CN115290779A - Method for determining levocarnitine related substances by high performance liquid chromatography - Google Patents
Method for determining levocarnitine related substances by high performance liquid chromatography Download PDFInfo
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- CN115290779A CN115290779A CN202210892284.4A CN202210892284A CN115290779A CN 115290779 A CN115290779 A CN 115290779A CN 202210892284 A CN202210892284 A CN 202210892284A CN 115290779 A CN115290779 A CN 115290779A
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- PHIQHXFUZVPYII-ZCFIWIBFSA-N (R)-carnitine Chemical compound C[N+](C)(C)C[C@H](O)CC([O-])=O PHIQHXFUZVPYII-ZCFIWIBFSA-N 0.000 title claims abstract description 132
- 229960001518 levocarnitine Drugs 0.000 title claims abstract description 115
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 76
- 238000000034 method Methods 0.000 title claims abstract description 38
- 239000000126 substance Substances 0.000 title claims abstract description 33
- 239000000243 solution Substances 0.000 claims abstract description 113
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonium chloride Substances [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims abstract description 60
- 239000012085 test solution Substances 0.000 claims abstract description 59
- 238000001514 detection method Methods 0.000 claims abstract description 41
- 230000035945 sensitivity Effects 0.000 claims abstract description 31
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 claims abstract description 26
- 239000012488 sample solution Substances 0.000 claims abstract description 16
- 238000010812 external standard method Methods 0.000 claims abstract description 8
- 239000012535 impurity Substances 0.000 claims description 53
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 42
- 239000012046 mixed solvent Substances 0.000 claims description 34
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 16
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 15
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 15
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 15
- 239000000523 sample Substances 0.000 claims description 10
- 238000002013 hydrophilic interaction chromatography Methods 0.000 claims description 9
- 239000002245 particle Substances 0.000 claims description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
- 239000000741 silica gel Substances 0.000 claims description 5
- 229910002027 silica gel Inorganic materials 0.000 claims description 5
- MPNXSZJPSVBLHP-UHFFFAOYSA-N 2-chloro-n-phenylpyridine-3-carboxamide Chemical compound ClC1=NC=CC=C1C(=O)NC1=CC=CC=C1 MPNXSZJPSVBLHP-UHFFFAOYSA-N 0.000 claims description 4
- -1 dihydroxypropyl Chemical group 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 4
- 239000013558 reference substance Substances 0.000 claims description 4
- 238000010521 absorption reaction Methods 0.000 claims description 2
- 238000000926 separation method Methods 0.000 abstract description 15
- 238000004458 analytical method Methods 0.000 abstract description 2
- 239000012088 reference solution Substances 0.000 abstract description 2
- 238000002360 preparation method Methods 0.000 description 18
- AAEQXEDPVFIFDK-UHFFFAOYSA-N 3-(4-fluorobenzoyl)-2-(2-methylpropanoyl)-n,3-diphenyloxirane-2-carboxamide Chemical compound C=1C=CC=CC=1NC(=O)C1(C(=O)C(C)C)OC1(C=1C=CC=CC=1)C(=O)C1=CC=C(F)C=C1 AAEQXEDPVFIFDK-UHFFFAOYSA-N 0.000 description 11
- 239000007788 liquid Substances 0.000 description 7
- QGQXAMBOYWULFX-LZWSPWQCSA-N 2-morpholin-4-ylethyl (e)-6-(4,6-dihydroxy-7-methyl-3-oxo-1h-2-benzofuran-5-yl)-4-methylhex-4-enoate Chemical compound OC=1C=2C(=O)OCC=2C(C)=C(O)C=1C\C=C(/C)CCC(=O)OCCN1CCOCC1 QGQXAMBOYWULFX-LZWSPWQCSA-N 0.000 description 6
- 230000009897 systematic effect Effects 0.000 description 6
- 239000012266 salt solution Substances 0.000 description 4
- 239000012224 working solution Substances 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000000337 buffer salt Substances 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000007975 buffered saline Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 208000031229 Cardiomyopathies Diseases 0.000 description 1
- 206010058892 Carnitine deficiency Diseases 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 208000031226 Hyperlipidaemia Diseases 0.000 description 1
- 208000001953 Hypotension Diseases 0.000 description 1
- 208000021642 Muscular disease Diseases 0.000 description 1
- 201000009623 Myopathy Diseases 0.000 description 1
- PUKNFWRLBQXPFL-FXRZFVDSSA-N [(e)-3-carboxyprop-2-enyl]-trimethylazanium;chloride Chemical compound [Cl-].C[N+](C)(C)C\C=C\C(O)=O PUKNFWRLBQXPFL-FXRZFVDSSA-N 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- OBFQBDOLCADBTP-UHFFFAOYSA-N aminosilicon Chemical compound [Si]N OBFQBDOLCADBTP-UHFFFAOYSA-N 0.000 description 1
- 206010003119 arrhythmia Diseases 0.000 description 1
- 230000006793 arrhythmia Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 208000020832 chronic kidney disease Diseases 0.000 description 1
- 208000022831 chronic renal failure syndrome Diseases 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000001631 haemodialysis Methods 0.000 description 1
- 230000000322 hemodialysis Effects 0.000 description 1
- 230000036543 hypotension Effects 0.000 description 1
- 229940090044 injection Drugs 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229940080535 levocarnitine injection Drugs 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000005245 sintering Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 208000016505 systemic primary carnitine deficiency disease Diseases 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- General Health & Medical Sciences (AREA)
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Abstract
The invention discloses a method for determining levocarnitine related substances by high performance liquid chromatography, which comprises the following steps: preparing a system applicability solution: preparing a reference solution from 3-carboxyl-N, N, N-trimethylpropene-2-ene-1-ammonium chloride, (R) -4-amino-2-hydroxy-N, N, N-trimethyl-4-oxobutyl-1-ammonium chloride, (3-carbamoylallyl) trimethylammonium and a levocarnitine reference; preparing a test solution; respectively carrying out HPLC detection on the system applicability solution and the sample solution; the content of 3-carboxyl-N, N, N-trimethylpropene-2-ene-1-ammonium chloride, (R) -4-amino-2-hydroxy-N, N, N-trimethyl-4-oxobutyl-1-ammonium chloride and (3-carbamoylallyl) trimethylammonium in the levocarnitine is determined by adopting an external standard method. The method has the advantages of high separation efficiency, high analysis speed and high detection sensitivity, and can effectively control the quality of the levocarnitine.
