mobile phase comprises: 72 percent, 28 percent to 82 percent and 18 percent.

For the volume ratio of the aqueous phase to the organic phase in the elution mobile phase, 72% to 28% to 82% to 18% means any ratio in the range of 72% to 28% to 82% to 18%, for example, 72% to 28%, 74% to 26%, 76% to 24%, 78% to 22%, 80% to 20% and 82% to 18%.

For example, the volume of the aqueous phase represents 77% of the volume of the elution mobile phase, and the volume of the organic phase represents 23% of the volume of the elution mobile phase; the volume of the aqueous phase represents 80% of the volume of the elution mobile phase and the volume of the organic phase represents 20% of the volume of the elution mobile phase.

Specifically, when the volume percentage of the aqueous phase in the elution mobile phase is less than 72%, the separation degree of the moclobemide and the internal standard substance is poor, and the detection accuracy of the moclobemide is influenced; when the volume ratio of the aqueous phase in the elution mobile phase is more than 82%, the detection time of the moclobemide is increased because the time of the appearance of the peaks of the moclobemide is later, so that the volume ratio of the aqueous phase to the organic phase in the elution mobile phase comprises 72% to 28% to 82% to 18%.

Preferably, the ultraviolet detection condition among the detection conditions includes:

the liquid chromatograph adopts an ultraviolet detector, and the detection wavelength is as follows: 230-250 nm.

For the detection wavelength in the ultraviolet detection condition, 230-250nm refers to any value in the range of 230nm to 250nm, for example, the detection wavelength may be 230nm, 235nm, 240nm, 245nm and 250 nm.

Preferably, the two variables of the standard curve equation are respectively: the ratio of the chromatographic peak area of the moclobemide in the standard solution to the chromatographic peak area of the internal standard substance, and the ratio of the concentration of the moclobemide in the standard solution to the concentration of the internal standard substance.

Specifically, if the ratio of the chromatographic peak area of the moclobemide to the chromatographic peak area of the internal standard substance is used as the x value (i.e., independent variable) of the standard curve equation, the ratio of the concentration of the moclobemide to the concentration of the internal standard substance is used as the y value (i.e., dependent variable) of the standard curve equation.

If the ratio of the chromatographic peak area of the moclobemide to the chromatographic peak area of the internal standard substance is used as the y value (i.e. dependent variable) of the standard curve equation, the ratio of the concentration of the moclobemide to the concentration of the internal standard substance is used as the x value (i.e. independent variable) of the standard curve equation.

Preferably, in order to better remove impurities and purify the target substance, the extracting agent for extracting the first supernatant after the internal standard substance is added comprises: n-hexane containing 0% -40% methyl tert-butyl ether.

For the extractant, 0% to 40% refers to any value in the range of 0% to 40%, for example, the extractant is n-hexane containing 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, and 40% of methyl t-butyl ether.

Preferably, in order to ensure sufficient extraction of the target object, the volume ratio of the first supernatant to the extracting agent is any one of 1:5 to 1: 15.

By volume ratio of the first supernatant to the extractant, 1:5 to 1:15 is meant any ratio in the range of 1:5 to 1:15, such as 1:5, 1:6, 1:8, 1:9, 1:10, 1:12 and 1: 15.

Specifically, when the volume of the first supernatant is 100. mu.L, the volume of the extractant may be any value in the range of 500. mu.L to 1500. mu.L.

Preferably, the first and second electrodes are formed of a metal,

adding an internal standard substance into the first supernatant, uniformly mixing by vortex, sequentially adding an extracting agent, and extracting the first supernatant to obtain a sample to be detected, wherein the method comprises the following steps:

adding an internal standard substance into the first supernatant, and carrying out vortex mixing for 0.5-1min at the rotating speed of 1500-2500 rpm;

adding the extracting agent in sequence, mixing for 8-12min at the rotation speed of 1500-;

blowing the second supernatant fluid by using nitrogen, sequentially adding the complex solution, carrying out vortex mixing for 0.5-1.5min at the rotating speed of 1500-charge 2500rpm, centrifuging for 4-6min at the rotating speed of 10000-charge 14000rpm, and taking the centrifuged third supernatant fluid as a sample to be detected.

Specifically, after the internal standard substance is added into the first supernatant, in order to enable the internal standard substance and the first supernatant to be mixed more uniformly, vortex mixing can be performed, then the extracting agent is added into the mixed first supernatant, vortex mixing is performed, extraction is performed, the mixed first supernatant is purified through the extracting agent, then centrifugation is performed, the centrifuged second supernatant is taken, and the purpose of separating impurities from a target object is achieved. Because the content of the target object is low after extraction by using the extracting agent, and the extracting agent is incompatible with the solution used by the detection system of the liquid chromatograph, in order to facilitate detection, nitrogen can be used for blow-drying so as to concentrate the second supernatant, a complex solution is added after concentration, the complex solution is compatible with the solution used by the detection system of the liquid chromatograph, the extracting agent is replaced by the complex solution, and vortex is performed so that the target object is uniformly distributed in the complex solution.

For the vortex rotation speed, 1500-.

For the vortex time after the internal standard substance is added, 0.5-1min refers to any time in the range of 0.5min to 1min, such as 0.5min, 0.6min, 0.7min, 0.8min, 0.9min and 1 min.

For the vortex time and the centrifugation time after adding the extractant, 8-12min refers to any time within the range of 8min to 12min, such as 8min, 9min, 10min, 11min and 12 min.

