Method for measuring nitrogen-containing heterocyclic compound in blood sample

Abstract

A method for measuring a nitrogen-containing heterocyclic compound in a blood sample. The method uses an LC-MS / MS approach, 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, the acetonitrile-methanol solution having an acetonitrile content of 85 + / − 5 vol%; and a gradient elution method is used. The measuring method can efficiently and accurately measure the content of small-molecule compounds, including nitrogen-containing heterocyclic compounds, in plasma.

Description

A method for detecting nitrogen-containing heterocyclic compounds in blood samples

[0001] Cross-references to related applications

[0002] This invention claims priority to Chinese Patent Application No. 2024110452738, filed on July 31, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of analytical chemistry, specifically relating to a method for detecting nitrogen-containing heterocyclic compounds in blood samples. Background Technology

[0004] EGFR2, also known as KDR or Flk-1, is identified as a receptor for VEGF and VEGFRC and is an early marker of endothelial progenitor cells, with its expression limited to endothelial cells in vivo. VEGFR2 has been shown to be a major signal transducer in the development of angiogenesis and pathological conditions such as cancer and diabetic retinopathy. Studies have demonstrated that anti-VEGF therapy can inhibit the expression and activation of pro-inflammatory factors, thereby alleviating ocular surface inflammation. VEGFR2 transduces major angiogenesis signals through its potent tyrosine kinase activity. However, unlike other representative tyrosine kinase receptors, VEGFR2 does not use the Ras pathway as its primary downstream signaling pathway; instead, it utilizes 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 pathway is an important therapeutic target for 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 the treatment of allergic diseases, autoimmune diseases, and inflammatory diseases, including but not limited to dry eye syndrome 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 vasculitis, idiopathic thrombocytopenic purpura (ITP), myasthenia gravis, allergic rhinitis, chronic obstructive pulmonary disease (COPD), adult respiratory distress syndrome (ARDS), and asthma.

[0006] However, drug metabolism studies are a crucial part of compound drug development. Current technology lacks guidance on methods for isolating and characterizing this compound, making it difficult to determine its metabolism in human body fluids, especially tears and plasma, during clinical trials. Therefore, there is a need to develop a method for detecting the content of this compound in human body fluids. Summary of the Invention

[0007] The first objective of this invention is to provide a method for detecting nitrogen-containing heterocyclic compounds, employing LC-MS / MS, wherein...

[0008] The chromatographic column is a C18 column;

[0009] 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. In the acetonitrile methanol solution, the acetonitrile content is 85 ± 5 vol%.

[0010] A gradient elution method was used. Initially, the content of mobile phase B was 30±5 vol%. During the gradient elution process, the content of mobile phase B gradually increased to 90±5 vol% and then decreased to 30±5 vol%.

[0011] A second object of the present invention is to provide a method for extracting nitrogen-containing heterocyclic compounds from tears, comprising:

[0012] The target component was extracted from tears using a solvent to obtain an extract.

[0013] 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 ammonia concentration being 5-15 wt%.

[0014] The method of this invention can efficiently and accurately detect the content of small molecule compounds, including nitrogen-containing heterocyclic compounds (especially those with poor water solubility), in tears and plasma, and has great application potential in the field of drug metabolism research of related compounds. Attached Figure Description

[0015] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used 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 invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0016] Figure 1 shows the standard curve mentioned in Embodiment 1 of the present invention.

[0017] Figure 2 shows the standard curve mentioned in Embodiment 2 of the present invention. Detailed Implementation

[0018] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the invention. Those skilled in the art can make various modifications and variations to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment can be used in another embodiment to produce further embodiments.

[0019] Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance is provided below for a better understanding of the teachings of this invention. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0020] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of 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 "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0021] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

[0022] In this invention, the numerical range represented by endpoints and positive and negative values ​​includes all numerical values ​​and fractions contained within that range, as well as the referenced endpoints.

[0023] This invention relates to concentration values, which include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% can fluctuate within ±0.1%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are permitted. For example, 100mM can fluctuate within ranges of ±1%, ±2%, ±5%, etc.

