阅读说明：本技术 盐酸右美托咪定原料或制剂中有关物质的检测方法 (Method for detecting related substances in dexmedetomidine hydrochloride raw material or preparation ) 是由 汤文星 贺珊珊 张现涛 何盛江 谭斌 吴沛桃 于 2020-12-24 设计创作，主要内容包括：本发明公开了一种盐酸右美托咪定原料或制剂中有关物质的检测方法,包括以下步骤：采用高效液相色谱法进行检测,流动相由流动相A和流动相B组成,所述流动相A为pH值为7.3～7.7的磷酸盐缓冲液,所述流动相B为乙腈,所述磷酸盐缓冲液中磷酸盐的浓度为8mmol/L～12mmol/L；采用梯度流动相洗脱。采用该流动相体系配合合适的梯度洗脱条件,可检测出盐酸右美托咪定原料或者制剂制备过程中带入的起始原料、中间体、副反应产物以及盐酸右美托咪定降解产物中的13个相关杂质,使杂质得到更好的分离,提高检测结果的准确性。(The invention discloses a method for detecting related substances in dexmedetomidine hydrochloride raw materials or preparations, which comprises the following steps: detecting by adopting a high performance liquid chromatography, wherein a mobile phase consists of a mobile phase A and a mobile phase B, the mobile phase A is a phosphate buffer solution with the pH value of 7.3-7.7, the mobile phase B is acetonitrile, and the concentration of phosphate in the phosphate buffer solution is 8-12 mmol/L; gradient mobile phase elution is used. By adopting the mobile phase system and matching with proper gradient elution conditions, 13 related impurities in dexmedetomidine hydrochloride raw materials or initial raw materials, intermediates, side reaction products and dexmedetomidine hydrochloride degradation products brought in the preparation process of the preparation can be detected, so that the impurities are better separated, and the accuracy of the detection result is improved.)

1. A method for detecting related substances in dexmedetomidine hydrochloride raw materials or preparations is characterized by comprising the following steps:

detecting by adopting a high performance liquid chromatography, wherein a mobile phase consists of a mobile phase A and a mobile phase B, the mobile phase A is a phosphate buffer solution with the pH value of 7.3-7.7, the mobile phase B is acetonitrile, and the concentration of phosphate in the phosphate buffer solution is 8-12 mmol/L; elution with a gradient mobile phase, the gradient of the mobile phase being set as follows, based on 100% of the volume of the mobile phase:

wherein the related substances are dexmedetomidine hydrochloride raw materials or initial raw materials, intermediates, side reaction products and dexmedetomidine hydrochloride degradation products brought in the preparation process of the preparation.

2. The method for detecting related substances in dexmedetomidine hydrochloride raw material or preparation as claimed in claim 1, characterized in that the phosphate is selected from one of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate and disodium hydrogen phosphate; preferably, the phosphate is selected from disodium hydrogen phosphate dihydrate.

3. The method for detecting related substances in dexmedetomidine hydrochloride raw material or preparation as claimed in claim 1, characterized in that octadecylsilane chemically bonded silica is used as filler in the chromatographic column.

4. The method for detecting related substances in dexmedetomidine hydrochloride raw material or preparation as claimed in claim 3, characterized in that the chromatographic column is Agilent Eclipse plus C18, 4.6 x 150mm, 3.5 μm.

5. The method for detecting related substances in dexmedetomidine hydrochloride raw material or preparation as claimed in claim 3, characterized in that the detection wavelength is 215 nm-225 nm, the column temperature is 25 ℃ -35 ℃, and the flow rate of elution of the mobile phase is 0.8 mL/min-1.2 mL/min.

6. The method for detecting dexmedetomidine hydrochloride raw material or formulation of claim 1, where a trapping cartridge is installed between the gradient mixer and the sample injector.

7. The method for detecting related substances in dexmedetomidine hydrochloride raw material or preparation as claimed in claim 1, wherein the preparation is dexmedetomidine hydrochloride injection.

