Method for detecting nitrogen-containing heterocyclic compound in blood sample

Abstract

The invention belongs to the field of analytical chemistry, and particularly relates to a method for detecting a nitrogen-containing heterocyclic compound in a blood sample, an LC-MS / MS method is adopted, and a chromatographic column is a C18 chromatographic column; the mobile phase A is an aqueous solution containing 0.1 + / -0.05 vol% of formic acid, the mobile phase B is an acetonitrile-methanol solution containing 0.1 + / -0.05 vol% of formic acid, and the content of acetonitrile in the acetonitrile-methanol solution is 85 + / -5 vol%; a gradient elution method is adopted. According to the method disclosed by the invention, the content of small molecular compounds (especially compounds with poorer solubility in water) including nitrogen-containing heterocyclic compounds in plasma can be efficiently and accurately detected, and the method has great application potential in the field of drug metabolism research of related compounds.

Description

[0001] Divisional application declaration

[0002] The present application is a divisional application of the Chinese Invention Patent Application No. 2024110452738, filed on July 31, 2024, entitled "A detection method of nitrogen-containing heterocyclic compound", the application number of which is 2024110452738. TECHNICAL FIELD

[0003] The present application belongs to the field of analytical chemistry, and particularly relates to a detection method of nitrogen-containing heterocyclic compound in blood samples. BACKGROUND

[0004] EGFR2, also known as KDR or Flk-1, is identified as the receptor of VEGF and VEGFC, and is an early marker of endothelial progenitor cells, whose expression is limited to endothelial cells in vivo. VEGFR2 is proved 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 signaling, 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, which makes 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 detection method for 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 a nitrogen-containing heterocyclic compound by using 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 the content of the mobile phase B is gradually increased to 90±5 vol% during the gradient elution process, and then decreased to 30±5 vol%.

[0011] The second object of the present application is to provide a method for extracting a nitrogen-containing heterocyclic compound 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 description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

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

[0017] Figure 2 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 of the terms "and / or", "or / and", "and / or" used herein includes any one of two or more associated listed items, and also includes any and all combinations of the associated listed items, including any two associated listed items, any more associated listed items, or all associated listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are combined to connect at least three items, 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 including listed features.

[0026] In the present application, "preferably", "more preferably", "more preferably", "preferably" only describe the embodiments or examples with better effects, 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 either of the two parallel schemes "have" or "have". 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 octyl (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 chains. 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 commercial route.

[0032] The present application relates to a method for detecting a nitrogen-containing heterocyclic compound by using 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 solution of acetonitrile and methanol containing 0.1±0.05vol% formic acid, wherein the content of acetonitrile in the acetonitrile and methanol solution is 85±5vol%; the 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 using 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]

[0036] 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:

[0037]

[0038] In some embodiments, the column temperature is 60±5℃.

[0039] In some embodiments, the injection temperature is 6±1℃.

[0040] 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.

[0041] In some embodiments, when the sample is a blood sample, the chromatographic column is a BEH C18 column, 50 x 2.1 mm, 1.7 μιη. In some specific embodiments, the chromatographic column is a Waters BEH C18 column, 50 x 2.1 mm, 1.7 μιη.

[0042] One skilled in the art can combine the above-mentioned conditions with common sense to obtain better embodiments of LC operating conditions according to the present application.

[0043] The above-mentioned LC operating conditions can be modified if necessary. The retention times of the analytes and / or internal standards can vary depending on the use and performance of the chromatographic column, as long as the elution order remains unchanged, which is considered acceptable.

[0044] In some embodiments, the mass spectrometry conditions comprise: ionization mode: ESI+; scan mode: MRM; ion source voltage: 3000 ± 500 V; ion source temperature (TEM): 550 ± 20 °C.

[0045] In some embodiments, the mass spectrometry conditions further comprise: curtain gas (CUR) gas pressure: 30 ± 5 psi; ion source gas 1 (GS1) gas pressure: 50 ± 5 psi; ion source gas 2 (GS2) gas pressure: 60 ± 5 psi; collision gas (CAD) gas pressure: 10 ± 2 psi.

