Quantitative analysis method for potential genotoxic impurities in cariprazine hydrochloride

Abstract

The invention discloses a quantitative analysis method of potential genotoxic impurities in cariprazine hydrochloride, which adopts a liquid chromatography-mass spectrometry method, takes a 20mM ammonium acetate aqueous solution as a mobile phase A, takes acetonitrile as a mobile phase B, and adopts a reversed-phase liquid chromatographic column with octadecylsilane bonded silica gel filler as a filler for elution. According to the method, bis (2-chloroethyl) amine can be effectively eluted, separated and quantitatively analyzed, the quality of a cariprazine hydrochloride product can be better controlled, and a simple, convenient, accurate, rapid and reliable detection method is provided for industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of compound quantitative analysis method development, and relates to a quantitative analysis method for potential genotoxic impurities in cariprazine hydrochloride. Background Art

[0002] Cariprazine hydrochloride is an atypical antipsychotic drug and a partial agonist at both the dopamine D2 and D3 receptors. It is primarily used to treat schizophrenia and bipolar mania in adults. Cariprazine is a D3 / D2 receptor partial agonist that preferentially binds to the D3 receptor, offering advantages over traditional psychotropics (which act solely on the D2 receptor) such as a lower incidence of extrapyramidal side effects and improved treatment of negative symptoms. Bis(2-chloroethyl)amine was a key genotoxic impurity in the quality studies of cariprazine hydrochloride.

[0003] The genotoxic impurity bis(2-chloroethyl)amine (structural formula is as follows) may be produced during the synthesis of cariprazine hydrochloride.

[0004] This impurity has a potential carcinogenic risk and must be strictly controlled below 0.025%.

[0005] Existing detection methods using ultraviolet spectroscopy have limitations. They lack characteristic absorption groups and are not sensitive enough. Bis(2-chloroethyl)amine has a simple molecular structure and does not contain a conjugated system or strong chromophores. It has no significant characteristic absorption peak in the ultraviolet-visible light region (200-400nm), making it impossible to effectively detect it using ultraviolet spectroscopy. Furthermore, the permitted limit for this impurity in pharmaceuticals is usually below the ppm level (for example, the ICH M7 guideline requires that its content be strictly controlled below 0.025%). However, the limit of quantification (LOQ) of ultraviolet spectroscopy is usually 0.03% to 1%, which is difficult to meet the needs of trace detection. Gas chromatography also has limitations in its applicability. Bis(2-chloroethyl)amine has high polarity and a small molecular weight (142.03 g / mol). Direct injection can easily lead to decomposition due to poor thermal stability, making it difficult to obtain stable chromatographic peaks using conventional GC analysis. Furthermore, the complex matrix components in the cariprazine hydrochloride API (such as the main component and its degradation products) can mask the chromatographic signal of the target impurity, necessitating the use of high-resolution columns or derivatization pretreatment, conditions that are typically not optimized in conventional GC methods.

[0006] In summary, conventional structural analysis methods such as UV spectroscopy and gas chromatography are unable to quantitatively analyze this impurity at low concentrations, resulting in product quality control deficiencies that require improvement. Therefore, there is an urgent need to develop a highly sensitive, selective, and stable analytical method. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for quantitative analysis of potential genotoxic impurities in cariprazine hydrochloride, so as to overcome the problems existing in the background technology and facilitate the quantitative analysis of the content of the compound bis(2-chloroethyl)amine.

[0008] A method for quantitative analysis of potential genotoxic impurities in cariprazine hydrochloride, using a liquid chromatography-mass spectrometry method, comprises the following steps: (1) Preparation of sample solution, specifically: Test solution: Take cariprazine hydrochloride, dissolve it in methanol and dilute it quantitatively to make a solution containing 5.00 mg of cariprazine hydrochloride per 1 ml, shake well and set aside; Recovery solution: Take appropriate amounts of bis(2-chloroethyl)amine and cariprazine hydrochloride samples, dissolve them in methanol and dilute them quantitatively to prepare a solution containing 5 mg of cariprazine hydrochloride and 1.25 μg of bis(2-chloroethyl)amine per 1 ml, shake well and set aside; Impurity localization solution: Take an appropriate amount of bis(2-chloroethyl)amine sample, dissolve it in methanol and quantitatively dilute it to make a solution containing 1.25 μg of bis(2-chloroethyl)amine per 1 ml, shake well and set aside; (2) Detection: A reversed-phase liquid chromatography column was used, with ammonium acetate aqueous solution as mobile phase A and acetonitrile as mobile phase B, with gradient elution, and a liquid chromatography-mass spectrometry detector was used as the detector; (3) Result analysis: Record the chromatogram and calculate the content of bis(2-chloroethyl)amine using the external standard method.

