High performance liquid chromatography detection method for impurities in oral solution

Abstract

The invention belongs to the technical field of pharmaceutical analysis and quality control, and particularly relates to a high performance liquid chromatography detection method for impurities in an oral solution. According to the detection method, high performance liquid chromatography is adopted, and the impurity is glyceryl p-hydroxybenzoate. Specifically, octadecyl bonded silica gel is adopted as a reversed-phase chromatographic column of a filler, a glacial acetic acid aqueous solution is adopted as a mobile phase A, methanol is adopted as a mobile phase B, and elution is performed according to a specified gradient procedure. The method comprises the following steps: respectively preparing a reference solution and a test solution by taking methanol-glacial acetic acid as a diluent, and calculating the content of glyceryl p-hydroxybenzoate in a sample by peak area according to an external standard method. Compared with a traditional detection method, the method has the advantages that specificity is better, sensitivity is higher, interference of auxiliary materials such as glycerin is effectively avoided, and the method is suitable for conventional quality control and stability research of the oral solution and has great significance in guaranteeing clinical medication safety.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis and quality control, and relates to a high performance liquid chromatography detection method for impurities in an oral solution, and is particularly suitable for detecting impurities in a memantine hydrochloride oral solution. Background Art

[0002] Memantine hydrochloride is a non-competitive NMDA receptor antagonist, mainly used to treat moderate to severe Alzheimer's dementia. In order to meet the dosing needs of patients, memantine hydrochloride is usually prepared as an oral solution dosage form, and parabens are often added to the prescription as antibacterial agents, such as methylparaben, propylparaben, etc. This type of substance is widely used in the fields of medicine, cosmetics and food because of its broad antibacterial spectrum, good stability and strong applicability, and can extend the shelf life while ensuring the quality of the preparation. At the same time, in order to improve the viscosity and taste of the solution, glycerol and other ingredients are often added to the excipients, but these excipients may react chemically with parabens under certain conditions to generate new impurities such as glyceryl parahydroxybenzoate, thereby posing potential risks to the quality and safety of the oral solution.

[0003] At present, the commonly used methods for detecting the content of parabens in the prior art include high performance liquid chromatography (HPLC), thin layer chromatography (TLC) and gas chromatography (GC), etc., but most of them are only for the conventional content determination of methylparaben or propylparaben itself. When these methods are directly applied to the analysis of antibacterial agent impurities in memantine hydrochloride oral solution, the following problems often occur: first, auxiliary materials such as glycerol may overlap with the peak of the target compound in the chromatographic separation, resulting in insufficient separation; second, the interference of auxiliary materials makes it difficult to accurately distinguish and quantify the components in the determination results; third, there is a lack of specific detection of potential new impurities (such as glyceryl parahydroxybenzoate), which makes it difficult to meet the requirements for comprehensive quality control of memantine hydrochloride oral solution.

[0004] Considering that during storage and use, paraben antibacterial agents may undergo ester exchange or other chemical reactions with excipients such as glycerol to produce new impurities, and the existing detection methods are difficult to take into account the separation, sensitivity and specific determination of new impurities. Therefore, it is necessary to provide a detection method for antibacterial agent impurities in memantine hydrochloride oral solution based on high performance liquid chromatography technology. Summary of the invention

[0005] According to the technical problems raised above, a high performance liquid chromatography detection method for impurities in an oral solution is provided. The purpose of the present invention is to effectively separate glyceryl parahydroxybenzoate, methylparaben, propylparaben and blank auxiliary materials, and to quickly, sensitively and accurately detect the content of antibacterial agent impurities, so as to make up for the deficiencies of the prior art and ensure the quality control of antibacterial agent impurities in memantine hydrochloride oral solution.

[0006] The technical means adopted by the present invention are as follows:

[0007] A high performance liquid chromatography method for detecting impurities in an oral solution, the detection method comprising the following detection conditions:

[0008] The chromatographic column uses octadecylsilane bonded silica gel as filler, mobile phase A is a glacial acetic acid aqueous solution with a volume ratio of 0.8% to 1.2%, and mobile phase B is methanol for elution; the detection wavelength is 252 to 256 nm, the flow rate is 0.9 to 1.1 ml / min, the column temperature is 27 to 33° C., and the injection volume is 20 to 50 μl.

