CN113848271B - Method for detecting related substances in levocetirizine hydrochloride oral solution - Google Patents
Method for detecting related substances in levocetirizine hydrochloride oral solution Download PDFInfo
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Abstract
The invention provides a method for detecting related substances in a levocetirizine hydrochloride oral solution, which belongs to the technical field of medicine analysis. According to the invention, acetonitrile and potassium dihydrogen phosphate buffer solution are used as mobile phases, gradient elution is carried out on a high performance liquid chromatograph, and p-chlorobenzol, p-chlorobenzophenone, p-chlorobenzomethylpiperazine, levocetirizine amide and p-hydroxybenzoic acid can be accurately and efficiently detected at one time under a liquid phase condition.
Description
Technical Field
The invention relates to the technical field of medicine analysis, in particular to a method for detecting related substances in levocetirizine hydrochloride oral solution.
Background
The levocetirizine hydrochloride is an active R-type single optical isomer of the second generation antihistamine cetirizine widely used in clinic, is a new generation of high-efficiency oral selective histamine H1 receptor antagonist without sedative side effects, has no obvious anticholinergic and 5-hydroxytryptamine resisting effects, has smaller central inhibition effect, and is used for treating allergic diseases such as urticaria, allergic rhinitis, eczema, dermatitis, skin pruritus and the like.
5 impurities are generated in the synthesis process of the levocetirizine hydrochloride bulk drug and in the production and storage processes of oral solution, wherein the p-chlorobenzhydryl piperazine and the levocetirizine amide are important intermediates for the synthesis of the levocetirizine hydrochloride, and belong to process impurities; the p-chlorobenzophenone and the p-chlorobenzol are not only important intermediates for synthesizing the levocetirizine hydrochloride, but also main degradation impurities of the oral solution under the oxidation or high temperature conditions, and belong to process impurities and degradation impurities; parahydroxybenzoic acid is a degradation product of the antibacterial agent, the hydroxybenzoate in the oral solution. In the known literature, the detection method of the United states Pharmacopeia USP41 can detect two impurities, namely levocetirizine amide and p-chlorobenzhydryl piperazine; inlet drug registration Standard Inlet registration Standard JX20110262 the detection method of levocetirizine hydrochloride drops can detect p-chlorobenzol alcohol and p-chlorobenzophenone. At present, a detection method capable of simultaneously detecting the above 5 impurities at one time has not been found.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for detecting related substances in an oral solution of levocetirizine hydrochloride, which can accurately and efficiently detect the above 5 impurities at one time under one liquid phase condition.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for detecting related substances in a levocetirizine hydrochloride oral solution, which adopts a high performance liquid chromatography to measure the related substances in the levocetirizine hydrochloride oral solution and uses an external standard method to calculate the content of the related substances in the levocetirizine hydrochloride oral solution;
in the high performance liquid chromatography, a mobile phase A is potassium dihydrogen phosphate buffer solution, and a mobile phase B is acetonitrile;
the gradient elution procedure of the mobile phase is:
0～t 1 min, mobile phase A65% and mobile phase B35%;
t 1 ～t 2 min, the mobile phase A is reduced from 65% to 30% at constant speed, and the mobile phase B is increased from 35% to 70% at constant speed;
t 2 ～t 3 min, mobile phase A30% and mobile phase B70%;
t 3 ～t 4 min, the mobile phase A is increased from 30% to 65% at a constant speed, and the mobile phase B is decreased from 70% to 35% at a constant speed;
t 4 ～t 5 min, mobile phase A65% and mobile phase B35%;
the t is 1 13 to 15, t 2 34 to 36, t 3 37 to 39, t 4 39 to 41, t 5 49 to 51
Preferably, said t 1 Is 14, t 2 Is 35, t 3 Is 38, t 4 40, t 5 50.
Preferably, the concentration of the monopotassium phosphate in the monopotassium phosphate buffer solution is 25-35 mmol/L, and the pH value of the monopotassium phosphate buffer solution is 2.8-3.2.
Preferably, the chromatographic column in the high performance liquid chromatography is a chromatographic column with octane-bonded silica gel as a filler.
Preferably, the chromatography column is of the type (Agilent SB-C 8 250mm 4.6mm,5 μm), (Agilent ZORBAX RX-C8 250mm 4.6mm,5 μm), (YMC-Pack C8 mm 4.6mm,5 μm) or (COSMOSIL Packed Column C8-MS250mm 4.6 mm).
