Method for detecting isomer of nebivolol intermediate

Abstract

The invention discloses a method for detecting an isomer of a nebivolol intermediate, which solves the problem of sample dissolution and improves the peak shape and separation degree of a chromatographic peak by optimizing a solvent for preparing a sample and liquid chromatographic conditions. A convenient and reliable detection method is provided for industrial production of the nebivolol intermediate, and the nebivolol bulk drug is high in chiral purity and more stable in quality through effective control of upstream quality.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for detecting isomers of a nebivolol intermediate, in particular to a method for detecting isomers of a nebivolol intermediate capable of quality control. BACKGROUND

[0002] Nebivolol Hydrochloride is developed by Johnson & Johnson Company in the United States, which is a strong, selective third-generation liposoluble beta receptor blocker with vasodilator effect, and can protect the cardiovascular while reducing blood pressure.

[0003] The stereochemical configuration of chiral drugs is closely related to pharmacodynamics, pharmacokinetics and toxicology, and the biological activity of stereoisomers of different configurations often has differences. Therefore, the chiral impurity control of chiral drugs is very important, and is also a key point of distinguishing from other drug quality research and control. For drugs containing multiple chiral centers, it is difficult to control multiple chiral impurities in the final product, and it is necessary to control them in stages. In addition, diastereomeric impurities are more likely to occur than enantiomeric impurities. Therefore, in addition to monitoring enantiomeric impurities, it is necessary to study and monitor diastereomeric impurities.

[0004]

[0005] Nebivolol contains four chiral centers and ten stereoisomers, which are chiral drugs constructed by two structural units of SSSR and RRRS. According to the synthesis process of nebivolol, the four chiral centers are derived from the intermediate (N-benzyl-L / D-nebivolol) of the previous step of the final product. Among them, the intermediate of (S, S, S, R)-configuration is N-benzyl-L-nebivolol, and the intermediate of (R, R, R, S,)-configuration is N-benzyl-D-nebivolol. The optical purity of the above intermediate needs to be controlled in the synthesis process of the drug substance, so as to ensure the quality of the final product, and the existing detection method has not realized effective separation and quantitative detection of N-benzyl-L / D-nebivolol isomer impurities. SUMMARY

[0006] The present application aims to provide a detection method which is specifically for the isomer impurities of nebivolol intermediate N-benzyl-L / D-nebivolol and can effectively elute, separate and quantitatively detect.

[0007] The technical scheme of the present application is a method for detecting isomers of a nebivolol intermediate having the following structure,

[0008]

[0009] comprising the following steps:

[0010] (1) Preparation of sample: the sample is prepared into a set concentration with alcohol solvent;

[0011] (2) Detection: a normal phase chiral column is used, n-hexane is used as mobile phase A, and a mixed solution of basic charge transfer agent and low alkyl alcohol 1, low alkyl alcohol 2 is used as mobile phase B for elution, and the volume fraction of the mobile phase A is not less than 85%;

[0012] (3) Analysis: the chromatogram is recorded, and the content of isomers is calculated.

[0013] In step (1), the prepared sample specifically comprises:

[0014] a. Test solution: after N-benzyl-nepib is dissolved in alcohol solvent, the same alcohol solvent is added for quantitative dilution to a set concentration to obtain a test solution, and the specific concentration of the test solution is 0.5-3.0 mg / ml, preferably 2.5 mg / ml;

[0015] b. Positioning solution: after the control sample of N-benzyl-nepib and its isomers is dissolved in alcohol solvent, the same alcohol solvent is added for quantitative dilution to a set concentration to obtain a positioning solution, and the specific concentration of the positioning solution is 0.05-3.0 mg / ml, preferably 0.1 mg / ml.

[0016] In step (3), the test solution and the positioning solution are injected respectively, the chromatogram is recorded, and the content of isomer impurities of the nepib intermediate is calculated by area normalization method.