For the centrifugal rotation speed, 10000-14000rpm refers to any rotation speed within the range of 10000rpm to 14000rpm, such as 10000rpm, 11000rpm, 12000rpm, 13000rpm and 14000 rpm.

For the vortex time after adding the redissolution, 0.5-1.5min refers to any time within the range of 0.5min to 1.5min, such as 0.5min, 0.6min, 0.8min, 1.0min, 1.2min, 1.4min and 1.5 min.

For the centrifugation time after adding the reconstitution liquid, 4-6min refers to any time within the range of 4min to 6min, such as 4min, 4.5min, 5min, 5.5min and 6 min.

Preferably, the double solution comprises: methanol solution containing 0% -70% water.

For a reconstituted solution, 0% to 70% refers to any value in the range of 0% to 70%, for example, a reconstituted solution is a methanol solution containing 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70% water.

The invention provides a detection method of moclobemide, which can obtain a first detection result corresponding to a standard solution with each concentration by detecting the standard solution containing the moclobemide with different concentrations through a liquid chromatograph. The serum or the plasma after centrifugation can be obtained by carrying out centrifugation treatment on the sample to be treated, and the internal standard substance and the extracting agent are sequentially added for extraction, so that the interference of impurities on sample detection can be reduced, and the sample to be detected which can be detected can be obtained. And detecting the sample to be detected by using the liquid chromatograph under the same detection condition with the standard solution to obtain a second detection result, and obtaining the content of the moclobemide in the sample to be detected based on the standard curve equation and the second detection result. Because alkaline solution is not required to be added before extraction, high-temperature nitrogen blow-drying is not required, and the pretreatment operation is simpler, the detection time of the sample to be detected can be shortened.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

Fig. 1 is a flowchart of a detection method of moclobemide according to an embodiment of the present invention;

fig. 2 is a chromatogram of moclobemide and an internal standard in a sample to be tested, provided by an embodiment of the invention;

FIG. 3 is a chromatogram of moclobemide and an internal standard in a standard solution provided by an embodiment of the invention;

FIG. 4 is a graph of the linear relationship of moclobemide provided by one embodiment of the invention;

FIG. 5 is a chromatogram for a flow rate of 1.0mL/min at a column temperature of 20 ℃ according to an embodiment of the present invention;

FIG. 6 is a chromatogram for a column temperature of 50 ℃ and a flow rate of 1.0mL/min, according to an embodiment of the present invention;

FIG. 7 is a chromatogram for a flow rate of 0.2mL/min at a column temperature of 40 ℃ according to an embodiment of the present invention;

FIG. 8 is a chromatogram for a flow rate of 0.3mL/min at a column temperature of 40 ℃ according to an embodiment of the present invention;

FIG. 9 is a chromatogram for a flow rate of 0.5mL/min at a column temperature of 40 ℃ according to an embodiment of the present invention;

FIG. 10 is a chromatogram for a flow rate of 0.8mL/min at a column temperature of 40 ℃ according to an embodiment of the present invention;

FIG. 11 is a chromatogram for a flow rate of 1.5mL/min at a column temperature of 40 ℃ according to an embodiment of the present invention;

FIG. 12 is a chromatogram for a flow rate of 2.0mL/min at a column temperature of 40 ℃ according to an embodiment of the present invention;

FIG. 13 is a chromatogram of an elution mobile phase according to an embodiment of the present invention, wherein the volume ratio of the aqueous phase to the organic phase is 87%: 13%;

FIG. 14 is a chromatogram of an elution mobile phase according to an embodiment of the present invention, wherein the volume ratio of the aqueous phase to the organic phase is 82%: 18%;

FIG. 15 is a chromatogram of an elution mobile phase according to an embodiment of the present invention, wherein the volume ratio of the aqueous phase to the organic phase is 72% to 28%;

FIG. 16 is a chromatogram of an elution mobile phase according to an embodiment of the present invention, wherein the volume ratio of the aqueous phase to the organic phase is 67% to 33%;

FIG. 17 is a chromatogram of an extractant of methyl tert-butyl ether according to an embodiment of the present invention;

FIG. 18 is a chromatogram of an extraction reagent provided in accordance with an embodiment of the present invention when ethyl acetate is used;

FIG. 19 is a chromatogram of an extractant in dichloromethane provided by an embodiment of the invention;

FIG. 20 is a chromatogram of an extractant of n-hexane containing 50% methyl-t-butyl ether according to an embodiment of the present invention;

FIG. 21 is a chromatogram of an extractant of n-hexane containing 40% methyl-t-butyl ether according to an embodiment of the present invention;

FIG. 22 is a chromatogram of an extractant of n-hexane containing 20% methyl-t-butyl ether according to an embodiment of the present invention;

FIG. 23 is a chromatogram of an extractant of n-hexane containing 10% methyl-t-butyl ether according to an embodiment of the present invention;

FIG. 24 is a chromatogram of an extractant of n-hexane in accordance with an embodiment of the present invention;

FIG. 25 is a chromatogram of a 1:5 volume ratio of first supernatant to extractant provided in accordance with an embodiment of the present invention;

FIG. 26 is a chromatogram of a 1:8 volume ratio of first supernatant to extractant provided by an embodiment of the invention;

FIG. 27 is a chromatogram of a first supernatant and an extractant at a 1:12 volume ratio provided by an embodiment of the invention;

FIG. 28 is a chromatogram of a 1:15 volume ratio of first supernatant to extractant provided in accordance with an embodiment of the present invention;

FIG. 29 is a chromatogram of an Agilent Eclipse Plus C18 column according to an embodiment of the present invention;

