Method for separating and purifying impurities in tolterodine tartrate tablet

Abstract

The invention provides a method for separating and purifying impurities in a tolterodine tartrate tablet, the impurity 4, the impurity 9 and the impurity 10 with higher purity are prepared by twice preparative chromatographic separation from the tablet and combining high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) technologies, and the impurity separation efficiency and the structure identification accuracy are remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a method for separating and identifying impurities in tolterodine tartrate tablets, which is suitable for drug quality control and impurity tracing research. Background Art

[0002] Tolterodine tartrate tablets are a drug used to treat overactive bladder. Its chemical name is: 2-[3-[di(1-methylethyl)amino]-1-phenylpropyl]-4-methylphenol, chemical formula I,

[0003]

[0004] During the storage process, this product is affected by factors such as excipients and the external environment, and degradation produces a variety of trace impurities, which may affect the safety and effectiveness of the drug. The European Pharmacopoeia EP11.0 discloses several impurities related to the raw material of this product, among which impurity 10 is included, and impurity 4 and impurity 9 have not yet been studied and included. Except for isomers, the impurities related to the preparation of this product have not been included in the pharmacopoeia, and the impurity research is insufficient. The degradation of the preparation involves various polymer excipients in the prescription, and the degradation pathway is more complicated than that of the raw material. Studies have found that the potential impurities in the preparation of this product include at least impurities 4, impurity 9 and impurity 10, and their structures are shown below:

[0005]

[0006] Among them, impurity 4 has a potential toxic structure of aromatic aldehyde, impurity 9 has a potential toxic structure of aromatic halogen, and impurity 10 has a potential toxic structure of NO compound.

[0007] In the prior art, for impurity research, the API or preparation is often scanned by the LCMS method. After determining the molecular weight information of potential impurities, the potential molecular structure is inferred based on possible degradation pathways, and then the synthesis route is designed for quantitative synthesis. This method has the advantage of a large amount of impurity preparation, but there is often the possibility of reasoning errors, resulting in the synthesized impurities being similar to the real potential impurities in the sample only in mass spectrum information, but the chemical structure is indeed different, resulting in a waste of resources. Even in some extreme cases, the retention time of the liquid chromatography is basically the same, which brings fundamental misjudgment to the structural reasoning of the impurities and the reasoning of the degradation pathways.

[0008] In recent years, drug regulatory authorities have further strengthened the analysis and control of impurity research. For earlier-marketed products such as Tolterodine Tartrate Tablets, quality research must be conducted again in accordance with the "Technical Guidelines for Impurity Research of Chemical Drugs" and the requirements of ICHM7 genotoxic impurity research to reduce product quality risks.

[0009] The specific degradation impurities in the three preparations involved in the present invention, among which impurities 4 and 9 have not yet been included in the pharmacopoeia, have been detected in the preparations. At present, the quality inspection and management department of my country is carrying out the quality improvement work of tartrate tertorodine preparations, and the relevant impurity reference substances are the necessary prerequisite for conducting such research. At present, there is no report on the separation and purification method of these three impurities. Therefore, directly separating and purifying these three impurities from the preparations has positive practical significance for the study and quality control of genotoxic impurities of tolterodine. Summary of the invention

[0010] The present invention provides a method for separating and purifying impurities in tolterodine tartrate tablets, which significantly improves the impurity separation efficiency and structure identification accuracy by performing two preparative chromatographic separations from the tablets and combining high resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) technology. The technical solution of the present invention is as follows:

[0011] Step 1) Sample extraction: grinding the tolterodine tartrate tablets, then ultrasonically extracting with an acetonitrile aqueous solution, filtering, combining the extracts and freeze-drying to obtain a concentrated sample containing the target impurities after removing the excipients;

[0012] Step 2) Primary preparation and separation

[0013] Chromatographic conditions: Shimadzu LC 20AP system was used, and the chromatographic column was Luna C18 (50×250 mm, 10 μm);

[0014] Mobile phase: Phase A: 0.1% trifluoroacetic acid in water, phase B: acetonitrile;

[0015] The fractions of impurities 4, 9 and 10 were collected respectively and freeze-dried to obtain crude impurities 4, 9 and 10.

