CN113848271A - Method for detecting related substances in levocetirizine hydrochloride oral solution - Google Patents

Method for detecting related substances in levocetirizine hydrochloride oral solution Download PDF

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CN113848271A
CN113848271A CN202111191362.XA CN202111191362A CN113848271A CN 113848271 A CN113848271 A CN 113848271A CN 202111191362 A CN202111191362 A CN 202111191362A CN 113848271 A CN113848271 A CN 113848271A
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mobile phase
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levocetirizine
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CN113848271B (en
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张文杰
李耀湘
刘佳伟
陈刘进
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Zhejiang Heli Xinjian Pharmaceutical Co ltd
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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
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • 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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
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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
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Abstract

The invention provides a method for detecting related substances in a levocetirizine hydrochloride oral solution, belonging to the technical field of pharmaceutical analysis. The invention takes acetonitrile and potassium dihydrogen phosphate buffer solution as mobile phase, carries out gradient elution on a high performance liquid chromatograph, and can accurately and efficiently detect p-chlorobenzyl alcohol, p-chlorobenzophenone, p-chlorobenzhydrylpiperazine, levocetirizine amide and p-hydroxybenzoic acid at one time under a liquid phase condition.

Description

Method for detecting related substances in levocetirizine hydrochloride oral solution
Technical Field
The invention relates to the technical field of drug analysis, in particular to a method for detecting related substances in levocetirizine hydrochloride oral solution.
Background
Levocetirizine hydrochloride is an active R-type single optical isomer of a second-generation antihistamine cetirizine which is widely used in clinic at present, is a new-generation high-efficiency oral selective histamine H1 receptor antagonist without sedative side effects, has no obvious anticholinergic and anti-5-hydroxytryptamine effects, has small central inhibition effect, and is used for treating allergic diseases such as urticaria, allergic rhinitis, eczema, dermatitis, cutaneous pruritus and the like.
In the synthesis process of the levocetirizine hydrochloride raw material drug and the production and storage processes of oral solution, 5 impurities can be generated, wherein the p-chlorobenzhydrylpiperazine and the levocetirizine amide are important intermediates for synthesizing the levocetirizine hydrochloride and belong to process impurities; the p-chlorobenzophenone and the p-chlorobenzyl alcohol are not only important intermediates for synthesizing levocetirizine hydrochloride, but also main degradation impurities of oral solution under the condition of oxidation or high temperature, and belong to process impurities and degradation impurities; p-hydroxybenzoic acid is a degradation product of parabens in the oral solution as a bacteriostatic agent. In known literature, the detection method of the United states pharmacopoeia USP41 can detect two impurities of levocetirizine amide and p-chlorobenzhydrylpiperazine; imported drug registration standard the detection method of imported registration standard JX20110262 levocetirizine hydrochloride drop can detect p-chlorobenzyl alcohol and p-chlorobenzophenone. At present, a detection method capable of simultaneously detecting the above 5 impurities at one time is not found.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for detecting related substances in levocetirizine hydrochloride oral solution, which can accurately and efficiently detect the above 5 impurities at one time under a liquid phase condition.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides a method for detecting related substances in a levocetirizine hydrochloride oral solution, which comprises the steps of determining the related substances in the levocetirizine hydrochloride oral solution by adopting a high performance liquid chromatography, and calculating the content of the related substances in the levocetirizine hydrochloride oral solution by using an external standard method;
in the high performance liquid chromatography, a mobile phase A is a potassium dihydrogen phosphate buffer solution, and a mobile phase B is acetonitrile;
the gradient elution procedure of the mobile phase is as follows:
0～t1min, mobile phase A65% and mobile phase B35%;
t1～t2min, uniformly reducing the mobile phase A from 65% to 30%, and uniformly increasing the mobile phase B from 35% to 70%;
t2～t3min, mobile phase A30 percent and mobile phase B70 percent;
t3～t4min, uniformly increasing the mobile phase A from 30% to 65%, and uniformly decreasing the mobile phase B from 70% to 35%;
t4～t5min, mobile phase A65% and mobile phase B35%;
said t is1Is 13 to 15, t 234 to 36, t337 to 39, t439 to 41, t5Is 49 to 51
Preferably, said t1Is 14, t2Is 35, t3Is 38, t4Is 40, t5Is 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 chromatographic column is of type (Agilent SB-C)8250 mm. times.4.6 mm, 5 μm), (Agilent ZORBAX RX-C8250 mm. times.4.6 mm, 5 μm), (YMC-Pack C8250 mm. times.4.6 mm, 5 μm) or (COSMOSIL Packed Column 5C8-MS250 mm. times.4.6 mm).
