A method for separating and detecting 4-bromo-2-nitro-1-phenoxybenzene by derivatization liquid chromatography-mass spectrometry

Abstract

This invention provides a method for the separation and detection of 4-bromo-2-nitro-1-phenoxybenzene using derivatized liquid chromatography-mass spectrometry (LC-MS), belonging to the field of analytical chemistry. The method includes: mixing a solution of the sample to be tested with a proton donor and zinc powder, reacting, filtering or centrifuging to obtain a test solution, detecting the content of 4-bromo-2-amino-1-phenoxybenzene in the test solution, and then calculating the content of 4-bromo-2-nitro-1-phenoxybenzene in the sample to be tested. This invention overcomes the technical problem of low response and inability to quantitatively detect 4-bromo-2-nitro-1-phenoxybenzene when using LC-MS to detect the prototype. Furthermore, it is simple to operate, highly sensitive, accurate, linear, and has good separation, exhibiting excellent unexpected technical effects.

Description

Technical Field

[0001] This invention relates to the field of analytical chemistry, and specifically to a method for separating and detecting 4-bromo-2-nitro-1-phenoxybenzene using derivatized liquid chromatography-mass spectrometry. Background Technology

[0002] 4-Bromo-2-nitro-1-phenoxybenzene is a raw material or intermediate for the synthesis of various pharmaceutical compounds. For example, in US patent application US20090286768A, 4-bromo-2-nitro-1-phenoxybenzene is used to synthesize cis-3-[8-amino-1-(3-amino-4-phenoxy-phenyl)-imidazo[1,5-a]pyrazin-3-yl]-1-methyl-cyclobutanol (i.e., the product of Example 259 in the US patent application) (which has good ACK1 inhibitory activity and can be used to prevent or treat cancerous tumors mediated by or overexpressing ACK1).

[0003]

[0004] Because 4-bromo-2-nitro-1-phenoxybenzene contains an aromatic nitro group, which is a genotoxicity warning structure (ICH S2(R1)), this compound requires strict control in pharmaceuticals. According to ICH M7 regulations, the threshold for toxicological concern (TTC) for impurities containing genotoxicity warning structures must be controlled below 1.5 μg / day. Based on the maximum daily dose, the limit for impurities containing genotoxicity warning structures in pharmaceuticals is at the ppm level. Although 4-bromo-2-nitro-1-phenoxybenzene exhibits UV absorption, its response is insufficient when detected by HPLC. Furthermore, 4-bromo-2-nitro-1-phenoxybenzene has a very high boiling point (210℃ (Press: 15 Torr)), resulting in poor response when detected by gas chromatography-mass spectrometry (GC-MS) due to its inability to vaporize. When detected by liquid chromatography-mass spectrometry (LC-MS), the prototype 4-bromo-2-nitro-1-phenoxybenzene shows virtually no response.

[0005] Currently, no analytical methods for the detection of 4-bromo-2-nitro-1-phenoxybenzene have been reported. Therefore, there is an urgent need for a highly sensitive and simple method for the detection of 4-bromo-2-nitro-1-phenoxybenzene. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention provides the following technical solutions.

[0007] In a first aspect, the present invention provides a method for detecting 4-bromo-2-nitro-1-phenoxybenzene.

[0008] A method for detecting the content of 4-bromo-2-nitro-1-phenoxybenzene in a sample includes: mixing a solution containing the sample with a proton donor and zinc powder, reacting, filtering or centrifuging to obtain a test solution, detecting the content of 4-bromo-2-amino-1-phenoxybenzene in the test solution, and then calculating the content of 4-bromo-2-nitro-1-phenoxybenzene in the sample. This technical solution overcomes the technical problem of low response and inability to quantitatively detect 4-bromo-2-nitro-1-phenoxybenzene when using liquid chromatography-mass spectrometry (LC-MS) to detect the original form of 4-bromo-2-nitro-1-phenoxybenzene, achieving unexpectedly superior technical results.

[0009] In some embodiments, the sample to be tested contains 4-bromo-2-nitro-1-phenoxybenzene, and the reaction is to reduce the 4-bromo-2-nitro-1-phenoxybenzene in the sample to 4-bromo-2-amino-1-phenoxybenzene.

[0010]

[0011] In some embodiments, the proton donor in the preparation of the test solution is at least one of acetic acid (analytical grade), hydrochloric acid (analytical grade concentrated hydrochloric acid with a mass fraction of approximately 37%), and 20-50 mg / ml ammonium chloride aqueous solution.

[0012] In some embodiments, the solvent of the solution containing the sample to be tested is at least one of methanol, ethanol, and isopropyl acetate.

[0013] In some embodiments, the mass ratio of the sample to zinc powder in the solution containing the sample to be tested is 1.0:3.0-1.0:10.0.

[0014] In some embodiments, the mass ratio of the test sample to zinc powder in the solution containing the test sample is 1.0:3.0, 1.0:3.1, 1.0:3.2, 1.0:3.3, 1.0:3.4, 1.0:3.5, 1.0:3.6, 1.0:3.7, 1.0:3.8, 1.0:3.9, 1.0:4.0, 1.0:4.5, 1.0:5.0, 1.0:5.5, 1.0:6.0, 1.0:6.5, 1.0:7.0, 1.0:7.5, 1.0:8.0, 1.0:8.5, 1.0:9.0, 1.0:9.5, or 1.0:10.0.

[0015] In some embodiments, the mass ratio of the sample to zinc powder in the solution containing the sample to be tested is 1.0:5.0.

[0016] In some embodiments, the ratio of the mass of the sample to be tested in the solution containing the sample to the volume of the proton donor is 10 mg:20 μl to 50 mg:20 μl.

