CN113189228B - Method for detecting related substances in terbutaline sulfate - Google Patents

Abstract

The invention provides a method for detecting related substances in terbutaline sulfate, which adopts high performance liquid chromatography for detection. The method can well realize the retention of the strong-polarity impurities and the effective elution of the weak-polarity impurities, and can also reduce the damage of the solvent to chromatographic facilities.

Description

Method for detecting related substances in terbutaline sulfate

Technical Field

The invention relates to a method for detecting related substances in terbutaline sulfate.

Background

Terbutaline sulfate, chemical name (+/-) -alpha- [ (tert-butylamino) methyl ] -3, 5-dihydroxy benzyl alcohol sulfate (2: 1), structural formula as follows, is an adrenergic agonist, can selectively stimulate beta 2-receptor, relax bronchial smooth muscle, inhibit release of endogenous spasmodic substances and edema caused by endogenous mediators, improve the clearance capability of bronchial mucosa ciliated epithelium, and can also relax uterine smooth muscle. The traditional Chinese medicine composition is mainly used for treating bronchial asthma, chronic bronchitis and other lung diseases accompanied with bronchospasm, can also be used for preventing premature delivery and fetal asphyxia, has the characteristics of high selectivity, less side effect and the like, and is widely applied at home and abroad. The strain is currently recorded in China, america, japan, europe, british pharmacopoeia and the like. At present, the preparation mainly comprises terbutaline sulfate tablets, terbutaline sulfate inhalation aerosol and terbutaline sulfate atomized liquid.

Terbutaline sulfate structural formula

Terbutaline sulfate is collected in USP, EP, BP, JP and ChP, where the control methods for the relevant substances are essentially identical: the system adopts a mixed solution of a main component and an impurity C to investigate the separation degree, and a mobile phase system adopts an ammonium formate buffer solution containing high-concentration sodium hexanesulfonate and methanol to perform isocratic elution according to the proportion of 77.

However, during the application process, the pharmacopoeia method has the defects that: the isocratic elution mode ensures that the impurities with strong polarity are not retained, and the impurities with weak polarity cannot be completely eluted. In order to take account of the polarity of each impurity, a gradient elution mode is needed, the organic phase ratio is gradually increased in the elution process, but the high-concentration ammonium formate and sodium hexanesulfonate in the mobile phase can be salted out along with the increase of the organic phase ratio, so that the chromatographic column and the instrument are damaged. In other non-pharmacopeial methods, solvents which damage the column, such as triethylamine, are also used to improve the separation.

Disclosure of Invention

The inventor researches to show that: process impurities of widely varying polarity may be produced in the terbutaline sulfate synthesis process, including but not limited to:

the isocratic elution mode ensures that the impurities with strong polarity are not retained, and the impurities with weak polarity cannot be completely eluted. In order to better realize the retention of strong polar impurities and the effective elution of weak polar impurities and reduce the damage of a solvent to chromatographic facilities, the invention provides a liquid chromatographic detection method which does not contain ion pair reagents of sodium hexanesulfonate and triethylamine.

Specifically, the invention provides a method for detecting related substances in terbutaline sulfate, wherein the related substances at least comprise TBS4-Z5:

the detection is carried out by adopting high performance liquid chromatography, and comprises the following contents:

a chromatographic column: octadecylsilane chemically bonded silica gel column

Mobile phase: the method comprises the following steps of (1) preparing a mobile phase A and a mobile phase B, wherein the mobile phase A is an ammonium formate buffer solution, and the pH value of the mobile phase A is adjusted to 2.5-3.5 by trifluoroacetic acid; the mobile phase B is methanol or/and acetonitrile; the elution procedure was followed with a gradient from 0 to 50 minutes as follows:

the size of the chromatographic column can be conventional, the length of the chromatographic column is 50-250 mm, the inner diameter of the chromatographic column is 2.1-4.6 mm, and the particle size of the filler is 3-5 mu m. For example, the length is 250mm, the inner diameter is 4.6mm, and the particle diameter is 5 μm.

Wherein the mobile phase A is an ammonium formate buffer solution, and the concentration of ammonium formate in the buffer solution is 0.04-0.06 mol/L, preferably 0.05mol/L. Taking ammonium formate solution with proper concentration, and adjusting the pH value with trifluoroacetic acid. The pH is 2.5 to 3.5, and further 2.8 to 3.2, for example 3.0, may be selected.

