Method for detecting NMV-B00 enantiomer of initial material of nemategravir

Abstract

The invention provides a method for detecting an NMV-B00 enantiomer of a nemategravir starting material, and belongs to the field of pharmaceutical analysis. According to the detection method, high performance liquid chromatography is adopted for detection, and an amylose-tri-(4-chlorine-3-methyl phenyl carbamate) chromatographic column, an amylose-tri-((s)-alpha-methyl benzyl carbamate) chromatographic column, a cellulose-tri-(4-methyl benzoate) chromatographic column or a cellulose-tri-(3, 4-dichlorophenyl)-3-(4-chloro-3-methyl phenyl carbamate) chromatographic column is adopted as a chromatographic column. The method comprises the following steps: taking a covalent bond bonded chromatographic column of 2, 5-dichlorophenyl carbamate as a separation chromatographic column, and taking a mixed solution of n-hexane and an alcoholic solution of acid as a mobile phase for elution. The detection method is good in separation degree, high in sensitivity, high in accuracy, good in reproducibility and high in durability.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical analysis, and particularly relates to a method for detecting the enantiomers of NMV-B00, the starting material of nirmatrelvir. Background Art

[0002] Nirmatrelvir has the following structural formula:

[0003]

[0004] NMV-B00 is the starting material for the synthesis of nirmatrelvir. NMV-B00 contains a chiral center and is an important constituent fragment of the structure of the anti-COVID-19 drug nirmatrelvir. The control of the enantiomers of this material is one of the key points for controlling the chiral impurities of nirmatrelvir API.

[0005]

[0006] Currently, there are no reported literatures and patents on the method for determining the content of the enantiomers of NMV-B00, the starting material of nirmatrelvir.

[0007] Therefore, there is an urgent need for a method for determining the content of the enantiomers of NMV-B00 with good resolution, high sensitivity, high accuracy, good reproducibility, and high durability. Summary of the Invention

[0008] To solve the above technical problems, the present invention provides the following technical solutions.

[0009] A method for detecting the enantiomers of NMV-B00 or the enantiomers of the salt of NMV-B00 in NMV-B00 or its salt, characterized in that high performance liquid chromatography is used for detection, and a amylose tris(4-chloro-3-methylphenylcarbamate) chromatographic column, an amylose tris((S)-α-methylbenzylcarbamate) chromatographic column, a cellulose tris(4-methylbenzoate) chromatographic column, or a cellulose tris(3,5-dichlorophenylcarbamate) covalently bonded chromatographic column is used as the separation chromatographic column, and a mixture of n-hexane and an alcoholic solution of an acid is used as the mobile phase for elution;

[0010]

[0011] In some embodiments, the amylose tris(4-chloro-3-methylphenylcarbamate) chromatographic column is the Phenomenex Chiral NX(2) chromatographic column.

[0012] In some embodiments, the amylose tris((S)-α-methylbenzylcarbamate) chromatographic column is the Phenomenex Chiral NS(2) chromatographic column.

[0013] In some embodiments, the cellulose tris(4-methylbenzoate) chromatographic column is a Phenomenex Chiral MJ(2) chromatographic column.

[0014] In some embodiments, the cellulose tris(3,5-dichlorophenylcarbamate) covalently bonded chromatographic column is a Phenomenex Chiral INC chromatographic column.

[0015] In some embodiments, the inner diameter of the separation chromatographic column is 2.0 mm - 10.0 mm.

[0016] In some embodiments, the length of the separation chromatographic column is 100 mm - 250 mm.

[0017] In some embodiments, the particle size of the packing material of the separation chromatographic column is 3 μm - 5 μm.

[0018] In some embodiments, the acid content in the acid-containing alcohol solution is 0.05% v - 0.15% vol or 0.10% vol.

[0019] In some embodiments, the acid in the acid-containing alcohol solution includes trifluoroacetic acid.

[0020] In some embodiments, the alcohol in the acid-containing alcohol solution includes at least one of isopropanol and ethanol.

[0021] In some embodiments, the volume ratio of n-hexane to the acid-containing alcohol solution mixture in the mobile phase is 95:5 - 80:20, preferably 90:10.

[0022] In some embodiments, the detection wavelength of the high performance liquid chromatography is 210 nm ± 5 nm.

[0023] In some embodiments, the column temperature of the high performance liquid chromatography is 15°C - 40°C or 25°C.