[0017] In some embodiments, the ratio of the mass of the sample to be tested in the solution containing the sample to the volume of the proton donor is 10 mg:20 μl, 15 mg:20 μl, 16 mg:20 μl, 17 mg:20 μl, 18 mg:20 μl, 19 mg:20 μl, 20 mg:20 μl, 21 mg:20 μl, 22 mg:20 μl, 23 mg:20 μl, 24 mg:20 μl, 25 mg:20 μl, 30 mg:20 μl, 35 mg:20 μl, 40 mg:20 μl, 45 mg:20 μl, or 50 mg:20 μl.

[0018] In some embodiments, the sample to be tested is cis-3-[8-amino-1-(3-amino-4-phenoxy-phenyl)-imidazo[1,5-a]pyrazin-3-yl]-1-methyl-cyclobutanol.

[0019] In some embodiments, the reaction time in the preparation of the test solution is 1-5 hours.

[0020] In some embodiments, the reaction time in the preparation of the test solution is 1 h, 2 h, 3 h, 4 h, or 5 h.

[0021] In some embodiments, the reaction time in the preparation of the test solution is 1-2 hours.

[0022] In some embodiments, the method for detecting the content of 4-bromo-2-amino-1-phenoxybenzene in the test solution is to use high performance liquid chromatography-mass spectrometry (HPLC-MS).

[0023] In some embodiments, the chromatographic column used in the high-performance liquid chromatography is an XSelect-HSS T3 column.

[0024] In some embodiments, the mobile phase of the high-performance liquid chromatography is a gradient elution using 0.1% vol formic acid aqueous solution or 10 mM ammonium formate aqueous solution as mobile phase A and acetonitrile as mobile phase B.

[0025] In some embodiments, the gradient elution procedure is as follows:

[0026] time Mobile phase A (%) Mobile phase B (%) 0min 95 5 15min-19min 10-15 90-85 20min-30min 10-15 90-85 .

[0027] In some embodiments, the gradient elution procedure is as follows:

[0028] time Mobile phase A (%) Mobile phase B (%) 0min 95 5 15min 10-15 90-85 20min-30min 10-15 90-85 .

[0029] In some embodiments, the gradient elution procedure is as follows:

[0030] time Mobile phase A (%) Mobile phase B (%) 0min 95 5 15min 15 85 20min-30min 15 85 .

[0031] In some embodiments, the gradient elution procedure is as follows:

[0032] time Mobile phase A (%) Mobile phase B (%) 0min 95 5 15min 10 90 20min 10 90 .

[0033] In some embodiments, the gradient elution procedure is as follows:

[0034] time Mobile phase A (%) Mobile phase B (%) 0min 95 5 15min 15 85 20min 15 85 .

[0035] In some embodiments, the gradient elution procedure is as follows:

[0036]

[0037]

[0038] In some embodiments, the high-performance liquid chromatography method further includes a 5-15 minute post-run after each gradient elution.

[0039] In some embodiments, the high-performance liquid chromatography method further includes a post-run of 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, or 15 min after each gradient elution.

[0040] In some embodiments, the inner diameter of the chromatographic column is 2 mm to 5 mm.

[0041] In some embodiments, the inner diameter of the chromatographic column is 2 mm, 3 mm, 4 mm, 4.6 mm, or 5 mm. In some embodiments, the inner diameter of the chromatographic column is 4.6 mm.

[0042] In some embodiments, the column length of the chromatographic column is 100mm-250mm.

[0043] In some embodiments, the column length of the chromatographic column is 100 mm, 150 mm, 200 mm or 250 mm.

[0044] In some embodiments, the column length is 100 mm.

[0045] In some embodiments, the particle size of the chromatographic column packing material is 2.5 μm-5.0 μm.

[0046] In some embodiments, the particle size of the chromatographic column packing material is 2.5 μm, 3.0 μm, 3.5 μm, 4.0 μm, 4.5 μm, or 5.0 μm.

[0047] In some embodiments, the particle size of the chromatographic column packing material is 2.5 μm-3.0 μm.

[0048] In some embodiments, the chromatographic column used in the high-performance liquid chromatography is an XSelect-HSS T3 column, which has an inner diameter of 4.6 mm, a column length of 100 mm, and a packing particle size of 2.5 μm.

[0049] In some embodiments, the column temperature of the high-performance liquid chromatography is 25℃-35℃.

[0050] In some embodiments, the column temperature of the high-performance liquid chromatography is 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, or 35°C.

[0051] In some embodiments, the elution flow rate of the high performance liquid chromatography is 0.8 ml / min to 1.2 ml / min.

[0052] In some embodiments, the elution flow rate of the high-performance liquid chromatography is 0.8 ml / min, 0.9 ml / min, 1.0 ml / min, 1.1 ml / min, or 1.2 ml / min.

[0053] In some embodiments, the mass spectrometry method employs an electrospray ionization (ESI) source.

[0054] In some embodiments, the mass spectrometry scanning mode is selected ion monitoring (SIM).

[0055] In some embodiments, the scan settings for selected ion monitoring are: positive ion mode, m / z = 264.

[0056] In some embodiments, the drying gas for the mass spectrometry method is nitrogen.

[0057] In some embodiments, the drying gas temperature for the mass spectrometry method is 300°C-350°C.

[0058] In some embodiments, the drying gas temperature for the mass spectrometry method is 300°C, 310°C, 320°C, 330°C, 340°C, or 350°C.

[0059] In some embodiments, the drying gas temperature for the mass spectrometry method is 300°C.

[0060] In some embodiments, the drying gas flow rate of the mass spectrometry method is 10.0 L / min to 13.0 L / min.

[0061] In some embodiments, the drying gas flow rate of the mass spectrometry method is 10.0 L / min, 11.0 L / min, 12.0 L / min, or 13.0 L / min.

[0062] In some embodiments, the drying gas flow rate of the mass spectrometry method is 11.0 L / min.

