Liquid chromatographic analysis method for related substances in (R)-N-((S)-1-amino-1-oxobutane-2-yl)-3-(chloromethyl) hexanamide

Abstract

The invention discloses a method for detecting related substances in (R)-N-((S)-1-amino-1-oxobutane-2-yl)-3-(chloromethyl) hexanamide, which comprises the following steps: step 1, mixing an (R)-N-((S)-1-amino-1-oxobutane-2-yl)-3-(chloromethyl) hexanamide (compound 1) sample with a diluent to prepare a sample solution; and 2, detecting the sample solution by using high performance liquid chromatography so as to determine the content of the related substances in the sample of the compound 1. A phosphate aqueous solution is used as a mobile phase, so that the separation capacity of each substance is improved; a phosphate aqueous solution and acetonitrile are used as mobile phases, so that baseline interference is reduced; a ghost peak trapping column is adopted to reduce ghost peak interference and optimize a blank baseline; by adopting gradient elution, the separation capacity is improved, the peak shape is improved, and the sensitivity is increased.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemical industry, and specifically relates to a method for detecting related substances in (R)-N-((S)-1-amino-1-oxobutane-2-yl)-3-(chloromethyl)hexanamide. Background Art

[0002] Brivaracetam is an anti-epileptic drug, mainly used to treat partial seizures in adults and children over 12 years old with epilepsy. Compared with levetiracetam, brivaracetam has higher affinity and ability to inhibit synaptic transmission and vesicle release, as well as higher lipophilicity and faster brain penetration. Its structural formula is as follows:

[0003]

[0004] (R)-configured N-((S)-1-amino-1-oxo-2-butyl)-3-chloromethylhexanamide (hereinafter referred to as "Compound 1") is the core precursor for the preparation of the active pharmaceutical ingredient of brivaracetam, and its purity is crucial. If the isomeric impurities are not fully removed in this step, these impurities will inevitably be transferred to the final drug product, thereby adversely affecting the chiral purity and overall quality of the drug. Therefore, accurate detection and effective management of the content of these isomeric impurities is a key measure to prevent the generation of more impurities in subsequent synthesis steps, which is of great significance for ensuring the quality control of brivaracetam raw materials. The following is the structural formula of Compound 1:

[0005]

[0006] Invention patent CN 106432030A discloses a method for generating (R)-N-((S)-1-amino-1-oxobutane-2-yl)-3-(chloromethyl)hexanamide (Compound 1) by reacting (R)-3-(chloromethyl)hexanyl chloride with (S)-2-aminobutanamide hydrochloride. The synthetic route is as follows:

[0007]

[0008] The following impurities are easily introduced into this compound during the preparation process, and quantitative studies of these impurities are required:

[0009]

[0010]

[0011] There are a large number of target related substances in compound 1, with different PKa and weak ultraviolet absorption, and liquid chromatography separation is very challenging. At present, there is no public report on the analytical detection method for the above related substances in (R)-N-((S)-1-amino-1-oxobutane-2-yl)-3-(chloromethyl)hexanamide. Therefore, it is necessary to study an analytical method that can accurately and sensitively detect the content of (R)-N-((S)-1-amino-1-oxobutane-2-yl)-3-(chloromethyl)hexanamide and its related substances, so as to effectively control the content of impurities in the subsequent buvacetam synthesis process. Summary of the invention

[0012] In view of the above problems existing in the prior art, the object of the present invention is to provide a sensitive and efficient method for detecting related substances in (R)-N-((S)-1-amino-1-oxobutane-2-yl)-3-(chloromethyl)hexanamide.

[0013] From the structures of the target compound and its related substances, it can be seen that they all have ultraviolet absorption at the end, so the phosphate-acetonitrile system with less absorption interference at the end was selected as the buffer system for its method development.

[0014] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0015] The present invention provides a method for detecting related substances in (R)-N-((S)-1-amino-1-oxobutane-2-yl)-3-(chloromethyl)hexanamide (compound 1), comprising the following steps:

[0016] Step 1: Mix the compound 1 sample with a diluent to prepare a sample solution;

[0017] Step 2: Use high performance liquid chromatography to detect the sample solution to determine the content of related substances in the compound 1 sample; the detection conditions of the high performance liquid chromatography are as follows:

[0018] The mobile phase A of the high performance liquid chromatography is a phosphate aqueous solution, and the mobile phase B is acetonitrile; the elution method is a gradient elution;

[0019] Step 3: Determine the content of related substances in the sample solution and calculate the content of related substances in the compound 1 sample based on the peak area.

