Method for measuring nitrogen-containing heterocyclic compound

Abstract

A method for measuring a nitrogen-containing heterocyclic compound, which uses an LC-MS / MS method, wherein a chromatographic column is a C18 chromatographic column; a mobile phase A is an aqueous solution containing 0.1±0.05 vol% formic acid, a mobile phase B is an acetonitrile methanol solution containing 0.1±0.05 vol% formic acid, and in the acetonitrile methanol solution, the content of acetonitrile is 85±5 vol%; and a gradient elution method is used. By means of the measurement method, the content of a small molecule compound including the nitrogen-containing heterocyclic compound in tears and plasma can be efficiently and accurately measured.

Description

Method for detecting nitrogen-containing heterocyclic compounds

[0001] Cross-reference to related applications

[0002] The present application claims priority to the Chinese patent application No. 2024110452738 filed on July 31, 2024, the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0003] The present application belongs to the field of analytical chemistry, and specifically relates to a method for detecting nitrogen-containing heterocyclic compounds. BACKGROUND

[0004] EGFR2, also known as KDR or Flk-1, is identified as the receptor for VEGF and VEGFC, an early marker for endothelial progenitor cells, whose expression is restricted to endothelial cells in vivo. VEGFR2 has been shown to be a major signal transducer for angiogenesis and the development of pathological conditions such as cancer and diabetic retinopathy. Studies have shown that anti-VEGF can inhibit the expression and activation of pro-inflammatory factors, thereby reducing ocular surface inflammation. VEGFR2 transduces the main signals of angiogenesis through its strong tyrosine kinase activity. However, unlike other representative tyrosine kinase receptors, VEGFR2 does not use the Ras pathway as the main downstream signal transduction, but uses the phospholipase C protein kinase C pathway to express mitogen-activated protein (MAP) kinase activation and DNA synthesis. Therefore, inhibiting VEGFR2 activity and its downstream signaling is an important target for treating diseases involving angiogenesis and inflammation.

[0005] CN114364679A discloses a novel compound with a nitrogen-containing heterocyclic structure, and mentions that the compound can be used as a VEGFR2 target inhibitor for treating allergic diseases, autoimmune diseases and inflammatory diseases, including but not limited to dry eye and allergic conjunctivitis, retinal inflammatory diseases, age-related macular degeneration (AMD), proliferative diabetic retinopathy (PDR) and retinopathy of prematurity (ROP), cancer, rheumatoid arthritis, glomerulonephritis, multiple vasculitides, idiopathic thrombocytopenic purpura (ITP), myasthenia gravis, allergic rhinitis, chronic obstructive pulmonary disease (COPD), adult respiratory distress syndrome (ARDs) and asthma, etc.

[0006] However, in the research of compound drug, drug metabolism research is an important link. The prior art lacks teaching on the separation and characterization method of the compound, making it difficult to judge the metabolism of the compound in human body fluids, especially in tears and plasma, in clinical research. Therefore, it is necessary to develop a method for detecting the content of the compound in human body fluids. SUMMARY

[0007] The first object of the present application is to provide a method for detecting nitrogen-containing heterocyclic compounds by LC-MS / MS, wherein,

[0008] The chromatographic column is a C18 chromatographic column.

[0009] The mobile phase A is an aqueous solution containing 0.1±0.05 vol% formic acid, and the mobile phase B is an acetonitrile-methanol solution containing 0.1±0.05 vol% formic acid, wherein the acetonitrile content in the acetonitrile-methanol solution is 85±5 vol%.

[0010] The gradient elution method is used, wherein the content of the mobile phase B is 30±5 vol% at the beginning, and gradually increases to 90±5 vol% during the gradient elution, and then decreases to 30±5 vol%.

[0011] The second object of the present application is to provide a method for extracting nitrogen-containing heterocyclic compounds from tear fluid, which comprises:

[0012] The target component is extracted from the tear fluid by using a solvent to obtain an extract.

[0013] The solvent is a mixture of ethyl acetate, methanol, water and ammonia water in a volume ratio of 100:15-25:3.0-4.0:6.0-7.0, and the concentration of the ammonia water is 5-15 wt%.

[0014] By the method of the present application, small molecular compounds including nitrogen-containing heterocyclic compounds (especially compounds with poor solubility in water) can be efficiently and accurately detected in tear fluid and plasma, and the method has great application potential in the field of drug metabolism research of related compounds. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the specific embodiments or prior art description will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0016] Figure 1 is the standard curve mentioned in Example 1 of the present application.

[0017] Figure 2 is the standard curve mentioned in Example 2 of the present application. DETAILED DESCRIPTION

[0018] The specific embodiments of the present application are described herein. It should be understood that the described embodiments are merely for the purpose of illustration and explanation and are not intended to limit the present application in any way. Various modifications and changes can be made to the present application by those skilled in the art which fall within the scope of the present application without departing from the spirit of the present application. For example, features described or illustrated as part of one embodiment can be used with another embodiment to yield still a further embodiment.

[0019] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. By further guidance, the following definitions are set forth to better define the present teachings. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0020] The alternative ranges of the terms "and / or", "or / and", "and / or" used herein include any one of the two or more related listed items, and also include any and all combinations of the related listed items, including any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", "and / or", it should be understood that in this application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B, and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (i.e., the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C, and D, i.e., includes the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (i.e., the technical solution connected by "logical and").