Preferably, the temperature of the chromatographic column is 25-35 ℃, and the flow rate of the mobile phase is 1.0mL/min.
Preferably, the detector in the high performance liquid chromatography is an ultraviolet detector, and the detection wavelength of the ultraviolet detector is 228-232 nm.
Preferably, the related substances are p-chlorobenzoyl alcohol, p-chlorobenzophenone, p-chlorobenzoyl piperazine, levocetirizine amide and p-hydroxybenzoic acid.
Preferably, the impurity reference substance solution in the high performance liquid chromatography is a mixture of p-chlorobenzol, p-chlorobenzophenone, p-chlorobenzoyl piperazine, levocetirizine amide, p-hydroxybenzoic acid and a solvent, wherein the solvent is a mixed solution of acetonitrile and water in a volume ratio of 1:1;
the mass of each 1mL of impurity reference substance solution is 0.4 mug, 0.4 mug and 1.0 mug of p-chlorodiphenyl methanol, p-chlorobenzophenone, p-chlorodiphenyl methylpiperazine, levocetirizine amide and p-hydroxybenzoic acid respectively.
Preferably, the sample solution in the high performance liquid chromatography is a mixture of levocetirizine hydrochloride oral solution and a solvent, wherein the solvent is a mixed solution of acetonitrile and water in a volume ratio of 1:1;
the concentration of the levocetirizine hydrochloride oral solution in the sample solution is 0.1mg/mL.
The beneficial technical effects are as follows: the invention provides a method for detecting related substances in a levocetirizine hydrochloride oral solution, which adopts a high performance liquid chromatography to measure the related substances in the levocetirizine hydrochloride oral solution and calculates the content of the related substances in the levocetirizine hydrochloride oral solution by an external standard method. According to the invention, acetonitrile and potassium dihydrogen phosphate buffer solution are used as mobile phases, gradient elution is carried out on a high performance liquid chromatograph, and p-chlorobenzol, p-chlorobenzophenone, p-chlorobenzomethylpiperazine, levocetirizine amide and p-hydroxybenzoic acid can be accurately and efficiently detected at one time under a liquid phase condition.
Drawings
FIG. 1 is an HPLC chart of a mixed solution of an impurity localization and separation control in example 1; wherein the impurities represented by the peaks are respectively: 1-p-hydroxybenzoic acid; 2-hydroxybenzoic acid methyl ester; 3-levocetirizine amide; 4-p-chlorobenzhydryl piperazine; 5-levocetirizine; 6-hydroxypropyl-hydroxyphenyl; 7-p-chlorobenzhydrol; 8-p-chlorobenzophenone;
FIG. 2 is an HPLC chart of a p-hydroxybenzoic acid control solution in example 2, wherein the impurities represented by the peaks are respectively: 1-p-hydroxybenzoic acid;
FIG. 3 is an HPLC plot of a blank solution of parahydroxybenzoic acid accuracy in example 2;
FIG. 4 is an HPLC chart of a sample solution of 50% parahydroxybenzoic acid accuracy in example 2, wherein the individual peaks represent the impurities as follows: 1-p-hydroxybenzoic acid;
FIG. 5 is an HPLC chart of a sample solution of 100% parahydroxybenzoic acid accuracy in example 2, wherein the respective peaks represent the impurities: 1-p-hydroxybenzoic acid;
FIG. 6 is an HPLC chart of a sample solution of example 2 with 150% parahydroxybenzoic acid accuracy, wherein the individual peaks represent the impurities: 1-p-hydroxybenzoic acid;
FIG. 7 is an HPLC chart of an accuracy control solution of the related substances in example 2, wherein the impurities represented by the respective peaks are respectively: 1-levocetirizine amide, 2-p-chlorobenzhydryl piperazine, 3-p-chlorobenzhydrol, 4-p-chlorobenzophenone;
FIG. 8 is an HPLC chart of a blank solution with respect to the accuracy of the substance of example 2;
FIG. 9 is an HPLC chart of a sample solution of 50% concentration with respect to the accuracy of the substance in example 2, wherein the respective peaks represent the impurities: 1-levocetirizine amide, 2-p-chlorobenzhydryl piperazine, 3-p-chlorobenzhydrol, 4-p-chlorobenzophenone;