[0024] In some embodiments, the flow rate of the high performance liquid chromatography is 0.3 ml / min - 1.0 ml / min or 0.5 ml / min.

[0025] In some embodiments, the acid-containing alcohol solution is an isopropanol solution containing 0.1% vol trifluoroacetic acid or an ethanol solution containing 0.1% vol trifluoroacetic acid.

[0026] In some embodiments, the model of the separation chromatographic column is 4.6 mm * 250 mm, 5 μm.

[0027] In some embodiments, the detection method is performed by high performance liquid chromatography (HPLC). An amylose tris(4-chloro-3-methylphenylcarbamate) chromatographic column (e.g., Phenomenex Chiral NX(2) chromatographic column), an amylose tris((S)-α-methylbenzylcarbamate) chromatographic column (e.g., Phenomenex Chiral NS(2) chromatographic column), a cellulose tris(4-methylbenzoate) chromatographic column (e.g., Phenomenex Chiral MJ(2) chromatographic column), or a cellulose tris(3,5-dichlorophenylcarbamate) covalently bonded chromatographic column (e.g., Phenomenex Chiral INC chromatographic column) is used as the separation chromatographic column. A mixture of n-hexane and an isopropanol solution containing 0.1% vol trifluoroacetic acid or a mixture of n-hexane and an ethanol solution containing 0.1% vol trifluoroacetic acid is used as the mobile phase for elution. The volume ratio of n-hexane to the acid-containing alcohol solution in the mobile phase is 90:10. The column temperature of the HPLC is 20°C - 30°C or 25°C; the flow rate of the HPLC is 0.4 ml / min - 0.6 ml / min or 0.5 ml / min.

[0028] In some embodiments, the detection method further includes the preparation of a test solution. The preparation method of the test solution is to dilute the sample to be tested with a diluent to a concentration of NMV-C00 of 5 mg / mL - 30 mg / mL or 15 mg / mL.

[0029] In some embodiments, the diluent is the mobile phase, ethanol, or isopropanol.

[0030] Advantageous Effects

[0031] Compared with the prior art, the present invention has the following advantageous effects:

[0032] The method provided by the present invention has good resolution, high sensitivity, high accuracy, good reproducibility, and high durability.

[0033] Term Explanation

[0034] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0035] The term "%vol" represents volume percentage.

[0036] The term "wt%" represents mass percentage.

[0037] The term "M" represents molar concentration mol / L.

[0038] In the following content, whether or not words such as "about" or "approximately" are used, it means within 1%, 2%, 5%, 7%, 8%, 10%, 15% or 20% of a given value or range, etc. Or, for those of ordinary skill in the art, the term "about" or "approximately" means within the acceptable standard error range of the average value. Whenever a number with an N value is disclosed, any number with an N+ / -1%, N+ / -2%, N+ / -3%, N+ / -5%, N+ / -7%, N+ / -8%, N+ / -10%, N+ / -15% or N+ / -20% value will be explicitly disclosed, where "+ / -" means plus or minus. Description of the Drawings

[0039] Figure 1 It is the chromatogram of the blank solution for Example 1.

[0040] Figure 2 It is the chromatogram of the reference solution for Example 1.

[0041] Figure 3 It is the chromatogram of the test solution for Example 1.

[0042] Figure 4 It is the chromatogram of the spiked test solution for Example 1.

[0043] Figure 5 It is the chromatogram of the blank solution for Example 2.

[0044] Figure 6 It is the chromatogram of the test solution for Example 2.

[0045] Figure 7 It is the chromatogram of the spiked test solution for Example 2.

[0046] Figure 8 It is the chromatogram of the blank solution for Example 3.

[0047] Figure 9 It is the chromatogram of the test solution for Example 3.

[0048] Figure 10 It is the chromatogram of the spiked test solution for Example 3.

[0049] Figure 11 It is the chromatogram of the blank solution for Example 4.

[0050] Figure 12 Chromatogram of the spiked solution of the test sample in Example 4.

[0051] Figure 13 Chromatogram of the blank solution in Example 5.

[0052] Figure 14 Chromatogram of the spiked solution of the test sample in Example 5.

[0053] Figure 15 Chromatogram of the blank solution in Example 6.

[0054] Figure 16 Chromatogram of the spiked solution of the test sample in Example 6.

[0055] Figure 17 Chromatogram of the blank solution in Example 7.