[0024] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0025] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0026] In this invention, terms such as "preferred," "better," "more suitable," and "ideal" are merely descriptions of more effective implementation methods or embodiments, and should be understood not to limit the scope of protection of this invention.

[0027] In this invention, "optionally," "optionally," "optionally," "optionally," "optionally," and "optional" mean that they are optional, that is, they are selected from either "with" or "without." If multiple "optional" or "optional" terms appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, then each "optional" or "optional" term is independent.

[0028] In this invention, the term "LC-MS / MS method" is an abbreviation 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 analyzed by mass spectrometry.

[0029] In this invention, the term "C18 column" refers to a chromatographic column whose stationary phase surface is modified with octyl (C18) chains. In the term "BEH C18 column, 50 × 2.1 mm, 1.7 μm," "BEH" stands for "Ethylene Bridged Hybrid," characterized by the use of ethylene bridging between the silica gel and the alkyl chains. This design provides higher column packing stability and inertness, as well as a more uniform carbon loading distribution; "50 × 2.1 mm" indicates that the column length is 50 mm and the inner diameter is 2.1 mm; "1.7 μm" indicates that the particle size of the column packing material is 1.7 μm.

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

[0031] In this invention, "SLE plate" refers to "solid-liquid extraction plate," also known as a solid-phase supported liquid-liquid extraction plate. It uses porous diatomaceous earth filler with high specific surface area and strong chemical inertness as the liquid-liquid distribution carrier and employs an organic solvent immiscible with water for elution. This effectively removes matrix interferences, such as proteins and phospholipids, from viscous samples (e.g., plasma, serum, or whole blood). The SLE plate described in this invention is commercially available.

[0032] This invention relates to a method for detecting nitrogen-containing heterocyclic compounds, employing LC-MS / MS, wherein the chromatographic column is a C18 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, initially the content of mobile phase B is 30±5 vol%, and during the gradient elution process, the content of mobile phase B gradually increases to 90±5 vol%, and then decreases to 30±5 vol%.

[0033] The present invention has found that the above method can effectively improve the separation effect of nitrogen-containing heterocyclic compounds in the sample, and is beneficial to improving the intensity and stability of mass spectrometry signals.

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

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

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

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

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

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

[0040] Those skilled in the art can combine the conditions mentioned above with common sense to obtain a preferred embodiment of the LC operating conditions of the present invention.

[0041] If necessary, the above LC operating conditions can be modified. The retention times of the analyte and / or internal standard may vary depending on the use and performance of the column; as long as the elution order remains unchanged, this is considered acceptable.

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

[0043] In some implementations, the mass spectrometry conditions also 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 conditions mentioned above with common sense to obtain a preferred embodiment of the MS operating conditions of the present invention.

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

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

[0047] In some embodiments, when the sample is a tear sample, the method for extracting a solution containing the target compound from the tear sample includes: extracting the target component from the tear sample using a solvent to obtain an extract; 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, wherein the concentration of ammonia is 5-15 wt%, more preferably 10 ± 2 wt%. Compared to other solvents, using the above-mentioned solvent allows for efficient extraction of nitrogen-containing heterocyclic compounds from tear samples with small sample volumes and helps to eliminate the influence of individual tear matrix differences.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] In some specific embodiments, the following solvents may also be considered: a mixture of ammonia and acetonitrile in a volume ratio of 1:4 to 6; or an acetonitrile solution containing 5 to 10 vol% ammonia (concentration of 10 wt%); or a mixture of ammonia, zinc sulfate solution, and acetonitrile. While the above solvents also provide generally satisfactory dissolution effects, the solvent provided by this invention is significantly superior in dissolution performance to the aforementioned solvents.

[0052] In some embodiments, the mixture of solvent and tear sample is vortexed for 8–15 minutes and then left to stand for 15–25 minutes to obtain an extract. This significantly shortens the extraction time while ensuring extraction efficiency.

[0053] In some embodiments, the method for extracting a solution containing the target compound from a tear sample further includes: extracting the sample extract with n-hexane, centrifuging to obtain a first supernatant and a precipitate; then, using a mixed solvent of ethyl acetate and n-hexane at a volume ratio of 1:0.8–1.2 (more preferably 1:1), extracting 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. This is beneficial for further improving the extraction effect of nitrogen-containing heterocyclic compounds and reducing the influence of individual matrix differences.

