CN108802211B

CN108802211B - Liquid phase detection method for related substances in cefquinome sulfate breast injectant

Info

Publication number: CN108802211B
Application number: CN201810354163.8A
Authority: CN
Inventors: 周淑贞; 林孝崇; 陶映娴; 蒲静君; 张庆
Original assignee: Foshan Nanhai Eastern Along Pharmaceutical Co ltd
Current assignee: Foshan Nanhai Eastern Along Pharmaceutical Co ltd
Priority date: 2018-04-19
Filing date: 2018-04-19
Publication date: 2021-03-19
Anticipated expiration: 2038-04-19
Also published as: CN108802211A

Abstract

The invention provides a liquid phase detection method for related substances in cefquinome sulfate breast injectant. The method has strong specificity and good reproducibility, and can ensure that each impurity peak has better separation effect and more accurate detection result.

Description

Liquid phase detection method for related substances in cefquinome sulfate breast injectant

Technical Field

The invention relates to the technical field of detection, and particularly relates to a liquid phase detection method for related substances in a cefquinome sulfate breast injectant.

Background

The cefquinome sulfate breast injectant is a medicine for treating the mastitis of the dairy cows, in particular for treating the mastitis of the dairy cows in a dry period, wherein the cefquinome sulfate serving as an effective component is aminothiazolone oximidone cephalosporin, has good spectrum antibacterial activity on a plurality of gram-positive and gram-negative bacteria, is quick to absorb and reaches a peak time period, has high bioavailability, and can reach high tissue concentration in lung and breast tissues.

In the existing liquid phase detection method for related substances in cefquinome sulfate breast injectant, although the separation degree between various chromatographic peaks accords with the specification, the separation degree is relatively small, so that the reproducibility of the peak area of an impurity peak is poor, and the related substance content is calculated by adopting an area normalization method, so that the related substance result is easily influenced by the tiny fluctuation of chromatographic conditions, and the accuracy of the detection result is difficult to ensure.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first purpose of the invention is to provide a liquid phase detection method for related substances in cefquinome sulfate breast injectant. The method has strong specificity and good reproducibility, and can ensure that each impurity peak has better separation effect and more accurate detection result.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

a liquid phase detection method for related substances in cefquinome sulfate breast injectant comprises the following steps:

detecting related substances in the cefquinome sulfate breast injection by adopting a high performance liquid chromatography;

wherein, in the high performance liquid chromatography, gradient elution is carried out by adopting the following conditions:

wherein the mobile phase A is a mixed solution of buffer solution and acetonitrile with the pH value of 3.6-4.0, and the ratio of the buffer solution to the acetonitrile is 85-95: 5-15;

the mobile phase B is a mixed solution of buffer solution and acetonitrile with the pH value of 3.6-4.0, and the ratio of the buffer solution to the acetonitrile is 40-60: 40-60.

Preferably, in the liquid phase detection method of the present invention, the high performance liquid chromatography is performed by gradient elution under the following conditions:

preferably, in the liquid phase detection method of the present invention, the mobile phase a is a mixture of a buffer solution having a pH of 3.6 and acetonitrile, and the ratio of the buffer solution to the acetonitrile is 90: 10;

and/or the mobile phase B is a mixed solution of buffer solution and acetonitrile with the pH value of 3.6, and the ratio of the buffer solution to the acetonitrile is 50: 50.

Preferably, in the liquid phase detection method of the present invention, the high performance liquid chromatography employs the following conditions:

flow rate: 0.8-1.2 ml/min;

column temperature: 25-35 ℃;

detection wavelength: 230-280 nm;

and/or the number of theoretical plates is not less than 2000 calculated according to cefquinome sulfate.

flow rate: 1.0 ml/min;

column temperature: 30 ℃;

detection wavelength: 245 nm.