[0009] In some embodiments, the mobile phase elution method is gradient elution, and the gradient program is: at 0 to 12 minutes, maintain mobile phase A:B = 70%:30%, at 12 to 13 minutes, adjust mobile phase A to 38%, and mobile phase B to 62%, maintain A:B = 38%:62% for 13 to 25 minutes, and adjust back to A:B = 70%:30% within 25 to 26 minutes, and then maintain this ratio for 26 to 35 minutes.

[0010] In some embodiments, the detection wavelength is 254 nm.

[0011] In some embodiments, the flow rate is 1.0 mL / min.

[0012] In some embodiments, the column temperature is 30°C.

[0013] In some embodiments, the injection volume is 30 μl.

[0014] In some embodiments, the mobile phase A is a 1% by volume aqueous solution of glacial acetic acid.

[0015] In some embodiments, the impurity is glyceryl paraben.

[0016] In some embodiments, the detection method comprises the following steps:

[0017] 1) Prepare the test solution of memantine hydrochloride oral solution and the glyceryl p-hydroxybenzoate impurity reference solution respectively;

[0018] 2) The test solution and the reference solution obtained in step 1) are respectively injected into a high performance liquid chromatograph, the chromatogram is recorded, and the content of glyceryl parahydroxybenzoate is calculated by the peak area according to the external standard method.

[0019] In some embodiments, the chromatographic column is an Inertsil ODS-3 model, or a reverse phase chromatographic column having a separation degree and column efficiency equivalent thereto.

[0020] In some embodiments, the structure of the glyceryl paraben is the following formula 1 and the following formula 2, or any one of the following formula 1 and the following formula 2,

[0021]

[0022] Compared with the prior art, the high performance liquid chromatography analysis method of the present invention can achieve effective separation of antibacterial agent impurities, which is beneficial for detection.

[0023] The present invention has the following beneficial technical effects:

[0024] 1. The present invention uses high performance liquid chromatography combined with an optimized gradient elution program, which can quickly and effectively separate the components in an acidic mobile phase environment, avoid interference from auxiliary materials, and ensure the peak shape integrity and specificity of the antibacterial agent impurities. Through the fine regulation of targeted detector parameters and mobile phase ratios, the present invention can achieve higher sensitivity, with a detection limit of 0.12ng and a quantitative limit of 0.39ng, significantly improving the detection ability of trace impurities.

[0025] 2. In the concentration range of 0.02-10 μg / ml, the method of the present invention shows a good linear relationship, which can meet the detection requirements of impurity content in memantine hydrochloride oral solution from 0.01% to 5%. This wide linear range provides greater operability for quantitative analysis, and can maintain accurate and stable measurement results at different impurity levels, which is suitable for a variety of quality control scenarios.

[0026] 3. The recovery rate of the present invention is maintained in the range of 95.0-105.0%. It can maintain a high accuracy in the preparation of sample solution, selection of chromatographic conditions, elution separation and detection. The determination of target impurities is highly consistent and reliable under different batches and different experimental conditions.

[0027] 4. The present invention achieved a relative standard deviation (RSD) of 0.59% and 0.95% in the repeatability test (n=6), which reflects the high stability and reproducibility in the experimental process. This result further verifies the effectiveness of the selected chromatographic column, mobile phase ratio, elution procedure and detection conditions, which can greatly reduce the errors inside and outside the laboratory.

[0028] 5. In the durability test of the present invention, the mobile phase A is a glacial acetic acid aqueous solution with a volume ratio of 0.8% to 1.2%, the detection wavelength is 252 to 256 nm, the flow rate is 0.9 to 1.1 ml / min, the column temperature is 27 to 33 ° C, the injection volume is 20 to 50 μl, and the brand and model of the chromatographic column are investigated. Compared with the test results without changing the conditions, the absolute values ​​of the target impurity contents are all within ± 0.05%, indicating that the method has good durability, and slight changes in the chromatographic conditions will not affect the test results, greatly reducing the result errors caused by slight changes in the chromatographic conditions.

