阅读说明：本技术 甲氨蝶呤的检测方法 (Method for detecting methotrexate ) 是由 贾永娟 刘杏立 刘春冉 倪君君 于 2020-12-04 设计创作，主要内容包括：本发明提供了甲氨蝶呤的检测方法,包括：制备具有甲氨蝶呤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；利用液质联用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、标准溶液中甲氨蝶呤的浓度和内标物的浓度,拟合甲氨蝶呤的标准曲线方程；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和沉淀蛋白试剂,涡旋混匀,并离心,取离心后的第二上清液作为待测样品；利用液质联用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,得到待测样品中甲氨蝶呤的浓度。本方案能够缩短样品检测时间。(The invention provides a detection method of methotrexate, which comprises the following steps: preparing standard solutions having at least three concentrations of methotrexate and an internal standard, the amount of internal standard in the standard solutions being the same; detecting each standard solution by using a liquid chromatograph-mass spectrometer under a detection condition to obtain a first detection result corresponding to the standard solution; fitting a standard curve equation of the methotrexate according to each first detection result, the concentration of the methotrexate 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 and a precipitated protein reagent into the first supernatant, uniformly mixing by vortex, centrifuging, and taking the centrifuged second supernatant as a sample to be detected; detecting the sample to be detected by using a liquid chromatograph-mass spectrometer under the detection condition to obtain a second detection result of the sample to be detected; and obtaining the concentration of the methotrexate 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 methotrexate is characterized by comprising the following steps:

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

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

fitting a standard curve equation of methotrexate according to each first detection result, the concentration of methotrexate 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 and a precipitated protein reagent into the first supernatant, uniformly mixing by vortex, centrifuging, and taking the centrifuged second supernatant as a sample to be detected;

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

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

2. The method for detecting methotrexate according to claim 1,

the liquid phase condition among the detection conditions includes:

a reverse phase chromatography column;

the aqueous phase in the elution mobile phase comprises: an aqueous solution containing 0% to 0.5% formic acid and 0 to 10mM of a buffer salt;

eluting the organic phase in the mobile phase comprises: methanol solution and/or acetonitrile solution, wherein the organic phase contains 0% -0.5% formic acid and 0-1mM buffer salt;

the column temperature is 18-60 ℃;

flow rates included 0.25-0.6 mL/min.

3. The method for detecting methotrexate according to claim 2,

when the organic phase and the aqueous phase are eluted isocratically, the volume ratio of the organic phase to the aqueous phase comprises:

22％:78％-40％:60％；

when the organic phase and the aqueous phase are eluted by gradient, the volume ratio of the organic phase to the aqueous phase comprises:

0.00min：10％:90％-40％:60％；

0.01min：30％:70％-100％:0％；

1.00min：30％:70％-100％:0％；

1.01min：10％:90％-40％:60％；

3.00min：10％:90％-40％:60％。

4. the method for detecting methotrexate according to claim 1,

mass spectrometry conditions in the detection conditions comprising:

the mass spectrometer in the LC-MS is in an ESI (+) detection mode;

the ion source parameters in the LC-MS are as follows: heating air flow rate (L/min) is 8-12, atomizing air flow rate (L/min) is 25-35, heating air temperature (DEG C) is 300-.

5. The method for detecting methotrexate according to claim 1,

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

6. The method for detecting methotrexate according to claim 1,

the precipitated protein reagent comprises: at least one of a methanol solution, an acetonitrile solution, trichloroacetic acid, sulfosalicylic acid, and perchloric acid.

7. The method for detecting methotrexate according to claim 1,

the volume ratio of the first supernatant to the precipitated protein reagent comprises 1:1 to 1: 240.

8. The method for detecting methotrexate according to claim 1,

the centrifugation treatment is carried out on the sample to be treated, and the centrifuged first supernatant is taken, which comprises the following steps:

centrifuging the sample to be treated at 3000-4000rpm for 8-12min, and taking the centrifuged supernatant as a first supernatant.

9. The method for detecting methotrexate according to claim 1,

adding an internal standard substance and a precipitated protein reagent into the first supernatant, uniformly mixing by vortex, centrifuging, taking the centrifuged second supernatant as a sample to be detected, and comprising the following steps:

adding an internal standard substance into the first supernatant, and carrying out vortex mixing for 0.5-1.5min at the rotating speed of 1500-2000 rpm;

adding the precipitated protein reagent in sequence, carrying out vortex mixing for 4-6min at the rotating speed of 1500-2000rpm, carrying out high-speed centrifugation for 8-12min at the rotating speed of 10000-15000rpm, and taking the centrifuged second supernatant as a sample to be detected.

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

the internal standard comprises methotrexate-d 3.

Technical Field

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

Background

Methotrexate is a folate reductase inhibitor, which is orange yellow crystalline powder, is easily soluble in dilute alkali, acid or alkali carbonate solution, is slightly soluble in dilute hydrochloric acid, and is hardly soluble in water, ethanol, chloroform or diethyl ether.

Currently, the method generally adopted for detecting the methotrexate content in a sample is high performance liquid chromatography. The high performance liquid chromatography detection usually requires complex pretreatment of a sample to be detected, and consumes more time, thereby resulting in longer sample detection time.

Disclosure of Invention

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

In order to solve the above problem, an embodiment of the present invention provides a method for detecting methotrexate, including:

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

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

fitting a standard curve equation of methotrexate according to each first detection result, the concentration of methotrexate 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 and a precipitated protein reagent into the first supernatant, uniformly mixing by vortex, centrifuging, and taking the centrifuged second supernatant as a sample to be detected;

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

and obtaining the concentration of methotrexate 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 methotrexate 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 methotrexate-d 3, and the isotope of the target substance is used as the internal standard substance, so that the influence on the detection of the target substance caused by the reaction between the internal standard substance and the target substance during the detection of the target substance can be avoided, and meanwhile, the influence of matrix effect is reduced to the greatest extent, and the accuracy of the detection result is improved.

It is noted that the first supernatant comprises serum or plasma.

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

(1) preparation of standard stock solution

The methotrexate standard is accurately weighed and placed in a volumetric flask, dissolved by using a sodium hydroxide aqueous solution, and subjected to constant volume until reaching the marked line of the volumetric flask to obtain a standard stock solution, and the standard stock solution is stored at the temperature of minus 80 ℃ and has the validity period of 24 months.

(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 an aqueous solution containing 40-60% of methanol as a diluent to obtain a standard working solution containing 100-50000ng/mL methotrexate, and storing the standard working solution at the temperature of-80 ℃ for 6 months.

(3) Preparation of internal standard stock solution

Taking 100 mu g/mL standard methotrexate-d 3 as internal standard stock solution, and storing at-80 ℃ for 24 months.

(4) Preparation of internal standard working solution

And (4) taking the internal standard stock solution in the step (3), diluting the internal standard stock solution with a diluent to obtain an internal standard working solution containing methotrexate-d 3, and storing the internal standard working solution at-80 ℃ for 12 months.

(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 centrifugal tubes, respectively adding the precipitated protein reagent into each centrifugal tube, mixing to prepare at least three mixed solutions with different concentrations, uniformly mixing the mixed solutions in a vortex manner at the rotation speed of 1500-2000rpm for 0.5-1.5min, centrifuging, and transferring the centrifuged second supernatant as the standard solution.

In order to reduce the volatility of the working solutions of methotrexate and methotrexate-d 3, the diluent was an aqueous solution containing 40-60% methanol.