[0063] In some embodiments, the nebulizer gas pressure of the mass spectrometry is 35 psi-55 psi.

[0064] In some embodiments, the nebulizer gas pressure of the mass spectrometry is 35 psi, 40 psi, 45 psi, 50 psi, or 55 psi.

[0065] In some embodiments, the nebulizer gas pressure of the mass spectrometry is 35 psi.

[0066] In some embodiments, the capillary voltage of the mass spectrometry method is a positive voltage of 3000V-5000V.

[0067] In some embodiments, the capillary voltage of the mass spectrometry method is a positive voltage of 3000V, 3100V, 3200V, 3300V, 3400V, 3500V, 3600V, 3700V, 3800V, 3900V, 4000V, 4100V, 4200V, 4300V, 4400V, 4500V, or 5000V.

[0068] In some embodiments, the capillary voltage of the mass spectrometry method is a positive voltage of 4000V.

[0069] In some embodiments, the fragmentation voltage of the mass spectrometry method is 65V-150V.

[0070] In some embodiments, the fragmentation voltage of the mass spectrometry method is 65V, 66V, 67V, 68V, 69V, 70V, 71V, 72V, 73V, 74V, 75V, 80V, 85V, or 90V.

[0071] In some embodiments, the fragmentation voltage of the mass spectrometry method is 70V.

[0072] In some embodiments, the gain factor of the mass spectrometry method is 1.0-5.0.

[0073] In some embodiments, the gain factor of the mass spectrometry method is 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, or 5.0.

[0074] In some embodiments, the mass spectrometry method employs a single quadrupole or triple quadrupole detector.

[0075] In some embodiments, the detection method further includes the preparation of a reference solution, which comprises: mixing a solution containing 4-bromo-2-nitro-1-phenoxybenzene with a proton donor and zinc powder, reacting, filtering or centrifuging to obtain the reference solution; the proton donor in the preparation of the reference solution is the same as the proton donor in the preparation of the test solution, the solvent in the solution containing 4-bromo-2-nitro-1-phenoxybenzene is the same as the solvent in the solution containing the test sample, and the reaction time in the preparation of the reference solution is the same as the reaction time in the preparation of the test solution. This technical solution involves a derivatization reaction of 4-bromo-2-nitro-1-phenoxybenzene with a proton donor and zinc powder to obtain a derivative (4-bromo-2-amino-1-phenoxybenzene). The content of 4-bromo-2-nitro-1-phenoxybenzene can then be detected by detecting the derivative. This overcomes the technical problem of low response and inability to quantitatively detect 4-bromo-2-nitro-1-phenoxybenzene when using liquid chromatography-mass spectrometry to detect the prototype, and has excellent unexpected technical effects.

[0076] In some embodiments, in the preparation of the reference solution, the mass ratio of 4-bromo-2-nitro-1-phenoxybenzene to zinc powder in the solution containing 4-bromo-2-nitro-1-phenoxybenzene is 1:1000000-30:1000000.

[0077] In some embodiments, the mass ratio of 4-bromo-2-nitro-1-phenoxybenzene to zinc powder in the solution containing 4-bromo-2-nitro-1-phenoxybenzene during the preparation of the reference solution is 1:1000000, 2:1000000, 2.9:1000000, 3:1000000, 4:1000000, 5:1000000, 6:1000000, 7:1000000, 8:1000000, 9:1000000, 10:1000000, 11:1000000, and 12:100000. 0, 13:1000000, 14:1000000, 15:1000000, 16:1000000, 17:1000000, 18:1000000, 19:1000000, 20:1000000, 21:1000000, 22:1000000, 23:1000000, 24:1000000, 25:1000000, 26:1000000, 27:1000000, 28:1000000, 29:1000000 or 30:1000000.

[0078] In some embodiments, in the preparation of the reference solution, the mass ratio of 4-bromo-2-nitro-1-phenoxybenzene to zinc powder in the solution containing 4-bromo-2-nitro-1-phenoxybenzene is 1:1000000-5:1000000.

[0079] In some embodiments, in the preparation of the reference solution, the mass ratio of 4-bromo-2-nitro-1-phenoxybenzene to zinc powder in the solution containing 4-bromo-2-nitro-1-phenoxybenzene is 3:1000000.

[0080] In some embodiments, in the preparation of the reference solution, the ratio of the mass of 4-bromo-2-nitro-1-phenoxybenzene in the solution containing 4-bromo-2-nitro-1-phenoxybenzene to the volume of the proton donor is 0.3 μg:20 μl to 5.0 μg:20 μl.

[0081] In some embodiments, in the preparation of the reference solution, the ratio of the mass of 4-bromo-2-nitro-1-phenoxybenzene in the solution containing 4-bromo-2-nitro-1-phenoxybenzene to the volume of the proton donor is 0.3 μg:20 μl, 0.4 μg:20 μl, 0.5 μg:20 μl, 0.6 μg:20 μl, 0.8 μg:20 μl, 1.0 μg:20 μl, 1.5 μg:20 μl, 2.0 μg... : 20μl, 2.5μg: 20μl, 2.6μg: 20μl, 2.7μg: 20μl, 2.8μg: 20μl, 2.9μg: 20μl, 3.0μg: 20μl, 3.1μg: 20 μl, 3.2μg: 20μl, 3.3μg: 20μl, 3.4μg: 20μl, 3.5μg: 20μl, 4.0μg: 20μl, 4.5μg: 20μl or 5.0μg: 20μl.

[0082] In some embodiments, the concentration of 4-bromo-2-nitro-1-phenoxybenzene in the solution containing 4-bromo-2-nitro-1-phenoxybenzene is 0.3 μg / ml to 5.0 μg / ml.