[0020] The structures of compound 1 and its related substances are as follows:

[0021]

[0022]

[0023] Furthermore, the chromatographic column uses octadecylsilane bonded silica gel as a filler.

[0024] In a preferred embodiment, the chromatographic column is Waters xbridge RP18150×4.6mm, 3.5um.

[0025] Furthermore, a ghost peak trapping column was used in liquid phase detection.

[0026] In a preferred embodiment, the ghost peak trapping column is Ghost-Buster 4.6×50 mm.

[0027] Furthermore, the mobile phase A is an aqueous solution of potassium dihydrogen phosphate with a concentration of 0.005 to 0.02 mol / L.

[0028] In a preferred embodiment, the concentration of the potassium dihydrogen phosphate aqueous solution is 0.01 mol / L.

[0029] Furthermore, the mobile phase flow rate of the high performance liquid chromatograph is 0.8 to 1.2 mL / min.

[0030] In a preferred embodiment, the flow rate is 1.0 mL / min.

[0031] Furthermore, the HPLC detection wavelength is 190-220 nm.

[0032] In a preferred embodiment, the wavelength is 200 nm.

[0033] Furthermore, the column temperature of the chromatographic column is 10°C to 30°C.

[0034] In a preferred embodiment, the column temperature is 20°C.

[0035] Furthermore, the injection volume is 8 μL to 12 μL.

[0036] In a preferred embodiment, the injection volume is 10 μL.

[0037] Further, the liquid chromatography detection method is optimized, and the mobile phase A and the mobile phase B are eluted using the elution program in the following table, wherein the percentages of the mobile phase A and the mobile phase B are volume percentages:

[0038] Time / min Mobile phase A / % Mobile phase B / % 0 70～90 10～30 25~35(after switching) 50～70 30～50 35~45(after switching) 20～40 60～80 45~55(after switching) 70～90 10～30 60 70～90 10～30

[0039] During the liquid phase analysis process, the proportion of solvent components is switched, the proportion of acetonitrile is gradually increased and the proportion of potassium dihydrogen phosphate aqueous solution is reduced, the elution capacity of the solvent components is slowly increased, so that potential related substances of different polarities can be fully eluted, and finally the initial solvent components are switched back to balance the chromatographic column before the next injection is tested.

[0040] Preferably, the mobile phase A and the mobile phase B are eluted using the elution program in the following table, wherein the percentages of the mobile phase A and the mobile phase B are volume percentages:

[0041] Time / min Mobile phase A / % Mobile phase B / % 0 70 30 30(after switching) 60 40 40(after switching) 30 70 50 30 70 50.1 (after switching) 70 30 60 70 30

[0042] During the liquid phase analysis process, the polarity of the solvent component was slowly increased, and the elution capacity was gradually improved, so that related substances of different polarities were eluted in sequence. At 30 minutes, all the target related substances had elute. By further increasing the proportion of acetonitrile and reducing the proportion of potassium dihydrogen phosphate aqueous solution, some other weakly polar substances in the composition were eluted. At 50 minutes, no potential impurities had elute, so the initial solvent component was switched back to balance the chromatographic column and wait for the next injection to be detected.

[0043] In summary, the beneficial effects of the present invention are:

[0044] The present invention increases the separation ability of each substance by using phosphate aqueous solution as mobile phase; using phosphate aqueous solution and acetonitrile as mobile phase, reducing baseline interference; using ghost peak capture column is to reduce ghost peak interference and optimize blank baseline; using gradient elution, improving separation ability, improving peak shape and increasing sensitivity. In the liquid phase analysis process, the proportion of acetonitrile is gradually increased by switching twice to reduce the proportion of potassium dihydrogen phosphate aqueous solution, so that some other weakly polar substances in the components are eluted; subsequently switching back to the initial solvent component, balancing the chromatographic column. The liquid chromatography analysis method provided by the present invention uses 0.01mol / L potassium dihydrogen phosphate aqueous solution and acetonitrile as mobile phase for gradient elution, which can achieve full separation between the target compound and related substances, and the separation between the main peak and the adjacent impurity peaks and between each impurity peak is ≥1.5, and it has been verified by methods such as specificity, precision, accuracy, linearity, quantitative limit and detection limit, and can accurately detect the percentage content of related substances in the target compound. The method of the present invention is fast and simple, has good specificity, high sensitivity, high accuracy, wide application range, low cost, and is suitable for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 HPLC spectrogram of the blank solution provided in Example 1 of the present invention;