Preferably, the temperature of the chromatographic column is 25-35 ℃, and the flow rate of the mobile phase is 1.0 mL/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-chlorobenzyl alcohol, p-chlorobenzophenone, p-chlorobenzhydrylpiperazine, levocetirizine amide and p-hydroxybenzoic acid.
Preferably, the impurity control solution in the high performance liquid chromatography is a mixture of p-chlorobenzyl alcohol, p-chlorobenzophenone, p-chlorobenzhydrylpiperazine, 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-chlorobenzyl alcohol, p-chlorobenzophenone, p-chlorobenzhydrylpiperazine, levocetirizine amide and p-hydroxybenzoic acid in each 1mL of impurity control solution was 0.4. mu.g, and 1.0. mu.g, respectively.
Preferably, 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 test solution is 0.1 mg/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 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. The invention takes acetonitrile and potassium dihydrogen phosphate buffer solution as mobile phase, carries out gradient elution on a high performance liquid chromatograph, and can accurately and efficiently detect p-chlorobenzyl alcohol, p-chlorobenzophenone, p-chlorobenzhydrylpiperazine, levocetirizine amide and p-hydroxybenzoic acid at one time under a liquid phase condition.
Drawings
FIG. 1 is a HPLC chart of a control mixed solution for impurity localization and separation in example 1; wherein the impurities represented by each peak are respectively: 1-p-hydroxybenzoic acid; 2-hydroxybenzyl methyl ester; 3-levocetirizine amide; 4-p-chlorobenzhydrylpiperazine; 5-levocetirizine; 6-propylparaben; 7-p-chlorobenzhydrol; 8-p-chlorobenzophenone;
FIG. 2 is an HPLC chart of a p-hydroxybenzoic acid control solution of example 2, in which the peaks represent the following impurities: 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 p-hydroxybenzoic acid at a concentration of 50% in example 2, wherein the peaks represent the following impurities: 1-p-hydroxybenzoic acid;
FIG. 5 is an HPLC chart of a sample solution of 100% concentration of p-hydroxybenzoic acid in example 2, wherein the peaks represent the following impurities: 1-p-hydroxybenzoic acid;
FIG. 6 is an HPLC plot of a sample solution of example 2 at a concentration of 150% p-hydroxybenzoic acid accuracy, wherein the peaks represent the following impurities: 1-p-hydroxybenzoic acid;
FIG. 7 is an HPLC chart of a control solution of substance accuracy in example 2, wherein each peak represents the following impurities: 1-levocetirizine amide, 2-p-chlorobenzhydrylpiperazine, 3-p-chlorobenzyl alcohol, 4-p-chlorobenzophenone;
FIG. 8 is an HPLC chart of a blank solution of example 2 with respect to substance accuracy;
FIG. 9 is an HPLC chart of a sample solution of 50% concentration in example 2 with respect to substance accuracy, in which each peak represents impurities: 1-levocetirizine amide, 2-p-chlorobenzhydrylpiperazine, 3-p-chlorobenzyl alcohol, 4-p-chlorobenzophenone;
FIG. 10 is an HPLC chart of a test solution of 100% concentration with respect to substance accuracy, in which each peak represents an impurity: 1-levocetirizine amide, 2-p-chlorobenzhydrylpiperazine, 3-p-chlorobenzyl alcohol, 4-p-chlorobenzophenone;
FIG. 11 is an HPLC chart of a test solution of 150% concentration in example 2 with respect to substance accuracy, in which the peaks represent the following impurities: 1-levocetirizine amide, 2-p-chlorobenzhydrylpiperazine, 3-p-chlorobenzyl alcohol, 4-p-chlorobenzophenone;