Methotrexate is soluble in 0.1% aqueous formic acid, 0.1M aqueous hydrochloric acid, and aqueous sodium hydroxide at room temperature, but methotrexate dissolved in acidic conditions is likely to precipitate at low temperature, and further, the re-dissolution of methotrexate is not guaranteed even after returning to room temperature, and therefore, in order to ensure sufficient dissolution of methotrexate, aqueous sodium hydroxide is selected to dissolve and dilute methotrexate.

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

the aqueous phase in the elution mobile phase comprises: an aqueous solution containing 0-0.5% formic acid, 0-10mM of a buffer salt;

eluting the organic phase in the mobile phase comprises: acetonitrile solution containing 0-0.5% formic acid, 0-1mM buffer salt or methanol solution containing 0-0.5% formic acid, 0-1mM buffer salt;

the column temperature is 18-60 ℃; the flow rate is 0.25-0.6 mL/min.

Preferably, the sample to be tested is introduced in an amount of 0.1-20. mu.L. When the sample volume is 0.1 muL, the signal-to-noise ratio is 32.2, the quantitative requirement can be met, and when the sample volume exceeds 20 muL, the test signal is saturated.

Specifically, the column under liquid phase conditions includes Waters Atlantis-dC18 column (inner diameter 2.1 mm. times. column length 50mm, particle size of packing 5 μm), Agilent ZORBAX-extended-C18 (inner diameter 2.1 mm. times. column length 50mm, particle size of packing 5 μm), Agilent ZORBAX XDB-C8 (inner diameter 2.1 mm. times. column length 50mm, particle size of packing 5 μm), SHIMADZU-SP-C18 (inner diameter 2.1 mm. times. column length 50mm, particle size of packing 2.6 μm), Agilent SB-Aq-C18 (inner diameter 2.1 mm. times. column length 50mm, particle size of packing 5 μm), Waters Atlantis-T3 (inner diameter 2.1 mm. times. column length 50mm, particle size of packing 5 μm), Agilent PosiloShell 120C 18 (inner diameter 2.1. times. column length 50mm, particle size of packing 8 mm), and inner diameter of packing 2.1 mm. times. length 2.1mm, particle size of packing 2.84 mm, particle size of Sustan-C18 mm.

The elution mobile phase may be composed of a methanol solution and an aqueous phase, may be composed of an acetonitrile solution and an aqueous phase, and may be composed of an aqueous phase mixed with a methanol solution and an acetonitrile solution at an arbitrary ratio.

Specifically, formic acid may or may not be added to the aqueous phase or the organic phase of the elution mobile phase. When formic acid is added into the elution mobile phase, the mass spectrum detector is in an ESI (+) detection mode, so that the ionization degree of the sample to be detected can be increased and the intensity of the target substance spectrum peak can be improved by detecting the sample to be detected through the elution mobile phase.

Specifically, when the formic acid content in the aqueous phase is more than 0.5% and the formic acid content in the organic phase is more than 0.5%, the formic acid content in the elution mobile phase is too high, resulting in too low a pH of the elution mobile phase, which causes irreversible damage to the chromatography column. Thus, the aqueous phase in the eluting mobile phase comprises: contains 0-0.5% formic acid; the organic phase comprises: contains 0-0.5% formic acid.

Specifically, the organic phase of the elution mobile phase may or may not be added with a buffer salt, and when added, the amount of buffer salt should be less than 1mM to prevent the buffer salt from precipitating, depositing and even damaging the column.

With respect to formic acid in the elution mobile phase, 0% -0.5% formic acid is meant formic acid having any value in the range of 0% to 0.5%, e.g., 0%, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, and 0.5% formic acid in the aqueous and/or organic phase.

With respect to the buffer salt in the aqueous phase, a buffer salt containing 0-10mM means a buffer salt containing any value in the range of 0mM to 10mM in the aqueous phase, for example, 0mM, 2mM, 4mM, 6mM, 8mM, and 10 mM.

With respect to the buffer salt in the organic phase, a buffer salt containing 0-1mM means a buffer salt containing any value in the range of 0mM to 1mM in the organic phase, for example, 0mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM, and 1 mM.

Preferably, the buffer salt in the elution mobile phase comprises ammonium formate or ammonium acetate.

The column temperature of 18-60 ℃ refers to any value in the range of 18 ℃ to 60 ℃, such as 18 ℃, 20 ℃, 23 ℃, 25 ℃, 28 ℃, 30 ℃, 33 ℃, 35 ℃, 38 ℃, 40 ℃, 43 ℃, 45 ℃, 48 ℃, 50 ℃, 53 ℃, 55 ℃, 58 ℃ and 60 ℃.

With respect to the flow rate, 0.25-0.6mL/min refers to any value in the range of 0.25mL/min to 0.6mL/min, such as 0.25mL/min, 0.3mL/min, 0.35mL/min, 0.4mL/min, 0.45mL/min, 0.5mL/min, 0.55mL/min, and 0.6 mL/min.

Preferably, when the organic phase and the aqueous phase are eluted isocratically, the volume ratio of the organic phase to the aqueous phase comprises: 22 percent, 78 percent to 40 percent and 60 percent; when the organic phase and the aqueous phase are eluted by gradient, the volume ratio of the organic phase to the aqueous phase comprises: 0.00 min: 10 percent, 90 percent to 40 percent, 60 percent; 0.01 min: 30 percent, 70 percent to 100 percent and 0 percent; 1.00 min: 30 percent, 70 percent to 100 percent and 0 percent; 1.01 min: 10 percent, 90 percent to 40 percent, 60 percent; 3.00 min: 10 percent, 90 percent to 40 percent and 60 percent.

Specifically, the elution mobile phase may employ an analytical method of isocratic elution or gradient elution.

The volume ratio of the organic phase and the aqueous phase for isocratic elution is 22% to 78% -40% to 60%, and refers to any ratio in the range of 22% to 78% to 40% to 60%, such as 22% to 78%, 30% to 70%, 35% to 65%, and 40% to 60%.

For the volume ratio of the organic phase to the aqueous phase in the gradient elution at 0.00min, 1.01min and 3.00min, 10%: 90% -40%: 60% means any ratio in the range of 10%: 90% to 40%: 60%, for example, 10%: 90%, 15%: 85%, 20%: 80%, 25%: 75%, 30%: 70%, 35%: 65% and 40%: 60%.

For the volume ratio of the organic phase to the aqueous phase in the gradient elution at 0.01min and 1.00min, 30%: 70% -100%: 0% means any ratio in the range of 30%: 70% to 100%: 0%, for example, 30%: 70%, 40%: 60%, 50%: 50%, 60%: 40%, 70%: 30%, 80%: 20%, 90%: 10% and 100%: 0%.

For example, when the volume ratio of the 0min organic phase to the aqueous phase is 10% to 90% and the ratio of the 0.01min organic phase to the aqueous phase is 80% to 20%, the organic phase gradually increases from 10% to 80% and the aqueous phase gradually decreases from 90% to 10% in the time period from 0min to 0.01 min.

Since the sum of the proportions of the organic phase and the aqueous phase in the elution mobile phase is 1, the proportion of the aqueous phase in the elution mobile phase decreases correspondingly when the proportion of the organic phase in the elution mobile phase increases.

Specifically, when isocratic elution is used, if the volume ratio of the organic phase in the elution mobile phase is less than 22%, the retention time of methotrexate increases, which increases the detection time of methotrexate; when the volume ratio of the organic phase in the elution mobile phase is more than 40 percent, the retention time of methotrexate is small, peaks before 0.5min, and matrix effect is easy to generate, so that the volume ratio of the organic phase to the aqueous phase in the elution mobile phase is 22 percent to 78 percent to 40 percent to 60 percent by adopting isocratic elution.

