A method for determining the degradation impurity L-2-aminobutyric acid in a concentrated solution of levetiracetam for injection

Abstract

This invention relates to the field of pharmaceutical analysis technology, specifically to a method for determining the content of L-2-aminobutyric acid (GABA), a degradation impurity, in a concentrated solution of levetiracetam for injection. The method includes the following steps: diluting the concentrated levetiracetam for injection with a 50% acetonitrile aqueous solution, and then determining the content using high-performance liquid chromatography (HPLC) with a UV detector. C0.05 is also used. 18 The chromatographic column uses an aqueous solution of ammonium dihydrogen phosphate and acetonitrile as the mobile phase. This invention provides an accurate method for determining the degradation impurity L-2-aminobutyric acid in levetiracetam, which is highly specific, sensitive, accurate, and easy to operate, effectively controlling the quality of concentrated levetiracetam injection solutions.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis, specifically relating to a method for determining the degradation impurity L-2-aminobutyric acid in a concentrated solution of levetiracetam for injection. Background Technology

[0002] Levetiracetam is primarily used clinically as adjunctive therapy for partial-onset seizures in adults and children with epilepsy. Its molecular formula is C8H2O. 14 N2O2 has a molecular weight of 170.21.

[0003] L-2-Aminobutanoic acid (2-aminobutanoic acid), a degradation impurity of levetiracetam, is an organic compound with the chemical formula C4H9NO2, and is an off-white powder. It is mainly used as a chemical raw material and pharmaceutical intermediate, and has been widely applied in drug synthesis.

[0004] According to the "Technical Requirements for Consistency Evaluation of Quality and Efficacy of Generic Chemical Tablets," the focus should be on studying the degradation products of the formulation, including degradation products of the active pharmaceutical ingredient (API) or reaction products of the API with excipients and / or inner packaging materials. These degradation impurities are compounds with ultraviolet absorption. Therefore, ultraviolet detectors with high sensitivity, low noise, and a wide linear range should be preferred.

[0005] Levetiracetam is highly soluble in water and also soluble in acetone and acetonitrile. To minimize the interference of solvent effects on the detection, a 50% aqueous solution of acetonitrile was chosen as the solvent for the method.

[0006] The method used in this study is liquid chromatography, controlled by an ultraviolet detector. It is simple to operate and has a sensitivity of up to 4 μg / ml, which meets the minimum limit requirements. Summary of the Invention

[0007] The purpose of this invention is to provide a liquid chromatography method for determining degradation impurities in concentrated levetiracetam injection solutions. This method employs a UV detector and uses a 50% acetonitrile aqueous solution as the solvent. The method has been validated and can effectively achieve quantitative detection of the degradation impurity L-2-aminobutyric acid (GABA), thereby enabling effective quality control of concentrated levetiracetam injection solutions. This method exhibits high sensitivity and specificity.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A method for controlling the content of degradation impurities in concentrated levetiracetam injection solution, characterized by: high performance liquid chromatography (HPLC) for detection, under the following conditions:

[0010] Chromatographic column: The column is packed with a mixture of octadecylsilane bonds, silica gel, and sulfonic acid groups, with dimensions of 4.6 × 250 mm and a diameter of 5 μm.

[0011] Mobile phase: aqueous solution of ammonium dihydrogen phosphate and acetonitrile.

[0012] Detector: Ultraviolet detector, with a preferred detection wavelength of 190nm~230nm.

[0013] The flow rate of the mobile phase is 0.8~1.2 ml / min.

[0014] The column temperature of the chromatographic column is 30~50℃.

[0015] The concentration of levetiracetam in the test sample is 1 mg / ml-20 mg / ml, preferably 5 mg / ml-15 mg / ml.

[0016] The specific method for determining the content of L-2-aminobutyric acid (GABA), a degradation impurity, in levetiracetam is as follows:

[0017] I. Instruments and Reagents

[0018] Agilent 1260 high performance liquid chromatograph, acetonitrile (chromatographic grade), ammonium dihydrogen phosphate (analytical grade), phosphoric acid (analytical grade), SATORIOUS analytical balance.

[0019] Diluent: 50% acetonitrile aqueous solution.

[0020] Ammonium dihydrogen phosphate aqueous solution: Ammonium dihydrogen phosphate aqueous solution (0.1% phosphoric acid).

[0021] Reference solution: Take an appropriate amount of L-2-aminobutyric acid reference standard, dissolve and quantitatively dilute it with 50% acetonitrile aqueous solution, shake well, and the reference solution is obtained.

[0022] II. Sample Determination:

[0023] (1) Sample preparation: Accurately measure an appropriate amount of levetiracetam injection concentrated solution, dilute quantitatively to the mark with 50% acetonitrile water, and shake well.

[0024] (2) Chromatographic conditions:

[0025] Mobile phase: Ammonium dihydrogen phosphate aqueous solution is the aqueous phase, and acetonitrile is the organic phase, for elution.

[0026] The determination was performed using high performance liquid chromatography, with an ultraviolet detection wavelength of 205 nm.

