Method for determination of related substances in L-carnitine and its injection by high performance liquid chromatography

Abstract

The invention discloses a method for determining related substances in L-carnitine and its injection by high performance liquid chromatography, wherein the mobile phase used is a potassium dihydrogen phosphate buffer-acetonitrile mixed solution with a pH of 4.0 to 6.0; a diluent is used to prepare the solution; the diluent is a mobile phase with a pH of 2.0 to 3.5 adjusted by a hydrochloric acid solution; and the volume ratio of potassium dihydrogen phosphate buffer to acetonitrile in the mobile phase is 35:65. The invention finally finds through a large number of experiments that the use of a mobile phase with a certain pH as a diluent to prepare various solutions can effectively avoid the interference of a 2-5min solvent peak, so that the content of L-carnitine can be accurately calculated for L-carnitine that does not contain 2-5min impurities in the sample itself, and the content of L-carnitine and 2-5min impurities can be accurately calculated simultaneously for L-carnitine that contains 2-5min impurities in the sample itself.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a method for determining related substances in L-carnitine and its injection by high performance liquid chromatography. Background Art

[0002] L-carnitine, also known as L-carnitine, has the chemical name (L-3-carboxy-2-hydroxypropyl) trimethylammonium salt. It is an amino acid that promotes the conversion of fat into energy. It has no toxic side effects on the human body and is necessary for fat metabolism. It can be used to treat chronic renal failure, cardiomyopathy, coronary heart disease and organic acidemia, etc. It can also be used as an auxiliary treatment for chronic liver disease. It is currently widely used in nutritional health products, medical treatment, food additives, feed additives and other fields.

[0003] At present, when high performance liquid chromatography is used to determine L-carnitine and its related substances for injection, there is a problem of solvent peak interference, that is, no matter whether it is the sample solution or the blank solution, a chromatographic peak appears at 2-5 min, but the peak area of ​​the sample solution is significantly larger than that of the blank solution. This will cause the following two problems: (1) If there is no 2-5 min impurity in the sample, the extra peak area will be calculated as the 2-5 min impurity when calculating the L-carnitine content, thereby affecting the correct calculation of the L-carnitine content; (2) If the sample itself contains 2-5 min impurities, this will not only affect the correct calculation of the L-carnitine content, but also the calculation of the impurity content.

[0004] At present, no literature reports on the above-mentioned 2-5 min solvent peak interference have been found. Summary of the invention

[0005] The object of the present invention is to solve the above problems and provide a method for determining related substances in L-carnitine and its injection by high performance liquid chromatography, which can effectively avoid the 2-5min solvent peak interfering with the detection of L-carnitine.

[0006] The technical scheme for achieving the purpose of the present invention is: a method for determining related substances in L-carnitine and its injection by high performance liquid chromatography, wherein the mobile phase used is a potassium dihydrogen phosphate buffer solution-acetonitrile mixed solution with a pH value of 4.0 to 6.0; a diluent is used to prepare the solution; and the diluent is a mobile phase with a pH value of 2.0 to 3.5 adjusted by a hydrochloric acid solution.

[0007] The volume ratio of potassium dihydrogen phosphate buffer to acetonitrile in the mobile phase is 20:80 to 50:50, preferably 35:65.

[0008] The mobile phase is prepared as follows: 0.01 mol / L to 0.1 mol / L potassium dihydrogen phosphate solution is first adjusted to pH=4.0-6.0 with an acid-base regulator to obtain a potassium dihydrogen phosphate buffer solution, and then mixed with acetonitrile according to the above volume ratio; or 0.01 mol / L to 0.1 mol / L potassium dihydrogen phosphate solution is first mixed with acetonitrile according to the above volume ratio, and then adjusted to pH=4.0-6.0 with an acid-base regulator.

[0009] The concentration of the potassium dihydrogen phosphate solution is preferably 0.05 mol / L.

[0010] The other conditions of the HPLC are as follows:

[0011] Chromatographic column: Chromatographic column with amino-bonded silane as filler; specification: 250mm×4.6mm×5μm.

[0012] Column temperature: 10~30℃.

[0013] UV detection wavelength: 205nm.

[0014] Injection volume: 10~100μL.

[0015] Mobile phase flow rate: 0.8~1.2mL / min.