[0046] Figure 2 HPLC spectrum of the impurity A localization solution provided in Example 1 of the present invention;

[0047] Figure 3 HPLC spectrum of the impurity B localization solution provided in Example 1 of the present invention;

[0048] Figure 4 HPLC spectrum of the impurity C localization solution provided in Example 1 of the present invention;

[0049] Figure 5 HPLC spectrum of the impurity D localization solution provided in Example 1 of the present invention;

[0050] Figure 6 HPLC spectrum of the sample solution of compound 1 provided in Example 1 of the present invention;

[0051] Figure 7 This is the HPLC spectrum of the mixed solution provided in Example 1 of the present invention.

[0052] Figure 8 This is the HPLC spectrum of the mixed solution provided in Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0053] The present invention will now be further described in detail with reference to the embodiments. The application of the present invention is not limited to the following embodiments, and any modifications made to the present invention will fall within the protection scope of the present invention.

[0054] The object of the present invention is to provide a sensitive, efficient, simple to operate, reliable and accurate method for detecting related substances in (R)-N-((S)-1-amino-1-oxobutane-2-yl)-3-(chloromethyl)hexanamide.

[0055] Example 1

[0056] In the embodiment of the analytical method of the present invention, the instruments and operating conditions are as follows:

[0057] Chromatographic column: Waters xbridge RP18150*4.6mm, 3.5um

[0058] Trapping column: Ghost-Buster4.6×50mm

[0059] Detection wavelength: 200nm

[0060] Flow rate: 1.0mL / min

[0061] Column temperature: 20℃

[0062] Injection volume: 10 μL

[0063] Mobile phase A: 0.01 mol / L potassium dihydrogen phosphate aqueous solution

[0064] Mobile phase B: acetonitrile

[0065] Running time: 60 minutes

[0066] Follow the gradient elution as shown below:

[0067] Time / min Mobile phase A / % Mobile phase B / % 0 70 30 30 60 40 40 40 60 50 40 60 50.1 70 30 60 70 30

[0068] Solution preparation:

[0069] Diluent: mobile phase A: acetonitrile = 50:50.

[0070] Blank solution: diluent.

[0071] Impurity A stock solution: Take about 4 mg of impurity A standard, place it in a 10 ml volumetric flask, add an appropriate amount of diluent, ultrasonically dissolve it, and dilute it to the scale with the diluent.

[0072] Impurity B stock solution: Take about 4 mg of impurity B standard, place it in a 10 ml volumetric flask, add an appropriate amount of diluent, ultrasonically dissolve it, and dilute it to the scale with diluent.

[0073] Impurity C stock solution: Take about 4 mg of impurity C standard, place it in a 10 ml volumetric flask, add an appropriate amount of diluent, ultrasonically dissolve it, and dilute it to the scale with diluent.

[0074] Impurity D stock solution: Take about 4 mg of impurity D standard, place it in a 10 ml volumetric flask, add an appropriate amount of diluent, ultrasonically dissolve it, and dilute it to the scale with the diluent.

[0075] System suitability solution: Take about 40 mg of compound 1 standard and place it in a 10 ml volumetric flask. Add 2.0 ml each of impurity A, impurity B and impurity D stock solutions and 0.15 ml of impurity C stock solution. Add appropriate amount of diluent and dissolve by ultrasonication. Dilute to the scale with diluent.

[0076] Control solution: Take about 40 mg of the standard compound 1, place it in a 10 ml volumetric flask, add an appropriate amount of diluent, dissolve it by ultrasonic, dilute it to the mark with the diluent, and shake it well. Accurately transfer 1.0 ml of the above solution to a 100 ml volumetric flask, and dilute it to the mark with the diluent.

[0077] Sample solution: Take 40 mg of compound 1 sample, weigh accurately, place in a 10 mL volumetric flask, add appropriate amount of diluent, dissolve by ultrasonication, and dilute to the scale with diluent.

[0078] Test process: Perform the test according to the injection sequence in the table below.

[0079] Solution name Injection times blank solution At least 2 times System Suitability Solution 1 blank solution 1 Control solution 1 Sample solution 1

[0080] System suitability:

[0081] (1) The blank solution has no interference with the relevant chromatographic peaks;

[0082] (2) In the system suitability solution, the separation between known substances should be ≥1.5.