[0021] The terms "containing", "including", and "comprising" used in the present application are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps.

[0022] The numerical ranges used in the present application expressed in endpoints and positive values include all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0023] In the present application, the concentration values are intended to include fluctuations within a certain range. For example, they can fluctuate within a corresponding range of accuracy. For example, 2% can fluctuate within a range of ±0.1%. For values that are larger or do not need to be controlled too precisely, the values are intended to include larger fluctuations. For example, 100 mM can fluctuate within a range of ±1%, ±2%, ±5%, etc.

[0024] In the present application, the descriptions such as "a plurality of" and "a plurality of" refer to greater than or equal to 2 in number, unless otherwise specified.

[0025] In the present application, the technical features described in an open manner include both closed technical solutions consisting of listed features and open technical solutions containing listed features.

[0026] In the present application, "preferably", "better", "better", "as appropriate" only describe the implementation manner or embodiment with better effect, and it should be understood that it does not constitute a limitation on the protection scope of the present application.

[0027] In the present application, "optionally", "optional", "optional", "optionally", "optional", "optional" means optional, that is, selected from any one of the two parallel schemes of "yes" or "no". If there are multiple "optional" or "optional" in a technical solution, unless otherwise specified, and there is no contradictory relationship or mutual restriction, each "optional" or "optional" is independent.

[0028] In the present application, the term "LC-MS / MS method" is a short name for liquid chromatography-mass spectrometry / mass spectrometry. In the LC-MS / MS method, the sample is first separated into components by liquid chromatography, and then subjected to mass spectrometry by mass spectrometer.

[0029] In the present application, the term "C18 chromatographic column" refers to a chromatographic column with octane (C18) chains on the surface of the stationary phase. In the term "BEH C18 chromatographic column, 50x2.1mm, 1.7μm", "BEH" means "Ethylene Bridged Hybrid", which is characterized by using ethylene to bridge between silica gel and alkyl chain. This design can provide higher column packing stability and inertness, as well as more uniform carbon loading distribution; "50x2.1mm" means the length of the chromatographic column is 50 millimeters (mm), and the inner diameter is 2.1 millimeters (mm); "1.7μm" means the particle size of the chromatographic column packing material is 1.7 micrometers (μm).

[0030] In the present application, "vol%" means the percentage of the volume of the solute to the total volume of the solution, and "%w / v" means the percentage of the mass (g) of the solute to the total volume (mL) of the solution.

[0031] In the present application, the "SLE plate" refers to a "solid-liquid extraction plate", the full name of which is a solid-phase supported liquid-liquid extraction plate. The SLE plate uses a porous diatomite filler with high specific surface area and strong chemical inertness as a liquid-liquid distribution carrier, and uses a water-immiscible organic solvent for elution, which can effectively remove matrix interferents such as proteins and phospholipids in viscous samples (such as plasma, serum or whole blood). The SLE plate described in the present application can be obtained by a commercially available route.

[0032] The present application relates to a method for detecting a nitrogen-containing heterocyclic compound by LC-MS / MS, wherein the chromatographic column is a C18 chromatographic column; the mobile phase A is a water solution containing 0.1±0.05vol% formic acid, and the mobile phase B is a methanol solution of acetonitrile containing 0.1±0.05vol% formic acid, wherein the content of acetonitrile in the methanol solution of acetonitrile is 85±5vol%; a gradient elution method is used, and the content of the mobile phase B is 30±5vol% at the beginning, and gradually increases to 90±5vol% during the gradient elution process, and then decreases to 30±5vol%.

[0033] The present application finds that by the above method, the separation effect of the nitrogen-containing heterocyclic compound in the sample can be effectively improved, and the intensity and stability of the mass spectrum signal can be improved.

[0034] In some embodiments, when the sample is a tear sample, the flow rate is 0.5mL / min, and the gradient elution process is as follows:

[0035] In some embodiments, when the sample is a blood sample, the flow rate is 0.5mL / min, and the gradient elution process is as follows:

[0036] In some embodiments, the column temperature is 60±5℃.

[0037] In some embodiments, the injection temperature is 6±1℃.

[0038] In some embodiments, when the sample is a tear sample, the chromatographic column is a BEH C18 chromatographic column, 100×2.1mm, 1.7μm. In some specific embodiments, the chromatographic column is a Waters BEH C18 chromatographic column, 100×2.1mm, 1.7μm.

[0039] In some embodiments, when the sample is a blood sample, the chromatographic column is a BEH C18 chromatographic column, 50×2.1mm, 1.7μm. In some specific embodiments, the chromatographic column is a Waters BEH C18 chromatographic column, 50×2.1mm, 1.7μm.

[0040] Those skilled in the art can combine the above-mentioned conditions according to common sense to obtain a better embodiment of the LC operating conditions of the present application.

[0041] The above-mentioned LC operating conditions can be modified if necessary. The retention time of the analyte and / or the internal standard can vary due to the use and performance of the chromatographic column, as long as the elution order remains unchanged, which is considered acceptable.

[0042] In some embodiments, the mass spectrometry conditions include: ionization mode: ESI+; scanning mode: MRM; ion source voltage: 3000±500 V; ion source temperature (TEM): 550±20℃.