[0083] In some embodiments, the concentration of 4-bromo-2-nitro-1-phenoxybenzene in the solution containing 4-bromo-2-nitro-1-phenoxybenzene is 0.3 μg / ml, 0.4 μg / ml, 0.5 μg / ml, 0.6 μg / ml, 0.8 μg / ml, 1.0 μg / ml, 1.5 μg / ml, 2.0 μg / ml, 2.5 μg / ml, 2.6 μg / ml, 2.7 μg / ml, 2.8 μg / ml, 2.9 μg / ml, 3.0 μg / ml, 3.1 μg / ml, 3.2 μg / ml, 3.3 μg / ml, 3.4 μg / ml, 3.5 μg / ml, 4.0 μg / ml, 4.5 μg / ml, or 5.0 μg / ml.

[0084] In some embodiments, the preparation of the reference solution and the reference solution further includes filtration after the reaction.

[0085] Secondly, the present invention provides an application of the detection method described in the first aspect.

[0086] In some embodiments, the use includes detecting the content of 4-bromo-2-nitro-1-phenoxybenzene in cis-3-[8-amino-1-(3-amino-4-phenoxy-phenyl)-imidazo[1,5-a]pyrazin-3-yl]-1-methyl-cyclobutanol.

[0087] In some embodiments, the use includes detecting the conversion of 4-bromo-2-nitro-1-phenoxybenzene in a reaction using 4-bromo-2-nitro-1-phenoxybenzene as a starter or intermediate to synthesize cis-3-[8-amino-1-(3-amino-4-phenoxy-phenyl)-imidazo[1,5-a]pyrazin-3-yl]-1-methyl-cyclobutanol or an intermediate thereof.

[0088] Beneficial effects

[0089] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0090] (1) The present invention uses a derivatization reaction of 4-bromo-2-nitro-1-phenoxybenzene with a proton donor and zinc powder to obtain a derivative (4-bromo-2-amino-1-phenoxybenzene). The content of 4-bromo-2-nitro-1-phenoxybenzene can be detected by detecting its derivative. This invention overcomes the technical problem that 4-bromo-2-nitro-1-phenoxybenzene has a low response and cannot be quantitatively detected when using liquid chromatography-mass spectrometry to detect the prototype. It has excellent unexpected technical effects.

[0091] (2) The method provided by the present invention is simple to operate, highly sensitive, highly accurate and linear, and has excellent unexpected technical effects.

[0092] (3) Compared with other chromatographic columns, using the XSelect-HSS T3 column for separation is more conducive to improving the accuracy of the results and has excellent unexpected technical effects.

[0093] Terminology Explanation

[0094] The term “room temperature” refers to ambient temperature, which is between approximately 10°C and approximately 35°C, or approximately 20°C and approximately 30°C, or approximately 25°C.

[0095] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0096] The term "post-run" refers to the elution process that continues at the settings at the start of the elution program after each HPLC analysis has finished and before the next analysis begins.

[0097] The term “mM” means millimoles per liter; “min” means minutes; “h” means hours; and “%vol” means volume percentage.

[0098] In the following content, all figures disclosed herein, whether or not they use words such as "approximately" or "about," are approximate values. The value of each figure may vary by 1%, 2%, 5%, 7%, 8%, 10%, 15%, or 20%. Whenever a figure with a value of N is disclosed, any figure with a value of N+ / -1%, N+ / -2%, N+ / -3%, N+ / -5%, N+ / -7%, N+ / -8%, N+ / -10%, N+ / -15%, or N+ / -20% will be explicitly disclosed, where "+ / -" indicates addition or subtraction.

[0099] cis-3-[8-amino-1-(3-amino-4-phenoxy-phenyl)-imidazo[1,5-a]pyrazin-3-yl]-1-methyl-cyclobutanol: Attached Figure Description

[0100] Figure 1 This is the mass spectrum of the reference solution in Example 1.

[0101] Figure 2 This is the mass spectrum of the spiked test solution in Example 1. Detailed Implementation

[0102] Those skilled in the art can refer to the content of this document to appropriately improve the chromatographic conditions and parameters. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The method of this invention has been described through preferred embodiments, and those skilled in the art will clearly be able to modify or appropriately change and combine the methods described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0103] To further understand the present invention, the present invention will be described in detail below with reference to embodiments and comparative examples.

[0104] Unless otherwise stated, the test samples described in the following examples and comparative examples are cis-3-[8-amino-1-(3-amino-4-phenoxy-phenyl)-imidazo[1,5-a]pyrazin-3-yl]-1-methyl-cyclobutanol prepared according to the method of US Patent Application US20090286768A.

[0105] ACE Excel 3C18-AR column, 4.6mm*100mm, 3.0μm. Manufacturer: ACE (UK) Chromatography Technology Ltd.

[0106] XSelect-HSS T3 column, 4.6mm*100mm, 2.5μm. Manufacturer: Waters.

[0107] Comparative Example 1: An ACE Excel 3C18-AR column was used as the chromatographic column.

[0108] 1. Chromatographic and mass spectrometry conditions:

[0109] Chromatographic column: ACE Excel 3C18-AR column, 4.6mm*100mm, 3.0μm;

[0110] Detector: MS (single quadrupole);

[0111] Ion source: ESI;

[0112] Scan mode: SIM, m / z = 264(+);

[0113] Flow rate: 0.4 ml / min;

[0114] Column temperature: 25℃;

[0115] Injection volume: 5.0 μl;

[0116] Mobile phase B: Acetonitrile;

[0117] Mobile phase A: 0.1% vol formic acid aqueous solution;

[0118] Elution procedure: see Table 1.

[0119] Table 1: Elution Procedure

[0120] Time (min) Mobile phase A Mobile phase B 0 80 20 10 10 90 18 10 90

[0121] Run for 5 minutes afterward;

[0122] Mass spectrometry parameters: see Table 2.