[0017] Preferably, the isomers of the nepib intermediate are (S, R, S, R)-N-benzyl-nepib, (R, S, S, R)-N-benzyl-nepib, (S, S, R, S)-N-benzyl-nepib, (S, S, S, S)-N-benzyl-nepib, (S, R, R, S)-N-benzyl-nepib, (R, R, S, R)-N-benzyl-nepib, and (R, R, R, R)-N-benzyl-nepib.

[0018]

[0019] Preferably, the alcohol solvent in step (1) is one or two of methanol, anhydrous ethanol, and isopropanol.

[0020] Further preferably, the alcohol solvent is methanol, anhydrous ethanol, isopropanol, methanol-anhydrous ethanol, methanol-isopropanol, and anhydrous ethanol-isopropanol, wherein the volume fraction of each alcohol solvent in the mixed alcohol solvent is 1%-99%.

[0021] More preferably, the alcohol solvent is methanol.

[0022] Preferably, the volume ratio of mobile phase A to mobile phase B in step (2) is 85:15.

[0023] Preferably, the basic charge-migrating agent in step (2) is at least one of diethylamine, triethylamine, ethanolamine.

[0024] Further preferably, the basic charge-migrating agent is diethylamine, triethylamine, ethanolamine, diethylamine-triethylamine, diethylamine-ethanolamine, triethylamine-ethanolamine, diethylamine-triethylamine-ethanolamine, wherein the volume fraction of each basic charge-migrating agent in the mixed basic charge-migrating agent solution is 1% to 99%.

[0025] More preferably, the basic charge-migrating agent is ethanolamine.

[0026] Preferably, the lower alkyl alcohol 1 and the lower alkyl alcohol 2 in step (2) are methanol, anhydrous ethanol, isopropanol.

[0027] Further preferably, the lower alkyl alcohol 1 and the lower alkyl alcohol 2 are different.

[0028] More preferably, the mixed solution of the lower alkyl alcohol 1 and the lower alkyl alcohol 2 is methanol-anhydrous ethanol, methanol-isopropanol, isopropanol-anhydrous ethanol.

[0029] Further more preferably, the mixed solution of the lower alkyl alcohol 1 and the lower alkyl alcohol 2 is isopropanol-anhydrous ethanol.

[0030] Preferably, in the mobile phase B, the volume ratio of the lower alkyl alcohol 1 to the lower alkyl alcohol 2 in step (2) is 15:0 to 0:15.

[0031] Further preferably, in the mobile phase B, the volume ratio of isopropanol to anhydrous ethanol is 10:5.

[0032] Preferably, in the mobile phase B, the added volume of the basic charge-migrating agent in step (2) is 0.1% to 0.5% of the total volume of the lower alkyl alcohol 1 and the lower alkyl alcohol 2.

[0033] Further preferably, the added volume of the basic charge-migrating agent is 0.4% of the total volume of the lower alkyl alcohol 1 and the lower alkyl alcohol 2.

[0034] Preferably, in the mobile phase, the mobile phase A is n-hexane, the mobile phase B is a 0.4% (V / V) ethanolamine (isopropyl alcohol: anhydrous ethanol = 10:5, V / V) mixed solution, and the mobile phase A:mobile phase B = 85:15 (V / V);the specific preparation method is as follows: first, prepare the isopropyl alcohol-anhydrous ethanol mixed solution according to the proportion of 10:5 (V / V), then add 0.4% (V / V) ethanolamine in the isopropyl alcohol-anhydrous ethanol mixed solution to obtain the mobile phase B, and finally mix the mobile phase A and the mobile phase B according to the proportion of 85:15 (V / V) to obtain the mobile phase.

[0035] Preferably, the chromatographic column in step (2) is filled with silica gel coated with amylose-tris (3, 5-dimethylphenyl carbamate) on the surface.

[0036] Further preferably, the specification of the chromatographic column is 4.6*250mm, 5μm.

[0037] More preferably, the chromatographic column is Daicel CHIRALPAK AD-H.