fig. 10 is an HPLC diagram of a sample solution with 100% concentration of the relevant substance accuracy, wherein the impurities represented by the peaks are respectively: 1-levocetirizine amide, 2-p-chlorobenzhydryl piperazine, 3-p-chlorobenzhydrol, 4-p-chlorobenzophenone;
FIG. 11 is an HPLC chart of a sample solution with 150% concentration of the relevant substance in example 2, wherein the respective peaks represent the impurities: 1-levocetirizine amide, 2-p-chlorobenzhydryl piperazine, 3-p-chlorobenzhydrol, 4-p-chlorobenzophenone;
FIG. 12 is an HPLC chart of an impurity control solution in example 4; wherein the impurities represented by the peaks are respectively: 1-p-hydroxybenzoic acid; 2-levocetirizine amide; 3-p-chlorobenzhydryl piperazine; 4-p-chlorobenzhydrol; 5-p-chlorobenzophenone;
FIG. 13 is an HPLC plot of the control solution of example 4; the impurities represented by the peaks in (a) are respectively: 1-hydroxybenzoic acid methyl ester; 2-levocetirizine; 3-hydroxypropyl-hydroxyphenyl;
fig. 14 is an HPLC diagram of a sample solution of 200501 batches of levocetirizine hydrochloride oral solution in example 4; the impurities represented by the peaks in (a) are respectively: 1-p-hydroxybenzoic acid; 2-hydroxybenzoic acid methyl ester; 3-levocetirizine amide; 4-p-chlorobenzhydryl piperazine; 5-levocetirizine; 6-hydroxypropyl-hydroxyphenyl; 7-p-chlorobenzhydrol; 8-p-chlorobenzophenone;
fig. 15 is an HPLC diagram of a sample solution of 200502 batches of levocetirizine hydrochloride oral solution in example 4; the impurities represented by the peaks in (a) are respectively: 1-p-hydroxybenzoic acid; 2-hydroxybenzoic acid methyl ester; 3-levocetirizine amide; 4-p-chlorobenzhydryl piperazine; 5-levocetirizine; 6-hydroxypropyl-hydroxyphenyl; 7-p-chlorobenzhydrol; 8-p-chlorobenzophenone;
FIG. 16 is an HPLC chart of 200503 batches of test solutions of levocetirizine hydrochloride oral solution in example 4; the impurities represented by the peaks in (a) are respectively: 1-p-hydroxybenzoic acid; 2-hydroxybenzoic acid methyl ester; 3-levocetirizine amide; 4-p-chlorobenzhydryl piperazine; 5-levocetirizine; 6-hydroxypropyl-hydroxyphenyl; 7-p-chlorobenzhydrol; 8-p-chlorobenzophenone;
FIG. 17 is a HPLC chart of a control mixed solution of the relevant substances of the levocetirizine hydrochloride drops of the import registration standard JX 20110262; the impurities represented by the peaks in (a) are respectively: 1-p-hydroxybenzoic acid; 2-levocetirizine amide; 3-levocetirizine; 4-hydroxybenzoic acid methyl ester; 5-p-chlorobenzhydryl piperazine; 6-hydroxypropyl-hydroxyphenyl; 7-p-chlorobenzhydrol; 8-p-chlorobenzophenone.
Detailed Description
The invention provides a method for detecting related substances in a levocetirizine hydrochloride oral solution, which adopts a high performance liquid chromatography to measure the related substances in the levocetirizine hydrochloride oral solution and calculates the content of the related substances in the levocetirizine hydrochloride oral solution by using an external standard;
in the high performance liquid chromatography, a mobile phase A is potassium dihydrogen phosphate buffer solution, and a mobile phase B is acetonitrile;
the gradient elution procedure of the mobile phase is:
0～t 1 min mobile phase A65% mobile phase B35%;
t 1 ～t 2 min, the mobile phase A is reduced from 65% to 30% at constant speed, and the mobile phase B is increased from 35% to 70% at constant speed;
t 2 ～t 3 min, mobile phase A30% and mobile phase B70%;
t 3 ～t 4 min, the mobile phase A is increased from 30% to 65% at a constant speed, and the mobile phase B is decreased from 70% to 35% at a constant speed;
t 4 ～t 5 min, mobile phase A65% and mobile phase B35%;
the t is 1 13 to 15, t 2 34 to 36, t 3 37 to 39, t 4 39 to 41, t 5 49 to 51
Preferably, said t 1 Is 14, t 2 Is 35, t 3 Is 38, t 4 40, t 5 50.