[0009] Wherein, bis(2-chloroethyl)amine structure is as follows: Preferably, in step (2), an aqueous ammonium acetate solution is used as mobile phase A, and more preferably, a 20 mM ammonium acetate solution is used as mobile phase A.

[0010] Preferably, the chromatographic column has an inner diameter of 3.0 to 5.0 mm, a length of 100 to 250 mm, and a filler particle size of 2 to 5 μm.

[0011] The reversed-phase liquid chromatography column is filled with octadecylsilane bonded silica gel.

[0012] Preferably, the temperature of the chromatographic column in step (2) is set to 28°C~32°C.

[0013] Preferably, the detection time of the liquid chromatography-mass spectrometry detector is 30 minutes.

[0014] Preferably, single ion monitoring (SIM) mode is used, the monitoring ion is m / z 141.7 (positive ion mode), the drying gas flow rate is 12.0 L / min, the temperature is 350° C., the nebulizer pressure is 55 psig, and the spray voltage is 4000 V. Beneficial effects

[0015] The liquid chromatography-mass spectrometry quantitative analysis method provided by the present invention sets the molecular weight in the mass spectrometry single ion monitoring (SIM) mode to m / z 141.7, and can obtain a single mass spectrum peak, which can be used to determine that the compound is bis(2-chloroethyl)amine, and detect this potential genotoxic impurity, thereby controlling its content to not exceed 0.025%, improving product quality control, and ensuring product safety and effectiveness. After comprehensive methodological verification, the present invention has strong specificity, high sensitivity, high accuracy, and good stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Positioning solution chromatogram for Example 1; Figure 2 The recovery solution chromatogram of Example 1 is shown; Figure 3 Positioning solution chromatogram for Example 2; Figure 4 The recovery solution chromatogram of Example 2 is shown; Figure 5 Positioning solution chromatogram for Example 3; Figure 6 The recovery solution chromatogram of Example 3 is shown. DETAILED DESCRIPTION

[0017] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0018] Example 1 (liquid phase, C18 column) Sample solution preparation: Positioning solution: Take an appropriate amount of bis(2-chloroethyl)amine sample, dissolve it in methanol and quantitatively dilute it to make a solution containing approximately 10 μg of bis(2-chloroethyl)amine per 1 ml, shake well and set aside.

[0019] Recovery solution: Take appropriate amount of bis(2-chloroethyl)amine and cariprazine hydrochloride samples respectively, dissolve them in methanol and dilute them quantitatively to make a solution containing approximately 40 mg of cariprazine hydrochloride and 10 μg of bis(2-chloroethyl)amine per 1 ml, shake well and set aside.

[0020] Diluent (blank solution): methanol Testing conditions: Liquid chromatograph: Agilent 1260 liquid chromatograph Mobile phase A: 0.1% heptafluorobutyric acid aqueous solution Mobile phase B: acetonitrile Chromatographic column: Agilent poroshel 120 SB-AQ 4.6*150mm 2.7μm Detection wavelength: CAD Flow rate: 1.0ml / min Column temperature: 30°C Injection volume: 10 μl Elution gradient: Elution time (min) Phase A (%) Phase B (%) 0 90 10 15 10 90 20 10 90 20.1 90 10 25 90 10 Sample determination: Measure 10 μl of blank solution and positioning solution respectively and inject them into liquid chromatograph.

[0021] Test results: Recovery solution test Figure 2 In order to achieve the limit detection of bis(2-chloroethyl)amine, the concentration of the sample to be tested needs to be greatly increased, exceeding the sample loading capacity of the liquid chromatography column. The chromatographic peak of bis(2-chloroethyl)amine disappears in the recovery rate detection solution. The liquid phase method has low sensitivity and cannot meet the detection requirements.