Preferably, the liquid phase detection method of the present invention includes:

preparing a test solution: adding an extraction solution saturated with a cefquinome sulfate reference substance into a cefquinome sulfate breast injectant sample, shaking uniformly, centrifuging, and collecting a precipitate; treating the precipitate for 1-3 times under the same conditions, drying the precipitate obtained from the last treatment, dissolving with mobile phase, ultrasonic diluting, shaking, and filtering to obtain test solution;

control solution 1 preparation: diluting the sample solution with mobile phase, and shaking to obtain reference solution 1;

control solution 2 preparation: dissolving 2, 3-cyclohexylpyrrole in a mobile phase, diluting and shaking uniformly to obtain a control solution 2;

the test solution, control solution 1, and control solution 2 were injected into the high performance liquid chromatography, respectively, and the chromatogram was recorded.

Preferably, in the liquid phase detection method of the present invention, the extraction solution saturated with the cefquinome sulfate control is a petroleum ether solution saturated with the cefquinome sulfate control;

preferably, the boiling range of the petroleum ether is 30-60 ℃.

Preferably, in the liquid phase detection method according to the present invention, the preparing of the control solution 1 includes:

placing 1ml of test solution into a 100ml measuring flask, then diluting with mobile phase to a constant volume, and shaking up to obtain the test solution.

Preferably, the liquid phase detection method of the present invention includes: if an impurity peak exists in a chromatogram of a test solution, the 2, 3-cyclohexylpyridine content is not more than 3 times of the main peak area of a contrast solution, other single impurities are not more than the main peak area of the contrast solution, and the total impurities are not more than 4 times of the main peak area of the contrast solution;

the peaks in the chromatogram of the test solution, which are smaller than 0.5 times the area of the main peak of the control solution, are ignored.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention establishes a method for detecting related substances of cefquinome sulfate breast injectant, which can reduce the influence of the micro fluctuation of chromatographic conditions on the detection results of the related substances, and the accuracy of the detection results can be further enhanced by changing the calculation method of the related substances.

2. The invention can ensure that each impurity peak has better separation effect, more accurate detection result and stronger specificity.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

In view of the problems of small impurity separation degree, poor reproducibility and the like in the existing liquid phase detection method for related substances in cefquinome sulfate breast injectant, the invention particularly provides a novel detection method.

Specifically, the detection method provided by the invention is a liquid phase detection method for related substances in a dry-period cefquinome sulfate breast injectant, and the steps can be referred to as follows:

preparing a test solution: adding an extraction solution saturated with a cefquinome sulfate reference substance into a cefquinome sulfate breast injection (in a dry milk period), shaking uniformly, centrifuging, and collecting a precipitate; treating the precipitate for 1-3 times under the same conditions, drying the precipitate obtained from the last treatment, dissolving with mobile phase, ultrasonic diluting, shaking, and filtering to obtain test solution.

Preferably, the preparation of the test solution comprises the steps of: placing a dry-milk-period cefquinome sulfate breast injectant sample into a centrifuge tube, and then adding a saturated cefquinome sulfate ether solution (namely, an ether solution saturated by a cefquinome sulfate standard substance, wherein the preparation method can be referred to as that about 9mg of cefquinome sulfate is added into 1000ml of petroleum ether and is shaken uniformly), wherein the ratio of the mass gram of the dry-milk-period cefquinome sulfate breast injectant sample to the volume milliliter of the saturated cefquinome sulfate ether solution is preferably 1-2: 85-115, and more preferably 1.5: 100;

then shaking up, centrifuging (rotating speed of 4500r/min, time of 10-30 min), and discarding the supernatant;

extracting the precipitate again according to the method, and repeatedly extracting for 1-3 times; drying the precipitate obtained after the last extraction and centrifugation (preferably blowing dry with protective gas such as nitrogen or argon);

then, adding a proper amount of mobile phase into a centrifuge tube filled with the dried precipitate, transferring the mixed system in the centrifuge tube into a measuring flask (preferably, the ratio of the mass gram of the cefquinome sulfate breast injectant in the dry period to the calibrated volume milliliter of the measuring flask is 1-2: 80-120, more preferably 1:100), performing ultrasonic treatment for 10-20 min (preferably 15min), cooling, diluting to a scale by using the mobile phase, shaking uniformly, and filtering to obtain a test solution.

preferably, the preparation of control solution 1 comprises: placing 1ml of sample solution in 100ml measuring flask, diluting with mobile phase to constant volume, and shaking to obtain control solution 1

2, 3-cyclohexylpyrrole is a known impurity in cefquinome sulfate breast injectant in dry period

Preferably, the preparation of control solution 2 comprises the following steps: taking a proper amount of 2, 3-cyclohexylpyrrole, and then dissolving and diluting with a mobile phase to ensure that the concentration of the 2, 3-cyclohexylpyrrole in the solution reaches 10-30 (preferably 20) mu g/ml.