[0029] 6. The present invention adopts gradient elution, which can effectively enhance the response signal and provide a higher proportion of organic phase when eluting the target impurity (such as glyceryl paraben), thereby improving the efficiency of the analyte concentration entering the detector within a relatively short elution time window.

[0030] In summary, the method of the present invention can be used to quickly, sensitively and accurately detect the content of antibacterial agent impurities, and has universal applicability. At the same time, it fills the technical gap of the method for determining antibacterial agent impurities in the field of memantine hydrochloride oral solution. Based on the above reasons, the present invention can be widely promoted in the field of drug analysis and quality control technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0032] Figure 1 It is the liquid chromatogram of the blank solution in Example 5 of the present invention.

[0033] Figure 2 It is the liquid chromatogram of the blank auxiliary material in Example 5 of the present invention.

[0034] Figure 3 It is the liquid chromatogram of the reference substance solution in Example 5 of the present invention.

[0035] Figure 4 It is the liquid chromatogram of the test solution in Example 5 of the present invention.

[0036] Figure 5 This is the detection limit chromatogram in Example 6 of the present invention.

[0037] Figure 6 This is the quantitative limit chromatogram in Example 6 of the present invention. DETAILED DESCRIPTION

[0038] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] Example 1

[0040] The invention provides a high performance liquid chromatography detection method for impurities in an oral solution, wherein the oral solution is a memantine hydrochloride oral solution.

[0041] First, prepare the sample solution:

[0042] Methanol–1% glacial acetic acid (60:40) was used as the diluent;

[0043] Preparation of reference solution: Take an appropriate amount of glyceryl parahydroxybenzoate reference substance, weigh accurately, dissolve it with the diluent and quantitatively dilute it to make a solution containing 1 μg per 1 ml;

[0044] Preparation of test solution: Accurately measure 5 ml of the memantine hydrochloride oral solution to be tested, place it in a 50 ml volumetric bottle, dilute to the scale with the diluent, and shake well;

[0045] Secondly, the reverse phase high performance liquid chromatography method was used, and the specific chromatographic conditions were as follows:

[0046] Chromatographic column: Octadecyl bonded silica gel is used as filler, the filler particle size is 3-5μm, and the column length is 250-300mm.

[0047] Mobile phase: 0.8% glacial acetic acid was used as mobile phase A and methanol was used as mobile phase B for gradient elution. The elution separation program is shown in Table 1.

[0048] Flow rate: 0.9ml / min.

[0049] Column temperature: 27°C.

[0050] Detection wavelength: 252nm.

[0051] Injection volume: 50μl.

[0052] Table 1 Gradient elution separation procedure

[0053]

[0054] That is, from 0 to 12 minutes, maintain mobile phase A:B=70%:30%, adjust mobile phase A to 38% and mobile phase B to 62% from 12 to 13 minutes, maintain A:B=38%:62% from 13 to 25 minutes, and adjust back to A:B=70%:30% within 25 to 26 minutes, and then maintain this ratio for 26 to 35 minutes.

[0055] Finally, the determination method and result calculation: the reference solution and the test solution were injected separately, and the detection was carried out under the above conditions. The content of glyceryl p-hydroxybenzoate in the memantine hydrochloride oral solution was calculated by the peak area according to the external standard method.

[0056] Example 2

[0057] This embodiment is the same as embodiment 1, except that the chromatographic column used in the chromatographic conditions is Inertsil ODS-3, 4.6 mm×250 mm, 5 μm.

[0058] Example 3

[0059] This embodiment is the same as embodiment 1, except that the chromatographic conditions are different, as follows:

[0060] Mobile phase: 1.2% glacial acetic acid as mobile phase A, methanol as mobile phase B, and gradient elution.

[0061] Flow rate: 1.1ml / min.

[0062] Column temperature: 33°C.

[0063] Detection wavelength: 256nm.