[0043] In some embodiments, the mass spectrometry conditions further include: curtain gas (CUR) pressure: 30±5 psi; ion source gas 1 (GS1) pressure: 50±5 psi; ion source gas 2 (GS2) pressure: 60±5 psi; collision gas (CAD) pressure: 10±2 psi.

[0044] Those skilled in the art can combine the above-mentioned conditions according to common sense to obtain a better embodiment of the MS operating conditions of the present application.

[0045] The MS operating conditions can be modified if necessary. To obtain higher sensitivity, the parameters can need to be re-optimized on different instruments or at different stages of use of the same instrument. As long as the important parameters remain unchanged (e.g. ion source voltage and TEM), no additional verification is required.

[0046] In some embodiments, the detection method further includes: extracting a solution containing the target compound from the sample, and then drying and re-dissolving to obtain the sample for detection in the LC-MS / MS method.

[0047] In some embodiments, when the sample is a tear sample, the method of extracting a solution containing the target compound from the tear sample includes: extracting the target component from the tear sample with a solvent to obtain an extract; the solvent is a mixture of ethyl acetate, methanol, water and ammonia water in a volume ratio of 100:15-25:3.0-4.0:6.0-7.0, and the concentration of ammonia water is 5-15 wt%, more preferably 10±2 wt%. Compared with other solvents, when the above-mentioned solvent is used, nitrogen-containing heterocyclic compounds can be efficiently extracted from a small amount of tear sample, and the influence of individual matrix differences in the tear sample can be excluded.

[0048] In some specific embodiments, the solvent is a mixture of ethyl acetate, methanol, water and ammonia water in a volume ratio of 100:20:3.5:6.5.

[0049] In some specific embodiments, the solvent is a mixture of ethyl acetate, methanol, water and ammonia water in a volume ratio of 100:15:4.0:7.0.

[0050] In some specific embodiments, the solvent is a mixture of ethyl acetate, methanol, water and ammonia water in a volume ratio of 100:25:3.0:6.0.

[0051] In some specific embodiments, the following solvents can also be considered: a mixture of ammonia water and acetonitrile in a volume ratio of 1:4-6; or an acetonitrile solution containing 5-10 vol% ammonia water (concentration of 10 wt%); or a mixture of ammonia water, zinc sulfate solution and acetonitrile. The above solvents also have a general dissolution effect, but the solvent provided by the present application is significantly superior to the above solvents in terms of dissolution effect.

[0052] In some embodiments, the mixture of the solvent and the tear sample is vortexed for 8-15 min, and then placed for 15-25 min to obtain the extraction solution. In this way, the extraction time can be greatly shortened while ensuring the extraction effect.

[0053] In some embodiments, the method for extracting a solution containing the target compound from the tear sample further comprises: after the sample extraction solution is extracted with n-hexane, centrifuging to obtain a first supernatant and a precipitate; and then re-extracting the precipitate with a mixed solvent of ethyl acetate and n-hexane in a volume ratio of 1:0.8-1.2 (more preferably 1:1), centrifuging to obtain a second supernatant, and mixing the first supernatant and the second supernatant to obtain a solution containing the target compound. In this way, the extraction effect of the nitrogen-containing heterocyclic compound can be further improved, and the influence of individual matrix differences can be reduced.

[0054] In some embodiments, when the sample is a tear sample, the re-dissolution is performed using a Trition X-100-containing acetonitrile aqueous solution, in which the concentration of Trition X-100 is 10±2 μg / mL, and the volume ratio of acetonitrile and water is 2:2.5-3.5, more preferably 2:3.

[0055] In some embodiments, when the sample is a tear sample, the upper sample is prepared using a glass container, and the volume of the upper sample is greater than or equal to 2 mL. Compared with using other containers (such as low-adsorption plastic tubes) or preparing less upper sample, the above scheme can significantly reduce the adverse effects of compound adsorption on detection.

[0056] The method of the present application can be used for qualitative detection (the result is usually presented in the form of "yes / no" or "presence / absence" etc.) and quantitative detection (the result is the concentration or content level of the target component) of nitrogen-containing heterocyclic compounds.

[0057] When the method of the present application is applied to quantitative detection, one skilled in the art can, in combination with common sense, establish a standard curve using standard substances of known concentrations under the same chromatographic and mass spectrometric conditions, and then detect the sample to be tested, and determine the concentration or content level of the target component in the sample to be tested by comparison with the standard substances.

[0058] In some embodiments, when the sample is a tear sample, artificial tears containing 0.4±0.05% w / v BSA are used as a surrogate matrix for preparing standard curves and / or quality control samples. The present application also provides the above-mentioned surrogate matrix, which has better intra-batch accuracy and precision when used to prepare standard curves and quality control samples.

[0059] In some specific embodiments, the surrogate matrix can also be used to prepare standard curves, and human blank tear fluid is used to prepare quality control samples, which still have better intra-batch accuracy and precision.