The test solution, control solution 1, and control solution 2 are injected into the HPLC, preferably in an amount of 20. mu.L, and the chromatogram is recorded.

If an impurity peak exists in a chromatogram of a test solution, the content of 2, 3-cyclohexyl pyridine is not more than 3 times (3.0%) of the area of a main peak of a control solution, other single impurities are not more than 1.0% of the area of the main peak of the control solution, and the total impurities are not more than 4 times (4.0%) of the area of the main peak of the control solution. The peaks in the chromatogram of the test solution, which are smaller than 0.5 times the area of the main peak of the control solution, are ignored.

Unlike the conventional detection method of external standard and area normalization, the present invention adopts self-control method, which is one of the improvement points of the present invention in the detection method of related substances in the traditional cefquinome sulfate breast injectant. By utilizing a self-contrast mode, the proportion content repeatability of a single impurity can be better, and meanwhile, the repeatability of total impurity detection is greatly improved.

The chromatographic conditions of the invention are as follows:

the type of the high performance liquid chromatograph: agilent 1260.

The type of the chromatographic column: kromasil 100-5C18 (4.6X 150mm, 5 μm)

Filling agent: octadecylsilane bonded, and silica gel.

Mobile phase: in the present invention, a gradient elution mode is adopted, and the gradient elution is another key point of the present invention.

The mobile phase comprises a mobile phase A and a mobile phase B;

wherein, the mobile phase A: a mixed solution of a buffer solution with a pH of 3.6-4.0 and acetonitrile, wherein the ratio of the buffer solution to the acetonitrile is 85-95: 5-15 (v/v);

preferably, the mobile phase A is a mixture of buffer solution with pH3.6 and acetonitrile, and the ratio of the buffer solution to the acetonitrile is 90:10 (v/v);

the preparation of the buffer in mobile phase a is preferably as follows: 1.42g of citric acid and 2.31g of disodium hydrogen phosphate are taken, dissolved in water and diluted to 1000ml (reaching the pH value of 3.6).

Mobile phase B: a mixed solution of a buffer solution with a pH of 3.6-4.0 and acetonitrile, wherein the ratio of the buffer solution to the acetonitrile is 40-60: 40-60 (v/v);

preferably, the mobile phase A is a mixture of buffer solution with pH3.6 and acetonitrile, and the ratio of the buffer solution to the acetonitrile is 50:50 (v/v);

Flow rate: 0.8-1.2 ml/min; preferably 1.0 ml/min.

Column temperature: 25-35 ℃; preferably, the following components are adopted: at 30 ℃.

Detection wavelength: 230-280 nm; preferably 245 nm.

The number of theoretical plates is not less than 2000 calculated according to cefquinome sulfate peak.

Meanwhile, compared with the traditional liquid chromatography detection method, the method does not need a system adaptability test, and the method is more convenient and faster to operate.

Example 1

Chromatographic conditions and system applicability test: the type of the high performance liquid chromatograph: agilent 1260; the type of the chromatographic column: kromasil 100-5C18(4.6 x 150mm, 5 μm); octadecylsilane chemically bonded silica is used as a filling agent; performing linear gradient elution with pH3.6 buffer solution (1.42 g citric acid and 2.31g disodium hydrogen phosphate, dissolved in water and diluted to 1000ml) -acetonitrile (90:10) as mobile phase A and pH3.6 buffer solution-acetonitrile (50:50) as mobile phase B according to the following table; the flow rate is 1.0 ml/min; the column temperature is 30 ℃; the detection wavelength was 254 nm. The number of theoretical plates is not less than 2000 calculated according to cefquinome sulfate peak.