[0016] The present invention has the positive effect that after a large number of experiments, the present invention finally finds that the use of a mobile phase with a certain pH as a diluent to prepare various solutions can effectively avoid the interference of the 2-5min solvent peak, so that the content of the levocarnitine in the sample itself without the 2-5min impurity can be accurately calculated, and the content of the levocarnitine and the 2-5min impurity can be accurately calculated at the same time in the sample containing the levocarnitine of the 2-5min impurity. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Chromatograms recorded and presented together for Example 1.

[0018] Figure 2 Chromatograms recorded for Comparative Example 1 and presented together.

[0019] Figure 3 Chromatograms recorded and presented together for Example 2.

[0020] Figure 4 Chromatograms recorded and presented together for Example 3. DETAILED DESCRIPTION

[0021] (Example 1)

[0022] 1. Chromatographic conditions.

[0023] Chromatographic column: A chromatographic column with amino-bonded silane as filler, specifically APS-2 Hypersil (250 mm × 4.6 mm × 5 μm).

[0024] Column temperature: 30℃.

[0025] UV detection wavelength: 205nm.

[0026] Injection volume: 50 μL.

[0027] Mobile phase flow rate: 1.0 mL / min.

[0028] Mobile phase: pH=5.5 potassium dihydrogen phosphate buffer-acetonitrile mixed solution (volume ratio of 35:65).

[0029] The preparation method is as follows: dissolve 6.8 g of potassium dihydrogen phosphate in 1 L of water to obtain a 0.05 mol / L potassium dihydrogen phosphate solution, then adjust the pH to 5.5 with a sodium hydroxide solution to obtain a potassium dihydrogen phosphate buffer solution, and then mix it with acetonitrile in a volume ratio of 35:65.

[0030] Diluent: Use hydrochloric acid to adjust the mobile phase pH to 2.0.

[0031] 2. Preparation of solution.

[0032] 1. Standard solution: Accurately weigh 0.05 g of L-carnitine standard (mass fraction is 100%) and place it in a 10 mL volumetric flask. Dilute to the mark with diluent as the standard solution.

[0033] 2. Blank solution: Take the diluent as the blank solution.

[0034] 3. Methods and results.

[0035] Inject the standard solution and blank solution into the liquid chromatograph respectively, record the chromatograms, and present the two chromatograms together. Figure 1 .

[0036] Depend on Figure 1 It can be seen that the chromatographic peaks of the standard solution prepared with a certain pH diluent of the present invention and the blank solution at 2 to 5 minutes are basically overlapping, which means that 2 to 5 minutes are all solvent peaks, and do not contain 2 to 5 minutes impurities. This result is completely consistent with the L-carnitine standard without impurities.

[0037] (Comparative Example 1)

[0038] 1. Chromatographic conditions.

[0039] Except that no diluent is prepared, the other steps are the same as those in Example 1.

[0040] 2. Preparation of solution.

[0041] 1. Comparative standard solution: Accurately weigh 0.05 g of L-carnitine standard (mass fraction is 100%) and place it in a 10 mL volumetric flask, and dilute to the scale with mobile phase as the comparative standard solution.

[0042] 2. Comparative blank solution: Take the mobile phase as the comparative blank solution.

[0043] 3. Methods and results.

[0044] Inject the comparison standard solution and the comparison blank solution into the liquid chromatograph respectively, record the chromatograms, and present the two chromatograms together. Figure 2 .

[0045] Depend on Figure 2 It can be seen that the chromatographic peaks appearing at 2-5 min between the reference standard solution prepared directly with the mobile phase of pH=5.5 and the reference blank solution are quite different. This result easily indicates that there are 2-5 min impurities in the reference standard solution, which is inconsistent with the L-carnitine standard that does not contain impurities.

[0046] (Example 2)

[0047] 1. Chromatographic conditions.

[0048] Same as Example 1.

[0049] 2. Preparation of solution.

[0050] 1. System suitability solution: Take an appropriate amount of the main impurity standard (mass fraction is 100%), dissolve it in water and dilute it to a concentration of 1 mg / mL, accurately measure 1 mL and place it in a 10 mL volumetric flask, then add 0.1 g of L-carnitine standard (mass fraction is 100%), and then dilute it to the scale with a diluent as the system suitability solution.

[0051] 2. L-carnitine API sample solution: Accurately weigh 0.05 g of L-carnitine API sample and place it in a 10 mL volumetric flask, and dilute to the mark with a diluent to serve as the L-carnitine API sample solution.