[0060] In some embodiments, when the sample is a blood sample, the method for extracting a solution containing the target compound from the blood sample comprises: sequentially mixing the blood sample with an aqueous acetonitrile solution, a zinc sulfate solution containing ammonia water, and then infiltrating the mixed solution into the column bed packing of the SLE plate, and then eluting the SLE plate with ethyl acetate to obtain a solution containing the target compound; in the aqueous acetonitrile solution, the volume ratio of acetonitrile to water is 1:0.8-1.2, more preferably 1:1; in the zinc sulfate solution containing ammonia water, the concentration of ammonia water is 10-30 wt%, the content of ammonia water is 8±1 vol%, and the concentration of zinc sulfate is 0.08±0.01 M. By the above-mentioned method, nitrogen-containing heterocyclic compounds can be efficiently extracted from a small amount of blood sample, and the influence of the blood matrix on the detection effect can be effectively controlled.

[0061] In some embodiments, when the sample is a blood sample, the reconstitution is performed using an aqueous acetonitrile solution containing formic acid; in the aqueous acetonitrile solution containing formic acid, the content of formic acid is 0.5±0.1 vol%, and the volume ratio of acetonitrile to water is 2:2.5-3.5, more preferably 2:3.

[0062] In some specific embodiments, when the sample is a blood sample, plasma containing an anticoagulant (such as K2EDTA) (such as human plasma, mouse plasma, etc.) is used to prepare standard curves and / or quality control samples.

[0063] In some specific embodiments, the drying is specifically N2 blowing drying at below 40°C.

[0064] In some embodiments, the nitrogen-containing heterocyclic compound has the structure of Formula (I):

[0065] wherein,

[0066] R1and R2are each independently selected from H and pyrazolyl, and R1and R2are not simultaneously pyrazolyl or H;

[0067] R3and R4are each independently selected from H, F, Cl, Br, I, OH, NH2, CN, C 1-3 alkyl and C 1-3 alkoxy, said C 1-3 alkyl and C 1-3 alkoxy are optionally substituted with 1, 2, or 3 halogen;

[0068] T1is selected from CH and N;

[0069] D1is selected from -O-, -C(R5)(R6)-, -N(R7)-, and

[0070] R5and R6are each independently selected from H, F, Cl, Br, I, OH, and C 1-3 alkyl, said C1 -3 alkyl are optionally substituted with 1, 2, or 3 halogen;

[0071] alternatively, R5and R6together with the carbon atom to which they are both attached form oxetanyl;

[0072] R7is selected from H, and C 1-3 alkyl, said C 1-3 alkyl are optionally substituted with 1, 2, or 3 halogen;

[0073] R8is selected from H and -C(=O)-C 1-3 alkyl;

[0074] n is selected from 1 and 2.

[0075] In some specific embodiments, the nitrogen-containing heterocyclic compound has any one of the following structures:

[0076] In some specific embodiments, the injection volume of the upper sample derived from a tear sample is 5 ± 1 μΐ^.

[0077] In some specific embodiments, the injection volume of the upper sample derived from a plasma sample is 8 ± 1 μΐ^.

[0078] In some embodiments, the mass spectrum collection time for the tear sample is 3.0-5.0 min.

[0079] In some embodiments, the mass spectrum collection time for the plasma sample is 4.5-8.0 min.

[0080] The skilled person can combine the above-mentioned embodiments according to common knowledge to obtain a better embodiment of the detection method of the present application.

[0081] The present application also relates to a method for extracting nitrogen-containing heterocyclic compounds from tear, comprising:

[0082] extracting the target component from the tear using a solvent to obtain an extract;

[0083] The solvent is a mixture of ethyl acetate, methanol, water and ammonia water in a volume ratio of 100:15-25:3.0-4.0:6.0-7.0, and the concentration of ammonia water is 5-15 wt%, more preferably 10±2 wt%.

[0084] In some embodiments, the solvent is a mixture of ethyl acetate, methanol, water and ammonia water in a volume ratio of 100:20:3.5:6.5.

[0085] In some embodiments, the solvent is a mixture of ethyl acetate, methanol, water and ammonia water in a volume ratio of 100:15:4.0:7.0.

[0086] In some embodiments, the solvent is a mixture of ethyl acetate, methanol, water and ammonia water in a volume ratio of 100:25:3.0:6.0.

[0087] In some embodiments, the method further comprises: after extracting the extract using n-hexane, centrifuging to obtain a first supernatant and a precipitate; and then using a mixture of ethyl acetate and n-hexane in a volume ratio of 1:0.8-1.2 (more preferably 1:1) to extract the precipitate again, centrifuging to obtain a second supernatant, and mixing the first supernatant and the second supernatant to obtain a solution containing the target compound.

[0088] In some embodiments, the method further comprises: drying the supernatant to obtain the nitrogen-containing heterocyclic compound. Preferably, the drying is performed by blowing N2 below 40℃.