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	100	0
5	100	0
			15	50	50
20	50	50
			21	100	0
23	100	0

The detection method comprises the following steps: taking about 1.5g of a dry-emulsion cefquinome injection sample, precisely weighing, placing the sample in a centrifuge tube, adding 20ml of petroleum ether (with a boiling range of 30-60 ℃) saturated by a cefquinome sulfate reference substance (taking about 9mg of cefquinome sulfate and adding 1000ml of petroleum ether), shaking uniformly, centrifuging at 4500r/min for 10 min, and discarding the supernatant; performing the same operation once, taking the precipitate, drying the precipitate by using nitrogen at room temperature, adding a proper amount of mobile phase, ultrasonically shaking the precipitate, transferring the precipitate into a 100ml measuring flask, ultrasonically shaking the precipitate for 15 minutes, cooling the precipitate, diluting the cooled precipitate to a scale by using the mobile phase, shaking the solution uniformly, and filtering the solution to be used as a test solution;

precisely measuring 1ml of a test solution, placing the test solution in a 100ml measuring flask, diluting with a mobile phase to a constant volume, and shaking up to obtain a control solution 1;

an appropriate amount of 2, 3-cyclohexylpyridine was dissolved in a mobile phase and diluted to give a solution containing 20. mu.g of 2, 3-cyclohexylpyridine per 1ml, as a control solution 2.

The sample solution, the control solution 1 and the control solution 2 were each 20. mu.l, and injected into a liquid chromatograph, respectively, to record the chromatogram. If an impurity peak exists in a chromatogram of a test solution, the content of 2, 3-cyclohexyl pyridine is not more than 3 times (3.0%) of the area of a main peak of a control solution, other single impurities are not more than 1.0% of the area of the main peak of the control solution, and the total impurities are not more than 4 times (4.0%) of the area of the main peak of the control solution. The peaks in the chromatogram of the test solution, which are smaller than 0.5 times the area of the main peak of the control solution, are ignored.

Investigation of related substance measurement methods:

1. determination of detection wavelength

Weighing 25mg to 250ml of cefquinome reference substance in a measuring flask, adding a mobile phase for dissolving, fixing the volume and shaking up to obtain the wavelength test solution. Scanning within the range of 190 nm-600 nm by using an ultraviolet spectrophotometry, and the result shows that the cefquinome has the maximum absorption at the wavelength of 254 nm.

2. Determination of impurity calculation method

Taking the same sample, repeating the step of 3 needles to examine the content of the impurity in the ratio (peak area%), respectively comparing the area normalization method with the self control method, preferably selecting a more appropriate impurity calculation method, and obtaining the results shown in the following table:

from the above experimental results, it can be seen that, when the area normalization method is applied, even if the same sample is repeatedly fed into 3 needles, the situation that the RSD of 2, 3-cyclohexylpyridine and other maximum single impurity content (peak area%) is larger can occur, the RSD of the total impurity content (peak area%) is very large, and the result of the self-comparison method is obviously better than that of the area normalization method, so that the application of the self-comparison method can lead the ratio content repeatability of the single impurity to be better, and simultaneously greatly improve the repeatability of the total impurity detection. Therefore, the self-control method is preferably the impurity calculation method of the present invention.

3. Specificity test

Preparing a blank auxiliary material solution: preparing blank auxiliary material solution of cefquinome sulfate breast injectant (in dry period) according to the formula process proportion.

Precisely sucking 20 mul of each of the sample solution, the reference solution, the blank auxiliary material solution and the mobile phase, and injecting samples according to the chromatographic condition of the related substance determination method established by the invention. As a result, chromatographic peaks appear on the corresponding positions of the sample and the reference, the base line in the chromatographic peaks of the sample is stable, and the separation degree of each impurity peak is good; the blank auxiliary material solution and the mobile phase have no chromatographic peak at each impurity position, which indicates that the mobile phase and the negative control have no interference on the retention time of each impurity.

4. Stability of sample solutions

Sample injection of 20 mul of the test solution is carried out for 0, 2, 4, 6 and 10 hours respectively under the same chromatographic conditions, and according to the measured chromatographic peak area, the RSD of the test sample group is 0.63 percent and less than 2.0 percent, which indicates that the stability of the reference sample and the test sample solution is good within 10 hours.