[0054] In some embodiments, when the sample is a tear sample, the resolution 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 to 3.5, more preferably 2:3.

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

[0056] The method of the present invention can be used for qualitative detection (results are usually presented in the form of "yes / no" or "present / absent") and quantitative detection (results are the concentration or content level of the target component) of nitrogen-containing heterocyclic compounds.

[0057] When the method of the present invention is applied to quantitative detection, those skilled in the art can, based on common sense, establish a standard curve using a standard substance of known concentration under the same chromatographic and mass spectrometric conditions, and then detect the sample to be tested, determining the concentration or content level of the target component in the sample to be tested by comparing it with the standard substance.

[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 substitute matrix for preparing standard curves and / or quality control samples. The present invention also provides the aforementioned substitute matrix, which exhibits superior intra-batch accuracy and precision when used to prepare standard curves and quality control samples.

[0059] In some specific implementations, the alternative matrix can also be used to prepare standard curves, and human blank tears can be 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 includes: sequentially mixing the blood sample with an aqueous acetonitrile solution and a zinc sulfate solution containing ammonia, then immersing the mixture in the column bed packing 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 to 1.2, more preferably 1:1; in the zinc sulfate solution containing ammonia, the concentration of ammonia is 10 to 30 wt%, the content of ammonia is 8 ± 1 vol%, and the concentration of zinc sulfate is 0.08 ± 0.01 M. By means of the above method, nitrogen-containing heterocyclic compounds can be efficiently extracted from blood samples of small quantities, and the influence of the blood matrix on the detection results can be effectively controlled.

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

[0062] In some specific embodiments, when the sample is a blood sample, a standard curve and / or quality control sample are prepared using plasma containing an anticoagulant (such as K2EDTA) (such as human plasma, mouse plasma, etc.).

[0063] In some specific embodiments, the drying process specifically involves drying with N2 at a temperature below 40°C.

[0064] In some embodiments, the structure of the nitrogen-containing heterocyclic compound is shown in formula (I):

[0065] in,

[0066] R1 and R2 are each independently selected from H and pyrazol group, and R1 and R2 are not simultaneously pyrazol group or H;

[0067] 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;

[0068] T1 is selected from CH and N;

[0069] D1 is selected from -O-, -C(R5)(R6)-, -N(R7)- and

[0070] R5 and R6 are each independently selected from H, F, Cl, Br, I, OH, and C. 1-3 Alkyl, the C1 -3 The alkyl group may be optionally replaced by one, two, or three halogens;

[0071] Alternatively, R5 and R6 together with the carbon atoms they are connected to form an oxobutyryl group;

[0072] R7 is selected from H, and C 1-3 Alkyl, the C 1-3 The alkyl group may be optionally replaced by one, two, or three halogens;

[0073] R8 is 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 of the following structures:

[0076] In some specific implementations, the injection volume of the sample derived from the tear fluid is 5 ± 1 μL.

[0077] In some specific implementations, the injection volume of the sample derived from the plasma sample is 8 ± 1 μL.

[0078] In some specific implementations, the mass spectrometry acquisition time for tear samples is 3.0–5.0 min.

[0079] In some specific implementations, the mass spectrometry acquisition time for plasma samples is 4.5 to 8.0 minutes.

[0080] 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.

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

[0082] The target component was extracted from tears using a solvent to obtain an extract.

[0083] 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%.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] In some embodiments, the method further includes: extracting the extract with n-hexane, centrifuging to obtain a first supernatant and a precipitate; then extracting the precipitate again with a mixed solvent of ethyl acetate and n-hexane at a volume ratio of 1:0.8 to 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.

[0088] In some embodiments, the method further includes drying the supernatant to obtain a nitrogen-containing heterocyclic compound. Preferably, the drying is performed by blowing with N2 at a temperature below 40°C.