[0056] Figure 18 Chromatogram of the spiked solution of the test sample in Example 7.

[0057] Figure 19 Chromatogram of the spiked solution of the test sample with different chromatographic columns in Example 8. Detailed implementation method

[0058] In order to enable those skilled in the art to better understand the technical solution of the present invention, some non-limiting embodiments are further disclosed below to further elaborate on the present invention.

[0059] All the reagents used in the present invention can be purchased from the market or can be prepared by the methods described in the present invention.

[0060] Example 1

[0061] 1. Chromatographic conditions:

[0062] Chromatographic column: Phenomenex Chiral NX(2), 4.6*250mm, 5μm

[0063] Detector: DAD detector, detection wavelength 210nm;

[0064] Flow rate: 0.5 mL / min;

[0065] Column temperature: 25°C;

[0066] Injection volume: 12 μL;

[0067] Running time: 30 min;

[0068] Mobile phase:

[0069] Phase A: n-Hexane;

[0070] Phase B: Isopropanol solution containing 0.1% trifluoroacetic acid (Add 1 mL of trifluoroacetic acid to 1000 mL of isopropanol and shake well).

[0071] Elution: Phase A: Phase B = 90:10 (V / V); Isocratic elution.

[0072] 2. Experimental procedures

[0073] Diluent / Blank solvent: Absolute ethanol

[0074] Test solution: Take about 150 mg of the test substance, accurately weigh it into a 10 mL volumetric flask, dissolve it with the diluent and dilute to the mark, then shake well.

[0075] Reference stock solution: Take 15 mg of the NMV-B00 enantiomer, accurately weigh it into a 100 mL volumetric flask, dissolve it with the diluent and dilute to the mark, then shake well.

[0076] Reference solution: Take 1.0 mL of the reference stock solution into a 10 mL volumetric flask, dissolve it with the diluent and dilute to the mark, then shake well.

[0077] Spiked test solution: Take about 150 mg of the test substance, accurately weigh it into a 10 mL volumetric flask, add 1.0 mL of the reference stock solution, then dissolve it with the diluent and dilute to the mark, then shake well.

[0078] Take the blank solution, reference solution, test solution and spiked test solution respectively, perform high performance liquid chromatography analysis under the above conditions, record the chromatogram, and the results are shown in Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 。

[0079] Result analysis: Figure 2 The chromatographic peak with a retention time of 12.997 minutes in Figure 3 is the chromatographic peak of the NMV-B00 enantiomer; Figure 4 The chromatographic peak with a retention time of 17.097 minutes in

[0080] is the chromatographic peak of NMV-B00. The chromatographic peak at 12.953 minutes in

[0081] is the chromatographic peak of the NMV-B00 enantiomer, and the chromatographic peak at 17.119 minutes is the chromatographic peak of NMV-B00. The resolution between the chromatographic peak of the NMV-B00 enantiomer and the chromatographic peak of NMV-B00 is 4.0. This method can be used for the detection of the NMV-B00 enantiomer in the starting material of nirmatrelvir.

[0082] Chromatographic column: Phenomenex Chiral NX(2), 4.6*250 mm, 5 μm

[0083] Detector: VWD detector, detection wavelength 210 nm;

[0084] Flow rate: 0.5 mL / min;

[0085] Column temperature: 35 °C;

[0086] Sample injection volume: 5 μL;

[0087] Running time: 40 min;

[0088] Mobile phase: Phase A: n-hexane;

[0089] Phase B: Ethanol solution containing 0.1% trifluoroacetic acid (Take 1 mL of trifluoroacetic acid and add it to 1000 mL of ethanol, shake well);

[0090] Elution: Phase A: Phase B = 95:5 (V / V); Isocratic elution.

[0091] 2. Experimental procedures

[0092] Diluent / Blank solvent: Absolute ethanol;

[0093] Test solution: Take about 300 mg of the test substance, accurately weigh it into a 10 mL volumetric flask, dissolve it with the diluent and dilute to the mark, shake well.

[0094] Reference stock solution: Take 15 mg of NMV-B00 enantiomer, accurately weigh it into a 100 mL volumetric flask, dissolve it with the diluent and dilute to the mark, shake well;

[0095] Spiked test solution: Take about 300 mg of the test substance, accurately weigh it into a 10 mL volumetric flask, add 1.0 mL of the reference stock solution, then dissolve it with the diluent and dilute to the mark, shake well.