Solution stability test results:

5. linear relation

Precisely weighing 18.75mg of cefquinome reference substance, putting the cefquinome reference substance into a 100ml measuring flask, adding a mobile phase to completely dissolve the cefquinome reference substance, fixing the volume, and shaking up the cefquinome reference substance to obtain a stock solution. The stock solutions 1, 2, 4, 8, 16, 24ml were weighed out into 100ml volumetric flasks, diluted to the scale with the mobile phase, shaken well and filtered through a 0.45 μm filter membrane to obtain solutions of 1.875 μ g/ml, 3.75 μ g/ml, 7.5 μ g/ml, 15 μ g/ml, 30 μ g/ml, 45 μ g/ml series of concentrations determined. According to the chromatographic conditions established by the invention, 20 mu l of sample is injected, and the chromatographic peak area of the sample is determined.

Results of the linearity test:

concentration (μ g/ml)	1.875	3.75	7.5	15	30	45
							Peak area	20.6	46.6	94.7	193.4	383.8	572.8

Establishing a standard curve by taking the peak area and the concentration as coordinates to obtain an equation: Y12.792X-1.2179 (R)²0.9999), indicating that the concentration of the related substance is in a range of 1.875 μ g/ml to 45 μ g/ml and has a good linear relationship with the peak area.

6. Precision test

The method comprises the steps of repeatedly injecting a sample solution (namely a solution containing cefquinome with the concentration of 0.75 mg/ml) for 0h under the item of 'sample solution stability' for 6 times under the same chromatographic condition, measuring the peak area of 2, 3-cyclohexylpyridine respectively by 20 mu l each time, inspecting the precision of the instrument, calculating the relative standard deviation RSD obtained by 6 groups of data, and obtaining the result that the precision is 0.61 percent less than 2.0 percent, thereby showing that the related substance measuring method established by the invention has good precision. The results of the precision test are as follows:

7. repeatability test

Selecting 6 parts of cefquinome sulfate breast injectant (in dry period) of the same batch number, precisely weighing, preparing a solution according to the operation of a test item, adopting the same chromatographic conditions, injecting 20 mu l of sample, collecting the chromatogram of 6 parts of sample, calculating the relative standard deviation RSD of the solution, and obtaining the result that the ratio of 1.61 percent to 2.0 percent. Therefore, the related substance determination method established by the invention has good repeatability. The results of the repeatability experiments are as follows:

8. reproducibility test

6 parts of cefquinome sulfate breast injectant (dry period) in the same batch number is selected, and another analyst uses different liquid chromatographs and different chromatographic columns in different laboratories and different working days to measure the content ratio of related substances in the same operation of a repeatability test. The results of the reproducibility test were as follows:

the results show that: in the detection results of the two analysts, the RSD of the 2, 3-cyclohexyl pyridine, the maximum single impurity and the total impurity is less than 2.0 percent, and the difference values are small. Therefore, the related substance measuring method established by the invention has good reproducibility.

9. Accuracy test

Accurately weighing 3 parts of cefquinome sulfate breast injectant (dry period) samples with known related substances according to 80%, 100% and 120% of the marked amount of the cefquinome sulfate breast injectant (dry period), respectively placing the cefquinome sulfate breast injectant (dry period) samples into 9 centrifuge tubes, preparing a test sample and a reference solution according to experimental operation, measuring the related substances according to the related substance measuring method established by the invention, and calculating the recovery rate. The accuracy test results are as follows:

from the above results, it can be seen that: the recovery rate and the average recovery rate of the invention are both 92-105%, and the RSD is within 2%, therefore, the related substance determination method established by the invention has good accuracy.

