Liquid chromatographic analysis method for isomer impurities in briracetam intermediate

Abstract

The invention discloses a liquid chromatographic analysis method for isomer impurities in a briracetam intermediate, which comprises the following steps: step 1, mixing a briracetam intermediate (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 isomer impurities in the sample of the compound 1. According to the method, normal-phase high-performance liquid chromatography is adopted, and normal hexane and isopropanol are used as mobile phases, so that the elution strength is improved, and the separation capacity is improved. By adopting gradient elution, the separation capacity is improved, the peak shape is improved, and the sensitivity is increased.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical and chemical technology, and specifically relates to a liquid chromatography method for the analysis of isomer impurities in bricetan intermediates. Background Technology

[0002] Brivaceratam is a third-generation antiepileptic drug developed by UCB (Universal Scientific Industrial Co., Ltd.) in Belgium. It is indicated for the treatment of partial seizures in epilepsy. Its structural formula is as follows:

[0003]

[0004] The briceracetam intermediate (compound 1) is a key intermediate in the synthesis of briceracetam active pharmaceutical ingredient (API). If isomer impurities are not completely removed, they will be introduced into the final API product, affecting the chiral purity and quality of the drug. Therefore, determining and controlling these isomer impurities can reduce their formation during subsequent reactions, which is of great significance for quality control. The structural formula of compound 1 is as follows:

[0005]

[0006] Compound 1 has two chiral centers, which makes it easy to introduce the following three isomer impurities during preparation. Quantitative studies of these impurities are necessary:

[0007]

[0008]

[0009] Currently, no publicly reported methods exist for the analysis and detection of isomer impurities in brivaracetam intermediate (compound 1). Structurally, compound 1 contains two chiral centers and has three isomers. Impurity A is an enantiomer of compound 1, while impurities B and C are another enantiomer pair. The large number of isomers, their similar structures, and their tendency to possess similar physicochemical properties make them difficult to separate and identify using conventional reversed-phase liquid chromatography. Therefore, it is necessary to investigate analytical methods that can accurately and sensitively detect the content of brivaracetam intermediate (compound 1) and its isomer impurities, in order to effectively control the impurity content in subsequent brivaracetam synthesis processes. Summary of the Invention

[0010] In view of the above-mentioned problems in the prior art, the purpose of this invention is to provide a sensitive and efficient liquid chromatography method for isomer impurities in bricetan intermediates.

[0011] Based on the structure of the target compound and its isomers, a covalently chiral column of a polysaccharide derivative with good solvent tolerance and a commonly used normal-phase mobile phase, n-hexane-isopropanol system, were selected for method development.

[0012] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0013] This invention provides a liquid chromatography method for the analysis of isomer impurities in bricetan intermediates, comprising the following steps:

[0014] Step 1: Mix the bricetan intermediate (compound 1) sample with diluent to prepare a sample solution;

[0015] Step 2: The sample solution was analyzed using high-performance liquid chromatography (HPLC) to determine the content of isomer impurities in compound 1. The HPLC detection conditions are as follows:

[0016] The mobile phases A and B of the high-performance liquid chromatography are both mixtures of n-hexane and isopropanol; the elution method is gradient elution.

[0017] Step 3: Determine the content of isomer impurities in the sample solution, and calculate the content of isomer impurities in compound 1 sample based on the peak area.

[0018] The structures of compound 1 and its isomer impurities are as follows:

[0019]

[0020]

[0021] Furthermore, the chromatographic column in the high-performance liquid chromatography is a chiral column. In the embodiment of this invention, the chromatographic column is a Daicel Chiral Pak IC 4.6×250mm, 5μm.

[0022] Furthermore, the ratio of n-hexane to isopropanol in mobile phase A is 60–100:0–40.

[0023] More preferably, the ratio of n-hexane to isopropanol in mobile phase A is 80:20.

[0024] Furthermore, the ratio of n-hexane to isopropanol in mobile phase B is 50–90:10–50.

[0025] More preferably, the ratio of n-hexane to isopropanol in mobile phase B is 60:40.

[0026] Furthermore, the flow rate of the mobile phase in the high-performance liquid chromatograph is 0.8–1.2 mL / min, and the flow rate in the embodiment of the present invention is 1.0 mL / min.