In the present invention, the concentration of potassium dihydrogen phosphate in the potassium dihydrogen phosphate buffer is preferably 25 to 35mmol/L, more preferably 30 mmol/L; the pH of the potassium dihydrogen phosphate buffer is preferably 2.8 to 3.2, more preferably 3.0.
In the invention, the chromatographic column in the high performance liquid chromatography is a chromatographic column with octane bonded silica gel as a filler; the column is preferably of the type (Agilent SB-C 8 250mm 4.6mm,5 μm), (Agilent ZORBAX RX-C8 mm 4.6mm,5 μm), (YMC-Pack C8 mm 4.6mm,5 μm) or (COSMOSIL Packed Column C8-MS250mm 4.6 mm), more preferably (Agilent SB-C) 8 250 mm. Times.4.6 mm,5 μm); the temperature of the chromatographic column is preferably 25-35 ℃, more preferably 30 ℃; the flow rate of the eluent in the chromatographic column is preferably 1.0mL/min; the elution time of the eluent in the chromatographic column is preferably 49-51 min.
In the present invention, the detector in the high performance liquid chromatography is preferably an ultraviolet detector; the detection wavelength of the ultraviolet detector is preferably 228 to 232nm, more preferably 230nm. In the present invention, the related substances are preferably p-chlorobenzoyl alcohol, p-chlorobenzophenone, p-chlorobenzoyl piperazine, levocetirizine amide and p-hydroxybenzoic acid.
In the invention, the impurity reference substance solution in the high performance liquid chromatography is preferably a mixture of p-chlorobenzol, p-chlorobenzophenone, p-chlorobenzopiperazine, levocetirizine amide and p-hydroxybenzoic acid and a solvent, and the solvent is preferably a mixed solution of acetonitrile and water in a volume ratio of 1:1; the mass of p-chlorobenzhydrol, p-chlorobenzophenone, p-chlorobenzhydryl piperazine, levocetirizine amide and p-hydroxybenzoic acid in each 1mL of the impurity reference solution is preferably 0.4 mug, 0.4 mug and 1.0 mug respectively.
In the invention, the sample solution in the high performance liquid chromatography is preferably a mixture of a levocetirizine hydrochloride oral solution and a solvent; the solvent is preferably a mixed solution of acetonitrile and water in a volume ratio of 1:1; the concentration of the levocetirizine hydrochloride oral solution in the test solution is preferably 0.1mg/mL.
For a better understanding of the present invention, the following examples are further illustrated, but are not limited to the following examples.
Example 1
Impurity positioning and separation:
respectively taking p-hydroxybenzoic acid, levocetirizine amide, p-chlorodiphenyl methylpiperazine, levocetirizine hydrochloride, methylparaben, propylparaben, p-chlorodiphenyl methanol and p-chlorobenzophenone, and preparing a reference substance mixed solution by acetonitrile-water (1:1). The concentration of each raw material in the obtained reference substance mixed solution is as follows: 1. Mu.g/ml of parahydroxybenzoic acid, 0.4. Mu.g/ml of levocetirizine amide, 0.4. Mu.g/ml of parachlorobenzopiperazine, 0.1mg/ml of levocetirizine hydrochloride, 0.135mg/ml of methylparaben, 0.015mg/ml of propylparaben, 0.4. Mu.g/ml of parachlorobenzoic acid, and 0.4. Mu.g/ml of parachlorobenzophenone.
Chromatographic conditions
The chromatographic conditions of the high performance liquid chromatography are as follows:
instrument: agilent 1260 high performance liquid chromatograph;
chromatographic column: octane bonded silica gel is used as filler, and the model of the chromatographic column is Agilent SB-C8, 250mm multiplied by 4.6mm,5 μm;
mobile phase: the potassium dihydrogen phosphate buffer solution is a mobile phase A, the concentration of potassium dihydrogen phosphate in the potassium dihydrogen phosphate buffer solution is 30mmol/L, and the pH value is regulated to 3.0 by phosphoric acid; acetonitrile is taken as a mobile phase B;
flow rate: 1.0ml/min;
wavelength: 230nm
Run time: 50min;
column temperature: 30 ℃;
sample injection amount: 20. Mu.L;
the gradient elution procedure was:
0-14 min, namely, a mobile phase A65% and a mobile phase B35%;
14-35 min, namely reducing the mobile phase A from 65% to 30% at a constant speed, and increasing the mobile phase B from 35% to 70% at a constant speed;
35-38 min, namely mobile phase A30% and mobile phase B70%;
38-40 min, namely the mobile phase A is increased from 30% to 65% at a constant speed, and the mobile phase B is reduced from 70% to 35% at a constant speed;
40-50 min, namely, a mobile phase A65% and a mobile phase B35%;
according to the chromatographic conditions, 20 μl of the reference mixed solution is precisely sucked, injected into a liquid chromatograph, and scanned by an Agilent DAD detector in the wavelength range of 190nm-400 nm. The results are shown in the following table and in FIG. 1:
TABLE 1 chromatographic peak information in impurity control solutions
Note that: relative retention time = impurity peak retention time/left cetirizine Li Qinfeng retention time
As can be seen from table 1 and fig. 1, the peak sequence is p-hydroxybenzoic acid, methylparaben, levocetirizine amide, p-chlorodibenzmethylpiperazine, levocetirizine, propylparaben, p-chlorodibenzenol, and p-chlorobenzophenone in sequence, the separation degree of each peak and adjacent peaks in the chromatogram of the reference mixed solution is greater than 2.0, and each peak is well separated.