10. Durability test

Taking a proper amount of cefquinome sulfate breast injectant (in a dry emulsion period) in the same batch, respectively inspecting the variation of flow rate +/-0.2 ml/min, the variation of column temperature +/-5 ℃, the variation of acetonitrile proportion in a mobile phase +/-2%, and the variation of instrument chromatographic behavior in different chromatographic columns, observing the separation condition of a base line, and calculating the average value and RSD (mean shift decomposition) value of data obtained under various conditions, wherein the durability test result is as follows:

test results show that in the durability test of the method for determining cefquinome sulfate related substances of the cefquinome sulfate breast injectant (in dry period), the flow rate, the column temperature, the chromatographic column and the mobile phase are combined, each impurity peak reaches baseline separation, and the RSD value of 2, 3-cyclohexylpyridine, other maximum single impurity and total impurity related substances is less than 2% under each condition, so that the method for determining related substances established by the invention is good in durability.

11. Detection limit

The cefquinome reference substance is dissolved by a mobile phase to prepare a test solution with 7.5 mu g/ml, the test solution is diluted step by step, the concentration of the test solution is adjusted to enable the response value of cefquinome to be about 3 times of the noise level, the detection limit of cefquinome is 1.5 mu g/ml, and the method provides that the peak which is less than 0.5 time of the main peak area of the reference solution is ignored (namely the peak which is less than 3.75 mu g/ml is ignored), so the detection limit of the related substance determination method established by the invention can meet the detection requirement.

12. Determination of cefquinome sulfate-related substances in sample

The method for determining related substances established by the invention respectively determines the related substances of cefquinome sulfate in three batches of cefquinome sulfate breast injectants (dry emulsion period), and the results are as follows:

from the above results, the RSD of the substances of interest in all three batches was less than 2%, and the intra-batch variation was small.

Comparative example 1

Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; using 0.345% sodium perchlorate monohydrate solution-phosphoric acid-acetonitrile (1000:12:115) (pH value is adjusted to 3.6 by triethylamine) as a mobile phase, and the flow rate is 1.0 ml/min; the column temperature is 30 ℃; the detection wavelength was 270 nm. Taking about 25mg of cefotaxime, adding 100ml of mobile phase to dissolve the cefotaxime, taking about 25mg of cefotaxime, putting the cefotaxime into a 25ml measuring flask, precisely adding 1ml of cefotaxime solution, and diluting the cefotaxime solution to the scale with the mobile phase. And precisely measuring 20 mul of the cefquinome, injecting the obtained product into a liquid chromatograph, and recording a chromatogram, wherein the separation degree of the cefquinome and the cefotaxime is more than 1.0.

The determination method comprises the following steps: taking about 1.5g of cefquinome sulfate breast injectant in a dry emulsion period, precisely weighing, placing the cefquinome sulfate breast injectant in a centrifuge tube, adding 20ml of petroleum ether (with a boiling range of 30-60 ℃) saturated by cefquinome sulfate reference substances (taking about 9mg of cefquinome sulfate and 1000ml of petroleum ether), shaking uniformly, centrifuging at 4500r/min for 10 min, and discarding supernatant; performing the same operation once, taking the precipitate, drying the precipitate at room temperature by using nitrogen, adding a proper amount of mobile phase, ultrasonically shaking the precipitate, transferring the precipitate into a 100ml measuring flask, ultrasonically shaking the precipitate for 15 minutes, cooling the precipitate, diluting the cooled precipitate to a scale by using the mobile phase, shaking the solution uniformly, and filtering the solution;

and dissolving appropriate amount of 2, 3-cyclohexyl pyridine in mobile phase, and diluting to obtain solution containing 20 μ g of 2, 3-cyclohexyl pyridine per 1ml as control solution.

And (4) respectively injecting 20 mu l of the test solution and the reference solution into a liquid chromatograph, and recording the chromatogram. If an impurity peak exists in a chromatogram of a sample solution, the content of 2, 3-cyclohexyl pyridine is not more than 3.0 percent, other single impurities are not more than 1.0 percent, and the total impurities are not more than 4.0 percent according to the calculation of a peak area normalization method.

1. Specificity test

(1) Preparing a blank auxiliary material solution: preparing blank auxiliary material solution of cefquinome sulfate breast injectant (in dry period) according to the formula process proportion.

However, the separation degree, peak purity and retention time stability among the impurity peaks of the sample are general and inferior to those of example 1, and the data comparison is as follows:

note: the peak purity standard-reaching proportion is that the number of impurity peaks with the peak purity reaching 98 percent/the number of total impurity peaks is 100 percent

As can be seen from the results of the above experimental comparison, the method for detecting a substance in example 1 is more specific and reproducible than that in comparative example 1.