[0027] Furthermore, the high-performance liquid chromatography detection wavelength is 200-240 nm, and the wavelength in the embodiment of this invention is 220 nm.

[0028] Furthermore, the column temperature of the chromatographic column is 20℃~40℃, and the column temperature in the embodiment of the present invention is 30℃.

[0029] Furthermore, the injection volume is 18 μL to 22 μL, and in the embodiment of the present invention, the injection volume is 20 μL.

[0030] To further optimize the detection method, mobile phase A and mobile phase B are eluted using the elution procedure shown in the table below, where the percentages of mobile phase A and mobile phase B are volume percentages:

[0031] Time / min Mobile phase A / % Mobile phase B / % 0 100 0 10～30 100 0 10-30 (after switching) 0 100 30～50 0 100 30-50 (after switching) 100 0 50 100 0

[0032] In liquid chromatography analysis, the initial solvent component has a low proportion of the highly polar solvent isopropanol and a high proportion of the weakly polar solvent n-hexane, resulting in a milder elution capability that allows impurities to be slowly eluted and separated. Subsequently, the solvent component is switched, and the proportion of isopropanol is increased to accelerate the elution rate, shorten the detection time, and improve the detection efficiency. Finally, the initial solvent component is switched back, the chromatographic column is equilibrated, and the next detection is performed.

[0033] More preferably, the mobile phase A and mobile phase B are eluted using the elution procedure shown in the table below, wherein the percentages of mobile phase A and mobile phase B are volume percentages:

[0034] Time / min Mobile phase A / % Mobile phase B / % 0 100 0 18 100 0 18.1 (Switch) 0 100 40 0 100 40.1 (Switch) 100 0 50 100 0

[0035] During the liquid chromatography analysis, it was found that impurities A and B exhibited similar retention behaviors on the column. The initial solvent component had a higher proportion of n-hexane and a lower proportion of isopropanol. The elution time of the initial solvent component was extended as much as possible to achieve baseline separation of impurities A and B. At 18 minutes, impurity A was eluted and was no longer affected by the peak of impurity B. The solvent component was then switched, and the elution capacity of the solvent component was improved by appropriately increasing the proportion of isopropanol and maintaining a certain elution time. Impurities B, C, and compound 1 eluted sequentially. At 40 minutes, all target related substances had eluted. The initial solvent component was switched back, the column was equilibrated, and the next injection was scheduled for detection.

[0036] In summary, the beneficial effects of this invention are:

[0037] This invention employs normal-phase high-performance liquid chromatography (HPLC) with hexane and isopropanol as the mobile phase, thereby improving elution intensity and separation capability. Gradient elution further enhances separation, peak shape, and sensitivity. During HPLC analysis, the initial solvent component has a higher proportion of hexane and a lower proportion of isopropanol, resulting in a gentler elution that allows impurities to be slowly eluted and separated. Subsequently, the solvent component is switched, increasing the proportion of isopropanol and decreasing the proportion of hexane to accelerate the elution rate. Finally, the initial solvent component is switched back to equilibrate the column. The HPLC method provided by this invention, using hexane and isopropanol as the mobile phase for gradient elution, achieves thorough separation between the target compound and its isomer impurities. The resolution between the main peak and adjacent impurity peaks, as well as between individual impurity peaks, is ≥1.5. The method has been validated through specificity, precision, accuracy, linearity, limit of quantitation, and limit of detection, accurately detecting the percentage content of related isomer impurities in the target compound. This method is rapid, simple, specific, sensitive, and accurate, with a wide range of applications and low cost, making it suitable for widespread application. Attached Figure Description

[0038] Figure 1 The HPLC spectrum of the blank solution provided in Example 1 of this invention;

[0039] Figure 2 This is the HPLC spectrum of the impurity A localization solution provided in Example 1 of the present invention;

[0040] Figure 3 This is the HPLC spectrum of the impurity B localization solution provided in Example 1 of the present invention;

[0041] Figure 4 This is the HPLC spectrum of the impurity C localization solution provided in Example 1 of the present invention;

[0042] Figure 5 The HPLC spectrum of the sample solution of compound 1 provided in Example 1 of this invention;

[0043] Figure 6 This is the HPLC spectrum of the mixed solution provided in Example 1 of the present invention.