[0089] In some embodiments, the structure of the nitrogen-containing heterocyclic compound is shown in formula (I):

[0090] in,

[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 The alkyl group may be optionally replaced by one, two, or three halogens;

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

[0097] R7 is selected from H, and C 1-3 Alkyl, the C 1-3 The alkyl group may be optionally replaced by 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 these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions should preferably be referred to the guidelines given in this invention, or may be performed according to experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

[0104] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0105] Example 1: Detection method and method validation of nitrogen-containing heterocyclic compounds in tears

[0106] 1. Preparation of standard curve samples

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

[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 of internal standard (nitrogenous heterocyclic compound -I-D4) to all samples except the blank sample (i.e., the substitute matrix for the blank). For the blank sample, add 3.2 mL of solvent. Vortex for 10 minutes, then let stand for 20 minutes. After vortexing to mix, the extract is ready. The solvent is a mixture of ethyl acetate, methanol, water, and ammonia in a volume ratio of 100:20:3.5:6.5, with an ammonia concentration of 10 wt%.

[0111] Vortex the extract until homogeneous, and add 500 μL of sample extract to the corresponding position on the 96-well plate (sample plate).

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

[0113] A second extraction was performed by adding 500 μL of ethyl acetate / n-hexane (1:1, v / v) to a 96-well plate (sample plate). Specifically, the plate was sealed with aluminum foil heat-sealing film and shaken at 1200 rpm for 5 minutes on a shaker. The plate was then centrifuged at 4000 rpm for 5 minutes at 4°C. 500 μL of the supernatant was transferred to a 96-well plate (2).

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

[0115] 3. Establishment of the standard curve

[0116] Samples of standard curve samples with different concentrations were taken and the peak areas were measured in an LC-MS / MS instrument. The concentration of the analyte in the standard curve sample was used as the abscissa (X), and the peak areas of the analyte and the internal standard were used as the ordinate (Y). Regression calculation was performed using the weighted least squares method (with a weighting factor of 1 / x^2) to confirm the linear regression model.

[0117] In the LC-MS / MS instrument, the liquid chromatography conditions were as follows: Column: Waters BEH C18 Column, 100 × 2.1 mm, 1.7 μm; Column temperature: 60℃; Injection temperature: 6℃; Injection volume: 5 μL; Mobile phase A: aqueous solution containing 0.1 vol% formic acid; Mobile phase B: acetonitrile / methanol (85:15, v / v) solution containing 0.1 vol% formic acid; Gradient elution was used at a flow rate of 0.5 mL / min, and the gradient elution process was as follows:

[0118] The mass spectrometry conditions were as follows: Ionization mode: ESI+; Scan 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; Acquisition time: 3.5min.

[0119] The obtained standard curve is shown in Figure 1. The linear regression equation is y = 0.2272x + 0.01292(R²). 2 =0.9977), indicating that the analyte exhibits good linearity in the range of 1–500 ng / mL.

[0120] 4. Sample testing

[0121] Take 5 μL of the sample to be tested and analyze it in an LC-MS / MS instrument. The liquid chromatography and mass spectrometry conditions are the same as those shown in "3. Establishment of Standard Curve". Use a blank sample to monitor for contamination during the operation. Confirm the content of the analyte in the sample according to the standard curve.

[0122] 5. Methodological Validation

[0123] (1) Recovery rate, precision and accuracy

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

[0125] Samples were pretreated according to the methods described in "2. Sample Pretreatment" to obtain LLOQ, LQC, GMQC, MQC, and HQC samples. Then, each sample was analyzed using liquid chromatography and mass spectrometry conditions as described in section 3. The results are shown in Table 2 below.

[0126] Table 2

[0127] The results show that the extraction recovery rate of the method of the present invention is 98.1% to 108% at various concentration levels, with good intra-batch accuracy and precision.

[0128] (2) Matrix effect

[0129] Four different tear matrix sources were collected, and LQC and HQC solutions with concentrations of 3.00 ng / mL and 375 ng / mL were prepared respectively. The samples were processed according to the sample pretreatment method in "2. Sample Pretreatment" and then injected for analysis. The results are shown in Table 3 below.