[0016] Since the content of the main drug is much greater than the content of impurities, removing the main component is also one of the main purposes of a single preparation and separation. The primary fractions of impurities 4, 9, and 10 collected by a single preparation and separation have relatively low purity, generally not more than 90%, and their purity is not yet sufficient to be used as impurity reference substances.

[0017] Step 3) Secondary preparation and separation

[0018] After the main component tertolodine was removed after the primary separation, the crude products of impurities 4, 9 and 10 with a purity of less than 90% were further purified using the following preparation conditions:

[0019] Chromatographic conditions: The chromatographic column was Synergi max-RP (30×150 mm, 4 μm);

[0020] Mobile phase: Phase A is 0.075% trifluoroacetic acid aqueous solution, phase B is 0.075% trifluoroacetic acid acetonitrile solution;

[0021] The fractions of impurities 4, 9, and 10 were collected respectively;

[0022] Impurities 4, 9 and 10 can be combined and then subjected to secondary preparation and separation, or impurities 4, 9 and 1 can be prepared and separated separately. It is preferred to combine the impurities for preparation and separation to improve the impurity separation efficiency.

[0023] Step 4) Take the fractions of impurity 4, impurity 9 and impurity 10 respectively and freeze-dry them to prepare impurities 4, impurity 9 and impurity 10, and their HPLC purity is greater than 98%. The impurities are used for HRMS and 1 Structural confirmation such as HNMR is used to confirm the chemical structure of impurities.

[0024] Furthermore,

[0025] In step 1), the mass volume ratio of the fine powder of tertolodine tartrate tablets to the acetonitrile aqueous solution is 1:2-10, preferably 1:5, and the concentration of the acetonitrile aqueous solution is selected from 30%-70%, preferably 50%, which is conducive to removing the insoluble excipients in the tablets and retaining the components to be separated as much as possible, thereby reducing the interference of the excipients.

[0026] In step 2), a gradient program is used for elution: 0-5 min (5% B→20% B), 5-45 min (20% B→50% B), 45-60 min (50% B→90% B);

[0027] Flow rate: 50-120 mL / min, preferably 90 mL / min;

[0028] Detection wavelength: 200nm~250nm, preferably 210nm, 220nm;

[0029] Sample concentration: using 50% acetonitrile as solvent, the sample concentration is selected from 0.5 mg to 50 mg / ml, preferably 10 mg / ml;

[0030] Injection volume: 0.5~5ml / time

[0031] In step 3), a gradient program was used for elution: 0 to 16 min (32% B → 37% B), 16.1 to 18 min (100% B);

[0032] Flow rate: 20-50 mL / min, preferably 30 mL / min,

[0033] Detection wavelength: 250-300 nm, preferably 281 nm;

[0034] Sample concentration: using 50% acetonitrile as solvent, the sample concentration is selected from 0.5 mg to 50 mg / ml, preferably 10 mg / ml;

[0035] Injection volume: 0.5~5ml / time

[0036] In each step of the present invention, the solvent is removed by freeze drying, and the conventional method of reduced pressure concentration is not used. Since the sample treatment and the fractions obtained by preparation contain a large proportion of water, the temperature required for direct concentration to remove the solvent is high and the time is long. Generally, the temperature of the concentrated aqueous solution needs to be close to 100° C., which leads to secondary degradation of impurities during the concentration process, which is not conducive to enriching high-purity impurity reference substances of impurities 4, impurities 9 and impurities 10.

[0037] The preparative liquid chromatography method provided by the present invention prepares specific potential genotoxic impurities in tolterodine tartrate tablets, directly separates and purifies them from tolterodine preparations, ensures the reliability of the impurity structure, avoids the possibility of misleading due to speculative directional synthesis, and has high impurity separation efficiency. The separation of three impurities can be achieved at the same time, and an impurity reference substance with high chromatographic purity can be obtained through secondary preparative separation, which meets the quality research and quality control of preparations and has positive practical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 : Impurity 4 after preparation and separation 1 HNMR spectrum

[0039] Figure 2 : Impurity 9 after preparation and separation 1 HNMR spectrum

[0040] Figure 3 : Impurity 10 after preparation and separation 1 HNMR spectrum

[0041] Figure 4 : Overlay HPLC chromatograms of impurities before and after preparation and separation DETAILED DESCRIPTION

[0042] To further illustrate the present invention, the method for separating and purifying impurities 4, 9 and 10 in tolterodine tartrate tablets by a preparative liquid phase method provided by the present invention is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0043] The tolterodine tartrate tablets, reagents, preparative liquid chromatograph, electronic analytical balance, etc. used in the specific implementation of the present invention are all known products.