[0096] Respectively take the blank solution, the test solution and the spiked test solution, perform high performance liquid chromatography analysis under the above conditions, record the chromatogram, and the results are shown in Figure 5 、 Figure 6 、 Figure 7 。

[0097] Result analysis: Figure 6 The chromatographic peak with a retention time of 15.819 minutes is the chromatographic peak of the NMV-B00 enantiomer, the chromatographic peak with a retention time of 17.242 minutes is the chromatographic peak of NMV-B00, and the resolution between the chromatographic peak of the NMV-B00 enantiomer and the chromatographic peak of NMV-B00 is 1.7; Figure 7The chromatographic peak with a retention time of 15.693 minutes is the chromatographic peak of the NMV-B00 enantiomer, and the chromatographic peak at 17.183 minutes is the chromatographic peak of NMV-B00. The resolution between the chromatographic peak of the NMV-B00 enantiomer and the chromatographic peak of NMV-B00 is 1.8. This method can be used for the detection of the NMV-B00 enantiomer in the starting material of nirmatrelvir.

[0098] Example 3:

[0099] 1. Chromatographic conditions:

[0100] Chromatographic column: Phenomenex Chiral NX(2), 4.6*250mm, 5μm

[0101] Detector: VWD detector, detection wavelength 210nm;

[0102] Flow rate: 0.6 mL / min;

[0103] Column temperature: 30°C;

[0104] Injection volume: 5 μL;

[0105] Running time: 40 min;

[0106] Mobile phase:

[0107] Phase A: n-hexane;

[0108] Phase B: ethanol solution containing 0.1% trifluoroacetic acid (take 1 mL of trifluoroacetic acid and add it to 1000 mL of ethanol, shake well);

[0109] Elution: Phase A: Phase B = 95:5 (V / V); isocratic elution.

[0110] 2. Experimental procedure

[0111] Diluent / blank solvent: absolute ethanol;

[0112] Test solution: Take about 300 mg of the test substance, accurately weigh it into a 10 mL volumetric flask, dissolve it with the diluent and dilute it to the mark, and shake well.

[0113] Reference stock solution: Take 15 mg of the NMV-B00 enantiomer, accurately weigh it into a 100 mL volumetric flask, dissolve it with the diluent and dilute it to the mark, and shake well;

[0114] Spiked test solution: Take about 300 mg of the test substance, accurately weigh it into a 10 mL volumetric flask, add 1.0 mL of the reference stock solution, then dissolve it with the diluent and dilute it to the mark, and shake well.

[0115] Separate the blank solution, the test solution, and the spiked test solution, and perform high-performance liquid chromatography analysis under the above conditions. Record the chromatogram, and the results are shown in Figure 8 , Figure 9 , Figure 10 .

[0116] Result analysis: Figure 9 The chromatographic peak with a retention time of 13.640 minutes in Figure 10 is the chromatographic peak of the NMV-B00 enantiomer, and the chromatographic peak with a retention time of 15.156 minutes is the chromatographic peak of NMV-B00. The resolution between the chromatographic peak of the NMV-B00 enantiomer and the chromatographic peak of NMV-B00 is 2.0;

[0117] Example 4:

[0118] 1. Chromatographic conditions:

[0119] Chromatographic column: Phenomenex Chiral NX(2), 4.6*250mm, 5μm

[0120] Detector: VWD detector, detection wavelength 210nm;

[0121] Flow rate: 0.5 mL / min;

[0122] Column temperature: 20°C;

[0123] Injection volume: 12 μL;

[0124] Run time: 30 min;

[0125] Mobile phase:

[0126] Phase A: n-hexane;

[0127] Phase B: isopropanol solution containing 0.1% trifluoroacetic acid (take 1 mL of trifluoroacetic acid and add it to 1000 mL of isopropanol, shake well);

[0128] Elution: Phase A: Phase B = 90:10 (V / V); isocratic elution.

[0129] 2. Experimental procedure

[0130] Diluent / blank solvent: absolute ethanol;

[0131] Reference stock solution: Accurately weigh 15 mg of the NMV-B00 enantiomer and transfer it to a 100 mL volumetric flask. Dissolve and dilute to the mark with the diluent, and mix well.