[0046] One skilled in the art can combine the above-mentioned conditions with common sense to obtain better embodiments of MS operating conditions according to the present application.

[0047] 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. No additional validation is required as long as the important parameters remain unchanged (e.g. ion source voltage and TEM).

[0048] In some embodiments, the detection method further comprises: 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.

[0049] In some embodiments, when the sample is a tear sample, the method for extracting a solution containing the target compound from the tear sample comprises: 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 with 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 tear sample with a small sample amount, and the influence of individual matrix differences can be reduced.

[0050] In some specific embodiments, the solvent is a mixture of ethyl acetate, methanol, water and ammonia water with a volume ratio of 100:20:3.5:6.5.

[0051] In some specific embodiments, the solvent is a mixture of ethyl acetate, methanol, water and ammonia water with a volume ratio of 100:15:4.0:7.0.

[0052] In some specific embodiments, the solvent is a mixture of ethyl acetate, methanol, water and ammonia water with a volume ratio of 100:25:3.0:6.0.

[0053] In some specific embodiments, the following solvents can also be considered: a mixture of ammonia water and acetonitrile with 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. When the above-mentioned solvents are used, the dissolution effect is also in line with the general requirements, but the solvent provided by the present application is significantly superior to the above-mentioned solvents in terms of dissolution effect.

[0054] 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 extract. In this way, the extraction time can be greatly shortened while ensuring the extraction effect.

[0055] In some embodiments, the method for extracting a solution containing the target compound from the tear sample further comprises: after the sample extract is extracted using n-hexane, centrifugation is performed to obtain a first supernatant and a precipitate; and then the precipitate is re-extracted using a mixed solvent of ethyl acetate and n-hexane with a volume ratio of 1:0.8-1.2 (more preferably 1:1), and then 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. In this way, the extraction effect of nitrogen-containing heterocyclic compounds can be further improved, and the influence of individual matrix differences can be reduced.

[0056] In some embodiments, when the sample is a tear sample, the reconstitution is performed using an aqueous solution of acetonitrile containing Triton X-100; in the aqueous solution of acetonitrile 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, more preferably 2:3.

[0057] 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 equal to or greater than 2 mL. Compared with using other containers (e.g., low-adsorption plastic tubes) or preparing a smaller upper sample, the above-mentioned scheme can significantly reduce the adverse effects of adsorption of compounds on the detection.

[0058] The method of the present application can be used for qualitative detection (the result is usually presented in the form of "yes / no" or "present / absent", etc.) and quantitative detection (the result is the concentration or content level of the target component) of nitrogen-containing heterocyclic compounds.

[0059] When the method of the present application is used for quantitative detection, one skilled in the art can, based on common sense, first establish a standard curve using a standard substance of known concentration under the same chromatographic and mass spectrometric conditions, and then detect the test sample, and determine the concentration or content level of the target component in the test sample by comparison with the standard substance.

[0060] In some embodiments, when the sample is a tear sample, an artificial tear containing 0.4±0.05% w / v BSA is used as a substitute matrix for preparing a standard curve and / or a quality control sample. The present application also provides the above-mentioned substitute matrix, which has better intra-batch accuracy and precision when used for preparing a standard curve and a quality control sample.

[0061] In some specific embodiments, the substitute matrix can also be used for preparing a standard curve, and human blank tear is used for preparing a quality control sample, which still has better intra-batch accuracy and precision.

[0062] In some embodiments, when the sample is a blood sample, the method for extracting a solution containing a target compound from a blood sample comprises: sequentially mixing the blood sample with an aqueous acetonitrile solution, an aqueous ammonia-containing zinc sulfate solution, and then infiltrating the mixed solution into the column bed packing of an SLE plate, and then eluting the SLE plate with ethyl acetate to obtain a solution containing a 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 aqueous ammonia-containing zinc sulfate solution, the concentration of aqueous ammonia is 10-30 wt%, the content of aqueous ammonia 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 blood sample with a small amount of sample, and the influence of the blood matrix on the detection effect can be effectively controlled.

[0063] In some embodiments, when the sample is a blood sample, the reconstitution is performed using an aqueous formic acid-containing acetonitrile solution, in which 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.