[0089] In some embodiments, the nitrogen-containing heterocyclic compound has the following structure (I):

[0090] wherein,

[0091] R1 and R2 are each independently selected from H and pyrazol group, and R1 and R2 are not simultaneously pyrazol group or H;

[0092] R3 and R4 are each independently selected from H, F, Cl, Br, I, OH, NH2, CN, C 1-3 Alkyl and C 1-3 Alkoxy, the C 1-3 Alkyl and C 1-3 The alkoxy group may be optionally replaced by one, two, or three halogens;

[0093] T1 is selected from CH and N;

[0094] D1 is selected from -O-, -C(R5)(R6)-, -N(R7)- and

[0095] R5 and R6 are each independently selected from H, F, Cl, Br, I, OH, and C. 1-3 Alkyl, the C1 -3 Alkyl groups may be optionally substituted with one, two, or three halogens;

[0096] Alternatively, R5 and R6 together with the carbon atom they are connected to form an oxobutyryl group;

[0097] R7 is selected from H, and C 1-3 Alkyl, the C 1-3 Alkyl groups may be optionally substituted with one, two, or three halogens;

[0098] R8 is selected from H and -C(=O)-C 1-3 alkyl;

[0099] n is selected from 1 and 2.

[0100] In some embodiments, the nitrogen-containing heterocyclic compound has any of the following structures:

[0101] The embodiments of the present invention will now be described in detail with reference to examples.

[0102] For ease of comparison, nitrogen-containing heterocyclic compound-I (whose structure is shown below, and whose crystal form is the A crystal form disclosed in CN116648247A) is used as the analyte in the following examples. However, this does not mean that the method of the present invention can only be applied to this compound. In fact, the method of the present invention has better detection effect on compounds within the scope of the present invention.

[0103] It should be understood that the examples are only used for illustrating the present application but not for limiting the scope of the present application. The experimental methods in the following examples, if not otherwise specified, are preferred to be in accordance with the instructions given in the present application, and can also be in accordance with the experimental manuals or conventional conditions in the art, or in accordance with other experimental methods known in the art, or in accordance with the conditions suggested by the manufacturers.

[0104] In the following specific examples, the measurement parameters of the raw material components, if not otherwise specified, can have slight deviations within the weighing accuracy range. The temperature and time parameters allow acceptable deviations caused by the instrument testing accuracy or operation accuracy.

[0105] Example 1 Method for detecting nitrogen-containing heterocyclic compounds in tears and methodological validation

[0106] 1. Preparation of standard curve samples

[0107] The standard curve samples were prepared according to the analyte concentrations shown in Table 1, using artificial tears containing 0.4% w / v BSA as the surrogate matrix.

[0108] Table 1

[0109] 2. Sample pretreatment

[0110] Take 20 μL of each sample (n = 6), add 3.2 mL of solvent containing 40 pg / mL internal standard (nitrogen-containing heterocyclic compound-I-D4) to all samples except the blank sample (i.e. the blank surrogate matrix), and add 3.2 mL of solvent to the blank sample, respectively. Vortex for 10 minutes and then stand for 20 minutes. After vortexing, the extraction solution preparation is completed. The solvent is a mixture of ethyl acetate, methanol, water and ammonia water in a volume ratio of 100:20:3.5:6.5, and the concentration of ammonia water is 10 wt%.

[0111] Mix the extraction solution well by vortexing, and add 500 μL of sample extraction solution to the corresponding position of the 96-well plate (sample plate).

[0112] Add 400 μL of n-hexane. Seal the plate with an aluminum foil heat-seal film, and shake at 1200 rpm for 5 minutes on a plate shaker. Centrifuge at 4000 rpm for 5 minutes at 4°C. Take 500 μL of supernatant to a new 96-well plate (2).

[0113] Add 500 μL of ethyl acetate / n-hexane (1:1, v / v) to the 96-well plate (sample plate) for the second extraction, specifically seal the plate with an aluminum foil heat-seal film, and shake at 1200 rpm for 5 minutes on a plate shaker. Centrifuge at 4000 rpm for 5 minutes at 4°C. Take 500 μL of supernatant to the 96-well plate (2).

[0114] The 96-well plate (2) was dried under N2 at 40°C. It was reconstituted with 150 μL of acetonitrile / water (2:3, v / v) containing 10 μg / mL Triton X-100. The plate was sealed with a silicone mat and shaken at 1200 rpm for 3 min on a plate shaker to obtain the injection sample of each sample.

[0115] 3. Establishment of standard curve

[0116] The injection sample of the standard curve sample of different concentrations was taken respectively, and the peak area was determined in the LC-MS / MS instrument. The concentration of the analyte in the standard curve sample was taken as the abscissa (X), and the peak area of the analyte and the internal standard was taken as the ordinate (Y). The linear regression model was confirmed by regression calculation with weighted least squares (weight factor was 1 / x2).

[0117] In the LC-MS / MS instrument, the liquid chromatography conditions were as follows: chromatographic column: Waters BEH C18 Column, 100 x 2.1 mm, 1.7 μm; column temperature: 60°C; injection temperature: 6°C; injection volume: 5 μL; mobile phase A: 0.1 vol% formic acid aqueous solution; mobile phase B: 0.1 vol% formic acid acetonitrile / methanol (85:15, v / v) solution; gradient elution was used, the flow rate was 0.5 mL / min, and the gradient elution process was as follows:

[0118] The mass spectrometry conditions were as follows: ionization mode: ESI+; scanning mode: MRM; ion source voltage: 3000 V; ion source temperature: 550°C; curtain gas (CUR) pressure: 30 psi; ion source gas 1 (GS1) pressure: 50 psi; ion source gas 2 (GS2) pressure: 60 psi; collision gas (CAD) pressure: 10 psi; collection time: 3.5 min.