(2) Comparing example 1 with comparative example 1, recording chromatograms, observing the separation degree and the peak purity standard proportion of each impurity peak, and taking the same batch of samples to examine the stability of the retention time of the impurity peak.

The detection results of the cefquinome sulfate breast injectant (dry period) under the conditions of the example 1 and the comparative example 1 are as follows:

From the comparison of the above experimental results, it can be seen that: the embodiment 1 can better ensure better separation effect, each impurity component can be better separated, and the stability of the retention time of the impurity peak is better, so that the specificity and the reproducibility of the detection method are stronger, and the detection result is more accurate.

In summary, both example 1 and comparative example 1 can make the cefquinome peak not interfered by the mobile phase and the blank preparation control, and both meet the basic requirement of specificity. However, example 1 can test the sample each peak separation degree effect, peak purity is better, and impurity peak retention time is more stable, prove the preferred example 1 than the comparative example 1 better.

2. The determination results of cefquinome related substances in three samples:

as can be seen from the above experimental results, the RSD of the related substances of the three batches is less than 2%, but the reproducibility result of example 1 is better than that of comparative example 1.

From the results, the specificity and the reproducibility of the detection method for the liquid phase related substances of the optimized cefquinome sulfate breast injectant (in the dry period) are improved.

While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims

1. A liquid phase detection method for related substances in cefquinome sulfate breast injectant is characterized by comprising the following steps:

the related substance is 2, 3-cyclohexyl pyridine;

the types of chromatographic columns adopted in the high performance liquid chromatography are as follows: kromasil 100-5C18,4.6 x 150mm, 5 μm;

the mobile phase A is a mixed solution of buffer solution with pH of 3.6 and acetonitrile, and the ratio of the buffer solution to the acetonitrile is 90: 10;

the mobile phase B is a mixed solution of buffer solution with pH of 3.6 and acetonitrile, and the ratio of the buffer solution to the acetonitrile is 50: 50;

the preparation method of the buffer solution in the mobile phase A is as follows: 1.42g of citric acid and 2.31g of disodium hydrogen phosphate are taken, dissolved in water and diluted to 1000 ml.

2. The liquid phase detection method according to claim 1, wherein the high performance liquid chromatography is performed by gradient elution under the following conditions:

。

3. the liquid phase detection method according to claim 2, wherein the high performance liquid chromatography is performed by gradient elution under the following conditions:

。

4. the liquid phase detection method according to claim 1, wherein the high performance liquid chromatography employs the following conditions:

flow rate: 0.8-1.2 ml/min;

column temperature: 25-35 ℃;

detection wavelength: 230-280 nm;

5. The liquid phase detection method according to claim 4, wherein the high performance liquid chromatography employs the following conditions:

flow rate: 1.0 ml/min;

column temperature: 30 ℃;

detection wavelength: 245 nm.

6. The liquid phase detection method according to claim 1, comprising:

control solution 2 preparation: dissolving 2, 3-cyclohexyl pyridine with a mobile phase, diluting and shaking up to obtain a control solution 2;

injecting the test solution, the control solution 1 and the control solution 2 into a high performance liquid chromatography respectively, and recording the chromatography;

the extraction solution saturated by the cefquinome sulfate reference substance is petroleum ether solution saturated by the cefquinome sulfate reference substance.

7. The liquid phase detection method according to claim 6, wherein the boiling range of the petroleum ether is 30 to 60 ℃.

8. The liquid phase detection method of claim 6, wherein the preparing of the control solution 1 comprises:

placing 1ml of the test solution into a 100ml measuring flask, then diluting with a mobile phase to a constant volume, and shaking up to obtain a control solution 1.

9. The liquid phase detection method according to claim 8, comprising: if an impurity peak exists in a chromatogram of a test solution, the 2, 3-cyclohexylpyridine content is not more than 3 times of the main peak area of a contrast solution, other single impurities are not more than the main peak area of the contrast solution, and the total impurities are not more than 4 times of the main peak area of the contrast solution;