FIG. 12 is an HPLC plot of a control solution of the impurity in example 4; wherein the impurities represented by each peak are respectively: 1-p-hydroxybenzoic acid; 2-levocetirizine amide; 3-p-chlorobenzhydrylpiperazine; 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 the formula are respectively: 1-hydroxybenzyl methyl ester; 2-levocetirizine; 3-propylparaben;
FIG. 14 is an HPLC chart of the test solution of 200501 lots of levocetirizine hydrochloride oral solution in example 4; the impurities represented by the peaks in the formula are respectively: 1-p-hydroxybenzoic acid; 2-hydroxybenzyl methyl ester; 3-levocetirizine amide; 4-p-chlorobenzhydrylpiperazine; 5-levocetirizine; 6-propylparaben; 7-p-chlorobenzhydrol; 8-p-chlorobenzophenone;
FIG. 15 is a HPLC chart of the test solution of the oral solution 200502 lot of levocetirizine hydrochloride in example 4; the impurities represented by the peaks in the formula are respectively: 1-p-hydroxybenzoic acid; 2-hydroxybenzyl methyl ester; 3-levocetirizine amide; 4-p-chlorobenzhydrylpiperazine; 5-levocetirizine; 6-propylparaben; 7-p-chlorobenzhydrol; 8-p-chlorobenzophenone;
FIG. 16 is an HPLC chart of the test solution of 200503 lots of levocetirizine hydrochloride oral solution in example 4; the impurities represented by the peaks in the formula are respectively: 1-p-hydroxybenzoic acid; 2-hydroxybenzyl methyl ester; 3-levocetirizine amide; 4-p-chlorobenzhydrylpiperazine; 5-levocetirizine; 6-propylparaben; 7-p-chlorobenzhydrol; 8-p-chlorobenzophenone;
FIG. 17 is an HPLC chart of a control mixed solution of substances related to imported registration standard JX20110262 levocetirizine hydrochloride drops; the impurities represented by the peaks in the formula are respectively: 1-p-hydroxybenzoic acid; 2-levocetirizine amide; 3-levocetirizine; 4-hydroxybenzyl methyl ester; 5-p-chlorobenzhydrylpiperazine; 6-propylparaben; 7-p-chlorobenzhydrol; 8-p-chlorobenzophenone.
Detailed Description
The invention provides a method for detecting related substances in a levocetirizine hydrochloride oral solution, which comprises the steps of determining the related substances in the levocetirizine hydrochloride oral solution by adopting a high performance liquid chromatography, and calculating 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 a potassium dihydrogen phosphate buffer solution, and a mobile phase B is acetonitrile;
the gradient elution procedure of the mobile phase is as follows:
0～t1min, mobile phase A65% and mobile phase B35%;
t1～t2min, uniformly reducing the mobile phase A from 65% to 30%, and uniformly increasing the mobile phase B from 35% to 70%;
t2～t3min, mobile phase A30 percent and mobile phase B70 percent;
t3～t4min, uniformly increasing the mobile phase A from 30% to 65%, and uniformly decreasing the mobile phase B from 70% to 35%;
t4～t5min, mobile phase A65% and mobile phase B35%;
said t is1Is 13 to 15, t 234 to 36, t337 to 39, t439 to 41, t5Is 49 to 51
Preferably, said t1Is 14, t2Is 35, t3Is 38, t4Is 40, t5Is 50.
In the invention, the concentration of the monopotassium phosphate in the monopotassium phosphate buffer solution is preferably 25-35 mmol/L, and more preferably 30 mmol/L; the pH value of the potassium dihydrogen phosphate buffer solution is preferably 2.8-3.2, and more preferably 3.0.
