A method for determining degradation impurities and excipients in pregabalin oral solution by HPLC

Abstract

The application discloses a method for determining degradation impurities and excipients in Pregabalin oral solution by HPLC, selects octadecylsilane bonded silica gel as a stationary phase, and uses a mixed solvent of an organic phase and a buffer as a mobile phase for gradient elution. The method can separate the degradation impurities and excipients in the Pregabalin oral solution under the same chromatographic condition, and the separation is fast and efficient, so that the detection result of the related substances of the product is exclusive and reliable, and the quality of the preparation can be effectively controlled. The detection method has strong specificity, high precision, good accuracy and convenient operation, and can effectively control the quality of the medicine.

Description

Technical Field

[0001] This invention relates to a method for detecting related substances in pharmaceutical products, specifically a method for determining degradation impurities and excipients in pregabalin oral solution using HPLC. Background Technology

[0002] Pregabalin is a non-γ-aminobutyric acid (GABA) receptor agonist or antagonist, a novel calcium channel modulator that blocks voltage-dependent calcium channels and reduces neurotransmitter release. Pregabalin has a high affinity for the α2-δ site (an auxiliary subunit of a voltage-gated calcium channel) in central nervous system tissues, although its mechanism of action is not fully understood. However, results from modified mouse studies and experiments with compounds structurally similar to pregabalin (such as gabapentin) suggest that binding to the α2-δ site is associated with its analgesic and anti-seizure effects in animal models. In vitro studies indicate that pregabalin may reduce the release of various calcium-dependent neurotransmitters by modulating calcium channel function.

[0003] Pregabalin oral solution (trade name: Manufactured by Pfizer, pregabalin was approved by the European Union in July 2004 for the treatment of partial seizures and by the US FDA in 2005 for the relief and treatment of neuropathic pain, including diabetic peripheral neuropathy (DPN), postherpetic neuralgia (PHN), spinal cord injury-related neuralgia, fibromyalgia-related neuralgia, and partial seizures in patients aged 4 years and older. Pregabalin was the first drug approved by the US FDA for diabetic peripheral neuropathy and postherpetic neuralgia, and is currently one of the best-selling analgesics.

[0004] Pregabalin structural formula:

[0005] The prescription ingredients of pregabalin oral solution are: pregabalin, sodium dihydrogen phosphate, disodium hydrogen phosphate, sucralose, methylparaben, propylparaben, strawberry flavoring, and purified water.

[0006] The degradation products and excipients in pregabalin oral solution originate from the following sources: First, degradation impurities generated during the storage process of pregabalin; second, degradation impurities generated by the reaction of pregabalin with degradation products of preservatives (methylparaben, propylparaben) in the formulation; and third, excipients introduced into the formulation, mainly including:

[0007] 1. Degradation of impurities:

[0008] (1) Lactam: (S)-4-isobutyl-2-one, the specific structural formula is shown in Formula I:

[0009]

[0010] (2) Impurity A: (S)-3-(4-hydroxybenzoic acid amino)5-methylhexanoic acid, the specific structural formulas are shown in the formula below.

[0011]

[0012] (3) Impurity B: 2,5-((s)-2-isobutyl-3-carboxypropylamino)-1,4-benzoquinone, the specific structural formulas are shown in Formula III:

[0013]

[0014] 2. Auxiliary materials:

[0015] (1) Methyl p-hydroxybenzoate, the specific structural formulas are shown in Formula IV:

[0016]

[0017] (2) Propyl p-hydroxybenzoate, the specific structural formulas are shown in Formula V:

[0018]

[0019] (3) Sucralose, the specific structural formulas are shown in Formula VI:

[0020]

[0021] (4) Strawberry flavoring.

[0022] Strict control is required to address potential degradation impurities that may arise during the production and storage of pregabalin oral solution to ensure product quality. Furthermore, the presence of numerous excipients in addition to the active pharmaceutical ingredient (API) in the oral solution can easily interfere with impurity determination. Therefore, achieving the detection and separation of degradation impurities in pregabalin oral solution while ensuring that the excipients in the formulation do not interfere with the determination of these degradation impurities, and maintaining the specificity of the method, is of significant practical importance for weight control in the formulation process.