FIG. 30 is a chromatogram of a Waters Xbridge C18 column according to an embodiment of the present invention;

FIG. 31 is a chromatogram of propranolol as an internal standard provided by an embodiment of the invention;

FIG. 32 is a chromatogram of sulfamethoxazole as an internal standard provided by an embodiment of the present invention;

FIG. 33 is a chromatogram for a detection wavelength of 220nm provided by an embodiment of the present invention;

FIG. 34 is a chromatogram for a detection wavelength of 230nm provided by an embodiment of the present invention;

FIG. 35 is a chromatogram for a detection wavelength of 250nm provided by an embodiment of the present invention;

FIG. 36 is a chromatogram for a detection wavelength of 260nm provided by an embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer and more complete, the technical solutions in the embodiments of the present invention will be described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention, and based on the embodiments of the present invention, all other embodiments obtained by a person of ordinary skill in the art without creative efforts belong to the scope of the present invention.

At present, a liquid chromatograph is used for detecting a sample to be detected, the antibiotic chloramphenicol is used as an internal standard substance for the sample to be detected, but the antibiotic chloramphenicol and moclobemide are jointly used, so that the quantification is inaccurate, and the accuracy of the detection of the sample to be detected is influenced.

Also, the sample to be tested needs at least 500 μ L, so that the user's compliance is poor.

In addition, in the pretreatment process, an alkaline solution (such as a sodium hydroxide solution, a saturated sodium carbonate solution and a saturated trisodium phosphate solution) needs to be added before an extracting agent is added to the sample to be detected, and nitrogen is required to be blown dry in a high-temperature water bath after extraction, so that the complexity of the pretreatment process is increased, the time required in the whole detection process is increased, and the analysis time is longer, so that the overall detection time of the moclobemide in the sample to be detected is longer.

Based on the above problem, an embodiment of the present invention provides a detection method of moclobemide, as shown in fig. 1, including:

step 101: preparing at least three concentrations of standard solutions, wherein the standard solutions are solutions with moclobemide and internal standard substances, and the amount of the internal standard substances in the at least three concentrations of standard solutions is the same;

step 102: respectively detecting each standard solution by using a liquid chromatograph under a preset detection condition to obtain a first detection result corresponding to each standard solution;

step 103: fitting a standard curve equation of the moclobemide according to each first detection result, the concentration of the moclobemide in the standard solution and the concentration of an internal standard substance;

step 104: centrifuging a sample to be processed, and taking a centrifuged first supernatant;

step 105: adding an internal standard substance into the first supernatant, uniformly mixing by vortex, sequentially adding an extracting agent, and extracting the first supernatant to obtain a sample to be detected;

step 106: detecting the sample to be detected by using a liquid chromatograph under the detection condition to obtain a second detection result of the sample to be detected;

step 107: and obtaining the concentration of the moclobemide in the sample to be detected based on the standard curve equation and the second detection result.

In the embodiment of the invention, the liquid chromatograph is used for detecting the standard solutions containing the moclobemide with different concentrations, so that the first detection result corresponding to the standard solution with each concentration can be obtained, and the standard solution contains the internal standard substance pirfenidone, so that the standard curve equation of the moclobemide is obtained by fitting based on the concentration of the moclobemide in the standard solutions with various concentrations, the concentration of the internal standard substance and a plurality of first detection results. The serum or the plasma after centrifugation can be obtained by carrying out centrifugation treatment on the sample to be treated, and the internal standard substance and the extracting agent are sequentially added for extraction, so that the interference of impurities on sample detection can be reduced, and the sample to be detected which can be detected can be obtained. And detecting the sample to be detected by using the liquid chromatograph under the same detection condition with the standard solution to obtain a second detection result, and obtaining the content of the moclobemide in the sample to be detected based on the standard curve equation and the second detection result. Because alkaline solution is not required to be added before extraction, high-temperature nitrogen blow-drying is not required, and the pretreatment operation is simpler, the detection time of the sample to be detected can be shortened.

The detection method of moclobemide is described in detail below by using several examples.

Example 1: preparation of Standard solutions of series of concentrations

(a) Preparation of standard stock solution

Accurately weighing 1.47mg of moclobemide standard substance, placing the moclobemide standard substance in a 2mL volumetric flask, dissolving the moclobemide standard substance in a methanol solution containing 30% of water, fixing the volume to 2mL to obtain a standard stock solution, and storing the standard stock solution at-80 ℃.

(b) Preparation of standard working solution

Taking a proper amount of the standard stock solution in the step (a), diluting and mixing the standard stock solution by using a methanol solution containing 30% of water as a diluent to obtain a standard working solution containing 1000-plus 30000ng/mL moclobemide, and storing the standard working solution at-80 ℃;

wherein, the standard working solution with different concentrations contains moclobemide: 1000ng/mL, 2000ng/mL, 3000ng/mL, 6000ng/mL, 10000ng/mL, 20000ng/mL, 30000 ng/mL.

(c) Preparation of internal standard stock solution

Putting 10mg of pirfenidone standard substance into a 10mL volumetric flask, dissolving by using a methanol solution, and fixing the volume to 10mL to obtain an internal standard stock solution containing 1mg/mL pirfenidone, and storing at-80 ℃.

(d) Preparation of internal standard working solution

Taking a proper amount of the internal standard stock solution in the step (c), diluting the internal standard stock solution by using a methanol solution containing 30% of water as a diluent to obtain an internal standard working solution containing 10 mu g/mL pirfenidone, and storing the internal standard working solution at-80 ℃.