Example 2
Accuracy of
Chromatographic conditions
The chromatographic conditions of the high performance liquid chromatography are as follows:
instrument: agilent 1260 high performance liquid chromatograph;
chromatographic column: octane bonded silica gel is used as filler, and the model of the chromatographic column is Agilent SB-C8, 250mm multiplied by 4.6mm,5 μm;
mobile phase: the potassium dihydrogen phosphate buffer solution is a mobile phase A, the concentration of potassium dihydrogen phosphate in the potassium dihydrogen phosphate buffer solution is 30mmol/L, and the pH value is regulated to 3.0 by phosphoric acid; acetonitrile is taken as a mobile phase B;
flow rate: 1.0ml/min;
wavelength: 230nm
Run time: 50min;
column temperature: 30 ℃;
sample injection amount: 20. Mu.L;
the gradient elution procedure was:
0-14 min, namely, a mobile phase A65% and a mobile phase B35%;
14-35 min, namely reducing the mobile phase A from 65% to 30% at a constant speed, and increasing the mobile phase B from 35% to 70% at a constant speed;
35-38 min, namely mobile phase A30% and mobile phase B70%;
38-40 min, namely the mobile phase A is increased from 30% to 65% at a constant speed, and the mobile phase B is reduced from 70% to 35% at a constant speed;
40-50 min, namely, a mobile phase A65% and a mobile phase B35%;
test solution: accurately weighing 12.41mg of parahydroxybenzoic acid reference substance, placing into a 100ml measuring flask, dissolving with acetonitrile-water (volume ratio 1:1), diluting to scale, shaking, and collecting as sample-adding stock solution. Precisely measuring 0.1ml, 0.2ml and 0.3ml of stock solution for sample addition, respectively placing in three parallel portions into 25ml measuring flask, adding 5ml of blank sample (without bacteriostat), diluting to scale with acetonitrile-water (1:1), and shaking.
P-hydroxybenzoic acid control solution: mixing parahydroxybenzoic acid and a solvent to obtain a parahydroxybenzoic acid reference solution; the solvent is a mixed solution of acetonitrile and water in a volume ratio of 1:1;
the mass of p-hydroxybenzoic acid in each 1mL of the impurity control solution was 1.0. Mu.g.
Blank solution: 5ml of a blank sample (without a bacteriostat) is taken, diluted to a scale by acetonitrile-water (volume ratio is 1:1), and uniformly shaken to be used as a blank solution.
According to the chromatographic conditions, 20 mu l of each solution is precisely measured, injected into a liquid chromatograph, the chromatogram is recorded, and the recovery rate and the RSD value are calculated by an external standard method. The results are shown in Table 2 and FIGS. 2-6.
TABLE 2 accuracy test results of parahydroxybenzoic acid
2 related substances
Test solution: precisely weighing 10.38mg of levocetirizine amide reference substance, 10.50mg of p-chlorobenzylpiperazine reference substance, 10.35mg of p-chlorobenzoyl alcohol reference substance and 10.84mg of p-chlorobenzophenone reference substance, placing into a 25ml measuring flask, dissolving with acetonitrile-water (1:1) and diluting to scale, shaking up, precisely weighing 5ml, placing into a 50ml measuring flask, diluting with acetonitrile-water (1:1) to scale, shaking up, and taking as a sample adding stock solution. Measuring 0.125ml, 0.250ml and 0.375ml of sample-adding stock solution, placing in parallel three portions respectively in 25ml measuring flask, adding 5ml of blank auxiliary material, diluting to scale with acetonitrile-water (1:1), and shaking.