[0064] In some specific embodiments, when the sample is a blood sample, plasma (e.g., human plasma, mouse plasma, etc.) containing an anticoagulant (e.g., K2EDTA) is used to prepare the standard curve and / or quality control sample.

[0065] In some specific embodiments, the drying is performed by blowing N2 at below 40°C.

[0066] In some embodiments, the nitrogen-containing heterocyclic compound has the following structure (I):

[0067]

[0068] wherein,

[0069] R1and R2are each independently selected from H and pyrazolyl, and R1and R2are not simultaneously pyrazolyl or H;

[0070] 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 is optionally substituted with 1, 2, or 3 halogens;

[0071] T1is selected from CH and N;

[0072] D1is selected from -O-, -C(R5)(R6)-, -N(R7)-, and

[0073] R5and R6are each independently selected from H, F, Cl, Br, I, OH, and C 1-3 alkyl, said C 1-3 alkyl is optionally substituted with 1, 2, or 3 halogens;

[0074] Alternatively, R5and R6, together with the carbon atom to which they are commonly attached, form an oxetanyl group;

[0075] R7is selected from H, and C 1-3 alkyl, said C 1-3 alkyl is optionally substituted with 1, 2, or 3 halogens;

[0076] R8is selected from H and -C(=O)-C1-3 alkyl;

[0077] n is selected from 1 and 2.

[0078] In some specific embodiments, the nitrogen-containing heterocyclic compound has any of the following structures:

[0079]

[0080]

[0081] In some specific implementations, the injection volume of the sample derived from the tear fluid is 5 ± 1 μL.

[0082] In some specific implementations, the injection volume of the sample derived from the plasma sample is 8 ± 1 μL.

[0083] In some specific implementations, the mass spectrometry acquisition time for tear samples is 3.0–5.0 min.

[0084] In some specific implementations, the mass spectrometry acquisition time for plasma samples is 4.5 to 8.0 minutes.

[0085] Those skilled in the art can combine the above-mentioned embodiments with common sense to obtain preferred embodiments of the detection method of the present invention.

[0086] The present invention also relates to a method for extracting nitrogen-containing heterocyclic compounds from tears, comprising:

[0087] The target component was extracted from tears using a solvent to obtain an extract.

[0088] The solvent is a mixture of ethyl acetate, methanol, water and ammonia in a volume ratio of 100:15-25:3.0-4.0:6.0-7.0, with the concentration of ammonia being 5-15 wt%, more preferably 10 ± 2 wt%.

[0089] In some specific embodiments, the solvent is a mixture of ethyl acetate, methanol, water and ammonia in a volume ratio of 100:20:3.5:6.5.

[0090] In some specific embodiments, the solvent is a mixture of ethyl acetate, methanol, water and ammonia in a volume ratio of 100:15:4.0:7.0.

[0091] In some specific embodiments, the solvent is a mixture of ethyl acetate, methanol, water and ammonia in a volume ratio of 100:25:3.0:6.0.

[0092] In some embodiments, the method further comprises: after the extraction of the extraction solution using n-hexane, centrifuging to obtain a first supernatant and a precipitate; and then after the re-extraction of the precipitate using 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.

[0093] In some embodiments, the method further comprises: drying the supernatant to obtain the nitrogen-containing heterocyclic compound. Preferably, the drying is specifically blowing dry under N2at 40°C or below.

[0094] In some embodiments, the nitrogen-containing heterocyclic compound has the structure shown in the following formula (I):

[0095]

[0096] wherein,

[0097] R1and R2are each independently selected from H and pyrazolyl, and R1and R2are not simultaneously pyrazolyl or H;

[0098] R3and R4are 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 alkoxy are optionally substituted with 1, 2, or 3 halogens;

[0099] T1is selected from CH and N;

[0100] D1is selected from -O-, -C(R5)(R6)-, -N(R7)-, and

[0101] R5and R6are each independently selected from H, F, Cl, Br, I, OH, and C 1-3 alkyl, the C 1-3 alkyl is optionally substituted with 1, 2, or 3 halogens;

[0102] alternatively, R5and R6together with the carbon atom to which they are both attached form an oxetanyl group;

[0103] R7is selected from H, and C 1-3 alkyl, the C 1-3 alkyl is optionally substituted with 1, 2, or 3 halogens;

[0104] R8is selected from H and -C(=O)-C 1-3 alkyl;

[0105] n is selected from 1 and 2.