Specifically, when gradient elution is adopted, if the volume ratio of the organic phase in the elution mobile phase is less than 10% in 0.00min, the column pressure cannot be recovered in a short time, so that the analysis time of the sample to be detected is too long; if the organic phase accounts for more than 40% by volume of the elution mobile phase at 0.00min, the retention time of methotrexate is small, and peaks before 0.5min, which is likely to cause matrix effect.

Preferably, the mass spectrometric conditions in the detection conditions comprise:

the mass spectrometer in the LC-MS is in an ESI (+) detection mode;

the ion source parameters in the LC-MS are as follows: heating air flow rate (L/min) is 8-12, atomizing air flow rate (L/min) is 25-35, heating air temperature (DEG C) is 300-.

With respect to the heating gas flow rate, 8-12L/min refers to any value in the range of 8L/min to 12L/min, such as 8L/min, 9L/min, 10L/min, 11L/min, and 12L/min.

With respect to the atomizing gas flow rate, 25-35L/min refers to any value in the range of 25L/min to 35L/min, such as, for example, 25L/min, 27L/min, 30L/min, 32L/min, and 35L/min.

For the heating gas temperature, 300-350 ℃ refers to any value in the range of 300 ℃ to 350 ℃, such as 300 ℃, 310 ℃, 320 ℃, 330 ℃, 340 ℃ and 350 ℃.

For the capillary voltage, 3000-4000V means any value in the range of 3000V to 4000V, for example, 3000V, 3100V, 3200V, 3300V, 3400V, 3500V, 3600V, 3700V, 3800V, 3900V, and 4000V.

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

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

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

Preferably, in order to better remove impurities and purify the target substance, the protein precipitation reagent for performing protein precipitation on the first supernatant after the internal standard substance is added comprises: at least one of a methanol solution, an acetonitrile solution, trichloroacetic acid, sulfosalicylic acid, and perchloric acid.

Preferably, for better removal of impurities, the volume ratio of the first supernatant to the precipitated protein reagent comprises 1:1 to 1: 240.

By volume ratio of 1:1 to 1:240 is meant any ratio in the range of 1:1 to 1:240, such as 1:1, 1:3, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:50, 1:100, 1:150, 1:200, and 1: 240.

Specifically, when the volume of the first supernatant is 100. mu.L, the volume of the precipitated protein reagent may be any value in the range of 100. mu.L to 24000. mu.L. When the volume ratio of the first supernatant to the precipitated protein reagent is 1:240, the signal-to-noise ratio is 16.0, and the quantitative requirement can be met.

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

the centrifugation treatment is carried out on the sample to be treated, and the centrifuged first supernatant is taken, which comprises the following steps:

centrifuging the sample to be treated at 3000-4000rpm for 8-12min, and taking the centrifuged supernatant as a first supernatant.

Specifically, the sample to be treated is treated by centrifugation to remove a part of impurities. It is understood that the first supernatant comprises serum, plasma, or other samples.

Preferably, the adding an internal standard substance and a precipitated protein reagent into the first supernatant, mixing uniformly by vortex, centrifuging, and taking the centrifuged second supernatant as a sample to be detected comprises:

adding an internal standard substance into the first supernatant, and carrying out vortex mixing for 0.5-1.5min at the rotating speed of 1500-2000 rpm;

adding the precipitated protein reagent in sequence, carrying out vortex mixing for 4-6min at the rotating speed of 1500-2000rpm, carrying out high-speed centrifugation for 8-12min at the rotating speed of 10000-15000rpm, and taking the centrifuged second supernatant as a sample to be detected.

Specifically, after the internal standard substance is added into the first supernatant, in order to mix the two solutions more uniformly, the two solutions may be mixed by swirling, and then the precipitated protein reagent is added into the mixed first supernatant, and the mixed first supernatant is mixed by swirling to precipitate the protein, so that the mixed first supernatant is purified by the precipitated protein reagent. And then high-speed centrifugation is carried out, and the centrifuged second supernatant is taken to realize the purpose of separating impurities from the target object. Because the sample to be detected can be purified without liquid-liquid extraction, the operation of the pretreatment process of the sample to be detected is simpler, and the time required by pretreatment is reduced.

For the vortex rotation speed, 1500-2000rpm refers to any rotation speed in the range of 1500rpm to 2000rpm, such as 1500rpm, 1600rpm, 1700rpm, 1800rpm, 1900rpm and 2000 rpm.

For the vortex time after the internal standard substance is added, 0.5-1.5min means any time within the range of 0.5min to 1.5min, such as 0.5min, 0.6min, 0.7min, 0.8min, 0.9min, 1.1min, 1.2min, 1.3min, 1.4min and 1.5 min.

For the vortex time after adding the protein precipitation reagent, 4-6min refers to any time within the range of 4min to 6min, such as 4min, 4.5min, 5min, 5.5min and 6 min.

For the centrifugal rotation speed, 10000-15000rpm refers to any rotation speed within the range of 10000rpm to 15000rpm, such as 10000rpm, 11000rpm, 12000rpm, 13000rpm, 14000rpm and 15000 rpm.

For the centrifugation time after adding the precipitated protein reagent, 8-12min refers to 8min, 8.5min, 9min, 9.5min, 10min, 10.5min, 11min, 11.5min and 12 min.

The invention provides a method for detecting methotrexate, which is characterized in that a first detection result corresponding to standard solutions with different concentrations can be obtained by detecting standard solutions containing methotrexate through a liquid chromatograph-mass spectrometer, and a standard curve equation of the methotrexate is obtained by fitting based on the concentration of the methotrexate in the standard solutions with various concentrations, the concentration of an internal standard substance and a plurality of detection results because the standard solutions contain the internal standard substance methotrexate-d 3. Centrifuging a sample to be treated to obtain centrifuged serum or plasma, and sequentially adding an internal standard substance and a precipitated protein reagent to perform protein precipitation to obtain a sample to be detected. And detecting by using a liquid chromatograph-mass spectrometer under the same detection condition with the standard solution to obtain a second detection result of the sample to be detected, and obtaining the content of the methotrexate in the sample to be detected based on the standard curve equation and the second detection result. Because the purification of the target object can be completed through protein precipitation, and the detection can be completed without complex pretreatment processes such as liquid-liquid extraction and the like, 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 method of detecting methotrexate according to an embodiment of the present invention;

FIG. 2 is a chromatogram of methotrexate and an internal standard in a standard solution provided by an embodiment of the invention;

FIG. 3 is a chromatogram of methotrexate and an internal standard in a test sample provided by an embodiment of the invention;

FIG. 4 is a chromatogram of a sample volume of 0.1 μ L of a sample to be tested according to an embodiment of the present invention;

FIG. 5 is a chromatogram of a sample volume of 10 μ L of a sample to be tested according to an embodiment of the present invention;

FIG. 6 is a chromatogram of 20 μ L of a sample volume of a sample to be tested 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 18 ℃ according to an embodiment of the present invention;

FIG. 8 is a chromatogram for a flow rate of 0.25mL/min at a column temperature of 18 ℃ according to an embodiment of the present invention;

FIG. 9 is a chromatogram for a flow rate of 0.3mL/min at a column temperature of 20 ℃ according to an embodiment of the present invention;

FIG. 10 is a chromatogram for a flow rate of 0.3mL/min at a column temperature of 25 ℃ according to an embodiment of the present invention;