[0130] Table 3

[0131] The results show that the effect of tear matrix effect on the detection of nitrogen-containing heterocyclic compounds can be ignored in the detection method of the present invention.

[0132] The above experimental results show that the method of the present invention has been validated by methodology, and the established method has high sensitivity, good accuracy and precision, good stability and good linearity.

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

[0134] 1. Preparation of standard curve samples

[0135] Using blank human plasma containing 0.1M K2EDTA anticoagulation as the blank matrix, standard curve samples were prepared according to the analyte concentrations shown in Table 4.

[0136] Table 4

[0137] 2. Sample pretreatment

[0138] Vortex mix the sample thoroughly, and add 100 μL of sample to the corresponding position in the 96-well plate.

[0139] To all samples except the blank sample (i.e., the blank matrix), add 50 mL of acetonitrile / water (1:1, v / v) containing the internal standard working solution. For the blank sample, add 50 mL of acetonitrile / water (1:1, v / v). Shake at 1200 rpm for 1 minute on a shaker. To each sample mixture, add 150 μL of a solution containing 8% ammonia (10 wt%) and 0.08 M zinc sulfate. Shake at 1200 rpm for 5 minutes on a shaker.

[0140] The shaken sample was loaded onto a 96-well SLE plate. Positive pressure (≤2 psi) was applied to ensure the sample was completely wetted into the column bed packing material, and then the SLE plate was allowed to stand for 5 minutes.

[0141] Elute the SLE plate with ethyl acetate, collect the eluent in a new 96-well plate, and dry the resulting solution with N2 at 40°C.

[0142] Reconstitute with 200 μL of acetonitrile / water (2:3, v / v) solution containing 0.5% formic acid. Seal the plate with a silicone pad and shake at 1200 rpm for 3 minutes on a shaker to obtain the upper sample of each sample.

[0143] 3. Establishment of the standard curve

[0144] Samples of standard curve samples with different concentrations were taken and the peak areas were measured in an LC-MS / MS instrument. The concentration of the analyte in the standard curve sample was used as the abscissa (X), and the peak areas of the analyte and the internal standard were used as the ordinate (Y). Regression calculation was performed using the weighted least squares method (with a weighting factor of 1 / x^2) to confirm the linear regression model.

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

[0146] The mass spectrometry conditions were as follows: Ionization mode: ESI+; Scan 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; Acquisition time: 5min.

[0147] The obtained standard curve is shown in Figure 2. The linear regression equation is y = 0.5913x + 0.008497 (R²). 2 =0.9978), indicating that the analyte exhibits good linearity in the range of 0.05–25 ng / mL.

[0148] 4. Sample testing

[0149] Take 8 μL of the sample to be tested and analyze it in an LC-MS / MS instrument. The liquid chromatography and mass spectrometry conditions are the same as those shown in "3. Establishment of Standard Curve". Use a blank sample to monitor for contamination during the operation. Confirm the content of the analyte in the sample according to the standard curve.

[0150] 5. Methodological Validation

[0151] (1) Precision and accuracy

[0152] Using blank human plasma containing 0.1M K2EDTA anticoagulation as the blank matrix, quality control samples with concentrations of 0.05 ng / mL, 0.150 ng / mL, 1.00 ng / mL, 10.0 ng / mL, and 19.0 ng / mL were prepared, corresponding to LLOQ, LQC, GMQC, MQC, and HQC (n=6), respectively.

[0153] Samples were pretreated according to the methods described in "2. Sample Pretreatment" to obtain LQC, MQC, and HQC samples. Then, each sample was analyzed using liquid chromatography and mass spectrometry conditions as described in section 3. The results are shown in Table 5 below.

[0154] Table 5

[0155] The results show that the method of the present invention has good accuracy and precision at various concentration levels, and good stability.

[0156] (2) Matrix effect

[0157] 1) Six blank human plasma samples from different sources were collected, and quality control samples were prepared at concentrations of 0.150 ng / mL, 10.0 ng / mL, and 19.0 ng / mL, corresponding to LQC, MQC, and HQC, respectively. The samples were processed according to the sample pretreatment method in "2. Sample Pretreatment" and then injected for analysis. The ratio of the analyte area to the internal standard area was obtained by comparing the biological matrix area and the solvent area, thus obtaining the normalized matrix effect factor. The detection results are shown in Table 6 below.