8. The method for detecting related substances in dexmedetomidine hydrochloride raw material or preparation as claimed in claim 1, characterized in that the sample dissolution solvent is water.

9. The method for detecting dexmedetomidine hydrochloride raw material or related substances in the preparation as claimed in claim 8, characterized by the step of formulating the dexmedetomidine hydrochloride raw material as a sample aqueous solution with a concentration of 0.1 mg/ml.

10. The method for detecting related substances in dexmedetomidine hydrochloride raw material or preparation according to any of claims 1 to 9, characterized in that the related substances include at least one of the following substances:

Technical Field

The invention relates to the technical field of pharmaceutical analysis technology, in particular to a method for detecting related substances in dexmedetomidine hydrochloride raw materials or preparations.

Background

Dexmedetomidine hydrochloride is a novel high-selectivity alpha 2-adrenoceptor agonist, which is a dextroisomer of medetomidine, and compared with medetomidine, the dexmedetomidine hydrochloride has stronger selectivity on central alpha 2-adrenoceptor agonism, short half-life and small dosage. Dexmedetomidine has sedative and anxiolytic effects, is clinically suitable for sedation of patients who begin intubation and use a respirator during intensive care therapy, and also has analgesic and anti-sympathetic inhibition effects on perioperative stress reactions, and can reduce the dosage of anesthetic drugs and maintain the stability of hemodynamics. Dexmedetomidine hydrochloride injection was developed by the cooperation of Orion Pharma (Finland) and Abott (USA), and was first marketed in the United states in 3 months in 2000, in Japan in 1 month in 2004, and in 2009 in China. The device is mainly used for starting intubation and calming a patient using a breathing machine during intensive care treatment in clinic. Clinical experience of domestic and foreign application for years shows that the dexmedetomidine hydrochloride injection has strong sedative effect, particularly has unique psychological and physiological synergistic effect on severe patients, and has good clinical effect.

The related substances are initial raw materials, intermediates, side reaction products, degradation products and the like brought in the process of synthesizing the medicine, and the quality and the safety of the medicine can be controlled by detecting the related substances.

Currently, only the standard of dexmedetomidine hydrochloride raw material is recorded in the united states pharmacopeia 43 th edition (USP43), and related preparations are not recorded in the pharmacopeia. The related substance method comprises the following steps: using Merck Lichrospher 100RP-18(4 mm. times.12.5 cm, 5 μm) chromatography column, 0.89g/L disodium hydrogen phosphate dihydrate (prepared by dissolving disodium hydrogen phosphate dihydrate with 90% water, adjusting pH to 7.0 with 16g/L sodium dihydrogen phosphate dihydrate, and diluting with water to scale) -methanol (40:60) as mobile phase; the wavelength was 220nm and the flow rate was 1.0ml per minute. Due to different synthesis process routes, the method for collecting related substances in USP43 is not suitable, and practice shows that the method is poor in specificity, cannot effectively separate impurities of dexmedetomidine hydrochloride, and cannot meet the requirement for detection of the related substances of dexmedetomidine hydrochloride.

The related substance detection of dexmedetomidine hydrochloride is also reported in related patents and literatures, and through comparison, the composition and the elution procedure of a mobile phase are different. The summary has the following problems, such as complicated preparation of mobile phase, long gradient elution time, low efficiency, low sensitivity, or poor tolerance of chromatographic column.

Disclosure of Invention

Based on the method, the method for detecting the related substances in the dexmedetomidine hydrochloride raw material or preparation has strong detection capability, specificity and sensitivity.