Reference substance solutions of related substances: mixing p-chlorobenzoyl alcohol, p-chlorobenzophenone, p-chlorobenzoyl piperazine, levocetirizine amide and a solvent to obtain a reference substance solution of related substances; the solvent is a mixed solution of acetonitrile and water in a volume ratio of 1:1;
the mass of each 1mL of the impurity reference solution is 0.4 mug of p-chlorobenzol, p-chlorobenzophenone, p-chlorobenzoylpiperazine and levocetirizine amide.
Blank solution: taking 5ml of blank auxiliary materials, diluting to the scale with acetonitrile-water (1:1), shaking uniformly to obtain blank solution
According to the chromatographic conditions, 20 mu l of each solution is precisely measured, injected into a liquid chromatograph, the chromatogram is recorded, and the recovery rate and the RSD value are calculated by an external standard method. The results are shown in the following tables and FIGS. 7-11.
TABLE 3 results of accuracy test of substances
Note that: 1. the three concentration points are respectively 50%, 100% and 150% of the impurity limit
2. Addition = sample size x purity/dilution x addition volume
The accuracy test results show that the recovery rates of 9 samples with high, medium and low concentrations of p-hydroxybenzoic acid, levocetirizine amide, p-chlorodibenzmethylpiperazine, p-chlorodibenzoate and p-chlorobenzophenone are 80% -120%, the average recovery rates are respectively 98.90%, 103.83%, 100.93%, 99.82%, 100.68%, and RSD is respectively 1.37%, 2.35%, 2.40%, 0.79% and 1.30%, and the accuracy of the method is good.
Example 3
Precision of
The chromatographic conditions of the high performance liquid chromatography are as follows:
instrument: agilent 1260 high performance liquid chromatograph;
chromatographic column: octane bonded silica gel is used as filler, and the model of the chromatographic column is Agilent SB-C8, 250mm multiplied by 4.6mm,5 μm;
mobile phase: the potassium dihydrogen phosphate buffer solution is a mobile phase A, the concentration of potassium dihydrogen phosphate in the potassium dihydrogen phosphate buffer solution is 30mmol/L, and the pH value is regulated to 3.0 by phosphoric acid; acetonitrile is taken as a mobile phase B;
flow rate: 1.0ml/min;
wavelength: 230nm
Run time: 50min;
column temperature: 30 ℃;
sample injection amount: 20. Mu.L;
the elution gradient is:
0-14 min, namely, a mobile phase A65% and a mobile phase B35%;
14-35 min, namely reducing the mobile phase A from 65% to 30% at a constant speed, and increasing the mobile phase B from 35% to 70% at a constant speed;
35-38 min, namely mobile phase A30% and mobile phase B70%;
38-40 min, namely the mobile phase A is increased from 30% to 65% at a constant speed, and the mobile phase B is reduced from 70% to 35% at a constant speed;
40-50 min, namely, a mobile phase A65% and a mobile phase B35%;
impurity control solution: mixing p-chlorobenzoyl alcohol, p-chlorobenzophenone, p-chlorobenzoyl piperazine, levocetirizine amide, p-hydroxybenzoic acid and a solvent to obtain an impurity reference substance solution; the solvent is a mixed solution of acetonitrile and water in a volume ratio of 1:1;
the mass of p-chlorobenzoyl alcohol, p-chlorobenzophenone, p-chlorobenzoyl piperazine, levocetirizine amide and p-hydroxybenzoic acid in each 1mL of the impurity reference solution was 0.4 mug, 0.4 mug and 1.0 mug, respectively.
Test solution: mixing the levocetirizine hydrochloride oral solution with a solvent to obtain a sample solution, and preparing 6 parts in parallel; the solvent is a mixed solution of acetonitrile and water in a volume ratio of 1:1;
the concentration of the levocetirizine hydrochloride oral solution in the test sample solution is 0.1mg/mL.
Control solution: diluting a proper amount of sample solution with acetonitrile water solution to 100 times of the volume of the sample solution; the acetonitrile aqueous solution is a mixed solution of acetonitrile and water in a volume ratio of 1:1.
1 repeatability
And (3) respectively injecting an impurity reference substance solution, a reference solution and a test substance solution according to the chromatographic conditions by an experimenter A, and detecting relevant substances of the levocetirizine hydrochloride oral solution with the lot number of 200501 provided by Zhejiang Kang Enbei pharmaceutical Co., ltd, wherein the results are shown in the following table.