[0044] Figure 7 This is the HPLC spectrum of the mixed solution provided in Comparative Example 1 of the present invention. Detailed Implementation

[0045] The present invention will now be described in further 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.

[0046] The purpose of this invention is to provide a sensitive, efficient, simple, reliable and accurate liquid chromatography method for the analysis of isomer impurities in bricetan intermediate (compound 1).

[0047] Example 1

[0048] In an embodiment of the analytical method of the present invention, the instruments and operating conditions are as follows:

[0049] Column: Daicel Chiral Pak IC 4.6×250mm, 5μm

[0050] Mobile phase A: n-hexane: isopropanol = 80:20

[0051] Mobile phase B: n-hexane: isopropanol = 60:40

[0052] Detection wavelength: 220nm

[0053] Flow rate: 1.0 mL / min

[0054] Column temperature: 30℃

[0055] Injection volume: 20 μL

[0056] Running time: 50min

[0057] Gradient procedure:

[0058]

[0059]

[0060] Solution preparation:

[0061] Diluent: Isopropanol.

[0062] Blank solution: diluent.

[0063] Control solution: Weigh 15 mg of impurity C reference standard, 20 mg each of impurity A and impurity B reference standards accurately, place them in the same 100 mL volumetric flask, add an appropriate amount of diluent and sonicate to dissolve, dilute to the mark with diluent, shake well, filter, and use as control stock solution. Accurately measure 1.0 mL of control stock solution, place it in a 10 mL volumetric flask, dilute to the mark with diluent, and shake well.

[0064] Systemic adaptability solution: Take 100 mg of compound 1 reference standard, place it in a 10 mL volumetric flask, add an appropriate amount of diluent and sonicate to dissolve, add 1.0 mL of reference stock solution, dilute to the mark with diluent, shake well, and filter.

[0065] Sample solution: Weigh 100 mg of compound 1 sample accurately, place it in a 10 mL volumetric flask, add an appropriate amount of diluent and sonicate to dissolve, dilute to the mark with diluent, shake well, and filter.

[0066] Testing procedure: Perform the testing according to the sample injection sequence in the table below.

[0067] Solution name Number of injections blank solution At least 2 times System Adaptation Solution 1 control solution 3 blank solution 1 Sample solution 1

[0068] System Applicability:

[0069] (1) The blank solution did not interfere with the relevant chromatographic peaks;

[0070] (2) In the system adaptability solution, the separation degree between each known substance should be ≥1.5.

[0071] (3) The control solution was injected three times consecutively, and the peak area RSD of each impurity should be ≤5.0%.

[0072] Calculation: Calculate the percentage content of impurities using the following formula:

[0073]

[0074] Wr: Weight of the impurity standard, in mg

[0075] Ws: Weight of sample of compound 1, mg

[0076] Ar: Peak area of ​​impurities in the control solution

[0077] As: Peak area of ​​impurities in the sample solution

[0078] P: Content of impurity standard

[0079] Authenticity Verification

[0080] Solution preparation:

[0081] Diluent: Isopropanol.

[0082] Blank solution: diluent.

[0083] Impurity A positioning solution: Weigh 20 mg of impurity A reference standard accurately, place it in a 100 ml volumetric flask, add an appropriate amount of diluent and sonicate to dissolve, dilute to the mark with diluent, shake well, and filter.

[0084] Impurity B positioning solution: Weigh 20 mg of impurity B reference standard accurately, place it in a 100 ml volumetric flask, add an appropriate amount of diluent and sonicate to dissolve, dilute to the mark with diluent, shake well, and filter.

[0085] Impurity C positioning solution: Weigh 15 mg of impurity C reference standard accurately, place it in a 100 ml volumetric flask, add an appropriate amount of diluent and sonicate to dissolve, dilute to the mark with diluent, shake well, and filter.

[0086] Sample solution: Weigh 100 mg of compound 1 sample accurately, place it in a 10 mL volumetric flask, add an appropriate amount of diluent and sonicate to dissolve, dilute to the mark with diluent, shake well, and filter.