FIG. 11 is a chromatogram for a flow rate of 0.3mL/min at a column temperature of 30 ℃ according to an embodiment of the present invention;

FIG. 12 is a chromatogram for a flow rate of 0.4mL/min at a column temperature of 30 ℃ according to an embodiment of the present invention;

FIG. 13 is a chromatogram for a flow rate of 0.4mL/min at a column temperature of 35 ℃ according to an embodiment of the present invention;

FIG. 14 is a chromatogram for a flow rate of 0.5mL/min at a column temperature of 35 ℃ according to an embodiment of the present invention;

FIG. 15 is a chromatogram for a flow rate of 0.6mL/min at a column temperature of 35 ℃ according to an embodiment of the present invention;

FIG. 16 is a chromatogram for a flow rate of 0.6mL/min at a column temperature of 40 ℃ according to an embodiment of the present invention;

FIG. 17 is a chromatogram for a flow rate of 0.6mL/min at a column temperature of 50 ℃ according to an embodiment of the present invention;

FIG. 18 is a chromatogram for a flow rate of 0.6mL/min at a column temperature of 60 ℃ according to an embodiment of the present invention;

FIG. 19 is a chromatogram for a flow rate of 0.4mL/min at a column temperature of 60 ℃ according to an embodiment of the present invention;

FIG. 20 is a chromatogram of a methanol solution of a precipitated protein reagent provided in accordance with an embodiment of the present invention;

FIG. 21 is a chromatogram of an acetonitrile solution of a precipitated protein reagent provided by an embodiment of the present invention;

FIG. 22 is a chromatogram of an aqueous solution containing 5% trichloroacetic acid provided as a protein precipitation reagent according to an embodiment of the present invention;

FIG. 23 is a chromatogram of an aqueous solution containing 6% perchloric acid of a precipitated protein reagent according to an embodiment of the present invention;

FIG. 24 is a chromatogram of an elution mobile phase having a volume ratio of 20% organic phase to 80% aqueous phase according to an embodiment of the present invention;

FIG. 25 is a chromatogram of an elution mobile phase having a volume ratio of organic phase to aqueous phase of 25% to 75%, according to an embodiment of the present invention;

FIG. 26 is a chromatogram of an elution mobile phase having a volume ratio of organic phase to aqueous phase of 22%: 78%, according to an embodiment of the present invention;

FIG. 27 is a chromatogram of an elution mobile phase having a volume ratio of organic phase to aqueous phase of 40% to 60% according to an embodiment of the present invention;

FIG. 28 is a chromatogram of a gradient elution provided by an embodiment of the present invention;

FIG. 29 is a chromatogram of a gradient elution provided by an embodiment of the present invention;

FIG. 30 is a chromatogram of a gradient elution provided by an embodiment of the present invention;

FIG. 31 is a chromatogram of a gradient elution provided by an embodiment of the present invention;

FIG. 32 is a chromatogram of an elution mobile phase provided by an embodiment of the present invention;

FIG. 33 is a chromatogram of an elution mobile phase provided by an embodiment of the present invention;

FIG. 34 is a chromatogram of an elution mobile phase provided by an embodiment of the present invention;

FIG. 35 is a chromatogram of an elution mobile phase provided by an embodiment of the present invention;

FIG. 36 is a chromatogram of an elution mobile phase provided by an embodiment of the present invention;

FIG. 37 is a chromatogram of an elution mobile phase provided by an embodiment of the present invention;

FIG. 38 is a chromatogram of an Agilent ZORBAX-XDB-C8 column provided in accordance with an embodiment of the present invention;

FIG. 39 is a chromatogram of an Agilent ZORBAX-extended-C18 column provided in accordance with an embodiment of the present invention;

FIG. 40 is a chromatogram from a Waters Atlantis-T3 column according to an embodiment of the present invention;

FIG. 41 is a chromatogram from a Waters Atlantis-dC18 column according to one embodiment of the present invention;

FIG. 42 is a chromatogram from an Agilent poroshell 120SB-C18 column according to one embodiment of the present invention;

FIG. 43 is a chromatogram from a column of SHIMADZU-SP-C18 according to an embodiment of the present invention;

FIG. 44 is a chromatogram of an IncertSustain-C8 column according to an embodiment of the 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 sample to be detected is generally detected by adopting high performance liquid chromatography, and the sample to be detected adopts ticlopidine or aminopterin as an internal standard substance, but both the ticlopidine and the aminopterin cannot eliminate the influence of matrix effect to the maximum extent, so that the accuracy of the detection of the sample to be detected is influenced, and further the actual application and popularization of the detection method of the methotrexate are influenced.

In addition, no alkali is added in the preparation process of the stock solution of the sample to be detected, so that the stock solution of the sample to be detected is difficult to store stably, and the detection accuracy of the sample to be detected is influenced. And the pretreatment of the sample to be detected generally adopts liquid-liquid extraction, so that the pretreatment process is relatively complex, the time required by the pretreatment process is relatively long, and the overall detection time of the methotrexate in the sample to be detected is relatively long.

Based on the above problems, an embodiment of the present invention provides a method for detecting methotrexate, as shown in fig. 1, including:

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

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

step 103: fitting a standard curve equation of methotrexate according to each first detection result, the concentration of methotrexate 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 and a precipitated protein reagent into the first supernatant, uniformly mixing by vortex, centrifuging, and taking the centrifuged second supernatant as a sample to be detected;

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

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

In the embodiment of the invention, the first detection result corresponding to each concentration of standard solution can be obtained by detecting standard solutions containing methotrexate with different concentrations through a LC-MS, and the standard curve equation of methotrexate is obtained by fitting based on the concentration of methotrexate in standard solutions with various concentrations, the concentration of the internal standard substance and a plurality of detection results because the standard solutions contain the internal standard substance. Centrifuging a sample to be treated to obtain centrifuged serum or plasma, and sequentially adding an internal standard substance and a precipitated protein reagent to perform protein precipitation to obtain a sample to be detected. And detecting by using a liquid chromatograph-mass spectrometer under the same detection condition with the standard solution to obtain a second detection result of the sample to be detected, and obtaining the content of the methotrexate in the sample to be detected based on the standard curve equation and the second detection result. Because the purification of the target object can be completed through protein precipitation, and the detection can be completed without liquid-liquid extraction, the detection time of the sample to be detected can be shortened.

The following examples illustrate the detection of methotrexate in more detail.

Example 1: preparation of Standard solutions of series of concentrations

(a) Preparation of a standard stock solution:

methotrexate standard 4.45mg was weighed accurately into a 2mL volumetric flask, dissolved in 0.1M aqueous sodium hydroxide and taken up in 2mL to give a standard stock solution, which was stored at-80 ℃ for 24 months.

(b) Preparation of standard working solution

Taking a proper amount of the standard stock solution in the step (a), diluting and mixing by using a water solution containing 50% methanol as a diluent to obtain a standard working solution containing 100-50000ng/mL methotrexate, and storing at-80 ℃ for 6 months;

wherein, the standard working solution with different concentrations contains methotrexate: 100ng/mL, 400ng/mL, 1000ng/mL, 2000ng/mL, 5000ng/mL, 10000ng/mL, 20000ng/mL, 50000 ng/mL.

(c) Preparation of internal standard stock solution

Taking 100 mu g/mL standard methotrexate-d 3 as internal standard stock solution, and storing at-80 ℃ for 24 months.

(d) Preparation of internal standard working solution

Taking 400 μ L of the stock solution of the internal standard in the step (c), diluting with 9600 μ L of 50% methanol-containing aqueous solution as a diluent to obtain 4000ng/mL of the working solution of the internal standard containing methotrexate-d 3, and storing at-80 ℃ for 12 months.

