Detection method of DCU in indapamide bulk drug
阅读说明：本技术 吲达帕胺原料药中dcu的检测方法 (Detection method of DCU in indapamide bulk drug ) 是由 练城 张萍 朱婷 王芝 龚明峰 陈小波 于 2019-11-27 设计创作，主要内容包括：本发明涉及一种吲达帕胺原料药中DCU的检测方法。其是利用气相色谱对吲达帕胺原料药中DCU进行检测,包括以下步骤：(1)配制吲达帕胺原料药供试品溶液；(2)配制DCU的对照品溶液；(3)进样,按以下条件对步骤(1)供试品溶液和步骤(2)中的对照品溶液分别进行检测：色谱条件：固定相为100％聚二甲基硅氧烷的毛细管柱,规格：30m×0.25mm×0.25μm,载气为惰性气体,进样口温度为310-350℃,检测器温度为330℃,分流比为1-50：1,初始柱流速为1.0-2.0ml/min,升温程序为：初始温度45-80℃,保持2min,以25-70℃/min的速率升温至230℃,保持5min,再继续以40℃/min的速率升温至280℃,保持15min。本发明使用的试剂毒性小,杂质干扰少,检测方法可靠稳定,对吲达帕胺原料药的质量控制具有重要意义。(The invention relates to a method for detecting DCU in indapamide bulk drug. The method is used for detecting DCU in indapamide bulk drug by using gas chromatography, and comprises the following steps: (1) preparing a test solution of the indapamide bulk drug; (2) preparing a DCU reference substance solution; (3) and (3) injecting samples, and respectively detecting the sample solution in the step (1) and the reference substance solution in the step (2) according to the following conditions: chromatographic conditions are as follows: the stationary phase is a capillary column of 100% polydimethylsiloxane, and the specification is as follows: 30m is multiplied by 0.25mm is multiplied by 0.25 mu m, the carrier gas is inert gas, the injection port temperature is 310-: 1, the initial column flow rate is 1.0-2.0ml/min, and the temperature rise program is as follows: the initial temperature is 45-80 deg.C, maintaining for 2min, heating to 230 deg.C at 25-70 deg.C/min, maintaining for 5min, heating to 280 deg.C at 40 deg.C/min, and maintaining for 15 min. The reagent used in the invention has low toxicity, less impurity interference and reliable and stable detection method, and has important significance for quality control of the indapamide bulk drug.)
1. The method for detecting DCU in the indapamide bulk drug is characterized in that the DCU in the indapamide bulk drug is detected by using gas chromatography, and comprises the following steps:
(1) preparing a test solution of the indapamide bulk drug;
(2) preparing a DCU reference substance solution;
(3) and (3) injecting samples, and respectively detecting the sample solution in the step (1) and the reference substance solution in the step (2) according to the following conditions:
chromatographic conditions are as follows: the stationary phase is a capillary column of 100% polydimethylsiloxane, and the specification is as follows: 30 m.times.0.25 mm.times.0.25 μm,
the carrier gas is an inert gas, and the carrier gas is,
the temperature of the sample inlet is 310-350 ℃,
the temperature of the detector was 330 c,
the split ratio is 1-50: 1,
the initial column flow rate is 1.0-2.0ml/min,
the temperature rising procedure is as follows: the initial temperature is 45-80 deg.C, maintaining for 2min, heating to 230 deg.C at 25-70 deg.C/min, maintaining for 5min, heating to 280 deg.C at 40 deg.C/min, and maintaining for 15 min.
2. The method for detecting DCU in an indapamide bulk drug according to claim 1, wherein the column flow rate is 1.0-2.0ml/min for 11.5min, and is increased from 2ml/min to 3.5ml/min for 15 min.
3. The method for detecting DCU in indapamide bulk drug according to claim 1, wherein the injection port temperature is 320 ℃.
4. The method for detecting DCU in indapamide bulk drug according to claim 1, wherein the split ratio is 2-4: 1.
5. the method for detecting DCU in indapamide bulk drug according to claim 1, wherein the initial column temperature is 45-80 ℃.
6. The method for detecting DCU in an indapamide bulk drug according to claim 5, wherein the initial column temperature is 45-50 ℃.
7. The method for detecting DCU in an indapamide bulk drug according to claim 1, wherein the sample size in the chromatographic condition is 1 μ L.
8. The method for detecting DCU in an indapamide drug substance as claimed in claim 1, wherein the concentration of the indapamide drug substance in step (1) is 1-10 mg/ml.
9. The method for detecting DCU in an indapamide drug substance as claimed in claim 5, wherein the concentration of DCU in step (2) is 1.5-15 μ g/ml.