[0123] Table 2: Mass Spectrometry Parameters

[0124]

[0125]

[0126] 2. Solution preparation

[0127] Reference stock solution 1: Weigh approximately 23 mg of 4-bromo-2-nitro-1-phenoxybenzene into a 25 ml volumetric flask, dilute to volume with methanol, and shake well.

[0128] Reference stock solution 2: Transfer 1.0 ml of reference stock solution 1 to a 25 ml volumetric flask, dilute to volume with methanol, and shake well to obtain the solution;

[0129] Reference stock solution 3: Transfer 1.0 ml of reference stock solution 2 to a 50 ml volumetric flask, dilute to volume with methanol, and shake well to obtain the solution;

[0130] Blank solution: Transfer 1.0 ml of methanol to a 5 ml EP tube, add about 100 mg of zinc powder, shake well, then add 20 μL of acetic acid, vortex for 3 min, let stand for 1 h, and then filter to obtain the blank solution.

[0131] Reference solution: Transfer 1.0 ml of reference stock solution 3 into a 5 ml EP tube, add about 100 mg of zinc powder, shake well, then add 20 μL of acetic acid, vortex for 3 min, let stand for 1 h, and then filter to obtain the solution.

[0132] Test solution: Weigh about 25 mg of the test sample into a 5 ml EP tube, add 1 ml of methanol to dissolve it, then add about 100 mg of zinc powder, shake well, then add 20 μl of acetic acid, vortex for 3 min, let stand for 1 h and filter to obtain the solution.

[0133] Spiked solution for test sample: Weigh about 25 mg of the test sample into a 5 ml EP tube, add 1 ml of reference stock solution 3 to dissolve it, then add about 100 mg of zinc powder, shake well, then add 20 μl of acetic acid, vortex for 3 min, let stand for 1 h and then filter to obtain the solution.

[0134] 3. Operation

[0135] Take blank solution, reference solution, test solution, and spiked test solution and perform LC-MS / MS analysis according to the chromatographic and mass spectrometric conditions of this comparative example (three parallel injections of reference solution), and record the chromatograms.

[0136] Calculate the recovery (R) of the compound obtained after derivatization of 4-bromo-2-nitro-1-phenoxybenzene in the spiked solution of the test sample. The formula for calculating the R value is as follows:

[0137]

[0138] In the formula: A T+S The peak area is the peak area of ​​the compound obtained after derivatization of 4-bromo-2-nitro-1-phenoxybenzene in the spiked solution of the test sample;

[0139] A T The peak area of ​​the compound obtained after derivatization of 4-bromo-2-nitro-1-phenoxybenzene in the test solution;

[0140] AS The average peak area of ​​the compound obtained by derivatization of 4-bromo-2-nitro-1-phenoxybenzene in reference solution is given in 3.

[0141] W T The sample weight in the test solution is expressed in mg.

[0142] W T+S The amount of the test sample in the spiked solution is expressed in mg.

[0143] Note: Since the effect of the test sample dissolving in methanol on the solution volume is minimal, the effect of the test sample on the change in solution volume is ignored when calculating the spiked recovery rate for ease of calculation.

[0144] 4. Results

[0145] The spiked recovery rate was 71.4%, which is a poor result (the spiked recovery rate should be between 70% and 130%).

[0146] Comparative Example 2: Investigation of flow rate and elution program under conditions using an ACE Excel 3C18-AR column as the chromatographic column.

[0147] 1. Chromatographic and mass spectrometry conditions:

[0148] Chromatographic column, detector, ion source, scan mode, column temperature, sample pan temperature, mobile phase B, mobile phase A: same as comparative example 1;

[0149] Flow rate: 1.0 ml / min;

[0150] Injection volume: 1.0 μl;

[0151] Washing procedure: see Table 3.

[0152] Table 3: Elution Procedure

[0153] Time (min) Mobile phase A Mobile phase B 0 95 5 15 10 90 20 10 90

[0154] Subsequent run: Same as comparative example 1;

[0155] Mass spectrometry parameters: Same as comparative example 1.

[0156] 2. Solution preparation

[0157] Reference stock solution 1, reference stock solution 2, reference stock solution 3, blank solution, reference solution, test solution, and test spiked solution: preparation method is the same as comparative example 1.

[0158] 3. Operation

[0159] Take blank solution, reference solution, test solution, and spiked test solution and perform LC-MS / MS analysis according to the chromatographic and mass spectrometric conditions of this comparative example (three parallel injections of reference solution), and record the chromatograms.

[0160] Calculate the recovery (R) of the compound obtained after derivatization of 4-bromo-2-nitro-1-phenoxybenzene in the spiked solution of the test sample. The formula for calculating the R value is the same as that for Comparative Example 1.

[0161] 4. Results

[0162] The spiked recovery rate was 76.5%, which is a poor result (the spiked recovery rate should be between 70% and 130%).

[0163] Comparative Example 3: Investigation of elution conditions using an ACE Excel 3C18-AR column as the chromatographic column

[0164] 1. Chromatographic and mass spectrometry conditions:

[0165] Column, detector, ion source, scan mode, flow rate, column temperature, sample pan temperature, mobile phase B, mobile phase A: same as comparative example 2, injection volume: 1.0 μl;

[0166] Washing procedure: see Table 4.

[0167] Table 4: Elution Procedure

[0168] Time (min) Mobile phase A Mobile phase B 0 95 5 15 15 85 20 15 85

[0169] Subsequent run: Same as comparative example 2;

[0170] Mass spectrometry parameters: Same as comparative example 2.

[0171] 2. Solution preparation

[0172] Reference stock solution 1, reference stock solution 2, reference stock solution 3, blank solution, reference solution, test solution, and test spiked solution: preparation method is the same as comparative example 1.