[0119] The obtained standard curve is shown in Figure 1, and the linear regression equation is y = 0.2272x + 0.01292 (R 2 = 0.9977). It can be seen that the analyte has a good linear relationship in the range of 1-500 ng / mL.

[0120] 4. Sample detection

[0121] 5 μL of the injection sample of the sample to be detected was taken, and was detected in the LC-MS / MS instrument. The liquid chromatography conditions and the mass spectrometry conditions were the same as those shown in "3. Establishment of standard curve". The blank sample was used to monitor whether there was pollution in the operation process, and the content of the analyte in the sample was confirmed according to the standard curve.

[0122] 5. Methodology verification

[0123] (1) Recovery, precision and accuracy

[0124] The control samples with concentrations of 1.00 ng / mL, 3.00 ng / mL, 20.0 ng / mL, 200 ng / mL and 375 ng / mL (n=6) were prepared respectively, corresponding to LLOQ (low limit of quantification control sample), LQC (low concentration control sample), GMQC (geometric intermediate concentration control sample), MQC (intermediate concentration control sample) and HQC (high concentration control sample) respectively, using artificial tears containing 0.4% w / v BSA as a substitute matrix.

[0125] The samples of LLOQ, LQC, GMQC, MQC and HQC were prepared according to the sample pretreatment method in 2. Sample pretreatment. Then, each sample was analyzed by injecting into a liquid chromatograph and mass spectrometer according to the conditions in 3. The results are shown in Table 2 below.

[0126] Table 2

[0127] According to the results, the extraction recovery of the method at each concentration level is 98.1-108%, and the within-batch accuracy and precision are good.

[0128] (2) Matrix effect

[0129] The LQC and HQC with concentrations of 3.00 ng / mL and 375 ng / mL were prepared respectively using four different sources of tear matrix, and the samples obtained by the sample pretreatment method in 2. Sample pretreatment were analyzed by injecting. The results are shown in Table 3.

[0130] Table 3

[0131] According to the results, the influence of tear matrix effect on the detection of nitrogen-containing heterocyclic compounds can be ignored in the detection method of the present application.

[0132] The above experimental results show that the method of the present application has been verified by methodological verification, and the established method has high sensitivity, good accuracy and precision, and good stability and linearity.

[0133] Example 2 Detection method of nitrogen-containing heterocyclic compounds in plasma and methodological verification

[0134] 1. Preparation of standard curve samples

[0135] The blank human plasma containing 0.1M K2EDTA anticoagulant was used as a blank matrix, and the standard curve samples were prepared according to the concentrations of the analyte shown in Table 4.

[0136] Table 4

[0137] 2. Sample pretreatment

[0138] The sample was vortexed and 100 μL of the sample was added to the corresponding position of the 96-well plate.

[0139] 50 mL of acetonitrile / water (1:1, v / v) containing internal standard working solution was added to each sample except the blank sample (i.e. blank matrix), and 50 mL of acetonitrile / water (1:1, v / v) was added to the blank sample. The plate was shaken at 1200 rpm for 1 min. 150 μL of 8% ammonia water (10 wt% concentration) and 0.08 M zinc sulfate solution was added to each sample mixture. The plate was shaken at 1200 rpm for 5 min.

[0140] The shaken sample was loaded on the 96-well SLE plate. The sample was completely infiltrated into the column bed using positive pressure (≤2 psi), and then the SLE plate was left to stand for 5 min.

[0141] The SLE plate was eluted with ethyl acetate, and the eluate was collected in a new 96-well plate. The resulting solution was blown dry with N2 at 40°C.

[0142] The sample was reconstituted with 200 μL of acetonitrile / water (2:3, v / v) containing 0.5% formic acid. The plate was sealed with a silica gel pad, and shaken at 1200 rpm for 3 min to obtain the sample for loading.

[0143] 3. Establishment of standard curve

[0144] The sample for loading of the standard curve sample of different concentrations was taken, and the peak area was determined in the LC-MS / MS instrument. The concentration of the analyte in the standard curve sample was taken as the abscissa (X), and the peak area of the analyte and internal standard was taken as the ordinate (Y). The linear regression model was confirmed by regression calculation using the weighted least squares method (weight factor: 1 / x2).

[0145] In the LC-MS / MS instrument, the liquid chromatography conditions were as follows: chromatographic column: Waters BEH C18 Column, 100 x 2.1 mm, 1.7 μm; column temperature: 60°C; injection temperature: 6°C; injection volume: 8 μL; mobile phase A: 0.1 vol% formic acid aqueous solution; mobile phase B: 0.1 vol% formic acid acetonitrile / methanol (85:15, v / v) solution; gradient elution was performed at a flow rate of 0.5 mL / min, and the gradient elution process was as follows:

[0146] The mass spectrometry conditions are as follows: ionization mode: ESI+; scanning mode: MRM; ion source voltage: 3000V; ion source temperature: 550℃; curtain gas (CUR) pressure: 30psi; ion source gas 1 (GS1) pressure: 50psi; ion source gas 2 (GS2) pressure: 60psi; collision gas (CAD) pressure: 10psi; collection time: 5min.

[0147] The obtained standard curve is shown in Fig. 2, and the linear regression equation is y=0.5913x+0.008497 (R 2 =0.9978), and it can be seen that the linear relationship of the tested substance is good in the range of 0.05-25ng / mL.