[0132] Test sample spiked solution: Accurately weigh about 150 mg of the test sample and transfer it to a 10 mL volumetric flask. Add 1.0 mL of the reference stock solution, then dissolve and dilute to the mark with the diluent, and mix well.

[0133] Respectively take the blank solution and the test sample spiked solution, perform high performance liquid chromatography analysis under the above conditions, record the chromatogram, and the results are shown in Figure 11 、 Figure 12 。

[0134] Result analysis: Figure 12 The chromatographic peak with a retention time of 14.142 minutes is the chromatographic peak of the NMV-B00 enantiomer, and the chromatographic peak with a retention time of 19.188 minutes is the chromatographic peak of NMV-B00. The resolution between the chromatographic peak of the NMV-B00 enantiomer and the chromatographic peak of NMV-B00 is 4.3. This method can be used for the detection of the NMV-B00 enantiomer in the starting material of nirmatrelvir.

[0135] Example 5:

[0136] 1. Chromatographic conditions:

[0137] Chromatographic column: Phenomenex Chiral NX(2), 4.6*250 mm, 5 μm

[0138] Detector: VWD detector, detection wavelength 210 nm;

[0139] Flow rate: 0.4 mL / min;

[0140] Column temperature: 25 °C;

[0141] Injection volume: 12 μL;

[0142] Running time: 30 min;

[0143] Mobile phase:

[0144] Phase A: n-hexane;

[0145] Phase B: isopropanol solution containing 0.1% trifluoroacetic acid (take 1 mL of trifluoroacetic acid and add it to 1000 mL of isopropanol, and mix well);

[0146] Elution: Phase A: Phase B = 90:10 (V / V); isocratic elution.

[0147] 2. Experimental procedure

[0148] Diluent / blank solvent: anhydrous ethanol;

[0149] Reference stock solution: Accurately weigh 15 mg of NMV-B00 enantiomer and transfer it to a 100 mL volumetric flask. Dissolve and dilute to the mark with diluent, and shake well.

[0150] Test sample spiked solution: Take about 150 mg of the test sample, accurately weigh it and transfer it to a 10 mL volumetric flask. Add 1.0 mL of the reference stock solution, then dissolve and dilute to the mark with diluent, and shake well.

[0151] Respectively take the blank solution and the test sample spiked solution, perform high performance liquid chromatography analysis under the above conditions, record the chromatogram, and the results are shown in Figure 13 、 Figure 14 。

[0152] Result analysis: Figure 14 The chromatographic peak with a retention time of 16.725 minutes in [[ ]] is the chromatographic peak of NMV-B00 enantiomer, and the chromatographic peak with a retention time of 22.281 minutes is the chromatographic peak of NMV-B00. The resolution between the chromatographic peak of NMV-B00 enantiomer and the chromatographic peak of NMV-B00 is 4.0. This method can be used for the detection of NMV-B00 enantiomer in the starting material of nirmatrelvir.

[0153] Example 6:

[0154] 1. Chromatographic conditions:

[0155] Chromatographic column: Phenomenex Chiral NX(2), 4.6*250 mm, 5 μm

[0156] Detector: VWD detector, detection wavelength 210 nm;

[0157] Flow rate: 0.5 mL / min;

[0158] Column temperature: 25 °C;

[0159] Injection volume: 1 μL;

[0160] Running time: 45 min;

[0161] Mobile phase:

[0162] Phase A: n-hexane;

[0163] Phase B: Ethanol solution containing 0.1% trifluoroacetic acid (take 1 mL of trifluoroacetic acid and add it to 1000 mL of ethanol, shake well);

[0164] Elution: Phase A: Phase B = 95:5 (V / V); isocratic elution.

[0165] 2. Experimental procedure

[0166] Diluent / blank solvent: anhydrous ethanol;

[0167] Reference stock solution: Accurately weigh 15 mg of NMV-B00 enantiomer and transfer it to a 100 mL volumetric flask. Dissolve and dilute to the mark with diluent, and shake well.

[0168] Test sample spiked solution: Take about 300 mg of the test sample, accurately weigh it and transfer it to a 10 mL volumetric flask. Add 1.0 mL of the reference stock solution, then dissolve and dilute to the mark with diluent, and shake well.