The mobile phase B mainly comprises methanol or/and acetonitrile. Methanol may be predominant.

The column temperature, flow rate, detection wavelength and other parameters of the invention can be selected in common ranges. The detection wavelength is selected from 210-400 nm, specifically 276nm; the flow rate of the mobile phase is selected from 0.8ml/min to 1.2ml/min, and is specifically 1.0ml/min; the column temperature is selected from 38-42 ℃, in particular 40 ℃.

The detection method of the invention also comprises other conventional steps:

(1) Preparing a blank solvent, a system applicability solution and an impurity reference substance solution;

(2) Preparing a test solution, a control solution and a sensitivity solution;

(3) Detecting a test article; and so on.

The experimental result shows that although the ion pair reagents sodium hexanesulfonate and triethylamine are not used in the detection by the method, the strong polar impurities TBS4-Z5 are effectively retained and other impurities are successfully eluted under the condition of ensuring good separation degree of main components and various impurities under the specific mobile phase condition, and the separation degree is good, so that the damage of the solvent to chromatographic facilities in the prior art is effectively avoided.

Based on the good retention and separation effects of the invention on compounds with large polarity difference, the invention also provides a method for simultaneously detecting the following two compounds, wherein the structural formulas of the two compounds are as follows:

the detection is carried out by adopting high performance liquid chromatography, and comprises the following contents:

a chromatographic column: octadecylsilane chemically bonded silica gel column

Mobile phase: the method comprises the following steps of (1) preparing a mobile phase A and a mobile phase B, wherein the mobile phase A is an ammonium formate buffer solution, and the pH value of the mobile phase A is adjusted to 2.5-3.5 by trifluoroacetic acid; the mobile phase B is methanol or/and acetonitrile; the elution procedure was followed with a gradient of 0-50 min:

according to this method, TBS4-Z5 and TBS3 can be retained and separated by the method of the present invention as long as the test sample contains both TBS4-Z5 and TBS3 compounds.

Drawings

FIG. 1 shows the substance verification-system applicability of terbutaline sulfate, as shown in FIG. 1, under the condition of ensuring good separation degree of main components and each impurity, the impurities TBS4-Z5 are effectively retained, and the separation degree is good

FIG. 2 chromatogram of comparative example 1

FIG. 3 chromatogram of comparative example 2

Detailed Description

The methanol used in the examples of the present invention was chromatographically pure, and the remaining reagents were analytically pure, unless otherwise indicated.

1. Instruments and reagents

The instrument comprises: thermo U3000 liquid chromatograph (with DAD detector); agilent 1260 II liquid chromatograph (with DAD detector); one part per million electronic balance (Mettler Toledo XPR 10); one-tenth-ten-thousandth electronic balance (XS 205DU, ME 204); PH meter (Mettler FE 28)

Reagent: ammonium formate, trifluoroacetic acid, methanol and water

2. Reference substance

Terbutaline sulfate reference substance (purity 99.8%, china institute for food and drug testing)

TBS3 and TBS4-Z5 controls were provided and standardized by Kyokuwa Teddy pharmaceutical Co., ltd, and the purities were 98.5% and 99.2%, respectively.

Example 1

Chromatographic conditions are as follows:

a chromatographic column: YMC Pack ODS-AQ,4.6 mm. Times.250mm, 5 μm

A mobile phase A:0.05mol/L ammonium formate buffer (pH adjusted to 3.0 with trifluoroacetic acid);

mobile phase B: methanol

Flow rate: 1.0ml/min; sample introduction amount: 20 mu l of the mixture; column temperature: at 40 ℃; detection wavelength: 276nm;

gradient elution procedure:

note: a Ghost peak trap column (Welch Ghost-Buster 4.6 x 50mm or Ghost peak trap column with equivalent performance) was connected in front of the active valve

1 System suitability survey

Terbutaline sulfate and appropriate amount of each impurity control were precisely weighed, dissolved and diluted with a diluent [ methanol-water (20) ], to prepare 1ml of magnesium mixed solution containing terbutaline sulfate at concentration of 1mg, TBS4-Z5, and TBS3 at concentration of 1.5 μ g, and analyzed by HPLC under the above chromatographic conditions, and the results of system applicability test are shown in table 1, as shown in fig. 1.