[0023] In addition, among the degradation impurities mentioned above, the 2,5-((s)-2-isobutyl-3-carboxypropylamino)-1,4-benzoquinone impurity contains the structure of 1,4-benzoquinone. According to the CPDB database (Carcinogenicity Database), substances with this structure have potential genotoxicity and a certain carcinogenicity rate. Furthermore, relevant research literature indicates that 1,4-benzoquinone can induce an increase in reactive oxygen species in K562 cells, leading to mitochondrial damage and increased apoptosis (Source: Original Article, July 2019, Vol. 31, No. 4, Effects of Camk2b Low Expression on 1,4-Benzoquinone-Induced Mitochondrial Toxicity in K562 Cells, Zhang Hong, Wang Tong, Wang Kun, Wang Boshen, Zhang Mengying, Zhou Yanhua, Pu Yuepu, Zhang Juan, Key Laboratory of Environmental Medical Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing, Jiangsu 210009, China). Furthermore, since the formation of this impurity is related to the structure of the excipient methylparaben in the drug solution, the peak times of this impurity and methylparaben in the liquid chromatography system are quite similar. This method can effectively separate and detect this impurity, which is of great practical significance for the quality control of this product. Summary of the Invention

[0024] The purpose of this invention is to provide an HPLC method for determining degradation impurities and excipients in pregabalin oral solution. This method can effectively separate and determine the content of degradation impurities in pregabalin oral solution, and the excipients do not interfere with the determination of impurities. The method has high sensitivity, good specificity, high precision, and good accuracy, and the detection results are accurate and reliable.

[0025] To achieve the above objectives, the technical solution of the present invention is as follows:

[0026] A method for determining degradation impurities in pregabalin oral solution using HPLC, simultaneously separating excipients from impurities to prevent interference with impurity determination. The method employs an octadecyl-bonded silica column as the packing material, using gradient elution with mobile phases A and B before detection by a detector.

[0027] The mobile phase A is a mixture of an organic solvent and a phosphate buffer solution, wherein the organic solvent is methanol and acetonitrile. The volume ratio of disodium hydrogen phosphate buffer to methanol to acetonitrile is 80:15:5. The phosphate buffer in the mobile phase A is preferably a disodium hydrogen phosphate buffer; the concentration range of the buffer is 0.005 mol / L to 0.015 mol / L, preferably 0.01 mol / L; the pH of the disodium hydrogen phosphate buffer is adjusted to 6.8 to 7.2 with phosphate, preferably 7.0.

[0028] The mobile phase B is acetonitrile.

[0029] The gradient elution settings are as follows:

[0030]

[0031] As a preferred embodiment, the gradient elution is configured as follows:

[0032]

[0033] The elution flow rate of the mobile phase is 0.7–0.9 ml / min, preferably 0.8 ml / min.

[0034] The column temperature of the chromatographic column is 20-30℃, preferably 25℃.

[0035] The detector has a detection wavelength of 210±5nm, preferably 210nm.

[0036] The method specifically includes the following steps:

[0037] 1) Test solution: The test solution is obtained by taking 10 μl of the sample solution.

[0038] 2) Reference solution: Weigh out the reference standards for lactam, impurity A and impurity B respectively, and dissolve and dilute them with solvent to prepare a reference solution;

[0039] 3) Blank excipient solution: Weigh out methylparaben, propylparaben, sucralose and strawberry flavoring according to the prescription ratio, and dissolve and dilute them with water to prepare a blank excipient solution;

[0040] 4) Inject the test solution from step 1) and the reference solution from step 2) into the sample respectively, perform high performance liquid chromatography analysis, record the chromatograms, determine the retention times of lactam, impurity A and impurity B, and calculate the content of impurities.

[0041] 5) Inject the blank excipient solution described in step 3) and perform high performance liquid chromatography analysis. Record the chromatogram, determine the peak positions of methylparaben, propylparaben, sucralose and strawberry flavor, and investigate the interference of excipient peaks on impurity detection.

[0042] The concentration of the reference solution was 0.04 mg / ml.

[0043] The solvent mentioned in step 2) is mobile phase A.