In the invention, a chromatographic column in the high performance liquid chromatography is a chromatographic column taking octane bonded silica gel as a filler; the type of the chromatographic column is preferably (Agilent SB-C)8250 mm. times.4.6 mm, 5 μm), (Agilent ZORBAX RX-C8250 mm. times.4.6 mm, 5 μm), (YMC-Pack C8250 mm. times.4.6 mm, 5 μm) or (COSMOSIL Packed Column 5C8-MS250 mm. times.4.6 mm), more preferably (Agilent SB-C)8250mm × 4.6mm, 5 μm); the temperature of the chromatographic column is preferably 25-35 ℃, and more preferably 30 ℃; the flow rate of the eluent in the chromatographic column is preferably 1.0 mL/min; the elution time of the eluent in the chromatographic column is preferably 49-51 min.
In the present invention, the detector in high performance liquid chromatography is preferably an ultraviolet detector; the detection wavelength of the ultraviolet detector is preferably 228-232 nm, and more preferably 230 nm. In the present invention, the related substances are preferably p-chlorobenzyl alcohol, p-chlorobenzophenone, p-chlorobenzhydrylpiperazine, 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-chlorobenzyl alcohol, p-chlorobenzophenone, p-chlorobenzhydrylpiperazine, levocetirizine amide, p-hydroxybenzoic acid and a solvent, wherein the solvent is preferably a mixed solution of acetonitrile and water in a volume ratio of 1: 1; the mass of p-chlorobenzyl alcohol, p-chlorobenzophenone, p-chlorobenzhydrylpiperazine, levocetirizine amide and p-hydroxybenzoic acid per 1mL of the impurity control solution is preferably 0.4. mu.g, and 1.0. mu.g, respectively.
In the invention, the test solution in the high performance liquid chromatography is preferably a mixture of 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 0.1 mg/mL.
In order to better understand the present invention, the following examples are further provided to illustrate the present invention, but the present invention is not limited to the following examples.
Example 1
Positioning and separating impurities:
p-hydroxybenzoic acid, levocetirizine amide, p-chlorobenzhydrylpiperazine, levocetirizine hydrochloride, methylparaben, propylparaben, p-chlorobenzyl alcohol and p-chlorobenzophenone are respectively taken and prepared into 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 p-hydroxybenzoic acid, 0.4 mu g/ml of levocetirizine amide, 0.4 mu g/ml of p-chlorobenzhydrylpiperazine, 0.1mg/ml of levocetirizine hydrochloride, 0.135mg/ml of methylparaben, 0.015mg/ml of propylparaben, 0.4 mu g/ml of p-chlorobenzophenone and 0.4 mu g/ml of p-chlorobenzophenone.
Chromatographic conditions
The chromatographic conditions of the high performance liquid chromatography are as follows:
the instrument comprises the following steps: agilent 1260 high performance liquid chromatograph;
a chromatographic column: octane bonded silica gel is used as filler, and the type of 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 the potassium dihydrogen phosphate in the potassium dihydrogen phosphate buffer solution is 30mmol/L, and the pH value is adjusted to 3.0 by phosphoric acid; acetonitrile is used as a mobile phase B;
flow rate: 1.0 ml/min;
wavelength: 230nm
Operating time: 50 min;
column temperature: 30 ℃;
sample introduction amount: 20 mu L of the solution;
the gradient elution procedure was:
0-14 min, wherein the mobile phase A is 65% and the mobile phase B is 35%;
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 for 14-35 min;
35-38 min, namely 30% of mobile phase A and 70% of mobile phase B;
increasing the mobile phase A from 30% to 65% at a constant speed, and reducing the mobile phase B from 70% to 35% at a constant speed for 38-40 min;
40-50 min, mobile phase A65% and mobile phase B35%;
precisely sucking 20 μ l of the control mixed solution according to the above chromatographic conditions, injecting into a liquid chromatograph, and scanning with an Agilent DAD detector at wavelength of 190nm-400 nm. The results are shown in the following table and attached figure 1:
TABLE 1 chromatographic Peak information in impurity control solutions
Figure BDA0003301236370000071
Note: relative retention time ═ impurity peak retention time/levocetirizine peak retention time
As can be seen from table 1 and fig. 1, the peak sequences are p-hydroxybenzoic acid, methylparaben, levocetirizine amide, p-chlorobenzhydrylpiperazine, levocetirizine, propylparaben, p-chlorobenzyl alcohol, and p-chlorobenzophenone in sequence, and the separation degree of each peak from the adjacent peak in the chromatogram of the mixed solution of the reference 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:
the instrument comprises the following steps: agilent 1260 high performance liquid chromatograph;
a chromatographic column: octane bonded silica gel is used as filler, and the type of 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 the potassium dihydrogen phosphate in the potassium dihydrogen phosphate buffer solution is 30mmol/L, and the pH value is adjusted to 3.0 by phosphoric acid; acetonitrile is used as a mobile phase B;
flow rate: 1.0 ml/min;
wavelength: 230nm
Operating time: 50 min;
column temperature: 30 ℃;
sample introduction amount: 20 mu L of the solution;
the gradient elution procedure was:
0-14 min, wherein the mobile phase A is 65% and the mobile phase B is 35%;
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 for 14-35 min;
35-38 min, namely 30% of mobile phase A and 70% of mobile phase B;
increasing the mobile phase A from 30% to 65% at a constant speed, and reducing the mobile phase B from 70% to 35% at a constant speed for 38-40 min;
40-50 min, mobile phase A65% and mobile phase B35%;
test solution: 12.41mg of p-hydroxybenzoic acid control was precisely weighed, placed in a 100ml measuring flask, dissolved and diluted to the scale with acetonitrile-water (volume ratio 1:1), shaken up, and used as a sample-adding stock solution. Precisely measuring the sample-adding stock solutions of 0.1ml, 0.2ml and 0.3ml, placing into 25ml measuring bottles, respectively, adding 5ml of blank sample (without bacteriostatic agent), diluting with acetonitrile-water (1:1) to scale, and shaking.
P-hydroxybenzoic acid control solution: mixing p-hydroxybenzoic acid with a solvent to obtain a p-hydroxybenzoic acid reference solution; the solvent is a mixed solution of acetonitrile and water in a volume ratio of 1: 1;
the mass of parahydroxybenzoic acid per 1mL of the impurity control solution was 1.0. mu.g.
Blank solution: taking 5ml of a blank sample (without bacteriostatic agent), diluting the blank sample to a scale with acetonitrile-water (volume ratio is 1:1), and shaking up to obtain a blank solution.
Precisely measuring the solutions respectively in 20 μ l under the chromatographic conditions, injecting into a liquid chromatograph, recording chromatogram, calculating recovery rate by external standard method, and calculating RSD value. The results are shown in Table 2 and FIGS. 2-6.
TABLE 2 para-hydroxybenzoic acid accuracy test results
Figure BDA0003301236370000091
2 related substances
Test solution: accurately weighing 10.38mg of levocetirizine amide reference substance, 10.50mg of p-chlorobenzhydrylpiperazine reference substance, 10.35mg of p-chlorobenzhydrol reference substance and 10.84mg of p-chlorobenzophenone reference substance, placing the reference substances in a 25ml measuring flask, dissolving and diluting the reference substances to scales by using acetonitrile-water (1:1), shaking up, accurately weighing 5ml, placing the reference substances in a 50ml measuring flask, diluting to scales by using acetonitrile-water (1:1), shaking up, and using the reference substances as sample-adding storage liquid. Weighing the sample-adding stock solutions 0.125ml, 0.250ml and 0.375ml, placing into 25ml measuring bottles respectively, adding blank adjuvant 5ml, diluting with acetonitrile-water (1:1) to scale, and shaking.