A method for detecting related substances in dexmedetomidine hydrochloride raw materials or preparations comprises the following steps:

detecting by adopting a high performance liquid chromatography, wherein a mobile phase consists of a mobile phase A and a mobile phase B, the mobile phase A is a phosphate buffer solution with the pH value of 7.3-7.7, the mobile phase B is acetonitrile, and the concentration of phosphate in the phosphate buffer solution is 8-12 mmol/L; elution with a gradient mobile phase, the gradient of the mobile phase being set as follows, based on 100% of the volume of the mobile phase:

wherein the related substances are dexmedetomidine hydrochloride raw materials or initial raw materials, intermediates, side reaction products and dexmedetomidine hydrochloride degradation products brought in the preparation process of the preparation.

In some of these embodiments, the phosphate is selected from one of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate; preferably, the phosphate is selected from disodium hydrogen phosphate dihydrate.

In some of these examples, the column is packed with octadecylsilane bonded silica.

In some of these examples, the column is Agilent Eclipse plus C18, 4.6X 150mm, 3.5 μm.

In some of these embodiments, the detection wavelength is 215nm to 225nm and the column temperature is 25 ℃ to 35 ℃.

In some of these embodiments, the mobile phase elutes at a flow rate of 0.8mL/min to 1.2 mL/min.

In some of these embodiments, a trapping cartridge is mounted between the gradient mixer and the injector.

In some of these embodiments, the formulation is dexmedetomidine hydrochloride injection.

In some of these embodiments, the sample dissolution solvent is water.

In some of these embodiments, a step is included of formulating the dexmedetomidine hydrochloride starting material as an aqueous sample solution having a concentration of 0.1 mg/ml.

In some of these embodiments, the related substances include at least one of:

in the invention, 8 mmol/L-12 mmol/L phosphate buffer solution is used as a mobile phase A, acetonitrile is used as a mobile phase B, the concentration of buffer salt is low, and the problem of micro-crystallization precipitation in the online mixing process is avoided, thus causing interference to the detection result and equipment abrasion; and the pressure of the mobile phase system is low, so that chromatographic columns and instruments can be effectively protected. The mobile phase system of the invention can simultaneously detect 13 related impurities in dexmedetomidine hydrochloride raw materials and preparations by matching with proper elution conditions, and has good separation degree and high sensitivity.

The invention can effectively detect the impurities possibly generated in the existing dexmedetomidine hydrochloride synthesis route process, and can also detect the impurities listed in USP 43. By adopting the detection method, the degradation substances generated by dexmedetomidine hydrochloride under the forced degradation condition can be well separated. The elution time is fast, the efficiency is high, and the chromatogram is excellent. The detection method can simultaneously detect the dexmedetomidine hydrochloride raw material and the injection, and is convenient for the comparison of the impurity spectrum. The detection method can be used for controlling the quality of dexmedetomidine hydrochloride raw materials and injection, and plays a good promoting role in controlling and promoting the quality of the products.

Drawings

FIG. 1 is a chromatogram for detecting 5 impurities listed in USP43 by using a USP43 related substance detection method in the background art;

FIG. 2 is a chromatogram for detecting impurities inferred from a synthetic process route by using a USP43 related substance detection method in the background art;

FIG. 3 is a chromatogram of total 13 impurities listed in USP43 and possible in the synthetic route of the product using the detection method according to one embodiment of the present invention;

FIG. 4 is a chromatogram of a sample of raw materials using a detection method according to an embodiment of the present invention;

FIG. 5 is a blank solvent-water chromatogram of a test method employing one embodiment of the invention;

FIG. 6 is a chromatogram of a sample of raw materials using a detection method according to an embodiment of the present invention;

FIG. 7 is a blank solvent-water chromatogram of a test method according to an embodiment of the invention;

FIG. 8 is a sensitive solution chromatogram for use in a detection method according to an embodiment of the invention;

FIG. 9 is a chromatogram of a sample for oxidative destruction of raw materials using a detection method according to an embodiment of the present invention;

FIG. 10 is a chromatogram of an injection sample using a detection method according to an embodiment of the present invention;

FIG. 11 is a chromatogram of blank excipients of injection using the detection method of one embodiment of the invention.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The embodiment of the invention provides a method for detecting related substances in dexmedetomidine hydrochloride raw materials or preparations. Wherein the related substances are dexmedetomidine hydrochloride raw materials or initial raw materials, intermediates, side reaction products and dexmedetomidine hydrochloride degradation products brought in the preparation process of the preparation.