[0044] The beneficial effects of this invention are as follows:

[0045] (1) This invention uses a chromatographic column with octadecyl-bonded silica gel as the stationary phase and a mixed solvent of organic phase and phosphate buffer as the mobile phase for gradient elution. This allows for rapid and efficient separation of impurities and excipients in pregabalin oral solution under the same chromatographic conditions, resulting in specific and reliable detection results for related substances in this product, and effectively controlling the quality of the preparation. This detection method is highly specific, precise, accurate, and easy to operate, and can effectively control drug quality.

[0046] (2) The HPLC method provided by the present invention can effectively separate and determine the content of pregabalin and its degradation impurities lactam, impurity A and impurity B. It has high sensitivity, strong specificity and good repeatability. Other excipients in the oral solution do not interfere with the determination of impurities. The test results are reliable and accurate. The separation degree between the peaks of degradation impurity lactam, impurity A and impurity B is greater than 1.5. Attached Figure Description

[0047] Figure 1 High-performance liquid chromatogram of pregabalin oral solution

[0048] Figure 2 The diagram shows a mixed solution of lactam, impurity A, and impurity B.

[0049] Figure 3 High performance liquid chromatogram of blank excipient

[0050] Figure 4 High-performance liquid chromatogram of pregabalin oral solution Detailed Implementation

[0051] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings. Test methods not specifically specified in the preferred embodiments are generally performed under conventional conditions. The examples provided are for better illustration of the invention and are not intended to limit the scope of the invention to the given embodiments. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0052] Example 1: Method for separating and determining degradation impurities and excipients in pregabalin oral solution

[0053] Instrument: Thermo U3000

[0054] Chromatographic column: octadecylsilane-bonded silica gel as the packing material (XB-C18, 250mm × 4.6mm, 5μm or equivalent column).

[0055] Mobile phase A: Buffer (0.01 mol / L disodium hydrogen phosphate solution, pH adjusted to 7.0 with phosphoric acid) - methanol - acetonitrile (80:15:5)

[0056] Mobile phase B: Acetonitrile

[0057] Table 1 shows the linear elution gradients as follows:

[0058]

[0059] Detector: UV

[0060] Detection wavelength: 210nm

[0061] Flow rate: 0.8 ml / min

[0062] Column temperature: 25℃

[0063] Injection volume: 10 μl

[0064] Chromatography Workstation: Chameleon

[0065] Take this product as the test solution; accurately weigh appropriate amounts of pregabalin, lactam, impurity A, and impurity B reference standards, dissolve and dilute them in mobile phase A to prepare a solution containing approximately 0.4 mg per ml, as the reference solution. Use octadecylsilane-bonded silica gel as the stationary phase (Welch Ultimate XB-C18, 4.6 mm × 250 mm, 5 μm); use 0.01 mol / L disodium hydrogen phosphate buffer (adjusted to pH 7.0 with phosphoric acid)-methanol-acetonitrile (80:15:5) as mobile phase A; use acetonitrile as mobile phase B; perform gradient elution according to Table 1 above; the detection wavelength is 210 nm; the column temperature is 25 °C; and the flow rate is 0.8 ml per minute. Accurately measure 10 μl each of the blank excipient positioning solution, reference solution, and test solution, inject them separately into the liquid chromatograph, and record the chromatograms.

[0066] The results showed that this method could effectively separate lactam, impurity A, impurity B, and all excipients in the blank excipient. The blank excipient did not interfere with impurity detection. The resolution between the peaks of lactam, impurity A, and impurity B was greater than 1.5. Figure 1 .

[0067] This method has high sensitivity. The LOD concentration of lactam is 0.4 μg / ml, equivalent to 0.002% of the finished product content; the LOD concentration of impurity A is 0.04 μg / ml, equivalent to 0.0002% of the finished product content; and the LOD concentration of impurity B is 0.05 μg / ml, equivalent to 0.0002% of the finished product content.

[0068] This method is highly specific. The peak positions of each excipient in the blank sample do not interfere with the determination of degradation impurities, demonstrating the high specificity of the method. See [link to documentation]. Figures 2-3 .