[0064] Injection volume: 30μl.

[0065] Example 4

[0066] This embodiment is the same as embodiment 1, except that the chromatographic conditions are different, as follows:

[0067] Mobile phase: 1% glacial acetic acid as mobile phase A, methanol as mobile phase B, and gradient elution.

[0068] Flow rate: 1.0ml / min.

[0069] Column temperature: 30℃.

[0070] Detection wavelength: 254nm.

[0071] Injection volume: 20 μl.

[0072] Performance test and verification of the present invention

[0073] The following examples 5-8 are used to test and verify the detection limit and quantification limit, linearity and range, sample recovery rate and repeatability, etc., to fully illustrate the technical effects of the method of the present invention in terms of sensitivity (detection ability of trace impurities), quantitative accuracy, linear range applicability, sample recovery and method precision, thereby proving that the method of the present invention can be effectively applied to the detection of antibacterial agent impurities in memantine hydrochloride oral solution, providing a reliable basis for quality control and stability research.

[0074] Main instruments: Shimadzu LC2030 series high performance liquid chromatograph; equipped with automatic sample injector and Labsolution liquid chromatography workstation.

[0075] Reagents and drugs: glacial acetic acid (analytical grade, Beijing Chemical Plant); methanol (chromatographic grade, Beijing Bailingwei Technology Co., Ltd.); glyceryl p-hydroxybenzoate (SINCO, CAS No. 93778-15-5; CAS No. 263552-76-7)

[0076] Reference substance stock solution: Take 2.5 mg of each glyceryl parahydroxybenzoate (Ester 1 and Ester 2), accurately weigh, place in 50 ml volumetric flasks, dissolve with diluent and dilute to the scale.

[0077] Reference solution: Accurately measure 1 ml of reference stock solution, combine and place in the same 50 ml volumetric flask, and dilute to the scale with diluent.

[0078] Example 5: Linearity and Range

[0079] like Figure 1-Figure 4 As shown, an appropriate amount of reference substance stock solution is accurately measured, and a series of standard solutions with concentrations of 0.02, 0.2, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, and 10.0 μg / ml are prepared with a diluent for linear test. The standard solution is tested according to the method of the present invention, and data regression analysis is performed on the standard concentration (X) and the peak area (Y). The results show that the peak area and the solution concentration have a good linear relationship in the range of 0.02 to 10.0 μg / mL. The linear range is relatively wide and is suitable for the detection of glyceryl p-hydroxybenzoate impurities from trace amounts to relatively high contents in memantine hydrochloride oral solution.

[0080] Example 6: Limit of Detection and Limit of Quantitation

[0081] A 1.0 μg / ml control solution under the linearity and range items was serially diluted and tested according to the chromatographic conditions of the present invention (including chromatographic column, mobile phase, gradient elution, etc.); the peak area of ​​each concentration solution and its corresponding signal-to-noise ratio (S / N) were recorded respectively.

[0082] The limit of detection (LOD) and limit of quantification (LOQ) were determined based on signal-to-noise ratios of approximately 3 and 10, respectively.

[0083] The results showed that (such as Figure 5 and Figure 6 The detection limit of glyceryl paraben is 0.12 ng and the quantification limit is 0.39 ng. This method has high sensitivity for the detection of trace impurities.

[0084] Example 7: Sample recovery rate

[0085] Accurately measure 0.5ml, 1ml, and 2ml of the reference stock solution, respectively, and place them in 50ml volumetric bottles, accurately measure 5ml of the test sample, dilute to the scale with diluent, and shake well. Inject and analyze, record the peak area, and the spike recovery rate is between 95.0% and 105.0%. The results show that this method can obtain an ideal recovery rate at different sample concentrations, and the accuracy of the method is good, as shown in Table 2.

[0086] According to the formula:

[0087] Recovery rate (%) = (measured amount - background amount) / added amount × 100%

[0088] Table 2 Recovery test results

[0089]

[0090] The results show that repeated additions are all within the normal allowable error range and can meet the actual quality analysis and control requirements.