The detection method of related substances in dexmedetomidine hydrochloride raw materials or preparations comprises the following steps:

detecting by adopting a high performance liquid chromatography, wherein a mobile phase consists of a mobile phase A and a mobile phase B, the mobile phase A is a phosphate buffer solution with the pH value of 7.3-7.7, the mobile phase B is acetonitrile, and the concentration of phosphate in the phosphate buffer solution is 8-12 mmol/L; gradient mobile phase elution is used.

In the invention, 8 mmol/L-12 mmol/L phosphate buffer solution is used as a mobile phase A, acetonitrile is used as a mobile phase B, the concentration of buffer salt is low, and the problem of micro-crystallization precipitation in the online mixing process is avoided, thus causing interference to the detection result and equipment abrasion; and the pressure of the mobile phase system is low, so that chromatographic columns and instruments can be effectively protected. The mobile phase system of the invention can simultaneously detect 13 related impurities in dexmedetomidine hydrochloride raw materials and preparations by matching with proper elution conditions, and has good separation degree and high sensitivity.

The invention can effectively detect the impurities possibly generated in the existing dexmedetomidine hydrochloride synthesis route process, and can also detect the impurities listed in USP 43. By adopting the detection method, the degradation substances generated by dexmedetomidine hydrochloride under the forced degradation condition can be well separated. The elution time is fast, the efficiency is high, and the chromatogram is excellent. The detection method can simultaneously detect the dexmedetomidine hydrochloride raw material and the preparation, and facilitates the comparison of the hybrid spectrum.

The detection method can be used for controlling the quality of dexmedetomidine hydrochloride raw materials and injection, and plays a good promoting role in controlling and promoting the quality of the products.

The gradient of the mobile phase is set as follows, based on 100% of the volume of the mobile phase:

the related substances which can be detected by adopting the method of the invention comprise the following impurities: USP43 lists impurities A, B, C, D and E. In addition, possible impurities are analyzed according to the synthetic process route: C. d, E, F, G, H, I and J. The chemical name and the structural formula are as follows:

in some embodiments, the gradient elution conditions are:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	75-85	15-25
20	55-65	35-45
			30	30-40	60-70
35	30-40	60-70
			35.01	75-85	15-25
45	75-85	75-85

The beneficial effects of the scheme are that the acetonitrile-phosphate mobile phase volume ratio and the elution procedure can better separate 13 related impurities of dexmedetomidine hydrochloride, and improve the accuracy of the detection result.

Preferably, the initial volume ratio of acetonitrile-phosphate buffer is 20: 80. Preferably, the pH value of the phosphate buffer is adjusted to 7.3-7.7 by using phosphoric acid, and the pH value is preferably 7.5. The initial proportion of the acetonitrile-phosphate mobile phase adopts the volume ratio, and the initial proportion is adjusted to the pH value range, so that impurities can be better separated.

In some embodiments, the gradient elution conditions are:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	80	20
20	60	40
			30	35	65
35	35	65
			35.01	80	80
45	80	20

When the impurity in the dexmedetomidine hydrochloride raw material is detected, the preparation method of the sample solution comprises the following steps:

taking a proper amount of dexmedetomidine hydrochloride raw material, adding water to dissolve the dexmedetomidine hydrochloride raw material, and quantitatively diluting the dexmedetomidine hydrochloride raw material into a solution.