Reference substance solution: mixing p-chlorobenzyl alcohol, p-chlorobenzophenone, p-chlorobenzhydrylpiperazine, levocetirizine amide and a solvent to obtain a related substance reference substance solution; the solvent is a mixed solution of acetonitrile and water in a volume ratio of 1: 1;
the mass of p-chlorobenzyl alcohol, p-chlorobenzophenone, p-chlorobenzhydrylpiperazine and levocetirizine amide in each 1mL of impurity control solution was 0.4. mu.g.
Blank solution: taking 5ml of blank auxiliary materials, diluting to scale with acetonitrile-water (1:1), shaking up to obtain blank solution
Precisely measuring the solutions respectively in 20 μ l under the chromatographic conditions, injecting into a liquid chromatograph, recording chromatogram, calculating recovery rate by external standard method, and calculating RSD value. The results are shown in the following table and in FIGS. 7-11.
TABLE 3 test results on accuracy of substances
Figure BDA0003301236370000101
Figure BDA0003301236370000111
Note: 1. the three concentration points are respectively 50%, 100% and 150% of the limit of impurities
2. The amount added is the weight of sample, purity/dilution times and volume added
The accuracy test result shows that the recovery rates of 9 samples with high, medium and low concentrations of p-hydroxybenzoic acid, levocetirizine amide, p-chlorobenzhydrylpiperazine, p-chlorobenzyl alcohol and p-chlorobenzophenone are between 80% and 120%, the average recovery rates are respectively 98.90%, 103.83%, 100.93%, 99.82% and 100.68%, and the RSD are respectively 1.37%, 2.35%, 2.40%, 0.79% and 1.30%, which indicates that the accuracy of the method is good.
Example 3
Precision degree
The chromatographic conditions of the high performance liquid chromatography are as follows:
the instrument comprises the following steps: agilent 1260 high performance liquid chromatograph;
a chromatographic column: octane bonded silica gel is used as filler, and the type of 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 the potassium dihydrogen phosphate in the potassium dihydrogen phosphate buffer solution is 30mmol/L, and the pH value is adjusted to 3.0 by phosphoric acid; acetonitrile is used as a mobile phase B;
flow rate: 1.0 ml/min;
wavelength: 230nm
Operating time: 50 min;
column temperature: 30 ℃;
sample introduction amount: 20 mu L of the solution;
the elution gradient was:
0-14 min, wherein the mobile phase A is 65% and the mobile phase B is 35%;
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 for 14-35 min;
35-38 min, namely 30% of mobile phase A and 70% of mobile phase B;
increasing the mobile phase A from 30% to 65% at a constant speed, and reducing the mobile phase B from 70% to 35% at a constant speed for 38-40 min;
40-50 min, mobile phase A65% and mobile phase B35%;
impurity control solution: mixing p-chlorobenzyl alcohol, p-chlorobenzophenone, p-chlorobenzhydrylpiperazine, 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-chlorobenzyl alcohol, p-chlorobenzophenone, p-chlorobenzhydrylpiperazine, levocetirizine amide and p-hydroxybenzoic acid per 1mL of the impurity control solution was 0.4. mu.g, and 1.0. mu.g, respectively.
Test solution: mixing the levocetirizine hydrochloride oral solution with a solvent to obtain a test 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 solution is 0.1 mg/mL.
Control solution: taking a proper amount of a sample solution, and diluting the sample solution to 100 times of the volume of the sample solution by using an acetonitrile aqueous solution; the acetonitrile water solution is a mixed solution of acetonitrile and water in a volume ratio of 1: 1.
1 repeatability
The laboratory technician A samples impurity reference solution, reference solution and test solution respectively according to the chromatographic conditions, and detects related substances of the levocetirizine hydrochloride oral solution with the batch number of 200501 provided by Zhejiang Conenginbi pharmaceutical Limited, and the results are shown in the table.
TABLE 4 results of the repeatability tests
Figure BDA0003301236370000131
The result shows that the error between the maximum value and the minimum value of the impurity content in the sample does not exceed 0.1 percent after the parallel measurement is carried out for 6 times, which indicates that the method has good repeatability.