[0069] Example 2: Method for separating and determining degradation impurities and excipients in pregabalin oral solution

[0070] Instrument: Thermo U3000

[0071] Chromatographic column: octadecylsilane-bonded silica gel as the packing material (XB-C18, 250mm × 4.6mm, 5μm or equivalent column).

[0072] Mobile phase A: Buffer (0.01 mol / L disodium hydrogen phosphate solution, pH adjusted to 7.0 with phosphoric acid) - methanol - acetonitrile (80:15:5)

[0073] Mobile phase B: Acetonitrile

[0074] Table 2 shows the linear elution gradients as follows:

[0075]

[0076] Detector: UV

[0077] Detection wavelength: 210nm

[0078] Flow rate: 0.8 ml / min

[0079] Column temperature: 25℃

[0080] Injection volume: 10 μl

[0081] Chromatography Workstation: Chameleon

[0082] Take this product as the test solution; accurately weigh appropriate amounts of pregabalin, lactam, impurity A, and impurity B reference standards, dissolve and dilute them in mobile phase A to prepare a solution containing approximately 0.4 mg per ml, as the reference solution. Use octadecylsilane-bonded silica gel as the stationary phase (Welch Ultimate XB-C18, 4.6 mm × 250 mm, 5 μm); use 0.01 mol / L disodium hydrogen phosphate buffer (adjusted to pH 7.0 with phosphoric acid)-methanol-acetonitrile (80:15:5) as mobile phase A; use acetonitrile as mobile phase B; perform gradient elution according to Table 2 above; the detection wavelength is 210 nm; the column temperature is 25 °C; and the flow rate is 0.8 ml per minute. Accurately measure 10 μl each of the blank excipient positioning solution, reference solution, and test solution, inject them separately into the liquid chromatograph, and record the chromatograms.

[0083] The results showed that under this elution gradient, pregabalin, impurity A, and lactam all emitted peaks and separated well from the blank excipient. However, impurity B did not emit a peak. Further analysis revealed that the peak times of impurity B overlapped with those of the excipient methylparaben, preventing separation. Therefore, under the above elution gradient, impurity B could not be effectively separated and detected. Figure 4 .

[0084] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred examples, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for determining degradation impurities and excipients in a pregabalin oral solution by HPLC, characterized in that: the method uses a chromatographic column with octadecylsilane bonded silica gel as the stationary phase, gradient elution is performed using mobile phase A and mobile phase B, the sample solution is injected into the detector for detection, and the gradient elution is set as follows: the degradation impurities in the pregabalin oral solution are lactam, impurity A, and impurity B, and their specific structural formulas are shown in the following formulas I, II, and III, respectively: the excipients are methyl paraben, propyl paraben, sucralose, and strawberry flavor; wherein mobile phase A is a mixed solution of an organic solvent and a phosphate buffer solution, and mobile phase B is acetonitrile; in the mobile phase A, the organic solvent is methanol and acetonitrile, the phosphate buffer solution is a sodium phosphate dibasic solution with a concentration ranging from 0.005 mol / L to 0.015 mol / L, and the pH value of the buffer solution is adjusted to a range of 6.8 to 7.2 using phosphoric acid; the volume ratio of the phosphate buffer solution, methanol, and acetonitrile is 80:15:

5. The concentration of the phosphate buffer solution ranges from 0.01 mol / L. The sodium phosphate dibasic buffer solution is adjusted to a pH value of 7.0 using phosphoric acid. The detection wavelength of the detector is 210 ± 5 nm. The detection wavelength of the detector is 210 nm.

2. The method of claim 1, wherein: The flow rate of the mobile phase is 0.6 to 0.9 ml / min.

3. The method of claim 1, wherein: The flow rate of the mobile phase is 0.8 ml / min.

4. The method of claim 1, wherein: The column temperature of the chromatographic column is 20 to 30°C.

5. The method of claim 4, wherein: The column temperature of the chromatographic column is 25°C.

6. The method of claim 1, wherein: The sample solution is directly injected without preparation, and the injection amount of the sample solution is 10 μl.

7. The method of claim 6, wherein: ​ 8. The method of claim 1, wherein: ​ 9. The method of claim 8, wherein: ​ 10. The method of claim 1, wherein: ​

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