[0027] (3) Specificity test

[0028] Under the above chromatographic conditions, blank solvent, L-2-aminobutyric acid reference solution, test solution, and solutions of other known impurities were injected sequentially for detection. The results showed that the resolution between L-2-aminobutyric acid and adjacent chromatographic peaks was greater than 1.5. The blank solvent, known impurities, and levetiracetam peak did not interfere with the detection of L-2-aminobutyric acid, and the specificity met the requirements.

[0029] (4) Limit of quantitation and limit of detection

[0030] Under the above chromatographic conditions, the limit of quantitation was determined based on the response concentration at a signal-to-noise ratio of 10:1, and the limit of detection was determined based on the response concentration at a signal-to-noise ratio of 3:1. The results showed that the limit of detection for L-2-aminobutyric acid was 2 μg / ml, and the limit of quantitation was 4 μg / ml.

[0031] (5) Accuracy

[0032] Thirteen samples were weighed, one of which was an unspecified test solution prepared according to the standard test solution preparation method. The other 12 samples were divided into four groups of three. Spikes were added at the limit of quantitation (LOQ) concentrations of L-2-aminobutyric acid (GABA), 50%, 100%, and 150% of the LOQ concentration, with three replicates prepared for each concentration. All samples were analyzed under the chromatographic conditions described above. The results were as follows: the average recovery rate at the LOQ was 107.7% (between 70% and 130%), with an RSD of 2.3% (less than 20.0%). The average recovery rate at the other concentrations was 103.4% (between 90% and 110%), with an RSD of 2.7% (less than 10.0%), meeting the requirements.

[0033] This method exhibits high specificity and sensitivity, and the instrument demonstrates good system applicability. It provides an effective method for determining L-2-aminobutyric acid, a degradation impurity, in concentrated levetiracetam injection solution.

[0034] The positive effects of this invention are as follows: This invention uses high performance liquid chromatography with an ultraviolet detector to determine the degradation impurity L-2-aminobutyric acid in levetiracetam injection concentrate, thereby realizing the determination of L-2-aminobutyric acid content in levetiracetam injection concentrate, and ultimately ensuring the safety and effectiveness of levetiracetam injection concentrate. Attached Figure Description

[0035] Figure 1 Here is the liquid chromatogram of the L-2-aminobutyric acid reference solution;

[0036] Figure 2 The liquid chromatogram of the test solution for levetiracetam injection concentrated solution. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0038] Example 1

[0039] Instrument: High performance liquid chromatograph: Agilent 1260, UV detector.

[0040] Column: CAPCELL PAK CR, 4.6×250mm, 5μm.

[0041] Mobile phase: Ammonium dihydrogen phosphate aqueous solution as the aqueous phase (0.1% phosphoric acid); acetonitrile as the organic phase.

[0042] Elution gradient:

[0043] Flow rate: 1.0 ml / min.

[0044] Column temperature: 40℃.

[0045] Detection wavelength: 205nm.

[0046] Injection volume: 100 μl.

[0047] Diluent: 50% acetonitrile solution.

[0048] Preparation of sample solution: Take an appropriate amount of L-2-aminobutyric acid reference solution, dissolve it with diluent and dilute quantitatively to prepare a solution of about 10 μg per ml, as the reference solution; accurately measure an appropriate amount of this product, add diluent and dilute quantitatively to prepare a solution containing about 10 mg of levetiracetam per ml, shake well, as the test solution.

[0049] Accurately measure the reference solution and test solution and inject them separately into the liquid chromatograph. Record the chromatograms as shown in the figure below. Figure 1 and Figure 2 The content of L-2-aminobutyric acid was calculated to be 0.03% using the external standard method.

Claims

1. An analytical method for determining the content of L-2-aminobutyric acid (GABA), a degradation impurity, in a concentrated solution of levetiracetam for injection, characterized in that... The content of L-2-aminobutyric acid, a degradation impurity in concentrated levetiracetam injection solution, was determined by high performance liquid chromatography using an ultraviolet detector.

2. The method according to claim 1, characterized in that... The concentrated solution of levetiracetam for injection was diluted with a 50% acetonitrile aqueous solution.

3. The method according to claim 1, characterized in that... The content of degradation impurities was determined by high performance liquid chromatography using an ultraviolet detector.

4. The method according to claim 1, characterized in that... Using octadecylsilane-bonded silica gel (C 18 () is a packed column.

5. The method according to claim 1, characterized in that... The method includes the following main components: (1) The diluent is 50% acetonitrile. (2) The concentration of levetiracetam in the test sample is 1 mg / ml-20 mg / ml, preferably 5 mg / ml-15 mg / ml. (3) The mobile phase is an aqueous solution of ammonium dihydrogen phosphate. (4) The column temperature of the chromatographic column is 30-50℃. (5) The flow rate of the mobile phase is 0.8-1.2 ml / min. (6) The method uses a high-performance liquid chromatograph and an ultraviolet detector for determination.

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