2 intermediate precision
The intermediate precision tests of the related substances were carried out by different experimenters (experimenters: B) at different times by different liquid chromatographs, the impurity content was determined according to the above determination method, and the results were analyzed and compared with the results of the determination of the repetitive tests (experimenters: A), and the results are shown in the following table.
TABLE 5 intermediate precision test results for the detection of related substances
Figure BDA0003301236370000132
Figure BDA0003301236370000141
The result shows that the same sample is taken by different persons at different time and with different instruments, the error between the maximum value and the minimum value of the measured impurity result is not more than 0.1 percent after 6 times of measurement, the number of the impurities is 7, and the intermediate precision of the method is good.
Example 4
The chromatographic conditions of the high performance liquid chromatography are as follows:
the instrument comprises the following steps: agilent 1260 high performance liquid chromatograph;
a chromatographic column: octane bonded silica gel is used as filler, and the type of 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 the potassium dihydrogen phosphate in the potassium dihydrogen phosphate buffer solution is 30mmol/L, and the pH value is adjusted to 3.0 by phosphoric acid; acetonitrile is used as a mobile phase B;
flow rate: 1.0 ml/min;
wavelength: 230nm
Operating time: 50 min;
column temperature: 30 ℃;
sample introduction amount: 20 mu L of the solution;
the gradient elution procedure was:
0-14 min, wherein the mobile phase A is 65% and the mobile phase B is 35%;
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 for 14-35 min;
35-38 min, namely 30% of mobile phase A and 70% of mobile phase B;
increasing the mobile phase A from 30% to 65% at a constant speed, and reducing the mobile phase B from 70% to 35% at a constant speed for 38-40 min;
40-50 min, mobile phase A65% and mobile phase B35%;
impurity control solution: mixing p-chlorobenzyl alcohol, p-chlorobenzophenone, p-chlorobenzhydrylpiperazine, 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-chlorobenzyl alcohol, p-chlorobenzophenone, p-chlorobenzhydrylpiperazine, levocetirizine amide and p-hydroxybenzoic acid per 1mL of the impurity control solution was 0.4. mu.g, and 1.0. mu.g, respectively.
Test solution: mixing the levocetirizine hydrochloride oral solution with a solvent to obtain a test 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 solution is 0.1 mg/mL.
Control solution: taking a proper amount of a test sample solution, and diluting the test sample solution to 100 times of the volume of the test sample solution by using an acetonitrile aqueous solution; the acetonitrile water solution is a mixed solution of acetonitrile and water in a volume ratio of 1: 1.
According to the chromatographic conditions, impurity reference solution and reference solution are respectively injected, and related substances of the test solution (the levocetirizine hydrochloride oral solution is provided by Zhejiang Connbie pharmaceutical company with the batch numbers of 200501, 200502 and 200503) are detected. The results are shown in the following table and in FIGS. 12, 13, 14, 15, and 16:
TABLE 6 content of related substances in levocetirizine hydrochloride oral solutions of different batches
Figure BDA0003301236370000161
Note: other total impurities refer to the sum of impurities other than known impurities
The contents of the relevant substances in Table 6 were calculated from the following formulas: formula for calculation
Figure BDA0003301236370000162
In the formula: sKnown impurities: peak area of known impurity in chromatogram of test solution
STo pair: peak area of known impurity in chromatogram of impurity control solution
CTo pair: concentration (mg/ml) of reference solution of known impurities in chromatogram of reference solution of impurities
N: dilution factor
T: the marked amount (when the p-hydroxybenzoic acid is calculated, T is calculated according to the marked amount of methylparaben, 0.675mg/ml, and other impurities T are calculated according to the marked amount of levocetirizine hydrochloride, 0.5 mg/ml)
Figure BDA0003301236370000163
In the formula: sSingle impurity: peak area of single impurity in chromatogram of test solution
SControl: peak area of levocetirizine in chromatogram of control solution
To sum up, the embodiments 1 to 4 show that the detection method of 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 levocetirizine hydrochloride oral solution.