Technical Field
The invention relates to a method for detecting DCU in indapamide bulk drug.
Background
Indapamide is a sulfonamide diuretic, has diuretic and calcium antagonistic effects, is a potent and long-acting hypotensive drug, and is clinically used for treating water-sodium retention in congestive heart failure. DCC (dicyclohexylcarbodiimide) and DCU (dicyclohexylurea) are residues in the indapamide synthesis process, the residual concentration of which may have serious quality consequences for the product. At present, only a DCC mass fraction determination method is disclosed in the national standard HG/T5320-2018, but a DCU content detection method is not established. Further, no other patents or documents are published in China about the detection method of the content of DCU. In the European pharmacopoeia and the United states pharmacopoeia, a detection method for the content of DCU in indapamide is not established, and a method for determining the content of DCU by adopting high performance liquid chromatography is only established in other types of medicines (lomustine bulk drugs). In summary, it is really necessary to establish a method for determining the content of DCU in the indapamide bulk drug.
Disclosure of Invention
The invention provides a method for detecting DCU in indapamide bulk drug, aiming at the technical problems in the prior art.
The technical scheme for solving the technical problems is as follows: the method for detecting DCU in the indapamide bulk drug by using gas chromatography comprises the following steps:
(1) preparing a test solution of the indapamide bulk drug;
(2) preparing a DCU reference substance solution;
(3) and (3) injecting samples, and respectively detecting the sample solution in the step (1) and the reference substance solution in the step (2) according to the following conditions:
chromatographic conditions are as follows: the stationary phase is a capillary column of 100% polydimethylsiloxane, and the specification is as follows: 30m × 0.25mm × 0.25 μm, inert gas as carrier, preferably nitrogen, 350 ℃ as injection port temperature, 330 ℃ as detector temperature, 1-50 as flow dividing ratio: 1, the initial column flow rate is 1.0-2.0ml/min, and the temperature rise program is as follows: the initial temperature is 45-80 deg.C, maintaining for 2min, heating to 230 deg.C at 25-70 deg.C/min, maintaining for 5min, heating to 280 deg.C at 40 deg.C/min, and maintaining for 15 min.
Wherein the column flow rate is 1.0-2.0ml/min for 11.5min, and 2ml/min to 3.5ml/min for 15 min.
Wherein the injection port temperature is 320 ℃.
Preferably, the split ratio is 2-4: 1.
preferably, the initial column temperature is 45-80 ℃.
More preferably, the initial column temperature is 45-50 ℃.
Wherein the sample amount in the chromatographic conditions is 1. mu.L.
Wherein, the concentration of the indapamide bulk drug in the step (1) is 1-10 mg/ml.
Wherein the concentration of DCU in the step (2) is 1.5-15 mu g/ml.
In the invention, the related calculation formula is as follows:
the DCU background content (mg) in the standard sample is multiplied by the sample weighing amount (mg) of the sample to be tested and the DCU content (mg/mg) in the background
Wherein: a. theTest articlePeak area of DCU in the background sample;
CcontrolConcentration of DCU in control solution, unit: mg/ml;
AcontrolControl solutionPeak area of medium DCU;
Wtest articleWeighing sample amount of a background sample, unit: mg;
Aadding standard sampleThe peak area of DCU in the standard sample is added;
v ═ dilution volume of spiked or background sample, units: and (3) ml.
1. The invention optimizes the initial column temperature in the gas chromatography condition:
during optimization: a chromatographic column: elite-1, 30 m.times.0.25 mm.times.0.25 μm; the carrier gas is nitrogen, the flow rate is 1.5ml/min and is kept for 11.5min, the flow rate is increased to 3.5ml/min at 2ml/min and is kept for 15 min; the temperature of a sample inlet is 320 ℃; the temperature of the detector is 330 ℃; the sample amount is 1 mul; split-flow sample injection, split-flow ratio 3: 1.
and (3) keeping the labeled sample solution for 2min at the initial column temperature of 40 ℃, 45 ℃, 50 ℃, 55 ℃ and 80 ℃, increasing the temperature to 230 ℃ at the speed of 40 ℃/min, keeping for 5min, continuing increasing the temperature to 280 ℃ at the speed of 40 ℃/min, keeping for 15min, respectively carrying out sample injection detection, wherein the temperature is slowly reduced when the initial column temperature is 40 ℃, and the peak type of the DCU chromatographic peak and the separation degree of other adjacent chromatographic peaks are good at other temperatures, and can meet the DCU detection requirement.