[0083] Calculation: Calculate the percentage of impurities according to the following formula:

[0084]

[0085] As: Peak area of ​​each impurity in the sample solution

[0086] Ar: Peak area of ​​compound 1 in the reference solution

[0087] Ws: weight of compound 1 sample, mg

[0088] Wr: The weight of the standard sample of compound 1, mg

[0089] P: content of compound 1 standard

[0090] Exclusiveness Verification

[0091] Solution preparation:

[0092] Diluent: Mobile phase A: acetonitrile = 50:50

[0093] Blank solution: diluent

[0094] Impurity A positioning solution: Take 40 mg of impurity A standard, accurately weigh it, place it in a 100 ml volumetric flask, add an appropriate amount of diluent, ultrasonically dissolve it, and dilute it to the scale with the diluent.

[0095] Impurity B localization solution: Take about 4 mg of impurity B standard, place it in a 10 ml volumetric flask, add an appropriate amount of diluent, ultrasonically dissolve it, and dilute it to the scale with diluent.

[0096] Impurity C positioning solution: Take about 4 mg of impurity C standard, place it in a 10 ml volumetric flask, add an appropriate amount of diluent, ultrasonically dissolve it, and dilute it to the scale with diluent.

[0097] Impurity D localization solution: Take about 4 mg of impurity D standard, place it in a 10 ml volumetric flask, add an appropriate amount of diluent, ultrasonically dissolve it, and dilute it to the scale with the diluent.

[0098] Sample solution: Take 40 mg of compound 1 sample, weigh accurately, place in a 10 mL volumetric flask, add appropriate amount of diluent, dissolve by ultrasonication, and dilute to the scale with diluent.

[0099] Mixed solution: Take 40 mg of compound 1 sample, weigh accurately, place in a 10 mL volumetric flask, add 2.0 mL each of impurity A positioning solution, impurity B positioning solution and impurity D positioning solution, and 0.15 mL of impurity C positioning solution, add appropriate amount of diluent, dissolve by ultrasonication, and dilute to the scale with diluent.

[0100] Take 10 μL each of the blank solution, positioning solution, system suitability solution, sample solution and mixed solution, inject them into the liquid chromatograph, and record the chromatogram.

[0101] The results are shown in Table 1 below, which provides specific data for Example 1 of the present invention.

[0102] Table 1 Specificity verification

[0103]

[0104]

[0105] From the above data, it can be concluded that the blank does not interfere with the substance to be tested, the impurities and the impurities and the main peak can be completely separated, and the specificity is good.

[0106] Related detection spectra can be found at Figures 1 to 7 .

[0107] Figure 1 HPLC spectrogram of the blank solution provided in Example 1 of the present invention;

[0108] Figure 2 HPLC spectrum of the impurity A localization solution provided in Example 1 of the present invention;

[0109] Figure 3 HPLC spectrum of the impurity B localization solution provided in Example 1 of the present invention;

[0110] Figure 4 HPLC spectrum of the impurity C localization solution provided in Example 1 of the present invention;

[0111] Figure 5 HPLC spectrum of the impurity D localization solution provided in Example 1 of the present invention;

[0112] Figure 6 HPLC spectrum of the sample solution of compound 1 provided in Example 1 of the present invention;

[0113] Figure 7 This is the HPLC spectrum of the mixed solution provided in Example 1 of the present invention.

[0114] Precision Verification

[0115] Solution preparation:

[0116] Diluent: Mobile phase A: acetonitrile = 50:50

[0117] Blank solution: diluent

[0118] Impurity A stock solution: Take the localization solution under the specificity item.

[0119] Impurity B stock solution: Take about 10 mg of impurity B standard, place it in a 25 ml volumetric flask, add an appropriate amount of diluent, ultrasonically dissolve it, and dilute it to the scale with diluent.

[0120] Impurity C stock solution: Take about 10 mg of impurity C standard, place it in a 25 ml volumetric flask, add an appropriate amount of diluent, ultrasonically dissolve it, and dilute it to the scale with diluent.

[0121] Impurity D stock solution: Take about 10 mg of impurity D standard, place it in a 25 ml volumetric flask, add an appropriate amount of diluent, ultrasonically dissolve it, and dilute it to the scale with the diluent.