Description
Technical Field
The invention belongs to the technical field of substance detection, relates to a method for determining related substances of levocarnitine by High Performance Liquid Chromatography (HPLC), and particularly relates to a method for determining impurities of 3-carboxyl-N, N, N-trimethacrylene-2-ene-1-ammonium chloride, (R) -4-amino-2-hydroxyl-N, N, N-trimethyl-4-oxobutyl-1-ammonium chloride and (3-carbamoylallyl) trimethylammonium in levocarnitine by high performance liquid chromatography.
Background
The levocarnitine is a medicine for treating a series of concurrent symptoms of chronic renal failure long-term hemodialysis patients caused by secondary carnitine deficiency, and is mainly used for treating cardiomyopathy, skeletal myopathy, arrhythmia, hyperlipidemia, hypotension, dialysis myospasm and the like.
The chemical name of levocarnitine is (3R) -hydroxy- (trimethyl ammonium) butyric acid inner salt, and main impurities are introduced in chemical synthesis: 3-carboxy-N, N, N-trimethylprop-2-en-1-aminium chloride (impurity A), (R) -4-amino-2-hydroxy-N, N, N-trimethyl-4-oxobutyl-1-aminium chloride (impurity C) ammonium and (3-carbamoylallyl) trimethylammonium (impurity D).
The levocarnitine is the main active ingredient in the levocarnitine injection, and the quality of the levocarnitine can be better reflected by measuring related substances in the levocarnitine. At present, the chromatographic conditions adopted by levocarnitine related substances in the 2020 edition of Chinese pharmacopoeia are as follows: using amino silicon sintering bonded silica gel as a filling agent; using 50mmol/L potassium dihydrogen phosphate solution (pH value is adjusted to 4.7 by sodium hydroxide test solution) -acetonitrile (35; the detection wavelength is 205nm; the column temperature is 30 ℃; the injection volume was 20. Mu.L. Under the chromatographic conditions, the system applicability solution chromatogram is shown in figure 1, and the impurities C and D have consistent retention time and cannot be effectively separated.
Therefore, a method for determining related substances in levocarnitine with high sensitivity is established, and the quality of the levocarnitine can be better controlled
Disclosure of Invention
The invention aims to establish a method for determining 3-carboxyl-N, N, N-trimethylpropene-2-ene-1-ammonium chloride, (R) -4-amino-2-hydroxy-N, N, N-trimethyl-4-oxobutyl-1-ammonium chloride and (3-carbamoylallyl) trimethylammonium chloride related to levocarnitine based on high performance liquid chromatography so as to better control the quality of levocarnitine.
The purpose of the invention is realized by the following technical scheme:
a method for determining levocarnitine related substances by high performance liquid chromatography comprises the following steps:
preparing a system applicability solution: taking 3-carboxyl-N, N, N-trimethylpropene-2-ene-1-ammonium chloride, (R) -4-amino-2-hydroxy-N, N, N-trimethyl-4-oxobutyl-1-ammonium chloride and (3-carbamoylallyl) trimethylammonium and a levocarnitine reference substance, and preparing a reference substance solution containing 15-30 mu g of 3-carboxyl-N, N, N-trimethylpropene-2-ene-1-ammonium chloride, 3-7 mu g of (R) -4-amino-2-hydroxy-N, N, N-trimethyl-4-oxobutyl-1-ammonium chloride, 3-7 mu g of (3-carbamoylallyl) trimethylammonium and 3-15 mg of levocarnitine per 1mL of mixed solvent by adopting a mixed solvent of a mobile phase and water;
preparing a test solution: preparing a test solution containing 4-6 mg of levocarnitine per 1mL of mixed solvent by using the mixed solvent of a mobile phase and water;
respectively carrying out HPLC detection on the system applicability solution and the sample solution, wherein the chromatographic conditions of the HPLC detection are as follows: adopting a hydrophilic chromatographic column which is chemically bonded with dihydroxypropyl on organic mixed silica gel particles, wherein the mobile phase is a mixed solution of acetonitrile and 50-60 mmol/L potassium dihydrogen phosphate solution with the volume ratio of 65-75, the column temperature is 25-35 ℃, the flow rate is 0.8-1.2 mL/min, and the detection wavelength is 200-210 nm; the sample injection amount is 5-100 mu L;
measuring the contents of impurities 3-carboxyl-N, N, N-trimethylpropene-2-ene-1-ammonium chloride, (R) -4-amino-2-hydroxy-N, N, N-trimethyl-4-oxobutyl-1-ammonium chloride and (3-carbamoylallyl) trimethylammonium in the levocarnitine by adopting an external standard method;
wherein, the mixed solvent is prepared from a mobile phase and water according to a volume ratio of 40-60; the pH value of the potassium dihydrogen phosphate solution is 4.3-5.5.