(e) Calibration of standard solutions

Transferring 10 mu L of the eight standard working solutions with different concentrations in the step (b) into 1.5mL centrifuge tubes, adding 10 mu L of the internal standard working solution in the step (d) into each centrifuge tube, adding 390 mu L of the aqueous solution containing 6% sulfosalicylic acid into each centrifuge tube, mixing to prepare eight mixed solutions with different concentrations, uniformly mixing the mixed solutions by vortex at the rotating speed of 1500-2000rpm for 0.5-1.5min, centrifuging, and transferring the centrifuged second supernatant as the standard solution.

Example 2: fitting standard curve equation

The eight standard solutions in example 1 were detected by a LC-MS to obtain chromatograms of the eight standard solutions of methotrexate with different concentrations.

The peak areas corresponding to the methotrexate and the internal standard substance in the eight standard solutions are respectively obtained from the chromatogram of the methotrexate standard solution, the ratio of the peak area of the methotrexate obtained from the chromatogram of the standard solution with each concentration to the chromatographic peak area of the internal standard substance is used as the ordinate y1 of a standard curve equation, the concentration of the methotrexate standard working solution and the concentration of the internal standard substance are used as the abscissa x1 of the standard curve equation, the data of different concentrations obtained by detection are subjected to linear regression, the standard curve equation is fitted to be y1 a x1+ b, and weight coefficients a and b are obtained, 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: agilent SB-Aq-C18, filler particle size 5 μm, inner diameter 2.1mm, length 50 mm;

with isocratic elution, the aqueous phase in the elution mobile phase comprises: an aqueous solution containing 0.1% formic acid;

eluting the organic phase in the mobile phase comprises: the methanol solution elutes the organic phase in the mobile phase and the water phase in a volume ratio of 30% to 70%, and the elution time is 2 min;

the column temperature was 35 ℃; the flow rate is 0.4 mL/min; sample introduction amount: 4 μ L.

Mass spectrum conditions: a mass spectrum detector in the LC-MS is in an ESI (+) detection mode;

the ion source parameters in the LC-MS are as follows: heating air flow rate (L/min) is 10, atomizing air flow rate (L/min) is 20, heating air temperature (DEG C) is 350, and capillary voltage (V) is 3500.

Wherein, the ion pair parameters of the mass spectrometer are shown in the following table 1:

TABLE 1

Name of substance	Parent ion	Daughter ions	Dwell	Fragmentor	CE	CAV
							Methotrexate (nature)	455.2	175.1	100	120	40	7
Methotrexate (quantitative)	455.2	308.2	100	120	20	7
							Methotrexate-d 3 (nature)	458	137.2	100	120	40	1
Methotrexate-d 3 (quantitative)	458	311.1	100	120	20	1

Where Dewll is the scan time, fragment is the fragmentation voltage, CE is the collision voltage, CAV: is a linear acceleration voltage.

It should be noted that standard solutions with different concentrations can be prepared according to the pretreatment operation during the treatment of the sample to be treated, that is, the vortex rotation speed time, the protein precipitation reagent, the vortex time and rotation speed after the protein precipitation reagent is added, and the centrifugal rotation speed and time in the standard solution are all consistent with the pretreatment of the sample to be detected in embodiment 3, so as to eliminate the system error and improve the accuracy of the detection result.

Example 3: pretreatment of sample to be tested

3.1 taking at least 2mL 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 ℃ for later use before analysis.

3.2 using a pipette to pipette 10 μ L of the internal standard working solution of example 1 into a 1.5mL centrifuge tube, then adding 100 μ L of the serum or plasma obtained in step 3.1, mixing by vortexing at 2000rpm for 0.5min, adding 300 μ L of the precipitated protein reagent aqueous solution containing 6% sulfosalicylic acid, mixing by vortexing at 2000rpm for 3min, and then centrifuging at 12000rpm for 10min at high speed to obtain a second supernatant, which is the sample to be measured.

Example 4: detection of a sample to be tested

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

The chromatographic peak area of the methotrexate in the sample to be detected and the chromatographic peak area of the internal standard substance in the sample to be detected can be obtained from the chromatogram of the sample to be detected, the chromatographic peak area of the methotrexate in the sample to be detected and the chromatographic peak area of the internal standard substance are taken as the ordinate y1 and are substituted into the standard curve equation y1 ═ a × 1+ b in the embodiment 2, and the weight coefficients a and b are known, so that the concentration of the methotrexate in the sample to be detected can be obtained.

In conclusion, the isotope of the sample to be detected is used as the internal standard substance, so that the internal standard substance and the sample to be detected are prevented from reacting to influence the detection of the sample to be detected when the sample to be detected is detected.

Example 5: linear relationship and quantitative limits of the detection method of methotrexate

Transferring 10 mu L of the eight methotrexate standard working solutions with different concentrations in the step (a), adding 10 mu L of the internal standard working solution in the step (b) and 390 mu L of methanol into each transferred methotrexate standard working solution with each concentration, uniformly mixing, and measuring by using a liquid chromatograph-mass spectrometer according to the pretreatment in the example 3 and the detection conditions in the example 2, wherein in the example, the detection is performed according to the sequence from low concentration to high concentration, so that the influence of the mixed liquid with high concentration on the mixed liquid with low concentration during the detection is avoided. And then, plotting the peak area-concentration of the quantitative chromatographic spectrum to obtain a standard curve, wherein the result shows that the linear range and the quantitative limit of the methotrexate are as follows:

(1) limit of detection (LOD): 0.083ng/mL

(2) Limit of quantitation (LOQ): 0.25ng/mL

(3) Linear range: methotrexate is in the range of 2.445ng/mL to 1219.51ng/mL, the linearity is good, and the correlation coefficient R is²﹥0.995。

According to the embodiment, the detection limit and the quantification limit of the methotrexate are respectively 0.083ng/mL and 0.25ng/mL, the sensitivity is very high, the accurate quantification can be performed on the biological sample with very low methotrexate content, and the high accuracy and the wide applicability of the detection method are ensured.

Example 6: recovery and precision of methotrexate assay

The methotrexate standard working solution in example 1 was prepared into high, medium and low concentrations of 3 for sample recovery and precision experiments, the detection conditions in example 2 were used for measurement, and the analysis and measurement were repeated for 3 batches, and the recovery of methotrexate is shown in table 2. The average recovery rate of the methotrexate in the range of 3 low, medium and high addition levels is 99.55 to 101.80 percent, and the precision is 0.41 to 1.87 percent.

TABLE 2 recovery and precision of methotrexate spiking

By combining 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 methotrexate in 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.

Fig. 2 is a chromatogram of methotrexate in a standard solution in example 2, and fig. 3 is a chromatogram of methotrexate in a test sample in example 3, wherein the retention times of methotrexate and an internal standard in fig. 2 and 3 are the same. In fig. 2 and 3, the chromatogram for internal standard methotrexate-d 3 is located on the top and the chromatogram for methotrexate is located on the bottom.

The unit length of the abscissa in FIG. 2 is 0.1, and the unit length of the ordinate in the chromatogram located above in FIG. 2 is 0.1X 10⁴The unit length of the ordinate of the chromatogram lying below is 0.5X 10³；

The unit length of the abscissa in FIG. 3 is 0.1, and the unit length of the ordinate of the chromatogram located above in FIG. 3 is 1X 10³The unit length of the ordinate of the chromatogram lying below is 0.1X 10³。

As can be seen from FIGS. 2 and 3, the retention time of methotrexate in the sample to be tested is consistent with that of the standard working solution, in the method, methotrexate-d 3 is used as an internal standard substance, the retention time of methotrexate is about 1.05min, and the retention time of the internal standard substance is about 1.05min, so that the identification of a target compound is more accurate, the analysis time is short, the interference is small, the internal standard quantification is suitable, the specificity is strong, and the accuracy and the sensitivity are high.