[0148] 4. Sample detection

[0149] 8μL of the sample to be tested is taken for detection in an LC-MS / MS instrument, and the liquid chromatography conditions and mass spectrometry conditions are the same as shown in “3. Establishment of standard curve”. The blank sample is used to monitor whether there is pollution in the operation process, and the content of the tested substance in the sample is confirmed according to the standard curve.

[0150] 5. Methodology verification

[0151] (1) Precision and accuracy

[0152] The blank human plasma containing 0.1M K2EDTA anticoagulant is used as a blank matrix, and the quality control samples with concentrations of 0.05ng / mL, 0.150ng / mL, 1.00ng / mL, 10.0ng / mL and 19.0ng / mL are respectively prepared, which correspond to LLOQ, LQC, GMQC, MQC and HQC (n=6) respectively.

[0153] The sample pretreatment method in “2. Sample pretreatment” is used to process and obtain the samples to be tested of LQC, MQC and HQC. Then, each sample to be tested is injected and analyzed according to the liquid chromatography conditions and mass spectrometry conditions in 3. The results are shown in the following table 5.

[0154] Table 5

[0155] It can be known from the results that the accuracy and precision of the method of the present application are good at each concentration level, and the stability is better.

[0156] (2) Matrix effect

[0157] 1) Take 6 different sources of blank human plasma, respectively, to prepare the concentration of 0.150 ng / mL, 10.0 ng / mL, 19.0 ng / mL quality control samples, which correspond to LQC, MQC, HQC respectively. According to the sample pretreatment method in "2. Sample pretreatment", the sample is pretreated and the sample is obtained, and then the sample is analyzed. The area ratio of the test substance and the internal standard is obtained by comparing the bio-matrix phase area and the solvent phase area, and the normalized matrix effect factor is obtained. The detection results are shown in Table 6 below.

[0158] Table 6

[0159] 2) Take hemolytic plasma samples and high-fat plasma samples, respectively, to prepare the concentration of 0.150 ng / mL, 19.0 ng / mL quality control samples, which correspond to LQC, HQC (n = 6) respectively. According to the sample pretreatment method in "2. Sample pretreatment", the sample is pretreated and the sample is obtained, and then the sample is analyzed. The detection results are shown in Table 7 below.

[0160] Table 7

[0161] From the results, it can be seen that in the detection method of the present application, the influence of blood matrix effect on the detection of nitrogen-containing heterocyclic compounds can be ignored.

[0162] The above experimental results show that the method of the present application has been verified by method, and the established method has high sensitivity, good accuracy and precision, and good stability and good linearity.

[0163] The above-described embodiments only express several embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as limiting the scope of the patent. It should be noted that for ordinary skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of protection of the present application.

Claims

1. A method for detecting a nitrogen-containing heterocyclic compound, using LC-MS / MS, wherein, the chromatographic column is a C18 chromatographic column; mobile phase A is a water solution containing 0.1 ± 0.05 vol% formic acid, and mobile phase B is a methanol-acetonitrile solution containing 0.1 ± 0.05 vol% formic acid, wherein the acetonitrile content in the methanol-acetonitrile solution is 85 ± 5 vol%; gradient elution is used, wherein the content of mobile phase B is 30 ± 5 vol% at the beginning, and gradually increases to 90 ± 5 vol% during the gradient elution, and then decreases to 30 ± 5 vol%.

2. The method for detecting a nitrogen-containing heterocyclic compound according to claim 1, wherein, When the sample is a tear sample, the flow rate is 0.5 mL / min, and the gradient elution procedure is as follows:

3. The method of detecting a nitrogen-containing heterocyclic compound according to claim 1, wherein, The mass spectrometry conditions further include at least one of the following: i) the column temperature is 60 ± 5℃; ii) the injection temperature is 6 ± 1℃; iii) when the sample is a tear sample, the chromatographic column is a BEH C18 chromatographic column, 100 × 2.1 mm, 1.7 μm.

4. The method of detecting a nitrogen-containing heterocyclic compound according to claim 1, wherein, The mass spectrometry conditions include: ionization mode: ESI+; scanning mode: MRM; ion source voltage: 3000 ± 500 V; ion source temperature: 550 ± 20℃. Preferably, the mass spectrometry conditions further include: curtain gas pressure: 30 ± 5 psi; ion source gas 1 pressure: 50 ± 5 psi; ion source gas 2 pressure: 60 ± 5 psi; collision gas pressure: 10 ± 2 psi.

5. The method of detecting a nitrogen-containing heterocyclic compound according to claim 1, wherein, The method further includes: extracting a solution containing the target compound from the sample, and then drying and reconstituting to obtain the sample for detection in the LC-MS / MS method.

6. The method of detecting a nitrogen-containing heterocyclic compound according to claim 5, wherein, When the sample is a tear sample, the method for extracting a solution containing the target compound from the tear sample includes: using a solvent to extract the target component from the tear sample to obtain an extract; the solvent is a mixture of ethyl acetate, methanol, water and ammonia water in a volume ratio of 100: 15-25: 3.0-4.0: 6.0-7.0, and the concentration of ammonia water is 5-15 wt%.