[0022] Example 2 (Gas Phase, Polar Column) Sample solution preparation: Impurity localization solution: Take an appropriate amount of bis(2-chloroethyl)amine sample, dissolve it in methanol and quantitatively dilute it to make a solution containing approximately 10 μg of bis(2-chloroethyl)amine per 1 ml, shake well and set aside.

[0023] Recovery solution: Take appropriate amounts of the impurity bis(2-chloroethyl)amine and cariprazine hydrochloride samples respectively, dissolve them in methanol and quantitatively dilute them to make a solution containing approximately 0.01 mg of bis(2-chloroethyl)amine and 40 mg of cariprazine hydrochloride per 1 ml, shake well and set aside.

[0024] Blank solution: methanol Testing conditions: Gas chromatograph: Agilent 8890 gas chromatograph Chromatographic column: Aqilent CAM 30m*0.32mm 0..5μm Detector: flame ionization detector (FID); Heating program: 60 ° C for 1 minute, then increase the temperature to 200 ° C at 10 ° C / min and hold for 2 minutes; injection port temperature: 260 ° C; Detector temperature: 270°C; Split ratio: 10:1; Column flow rate: 3.0 ml / min; Hydrogen flow rate: 30 ml / min; Air flow rate: 300ml / min; Nitrogen flow rate: 30 ml / min; Injection volume: 1 μl.

[0025] Sample determination: Measure 1 μl of blank solution and recovery solution respectively and inject them into gas chromatograph.

[0026] Test results: Recovery solution test Figure 4 The chromatographic peak of bis(2-chloroethyl)amine disappeared in the recovery solution chromatogram. Gas chromatography was unable to quantitatively analyze impurities, and other methods needed to be developed.

[0027] Example 3 (Liquid Chromatography-Mass Spectrometry, Reversed Phase Liquid Chromatography Column) Sample solution preparation: Positioning solution: Take an appropriate amount of bis(2-chloroethyl)amine sample, dissolve it in methanol and quantitatively dilute it to make a solution containing approximately 1.25µg of bis(2-chloroethyl)amine per 1ml, shake well and set aside.

[0028] Recovery solution: Take appropriate amounts of the impurity bis(2-chloroethyl)amine and cariprazine hydrochloride samples respectively, dissolve them in methanol and quantitatively dilute them to make a solution containing approximately 1.25µg of bis(2-chloroethyl)amine and 5mg of cariprazine hydrochloride per 1ml, shake well and set aside.

[0029] Diluent (blank solution): methanol Testing conditions: Liquid chromatograph: Agilent 6130 liquid-mass chromatograph Mobile phase A: 20 mM ammonium acetate aqueous solution Mobile phase B: acetonitrile Chromatographic column: Waters XBridge C18 4.6*150mm 3.5μm Detection wavelength: 210nm Flow rate: 0.8ml / min Column temperature: 30°C Injection volume: 5μ1.

[0030] Drying gas flow: 12.0 L / min; Drying gas temperature: 350°C; Atomizer pressure: 55psig; Spray voltage: 4000v; Ion mode: positive charge; Elution gradient: Elution time (min) Phase A (%) Phase B (%) 0 80 20 3 80 20 10 20 80 15 20 80 15.1 80 20 20 80 20 Sample determination: 5µl of blank solution and positioning solution were measured and injected into the liquid chromatograph respectively.

[0031] Test results: The test results of the recovery solution are shown in Table 2 and Figure 6 , there is no interference in the blank solution, no interference before the known peak, high sensitivity, good specificity, and meets the requirements.