[0038] The Daicel CHIRALPAK AD-H stationary phase contains a hydrophilic glycoside structure and a hydrophobic benzene ring structure. The molecular structure of N-benzyl-naratriptol contains groups such as F, OH, ether group, amino group and other hydrogen bonding sites, which can form hydrogen bonds with O in the carbonyl group and H in the amide group of the stationary phase structure; in addition, the molecular structure of N-benzyl-naratriptol also contains a benzene ring, which can produce π-π electron interaction with the benzene ring in the stationary phase. The naratriptol intermediate is a basic compound, which can cause secondary action with the exposed silanol group in the chiral column, resulting in poor peak shape, and therefore the addition of basic charge shift reagent in the mobile phase can improve the peak shape.

[0039] Preferably, the detection wavelength in step (2) is 250-290nm.

[0040] Further preferably, the detection wavelength is 280nm.

[0041] Preferably, the column temperature of the chromatographic column in step (2) is 20-35℃, the flow rate of the mobile phase is 0.8-1.2ml / min, and the injection volume is 5μl-10μl.

[0042] Further preferably, the column temperature of the chromatographic column is 23-28℃, the flow rate of the mobile phase is 1.0ml / min, and the injection volume is 10μl.

[0043] More preferably, the column temperature of the chromatographic column is 25℃.

[0044] Beneficial effects: Compared with the prior art, the present application has the following remarkable advantages:

[0045] The present application adopts liquid chromatography to control the quality of various isomers of nebivolol intermediates. Through optimization of the solvent for preparing the sample and the liquid chromatography conditions, the sample dissolution problem is solved, the chromatographic peak shape and resolution are improved, and effective separation of the chromatographic peaks of the intermediate and each isomer is achieved. A convenient and reliable detection method is provided for industrial production of nebivolol intermediates, and through effective control of the upstream quality, the chiral purity of nebivolol bulk drug is high, and the quality is more stable. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 HPLC detection chromatogram of the positioning solution of Comparative Example 1;

[0047] Figure 2 HPLC detection chromatogram of the positioning solution of Comparative Example 2;

[0048] Figure 3 HPLC detection chromatogram of the positioning solution-1 of Comparative Example 3;

[0049] Figure 4 HPLC detection chromatogram of the positioning solution-1 of Comparative Example 4;

[0050] Figure 5 HPLC detection chromatogram of the positioning solution-2 of Comparative Example 4;

[0051] Figure 6 HPLC detection chromatogram of the positioning solution-2 of Example 1;

[0052] Figure 7 HPLC detection chromatogram of the test sample solution of Example 2;

[0053] Figure 8 HPLC detection chromatogram of the test sample solution of Example 3. DETAILED DESCRIPTION

[0054] The technical solutions of the present application are further described below in combination with examples.

[0055] Comparative Example 1

[0056] 1. Detection method

[0057] (1) Preparation of the positioning solution: take N-benzyl-L-nebivolol (SSSR configuration), N-benzyl-D-nebivolol (RRRS configuration) and other isomers [SRSR configuration reference substance, RSSR configuration reference substance, SSRS configuration reference substance, SSSS configuration reference substance, SRRS configuration reference substance, RRSR configuration reference substance, RRRR configuration reference substance, respectively] each in an appropriate amount, dissolve in methanol and dilute quantitatively to about 0.25 mg per 1 ml of mixed solution.

[0058] (2) Chromatographic conditions were set as follows: The high performance liquid chromatograph used was an Agilent 1260 liquid chromatograph, the column was Daicel CHIRALPAKAD-H (4.6×250mm, 5μm), the injection volume was 20μl; the mobile phase was 0.1% diethylamine-anhydrous ethanol (V / V), isocratic elution, the flow rate was 1.0ml / min; the column temperature was 40℃; the detection wavelength of the ultraviolet detector was 280nm, and the running time was 150min.

[0059] (3) Determination and analysis: Inject the local solution, record the chromatogram, and calculate the content of N-benzyl-L / D-nebivolol isomers by area normalization method.