[0087] Mixed solution: Take 100 mg of compound 1 sample, place it in a 10 mL volumetric flask, add an appropriate amount of diluent and sonicate to dissolve, add 1.0 mL each of impurity A positioning solution, impurity B positioning solution and impurity C positioning solution, dilute to the mark with diluent, shake well and filter.

[0088] Take 20 μL each of blank solution, positioning solution, sample solution and mixed solution, inject them into the liquid chromatograph and record the chromatograms.

[0089] The results are shown in Table 1 below, providing specific data for Embodiment 1 of the present invention.

[0090] Table 1 Specificity Verification

[0091]

[0092] The data above shows that the blank does not interfere with the analyte, and the impurities can be completely separated from each other and from the main peak, indicating good specificity.

[0093] See relevant detection chromatograms Figures 1-6 .

[0094] Figure 1 The HPLC spectrum of the blank solution provided in Example 1 of this invention;

[0095] Figure 2 This is the HPLC spectrum of the impurity A localization solution provided in Example 1 of the present invention;

[0096] Figure 3 This is the HPLC spectrum of the impurity B localization solution provided in Example 1 of the present invention;

[0097] Figure 4 This is the HPLC spectrum of the impurity C localization solution provided in Example 1 of the present invention;

[0098] Figure 5 The HPLC spectrum of the sample solution of compound 1 provided in Example 1 of this invention;

[0099] Figure 6 This is the HPLC spectrum of the mixed solution provided in Example 1 of the present invention.

[0100] Precision verification

[0101] Solution preparation:

[0102] Diluent: Isopropanol.

[0103] Control solution: Weigh 15 mg of impurity C reference standard, 20 mg each of impurity A and impurity B reference standards accurately, place them in the same 100 mL volumetric flask, add an appropriate amount of diluent and sonicate to dissolve, dilute to the mark with diluent, shake well, filter, and use as control stock solution. Accurately measure 1.0 mL of control stock solution, place it in a 10 mL volumetric flask, dilute to the mark with diluent, and shake well.

[0104] Systemic adaptability solution: Take 100 mg of compound 1 reference standard, place it in a 10 mL volumetric flask, add an appropriate amount of diluent and sonicate to dissolve, add 1.0 mL of reference stock solution, dilute to the mark with diluent, shake well, and filter.

[0105] Sample spiking solution: Accurately weigh 100 mg of compound 1 sample, place it in a 10 mL volumetric flask, add an appropriate amount of diluent and sonicate to dissolve, add 1.0 mL of control stock solution, dilute to the mark with diluent, shake well, and filter. Prepare 6 parallel aliquots, labeled P1-P6.

[0106] Take 20 μL of each of the above solvents and inject them into the liquid chromatograph, and record the chromatograms.

[0107] The results are shown in Table 2 below, providing precision data for Embodiment 1 of the present invention.

[0108] Table 2 Precision verification results

[0109] project Impurity A% Impurities B% Impurities C% P1 0.2107％ 0.2197％ 0.1586％ P2 0.2075％ 0.2147％ 0.1573％ P3 0.2088％ 0.2153％ 0.1614％ P4 0.2080％ 0.2135％ 0.1603％ P5 0.2082％ 0.2124％ 0.1612％ P6 0.2093％ 0.2117％ 0.1617％ average value 0.21％ 0.21％ 0.16％ RSD 0.55％ 1.33％ 1.10％

[0110] The RSD of each impurity is less than 10.0%, therefore the method has good precision.

[0111] Limit of Quantification

[0112] Referring to the signal-to-noise ratios of impurities A, B, and C under the specificity category, dilute stepwise until the concentration is no more than 50% of the limit concentration and the signal-to-noise ratio is no less than 10. This concentration is the LOQ.

[0113] The results are shown in Table 3 below, providing the limit of quantitation data for Example 1 of the present invention.

[0114] Table 3. Limits of Quantification Data

[0115] LOQ μg / ml S / N relative to sample concentration % Impurity A 6.213 40.7 0.062％ Impurity B 6.054 25.2 0.061％ Impurity C 4.428 34.2 0.044％

[0116] The data above show that the sensitivity of the method meets the measurement requirements.