[0122] System suitability solution: Take about 40 mg of compound 1 standard and place it in a 10 ml volumetric flask. Add 2.0 ml each of impurity A stock solution, impurity B stock solution, impurity D stock solution, and 0.15 ml of impurity C stock solution. Add an appropriate amount of diluent and dissolve it by ultrasonication. Dilute to the scale with diluent.

[0123] Control solution: Take about 40 mg of the standard compound 1, place it in a 10 ml volumetric flask, add an appropriate amount of diluent, dissolve it by ultrasonic, dilute it to the mark with the diluent, and shake it well. Accurately transfer 1.0 ml of the above solution to a 100 ml volumetric flask, and dilute it to the mark with the diluent.

[0124] Sample spike solution: Take 40 mg of compound 1 sample, weigh accurately, place in a 10 mL volumetric flask, add 2.0 ml each of impurity A stock solution, impurity B stock solution and impurity D stock solution, and 0.15 ml of impurity C stock solution, add appropriate amount of diluent, ultrasonically dissolve, and dilute to scale with diluent. Prepare 6 portions in parallel and mark them as P1-P6.

[0125] Take 10 μL of each of the above solvents, inject them into the liquid chromatograph, and record the chromatogram.

[0126] The results are shown in Table 2 below, which provides precision data for Example 1 of the present invention.

[0127] Table 2 Precision verification results

[0128] project Impurity A% Impurity B% Impurity C% Impurity D% P1 2.495％ 2.246％ 0.144％ 2.009％ P2 2.510％ 2.345％ 0.140％ 1.870％ P3 2.469％ 2.212％ 0.145％ 1.857％ P4 2.506％ 2.386％ 0.152％ 1.876％ P5 2.476％ 2.393％ 0.153％ 1.854％ P6 2.517％ 2.453％ 0.157％ 1.876％ average value 2.496％ 2.339％ 0.148％ 1.890％ RSD 0.77％ 3.96％ 4.36％ 3.12％

[0129] The RSD of the content of each impurity was less than 10.0%, so the precision of this method was good.

[0130] Limit of Quantitation

[0131] Referring to the signal-to-noise ratio of impurity A, impurity B, impurity C, compound 1 and impurity D under the specificity item, dilute step by step until the concentration is no greater than the limit concentration and the signal-to-noise ratio is no less than 10. This concentration is the LOQ.

[0132] The results are shown in Table 3 below, which provides quantitative limit data for Example 1 of the present invention.

[0133] Table 3 Quantitation limit data

[0134] LOQ μg / ml S / N Relative to sample concentration% Impurity A 8.098 589.77 0.202％ Impurity B 8.371 487.50 0.209％ Impurity C 4.144 32.52 0.104％ Compound 1 2.027 89.90 0.051％ Impurity D 2.102 85.10 0.053％

[0135] From the above data, it can be seen that the sensitivity of the method meets the measurement requirements.

[0136] Detection limit

[0137] The LOQ solution is diluted until the concentrations of impurity A, impurity B, impurity C, compound 1 and impurity D are no greater than 50% of the LOQ concentration and the signal-to-noise ratio is no less than 3. This concentration is the LOD.

[0138] The results are shown in Table 4 below, which provides detection limit data for Example 1 of the present invention.

[0139] Table 4 Detection limit data

[0140] LOD μg / ml S / N Relative to sample concentration% Impurity A 4.049 326.93 0.101％ Impurity B 4.185 262.40 0.108％ Impurity C 2.072 23.46 0.052％ Compound 1 1.014 48.94 0.025％ Impurity D 1.051 55.86 0.027％

[0141] From the above data, it can be seen that the sensitivity of the method meets the measurement requirements.

[0142] Linear

[0143] Prepare linear solutions with impurity limit concentrations ranging from LOQ to 150%, accurately measure 10 μL of each linear solution, inject into the liquid chromatograph, record the chromatogram, and perform linear regression with concentration as the abscissa and peak area as the ordinate.

[0144] The results are shown in Table 5 below, which provides linear data for Example 1 of the present invention.

[0145] Table 5 Linear data

[0146] project Linear equations Correlation coefficient R(≥0.99) Impurity A y=11458x+7.1278 0.9999 Impurity B y=11659x+2.5663 0.9999 Impurity C y=3346.4x-3.1314 0.9961 Impurity D y=13460x-5.0702 0.9999 Compound 1 y=10240x+0.9008 0.9999

[0147] From the above data, it can be seen that the linearity of the method meets the measurement requirements.