[0091] Example 8: Repeatability

[0092] Accurately measure 5 ml of this product, place it in a 50 ml volumetric bottle, dilute it to the mark with solvent, and shake it well. According to the method of the present invention, 6 parallel samples were sampled and analyzed, and the peak area of ​​each antibacterial agent impurity was recorded. The content of glyceryl p-hydroxybenzoate in each test sample was calculated by external standard method. The content of glyceryl p-hydroxybenzoate 1 in the sample was 0.25% (RSD was 0.59%), and the content of glyceryl p-hydroxybenzoate 2 was 0.03% (RSD was 0.95%). The results showed that the repeatability was good, as shown in Table 3.

[0093] According to the formula:

[0094] Glyceryl paraben content (%) = (impurity peak area of ​​test sample / peak area of ​​reference sample) × (solubility of reference sample / concentration of test sample) × 100%

[0095] Table 3 Repeatability test results (%)

[0096]

[0097] The results showed that the method had good repeatability and was suitable for daily quality control and stability assessment.

[0098] Example 9: Durability

[0099] Accurately measure 5 ml of this product, place it in a 50 ml volumetric bottle, dilute it to the mark with solvent, and shake it well. Inject the sample according to the chromatographic conditions described in Table 4, record the peak area of ​​each antibacterial agent impurity, and calculate the content of glyceryl paraben in each test sample using the external standard method. The results show that the durability is good, as shown in Table 4.

[0100] According to the formula:

[0101] Glyceryl paraben content (%) = (impurity peak area of ​​test sample / peak area of ​​reference sample) × (solubility of reference sample / concentration of test sample) × 100%

[0102] Table 4 Durability test results

[0103]

[0104] In summary, the above embodiments fully demonstrate that the method of the present invention has good performance in terms of detection limit and quantification limit, linear range, recovery rate and repeatability, and can achieve rapid and accurate detection of glyceryl p-hydroxybenzoate impurities in memantine hydrochloride oral solution, providing a reliable analytical means for the quality control and stability study of the oral solution.

[0105] The above embodiments are only preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, a person of ordinary skill in the art should understand that any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A high performance liquid chromatography method for detecting impurities in an oral solution, characterized in that: The method includes the following detection conditions: The chromatographic column uses octadecylsilane bonded silica gel as filler, mobile phase A is a glacial acetic acid aqueous solution with a volume ratio of 0.8% to 1.2%, and mobile phase B is methanol for elution; the detection wavelength is 252 to 256 nm, the flow rate is 0.9 to 1.1 ml / min, the column temperature is 27 to 33° C., and the injection volume is 20 to 50 μl.

2. The detection method according to claim 1, characterized in that: The mobile phase elution method is gradient elution, and the gradient elution program is: at 0 to 12 minutes, the mobile phase A:B is maintained at 70%:30%, at 12 to 13 minutes, the mobile phase A is adjusted to 38%, the mobile phase B is adjusted to 62%, 13 to 25 minutes maintain A:B=38%:62%, and within 25 to 26 minutes, adjust back to A:B=70%:30%, and then maintain this ratio for 26 to 35 minutes.

3. The detection method according to claim 1, characterized in that: The detection wavelength is 254 nm.

4. The detection method according to claim 1, characterized in that: The flow rate was 1.0 mL / min.

5. The detection method according to claim 1, characterized in that: The column temperature was 30°C.

6. The detection method according to claim 1, characterized in that: The injection volume was 30 μl.

7. The detection method according to claim 1, characterized in that: The mobile phase A is a 1% by volume aqueous solution of glacial acetic acid.

8. The detection method according to any one of claims 1 to 7, characterized in that: The impurity is glyceryl parahydroxybenzoate.

9. The detection method according to claim 8, characterized in that: The chromatographic column is of Inertsil ODS-3 model, or a reverse phase chromatographic column with equivalent separation degree and column efficiency.

10. The detection method according to claim 8, characterized in that: The structure of the glyceryl parahydroxybenzoate is the following formula 1 and the following formula 2, or any one of the following formula 1 and the following formula 2.

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