Preferably, a system suitability solution is formulated: a control solution containing 25. Mu.g of 3-carboxy-N, N, N-trimethylpropene-2-ene-1-ammonium chloride, 5. Mu.g of (R) -4-amino-2-hydroxy-N, N, N-trimethylpropene-2-ene-1-ammonium chloride, 5. Mu.g of (3-carbamoylallyl) trimethylammonium chloride and 5. Mu.g of (3-carbamoylallyl) trimethylammonium and 5mg of levocarnitine per 1mL of mixed solvent is prepared by using a mixed solvent of a mobile phase and water.
Preferably, the test solution is prepared by: taking levocarnitine, and preparing a test solution containing 5mg of levocarnitine per 1mL of mixed solvent by adopting the mixed solvent of a mobile phase and water.
Preferably, the hydrophilic interaction chromatographic column with dihydroxypropyl groups chemically bonded on the organic mixed silica gel particles is a HILIC chromatographic column (250 mm in length, 4.6mm in inner diameter and 5 mu m in particle size).
Preferably, the mobile phase is a mixed solution of acetonitrile and 50-60 mmol/L potassium dihydrogen phosphate solution with the volume ratio of 75.
Preferably, the pH of the potassium dihydrogen phosphate solution is 4.7.
Preferably, the flow rate is 1.0mL/min.
Preferably, the detection wavelength is 205nm.
Preferably, the sample injection amount is 10 to 20. Mu.L.
Preferably, the mixed solvent is prepared from a mobile phase and water according to a volume ratio of 50.
The further preferable technical scheme of the method for measuring the levocarnitine related substances by the high performance liquid chromatography further comprises the following steps:
preparing a control solution: precisely measuring a proper amount of a test solution, and preparing the test solution containing 0.02-0.03 mg of levocarnitine per 1mL of a mixed solvent by adopting the mixed solvent of a mobile phase and water;
performing HPLC detection on the control solution, wherein the chromatographic condition of the HPLC detection is the same as that of the sample solution;
and (3) determining unknown impurities in the levocarnitine by adopting a self-control method.
Preferably, a control solution is prepared: precisely measuring a proper amount of test solution, and preparing the test solution containing 0.025mg of levocarnitine in each 1mL of mixed solvent by adopting the mixed solvent of a mobile phase and water.
The further preferable technical scheme of the method for measuring the levocarnitine related substances by the high performance liquid chromatography further comprises the following steps:
preparing a sensitivity solution: precisely measuring a proper amount of a reference solution, and preparing a test solution containing 0.002-0.003 mg of levocarnitine in 1m of mixed solvent by adopting the mixed solvent of a mobile phase and water;
and performing HPLC detection on the sensitivity solution, wherein the chromatographic conditions of the HPLC detection are the same as those of the HPLC detection of the test solution.
Preferably, a sensitivity solution is prepared: an appropriate amount of control solution was precisely measured, and a test solution containing 0.0025mg of levocarnitine per 1mL of mobile phase-water (50.
The detector adopted by the high performance liquid chromatography is an ultraviolet absorption detector.
The invention has the beneficial effects that:
the invention is based on high performance liquid chromatography, uses a hydrophilic action chromatographic column chemically bonded with dihydroxypropyl on organic mixed silica gel particles to measure levocarnitine related substances 3-carboxyl-N, N, N-trimethylpropene-2-ene-1-ammonium chloride, (R) -4-amino-2-hydroxy-N, N, N-trimethyl-4-oxobutyl-1-ammonium chloride and (3-carbamoylallyl) trimethylammonium, has high separation efficiency, high analysis speed and high detection sensitivity, controls the levocarnitine related substances 3-carboxyl-N, N, N-trimethylpropene-2-ene-1-ammonium chloride to be not more than 0.3 percent, controls the (R) -4-amino-2-hydroxy-N, N, N-trimethyl-4-oxobutyl-1-ammonium chloride and (3-carbamoylallyl) trimethylammonium to be not more than 0.10 percent, controls any single impurity to be not more than 0.10 percent and controls the total impurity of other any single impurity to be not more than 0.5 percent, and can effectively control the quality of levocarnitine.