[0148] Accuracy

[0149] Prepare an accuracy solution with an impurity limit concentration of LOQ to 150%, accurately measure 10 μL of each accuracy solution, inject it into the liquid chromatograph, record the chromatogram, and calculate the recovery rate of each impurity.

[0150] The results are shown in Table 6 below, which provides accuracy data for Example 1 of the present invention.

[0151] Table 6 Accuracy data

[0152]

[0153] At the spiked LOQ limit level, the mean recovery rate of each impurity was within the range of 80% to 120%. At the spiked 100% limit and 150% limit levels, the mean recovery rate of each impurity was within the range of 90% to 110%. This method has good accuracy.

[0154] Comparative Example 1

[0155] The difference between this comparative example and Example 1 is that the mobile phase ratio and gradient elution procedure of the method are changed.

[0156] In this comparative example 1, the instruments and operating conditions are as follows:

[0157] Chromatographic column: Waters xbridge RP18150*4.6mm, 3.5um

[0158] Trapping column: Ghost-Buster4.6×50mm

[0159] Detection wavelength: 200nm

[0160] Flow rate: 1.0mL / min

[0161] Column temperature: 20℃

[0162] Injection volume: 10 μL

[0163] Mobile phase A: 0.01 mol / L potassium dihydrogen phosphate aqueous solution

[0164] Mobile phase B: acetonitrile

[0165] Running time: 50min

[0166] Follow the gradient elution in the table below.

[0167] Time / min Mobile phase A / % Mobile phase B / % 0 80 20 5 80 20 40 60 40 40.1 80 20 50 80 20

[0168] Solution preparation:

[0169] Diluent: mobile phase A: acetonitrile = 50:50.

[0170] Blank solution: diluent

[0171] Impurity A stock solution: Take about 4 mg of impurity A standard, place it in a 10 ml volumetric flask, add an appropriate amount of diluent, ultrasonically dissolve it, and dilute it to the scale with the diluent.

[0172] Impurity B stock solution: Take about 4 mg of impurity B standard, place it in a 10 ml volumetric flask, add an appropriate amount of diluent, ultrasonically dissolve it, and dilute it to the scale with diluent.

[0173] Impurity C stock solution: Take about 4 mg of impurity C standard, place it in a 10 ml volumetric flask, add an appropriate amount of diluent, ultrasonically dissolve it, and dilute it to the scale with diluent.

[0174] Impurity D stock solution: Take about 4 mg of impurity D standard, place it in a 10 ml volumetric flask, add an appropriate amount of diluent, ultrasonically dissolve it, and dilute it to the scale with the diluent.

[0175] System suitability solution: Take about 40 mg of compound 1 standard and place it in a 10 ml volumetric flask. Add 2.0 ml each of impurity A, impurity B and impurity D stock solutions and 0.15 ml of impurity C stock solution. Add appropriate amount of diluent and dissolve by ultrasonication. Dilute to the scale with diluent.

[0176] Control solution: Take about 40 mg of the standard compound 1, place it in a 10 ml volumetric flask, add an appropriate amount of diluent, dissolve it by ultrasonic, dilute it to the mark with the diluent, and shake it well. Accurately transfer 1.0 ml of the above solution to a 100 ml volumetric flask, and dilute it to the mark with the diluent.

[0177] Sample solution: Take 40 mg of compound 1 sample, weigh accurately, place in a 10 mL volumetric flask, add appropriate amount of diluent, dissolve by ultrasonication, and dilute to the scale with diluent.

[0178] Mixed solution: Take 40 mg of compound 1 sample, weigh accurately, place in a 10 mL volumetric flask, add 2.0 mL each of impurity A positioning solution, impurity B positioning solution and impurity D positioning solution, and 0.15 mL of impurity C positioning solution, add appropriate amount of diluent, dissolve by ultrasonication, and dilute to the scale with diluent.

[0179] Test process: Perform the test according to the injection sequence in the table below.

[0180]

[0181]

[0182] System suitability:

[0183] (1) The blank solution has no interference with the relevant chromatographic peaks;

[0184] (2) In the system suitability solution, the separation between known substances should be ≥1.5.