(e) Calibration of standard solutions

And (3) transferring 10 mu L of the seven standard working solutions with different concentrations in the step (b), 10 mu L of the internal standard working solution in the step (d) and 80 mu L of methanol solution containing 30% of water into a 1.5mL centrifuge tube by using a liquid transfer machine, respectively, uniformly mixing for 1min in a vortex mode at the rotating speed of 2000rpm, and mixing to prepare seven standard solutions containing the moclobemide standard substances with different concentrations, wherein the amount of the internal standard substances in the seven standard solutions is the same.

It should be noted that the standard solutions with different concentrations do not need to be operated according to the pretreatment when the sample to be treated is treated, so that the operation is simpler, and the overall detection time of the moclobemide is shortened.

Example 2: fitting standard curve equation

The seven standard solutions in example 1 were respectively detected by a liquid chromatograph to obtain chromatograms of seven standard solutions of moclobemide with different concentrations.

The method comprises the steps of respectively obtaining peak areas corresponding to moclobemide and an internal standard substance in seven standard solutions from a chromatogram of the standard solution of the moclobemide, taking the ratio of the peak area of the moclobemide obtained from the chromatogram of the standard solution with each concentration to the chromatographic peak area of the internal standard substance as the ordinate y1 of a standard curve equation, taking the ratio of the concentration of the moclobemide in the standard solution to the concentration of the internal standard substance as the abscissa x1 of the standard curve equation, carrying out linear regression on seven kinds of data with different concentrations obtained by detection, and fitting to obtain a standard curve equation of y1 ═ x1+ b, and obtaining weight coefficients a and b, wherein the weight coefficient a is the slope of the standard curve equation, and the weight coefficient b is the intercept of the standard curve equation.

The detection conditions include:

a chromatographic column: phenomenex-C18, filler particle size 5 μm, internal diameter 4.6mm, length 100 mm;

the aqueous phase in the elution mobile phase comprises: an aqueous solution containing 10mM sodium dihydrogen phosphate and 5mM disodium hydrogen phosphate;

eluting the organic phase in the mobile phase comprises: acetonitrile solution, the volume ratio of the water phase to the organic phase in the elution mobile phase is 77% to 23%, and the analysis time is 6 min;

the column temperature was 40 ℃; the flow rate was 1.0mL/min and the amount of sample was 5. mu.L.

Ultraviolet detection conditions:

the liquid chromatograph adopts an UltiMate3000 Variable Wavelength Detector, and the detection Wavelength is as follows: 240 nm; response time: 0.6 s; acquisition frequency: 2 Hz.

Example 3: treatment of samples to be tested

3.1 taking at least 500 μ L of blood to be treated, centrifuging at 3500rpm for 10min, taking supernatant serum or plasma as first supernatant, and storing the serum or plasma at-20 deg.C until the serum or plasma is ready for analysis.

3.2 pipetting 10. mu.L of the internal standard working solution of example 1 with a pipette gun into a 1.5mL centrifuge tube, adding 100. mu.L of the serum or plasma of step 3.1, vortex mixing at 2000rpm for 1min, adding 1000. mu.L of an extractant (n-hexane containing 30% methyl t-butyl ether), vortex mixing at 2000rpm for 10min, then centrifuging at 12000rpm for 10min, taking 900. mu.L of the second supernatant (upper organic phase) and placing in another 1.5mL centrifuge tube, blow-drying the pipetted second supernatant at room temperature (25 ℃) with nitrogen, sequentially adding 100. mu.L of a redissolution (methanol solution containing 30% water), vortex mixing at 2000rpm for 1min, and centrifuging at 12000rpm for 5min, taking the centrifuged third supernatant as the sample to be tested.

Example 4: detection of a sample to be tested

And (3) detecting the sample to be detected by using the liquid chromatograph under the detection conditions in the embodiment 2 to obtain a chromatogram of the sample to be detected.

The chromatographic peak area of the moclobemide in the sample to be detected and the chromatographic peak area of the internal standard substance can be obtained from the chromatogram of the sample to be detected, the chromatographic peak area of the moclobemide in the sample to be detected and the chromatographic peak area of the internal standard substance are taken as ordinate y1', and are substituted into the standard curve equation of y1 a x1+ b in the embodiment 2, because the weight coefficients a and b are known, the ratio of the concentration of the moclobemide in the sample to be detected to the concentration of the internal standard substance can be obtained, and because the concentration of the internal standard substance in the sample to be detected is known, the concentration of the moclobemide in the sample to be detected can be obtained through calculation.

Example 5: description of sample Pre-processing and fitting of Standard Curve equations

Example 1 of the present application:

(1)10 mul of internal standard working solution plus 100 mul of serum or plasma to obtain mixed solution;

(2) adding 1000 μ L extractant (n-hexane containing 30% methyl tert-butyl ether), mixing for 10min, and centrifuging to obtain second supernatant;

(3) and taking 900 mu L of the second supernatant, drying the second supernatant by nitrogen at normal temperature (25 ℃), sequentially adding 100 mu L of redissolution (30% water-containing methanol solution), uniformly mixing for 1min, and centrifuging to obtain a third supernatant serving as a sample to be detected.

Comparative example 1: (reversed-phase high performance liquid chromatography for measuring concentration [ J ] of moclobemide in plasma. China Hospital J. Pharmacology, 2004,24(009):533-

(1)10 mu L of internal standard working solution +500 mu L of plasma to obtain mixed solution;

(2) sequentially adding 500 μ L saturated trisodium phosphate solution (pH 11.0) and 5000 μ L dichloromethane, mixing for 20min, and centrifuging to obtain second supernatant;

(3) and drying 3000 mu L of the second supernatant in a water bath at 80 ℃ by using high-purity nitrogen, and sequentially adding 200 mu L of complex solution (acetonitrile solution containing 0.067M potassium dihydrogen phosphate buffer solution) and uniformly mixing to obtain a third supernatant serving as a sample to be detected.