[0106] In some embodiments, the nitrogen-containing heterocyclic compound has any one of the following structures:

[0107]

[0108]

[0109] Embodiments of the present application will be described in detail below with reference to the examples.

[0110] For the purpose of comparison, the nitrogen-containing heterocyclic compound-I (the structure of which is shown below, and the crystal form thereof is the A crystal form disclosed in CN116648247A) is used as the test substance in the following examples, but this does not mean that the method of the present application can only be applied to this compound. In fact, the method of the present application has a better detection effect on compounds within the scope of the present application.

[0111]

[0112] It should be understood that these examples are only used to illustrate the present application and do not limit the scope of the present application. The experimental methods in the following examples are not specified, and the priority is given to the guidance given in the present application. The experimental methods can also be in accordance with the experimental manual 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 manufacturer.

[0113] In the following specific examples, the amount of the raw material components is measured, and if not specifically stated, there may be slight deviations within the weighing accuracy range. The temperature and time parameters allow for acceptable deviations caused by instrument testing accuracy or operation accuracy.

[0114] Example 1 Detection method of nitrogen-containing heterocyclic compounds in tears and methodological verification

[0115] 1. Preparation of standard curve samples

[0116] The standard curve samples were prepared according to the test substance concentrations shown in Table 1 using artificial tears containing 0.4% w / v BSA as the surrogate matrix.

[0117] Table 1

[0118] Test substance (ng / mL) C8 C7 C6 C5 C4 C3 C2 C1 Nitrogen-containing heterocyclic compound-I 1.00 2.00 5.00 25.0 120 250 400 500

[0119] 2. Sample pretreatment

[0120] Take 20 μL of each sample (n = 6), add 3.2 mL of solvent containing 40 pg / mL of internal standard (nitrogen-containing heterocyclic compound-I-D4) to all samples except the blank sample (i.e. 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 preparation of the extract 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%.

[0121] Vortex the extract to mix evenly, and add 500 μL of the sample extract to the corresponding position of the 96-well plate (sample plate).

[0122] 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 the supernatant to a new 96-well plate (2).

[0123] 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 the supernatant to the 96-well plate (2).

[0124] Dry the 96-well plate (2) under N2 at 40°C. Resuspend with 150 μL of acetonitrile / water (2:3, v / v) containing 10 μg / mL of Triton X-100. Seal the plate with a silica gel pad, and shake at 1200 rpm for 3 minutes on a plate shaker to obtain the sample for injection.

[0125] 3. Establishment of standard curve

[0126] Take the sample for injection of the standard curve sample with different concentrations, respectively, and measure the peak area in the LC-MS / MS instrument. Take the concentration of the analyte in the standard curve sample as the abscissa (X), and take the peak area of the analyte and the internal standard as the ordinate (Y), and perform regression calculation by weighted least squares method (weight factor is 1 / x^2) to confirm the linear regression model.

[0127] In the LC-MS / MS instrument, the liquid chromatography conditions are 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 is adopted, the flow rate is 0.5 mL / min, and the gradient elution process is as follows:

[0128]

[0129] The mass spectrometry conditions are 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.

[0130] The obtained standard curve is shown in Figure 1 The linear regression equation is y = 0.2272x + 0.01292 (R 2 = 0.9977), and it can be seen that the linear relationship of the analyte is good in the range of 1-500 ng / mL.

[0131] 4. Sample detection

[0132] 5 μL of the sample to be tested is taken for detection in the LC-MS / MS instrument, and the liquid chromatography conditions and mass spectrometry conditions are the same as those 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 analyte in the sample is confirmed according to the standard curve.