[0169] Respectively take the blank solution and the test sample spiked solution, perform high performance liquid chromatography analysis under the above conditions, record the chromatogram, and the results are shown in Figure 15 、 Figure 16 。

[0170] Result analysis: Figure 16 The chromatographic peak with a retention time of 17.749 minutes in

[0171] Example 7

[0172] 1. Chromatographic conditions:

[0173] Chromatographic column: Phenomenex Chiral NX(2), 4.6 * 250 mm, 5 μm

[0174] Detector: VWD detector, detection wavelength 210 nm;

[0175] Flow rate: 0.5 mL / min;

[0176] Column temperature: 25 °C;

[0177] Injection volume: 12 μL;

[0178] Running time: 30 min;

[0179] Mobile phase:

[0180] Phase A: n-hexane;

[0181] Phase B: Isopropanol solution containing 0.1% trifluoroacetic acid (take 1 mL of trifluoroacetic acid and add it to 1000 mL of isopropanol, and shake well);

[0182] Elution: Phase A: Phase B = 88:12 (V / V); isocratic elution.

[0183] 2. Experimental procedure

[0184] Diluent / Blank Solvent: Absolute ethanol;

[0185] Reference Stock Solution: Accurately weigh 15 mg of NMV-B00 enantiomer and transfer it to a 100 mL volumetric flask. Dissolve and dilute to the mark with the diluent, then shake well;

[0186] Test Sample Spiked Solution: Take about 150 mg of the test sample, accurately weigh it into a 10 mL volumetric flask, add 1.0 mL of the reference stock solution, then dissolve and dilute to the mark with the diluent, and shake well.

[0187] Take the blank solution and the test sample spiked solution respectively, perform high performance liquid chromatography analysis under the above conditions, record the chromatogram, and the results are shown in Figure 17 、 Figure 18 。

[0188] Result Analysis: Figure 18 The chromatographic peak with a retention time of 11.876 minutes in is the chromatographic peak of the NMV-B00 enantiomer; the chromatographic peak with a retention time of 15.422 minutes is the chromatographic peak of NMV-B00. The resolution between the chromatographic peak of the NMV-B00 enantiomer and the chromatographic peak of NMV-B00 is 4.0. This method can be used for the detection of the NMV-B00 enantiomer in the starting material of nirmatrelvir.

[0189] Example 8: Chromatographic Column Investigation

[0190] Diluent: Ethanol;

[0191] NMV-B00 Solution: Prepare it to 20 mg / ml with the diluent.

[0192] NMV-B00 Enantiomer Solution: Prepare it to 20 mg / ml with the diluent.

[0193] Mixed Solution: Take 0.5 mL of the NMV-B00 solution and the NMV-B00 enantiomer solution respectively, mix them, and shake well to obtain it.

[0194] Take the mixed solution and investigate different chromatographic columns according to the chromatographic conditions in Table 1.

[0195] Table 1: Chromatographic Conditions for Chromatographic Column Investigation

[0196]

[0197] Results: See Figure 19 and Table 2.

[0198] Table 2: Results of Chromatographic Column Investigation

[0199] Chromatographic column Resolution between NMV-B00 and its enantiomer Phenomenex Chiral MZ(2), 4.6 mm * 250 mm, 5 μm Co-elution Phenomenex Chiral NS(2), 4.6 mm * 250 mm, 5 μm 2.7 Phenomenex Chiral MJ(2), 4.6 mm * 250 mm, 5 μm 2.8 Phenomenex Chiral INC, 4.6 mm * 250 mm, 5 μm 2.4 Phenomenex Chiral NX(2), 4.6 mm * 250 mm, 5 μm 3.9 Phenomenex Chiral MX(2), 4.6 mm * 250 mm, 5 μm Co-elution

[0200] Conclusion:

[0201] Using Phenomenex Chiral NX(2), 4.6 * 250 mm, 5 μm chromatographic column, Phenomenex Chiral NS(2), 4.6 * 250 mm, 5 μm chromatographic column, Phenomenex Chiral MJ(2), 4.6 * 250 mm, 5 μm chromatographic column, and Phenomenex Chiral INC, 4.6 * 250 mm, 5 μm chromatographic column as the separation chromatographic columns, the separation of NMV-B00 and its enantiomer can be better achieved. Among them, the most preferred is Phenomenex Chiral NX(2), which has the highest resolution. Using Phenomenex Chiral MZ(2) and Phenomenex Chiral MX(2) as the separation chromatographic columns, NMV-B00 and its enantiomer co-elute, showing no separation trend.