[0117] Detection limit

[0118] Dilute the LOQ solution until the concentrations of impurities A, B, and C are no more than 50% of the LOQ concentration and the signal-to-noise ratio is no less than 3. This concentration is the LOD.

[0119] The results are shown in Table 4 below, providing detection limit data for Embodiment 1 of the present invention.

[0120] Table 4 Detection Limit Data

[0121] LOD μg / ml S / N relative to sample concentration % Impurity A 3.106 23.2 0.031％ Impurity B 3.027 12.8 0.030％ Impurity C 2.214 16.5 0.022％

[0122] The data above show that the sensitivity of the method meets the measurement requirements.

[0123] linear

[0124] Prepare linear solutions with impurity limits ranging from LOQ to 200%. Accurately measure 20 μL of each linear solution, inject it into the liquid chromatograph, record the chromatogram, and perform linear regression with concentration as the x-axis and peak area as the y-axis.

[0125] The results are shown in Table 5 below, providing linear data for Embodiment 1 of the present invention.

[0126] Table 5 Linear Data

[0127] project Linear equations Correlation coefficient R (≥0.99) Impurity A y = 2301.2x + 1.819 0.9995 Impurity B y = 1863.6x + 0.017 0.9997 Impurity C y = 1813.4x + 0.5014 0.9993

[0128] The data above show that the linearity of the method meets the measurement requirements.

[0129] Accuracy

[0130] Prepare accuracy solutions with LOQ~200% impurity limit concentrations. Accurately measure 20 μL of each accuracy solution, inject it into the liquid chromatograph, record the chromatogram, and calculate the recovery rate of each impurity.

[0131] The results are shown in Table 6 below, providing accuracy data for Embodiment 1 of the present invention.

[0132] Table 6 Accuracy Data

[0133]

[0134] At the LOQ (Lowest Level) spiked level, the average recovery rate of each impurity was in the range of 80% to 120%. At the 100%, 150%, and 200% LOQ levels, the average recovery rate of each impurity was in the range of 90% to 110%, indicating that the method has good accuracy.

[0135] Comparative Example 1

[0136] The difference between this comparative example and Example 1 is that the proportion of the mobile phase in the method is changed, and the elution method is changed from gradient elution to isocratic elution.

[0137] In this Comparative Example 1, the instruments and operating conditions are as follows:

[0138] Column: Daicel Chiral Pak IC 4.6×250mm, 5μm

[0139] Mobile phase: n-hexane:isopropanol = 70:30

[0140] Detection wavelength: 220nm

[0141] Flow rate: 1.0 mL / min

[0142] Column temperature: 30℃

[0143] Injection volume: 20 μL

[0144] Running time: 30 minutes

[0145] Solution preparation:

[0146] Diluent: Isopropanol.

[0147] Blank solution: diluent.

[0148] Control solution: Weigh 15 mg of impurity C reference standard, 20 mg each of impurity A and impurity B reference standards accurately, place them in the same 100 mL volumetric flask, add an appropriate amount of diluent and sonicate to dissolve, dilute to the mark with diluent, shake well, filter, and use as control stock solution. Accurately measure 1.0 mL of control stock solution, place it in a 10 mL volumetric flask, dilute to the mark with diluent, and shake well.

[0149] Systemic adaptability solution: Take 100 mg of compound 1 reference standard, place it in a 10 mL volumetric flask, add an appropriate amount of diluent and sonicate to dissolve, add 1.0 mL of reference stock solution, dilute to the mark with diluent, shake well, and filter.

[0150] Sample solution: Weigh 100 mg of compound 1 sample accurately, place it in a 10 mL volumetric flask, add an appropriate amount of diluent and sonicate to dissolve, dilute to the mark with diluent, shake well, and filter.

[0151] Mixed solution: Take 100 mg of compound 1 sample, place it in a 10 mL volumetric flask, add an appropriate amount of diluent and sonicate to dissolve, add 1.0 mL each of impurity A positioning solution, impurity B positioning solution and impurity C positioning solution, dilute to the mark with diluent, shake well and filter.

[0152] Testing procedure: Perform the testing according to the sample injection sequence in the table below.