[0133] 5. Methodology verification

[0134] (1) Recovery, precision and accuracy

[0135] The artificial tear liquid containing 0.4% w / v BSA is used as a substitute matrix, and quality 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) are prepared, respectively, corresponding to LLOQ (lower limit of quantification quality control sample), LQC (low concentration quality control sample), GMQC (geometric medium concentration quality control sample), MQC (medium concentration quality control sample) and HQC (high concentration quality control sample).

[0136] The sample pretreatment method in “2. Sample pretreatment” is used to process and obtain the samples to be tested of LLOQ, LQC, GMQC, MQC and HQC. Then, each sample to be tested is analyzed by injection according to the liquid chromatography conditions and mass spectrometry conditions in 3. The results are shown in Table 2 below.

[0137] Table 2

[0138]

[0139]

[0140] From the results, the extraction recovery of the method at each concentration level is 98.1-108%, and the batch accuracy and precision are good.

[0141] (2) Matrix effect

[0142] Take 4 different sources of tear matrix, respectively, prepare LQC, HQC with concentration of 3.00 ng / mL, 375 ng / mL, according to the sample pretreatment method in "2. Sample pretreatment" to process and get the sample, sample analysis. The results are shown in Table 3 below.

[0143] Table 3

[0144]

[0145] From the results, in the detection method of the application, the influence of tear matrix effect on the detection of nitrogen-containing heterocyclic compounds can be ignored.

[0146] The above experimental results show that: the method of the application is verified by method, the established method has high sensitivity, good accuracy and precision, and good stability, and good linearity.

[0147] Example 2 Detection method of nitrogen-containing heterocyclic compounds in plasma and method verification

[0148] 1. Preparation of standard curve sample

[0149] Take blank human plasma containing 0.1M K2EDTA anticoagulant as blank matrix, and prepare standard curve sample according to the concentration of the measured substance shown in Table 4.

[0150] Table 4

[0151] Test substance (ng / mL) C8 C7 C6 C5 C4 C3 C2 C1 Nitrogen-containing heterocyclic compound-I 0.05 0.1 0.25 1.25 6.25 12.5 20 25

[0152] 2. Sample pretreatment

[0153] Mix the sample by vortexing, and add 100 μL of the sample to the corresponding position of the 96-well plate.

[0154] Add 50 mL of acetonitrile / water (1:1, v / v) containing internal standard working solution to all samples except blank samples (i.e. blank matrix), and add 50 mL of acetonitrile / water (1:1, v / v) to blank samples. Shake on the plate shaker at 1200 rpm for 1 minute. Add 150 μL of 8% ammonia water (concentration of 10 wt%) and 0.08M zinc sulfate solution to each sample mixture, respectively. Shake on the plate shaker at 1200 rpm for 5 minutes.

[0155] The shaken samples were loaded onto a 96-well SLE plate. The samples were completely wetted into the bed packing using positive pressure (≤2 psi), and the SLE plate was allowed to sit for 5 minutes.

[0156] 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 under N2at 40°C.

[0157] The samples were reconstituted with 200 μL of a solution of 0.5% formic acid in acetonitrile / water (2:3, v / v). The plate was sealed with a silica gel mat and shaken on a plate shaker at 1200 rpm for 3 minutes to obtain the sample load.

[0158] 3. Preparation of a standard curve

[0159] The sample loads of the standard curve samples of different concentrations were taken separately, and the peak areas were 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 using the weighted least squares method (weight factor: 1 / x2).

[0160] In the LC-MS / MS instrument, the liquid chromatography conditions were as follows: 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: a solution of 0.1 vol% formic acid in water; mobile phase B: a solution of 0.1 vol% formic acid in acetonitrile / methanol (85:15, v / v); gradient elution was performed at a flow rate of 0.5 mL / min, and the gradient elution process was as follows:

[0161]

[0162]

[0163] 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; acquisition time: 5 min.

[0164] The obtained standard curve is shown in Figure 2 The linear regression equation was y = 0.5913x + 0.008497 (R 2 = 0.9978), and it can be seen that the analyte had a good linear relationship in the range of 0.05-25 ng / mL.