Drawings
FIG. 1 is HPLC chromatogram under the chromatographic condition adopted by relative substances of L-carnitine in the 2020 edition of Chinese pharmacopoeia.
FIG. 2 is an HPLC chromatogram of a solution suitable for use in the system of example 1 (peak 1 is impurity D, peak 2 is impurity C, peak 3 is levocarnitine, and peak 4 is impurity A).
FIG. 3 is an HPLC chromatogram of the test solution of example 1 (peak 1 is levocarnitine and peak 2 is impurity A).
FIG. 4 is an HPLC chromatogram of the sensitive solution of example 1 (peak 1 is levocarnitine).
FIG. 5 is an HPLC chromatogram of a system compatible solution of example 2 (peak 1 is impurity D, peak 2 is impurity C, peak 3 is levocarnitine, and peak 4 is impurity A).
FIG. 6 is an HPLC chromatogram of the test solution of example 2 (peak 1 is L-carnitine, and peak 2 is impurity A).
FIG. 7 is an HPLC chromatogram of a system suitability solution of example 3 (peak 1 is impurity D, peak 2 is impurity C, peak 3 is levocarnitine, and peak 4 is impurity A).
FIG. 8 is an HPLC chromatogram of the test solution of example 3 (peak 1 is L-carnitine, and peak 2 is impurity A).
FIG. 9 is an HPLC profile of a system suitability solution of example 4 (peak 1 is impurity D, peak 2 is impurity C, peak 3 is levocarnitine, and peak 4 is impurity A).
FIG. 10 is an HPLC chromatogram of the test solution of example 4 (peak 1 is L-carnitine, and peak 2 is impurity A).
FIG. 11 is an HPLC chromatogram of a system compatible solution of example 5 (peak 1 is impurity D, peak 2 is impurity C, peak 3 is levocarnitine, and peak 4 is impurity A).
FIG. 12 is an HPLC chromatogram of the test solution of example 5 (peak 3 is L-carnitine, and peak 4 is impurity A).
FIG. 13 is an HPLC chromatogram of the quantitative limiting solution of example 6 (peak 1 is levocarnitine).
FIG. 14 is an HPLC chromatogram of a detection-limited solution of example 6 (peak 1 is levocarnitine).
Detailed Description
The technical solutions of the present invention are further illustrated by the following examples, which are not intended to limit the present invention.
The reagents used in the examples are either commercially available or simply synthesized according to established methods.
The type of the high performance liquid chromatograph:
examples 1 to 6 the column was HILIC (250 mm. Times.4.6 mm,5 μm).
The reagents used were:
example 1
A method for determining related substances in levocarnitine by high performance liquid chromatography comprises the following steps:
preparation of system applicability solution: a systematic solution containing 25. Mu.g of 3-carboxy-N, N, N-trimethylpropene-2-ene-1-ammonium chloride, 5. Mu.g of (R) -4-amino-2-hydroxy-N, N, N-trimethyl-4-oxobutyl-1-ammonium chloride, 5. Mu.g of (3-carbamoylallyl) trimethylammonium, and 5mg of (3-carbamoylallyl) trimethylammonium and 5mg of levocarnitine per 1mL of mobile phase-water (50.
Preparing a test solution: a test solution containing 5mg of levocarnitine per 1mL of mobile phase-water (50.
Preparation of a control solution: precisely measuring a proper amount of the test solution, and preparing the test solution containing 0.025mg of levocarnitine per 1mL of mobile phase-water (50.
Preparation of a sensitivity solution: an appropriate amount of the control solution was precisely measured, and a test solution containing 0.0025mg of levocarnitine per 1mL of mobile phase-water (50.
And (3) determination: respectively sucking 10 μ L of system applicability solution, sample solution, control solution and sensitivity solution, injecting into liquid chromatograph for HPLC detection, and performing HPLC detection under the following chromatographic conditions: adopting a HILIC chromatographic column (250 mm multiplied by 4.6mm,5 mu m), wherein the mobile phase is a mixed solvent of acetonitrile and 50mmol/L potassium dihydrogen phosphate solution (pH is 4.7) with the volume ratio of 75, the flow rate is 1.0mL/min, and the detection wavelength is 205nm; the column temperature was 30 ℃.
The HPLC chromatogram of the system suitability solution of this example is shown in FIG. 2, and it can be seen that under the chromatographic conditions of this example, the degrees of separation between (3-carbamoylallyl) trimethylammonium (impurity D, peak 1) and (R) -4-amino-2-hydroxy-N, N, N-trimethyl-4-oxobutyl-1-ammonium chloride (impurity C, peak 2), (R) -4-amino-2-hydroxy-N, N, N-trimethyl-4-oxobutyl-1-ammonium chloride (impurity C) and L-carnitine (peak 3), L-carnitine and 3-carboxy-N, N, N-trimethylpropene-2-ene-1-ammonium chloride (impurity A, peak 4) are all greater than 1.5. And the separation degree of the levocarnitine and the 3-carboxyl-N, N, N-trimethylpropene-2-ene-1-ammonium chloride (impurity A) is more than 2.0, which is superior to that of the levocarnitine and the 3-carboxyl-N, N, N-trimethylpropene-2-ene-1-ammonium chloride (impurity A) which are specified in the 2020 edition of Chinese pharmacopoeia and is not less than 1.0. The method of the invention can more effectively control the amount of 3-carboxyl-N, N, N-trimethylpropene-2-ene-1-ammonium chloride (impurity A), thereby more effectively controlling the product quality.