7. The method of detecting a nitrogen-containing heterocyclic compound according to claim 6, wherein, The mixture of the solvent and the tear sample is vortexed for 8-15 min, and then placed for 15-25 min to obtain the extract.

8. The method of detection of a nitrogen-containing heterocyclic compound according to claim 6 or 7, wherein, The method for extracting a solution containing the target compound from the tear sample further includes: after the sample extract is extracted with n-hexane, centrifugation is performed to obtain a first supernatant and a precipitate; then the precipitate is extracted again with a mixed solvent of ethyl acetate and n-hexane in a volume ratio of 1: 0.8-1.2, and centrifugation is performed to obtain a second supernatant, and the first supernatant and the second supernatant are mixed to obtain a solution containing the target compound.

9. The method of detection of a nitrogen-containing heterocyclic compound according to any one of claims 6 to 8, wherein, When the sample is a tear sample, the reconstitution is performed using an aqueous acetonitrile solution containing Triton X-100; in the aqueous acetonitrile solution containing Triton X-100, the concentration of Triton X-100 is 10 ± 2 μg / mL, and the volume ratio of acetonitrile to water is 2: 2.5-3.

5.

10. The method of detecting a nitrogen-containing heterocyclic compound according to claim 5 or 6, wherein, When the sample is a tear sample, the sample is prepared using a glass container, and the volume of the sample is greater than or equal to 2 mL.

11. The method of detecting a nitrogen-containing heterocyclic compound according to claim 5 or 6, wherein, When the sample is a tear sample, an artificial tear solution containing 0.4 ± 0.05% w / v BSA is used as a surrogate matrix for preparing a standard curve and / or a quality control sample.

12. The method of detecting a nitrogen-containing heterocyclic compound according to claim 1, wherein, The nitrogen-containing heterocyclic compound has the following structure of formula (I): wherein, R1and R2are each independently selected from H and pyrazolyl, and R1and R2are not simultaneously pyrazolyl or H; R3and R4are each independently selected from H, F, CI, Br, I, OH, NH2, CN, C 1-3 alkyl and C 1-3 alkoxy, said C 1-3 alkyl and C 1-3 alkoxy optionally substituted with 1, 2 or 3 halogen; T1is selected from CH and N; D1is selected from -0-, -C(R5)(R6)-, -N(R7)- and R5and R6are each independently selected from the group consisting of H, F, Cl, Br, I, OH, and C1 1-3 alkyl, said C1 -3 alkyl is optionally substituted with 1, 2, or 3 halogen; or R5and R6together with the carbon atom to which they are both attached form oxetanyl; R7is selected from H, and C 1-3 alkyl, said C 1-3 alkyl is optionally substituted with 1, 2, or 3 halogen; R8is selected from H and -C(=0)-C 1-3 alkyl; n is selected from 1 and 2.

13. A method for extracting nitrogen-containing heterocyclic compounds from tear fluid, comprising: extracting target components from tear fluid using a solvent to obtain an extract; the solvent is a mixture of ethyl acetate, methanol, water and ammonia water in a volume ratio of 100: 15-25: 3.0-4.0: 6.0-7.0, and the concentration of ammonia water is 5-15 wt%.

14. The method of extracting nitrogen-containing heterocyclic compounds from tear fluid according to claim 13, wherein, The mixture of the solvent and tear fluid is vortexed for 8-15 min, and then left to stand for 15-25 min to obtain the extract.

15. The method for extracting nitrogen-containing heterocyclic compounds from tear fluid according to claim 13 or 14, further comprising: After the extract is extracted using n-hexane, centrifugation is performed to obtain a first supernatant and a precipitate; then the precipitate is re-extracted using a mixed solvent of ethyl acetate and n-hexane in a volume ratio of 1: 0.8-1.2, and centrifugation is performed to obtain a second supernatant, and the first supernatant and the second supernatant are mixed to obtain a solution containing target compounds.

16. The method for extracting nitrogen-containing heterocyclic compounds from tear fluid according to claim 15, further comprising: drying the supernatant to obtain nitrogen-containing heterocyclic compounds; Preferably, the drying is specifically blowing dry using N2at 40°C or below.

17. The method of extracting nitrogen-containing heterocyclic compounds from tear fluid according to claim 13, wherein, The nitrogen-containing heterocyclic compound has the following structure of formula (I): wherein, R1and R2are each independently selected from H and pyrazolyl, and R1and R2are not simultaneously pyrazolyl or H; R3and R4are each independently selected from H, F, CI, Br, I, OH, NH2, CN, C 1-3 alkyl and C 1-3 alkoxy, said C 1-3 alkyl and C 1-3 alkoxy optionally substituted with 1, 2 or 3 halogen; T1is selected from CH and N; D1is selected from -0-, -C(R5)(R6)-, -N(R7)- and R5and R6are each independently selected from the group consisting of H, F, Cl, Br, I, OH, and C1 1-3 alkyl, said C1 -3 alkyl is optionally substituted with 1, 2, or 3 halogen; or R5and R6together with the carbon atom to which they are both attached form oxetanyl; R7is selected from H, and C 1-3 alkyl, said C 1-3 alkyl is optionally substituted with 1, 2, or 3 halogen; R8is selected from H and -C(=0)-C 1-3 alkyl; n is selected from 1 and 2.

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