Comparative example 1
The impurity control solution in example 1 was tested according to the test method for the imported drug registration standard JX20110262 levocetirizine hydrochloride drops, and the results are shown in table 7 and fig. 17:
TABLE 7 Retention time and relative Retention time of control mixture in comparative example 1
Figure BDA0003301236370000171
As can be seen from table 7 and fig. 17, the 2 impurities of levocetirizine amide and p-chlorobenzhydrylpiperazine are basically consistent with the main peak and the peak emergence time of methylparaben in the oral solution, and are mixed together, so that the levocetirizine amide and p-chlorobenzhydrylpiperazine cannot be separated effectively, and the p-hydroxybenzoic acid peak emergence is too early and is easily interfered by the solvent peak, so that the method can only effectively detect 2 impurities of p-chlorobenzophenol and p-chlorobenzophenone.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (10)

1. A method for detecting related substances in a levocetirizine hydrochloride oral solution is characterized in that the related substances in the levocetirizine hydrochloride oral solution are measured by adopting a high performance liquid chromatography, and the content of the related substances in the levocetirizine hydrochloride oral solution is calculated by using an external standard method;
in the high performance liquid chromatography, a mobile phase A is a potassium dihydrogen phosphate buffer solution, and a mobile phase B is acetonitrile;
the gradient elution procedure of the mobile phase is as follows:
0～t1min is mobile phase A65%, flowMobile phase B35%;
t1～t2min, uniformly reducing the mobile phase A from 65% to 30%, and uniformly increasing the mobile phase B from 35% to 70%;
t2～t3min, mobile phase A30 percent and mobile phase B70 percent;
t3～t4min, uniformly increasing the mobile phase A from 30% to 65%, and uniformly decreasing the mobile phase B from 70% to 35%;
t4～t5min, mobile phase A65% and mobile phase B35%;
said t is1Is 13 to 15, t234 to 36, t337 to 39, t439 to 41, t549 to 51.
2. The method of claim 1, wherein the elution gradient of the mobile phase is:
said t is1Is 14, t2Is 35, t3Is 38, t4Is 40, t5Is 50.
3. The method according to claim 1, wherein the concentration of the potassium dihydrogen phosphate in the potassium dihydrogen phosphate buffer is 25 to 35mmol/L, and the pH value of the potassium dihydrogen phosphate buffer is 2.8 to 3.2.
4. The method as claimed in claim 1, wherein the high performance liquid chromatography column is a column filled with octane-bonded silica gel.
5. The method of claim 4, wherein the chromatography column is of type (Agilent SB-C)8250 mm. times.4.6 mm, 5 μm), (Agilent ZORBAX RX-C8250 mm. times.4.6 mm, 5 μm), (YMC-Pack C8250 mm. times.4.6 mm, 5 μm) or (COSMOSIL Packed Column 5C8-MS250 mm. times.4.6 mm).
6. The method of claim 1, 4 or 5, wherein the temperature of the chromatographic column is 25 to 35 ℃ and the flow rate of the mobile phase is 1.0 mL/min.
7. The method according to claim 1, wherein the detector in the high performance liquid chromatography is an ultraviolet detector, and the detection wavelength of the ultraviolet detector is 228-232 nm.
8. The method of claim 1, wherein the related substances are p-chlorobenzyl alcohol, p-chlorobenzophenone, p-chlorobenzhydrylpiperazine, levocetirizine amide and p-hydroxybenzoic acid.
9. The method of claim 1, wherein the impurity control solution in the high performance liquid chromatography is a mixture of p-chlorobenzyl alcohol, p-chlorobenzophenone, p-chlorobenzhydrylpiperazine, levocetirizine amide, p-hydroxybenzoic acid and a solvent, and the solvent is a mixed solution of acetonitrile and water in a volume ratio of 1: 1;
the mass of p-chlorobenzyl alcohol, p-chlorobenzophenone, p-chlorobenzhydrylpiperazine, levocetirizine amide and p-hydroxybenzoic acid in each 1mL of impurity control solution was 0.4. mu.g, and 1.0. mu.g, respectively.
10. 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 test solution is 0.1 mg/mL.
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