[0060] 2. Test Results

[0061] Following the normal-phase chromatographic method of the European Pharmacopoeia, the analysis time was extended to 150 min, and then... Figure 1 It can be seen that the resolution of the RRSR and RRRR isomers of N-benzyl-D-nebivolol and the co-eluted isomers is 1.29. The peak shape of the SSRS isomer is poor, and the SSSS isomer did not elute within 150 min, but eluted in the chromatogram of the next injection sample solution. The total elution time was about 260 min, and the peak shape was inverted triangle.

[0062] Comparative Example 2

[0063] 1. Detection Method

[0064] (1) Preparation of localization solution: Take appropriate amounts of N-benzyl-L-nebivolol (SSSR configuration) and its four isomers [RRRS configuration reference, SRSR configuration reference, SRRS configuration reference, and SSRS configuration reference, respectively], dissolve them in methanol and quantitatively dilute them to a mixed solution containing approximately 0.2 mg of each per 1 ml.

[0065] (2) Chromatographic conditions were set as follows: The high performance liquid chromatograph used was an Agilent 1260 liquid chromatograph, the column was Daicel CHIRALPAKAD-H (4.6×250mm, 5μm), the injection volume was 10μl; the mobile phase A was n-hexane, the mobile phase B was 0.4% ethanolamine-isopropanol, the isocratic elution was performed, the flow rate was 1.0ml / min, the mobile phase A: mobile phase B = 70:30 (V / V); the column temperature was 25℃; the detection wavelength of the ultraviolet detector was 280nm, and the run time was 110min.

[0066] (3) Determination and analysis: Inject the local solution, record the chromatogram, and calculate the content of N-benzyl-L / D-nebivolol isomers by area normalization method.

[0067] 2. Test results

[0068] By Figure 2 It can be seen that under this chromatographic condition, only three chromatographic peaks are eluted, in which the two isomers of SRSR configuration and SRRS configuration are co-eluted, and N-benzyl-L-nepibrol and the isomer of SSRS configuration are co-eluted.

[0069] Comparative Example 3

[0070] 1. Test method

[0071] The difference from Comparative Example 1 is that the mobile phase B of the chromatographic condition is 0.4% ethanolamine-isopropyl alcohol, and the mobile phase A: mobile phase B = 85:15 (V / V) when eluted isothermally, and the rest of the analysis conditions are the same as those of Comparative Example 1.

[0072] 2. Test results

[0073] By Figure 3 It can be seen that compared with Comparative Example 1, after reducing the elution strength of the mobile phase B, the co-eluted chromatographic peaks have a separation trend, in which the separation degree of the isomers of SRSR configuration and SRRS configuration is 0.94, and the separation degree of N-benzyl-L-nepibrol and the isomer of SSRS configuration is 0.70. It is illustrated that the alcohol substance in the mobile phase participates in the competition of hydrogen bond. Since the volume of isopropyl alcohol molecule is larger than that of ethanol molecule, the steric hindrance of isopropyl alcohol forming hydrogen bond is larger. When the addition amount of isopropyl alcohol in the mobile phase is reduced, the ability of the mobile phase molecule to participate in the competition of hydrogen bond is weaker, so the difference between the action of the isomers of SRSR configuration and SRRS configuration, N-benzyl-L-nepibrol and the isomer of SSRS configuration and the chiral stationary phase is larger, and the separation degree is increased.

[0074] Comparative Example 4

[0075] 1. Test method

[0076] (1) Preparation of positioning solution-1: take N-benzyl-L-nepibrol (SSSR configuration) and its four isomers [RRRS configuration reference substance, SRSR configuration reference substance, SRRS configuration reference substance, and SSRS configuration reference substance, respectively] in appropriate amounts, dissolve and quantitatively dilute with methanol to obtain a mixed solution containing about 0.2 mg per 1 ml.

[0077] (2) Preparation of positioning solution-2: take N-benzyl-L-nepibrol (SSSR configuration), N-benzyl-D-nepibrol (RRRS configuration), and other isomers [SRSR configuration reference substance, RSSR configuration reference substance, SSRS configuration reference substance, SSSS configuration reference substance, SRRS configuration reference substance, RRSR configuration reference substance, RRRR configuration reference substance, respectively] in appropriate amounts, dissolve and quantitatively dilute with methanol to obtain a mixed solution containing about 0.1 mg per 1 ml.