[0052] 3. Main impurity reference solution: Take an appropriate amount of the main impurity standard (mass fraction is 100%), dissolve it in water and dilute it to a concentration of 0.25 mg / mL, accurately measure 1 mL and place it in a 10 mL volumetric flask, and dilute it to the scale with a diluent as the main impurity reference solution.

[0053] 4. Blank solution: Take the diluent as the blank solution.

[0054] The chemical name of the main impurity is (E)-4-(trimethylamino)-2-butenoic acid inner salt [also known as (Z)-4-(trimethylamino)-2-butenoic acid inner salt]; the structural formula is as follows:

[0055] .

[0056] 3. Methods and results.

[0057] The system suitability solution, L-carnitine API sample solution, main impurity reference solution and blank solution were injected into the liquid chromatograph respectively, and the chromatograms were recorded. The four chromatograms obtained were presented together. Figure 3 .

[0058] Depend on Figure 3 It can be seen that the chromatographic peaks of various solutions prepared with the diluent of a certain pH of the present invention at 2 to 5 min are basically able to overlap, which means that various solutions do not contain 2 to 5 min impurities. This result is completely consistent with the system suitability solution containing only the main impurity standard and the L-carnitine standard and the main impurity control solution containing only the main impurity standard, and indicates that the L-carnitine bulk drug sample solution does not contain 2 to 5 min impurities and the main impurities are not obvious.

[0059] (Example 3)

[0060] 1. Chromatographic conditions.

[0061] Same as Example 1.

[0062] 2. Preparation of solution.

[0063] 1. System suitability solution: same as Example 2.

[0064] 2. L-Carnitine injection sample solution: Accurately measure 0.25 mL of L-Carnitine injection sample (specification: 5 mL, containing 1.0 g of L-Carnitine) and place it in a 10 mL volumetric flask. Dilute to the mark with a diluent as the L-Carnitine injection sample solution.

[0065] 3. Main impurity control solution: same as Example 2.

[0066] 4. Blank solution: Take the diluent as the blank solution.

[0067] 3. Methods and results.

[0068] Inject the system suitability solution, L-carnitine injection sample solution, main impurity reference solution and blank solution into the liquid chromatograph respectively, record the chromatograms, and present the four chromatograms together, as shown in the figure. Figure 4 .

[0069] Depend on Figure 4It can be seen that the chromatographic peaks of various solutions prepared with the diluent of a certain pH of the present invention at 2 to 5 min are basically able to overlap, which means that various solutions do not contain 2 to 5 min impurities. This result is completely consistent with the system suitability solution containing only the main impurity standard and the L-carnitine standard and the main impurity control solution containing only the main impurity standard, and indicates that the L-carnitine injection sample solution does not contain the 2 to 5 min impurity, but the main impurity is very obvious.

Claims

1. A method for determining related substances in L-carnitine and its injection by high performance liquid chromatography, wherein the mobile phase used is a potassium dihydrogen phosphate buffer-acetonitrile mixed solution; the pH of the potassium dihydrogen phosphate buffer is 4.0-6.0; characterized in that: A solution is prepared by using a diluent; the diluent is a mobile phase with a pH value of 2.0 adjusted by a hydrochloric acid solution; the volume ratio of potassium dihydrogen phosphate buffer to acetonitrile in the mobile phase is 35:65; and the chromatographic column used is a chromatographic column with amino-bonded silane as a filler, with a specification of 250 mm×4.6 mm×5 μm.

2. The method for determining related substances in L-carnitine and its injection by high performance liquid chromatography according to claim 1, characterized in that The mobile phase is prepared as follows: 0.01 mol / L to 0.1 mol / L potassium dihydrogen phosphate solution is first adjusted to pH 4.0 to 6.0 using an acid-base regulator to obtain a potassium dihydrogen phosphate buffer solution, and then mixed with acetonitrile in the above volume ratio to obtain the mobile phase.

3. The method for determining related substances in L-carnitine and its injection by high performance liquid chromatography according to claim 2, characterized in that: The concentration of the potassium dihydrogen phosphate solution is 0.05 mol / L.

4. The method for determining related substances in L-carnitine and its injection by high performance liquid chromatography according to claim 1, characterized in that The other conditions of the HPLC are as follows: Column temperature: 10-30°C; UV detection wavelength: 205nm; Injection volume: 10-100 μL; Mobile phase flow rate: 0.8~1.2mL / min.

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