The method can be used for detecting the excellent quality of the dexmedetomidine hydrochloride injection, for example, the problems about storage and quality guarantee period can be judged by judging the degradation rate and the degradation product impurities of dexmedetomidine hydrochloride in the dexmedetomidine hydrochloride injection along with the prolonging of the storage time and comparing the degradation rate and the degradation product impurities with the impurities in dexmedetomidine hydrochloride raw materials used by the injection. The more the injection liquid is relative to impurities in the feedstock, the higher the degree of degradation is likely to be. For example, the factory quality problem of the fresh dexmedetomidine hydrochloride injection can be judged by judging the impurity components and the content in the fresh dexmedetomidine hydrochloride injection.

As the concentration of the dexmedetomidine hydrochloride injection is 0.1mg/ml (calculated by dexmedetomidine), the detection concentration of the raw materials is consistent with the concentration of the preparation in order to meet the requirement of the detection concentration of the preparation. The method has the advantages that the concentration of the raw materials matched with the preparation is uniform, the detection method is uniform, and the operation and the comparison of impurity spectrums are convenient.

In one embodiment, the aqueous sample solution contains about 0.1mg dexmedetomidine per lml.

In some embodiments, the method further comprises detecting the control solution in the same manner as the sample solution. The preparation method of the reference solution can be as follows: and taking a proper amount of the sample solution, and adding water for dilution. For example, it may be diluted to a solution containing about 0.1. mu.g of dexmedetomidine per lml.

In some embodiments, the phosphate is selected from one of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate; preferably, the phosphate is selected from disodium hydrogen phosphate dihydrate.

Dexmedetomidine hydrochloride exists in the form of injection preparation, and in some embodiments, the preparation detected by the invention can be dexmedetomidine hydrochloride injection. Of course, if other preparations, such as emulsion, ointment, etc., are available, the method of the present invention may be used for detection, but it may be necessary to add a step of dissolving dexmedetomidine hydrochloride and related substances from the preparation to obtain an aqueous solution before detection.

In some embodiments, the sample dissolution solvent is water.

In some embodiments, the mobile phase A is a phosphate buffer adjusted to pH 2.5-4.0 by a pH adjusting agent. Preferably, the pH adjuster is phosphoric acid.

In some embodiments, the chromatography column uses octadecylsilane bonded silica gel as a packing material.

In some embodiments, the chromatography column is Agilent Eclipse plus C18, 4.6X 150mm, 3.5 μm. The chromatographic column with the specific type has higher sensitivity to dexmedetomidine hydrochloride and impurities thereof, and has better impurity separation effect.

In some embodiments, the detection wavelength is 215nm to 220nm, preferably 220 nm.

In some embodiments, the column temperature is from 25 ℃ to 35 ℃, preferably 30 ℃.

In some embodiments, the mobile phase elutes at a flow rate of 0.8mL/min to 1.2 mL/min. Preferably 1 mL/min.

In some embodiments, the sample volume is 45ul to 55ul, preferably 50ul, under chromatographic conditions.

The beneficial effects of the scheme are that the wavelength is the maximum absorption wavelength of dexmedetomidine hydrochloride, impurities can be better detected, and the column is mild in flow speed, so that a detection environment can be better provided; in order to cooperate with the injection detection, the sample injection volume can reach higher sensitivity.

In some embodiments, a capture column is installed between the gradient mixer and the injector. The small trapping column is arranged between the gradient mixer and the sample injector, so that secondary purification can be performed on the instrument and the mobile phase, ghost peaks are removed, the base line of the chromatogram is stable, a better impurity separation chromatogram is obtained, the gradient peaks are eliminated, the interference of the gradient peaks on impurity detection is reduced, and a more accurate detection result is provided. And has the protection function, and the service life of the chromatographic column and the instrument is prolonged. In one embodiment, the trap Column may be selected from the group consisting of the Ghost-Buster Column, 4.6mm by 50 mm.