[0173] 3. Operation

[0174] Take blank solution, reference solution, test solution, and spiked test solution and perform LC-MS / MS analysis according to the chromatographic and mass spectrometric conditions of this comparative example (three parallel injections of reference solution), and record the chromatograms.

[0175] Calculate the recovery rate (R) of the compound obtained by derivatization of 4-bromo-2-nitro-1-phenoxybenzene in the spiked solution of the test sample. The formula for calculating the R value is the same as that for Comparative Example 1.

[0176] 4. Results

[0177] The measured spiked recovery rate was 68.1%, which is poor and does not meet the requirements (the spiked recovery rate should be between 70% and 130%).

[0178] Example 1: Using an XSelect-HSS T3 column as the separation chromatographic column

[0179] 1. Chromatographic and mass spectrometry conditions:

[0180] The difference between the chromatographic and mass spectrometric conditions and those of Comparative Example 3 is that Example 1 used an XSelect-HSS T3 column, 4.6mm*100mm, 2.5μm.

[0181] 2. Solution preparation

[0182] Reference stock solution 1, reference stock solution 2, reference stock solution 3, blank solution, reference solution, test solution, and test spiked solution: preparation method is the same as comparative example 1.

[0183] 3. Operation

[0184] Take blank solution, reference solution, test solution, and spiked test solution and perform LC-MS / MS analysis according to the chromatographic and mass spectrometric conditions of this embodiment (parallel injection of reference solution three times), and record the chromatograms.

[0185] Calculate the recovery (R) of the compound obtained after derivatization of 4-bromo-2-nitro-1-phenoxybenzene in the spiked solution of the test sample. The formula for calculating the R value is the same as that for Comparative Example 1.

[0186] 4. Results

[0187] The spiked recovery rate was measured to be 96.0%, which meets the requirements (the spiked recovery rate should be between 70% and 130%).

[0188] Example 2: Investigation of the mobile phase using an XSelect-HSS T3 column as the separation column

[0189] 1. Chromatographic and mass spectrometry conditions:

[0190] The difference between the chromatographic and mass spectrometric conditions and those in Example 1 is that the mobile phase A in Example 2 is a 10 mM ammonium formate aqueous solution.

[0191] 2. Solution preparation

[0192] Reference stock solution 1, reference stock solution 2, reference stock solution 3, blank solution, reference solution, test solution, and test spiked solution: preparation method is the same as comparative example 1.

[0193] 3. Operation

[0194] Take blank solution, reference solution, test solution, and spiked test solution and perform LC-MS / MS analysis according to the chromatographic and mass spectrometric conditions of this embodiment (parallel injection of reference solution three times), and record the chromatograms.

[0195] Calculate the recovery (R) of the compound obtained after derivatization of 4-bromo-2-nitro-1-phenoxybenzene in the spiked solution of the test sample. The formula for calculating the R value is the same as that for Comparative Example 1.

[0196] 4. Results

[0197] The spiked recovery rate was measured to be 107.4%, which is acceptable and meets the requirements (the spiked recovery rate should be between 70% and 130%).

[0198] Example 3: Derivatization time study (2 hours)

[0199] 1. Chromatographic and mass spectrometry conditions:

[0200] The chromatographic and mass spectrometric conditions were the same as in Example 1.

[0201] 2. Solution preparation

[0202] The difference from Comparative Example 1 is that the derivatization time for the blank solution, reference solution, test solution, and spiked test solution is adjusted to 2 hours.

[0203] 3. Operation

[0204] Take blank solution, reference solution, test solution, and spiked test solution and perform LC-MS / MS analysis according to the chromatographic and mass spectrometric conditions of this embodiment (parallel injection of reference solution three times), and record the chromatograms.

[0205] Calculate the recovery (R) of the compound obtained after derivatization of 4-bromo-2-nitro-1-phenoxybenzene in the spiked solution of the test sample. The formula for calculating the R value is the same as that for Comparative Example 1.

[0206] 4. Results

[0207] The spiked recovery rate was measured to be 98.4%, which is qualified and meets the requirements (the spiked recovery rate should be between 70% and 130%).

[0208] Comparative Example 4: No derivatization

[0209] 1. Chromatographic and mass spectrometry conditions:

[0210] Except for the SIM ion collection setting of m / z 294, the rest is the same as in Example 1.

[0211] 2. Solution preparation

[0212] The difference from Example 1 is that zinc powder was not added to the blank solution, control solution, test solution and spiked test solution of Comparative Example 4.

[0213] 3. Operation

[0214] Take blank solution, reference solution, test solution, and spiked test solution and perform LC-MS / MS analysis according to the chromatographic and mass spectrometric conditions of this embodiment (parallel injection of reference solution three times), and record the chromatograms.

[0215] Calculate the recovery (R) of the compound obtained after derivatization of 4-bromo-2-nitro-1-phenoxybenzene in the spiked solution of the test sample. The formula for calculating the R value is the same as that for Comparative Example 1.

[0216] 4. Results

[0217] No response peaks were observed in the reference solution, test solution, and spiked test solution during the sampling period.

[0218] Example 4: Investigation of linearity, limit of detection, and limit of quantitation

[0219] 1. Chromatographic and mass spectrometry conditions:

[0220] The chromatographic and mass spectrometric conditions were the same as in Example 1.

[0221] 2. Solution preparation:

[0222] Blank solution: Transfer 1.0 ml of methanol to a 5 ml EP tube, add about 100 mg of zinc powder, shake well, then add 20 μl of acetic acid, vortex for 3 min, let stand for 1 h, and then filter to obtain the blank solution.

[0223] Reference stock solution 1: Weigh approximately 37.5 mg of 4-bromo-2-nitro-1-phenoxybenzene into a 50 ml volumetric flask, dilute to volume with methanol, and shake well.