The HPLC chromatogram of the test solution in this example is shown in FIG. 3, which shows that the sample contains impurity A (peak No. 2), and the content of impurity A in levocarnitine can be determined by an external standard method.
The HPLC spectrum of the sensitivity solution in this example is shown in FIG. 4, which shows that the signal-to-noise ratio (S/N) of L-carnitine is 12.75, the sensitivity meets the requirement, and the quality of L-carnitine can be effectively controlled.
Example 2
This example adjusted the pH of the buffered saline solution in the mobile phase to 4.3.
The method for determining related substances in levocarnitine by using the high performance liquid chromatography comprises the following steps:
preparation of system applicability solution: a systematic solution containing 25. Mu.g of 3-carboxy-N, N, N-trimethylpropene-2-ene-1-ammonium chloride, 5. Mu.g of (R) -4-amino-2-hydroxy-N, N, N-trimethyl-4-oxobutyl-1-ammonium chloride, 5. Mu.g of (3-carbamoylallyl) trimethylammonium, and 5mg of (3-carbamoylallyl) trimethylammonium and 5mg of levocarnitine per 1mL of mobile phase-water (50.
Preparing a test solution: the test solution containing 5mg L-carnitine per 1mL of mobile phase-water (50.
Preparation of a control solution: precisely measuring a proper amount of the test solution, and preparing the test solution containing 0.025mg of levocarnitine per 1mL of mobile phase-water (50.
Preparation of a sensitivity solution: an appropriate amount of control solution was precisely measured, and a test solution containing 0.0025mg of levocarnitine per 1mL of mobile phase-water (50.
And (3) determination: respectively sucking 10 mu L of system applicability solution, test solution, control solution and sensitivity solution, injecting into a liquid chromatograph for HPLC detection, wherein the chromatographic conditions of HPLC detection are as follows: adopting a HILIC chromatographic column (250 mm multiplied by 4.6mm,5 mu m), wherein the mobile phase is a mixed solvent with the volume ratio of acetonitrile-50 mmol/L potassium dihydrogen phosphate solution (pH is 4.3) being 75, the flow rate is 1.0mL/min, and the detection wavelength is 205nm; the column temperature was 30 ℃.
The HPLC chromatogram of the system suitability solution of this example is shown in FIG. 5, and it can be seen that after the pH of the buffered salt solution is adjusted to 4.3, the degrees of separation between (3-carbamoylallyl) trimethylammonium (impurity D, peak 1) and (R) -4-amino-2-hydroxy-N, N, N-trimethyl-4-oxobutyl-1-ammonium chloride (impurity C, peak 2), (R) -4-amino-2-hydroxy-N, N, N-trimethyl-4-oxobutyl-1-ammonium chloride (impurity C) and levocarnitine (peak 3), levocarnitine and 3-carboxy-N, N, N-trimethylpropene-2-ene-1-ammonium chloride (peak 4, impurity A) are greater than 1.5. It is shown that the pH of the buffered salt solution adjusted to 4.3 had no significant effect on the degree of separation between the components.
The HPLC chromatogram of the sample solution of this example is shown in fig. 6, which shows that the sample solution contains impurity a (peak No. 2), and the content of impurity a in levocarnitine can be determined by the external standard method.
According to the HPLC spectrogram of the sensitivity solution in the embodiment, the signal-to-noise ratio (S/N) of the levocarnitine is 19.62, the sensitivity meets the requirement, and the quality of the levocarnitine can be effectively controlled.
Example 3
This example adjusted the pH of the buffered saline solution in the mobile phase to 5.5.
A method for determining related substances in levocarnitine by high performance liquid chromatography comprises the following steps:
preparation of system applicability solution: a systematic working solution containing 25. Mu.g of 3-carboxy-N, N, N-trimethylpropene-2-ene-1-ammonium chloride, 5. Mu.g of (R) -4-amino-2-hydroxy-N, N, N-trimethyl-4-oxobutyl-1-ammonium chloride, 5. Mu.g of (3-carbamoylallyl) trimethylammonium, and 5mg of levocarnitine per 1mL of mobile phase-water (50.
Preparing a test solution: the test solution containing 5mg L-carnitine per 1mL of mobile phase-water (50.
Preparation of a control solution: precisely measuring a proper amount of the test solution, and preparing the test solution containing 0.025mg of levocarnitine per 1mL of mobile phase-water (50.
Preparation of a sensitivity solution: an appropriate amount of control solution was precisely measured, and a test solution containing 0.0025mg of levocarnitine per 1mL of mobile phase-water (50.