[0158] Table 6

[0159] 2) Take hemolyzed plasma samples and high-lipid plasma samples, and prepare quality control samples with concentrations of 0.150 ng / mL and 19.0 ng / mL, respectively, corresponding to LQC and HQC (n=6). Process the samples according to the sample pretreatment method in "2. Sample Pretreatment" and inject them for analysis. The detection results are shown in Table 7 below.

[0160] Table 7

[0161] The results show that the effect of blood matrix effect on the detection of nitrogen-containing heterocyclic compounds can be ignored in the detection method of the present invention.

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

[0163] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for detecting nitrogen-containing heterocyclic compounds, employing LC-MS / MS, wherein, The chromatographic column is a C18 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. In the acetonitrile methanol solution, the acetonitrile content is 85 ± 5 vol%. A gradient elution method was used. Initially, the content of mobile phase B was 30±5 vol%. During the gradient elution process, the content of mobile phase B gradually increased to 90±5 vol% and then decreased to 30±5 vol%. The sample was a blood sample, the flow rate was 0.5 mL / min, and the gradient elution process was as follows:

2. The method for detecting nitrogen-containing heterocyclic compounds according to claim 1, wherein, Mass spectrometry conditions also 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 was a BEH C18 column, 50×2.1mm, 1.7μm.

3. The method for detecting nitrogen-containing heterocyclic compounds according to claim 1, wherein, Mass spectrometry conditions included: ionization mode: ESI+; scan mode: MRM; ion source voltage: 3000±500V; ion source temperature: 550±20℃.

4. The method for detecting nitrogen-containing heterocyclic compounds according to claim 3, wherein, The mass spectrometry conditions also included: 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 for detecting nitrogen-containing heterocyclic compounds according to claim 1, wherein, The detection method further includes: A solution containing the target compound was extracted from the sample, then dried and reconstituted to obtain a top sample for detection in LC-MS / MS.

6. The method for detecting nitrogen-containing heterocyclic compounds according to claim 5, wherein, Methods for extracting solutions containing target compounds from blood samples include: The blood sample was mixed sequentially with an aqueous acetonitrile solution and a zinc sulfate solution containing ammonia. The mixture was then poured into the column bed packing of an SLE plate. The SLE plate was then eluted with ethyl acetate to obtain a solution containing the target compound. In the acetonitrile aqueous solution, the volume ratio of acetonitrile to water is 1:0.8 to 1.2; In the zinc sulfate solution containing ammonia, the concentration of ammonia is 10-30 wt%, the content of ammonia is 8 ± 1 vol%, and the concentration of zinc sulfate is 0.08 ± 0.01 M.

7. The method for detecting nitrogen-containing heterocyclic compounds according to claim 5 or 6, wherein, The resolution was performed using an aqueous solution of acetonitrile containing formic acid; In the formic acid-containing acetonitrile aqueous solution, the formic acid content is 0.5 ± 0.1 vol%, and the volume ratio of acetonitrile to water is 2:2.5 to 3.

5.

8. The method for detecting nitrogen-containing heterocyclic compounds according to claim 1, wherein, The structure of the nitrogen-containing heterocyclic compound is shown in formula (I): in, R1 and R2 are each independently selected from H and pyrazol group, and R1 and R2 are not simultaneously pyrazol group or H; 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; T1 is selected from CH and N; D1 is selected from -O-, -C(R5)(R6)-, -N(R7)- and R5 and R6 are each independently selected from H, F, Cl, Br, I, OH, and C. 1-3 Alkyl, the C1 -3 The alkyl group may be optionally replaced by one, two, or three halogens; Alternatively, R5 and R6 together with the carbon atom they are connected to form an oxobutyryl group; R7 is selected from H, and C 1-3 Alkyl, the C 1-3 The alkyl group may be optionally replaced by one, two, or three halogens; R8 is selected from H and -C(=O)-C 1-3 alkyl; n is selected from 1 and 2.

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