[0224] Reference stock solution 2: Transfer 1.0 ml of reference stock solution 1 to a 50 ml volumetric flask, dilute to volume with methanol, and shake well to obtain the solution;

[0225] Reference stock solution 3: Transfer 1.0 ml of reference stock solution 2 to a 50 ml volumetric flask, dilute to volume with methanol, and shake well to obtain the solution;

[0226] Reference solution: Transfer 1.0 ml of reference stock solution 3 into a 5 ml EP tube, add about 100 mg of zinc powder, shake well, then add 20 μl of acetic acid, vortex for 3 min, let stand for 1 h, and then filter to obtain the solution.

[0227] 3% Reference Solution (Detection Limit Solution): Transfer 3.0 ml of reference stock solution 3 to a 100 ml volumetric flask, dilute to volume with methanol, shake well, transfer 1 ml to a 5 ml EP tube, add about 100 mg of zinc powder, shake well, then add 20 μl of acetic acid, vortex for 3 min, let stand for 1 h, and then filter to obtain the solution.

[0228] 10% reference solution (limit of quantitation solution): Transfer 1.0 ml of reference stock solution 3 to a 10 ml volumetric flask, dilute to volume with methanol, shake well, transfer 1 ml to a 5 ml EP tube, add about 100 mg of zinc powder, shake well, then add 20 μl of acetic acid, vortex for 3 min, let stand for 1 h, and then filter to obtain the solution.

[0229] 30% reference solution: Transfer 3.0 ml of reference stock solution 3 to a 10 ml volumetric flask, dilute to volume with methanol, shake well, transfer 1 ml to a 5 ml EP tube, add about 100 mg of zinc powder, shake well, then add 20 μl of acetic acid, vortex for 3 min, let stand for 1 h, and then filter to obtain the solution.

[0230] 70% reference solution: Transfer 7.0 ml of reference stock solution 3 to a 10 ml volumetric flask, dilute to volume with methanol, shake well, transfer 1 ml to a 5 ml EP tube, add about 100 mg of zinc powder, shake well, then add 20 μl of acetic acid, vortex for 3 min, let stand for 1 h, and then filter to obtain the solution.

[0231] 150% reference solution: Transfer 1.5 ml of reference stock solution 2 to a 50 ml volumetric flask, dilute to volume with methanol, shake well, transfer 1 ml to a 5 ml EP tube, add about 100 mg of zinc powder, shake well, then add 20 μl of acetic acid, vortex for 3 min, let stand for 1 h, and then filter to obtain the solution.

[0232] 3. Operation

[0233] Injection: 3 injections of blank solution, 3 injections of 3% reference solution (limit of detection solution), 3 injections of 10% reference solution (limit of quantitation solution), 3 injections of blank solution, and 1 injection each of 10% reference solution, 30% reference solution, 70% reference solution, 100% reference solution, and 150% reference solution.

[0234] 4. Results:

[0235] The linearity results are shown in Table 5, the quantitation limit results are shown in Table 6, and the detection limit results are shown in Table 7.

[0236] Table 5: Linearity Results

[0237]

[0238] Table 6: Results of Limit of Quantitation

[0239]

[0240] Table 7: Detection Limit Results

[0241]

[0242]

[0243] 5. Conclusion

[0244] The method provided by this invention has good linearity, high accuracy, and high sensitivity, and has excellent technical effects.

[0245] Example 4: Sample Detection

[0246] 1. Chromatographic and mass spectrometric conditions

[0247] Same as Example 1.

[0248] 2. Solution preparation

[0249] Reference stock solution 1, reference stock solution 2, reference stock solution 3, blank solution, reference solution, and test solution: prepared in the same manner as comparative example 1.

[0250] Take blank solution, reference solution (6 consecutive injections), and test solution and perform LC-MS / MS analysis according to the chromatographic and mass spectrometric conditions of this embodiment, and record the chromatograms.

[0251] 3. Operation

[0252] The content of 4-bromo-2-nitro-1-phenoxybenzene in the test sample solution was calculated using the external standard method.

[0253] The external standard method calculation formula is:

[0254]

[0255] In the formula: A T The peak area of ​​derivative impurities in the test sample solution;

[0256] A S This is the average peak area of ​​the derivative impurity in the reference solution after six consecutive injections.

[0257] W T The sample weight is in mg.

[0258] W S The mass (mg) of 4-bromo-2-nitro-1-phenoxybenzene reference standard weighed for preparing the reference solution;

[0259] DT The dilution factor of the test solution;

[0260] D S This is the dilution factor of the reference solution;

[0261] P represents the content of 4-bromo-2-nitro-1-phenoxybenzene reference standard.

[0262] 4. Results

[0263] The content of 4-bromo-2-nitro-1-phenoxybenzene in the test sample was measured to be 46 ppm.

[0264] In summary, based on the above examples and comparative results, we can conclude that:

[0265] (1) The present invention uses a derivatization reaction of 4-bromo-2-nitro-1-phenoxybenzene with a proton donor and zinc powder, and then detects the content of 4-bromo-2-nitro-1-phenoxybenzene by detecting its derivatives. This overcomes the technical problem that 4-bromo-2-nitro-1-phenoxybenzene has a low response and cannot be quantitatively detected, and has excellent unexpected technical effects.

[0266] (2) The method provided by the present invention is simple to operate, highly sensitive, highly accurate and linear, and has excellent unexpected technical effects.

[0267] (3) Compared with other chromatographic columns, using the XSelect-HSS T3 column for separation is more conducive to improving the accuracy of the results and has excellent unexpected technical effects.

[0268] The method of this invention has been described through preferred embodiments. Those skilled in the art will readily be able to modify or appropriately alter and combine the methods and applications described herein within the scope, spirit, and context of this invention to implement and apply the technology of this invention. Those skilled in the art can refer to the content herein to appropriately improve the parameters. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of this invention.