[0044] Example 1 Separation and Preparation of Impurities 4, 9 and 10

[0045] 1) Take 2000 tolterodine tartrate tablets, grind them, take 100 g, add 500 ml of 50% acetonitrile, sonicate, filter, and freeze-dry to obtain a tolterodine concentrated sample rich in impurities.

[0046] 2) Primary separation and preparation

[0047] Chromatographic conditions: Shimadzu LC 20AP system was used, and the chromatographic column was Luna C18 (50×250 mm, 10 μm);

[0048] Mobile phase: Phase A is 0.1% trifluoroacetic acid aqueous solution, phase B is acetonitrile;

[0049] Gradient program: 0-5 min (5% B → 20% B), 5-45 min (20% B → 50% B), 45-60 min (50% B → 90% B);

[0050] Flow rate: 90 mL / min, detection wavelength 210 nm;

[0051] Sample concentration: 50% acetonitrile as solvent, sample concentration is 10 mg / ml;

[0052] Injection volume: 1ml / time

[0053] The fractions of impurities 4, 9 and 10 were collected and freeze-dried to obtain crude impurities 4, 9 and 10.

[0054] 3) Secondary separation and preparation

[0055] Impurities 4, 9 and 10 prepared by separation were combined and further purified. The preparation conditions were as follows:

[0056] Chromatographic conditions: The chromatographic column was Synergi max-RP (30×150 mm, 4 μm);

[0057] Mobile phase: Phase A is 0.075% trifluoroacetic acid aqueous solution, phase B is 0.075% trifluoroacetic acid acetonitrile solution;

[0058] Gradient program: 0-16 min (32% B → 37% B), 16.1-18 min (100% B);

[0059] Flow rate: 30 mL / min, detection wavelength 281 nm;

[0060] Sample concentration: 50% acetonitrile as solvent, sample concentration is 10 mg / ml;

[0061] Injection volume: 1ml / time

[0062] The target impurity fractions were collected and the purity after freeze-drying was greater than 99%.

[0063] Example 2 Structural Identification of Impurities 4, 9 and 10

[0064] Impurities 4, 9 and 10 were structurally confirmed by LCMS and NMR, and the results are as follows:

[0065] (1) High-resolution mass spectrometry (HRMS) analysis

[0066] Instrument: Agilent 6200 LC / MS system;

[0067] Chromatographic column: Shimpack CLC-ODS C18 column (6.0×150 mm, 5 μm);

[0068] Mobile phase: 20 mM ammonium formate (pH = 3.0)-acetonitrile, gradient elution (0-30 min, 10% B → 70% B);

[0069] Detection mode: positive ion mode, scanning range m / z 100~1000.

[0070] The results of the study showed that:

[0071] The mass spectrum signal of impurity 4 is m / e 340, which is [M+H] + , which is consistent with the molecular structure of this product

[0072] The mass spectrum signal of impurity 9 is m / e 346, which is [M+H] + , which is consistent with the molecular structure of this product

[0073] The mass spectrum signal of impurity 10 is m / e 342, which is [M+H] + , which is consistent with the molecular structure of this product

[0074] 2) Nuclear Magnetic Resonance ( 1 HNMR) analysis

[0075] Instrument: BrukerAVANCEIII 400MHz NMR instrument;

[0076] Sample treatment: The impurities were dissolved in deuterated reagents, and 1H NMR tests were performed on impurities 4, 9 and 10 respectively.

[0077] NMR attribution of impurity 4 (d-CD3OD): δ (ppm) 1.105~1.210 (12H, 4×-CH 3 ), 2.388 (2H, -CH 2 ), 2.795~2.993(2H,-CH 2 ), 3.582(2H, 2×-CH), 4.343(1H, -CH), 6.961~7.811(8H, Ar-H), 9.756(1H, -CHO), 11.010(1H, -OH), which is consistent with the molecular structure of this product.