Example 7: description of sample size

The tests corresponding to fig. 4 to 6 are parallel tests corresponding to the tests in examples 3 and 4, respectively, with the difference that the sample volumes are different. Among the chromatograms in fig. 4 to 6, the upper chromatogram is the internal standard methotrexate-d 3, and the lower chromatogram is the methotrexate.

FIG. 4 is a chromatogram showing the amount of sample of 0.1. mu.L taken by the sample to be measured, and a single bar on the abscissa in FIG. 4The bit length is 0.1, and the unit length of the ordinate is 0.025X 10²；

FIG. 5 is a chromatogram in which the amount of sample to be measured is 10. mu.L, the unit length on the abscissa of FIG. 5 is 0.1, and the unit length on the ordinate of the chromatogram located above in FIG. 5 is 0.2X 10⁴The unit length of the ordinate of the chromatogram lying below is 0.5X 10²；

FIG. 6 is a chromatogram in which the amount of sample to be measured is 20. mu.L, the unit length on the abscissa of FIG. 6 is 0.1, and the unit length on the ordinate of the chromatogram located above in FIG. 6 is 0.5X 10⁴The unit length of the ordinate of the chromatogram lying below is 1X 10²。

Specifically, the sample volume of the sample to be detected is 0.1-20 μ L, the signal-to-noise ratio when the sample volume is 0.1 μ L is 32.2, the quantitative requirement can be met, and the peak type of the chromatographic peak of the sample to be detected is influenced by the solvent effect when the sample volume exceeds 20 μ L, so that the sample volume of the sample to be detected is 0.1-20 μ L.

Example 8: description of flow Rate and column temperature

The experiments in fig. 7 to 19 correspond to parallel experiments corresponding to examples 3 and 4, respectively, with the difference between the flow rate and the column temperature, and in fig. 7 to 19, the chromatogram of the internal standard methotrexate-d 3 is located at the top and the chromatogram of methotrexate is located at the bottom.

FIG. 7 is a chromatogram at a column temperature of 18 ℃ and a flow rate of 0.2mL/min, in which the unit length on the abscissa of FIG. 7 is 0.1 and the unit length on the ordinate of the chromatogram located above in FIG. 7 is 0.5X 10³The unit length of the ordinate of the chromatogram lying below is 0.5X 10²；

FIG. 8 is a chromatogram at a column temperature of 18 ℃ and a flow rate of 0.25mL/min, in which the unit length on the abscissa of FIG. 8 is 0.1 and the unit length on the ordinate of the chromatogram located above in FIG. 8 is 1X 10³The unit length of the ordinate of the chromatogram lying below is 0.5X 10²；

FIG. 9 is a chromatogram at a column temperature of 20 ℃ and a flow rate of 0.3mL/min, with the unit length on the abscissa of FIG. 9 being 0.1 and the ordinate of the chromatogram located above in FIG. 9Has a unit length of 0.5X 10³The unit length of the ordinate of the chromatogram lying below is 0.5X 10²；

FIG. 10 is a chromatogram at a column temperature of 25 ℃ and a flow rate of 0.3mL/min, in which the unit length on the abscissa of FIG. 10 is 0.1 and the unit length on the ordinate of the chromatogram located above in FIG. 10 is 1X 10³The unit length of the ordinate of the chromatogram lying below is 0.5X 10²；

FIG. 11 is a chromatogram at a column temperature of 30 ℃ and a flow rate of 0.3mL/min, in which the unit length on the abscissa of FIG. 11 is 0.1 and the unit length on the ordinate of the chromatogram located above in FIG. 11 is 1X 10³The unit length of the ordinate of the chromatogram lying below is 0.5X 10²；

FIG. 12 is a chromatogram at a column temperature of 30 ℃ and a flow rate of 0.4mL/min, in which the unit length on the abscissa of FIG. 12 is 0.1 and the unit length on the ordinate of the chromatogram located above in FIG. 12 is 1X 10³The unit length of the ordinate of the chromatogram lying below is 1X 10²；

FIG. 13 is a chromatogram at a column temperature of 35 ℃ and a flow rate of 0.4mL/min, in which the unit length on the abscissa of FIG. 13 is 0.1 and the unit length on the ordinate of the chromatogram located above in FIG. 13 is 1X 10³The unit length of the ordinate of the chromatogram lying below is 1X 10²；

FIG. 14 is a chromatogram at a column temperature of 35 ℃ and a flow rate of 0.5mL/min, in which the unit length on the abscissa of FIG. 14 is 0.1 and the unit length on the ordinate of the chromatogram located above in FIG. 14 is 0.1X 10⁴The unit length of the ordinate of the chromatogram lying below is 1X 10²；

FIG. 15 is a chromatogram at a column temperature of 35 ℃ and a flow rate of 0.6mL/min, in which the unit length on the abscissa of FIG. 15 is 0.1 and the unit length on the ordinate of the chromatogram located above in FIG. 15 is 0.1X 10⁴The unit length of the ordinate of the chromatogram lying below is 1X 10²；

FIG. 16 is a chromatogram at a column temperature of 40 ℃ and a flow rate of 0.6mL/min, in which the unit length on the abscissa of FIG. 16 is 0.1 and the unit length on the ordinate of the chromatogram located above in FIG. 16 is 0.1×10⁴The unit length of the ordinate of the chromatogram lying below is 1X 10²；

FIG. 17 is a chromatogram at a column temperature of 50 ℃ and a flow rate of 0.6mL/min, in which the unit length on the abscissa of FIG. 17 is 0.1 and the unit length on the ordinate of the chromatogram located above in FIG. 17 is 0.2X 10⁴The unit length of the ordinate of the chromatogram lying below is 1X 10²；

FIG. 18 is a chromatogram at a column temperature of 60 ℃ and a flow rate of 0.6mL/min, in which the unit length on the abscissa of FIG. 18 is 0.05 and the unit length on the ordinate of the chromatogram located above in FIG. 18 is 0.2X 10⁴The unit length of the ordinate of the chromatogram lying below is 1X 10²；

FIG. 19 is a chromatogram at a column temperature of 60 ℃ and a flow rate of 0.4mL/min, in which the unit length on the abscissa of FIG. 19 is 0.1 and the unit length on the ordinate of the chromatogram located above in FIG. 19 is 0.2X 10⁴The unit length of the ordinate of the chromatogram lying below is 1X 10²。

As can be seen from fig. 7 to 19, when the flow rate is lower than 0.25mL/min and the column temperature is lower than 18 ℃, the retention time of methotrexate and the internal standard substance is 2.8min, which results in too long detection time of the whole sample to be detected, and affects the detection timeliness of the sample to be detected. And because the flow velocity of the mobile phase is too low, the ionization efficiency of the sample to be detected in the LC-MS is poor, so that the detection signal is poor, and the detection of the sample to be detected is not facilitated.