FIG. 2 is a chromatogram of moclobemide and an internal standard in a sample to be tested according to the present application; the unit length of the abscissa of fig. 2 is 0.25, and the unit length of the ordinate is 0.5;

referring to fig. 2 in detail, it can be seen from the present application example 1 and comparative example 1 that the analysis time of the sample of the present invention is 6min, wherein the retention time of moclobemide is about 5.2min, and the retention time of the internal standard substance pirfenidone is about 4.4min, which is shorter than the analysis time of 10min of comparative example 1. The using amount of the sample is only 20% of that of the comparative sample, and the sample better meets the scientific ethical and moral standard, so that the compliance of the personnel to be tested is higher; compared with the pretreatment mode of adding a saturated trisodium phosphate solution (the pH value is 11.0) and drying by high-temperature nitrogen in the comparative example 1, the preparation time of the saturated trisodium phosphate solution is saved, the error of the reagent preparation process is reduced, and meanwhile, the use amount of the solvent is less, so that the method is more environment-friendly; and high-temperature nitrogen is not needed for drying, so that the pretreatment operation is simpler, the consumed time is shorter, and the detection time of the sample to be detected is shortened.

In the case of fitting the standard curve equation in comparative example 1, it is also necessary to add 0.5mL of blank plasma without moclobemide and 10 μ L of internal standard solution to 10 μ L of standard solution with different concentrations, and to perform the operation in the manner of the pretreatment in comparative example 1 above, so as to measure moclobemide in the standard solution.

As can be seen from examples 1 and 2, in the present example, when the standard curve equation is fitted, it is not necessary to add a blank serum or a blank plasma that does not contain moclobemide, and the obtained measurement results achieve the same or even better results on the premise of not performing a pretreatment operation on the standard solution, simplifying the pretreatment mode, and saving the analysis time.

Example 6: detection limit and quantification limit of detection method of moclobemide

Preparing low-concentration standard solutions of moclobemide with different concentrations, respectively adding 10 μ L of internal standard working solution, adding 10 μ L of each low-concentration standard solution of moclobemide with each concentration and 90 μ L of blank sample (for example, serum or plasma not containing moclobemide), thereby preparing samples with different concentrations, and performing determination according to the pretreatment in embodiment 3 and the detection conditions in embodiment 2. The detection limit and the quantification limit of moclobemide are shown in the following, and the standard addition concentration is shown in the following table 1.

TABLE 1

Standard concentration (ng/mL)	10	15	20	25	30	40	45	50	55	60
											Signal to noise ratio	1.5	2.0	4.2	4.5	4.6	5.1	7.6	9.8	10.0	12.1

Wherein, the signal-to-noise ratio (S/N) is 10.0 as the limit of quantification, and the signal-to-noise ratio (S/N) is 4.2 as the limit of detection.

Moclobemide

(1) Limit of detection (LOD): 20 ng/mL.

(2) Limit of quantitation (LOQ): 55 ng/mL.

According to the embodiment, the detection limit and the quantification limit of the moclobemide are respectively 20ng/mL and 55ng/mL, the sensitivity is very high, the biological sample with very low moclobemide content can be accurately quantified, and the high accuracy and the wide applicability of the detection method are ensured.

Example 7: acquisition of linear equation of detection method of moclobemide and linear relation

Measuring seven standard solutions with different concentrations in example 1 by using a liquid chromatograph according to the detection conditions in example 2 to obtain chromatograms of the moclobemide and the internal standard substance with various concentrations, wherein the chromatograms of the moclobemide and the internal standard substance in the standard solutions are shown in fig. 3; the retention time of moclobemide is about 5.2min, and the retention time of the internal standard substance pirfenidone is about 4.4 min.

The unit length of the abscissa in fig. 3 is 0.25, and the unit length of the ordinate is 0.5.

Determining peak areas of various chromatographic peaks, taking the ratio of the chromatographic peak area of the moclobemide to the chromatographic peak area of an internal standard substance as a longitudinal coordinate y2 of a standard curve equation, taking the ratio of the concentration of the moclobemide in a standard solution to the chromatographic peak area of the internal standard substance as a horizontal coordinate x2 of the standard curve equation, performing linear regression on seven kinds of data with different concentrations obtained by detection, and fitting to obtain a standard curve equation of y2 a x2+ b and a coefficient c; the results of the linear equation measurements are shown in Table 2 and the linear equation is shown in FIG. 4.

TABLE 2

Detecting the index	Linear range	Linear equation of equations	Correlation coefficient	Weighting
					Moclobemide-a certain example 1	100-3000ng/mL	y2＝0.1937*x2-1.9815	0.9992	1/X2

Table 2 shows the linear relationship data in the first example, and it can be seen from Table 2 that the correlation coefficient R is greater than 0.9900 and the linear relationship is good when the moclobemide is in the linear range of 100-3000 ng/mL.

Example 8: recovery rate and precision of detection method of moclobemide

The moclobemide standard working solution in example 1 was prepared into high, medium and low concentrations of 3 to perform sample recovery and precision experiments, the detection conditions in example 2 were followed, and the analysis and determination were repeated for 3 batches, with the recovery and precision of moclobemide as shown in table 3. The average recovery rate of the moclobemide in the range of 3 addition levels of low, medium and high is 100.63-102.33%, and the precision is 0.83-1.96%.