[0153] Solution name Number of injections blank solution At least 2 times System Adaptation Solution 1 control solution 3 blank solution 1 Sample solution 1

[0154] System Applicability:

[0155] (1) The blank solution did not interfere with the relevant chromatographic peaks;

[0156] (2) In the system adaptability solution, the separation degree between each known substance should be ≥1.5.

[0157] (3) The control solution was injected three times consecutively, and the peak area RSD of each impurity should be ≤5.0%.

[0158] Calculation: Calculate the percentage content of impurities using the following formula:

[0159]

[0160] Wr: Weight of the impurity standard, in mg

[0161] Ws: Weight of sample of compound 1, mg

[0162] Ar: Peak area of ​​impurities in the control solution

[0163] As: Peak area of ​​impurities in the sample solution

[0164] P: Content of impurity standard

[0165] Conclusion: The results are as follows Figure 7 As shown, after changing the mobile phase ratio and changing the elution method from gradient elution to isocratic elution, the chromatographic peaks of impurity A and impurity B in the chromatogram could not be separated, and the peak shape of impurity C was poor. The resolution and sensitivity of this comparative method were not good.

[0166] Figure 7 This is the HPLC spectrum of the mixed solution provided in Comparative Example 1 of the present invention.

[0167] This invention utilizes liquid chromatography to achieve complete separation between the chromatographic peaks of the main component and its three structural isomers, with no interference from diluents or the main component in the detection of each impurity content. This method is highly specific, accurate, precise, and sensitive, with simple sample preparation and analytical procedures, and high detection efficiency.

[0168] 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 modifications made using the present invention are within the patent protection scope of the present invention.

Claims

1. A liquid chromatography method for the analysis of isomer impurities in briracetam intermediates, characterized in that: Includes the following steps: Step 1: Mix the bricetan intermediate (compound 1) sample with diluent to prepare a sample solution; Step 2: Use high performance liquid chromatography to detect the sample solution to determine the content of isomer impurities in the compound 1 sample; The detection conditions for the high-performance liquid chromatography are as follows: The mobile phases A and B of the high-performance liquid chromatography are both mixtures of n-hexane and isopropanol; the elution method is gradient elution. Step 3: Determine the content of isomer impurities in the sample solution, and calculate the content of isomer impurities A, B, and C in the compound 1 sample based on the peak area; The structures of compound 1 and its isomers, impurity A, impurity B, and impurity C are as follows:

2. The liquid chromatography method for isomer impurities in briracetam intermediates according to claim 1, characterized in that, The chromatographic column used in high performance liquid chromatography is a chiral column.

3. The liquid chromatography method for isomer impurities in briracetam intermediates according to claim 1, characterized in that, The solvent ratio in mobile phase A is 60-100:0-40 for n-hexane and isopropanol.

4. The liquid chromatography method for isomer impurities in briracetam intermediates according to claim 1, characterized in that, The solvent ratio in mobile phase B is 50-90:10-50 for hexane and isopropanol.

5. The liquid chromatography method for the analysis of related substances in briracetam intermediates according to claim 1, characterized in that, The mobile phase flow rate of the high performance liquid chromatograph is 0.8–1.2 mL / min.

6. The liquid chromatography method for isomer impurities in briracetam intermediates according to claim 1, characterized in that, The high-performance liquid chromatography detection wavelength is 200–240 nm.

7. The liquid chromatography method for isomer impurities in briracetam intermediates according to claim 1, characterized in that, The column temperature is 20℃~40℃.

8. The liquid chromatography method for isomer impurities in briracetam intermediates according to claim 1, characterized in that, The injection volume is 18 μL to 22 μL.

9. The liquid chromatography method for isomer impurities in bricetan intermediates according to claim 1, characterized in that, The mobile phase A and mobile phase B are eluted using the following elution procedure, wherein the percentages of mobile phase A and mobile phase B are volume percentages: Washing procedure: In the initial 0min stage of S1, the solvent ratio of the mobile phase is: mobile phase A is 100% and mobile phase B is 0%. At a certain time point in the 10-30 min stage, S2 switches the mobile phase solvent ratio to: mobile phase A is 0% and mobile phase B is 100%. At a certain point in the 30-50 min phase, S3 switches the mobile phase solvent ratio to: mobile phase A is 100% and mobile phase B is 0%; this continues until the liquid phase analysis ends after 50 min.

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