2. The invention optimizes the split ratio in the gas chromatography condition:
during optimization, the chromatographic column: elite-1, 30 m.times.0.25 mm.times.0.25 μm; the carrier gas is nitrogen, the flow rate is 1.5ml/min and is kept for 11.5min, the flow rate is increased to 3.5ml/min at 2ml/min and is kept for 15 min; the initial column temperature is 50 ℃, the column temperature is kept for 2min, the temperature is increased to 230 ℃ at the speed of 40 ℃/min, the column temperature is kept for 5min, the temperature is increased to 280 ℃ at the speed of 40 ℃/min, and the column temperature is kept for 15 min; the temperature of a sample inlet is 320 ℃; the temperature of the detector is 330 ℃; the amount of sample was 1. mu.l.
The split ratios are changed to be 1:1, 2:1, 3:1, 6:1, 10:1, 20:1, 40:1 and 50:1 respectively, the solutions of the added standard samples are taken for sample injection detection respectively, the peak type of the chromatographic peak of the DCU and the separation degree of other adjacent chromatographic peaks are good, the detection requirements of the DCU can be met, when the split ratio is 50:1, the signal-to-noise ratio of the DCU chromatographic peak is 18.9, the signal-to-noise ratio close to the quantitative requirements is 10, and therefore the split ratio is not larger than 50: 1.
3. The invention optimizes the initial column flow rate in the gas chromatography condition:
during optimization: a chromatographic column: elite-1, 30 m.times.0.25 mm.times.0.25 μm; the carrier gas is nitrogen; the initial column temperature is 50 ℃, the column temperature is kept for 2min, the temperature is increased to 230 ℃ at the speed of 40 ℃/min, the column temperature is kept for 5min, the temperature is increased to 280 ℃ at the speed of 40 ℃/min, and the column temperature is kept for 15 min; the temperature of a sample inlet is 320 ℃; the temperature of the detector is 330 ℃; the sample amount is 1 mul, and the split ratio is 3: 1.
And (3) keeping the initial flow of the carrier gas of the standard sample solution for 11.5min, increasing the initial flow of the carrier gas to 3.5ml/min and keeping the initial flow of the carrier gas for 15min, and respectively carrying out sample injection detection, wherein the peak type of the DCU chromatographic peak and the separation degree of other adjacent chromatographic peaks are good under the condition of each flow rate, and the DCU detection requirements can be met.
4. The invention optimizes the heating rate in the gas chromatography condition:
during optimization: a chromatographic column: elite-1, 30 m.times.0.25 mm.times.0.25 μm; the carrier gas is nitrogen, the flow rate is 1.5ml/min and is kept for 11.5min, the flow rate is increased to 3.5ml/min at 2ml/min and is kept for 15 min; the initial column temperature is 50 ℃, the column temperature is kept for 2min, the temperature is increased to 230 ℃ at the speed of 40 ℃/min, the column temperature is kept for 5min, the temperature is increased to 280 ℃ at the speed of 40 ℃/min, and the column temperature is kept for 15 min; the temperature of a sample inlet is 320 ℃; the temperature of the detector is 330 ℃; the sample amount is 1 mul, and the split ratio is 3: 1.
The initial column temperature is 50 ℃, the initial column temperature is kept for 2min, the temperature rising rate is changed to rise to 230 ℃ at the speed of 25 ℃/min, 30 ℃/min, 35 ℃/min, 40 ℃/min, 45 ℃/min, 50 ℃/min, 60 ℃/min and 70 ℃/min, the temperature is kept for 5min, the temperature is further raised to 280 ℃ at the speed of 40 ℃/min, the temperature is kept for 15min, the sample solution with the standard is taken to be injected and detected respectively, the peak type of the DCU chromatographic peak and the separation degree of other adjacent chromatographic peaks are good under the condition of each temperature rising rate, and the DCU detection requirements can be met.
The method for detecting the content of DCU in the indapamide bulk drug has the advantages that the linear range is between 0.752 and 15.04 mu g/ml, the detection limit is 0.376 mu g/ml, and the recovery rate is more than 98%. The detection method for the content of DCU in the indapamide bulk drug provided by the invention has the advantages of low toxicity of used reagents, less impurity interference and reliability and stability, and has important significance for quality control of the indapamide bulk drug.
Drawings
FIG. 1 is a chromatogram of a test solution numbered 1 in example 2;
FIG. 2 is a chromatogram of a test solution numbered 2 in example 2;
FIG. 3 is a chromatogram of test solution No. 3 from example 2;
FIG. 4 is a chromatogram of test solution No. 4 from example 2;
FIG. 5 is a chromatogram of a DCU control solution of example 2.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.