TABLE 3

By integrating the verification tests, the recovery rate, the precision and other technical indexes of the embodiment meet the requirements, and the method for detecting the concentration of the moclobemide in the blood has good reproducibility and good sample-adding recovery rate, so that the accuracy of the detection result is improved, and the system error is eliminated.

As can be seen from the graphs in FIGS. 2 and 3, the retention time of the moclobemide in the sample to be detected is consistent with that of the moclobemide in the standard solution, and the method takes pirfenidone as an internal standard substance, so that the identification of a target compound is more accurate, the analysis time is short, the interference is small, the internal standard is suitable for quantification, the specificity is strong, and the accuracy and the sensitivity are high.

Example 9: description of flow Rate and column temperature

The tests corresponding to fig. 5 to 12 are parallel tests corresponding to examples 3 and 4, respectively, with the difference that the flow rate and the column temperature are different.

FIG. 5 is a chromatogram at a column temperature of 20 ℃ and a flow rate of 1.0mL/min, the unit length on the abscissa of FIG. 5 being 0.25 and the unit length on the ordinate being 0.25; in FIG. 5, the chromatographic peak with the retention time of about 4.7min is the chromatographic peak of the internal standard substance, and the chromatographic peak with the retention time of about 5.25min is the chromatographic peak of moclobemide;

FIG. 6 is a chromatogram at a column temperature of 50 ℃ and a flow rate of 1.0mL/min, the unit length on the abscissa of FIG. 6 being 0.25 and the unit length on the ordinate being 0.25; in FIG. 6, the chromatographic peak with the retention time of about 4.3min is the chromatographic peak of the internal standard substance, and the chromatographic peak with the retention time of about 5.0min is the chromatographic peak of moclobemide;

FIG. 7 is a chromatogram at a column temperature of 40 ℃ and a flow rate of 0.2mL/min, the unit length on the abscissa of FIG. 7 being 1.0 and the unit length on the ordinate being 0.5; the chromatographic peaks in figure 7 without internal standard and moclobemide;

FIG. 8 is a chromatogram at a column temperature of 40 ℃ and a flow rate of 0.3mL/min, the unit length on the abscissa of FIG. 8 being 1.0 and the unit length on the ordinate being 0.5; in FIG. 8, the chromatographic peak with the retention time of about 14.8min is the chromatographic peak of the internal standard substance, and the chromatographic peak with the retention time of about 17.2min is the chromatographic peak of moclobemide;

FIG. 9 is a chromatogram at a column temperature of 40 ℃ and a flow rate of 0.5mL/min, the unit length on the abscissa of FIG. 9 being 0.5 and the unit length on the ordinate being 0.5; in FIG. 9, the chromatographic peak with the retention time of about 8.9min is the chromatographic peak of the internal standard substance, and the chromatographic peak with the retention time of about 10.4min is the chromatographic peak of moclobemide;

FIG. 10 is a chromatogram at a column temperature of 40 ℃ and a flow rate of 0.8mL/min, the unit length on the abscissa of FIG. 10 being 0.25 and the unit length on the ordinate being 0.5; in FIG. 10, the chromatographic peak with the retention time of about 5.7min is the chromatographic peak of the internal standard substance, and the chromatographic peak with the retention time of about 6.5min is the chromatographic peak of moclobemide;

FIG. 11 is a chromatogram at a column temperature of 40 ℃ and a flow rate of 1.5mL/min, the unit length on the abscissa of FIG. 11 being 0.25 and the unit length on the ordinate being 0.5; in FIG. 11, the chromatographic peak with the retention time of about 3.0min is the chromatographic peak of the internal standard substance, and the chromatographic peak with the retention time of about 3.4min is the chromatographic peak of moclobemide;

FIG. 12 is a chromatogram at a column temperature of 40 ℃ and a flow rate of 2.0mL/min, the unit length on the abscissa of FIG. 12 being 0.25 and the unit length on the ordinate being 0.5; in FIG. 12, the chromatographic peak with retention time of about 2.3min is the chromatographic peak of the internal standard substance, and the chromatographic peak with retention time of about 2.6min is the chromatographic peak of moclobemide.

As can be seen from fig. 2 and fig. 5 to 12, when the flow rate is 1.0mL/min and the column temperature is in the range of 20 ℃ to 50 ℃, the retention time of moclobemide is 4.3min to 4.7min, the retention time of the internal standard substance is about 5.0min to 5.25min, the column temperature has a small influence on the retention time of moclobemide and the internal standard substance, but the column temperature is higher, which influences the service life of the chromatographic column, so that the column temperature range for detection of moclobemide and the internal standard substance is set in the range of 20 ℃ to 50 ℃, so that the retention time of the target substance is relatively shortest.

When the flow rate is less than 0.3mL/min, the retention time of the moclobemide and the internal standard substance is increased, so that the detection time of the target substance is too long, and the timeliness of the sample to be detected is influenced. And when the flow rate is more than 2.0mL/min, the internal standard substance has an early peak appearance and a short retention time, so that the risk of matrix interference exists, and meanwhile, the column pressure is increased by overlarge flow rate and exceeds the pressure which can be borne by the chromatographic column, so that the irreversible damage is caused to the chromatographic column.

Example 10: description of eluting Mobile phase

The tests corresponding to figures 13 to 16, respectively, are parallel tests corresponding to examples 3 and 4, with the difference that the volume ratio of aqueous phase to organic phase in the elution mobile phase is different.