And (3) determination: respectively sucking 10 μ L of system applicability solution, sample solution, control solution and sensitivity solution, injecting into liquid chromatograph for HPLC detection, and performing HPLC detection under the following chromatographic conditions: adopting a HILIC chromatographic column (250 mm multiplied by 4.6mm,5 mu m), wherein the mobile phase is a mixed solvent with the volume ratio of acetonitrile-50 mmol/L potassium dihydrogen phosphate solution (pH is 5.5) being 75, the flow rate is 1.0mL/min, and the detection wavelength is 205nm; the column temperature was 30 ℃.
The HPLC chromatogram of the applicable solution of this example is shown in FIG. 7, and it can be seen that after the pH of the buffered salt solution in the mobile phase is adjusted to 5.5, the degrees of separation between (3-carbamoylallyl) trimethylammonium (impurity D, peak 1) and (R) -4-amino-2-hydroxy-N, N, N-trimethyl-4-oxobutyl-1-ammonium chloride (impurity C, peak 2), levocarnitine (peak 3) and 3-carboxy-N, N, N-trimethylpropene-2-ene-1-ammonium chloride (impurity A, peak 4) are each greater than 1.5, and the degrees of separation between (R) -4-amino-2-hydroxy-N, N, N-trimethyl-4-oxobutyl-1-ammonium chloride (impurity C) and levocarnitine are each greater than 1.0. It is shown that the separation degree of L-carnitine from 3-carboxy-N, N, N-trimethylpropene-2-ene-1-ammonium chloride (impurity A) is not affected after the pH of the buffer salt solution is adjusted to 5.5.
The HPLC chromatogram of the sample solution of this example is shown in FIG. 8, which shows that the sample solution contains impurity A (peak No. 2), and the content of impurity A in levocarnitine can be determined by the external standard method.
According to the HPLC spectrogram of the sensitivity solution in the embodiment, the signal-to-noise ratio (S/N) of the levocarnitine is 15.38, the sensitivity meets the requirement, and the quality of the levocarnitine can be effectively controlled.
Example 4
In this example, the sample amount was adjusted to 20. Mu.L.
A method for determining related substances in levocarnitine by high performance liquid chromatography comprises the following steps:
preparation of system applicability solution: a systematic working solution containing 25. Mu.g of 3-carboxy-N, N, N-trimethylpropene-2-ene-1-ammonium chloride, 5. Mu.g of (R) -4-amino-2-hydroxy-N, N, N-trimethyl-4-oxobutyl-1-ammonium chloride, 5. Mu.g of (3-carbamoylallyl) trimethylammonium, and 5mg of levocarnitine per 1mL of mobile phase-water (50.
Preparing a test solution: the test solution containing 5mg L-carnitine per 1mL of mobile phase-water (50.
Preparation of a control solution: precisely measuring a proper amount of the test solution, and preparing the test solution containing 0.025mg of levocarnitine per 1mL of mobile phase-water (50.
Preparation of a sensitivity solution: an appropriate amount of control solution was precisely measured, and a test solution containing 0.0025mg of levocarnitine per 1mL of mobile phase-water (50.
And (3) determination: respectively sucking 20 mu L of system applicability solution, test solution, control solution and sensitivity solution into a liquid chromatograph for HPLC detection, wherein the chromatographic conditions of the HPLC detection are as follows: the method comprises the following steps: HILIC chromatographic column (250 mm × 4.6mm,5 μm), mobile phase is acetonitrile-50 mmol/L potassium dihydrogen phosphate solution (pH is 4.7) mixed solvent with volume ratio of 75; the column temperature was 30 ℃.
The HPLC chromatogram of the applicable solution in this example is shown in FIG. 9, and it can be seen that, after the sample amount is increased, the separation degrees of (3-carbamoylallyl) trimethylammonium (impurity D, peak 1) and (R) -4-amino-2-hydroxy-N, N, N-trimethyl-4-oxobutyl-1-ammonium chloride (impurity C, peak 2), (R) -4-amino-2-hydroxy-N, N, N-trimethyl-4-oxobutyl-1-ammonium chloride (impurity C) and L-carnitine (peak 3), L-carnitine and 3-carboxy-N, N, N-trimethylpropene-2-ene-1-ammonium chloride (impurity A, peak 4) are all greater than 1.5. It is shown that increasing the sample size does not affect the degree of separation between the components.
The HPLC chromatogram of the sample solution of this example is shown in FIG. 10, which shows that the sample solution contains impurity A (peak No. 2), and the content of impurity A in levocarnitine can be determined by the external standard method.
According to the HPLC spectrogram of the sensitivity solution in the embodiment, the signal-to-noise ratio (S/N) of the levocarnitine is 13.21, the sensitivity meets the requirement, and the quality of the levocarnitine can be effectively controlled.
Example 5
This example adjusted the concentration of the buffer salt in the mobile phase to 60mmol/L.
The method for determining related substances in levocarnitine by using the high performance liquid chromatography comprises the following steps:
preparation of system applicability solution: a systematic working solution containing 25. Mu.g of 3-carboxy-N, N, N-trimethylpropene-2-ene-1-ammonium chloride, 5. Mu.g of (R) -4-amino-2-hydroxy-N, N, N-trimethyl-4-oxobutyl-1-ammonium chloride, 5. Mu.g of (3-carbamoylallyl) trimethylammonium, and 5mg of levocarnitine per 1mL of mobile phase-water (50.