Claims

1. A method for detecting the content of 4-bromo-2-nitro-1-phenoxybenzene in a sample, characterized in that, The solution of the sample to be tested is mixed with a proton donor and zinc powder, reacted, filtered or centrifuged to obtain a test solution, and the content of 4-bromo-2-amino-1-phenoxybenzene in the test solution is detected, thereby calculating the content of 4-bromo-2-nitro-1-phenoxybenzene in the sample to be tested.

2. The detection method according to claim 1, wherein the proton donor in the preparation of the test solution is acetic acid or concentrated hydrochloric acid; and / or The solvent of the solution of the sample to be tested is at least one of methanol, ethanol, and isopropyl acetate; and / or The mass ratio of the test sample to zinc powder in the solution is 1.0:3.0-1.0:10.0, or 1.0:5.0; and / or The ratio of the mass of the test sample in the solution to the volume of the proton donor is 10 mg:20 μl to 50 mg:20 μl, or 20 mg:20 μl; and / or The sample to be tested was cis-3-[8-amino-1-(3-amino-4-phenoxy-phenyl)-imidazo[1,5-a]pyrazin-3-yl]-1-methyl-cyclobutanol.

3. The detection method according to claim 1, wherein the reaction time is 1h-5h or 1h-2h.

4. The detection method according to claim 1, wherein the detection method for the content of 4-bromo-2-amino-1-phenoxybenzene in the test solution is to use high performance liquid chromatography-mass spectrometry (HPLC-MS) for detection.

5. The detection method according to claim 4, wherein the chromatographic column of the high-performance liquid chromatography is an XSelect-HSS T3 column; and / or The high-performance liquid chromatography method uses a gradient elution of mobile phase A (0.1% vol formic acid aqueous solution or 10 mM ammonium formate aqueous solution) and mobile phase B (acetonitrile); and / or The gradient elution procedure is as follows: 。 6. The detection method according to claim 5, wherein the gradient elution procedure is as follows: ； or The gradient elution procedure is as follows: or The gradient elution procedure is as follows: 。 7. The detection method according to claim 5, wherein the high-performance liquid chromatography further comprises a 5-15 minute post-run after each gradient elution; and / or The inner diameter of the chromatographic column is 2mm-5mm, or 4.6mm; and / or The column length is 100mm-250mm, or 100mm; and / or The chromatographic column has a packing particle size of 2.5 μm-5 μm, or 2.5 μm-3.0 μm; and / or The chromatographic column used in the high-performance liquid chromatography is an XSelect-HSS T3 column, which has an inner diameter of 4.6 mm, a column length of 100 mm, and a packing particle size of 2.5 μm.

8. The detection method according to claim 4, wherein the column temperature of the high-performance liquid chromatography is 25℃-35℃; and / or The elution flow rate of the high-performance liquid chromatography method is 0.8 ml / min-1.2 ml / min; and / or The mass spectrometry method employs an electrospray ionization (ESI) source; and / or The mass spectrometry scanning mode is selected ion monitoring (SIM); and / or The scan settings for selected ion monitoring are: positive ion mode, m / z of 264; and / or The drying gas for the mass spectrometry method is nitrogen; and / or The drying gas temperature for the mass spectrometry method is 300℃-350℃ or 300℃; and / or The drying gas flow rate for the mass spectrometry method is 10.0 L / min-13.0 L / min, or 11.0 L / min; and / or The nebulizer gas pressure for the mass spectrometry method is 35 psi-55 psi, or 35 psi; and / or The capillary voltage for the mass spectrometry method is a positive voltage of 3000V-5000V, or a positive voltage of 4000V. The mass spectrometry method employs a single quadrupole or triple quadrupole detector.

9. The detection method according to claim 1, further comprising the preparation of a reference solution, wherein the preparation of the reference solution comprises: A solution containing 4-bromo-2-nitro-1-phenoxybenzene is mixed with a proton donor and zinc powder, reacted, and then filtered or centrifuged to obtain a reference solution. The proton donor in the preparation of the reference solution is the same as that in the preparation of the test solution; the solvent in the solution containing 4-bromo-2-nitro-1-phenoxybenzene is the same as that in the solution containing the test sample; and the reaction time in the preparation of the reference solution is the same as that in the preparation of the test solution. And / or In the preparation of the reference solution, the mass ratio of 4-bromo-2-nitro-1-phenoxybenzene to zinc powder in the solution containing 4-bromo-2-nitro-1-phenoxybenzene is 1:100000-30:100000; and / or In the preparation of the reference solution, the ratio of the mass of 4-bromo-2-nitro-1-phenoxybenzene in the solution containing 4-bromo-2-nitro-1-phenoxybenzene to the volume of the proton donor is 0.03 μg:20 μl to 0.5 μg:20 μl; and / or The concentration of 4-bromo-2-nitro-1-phenoxybenzene in the solution containing 4-bromo-2-nitro-1-phenoxybenzene is 0.03 μg / ml to 0.5 μg / ml.

10. The use of the detection method according to any one of claims 1-9, characterized in that, Includes for: 1) Detect the content of 4-bromo-2-nitro-1-phenoxybenzene in cis-3-[8-amino-1-(3-amino-4-phenoxy-phenyl)-imidazo[1,5-a]pyrazin-3-yl]-1-methyl-cyclobutanol; or, 2) Detect the conversion rate of 4-bromo-2-nitro-1-phenoxybenzene in the reaction of synthesizing cis-3-[8-amino-1-(3-amino-4-phenoxy-phenyl)-imidazo[1,5-a]pyrazin-3-yl]-1-methyl-cyclobutanol or its intermediates using 4-bromo-2-nitro-1-phenoxybenzene as a raw material or intermediate.

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