TABLE 4 repeatability test results
The results show that the error between the maximum value and the minimum value of the impurity content in the sample is not more than 0.1% after the measurement is performed in parallel for 6 times, which shows that the repeatability of the method is good.
2 intermediate precision
The intermediate precision tests of the detection of the substances by different laboratory staff (laboratory staff: B) at different times and with different liquid chromatography were carried out, the impurity content was determined according to the above-mentioned determination method and was analyzed and compared with the determination result of the repeatability test (laboratory staff: A), the results are shown in the following table.
TABLE 5 results of intermediate precision test for the detection of substances
The results show that the maximum value and the minimum value of the measured impurity result have the error of not more than 0.1% and the number of the impurities is 7, so that the method has good intermediate precision.
Example 4
The chromatographic conditions of the high performance liquid chromatography are as follows:
instrument: agilent 1260 high performance liquid chromatograph;
chromatographic column: octane bonded silica gel is used as filler, and the model of the chromatographic column is Agilent SB-C8, 250mm multiplied by 4.6mm,5 μm;
mobile phase: the potassium dihydrogen phosphate buffer solution is a mobile phase A, the concentration of potassium dihydrogen phosphate in the potassium dihydrogen phosphate buffer solution is 30mmol/L, and the pH value is regulated to 3.0 by phosphoric acid; acetonitrile is taken as a mobile phase B;
flow rate: 1.0ml/min;
wavelength: 230nm
Run time: 50min;
column temperature: 30 ℃;
sample injection amount: 20. Mu.L;
the gradient elution procedure was:
0-14 min, namely, a mobile phase A65% and a mobile phase B35%;
14-35 min, namely reducing the mobile phase A from 65% to 30% at a constant speed, and increasing the mobile phase B from 35% to 70% at a constant speed;
35-38 min, namely mobile phase A30% and mobile phase B70%;
38-40 min, namely the mobile phase A is increased from 30% to 65% at a constant speed, and the mobile phase B is reduced from 70% to 35% at a constant speed;
40-50 min, namely, a mobile phase A65% and a mobile phase B35%;
impurity control solution: mixing p-chlorobenzoyl alcohol, p-chlorobenzophenone, p-chlorobenzoyl piperazine, levocetirizine amide, p-hydroxybenzoic acid and a solvent to obtain an impurity reference substance solution; the solvent is a mixed solution of acetonitrile and water in a volume ratio of 1:1;
the mass of p-chlorobenzoyl alcohol, p-chlorobenzophenone, p-chlorobenzoyl piperazine, levocetirizine amide and p-hydroxybenzoic acid in each 1mL of the impurity reference solution was 0.4 mug, 0.4 mug and 1.0 mug, respectively.
Test solution: mixing the levocetirizine hydrochloride oral solution with a solvent to obtain a sample solution; the solvent is a mixed solution of acetonitrile and water in a volume ratio of 1:1;
the concentration of the levocetirizine hydrochloride oral solution in the test sample solution is 0.1mg/mL.
Control solution: taking a proper amount of sample solution, and diluting the sample solution to 100 times of the volume of the sample solution by using an acetonitrile water solution; the acetonitrile aqueous solution is a mixed solution of acetonitrile and water in a volume ratio of 1:1.
And (3) respectively injecting an impurity reference substance solution, a reference solution and a test substance solution (the levocetirizine hydrochloride oral solution is prepared by Zhejiang Kang Enbei pharmaceutical Co., ltd.) according to the chromatographic conditions, and detecting the relevant substances. The results are shown in the following tables and figures 12, 13, 14, 15, 16:
TABLE 6 content of related substances in Levocetirizine hydrochloride oral solutions of different lot numbers
Note that: other total impurities refer to the sum of impurities other than known impurities
The contents of the related substances in table 6 were calculated from the following formulas: calculation formula
Wherein: s is S Known impurities : peak area of known impurities in sample solution chromatogram
S For a pair of : peak area of known impurities in impurity control solution chromatogram
C For a pair of : impurity control solution concentration (mg/ml) of known impurities in the chromatogram of the impurity control solution
N: dilution factor
T: indicating quantity (T is calculated according to the indicating quantity of methyl hydroxybenzoate of 0.675mg/ml when calculating the parahydroxybenzoic acid, and other impurities T are calculated according to the indicating quantity of levocetirizine hydrochloride of 0.5 mg/ml)
Wherein: s is S Single impurity : peak area of single impurity in sample solution chromatogram
S Control : peak area of levocetirizine in control solution chromatogram
In summary, examples 1-4 show that the method for detecting the levocetirizine hydrochloride oral solution provided by the invention has the characteristics of simple operation, high precision, high accuracy and good separation degree, and is suitable for detecting related substances of the Zuo Xiti cetirizine hydrochloride oral solution.