[0078] (2) Chromatographic conditions were set as follows: The high performance liquid chromatograph used was an Agilent 1260 liquid chromatograph, the column was Daicel CHIRALPAKAD-H (4.6*250mm, 5μm), the injection volume was 10μl; the mobile phase A was n-hexane, the mobile phase B was 0.4% ethanolamine (isopropanol: anhydrous ethanol = 12:3), the isocratic elution was performed with a flow rate of 1.0ml / min and a mobile phase A: mobile phase B = 85:15 (V / V); the column temperature was 25℃, the detection wavelength of the UV detector was 280nm, and the run time was 80min.

[0079] (3) Determination and analysis: The local solution was injected, the chromatogram was recorded, and the contents of the eight isomers of N-benzyl-L / D-nebivolol were calculated by the area normalization method.

[0080] 2. Test Results

[0081] Depend on Figure 4 As can be seen, after adding 20% ​​anhydrous ethanol to mobile phase B, the resolution between the SRSR and SRRS isomers was 1.73; while the resolution between the N-benzyl-L-nebivolol and the SSRS isomers was 1.37. If the main peak concentration is too high, it will affect the accurate quantification of the SSRS isomers. The addition of ethanol increases the resolution, and the participation of ethanol molecules is more conducive to the recognition of isomers by the chiral stationary phase. Figure 5 It is evident that the separation degree of N-benzyl-D nebivolol from its various isomers is greater than 1.5, with the separation degree of SRRS and RSSR configuration isomers being 0.96, which need to be combined for control.

[0082] Example 1

[0083] 1. Detection Method

[0084] The difference from Comparative Example 4 is that the chromatographic conditions are as follows: mobile phase B is 0.4% ethanolamine (isopropanol: anhydrous ethanol = 10:5), mobile phase A: mobile phase B = 85:15 (V / V) during isocratic elution, and the other analytical conditions are the same as those of Comparative Example 3.

[0085] 2. Test Results

[0086] Depend on Figure 6 As can be seen, when 33.3% anhydrous ethanol was added to mobile phase B, the resolution of N-benzyl-L-nebivolol and the SSRS configuration isomer was 1.90. Except for the SRRS and RSSR configuration isomers, which had a resolution of 0.46 (requiring combined control), all other isomer impurities could be effectively separated, with resolutions greater than 1.5 between them, and there was no detection interference.

[0087] Example 2

[0088] 1. Detection method

[0089] (1) Preparation of test solution: Take about 25 mg of N-benzyl-L-nepibrol and place it in a 10 ml volumetric flask. Dissolve and dilute to prepare a solution containing about 2.5 mg per 1 ml.

[0090] (2) Preparation of positioning solution: Take N-benzyl-L-nepibrol (SSSR configuration), N-benzyl-D-nepibrol (RRRS configuration) and other isomers [SRSR configuration reference substance, RSSR configuration reference substance, SSRS configuration reference substance, SSSS configuration reference substance, SRRS configuration reference substance, RRSR configuration reference substance, RRRR configuration reference substance, respectively] in appropriate amounts, dissolve in methanol and dilute to prepare a mixed solution containing about 0.1 mg per 1 ml.

[0091] (3) Set the chromatographic conditions: The high performance liquid chromatograph used is Agilent 1260 liquid chromatograph, the chromatographic column is Daicel CHIRALPAK AD-H (4.6x250mm, 5μm), the injection volume is 10μl; the mobile phase A is n-hexane, the mobile phase B is 0.4% ethanolamine (isopropyl alcohol: anhydrous ethanol = 10:5), isocratic elution, the flow rate is 1.0ml / min, the mobile phase A: mobile phase B = 85:15 (V / V); the column temperature is 25℃, the detection wavelength of the ultraviolet detector is 280nm, and the running time is 80min.