Preparing a test solution: a test solution containing 5mg of levocarnitine per 1mL of mobile phase-water (50.
Preparation of a control solution: precisely measuring a proper amount of a test solution, and preparing the test solution containing 0.025mg of levocarnitine per 1mL of mobile phase-water (50; .
Preparation of a sensitivity solution: precisely measuring a proper amount of a control solution, and preparing a test solution containing 0.0025mg of levocarnitine per 1mL of mobile phase-water (50;
and (3) determination: respectively sucking 10 mu L of system applicability solution, test solution, control solution and sensitivity solution into a liquid chromatograph for HPLC detection, wherein the chromatographic conditions of the HPLC detection are as follows: the method comprises the following steps: HILIC chromatographic column (250 mm × 4.6mm,5 μm), mobile phase is acetonitrile-60 mmol/L potassium dihydrogen phosphate solution (pH is 4.7) mixed solvent with volume ratio of 75; the column temperature was 30 ℃.
The HPLC chromatogram of the applicable solution in this example is shown in FIG. 11, and it can be seen that after the concentration of the buffered salt solution is adjusted to 60mmol/L, the degrees of separation between (3-carbamoylallyl) trimethylammonium (impurity D, peak 1) and (R) -4-amino-2-hydroxy-N, N, N-trimethyl-4-oxobutyl-1-ammonium chloride (impurity C, peak 2), levocarnitine (peak 3) and 3-carboxy-N, N, N-trimethylpropene-2-ene-1-ammonium chloride (impurity A, peak 4) are each greater than 1.5, and the degrees of separation between (R) -4-amino-2-hydroxy-N, N, N-trimethyl-4-oxobutyl-1-ammonium chloride (impurity C) and levocarnitine are each greater than 1.0. It shows that the separation degree of L-carnitine and 3-carboxy-N, N, N-trimethylpropene-2-ene-1-ammonium chloride (impurity A) is still more than 1.5 after the concentration of the buffer salt solution in the mobile phase is adjusted to 60mmol/L.
The HPLC chromatogram of the sample solution in this example is shown in fig. 12, which shows that the sample solution contains impurity a (peak 4) and unknown impurity (peak 1 and peak 2), the content of impurity a in levocarnitine can be determined by external standard method, and the unknown impurity in levocarnitine can be determined by self-control method.
According to the HPLC spectrogram of the sensitivity solution in the embodiment, the signal-to-noise ratio (S/N) of the levocarnitine is 14.56, the sensitivity meets the requirement, and the quality of the levocarnitine can be effectively controlled.
Example 6
The method for determining related substances in levocarnitine by using the high performance liquid chromatography comprises the following steps:
preparation of system applicability solution: a systematic working solution containing 25. Mu.g of 3-carboxy-N, N, N-trimethylpropene-2-ene-1-ammonium chloride, 5. Mu.g of (R) -4-amino-2-hydroxy-N, N, N-trimethyl-4-oxobutyl-1-ammonium chloride, 5. Mu.g of (3-carbamoylallyl) trimethylammonium, and 5mg of levocarnitine per 1mL of mobile phase-water (50.
Preparing a test solution: a test solution containing 5mg of levocarnitine per 1mL of mobile phase-water (50.
Preparation of a control solution: precisely measuring a proper amount of the test solution, and preparing the test solution containing 0.025mg of levocarnitine per 1mL of mobile phase-water (50.
Preparation of a quantitative limiting solution (sensitivity solution): a proper amount of control solution is precisely measured, and a test solution containing 0.00125mg of levocarnitine per 1mL of mobile phase-water (50. Corresponding to 0.025% of the concentration of the test sample.
Preparing a detection limiting solution: the control solution was measured precisely and an appropriate amount of mobile phase-water (50. Corresponding to 0.01% of the concentration of the test sample.
And (3) determination: respectively sucking 20 mu L of system applicability solution, test solution, control solution, quantitative limit solution, detection limit solution and sensitivity solution into a liquid chromatograph for HPLC detection, wherein the chromatographic conditions of the HPLC detection are as follows: adopting a HILIC chromatographic column (250 mm multiplied by 4.6mm,5 mu m), wherein the mobile phase is a mixed solvent with the volume ratio of acetonitrile-50 mmol/L potassium dihydrogen phosphate solution (pH is 4.7) being 75, the flow rate is 1.0mL/min, and the detection wavelength is 205nm; the column temperature was 30 ℃.
The HPLC chromatogram of the quantitation limit solution of this example is shown in FIG. 13, peak No. 1 is levocarnitine; the HPLC chromatogram of the detection limit solution of this example is shown in FIG. 14, and the peak No. 1 is L-carnitine.