[0202] The method of the present invention has been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate modifications and combinations to the methods and applications described herein within the content, spirit, and scope of the present invention to implement and apply the technology of the present invention. Those skilled in the art can draw on the content of this article and appropriately modify the method parameters for implementation. It should be particularly noted that all such similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.

Claims

1. A method for detecting an enantiomer of NMV-B00 or a salt thereof in NMV-B00 or a salt thereof, characterized in that, Detection is carried out by high performance liquid chromatography, using a amylose tris-(4-chloro-3-methylphenylcarbamate) chromatographic column, an amylose tris-((s)-α-methylbenzylcarbamate) chromatographic column, a cellulose tris-(4-methylbenzoate) chromatographic column or a cellulose tris-(3,5-dichlorophenylcarbamate) covalently bonded chromatographic column as the separation chromatographic column, and eluting with a mixture of n-hexane and an acid-containing alcohol solution as the mobile phase; 2. According to the detection method described in claim 1, the amylose tris-(4-chloro-3-methylphenylcarbamate) chromatographic column is a Phenomenex Chiral NX(2) chromatographic column; and / or the amylose tris-((s)-α-methylbenzylcarbamate) chromatographic column is a Phenomenex Chiral NS(2) chromatographic column; and / or the cellulose tris-(4-methylbenzoate) chromatographic column is a Phenomenex Chiral MJ(2) chromatographic column; and / or the cellulose tris-(3,5-dichlorophenylcarbamate) covalently bonded chromatographic column is a Phenomenex Chiral INC chromatographic column; and / or the inner diameter of the separation chromatographic column is 2.0 mm - 10.0 mm; and / or the length of the separation chromatographic column is 100 mm - 250 mm; and / or the particle size of the packing of the separation chromatographic column is 3 μm - 5 μm.

3. According to the detection method described in any one of claims 1-2, the acid content in the acid-containing alcohol solution is 0.05% v - 0.15% vol or 0.10% vol; and / or the acid in the acid-containing alcohol solution includes trifluoroacetic acid; and / or the alcohol in the acid-containing alcohol solution includes at least one of isopropanol and ethanol.

4. According to the detection method described in any one of claims 1-3, the volume ratio of n-hexane to the acid-containing alcohol solution mixture in the mobile phase is 95:5 - 80:20, preferably 90:

10.

5. According to the detection method described in any one of claims 1-4, the detection wavelength of the high performance liquid chromatography is 210 nm ± 5 nm; and / or the column temperature of the high performance liquid chromatography is 15°C - 40°C or 25°C; and / or the flow rate of the high performance liquid chromatography is 0.3 ml / min - 1.0 ml / min or 0.5 ml / min.

6. According to the detection method described in any one of claims 1-5, the acid-containing alcohol solution is an isopropanol solution containing 0.1% vol trifluoroacetic acid or an ethanol solution containing 0.1% vol trifluoroacetic acid.

7. According to the detection method described in any one of claims 1-6, the model of the separation chromatographic column is 4.6 mm * 250 mm, 5 μm.

8. The detection method according to any one of claims 1-7, which uses high performance liquid chromatography for detection. A straight-chain starch tris-(4-chloro-3-methylphenylcarbamate) chromatographic column, a straight-chain starch tris-((S)-α-methylbenzylcarbamate) chromatographic column, a cellulose tris-(4-methylbenzoate) chromatographic column or a cellulose tris-(3,5-dichlorophenylcarbamate) covalently bonded chromatographic column is used as the separation chromatographic column. A mixture of n-hexane and an isopropanol solution containing 0.1% vol trifluoroacetic acid is used as the mobile phase for elution or a mixture of n-hexane and an ethanol solution containing 0.1% vol trifluoroacetic acid is used as the mobile phase for elution. The volume ratio of n-hexane to the acid-containing alcohol solution in the mobile phase is 90:

10. The column temperature of the high performance liquid chromatography is 20°C - 30°C or 25°C; the flow rate of the high performance liquid chromatography is 0.4 ml / min - 0.6 ml / min or 0.5 ml / min.

9. The detection method according to any one of claims 1-8, wherein the detection method further includes the preparation of a test solution. The preparation method of the test solution is to dilute the sample to be tested with a diluent to a concentration of NMV-C00 of 5 mg / mL - 30 mg / mL or 15 mg / mL; and / or the diluent is the mobile phase, ethanol or isopropanol.

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