FIG. 13 is a chromatogram showing the volume ratio of the aqueous phase to the organic phase in the elution mobile phase of 87% to 13%, the length in units on the abscissa of FIG. 13 being 1.0 and the length in units on the ordinate being 0.5; the chromatographic peak with retention time of about 17.5min in fig. 13 is the chromatographic peak of the internal standard, and the chromatographic peak of moclobemide is absent;

FIG. 14 is a chromatogram showing the volume ratio of the aqueous phase to the organic phase in the elution mobile phase at 82%: 18%, the length unit on the abscissa of FIG. 14 being 0.5 and the length unit on the ordinate being 0.5; in FIG. 14, the chromatographic peak with the retention time of about 8.0min is the chromatographic peak of the internal standard substance, and the chromatographic peak with the retention time of about 10.4min is the chromatographic peak of moclobemide;

FIG. 15 is a chromatogram showing the volume ratio of the aqueous phase to the organic phase in the elution mobile phase at 72% to 28%, the length of the unit on the abscissa of FIG. 15 being 0.25 and the length of the unit on the ordinate being 0.25; in FIG. 15, the chromatographic peak with the retention time of about 3.2min is the chromatographic peak of the internal standard substance, and the chromatographic peak with the retention time of about 3.5min is the chromatographic peak of moclobemide;

FIG. 16 is a chromatogram showing the volume ratio of the aqueous phase to the organic phase in the elution mobile phase at 67% to 33%, the length unit on the abscissa of FIG. 16 being 0.25 and the length unit on the ordinate being 0.5; the chromatographic peak with the retention time of about 2.5min in FIG. 16 is the chromatographic peak obtained by coincidence of the internal standard and moclobemide.

As can be seen from fig. 2 and 13 to 16, when the volume percentage of the aqueous phase in the elution mobile phase is greater than 82%, the retention time of moclobemide is increased, the detection time of the target is too long, and the timeliness of the sample to be detected is affected; when the volume percentage of the water phase in the elution mobile phase is less than 72%, the moclobemide and the internal standard substance have poor separation degree and even overlap, so that the detection accuracy of the sample to be detected is influenced.

Example 11: description of the extractant

The runs corresponding to fig. 17 to 24 are parallel runs corresponding to examples 3 and 4, with the difference in extractant. In fig. 17 to 24, the unit length of the abscissa is 0.25, and the unit length of the ordinate is 0.5.

FIG. 17 is a chromatogram for an extractant of methyl tert-butyl ether; in FIG. 17, the chromatographic peak with the retention time of about 4.5min is the chromatographic peak of the internal standard substance, and the chromatographic peak with the retention time of about 5.25min is the chromatographic peak of moclobemide;

FIG. 18 is a chromatogram for ethyl acetate as extractant; in FIG. 18, the chromatographic peak with the retention time of about 4.5min is the chromatographic peak of the internal standard substance, and the chromatographic peak with the retention time of about 5.25min is the chromatographic peak of moclobemide;

FIG. 19 is a chromatogram for methylene chloride as the extractant; in FIG. 19, the chromatographic peak with the retention time of about 4.5min is the chromatographic peak of the internal standard substance, and the chromatographic peak with the retention time of about 5.25min is the chromatographic peak of moclobemide;

FIG. 20 is a chromatogram of an extractant in n-hexane containing 50% methyl t-butyl ether; in FIG. 20, the chromatographic peak with the retention time of about 4.4min is the chromatographic peak of the internal standard substance, and the chromatographic peak with the retention time of about 5.2min is the chromatographic peak of moclobemide;

FIG. 21 is a chromatogram of an extractant in n-hexane containing 40% methyl t-butyl ether; in FIG. 21, the chromatographic peak with the retention time of about 4.4min is the chromatographic peak of the internal standard substance, and the chromatographic peak with the retention time of about 5.2min is the chromatographic peak of moclobemide;

FIG. 22 is a chromatogram of an extractant in n-hexane containing 20% methyl t-butyl ether; in FIG. 22, the chromatographic peak with the retention time of about 4.4min is the chromatographic peak of the internal standard substance, and the chromatographic peak with the retention time of about 5.2min is the chromatographic peak of moclobemide;

FIG. 23 is a chromatogram of an extractant in n-hexane containing 10% methyl t-butyl ether; in FIG. 23, the chromatographic peak with the retention time of about 4.4min is the chromatographic peak of the internal standard substance, and the chromatographic peak with the retention time of about 5.2min is the chromatographic peak of moclobemide;

FIG. 24 is a chromatogram of an extractant in n-hexane (i.e., n-hexane containing 0% methyl t-butyl ether); in FIG. 24, the chromatographic peak with a retention time of about 4.4min is the chromatographic peak of the internal standard, and the chromatographic peak with a retention time of about 5.2min is the chromatographic peak of moclobemide.

As can be seen from fig. 2 and 17 to 24, when the extracting agents are methyl tert-butyl ether, ethyl acetate, dichloromethane and n-hexane containing 50% methyl tert-butyl ether, the chromatographic peak of the internal standard substance is affected by the impurity peak, which affects the detection accuracy of the sample to be detected. When the extracting agent is n-hexane containing 0-40% of methyl tert-butyl ether, the chromatographic peak of the internal standard substance and the chromatographic peak of moclobemide have good separation degree and have no impurity interference, so the n-hexane containing 0-40% of methyl tert-butyl ether can be used for the extracting agent in the embodiment.