When the flow rate is greater than 0.6mL/min and the column temperature is greater than 60 ℃, the column pressure of the chromatographic column exceeds the pressure which can be borne by the chromatographic column, and when the number of samples to be detected is large, irreversible damage can be caused to the chromatographic column, so that the accuracy of detecting the samples to be detected is reduced. Moreover, the column temperature at this time exceeds the temperature that the chromatographic column can bear, which can cause irreversible damage to the filler in the chromatographic column and affect the detection effect of the sample to be detected. Most importantly, the retention time of the methotrexate is less than 0.5min, so that the peak of the methotrexate is too fast, and the retention time of a solvent peak is usually about 0.5min, so that the solvent peak can interfere with the chromatographic peak of the methotrexate, and the identification of a sample to be detected is not facilitated.

In conclusion, the flow rate of the detection on the sample to be detected is in the range of 0.25-0.6mL/min, the column temperature is in the range of 18-60 ℃, the analysis time of methotrexate can be within 3.0min, the overall detection time of the sample to be detected is shortened, and the timeliness of the sample detection is improved.

Example 9: description of the Agents for precipitating proteins

The assays corresponding to FIGS. 20 to 23 are parallel assays corresponding to examples 3 and 4, respectively, with the difference being the precipitated protein reagent. In fig. 20 to 23, the chromatograms for methotrexate-d 3, which is an internal standard, are located at the top, and the chromatograms for methotrexate are located at the bottom.

FIG. 20 is a chromatogram of a methanol solution as a protein-precipitating reagent, in which the unit length on the abscissa of FIG. 20 is 0.1 and the unit length on the ordinate of the chromatogram located above in FIG. 20 is 0.4X 10³The unit length of the ordinate of the chromatogram lying below is 0.5X 10²；

FIG. 21 is a chromatogram showing the case where the protein-precipitating reagent is an acetonitrile solution, in which the unit length on the abscissa of FIG. 21 is 0.1 and the unit length on the ordinate of the chromatogram located above in FIG. 21 is 0.4X 10³The unit length of the ordinate of the chromatogram lying below is 0.4X 10²；

FIG. 22 is a chromatogram showing the case where the protein-precipitating reagent is an aqueous solution containing 5% trichloroacetic acid, in which the unit length on the abscissa of FIG. 22 is 0.1 and the unit length on the ordinate of the chromatogram located above in FIG. 22 is 1X 10²The unit length of the ordinate of the chromatogram lying below is 0.2X 10²；

FIG. 23 is a chromatogram showing the results of a 6% perchloric acid-containing aqueous solution as a protein-precipitating reagent, in which the unit length on the abscissa of FIG. 23 is 0.1 and the unit length on the ordinate of the chromatogram located above in FIG. 23 is 1X 10²The unit length of the ordinate of the chromatogram lying below is 0.2X 10²。

As can be seen from fig. 3 and 20 to 23, when at least one of methanol solution, acetonitrile solution, trichloroacetic acid, sulfosalicylic acid and perchloric acid is used as a protein precipitation reagent for performing protein precipitation on the first supernatant, the chromatographic peaks of the sample to be detected are not a leading edge peak and a tailing peak, and the peak width of the chromatographic peak is not too wide. And because methanol solution, acetonitrile solution, trichloroacetic acid, sulfosalicylic acid and perchloric acid are common reagents and are easy to obtain, the difficulty of carrying out protein precipitation on the first supernatant can be reduced.

Example 10: description of elution ratio for eluting Mobile phase

The tests corresponding to fig. 24 to 31 are parallel tests corresponding to examples 3 and 4, respectively, except that the volume ratio of the organic phase to the aqueous phase in the mobile phase is different. In fig. 24 to 31, the chromatogram of the internal standard methotrexate-d 3 is located at the upper side, and the chromatogram of methotrexate is located at the lower side.

FIGS. 24 to 27 show the chromatograms of the samples to be tested at the ratios of the organic to aqueous phases in the elution mobile phase by volume of 20%: 80%, 25%: 75%, 22%: 78% and 40%: 60% when eluted at equal intervals;

the unit length of the abscissa in fig. 24 is 0.2, and the unit length of the ordinate of the chromatogram located above in fig. 24 is 1 × 10³The unit length of the ordinate of the chromatogram lying below is 0.5X 10²；

The unit length of the abscissa in FIG. 25 is 0.2, and the unit length of the ordinate of the chromatogram located above in FIG. 25 is 1X 10³The unit length of the ordinate of the chromatogram lying below is 1X 10²；

The unit length on the abscissa of FIG. 26 was 0.2, and the unit length on the ordinate of the chromatogram located above in FIG. 26 was 1X 10³The unit length of the ordinate of the chromatogram lying below is 0.5X 10²；

The unit length on the abscissa of FIG. 27 is 0.2, and the unit length on the ordinate of the chromatogram located above in FIG. 27 is 2X 10³The unit length of the ordinate of the chromatogram lying below is 1X 10²。

Fig. 28 to fig. 31 show chromatograms of samples to be tested under different gradient elution mobile phase conditions during gradient elution;

in fig. 28, the volume ratio of the organic phase to the water is: 0.00 min: 10 percent to 90 percent; 0.01 min: 80 percent to 20 percent; 1.00 min: 80 percent to 20 percent; 1.01 min: 10 percent to 90 percent; 3.00 min: 10 percent to 90 percent; the unit length on the abscissa of FIG. 28 was 0.3, and the unit length on the ordinate of the chromatogram located above in FIG. 28 was 0.2X 10⁴The unit length of the ordinate of the chromatogram lying below is 0.2X 10³；

The volume ratio of the organic phase to water in fig. 29 is: 0.00 min: 10 percent to 90 percent; 0.01 min: 40 percent to 60 percent; 1.00 min: 40 percent to 60 percent; 1.01 min: 10 percent to 90 percent; 3.00 min: 10 percent to 90 percent; the unit length on the abscissa of fig. 29 is 0.2, and the unit length on the ordinate of the chromatogram located above in fig. 29 is 0.2 × 10⁴The unit length of the ordinate of the chromatogram lying below is 0.2X 10³；

The volume ratio of the organic phase to water in fig. 30 is: 0.00 min: 10 percent to 90 percent; 0.01 min: 30 percent to 70 percent; 1.00 min: 30 percent to 70 percent; 1.01 min: 10 percent to 90 percent; 3.00 min: 10 percent to 90 percent; the unit length on the abscissa of FIG. 30 is 0.3, and the unit length on the ordinate of the chromatogram located above in FIG. 30 is 0.2X 10⁴The unit length of the ordinate of the chromatogram lying below is 0.2X 10³；

The volume ratio of the organic phase to water in fig. 31 is: 0.00 min: 40 percent to 60 percent; 0.01 min: 100 percent to 0 percent; 1.00 min: 100 percent to 0 percent; 1.01 min: 40 percent to 60 percent; 3.00 min: 40 percent to 60 percent; the unit length on the abscissa of FIG. 31 was 0.2, and the unit length on the ordinate of the chromatogram located above in FIG. 31 was 1X 10²The unit length of the ordinate of the chromatogram lying below is 1X 10²。

As can be seen from fig. 24 to fig. 31, when the isocratic elution mode is adopted, if the proportion of the organic phase in the elution mobile phase is less than 22%, the retention time of methotrexate is increased, and the detection time of the sample to be detected is longer than 3min, resulting in an excessively long detection time of the sample to be detected; when the volume ratio of the organic phase in the elution mobile phase is more than 40%, the retention time of methotrexate is short, and peaks before 0.5min, so that matrix effect is easy to generate.