[0092] (4) Determination and analysis: The test solution and the positioning solution are injected respectively, the chromatogram is recorded, and the content of the eight isomers of N-benzyl-L / D-nepibrol is calculated by area normalization method.

[0093] 2. Detection results

[0094] From Figure 7 It can be seen that when the proportion of RSSR configuration isomer in the chromatogram of N-benzyl-L-nepibrol test solution is 0.03%, the S / N is 14.7; when the proportion of SSSS configuration isomer is 0.03%, the S / N is 4.7.

[0095] The methodological verification of the analysis and determination method provided in Example 2 is carried out, and the results are as follows:

[0096]

[0097] According to the above methodological verification results, it can be known that the determination method of N-benzyl-nepibrol and the content of eight isomers designed by the application has high sensitivity, strong specificity, high precision, and good chromatographic condition durability, and can be used for quality control of eight isomers in N-benzyl-nepibrol.

[0098] Example 3

[0099] 1. Detection method

[0100] The difference from Example 2 is that the test sample is replaced by N-benzyl-D-nepivolo with N-benzyl-L-nepivolo.

[0101] The rest of the analysis conditions are the same as those in Example 2.

[0102] 2. Detection results

[0103] From Figure 8 It can be seen that when the proportion of SRRS configuration isomer in the chromatogram of N-benzyl-D-nepivolo test sample solution is 0.03%, the S / N is 14.1; when the proportion of SSSS configuration isomer is 0.04%, the S / N is 5.7.

Claims

1. A method for detecting isomers of nebivolol intermediates, wherein the structure of the nebivolol intermediates is as follows: Its features are, Includes the following steps: (1) Sample preparation: Prepare the sample to the set concentration using an alcohol solvent; (2) Detection: A normal-phase chiral chromatographic column was used, with n-hexane as mobile phase A and a mixed solution of basic charge transfer agent and lower alkyl alcohol 1 and lower alkyl alcohol 2 as mobile phase B for elution. The volume fraction of mobile phase A was not less than 85%. (3) Analysis: Record the chromatogram and calculate the content of isomers.

2. The detection method according to claim 1, characterized in that, The isomers of the nebivolol intermediate are (S,R,S,R)-N-benzyl-nebivolol, (R,S,S,R)-N-benzyl-nebivolol, (S,S,R,S)-N-benzyl-nebivolol, (S,S,S,S)-N-benzyl-nebivolol, (S,R,R,S)-N-benzyl-nebivolol, (R,R,S,R)-N-benzyl-nebivolol, and (R,R,R,R)-N-benzyl-nebivolol.

3. The detection method according to claim 1, characterized in that, The alcohol solvent mentioned in step (1) is one or two of methanol, anhydrous ethanol, and isopropanol.

4. The detection method according to claim 1, characterized in that, The alkaline charge-transfer agent mentioned in step (2) is at least one of diethylamine, triethylamine, and ethanolamine.

5. The detection method according to claim 1, characterized in that, The lower alkyl alcohol 1 and lower alkyl alcohol 2 mentioned in step (2) are methanol, anhydrous ethanol, and isopropanol.

6. The detection method according to claim 1, characterized in that, In mobile phase B, the volume ratio of lower alkyl alcohol 1 to lower alkyl alcohol 2 described in step (2) is 15:0 to 0:

15.

7. The detection method according to claim 1, characterized in that, In mobile phase B, the volume of the basic charge transfer agent added in step (2) is 0.1% to 0.5% of the total volume of lower alkyl alcohol 1 and lower alkyl alcohol 2.

8. The detection method according to claim 1, characterized in that, The chromatographic column described in step (2) is packed with silica gel coated with amylose-tris(3,5-dimethylphenylcarbamate).

9. The detection method according to claim 1, characterized in that, The detection wavelength for step (2) is 250–290 nm.

10. The detection method according to claim 1, characterized in that, The column temperature of the chromatographic column in step (2) is 20-35℃, and the flow rate of the mobile phase is 0.8-1.2 ml / min.

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