[0032] Table 2 Test results of Example 3 name Retention time (min) Separation from the front peak Bis(2-chloroethyl)amine 7.683 22.5 The parameters of Example 3 were subjected to methodological verification, and the results were as follows: Verification Project result Exclusivity The blank space has no interference and good specificity. Limit of quantification / limit of detection The limit of quantification of bis(2-chloroethyl)amine was 0.3125µg / ml, and the relative content was 0.006%. The limit of detection was 0.1563µg / ml, and the relative content was 0.003%. The concentration of bis(2-chloroethyl)amine was in the range of 0.3125µg / ml~2.525µg / ml. Linear phase The linearity and range relationship coefficient r=0.9949, and the Y-intercept deviation is 1.2%. Recovery rate The results for the 50% spiked recovery solution were 92.8%, 90.2%, and 91.7%, respectively. The results for the 100% spiked recovery solution were 97.8%, 108.9%, and 100.3%, respectively. The results for the 150% spiked recovery solution were 96.2%, 94.7%, and 94.5%, respectively. Repeatability The RSD of the peak area of ​​bis(2-chloroethyl)amine was 1.5%, and the RSD of the retention time was 0.02%. The quantitative analysis method provided by the present invention has high sensitivity, strong specificity and high accuracy, and can be used for quality control of bis(2-chloroethyl)amine in cariprazine hydrochloride.

[0033] It is understood that the above embodiments are merely exemplary embodiments for illustrating the principles of the present disclosure, and the present disclosure is not limited thereto. Those skilled in the art may make various modifications and improvements without departing from the spirit and substance of the present disclosure, and such modifications and improvements are also considered to be within the scope of protection of the present disclosure.

Claims

1. A method for quantitative analysis of potential genotoxic impurities in cariprazine hydrochloride, characterized in that: The liquid chromatography-mass spectrometry method comprises the following steps: (1) Preparation of sample solution, specifically: Test solution: Dissolve cariprazine hydrochloride in methanol and dilute quantitatively to make a solution containing 5.00 mg of cariprazine hydrochloride per 1 ml. Shake well and set aside. Recovery solution: Take appropriate amount of bis(2-chloroethyl)amine and cariprazine hydrochloride samples, dissolve them in methanol and dilute quantitatively to make a solution containing 5 mg of cariprazine hydrochloride and 1.25 μg of bis(2-chloroethyl)amine per 1 ml, shake well and set aside; Impurity localization solution: Take an appropriate amount of bis(2-chloroethyl)amine sample, dissolve it in methanol and quantitatively dilute it to make a solution containing 1.25 μg of bis(2-chloroethyl)amine per 1 ml, shake well and set aside; (2) Detection: A reversed-phase liquid chromatography column was used, with ammonium acetate aqueous solution as mobile phase A and acetonitrile as mobile phase B, with gradient elution, and a liquid chromatography-mass spectrometry detector was used as the detector; (3) Result analysis: record the chromatogram and calculate the content of bis(2-chloroethyl)amine using the external standard method; Among them, the structure of bis(2-chloroethyl)amine is as follows: 。 2. The method for quantitative analysis of potential genotoxic impurities in cariprazine hydrochloride according to claim 1, characterized in that: The ammonium acetate aqueous solution in the mobile phase A of step (2) is 20 mM.

3. The method for quantitative analysis of potential genotoxic impurities in cariprazine hydrochloride according to claim 1, characterized in that: The reversed-phase liquid chromatography column has an inner diameter of 3.0 to 5.0 mm, a length of 100 to 250 mm, and a filler particle size of 2 to 5 μm.

4. The method for quantitative analysis of potential genotoxic impurities in cariprazine hydrochloride according to claim 1, characterized in that: The reversed-phase liquid chromatography column uses octadecylsilane bonded silica gel as a filler.

5. The method for quantitative analysis of potential genotoxic impurities in cariprazine hydrochloride according to claim 1, characterized in that: The temperature of the reverse phase liquid chromatography column is set at 28°C to 32°C.

6. The method for quantitative analysis of potential genotoxic impurities in cariprazine hydrochloride according to claim 1, characterized in that: The detection time of the liquid chromatography-mass spectrometry detector is 30 minutes.

7. The method for quantitative analysis of potential genotoxic impurities in cariprazine hydrochloride according to claim 1, characterized in that: Single ion monitoring (SIM) mode was used to monitor the positive ion mode at m / z 141.7, with a drying gas flow rate of 12.0 L / min, a temperature of 350°C, a nebulizer pressure of 55 psig, and a spray voltage of 4000 V.

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