When gradient elution is adopted, if the volume ratio of the organic phase in the elution mobile phase is less than 10% in 0.00min, the column pressure cannot be recovered in a short time, so that the analysis time of the sample to be detected is too long; if the volume ratio of the organic phase in the elution mobile phase is more than 40% in 0.00min, the retention time of methotrexate is short, peaks before 0.5min, and a matrix effect is easy to generate; if the volume ratio of the organic phase in the elution mobile phase is 10% in 0.00min, the volume ratio of the organic phase in the elution mobile phase in the gradient elution process cannot be less than 30%, otherwise, the retention time of the internal standard substance is too long, and the peak cannot be generated within 3min, so that the analysis time of the sample to be detected is too long.

Example 11: description of eluting Mobile phase

The tests of fig. 32 to 37 are parallel tests corresponding to those of examples 3 and 4, with the difference that formic acid is added to the elution mobile phase. In fig. 32, the chromatogram of methotrexate-d 3, which is an internal standard, is shown at the top of fig. 37, and the chromatogram of methotrexate is shown at the bottom of fig. 37.

FIG. 32 is a chromatogram in which the aqueous phase in the elution mobile phase is distilled water and the organic phase is a methanol solution, wherein the unit length on the abscissa of FIG. 32 is 0.1 and the unit length on the ordinate of the chromatogram located above in FIG. 32 is 1X 10³The unit length of the ordinate of the chromatogram lying below is 1X 10²；

FIG. 33 is a chromatogram showing elution of an aqueous solution containing 0.1% formic acid in an aqueous phase and 0.1% formic acid in a methanol solution in an organic phase in a mobile phase, wherein the unit length of the abscissa in FIG. 33 is 0.2 and the unit length of the ordinate in the chromatogram located above in FIG. 33 is 0.2X 10⁴The unit length of the ordinate of the chromatogram lying below is 1X 10²；

FIG. 34 is a chromatogram showing elution of an aqueous solution containing 10mM ammonium acetate in an aqueous phase and 0.1% formic acid in a methanol solution in an organic phase in a mobile phase, wherein the unit length on the abscissa of FIG. 34 is 0.2 and the unit length on the ordinate of the chromatogram located above in FIG. 34 is 1X 10²The unit length of the ordinate of the chromatogram lying below is 0.5X 10¹；

FIG. 35 is a chromatogram showing that the aqueous phase in the elution mobile phase is an aqueous solution containing 0.1% formic acid and 1mM ammonium acetate, and the organic phase is a methanol solution containing 0.1% formic acid, wherein the unit length on the abscissa of FIG. 35 is 0.2, and the unit length on the ordinate of the chromatogram located above in FIG. 35 is 0.2X 10⁴The unit length of the ordinate of the chromatogram lying below is 0.2X 10³；

FIG. 36 is a chromatogram showing that the aqueous phase in the elution mobile phase is an aqueous solution containing 0.1% formic acid and 1mM ammonium formate, and the organic phase is a methanol solution containing 0.1% formic acid, wherein the unit length on the abscissa of FIG. 36 is 0.2, and the unit length on the ordinate of the chromatogram located above in FIG. 36 is 0.2X 10⁴The unit length of the ordinate of the chromatogram lying below is 0.2X 10³；

FIG. 37 is a chromatogram showing elution of an aqueous solution containing 0.1% formic acid in an aqueous phase and 0.1% formic acid in an acetonitrile solution in an organic phase in a mobile phase, wherein the unit length of the abscissa in FIG. 37 is 0.2 and the unit length of the ordinate in the chromatogram located above in FIG. 37 is 0.2X 10⁴The unit length of the ordinate of the chromatogram lying below is 2X 10²。

As can be seen from fig. 3 and 32 to 37, when no formic acid, no buffer salt, or no buffer salt is added to the elution mobile phase, both the peak shape and the peak width of the chromatographic peak of the sample to be measured and the internal standard substance meet the test requirements.

Example 12: description of the column

The experiments in fig. 38 to 44 correspond to parallel experiments corresponding to example 3 and example 4, respectively, with the difference of the reverse phase chromatography columns. The chromatograms for internal standard methotrexate-d 3 in each of figures 38-44, located above, and for methotrexate, located below.

FIG. 38 is a chromatogram of Agilent ZORBAX-XDB-C8 in which the filler had a particle size of 5 μm, an inner diameter of 2.1mm and a length of 50mm, and the abscissa of FIG. 38 had a unit length of 0.2, which is shown in the figure38 the unit length of the ordinate of the chromatogram lying above was 0.2X 10⁴The unit length of the ordinate of the chromatogram lying below is 2X 10²；

FIG. 39 is a chromatogram of a column of Agilent ZORBAX-extended-C18 in which the filler has a particle diameter of 3.5 μm, an inner diameter of 2.1mm and a length of 50mm, the unit length on the abscissa of FIG. 39 is 0.2, and the unit length on the ordinate of the chromatogram located above in FIG. 39 is 0.2X 10⁴The unit length of the ordinate of the chromatogram lying below is 2X 10²；

FIG. 40 is a chromatogram obtained when the column used was Waters Atlantis-T3, in which the filler had a particle diameter of 5 μm, an inner diameter of 2.1mm and a length of 50mm, the unit length on the abscissa of FIG. 40 was 0.2, and the unit length on the ordinate of the chromatogram located above in FIG. 40 was 2X 10³The unit length of the ordinate of the chromatogram lying below is 1.5X 10²；

FIG. 41 is a chromatogram obtained when the column used was Waters Atlantis-dC18, in which the packing had a particle size of 5 μm, an inner diameter of 2.1mm and a length of 50mm, the unit length on the abscissa of FIG. 41 was 0.2, and the unit length on the ordinate of the chromatogram located above in FIG. 41 was 0.2X 10³The unit length of the ordinate of the chromatogram lying below is 0.2X 10²；

FIG. 42 is a chromatogram of a chromatographic column of Agilent poroshell 120SB-C18, in which the filler has a particle diameter of 2.7 μm, an inner diameter of 2.1mm and a length of 50mm, the unit length on the abscissa of FIG. 42 is 0.2, and the unit length on the ordinate of the chromatogram located above in FIG. 42 is 0.4X 10⁴The unit length of the ordinate of the chromatogram lying below is 0.4X 10³；

FIG. 43 is a chromatogram obtained when the column used was SHIMADZU-SP-C18, wherein the filler had a particle diameter of 2.6 μm, an inner diameter of 2.1mm and a length of 50mm, the unit length on the abscissa of FIG. 43 was 0.2, and the unit length on the ordinate of the chromatogram located above in FIG. 43 was 0.2X 10⁴The unit length of the ordinate of the chromatogram lying below is 2X 10²；

FIG. 44 is a chromatogram of a chromatography column of IncertSustain-C8 in which the filler particlesDiameter of 5 μm, inner diameter of 2.1mm, length of 50mm, unit length of abscissa in FIG. 44 of 0.2, unit length of ordinate of chromatogram located above in FIG. 44 of 1X 10³The unit length of the ordinate of the chromatogram lying below is 1X 10²。

As can be seen from FIGS. 3 and 38 to 44, the chromatographic peak of the target obtained by detecting the sample to be detected by utilizing Agilent SB-Aq-C18, Agilent ZORBAX XDB-C8, Agilent ZORBAX-extended-C18, Waters atlantatis-T3, Waters atlantatis-dC 18, Agilent poroshell 120SB-C18, SHIMADZU-SP-C18 and Incertsustain-C8 chromatographic columns has no leading edge or tailing, and the peak width also meets the detection requirement.

It should be noted that the abscissa of fig. 2 to fig. 44 is the acquisition time (min), the ordinate is the ion signal intensity, and the missing graph in the chromatogram does not affect the technical content of the present solution.

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 like 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.