[0185] Calculation: Calculate the percentage of impurities according to the following formula:

[0186]

[0187] As: Peak area of ​​each impurity in the sample solution

[0188] Ar: Peak area of ​​compound 1 in the reference solution

[0189] Ws: weight of compound 1 sample, mg

[0190] Wr: The weight of the standard sample of compound 1, mg

[0191] P: content of compound 1 standard

[0192] Conclusion: If Figure 8As shown, after changing the mobile phase ratio and gradient elution program of the method, the chromatographic peaks of impurity B and impurity C in the chromatogram cannot be separated, and the separation degree of this comparative method is poor.

[0193] Figure 8 This is the HPLC spectrum of the mixed solution provided in Comparative Example 1 of the present invention.

[0194] The present invention uses a liquid chromatography analysis method to achieve complete separation between the main component and its related substance chromatographic peaks, and the diluent and the main component have no interference in the inspection of the content of each impurity. The method has strong specificity, high accuracy, precision and sensitivity, simple sample preparation and analysis operations, and high detection efficiency.

[0195] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent changes made using the present invention are within the patent protection scope of the present invention.

Claims

1. A method for detecting related substances in (R)-N-((S)-1-amino-1-oxobutane-2-yl)-3-(chloromethyl)hexanamide, comprising the following steps: Step 1: Mixing a (R)-N-((S)-1-amino-1-oxobutane-2-yl)-3-(chloromethyl)hexanamide (Compound 1) sample with a diluent to prepare a sample solution; Step 2: Use high performance liquid chromatography to detect the sample solution to determine the content of related substances in the compound 1 sample; the detection conditions of the high performance liquid chromatography are as follows: The mobile phase A of the high performance liquid chromatography is a phosphate aqueous solution, and the mobile phase B is acetonitrile; the elution method is a gradient elution; Step 3: Determine the content of related substances in the sample solution, and calculate the content of related substances in the compound 1 sample: impurity A, impurity B, impurity C, and impurity D based on the peak area. The structures of compound 1 and its related substances are as follows:

2. The method for detecting related substances in (R)-N-((S)-1-amino-1-oxobutane-2-yl)-3-(chloromethyl)hexanamide according to claim 1, characterized in that: The chromatographic column uses octadecylsilane bonded silica gel as filler.

3. The method for detecting related substances in (R)-N-((S)-1-amino-1-oxobutane-2-yl)-3-(chloromethyl)hexanamide according to claim 1, characterized in that: A ghost peak trapping column was used for liquid phase detection.

4. The method for detecting related substances in (R)-N-((S)-1-amino-1-oxobutane-2-yl)-3-(chloromethyl)hexanamide according to claim 1, characterized in that: Mobile phase A is potassium dihydrogen phosphate aqueous solution with a concentration of 0.005-0.02 mol / L.

5. The method for detecting related substances in (R)-N-((S)-1-amino-1-oxobutane-2-yl)-3-(chloromethyl)hexanamide according to claim 1, characterized in that: The mobile phase flow rate of the high performance liquid chromatography is 0.8 to 1.2 mL / min.

6. The method for detecting related substances in (R)-N-((S)-1-amino-1-oxobutane-2-yl)-3-(chloromethyl)hexanamide according to claim 1, characterized in that: The detection wavelength of HPLC is 190~220nm.

7. The method for detecting related substances in (R)-N-((S)-1-amino-1-oxobutane-2-yl)-3-(chloromethyl)hexanamide according to claim 1, characterized in that: The column temperature is 10℃~30℃.

8. The method for detecting related substances in (R)-N-((S)-1-amino-1-oxobutane-2-yl)-3-(chloromethyl)hexanamide according to claim 1, characterized in that: The injection volume is 8μL~12μL.

9. The method for detecting related substances in (R)-N-((S)-1-amino-1-oxobutane-2-yl)-3-(chloromethyl)hexanamide according to claim 1, characterized in that: The mobile phase A and the mobile phase B are eluted using the following elution procedure, wherein the percentages of the mobile phase A and the mobile phase B are by volume: Elution procedure: In the initial 0min stage of S1, the ratio of mobile phase solvents is: mobile phase A is 70-90%, and mobile phase B is 10-30%; At a certain time point in the 25-35min stage, S2 switches the mobile phase solvent ratio to: mobile phase A is 50-70%, mobile phase B is 30-50%; At a certain time point in the 35-45 min stage, S3 switches the mobile phase solvent ratio to: mobile phase A is 20-40%, and mobile phase B is 60-80%; At a certain time point in the 45-60min stage, S4 switches the mobile phase solvent ratio to: mobile phase A is 70-90%, mobile phase B is 10-30%; and continues until 60min to end the liquid phase analysis.

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