The concentration of the sensitive solution in related substances of the levocarnitine in the 2020 th edition of Chinese pharmacopoeia is equivalent to 0.05 percent of the concentration of the solution of the test sample; in the detection method, the concentration of the sensitive solution is equal to 0.025 percent of that of the test sample solution, and the method has higher sensitivity. Therefore, the separation degree and the sensitivity of the method are better than those of the related substance detection in the current Chinese pharmacopoeia 2020 edition, which shows that the method can more effectively control the quality of products.
Claims (10)
1. A method for determining levocarnitine related substances by high performance liquid chromatography is characterized by comprising the following steps: the method comprises the following steps:
preparing a system applicability solution: taking 3-carboxyl-N, N, N-trimethylpropene-2-ene-1-ammonium chloride, (R) -4-amino-2-hydroxy-N, N, N-trimethyl-4-oxobutyl-1-ammonium chloride and (3-carbamoylallyl) trimethylammonium and a levocarnitine reference substance, and preparing a reference substance solution containing 15-30 mu g of 3-carboxyl-N, N, N-trimethylpropene-2-ene-1-ammonium chloride, 3-7 mu g of (R) -4-amino-2-hydroxy-N, N, N-trimethyl-4-oxobutyl-1-ammonium chloride, 3-7 mu g of (3-carbamoylallyl) trimethylammonium and 3-15 mg of levocarnitine per 1mL of mixed solvent by adopting a mixed solvent of a mobile phase and water;
preparing a test solution: preparing a test solution containing 4-6 mg of levocarnitine per 1mL of mixed solvent by using the mixed solvent of a mobile phase and water;
respectively carrying out HPLC detection on the system applicability solution and the sample solution, wherein the chromatographic conditions of the HPLC detection are as follows: adopting a hydrophilic action chromatographic column chemically bonded with dihydroxypropyl on organic mixed silica gel particles, wherein a mobile phase is a mixed solution of acetonitrile and 50-60 mmol/L potassium dihydrogen phosphate solution with the volume ratio of 65-75, the column temperature is 25-35 ℃, the flow rate is 0.8-1.2 mL/min, and the detection wavelength is 200-210 nm; the sample injection amount is 5-100 mu L;
measuring the contents of impurities 3-carboxyl-N, N, N-trimethylpropene-2-ene-1-ammonium chloride, (R) -4-amino-2-hydroxyl-N, N, N-trimethyl-4-oxobutyl-1-ammonium chloride and (3-carbamoylallyl) trimethylammonium chloride in the levocarnitine by adopting an external standard method;
wherein, the mixed solvent is prepared from a mobile phase and water according to a volume ratio of 40-60; the pH value of the 50mmol/L potassium dihydrogen phosphate solution is 4.3-5.5.
2. The method for determining levocarnitine related substances according to claim 1, which comprises the steps of:
preparing a system applicability solution: preparing a control solution containing 25 mu g of 3-carboxyl-N, N, N-trimethylpropene-2-ene-1-ammonium chloride, 5 mu g of (R) -4-amino-2-hydroxy-N, N, N-trimethylpropene-2-ene-1-ammonium chloride, 5 mu g of (3-carbamoylallyl) trimethylammonium chloride and 5 mu g of (3-carbamoylallyl) trimethylammonium and 5mg of levocarnitine per 1mL of mixed solvent by using a mixed solvent of a mobile phase and water;
preparing a test solution: taking levocarnitine, and preparing a test solution containing 5mg of levocarnitine per 1mL of mixed solvent by adopting the mixed solvent of a mobile phase and water.
3. The method for determining the levocarnitine related substance by high performance liquid chromatography according to claim 1, which comprises the following steps: the hydrophilic chromatographic column (column length 250mm × inner diameter 4.6mm, particle size 5 μm) is HILIC chromatographic column.
4. The method for determining levocarnitine related substances according to claim 1, which comprises the steps of: the mobile phase is a mixed solution of acetonitrile and 50-60 mmol/L potassium dihydrogen phosphate solution with the volume ratio of 75.
5. The method for determining levocarnitine related substances according to claim 1, which comprises the steps of: the pH of the potassium dihydrogen phosphate solution was 4.7.
6. The method for determining levocarnitine related substances according to claim 1, which comprises the steps of: the flow rate was 1.0mL/min.
7. The method for determining levocarnitine related substances according to claim 1, which comprises the steps of: the detection wavelength is 205nm.
8. The method for determining levocarnitine related substances according to claim 1, which comprises the steps of: the detector used in the high performance liquid chromatography is an ultraviolet absorption detector.
9. The method for determining the levocarnitine related substance by high performance liquid chromatography according to claim 1, which comprises the following steps: the mixed solvent is prepared from a mobile phase and water according to a volume ratio of 50.
10. The method for determining levocarnitine related substances according to claim 1, which comprises the steps of: further comprising:
preparing a control solution: precisely measuring a proper amount of a test solution, and preparing the test solution containing 0.02-0.03 mg of levocarnitine per 1mL of a mixed solvent by adopting the mixed solvent of a mobile phase and water;
respectively carrying out HPLC detection on the control solution and the sensitivity solution, wherein the chromatographic condition of the HPLC detection is the same as that of the sample solution;
the unknown impurities in the levocarnitine are determined by a self-control method.
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