[0165] 4. Sample detection

[0166] Take 8 μL of the sample to be tested, and detect in the LC-MS / MS instrument, with the liquid chromatography conditions and mass spectrometry conditions being the same as shown in "3. Establishment of standard curve". Use a blank sample to monitor whether there is contamination in the operation process, and confirm the content of the analyte in the sample according to the standard curve.

[0167] 5. Methodology verification

[0168] (1) Precision and accuracy

[0169] Take 8 μL of the sample to be tested, and detect in the LC-MS / MS instrument, with the liquid chromatography conditions and mass spectrometry conditions being the same as shown in "3. Establishment of standard curve". Use a blank sample to monitor whether there is contamination in the operation process, and confirm the content of the analyte in the sample according to the standard curve.

[0170] According to the sample pretreatment method in "2. Sample pretreatment", the LQC, MQC and HQC samples are pretreated and obtained. Then, each sample is injected and analyzed according to the liquid chromatography conditions and mass spectrometry conditions in 3. The results are shown in Table 5 below.

[0171] Table 5

[0172]

[0173]

[0174] From the results, it can be seen that the accuracy and precision of the method at each concentration level are good, and the stability is better.

[0175] (2) Matrix effect

[0176] 1) Take 6 different sources of blank human plasma, and prepare control samples with concentrations of 0.150 ng / mL, 10.0 ng / mL and 19.0 ng / mL, respectively, which correspond to LQC, MQC and HQC. The samples obtained by pretreatment according to the sample pretreatment method in "2. Sample pretreatment" are injected and analyzed. The area ratio of the analyte and the internal standard is obtained by comparing the biological 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.

[0177] Table 6

[0178]

[0179] 2) Take hemolytic plasma sample and high-fat plasma sample, respectively prepare quality control samples with concentrations of 0.150 ng / mL and 19.0 ng / mL, which correspond to LQC and HQC (n=6) respectively. The samples are treated according to the sample pretreatment method in "2. Sample pretreatment" to obtain the upper samples, which are injected for analysis. The detection results are shown in Table 7 below.

[0180] Table 7

[0181]

[0182] 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.

[0183] 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 linearity.

[0184] The above-described embodiments only express several embodiments of the present application, and the description is more specific and detailed, but it should not be understood as limiting the scope of the patent. It should be noted that for ordinary skilled persons 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 an aqueous solution containing 0.1 ± 0.05 vol% formic acid, and 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%; a gradient elution method 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 process, and then decreases to 30 ± 5 vol%; the sample is a blood sample, the flow rate is 0.5 mL / min, and the gradient elution process is as follows:

2. The method for 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) the chromatographic column is a BEH C18 chromatographic column, 50 × 2.1 mm, 1.7 μm.

3. 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℃.

4. The method of detecting a nitrogen-containing heterocyclic compound according to claim 3, wherein, 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 for detecting further includes: extracting a solution containing the target compound from the sample, and then drying and reconstituting to obtain a sample for detection in the LC-MS / MS method.

6. The method of detecting a nitrogen-containing heterocyclic compound according to claim 5, wherein, the method for extracting a solution containing the target compound from the blood sample includes: mixing the blood sample with an aqueous acetonitrile solution and an aqueous ammonia-containing zinc sulfate solution in sequence, and then infiltrating the mixed solution into the column bed filling of an 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; in the aqueous ammonia-containing zinc sulfate solution, the concentration of aqueous ammonia is 10-30 wt%, the content of aqueous ammonia is 8 ± 1 vol%, and the concentration of zinc sulfate is 0.08 ± 0.01 M.

7. The method of detection of a nitrogen-containing heterocyclic compound according to claim 5 or 6, wherein, 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.

8. The method of detecting a nitrogen-containing heterocyclic compound according to claim 1, wherein, the nitrogen-containing heterocyclic compound has the following structure (I): wherein, R1 and R2 are each independently selected from H and pyrazolyl, and R1 and R2 are 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; T1 is 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 C 1-3 alkyl, said C 1-3 alkyl is optionally substituted with 1, 2, or 3 halogen; alternatively, R5 and R6 together with the carbon atom to which they are commonly connected form an oxetanyl group; 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.

Images