[0078] NMR attribution of impurity 9 (d-CD3OD): δ (ppm) 1.122~1.215 (12H, 4×-CH 3 ), 2.363~2.480(2H,-CH 2 ), 2.7~3.0(2H, -CH 2 ), 3.589 (2H, 2×-CH), 4.286~4.306 (1H, -CH), 6.773~7.338 (8H, Ar-H), 9.899 (1H, -OH), which is consistent with the molecular structure of this product

[0079] NMR attribution of impurity 10 (DMSO-d6): δ (ppm) 0.918~1.359 (12H, 4×-CH 3 ), 2.264 (3H, -CH 3 ), 2.719~2.761(2H,-CH 2 ), 3.245~3.299(2H,-CH 2 ), 3.502~3.816(3H, 3×-CH), 6.670~6.796(3H, Ar-H), 7.333~7.746(5H, Ar-H) are consistent with the molecular structure of this product.

Claims

1. A method for separating and purifying impurities 4, 9 and 10 in tolterodine tartrate tablets, wherein the impurity structures are as follows: It is characterized in that The preparation process includes the following steps: Step 1): Sample extraction The tolterodine tartrate tablets are ground, then ultrasonically extracted with an acetonitrile aqueous solution, filtered, the extracts are combined, and freeze-dried to obtain a concentrated sample containing the target impurities after removing the excipients; Step 2): Primary preparation and separation Chromatographic conditions: Shimadzu LC 20AP system was used, and the chromatographic column was Luna C18 (50×250 mm, 10 μm); Mobile phase: Phase A is 0.1% trifluoroacetic acid aqueous solution, phase B is acetonitrile; The fractions of impurities 4, 9 and 10 were collected respectively and freeze-dried to obtain crude impurities 4, 9 and 10; Step 3): Secondary preparation and separation Chromatographic conditions: The chromatographic column was Synergi max-RP (30×150 mm, 4 μm); Mobile phase: Phase A is 0.075% trifluoroacetic acid aqueous solution, phase B is 0.075% trifluoroacetic acid acetonitrile solution; The fractions of impurities 4, 9, and 10 were collected respectively; Step 4): The fractions of impurity 4, impurity 9 and impurity 10 are taken and freeze-dried respectively to prepare impurities 4, impurity 9 and impurity 10, and the HPLC purity of impurities 4, impurity 9 and impurity 10 is greater than 98%.

2. The separation and purification method according to claim 1, characterized in that: In step 1), the mass volume ratio of the fine powder of tertolodine tartrate tablets to the acetonitrile aqueous solution is 1:2-10, and the concentration of the acetonitrile aqueous solution is selected from 30% to 70%; In step 2), a gradient program is used for elution: 0-5 min (5% B→20% B), 5-45 min (20% B→50% B), 45-60 min (50% B→90% B); The flow rate is 50-120 mL / min, the detection wavelength is 200 nm-250 nm, the injection volume is 0.5-5 ml / time, the sample is 50% acetonitrile as solvent, and the sample concentration is 0.5-50 mg / ml; In step 3), a gradient program was used for elution: 0 to 16 min (32% B → 37% B), 16.1 to 18 min (100% B); The flow rate is 20-50 mL / min, the detection wavelength is 250-300 nm, the injection volume is 0.5-5 ml / time, the sample uses 50% acetonitrile as solvent, and the sample concentration is 0.5-50 mg / ml.

3. The separation and purification method according to claim 2, characterized in that: In step 1), the mass volume ratio of tertolodine tartrate tablets fine powder to acetonitrile aqueous solution is 1:5, and the concentration of acetonitrile aqueous solution is 50%; in step 2), the flow rate is 90 mL / min, the detection wavelength is 210 nm, the injection volume is 1 ml / time, the sample uses 50% acetonitrile as solvent, and the sample concentration is 10 mg / ml; Step 3) The flow rate was 30 mL / min, the detection wavelength was 281 nm; the injection volume was 1 ml / time, the sample used 50% acetonitrile as solvent, and the sample concentration was 10 mg / ml.

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