Example 1

5 impurity positioning solutions listed in USP43 and impurity mixed solutions possibly generated according to a synthetic process route are respectively subjected to sample injection detection by adopting a USP43 related substance detection method in the background technology. Using Merck Lichrospher 100RP-18(4 mm. times.12.5 cm, 5 μm) chromatography column, 0.89g/L disodium hydrogen phosphate dihydrate (prepared by dissolving disodium hydrogen phosphate dihydrate with 90% water, adjusting pH to 7.0 with 16g/L sodium dihydrogen phosphate dihydrate, and diluting with water to scale) -methanol (40:60) as mobile phase; the wavelength is 220 nm; the column temperature was 30 ℃ and the flow rate was 1.0ml per minute.

The detection results are shown in Table 1, and the spectra are shown in figure 1 and figure 2.

TABLE 1

As can be seen from fig. 1 and table 1, among 5 impurities listed in USP43, impurity D and impurity E are not well separated; as can be seen from fig. 2, it is assumed that impurities C are not separated from the main component among impurities that may be generated according to the synthetic process route, and impurities E and F are difficult to elute under such conditions. The method is not applicable to the detection of dexmedetomidine hydrochloride related substances.

Example 2

By adopting the detection method, 5 impurity positioning solutions listed in USP43 and possibly generated impurities and impurity mixed solutions according to a synthesis process route are respectively subjected to sample injection detection. Instruments and conditions: shimadzu LC-20AT UV liquid chromatography system, column: agilent Eclipse plus C18 (4.6X 150mm, 3.5 μm); the detection wavelength is 220 nm; the column temperature is 30 ℃; the flow rate is 1.0 ml/min; gradient elution was performed according to the following table 2 using 10mmol/L disodium hydrogen phosphate dihydrate as mobile phase a and acetonitrile as mobile phase B:

TABLE 2

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	80	20
20	60	40
			30	35	65
35	35	65
			35.01	80	80
45	80	20

The results are shown in Table 3 and FIG. 3.

TABLE 3

As can be seen from Table 3 and FIG. 3, the separation degree between 13 impurities, the impurity and the main component is good, the dexmedetomidine retention time is 18.3min, the separation degree of the impurity H adjacent to the dexmedetomidine retention time can reach 3.3, and the detection requirements are met. Therefore, the detection method can be used for detecting the dexmedetomidine hydrochloride related substances.

Example 3

Instruments and conditions: shimadzu LC-20AT UV liquid chromatography system, column: agilent Eclipse plus C18 (4.6X 150mm, 3.5 μm); the detection wavelength is 220 nm; taking 10mmol/L disodium hydrogen phosphate dihydrate as a mobile phase A and acetonitrile as a mobile phase B, and keeping the column temperature at 30 ℃; the flow rate was 1.0ml/min and the gradient elution was carried out according to the following Table 4:

TABLE 4

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	80	20
20	60	40
			30	35	65
35	35	65
			35.01	80	80
45	80	20

Test samples: taking a proper amount of raw materials, adding water to dissolve and dilute the raw materials to prepare a solution containing 0.1mg/ml of dexmedetomidine as a raw material test solution; the solution and the blank solvent were measured precisely at 50ul each and injected into a liquid chromatograph. The results are shown in FIGS. 4 and 5.

As can be seen from FIGS. 4 and 5, when the mobile phase changes the gradient in 25-35min, the baseline is unstable, many gradient peaks appear, and multiple experiments show that the sizes of the gradient peaks are inconsistent, which occasionally affects the detection of impurities (the retention time is about 26min), and is not favorable for the accuracy of the detection result.

The peak results in fig. 4 are as follows:

example 4

Instruments and conditions: shimadzu LC-20AT UV liquid chromatography system, column: agilent Eclipse plus C18 (4.6X 150mm, 3.5 μm); the detection wavelength is 220 nm; taking 10mmol/L disodium hydrogen phosphate dihydrate as a mobile phase A and acetonitrile as a mobile phase B, and keeping the column temperature at 30 ℃; the flow rate was 1.0ml/min, and a trap Column was installed between the gradient mixer and the sample injector, and gradient elution was carried out using a Ghost-Buster Column, 4.6 mm. times.50 mm, according to the following Table 5:

TABLE 5

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	80	20
20	60	40
			30	35	65
35	35	65
			35.01	80	80
45	80	20

Taking a proper amount of raw materials, adding water to dissolve and dilute the raw materials to prepare a solution containing 0.1mg/ml of dexmedetomidine as a raw material test solution; taking a proper amount of dexmedetomidine hydrochloride reference substance, adding water to dissolve and dilute the dexmedetomidine hydrochloride reference substance to prepare a solution with the concentration of about 0.007 mu g/ml of dexmedetomidine per 1ml, taking the solution and a blank solvent as sensitivity solutions, precisely measuring 50 mu l of each solution and blank solvent, and injecting the solutions into a liquid chromatograph. The results are shown in FIGS. 6-8.

Compared with the graph of FIG. 4 (without the trapping small column), the graph of FIG. 6 has a stable baseline, no gradient peak between 25 and 35min, and is more favorable for detecting impurities. FIG. 8 is a chromatogram of a sensitive solution, which has an S/N of about 10, i.e., the method has a limit of quantitation of 0.007% of the detection concentration of the test sample, which is much lower than the limit of unknown impurities by 0.10%, and when the impurity is higher than 0.007%, the test sample can be quantitatively detected; the sensitivity of the detection method meets the detection requirement.

The peak results of fig. 8 are as follows:

example 5

Instruments and conditions: shimadzu LC-20AT UV liquid chromatography system, column: agilent Eclipse plus C18 (4.6X 150mm, 3.5 μm); the detection wavelength is 220 nm; taking 10mmol/L disodium hydrogen phosphate dihydrate as a mobile phase A and acetonitrile as a mobile phase B, and keeping the column temperature at 30 ℃; the flow rate was 1.0ml/min, and a trap Column was installed between the gradient mixer and the sample injector, and gradient elution was carried out using a Ghost-Buster Column, 4.6 mm. times.50 mm, according to the following Table 6:

TABLE 6

Test samples: taking a proper amount of raw materials, adding 2ml of 30% hydrogen peroxide solution, standing at room temperature for 8h, adding water for dissolving and diluting to prepare a solution containing 0.1mg/ml dexmedetomidine. The solution was measured precisely at 50ul and injected into a liquid chromatograph, and the results are shown in FIG. 9.

As can be seen from FIG. 9, the product is relatively unstable under oxidation conditions and has a small amount of degraded impurities, but the separation degree between the main peak and adjacent impurities and between the impurity peaks is good, the purity of the main peak meets the requirement, and the detection method of the invention can effectively detect the degradation product of dexmedetomidine hydrochloride. The method is proved to have good specificity.

Example 6

Instruments and conditions: shimadzu LC-20AT UV liquid chromatography system, column: agilent Eclipse plus C18 (4.6X 150mm, 3.5 μm); the detection wavelength is 220 nm; taking 10mmol/L disodium hydrogen phosphate dihydrate as a mobile phase A and acetonitrile as a mobile phase B, and keeping the column temperature at 30 ℃; the flow rate was 1.0ml/min, and a trap Column was installed between the gradient mixer and the sample injector, and gradient elution was carried out using a Ghost-Buster Column, 4.6 mm. times.50 mm, according to the following Table 7:

TABLE 7

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	80	20
20	60	40
			30	35	65
35	35	65
			35.01	80	80
45	80	20

Precisely measuring the above solutions 50ul each with injection and blank adjuvants, and injecting into liquid chromatograph. The results are shown in FIGS. 10 and 11.

As can be seen from FIGS. 10 and 11, the blank excipients did not peak and did not interfere with the detection of the injections. The detection method can be used for detecting related substances of dexmedetomidine hydrochloride injection, and the detection maps of the related substances of the raw materials and the injection are shown in the figure 6 and the figure 10 respectively, so that the detection methods are consistent, the operation is simple and convenient, and the comparison of the impurity mass spectrum is facilitated.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.