Comparative example 1
The impurity control solution in example 1 was tested according to the test method of the imported drug registration standard JX20110262 levocetirizine hydrochloride drop, the results of which are shown in table 7 and fig. 17:
TABLE 7 retention time and relative retention time of control mixture in comparative example 1
As shown in table 7 and fig. 17, the 2 impurities of levocetirizine amide and p-chlorobenzoyl piperazine are basically consistent with the main peak and the peak time of the methylparaben in the oral solution, and are mixed together, so that the effective separation cannot be performed, and the peak of the p-hydroxybenzoic acid is too early and is easily interfered by the solvent peak, so that the method can only effectively detect 2 impurities of p-chlorobenzoyl alcohol and p-chlorobenzophenone.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (4)
1. The method for detecting the related substances in the levocetirizine hydrochloride oral solution is characterized in that a high performance liquid chromatography is adopted to measure the related substances in the levocetirizine hydrochloride oral solution, and an external standard method is used to calculate the content of the related substances in the levocetirizine hydrochloride oral solution;
in the high performance liquid chromatography, a mobile phase A is potassium dihydrogen phosphate buffer solution, and a mobile phase B is acetonitrile;
the gradient elution procedure of the mobile phase is:
0-t 1min, namely a mobile phase A65% and a mobile phase B35%;
t 1-t 2 min, wherein the mobile phase A is uniformly reduced from 65% to 30%, and the mobile phase B is uniformly increased from 35% to 70%;
t 2-t 3 min, mobile phase A30% and mobile phase B70%;
t 3-t 4min, wherein the mobile phase A is increased from 30% to 65% at a constant speed, and the mobile phase B is reduced from 70% to 35% at a constant speed;
t 4-t 5min, namely a mobile phase A65% and a mobile phase B35%;
the elution gradient of the mobile phase is:
t1 is 14, t2 is 35, t3 is 38, t4 is 40, and t5 is 50;
the concentration of the potassium dihydrogen phosphate in the potassium dihydrogen phosphate buffer solution is 25-35 mmol/L, and the pH value of the potassium dihydrogen phosphate buffer solution is 2.8-3.2;
the chromatographic column in the high performance liquid chromatography is a chromatographic column with octane bonded silica gel as a filler;
the size of the chromatographic column is 250mm×4.6mm,5 μm;
the temperature of the chromatographic column is 25-35 ℃, and the flow rate of the mobile phase is 1.0mL/min;
the detector in the high performance liquid chromatography is an ultraviolet detector, and the detection wavelength of the ultraviolet detector is 228-232 nm;
the related substances are p-chlorobenzoyl alcohol, p-chlorobenzophenone, p-chlorobenzoyl piperazine, levocetirizine amide and p-hydroxybenzoic acid.
2. The method of claim 1, wherein the chromatography column is of the type Agilent SB-C8, agilent ZORBAX RX-C8, YMC-Pack C8 or COSMOSIL Packed Column C8-MS.
3. The method according to claim 1, wherein the impurity reference solution in the high performance liquid chromatography is a mixture of p-chlorobenzoyl alcohol, p-chlorobenzophenone, p-chlorobenzoyl piperazine, levocetirizine amide, p-hydroxybenzoic acid and a solvent, wherein the solvent is a mixed solution of acetonitrile and water in a volume ratio of 1:1;
the mass of p-chlorobenzoyl alcohol, p-chlorobenzophenone, p-chlorobenzoyl piperazine, levocetirizine amide and p-hydroxybenzoic acid in each 1mL of the impurity reference solution was 0.4 mug, 0.4 mug and 1.0 mug, respectively.
4. The method according to claim 1, wherein the sample solution in the high performance liquid chromatography is a mixture of levocetirizine hydrochloride oral solution and a solvent, and the solvent is a mixed solution of acetonitrile and water in a volume ratio of 1:1;
the concentration of the levocetirizine hydrochloride oral solution in the sample solution is 0.1mg/mL.
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