Example 12: description of volume of extractant

The tests corresponding to fig. 25 to 28 are parallel tests corresponding to examples 3 and 4, with the difference in the volume of the extractant. In fig. 25 to 28, the abscissa and the ordinate are each 0.25 and 0.5 in unit length, and in fig. 25 to 28, the chromatographic peaks at a retention time of about 4.4min and the chromatographic peaks at a retention time of about 5.2min are each the chromatographic peaks of moclobemide.

FIG. 25 is a chromatogram for a volume ratio of first supernatant to extractant of 1: 5;

FIG. 26 is a chromatogram for a volume ratio of first supernatant to extractant of 1: 8;

FIG. 27 is a chromatogram for a volume ratio of first supernatant to extractant of 1: 12;

fig. 27 is a chromatogram when the volume ratio of the first supernatant to the extractant is 1: 15.

As can be seen from fig. 2, 25 to 28, when the volume ratio of the first supernatant to the extractant is 1:5, 1:8, 1:12, or 1:15, the internal standard substance and the moclobemide in the sample to be processed can be sufficiently extracted, and thus the volume ratio of the first supernatant to the extractant in the embodiment of the present invention includes 1:5 to 1: 15.

Example 13: description of the column

The tests corresponding to fig. 29 and 30 are parallel tests corresponding to examples 3 and 4, differing mainly in the reverse phase C18 column. In fig. 29 and 30, the unit length of the abscissa is 0.5, and the unit length of the ordinate is 0.25.

FIG. 29 is a chromatogram of a column of Agilent Eclipse Plus C18, having an inner diameter of 4.6mm, a column length of 100mm and a packing particle size of 3.5 μm; in FIG. 29, the chromatographic peak with retention time of about 6.1min is the chromatographic peak of the internal standard substance, and the chromatographic peak with retention time of about 7.1min is the chromatographic peak of moclobemide;

FIG. 30 is a chromatogram of a column of Waters Xbridge C18, wherein the column has an inner diameter of 4.6mm, a column length of 150mm, and a packing particle size of 5.0 μm; in FIG. 30, the chromatographic peak with retention time of about 7.2min is the chromatographic peak of the internal standard, and the chromatographic peak with retention time of about 8.1min is the chromatographic peak of moclobemide.

As can be seen from FIGS. 2, 29 and 30, Phenomenex was usedThe chromatographic peak of the target substance obtained by detecting the sample to be detected by the chromatographic columns of-C18, Agilent Eclipse Plus C18 and Waters Xbridge C18 has no front edge or tailing, and the peak width also meets the detection requirement.

Example 14: description for internal standards

The assays corresponding to fig. 31 and 32 are parallel assays corresponding to examples 3 and 4, with the difference being the internal standard.

FIG. 31 is a chromatogram of propranolol as an internal standard, with a unit length of 0.5 on the abscissa and 0.25 on the ordinate of FIG. 31; in FIG. 31, the chromatographic peak with retention time of about 7.7min is the chromatographic peak of propranolol as an internal standard substance, and the chromatographic peak with retention time of about 5.2min is the chromatographic peak of moclobemide;

FIG. 32 is a chromatogram when the internal standard is sulfamethoxazole, in which FIG. 32 the unit length on the abscissa is 0.25 and the unit length on the ordinate is 0.5; in FIG. 32, the chromatographic peak with the retention time of about 1.4min is the chromatographic peak of sulfamethoxazole as the internal standard, and the chromatographic peak with the retention time of about 5.2min is the chromatographic peak of moclobemide.

As can be seen from fig. 31 and 32, when the internal standard substance is propranolol, the detection accuracy of the sample to be detected is affected due to the abnormal chromatographic peak shape of propranolol; when the internal standard substance is sulfamethoxazole, the retention time of sulfamethoxazole is short, the base line of the sample is uneven, and the chromatographic peak of sulfamethoxazole is easily interfered by impurities, so that the detection accuracy of the sample to be detected is influenced.

Example 15: description for detection wavelength

The tests corresponding to fig. 33 to 36 are parallel tests corresponding to examples 3 and 4, and differ in the detection wavelength. The unit lengths of the abscissa and ordinate in fig. 33 to 36 are all 0.25 and 0.5, respectively; in FIGS. 33 to 36, the chromatographic peaks with retention time of about 4.5min are all chromatographic peaks of propranolol as an internal standard substance, and the chromatographic peaks with retention time of about 5.2min are all chromatographic peaks of moclobemide.

FIG. 33 is a chromatogram at a detection wavelength of 220 nm;

FIG. 34 is a chromatogram at a detection wavelength of 230nm

FIG. 35 is a chromatogram at a detection wavelength of 250 nm;

FIG. 36 is a chromatogram at a detection wavelength of 260 nm.

As can be seen from fig. 2 and fig. 33 to fig. 36, when the detection wavelength is less than 230nm, the signal intensity of moclobemide decreases, which may affect the detection sensitivity of the sample to be detected; similarly, when the detection wavelength is greater than 250nm, the signal intensity of moclobemide is reduced, which affects the detection sensitivity of the sample to be detected.

It should be noted that the abscissa in fig. 2, fig. 3, and fig. 5 to fig. 36 is the acquisition time (min), and the ordinate is the signal intensity (mAU), and the missing graph in the chromatogram does not affect the technical content of the scheme.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising a" does not exclude the presence of other similar elements in a process, method, article, or apparatus that comprises the element.

Finally, it is to be noted that: the above description is only a preferred embodiment of the present invention, and is only used to illustrate the technical solutions of the present invention, and not to limit the protection scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.