TABLE 1 results of the detection of the specificity of the relevant substances

Name of impurity	RT(min)	Degree of separation
			TBS4-Z5	5.720	33.9
Terbutaline (main peak)	26.757	50.5
			TBS3	48.347	N/A

As can be seen from Table 1 and FIG. 1, in the method of the present invention, TBS4-Z5, which is a highly polar impurity, is effectively retained on the chromatographic column, TBS3, which is a less polar impurity, is effectively eluted, and the applicability of the system is satisfactory.

2 specificity of degradation

Degradation solutions were prepared as in table 2.

TABLE 2 preparation of terbutaline sulfate degradation solution

Under each degradation condition, blank solvents and the like do not interfere with the detection of related substances of the product. The purity of the main peak is more than 990, the minimum separation degree between the impurities and the main components is 3.9, and the degradation recovery rate is between 99.00 and 103.08 percent, which all meet the requirements. The method of the invention can meet the monitoring of related substances under various degradation conditions.

The system applicability and the degradation specificity are integrated, so that the detection method for the terbutaline sulfate related substances is good in specificity, and a foundation is laid for accurate quantitative analysis of each impurity.

3 confirmation of sensitivity

Precisely weighing a proper amount of each impurity reference substance, adding a diluent to dissolve the impurity reference substance, and continuously diluting until the S/N is more than 10, namely the quantitative limit of each impurity.

Experiments show that each impurity can be accurately quantified above about 0.02%, the repeatability of the quantitative limit is good, and the sensitivity of the method is high.

4 durability

The method of the invention has the advantages that when the flow rate changes by 0.1ml/min, the column temperature changes by 2 ℃, the pH value of the buffer solution changes by 0.2, and under the condition of changing different instruments and chromatographic columns, the durability test is carried out by detecting related substances of the solution of the added standard test sample, so that the durability result is good. The specific results are shown in Table 3.

TABLE 3 durability test results (%)

The detection method can be used for detecting degradation impurities of the terbutaline sulfate and process impurities brought in the terbutaline sulfate synthesis process; can separate chromatographic peaks of terbutaline sulfate related substances and has good chromatographic peak separation degree.

Comparative example 1: USP43 related substance analysis method

Chromatographic conditions

A chromatographic column: zinshengtang CAPCELL PAK C18 (4.6X 150mm,5 μm);

mobile phase: mixing the ion pair solution with methanol at a ratio of 77:23, filtering, and degassing. [ ion pair solution: weighing 3.15g of ammonium formate, placing the ammonium formate in a 1000ml measuring flask, adding 900ml of water to dissolve the ammonium formate, adjusting the pH value to 3.0 by using formic acid, adding 5.49g of sodium hexanesulfonate, diluting the ammonium formate to a scale by using water, and shaking up ];

column temperature: 30 ℃; flow rate: 1.0ml/min; detection wavelength: 276nm; sample introduction volume: 20 μ l.

HPLC analysis:

the sample system applicability solution was injected under the chromatographic conditions described above.

As a result:

as shown in FIG. 2, TBS3 could not be separated by elution, with no impurities TBS4-Z5 remaining.

Comparative example 2: changing pharmacopoeia isocratic elution into gradient elution

Chromatographic conditions are as follows:

and (3) chromatographic column: thermo Scientific Hypersil GOLD C18,4.6 x 100mm,3 μm

Mobile phase A: pH3.0 buffer (4.23 g sodium hexane sulfonate and 3.15g ammonium formate, adding water 900ml to dissolve, adjusting pH to 3.0 with 10% phosphoric acid, adding water to 4000ml, to get the final product.)

And (3) mobile phase B: methanol

Detection wavelength: 276nm; column temperature: 45 ℃; flow rate: 1.0ml/min; sample introduction amount: 20 μ l

Gradient elution procedure:

as a result:

as shown in FIG. 3, TBS3 was not separated from TBS4-Z5 as impurities.

Claims (10)

1. A method for detecting related substances in terbutaline sulfate, which is characterized by comprising the following steps: the related substances at least comprise TBS4-Z5:

the detection is carried out by adopting high performance liquid chromatography, which comprises the following steps:

a chromatographic column: octadecylsilane chemically bonded silica gel column

Mobile phase: the method comprises the following steps of (1) preparing a mobile phase A and a mobile phase B, wherein the mobile phase A is an ammonium formate buffer solution, and the pH value is adjusted to 2.5-3.5; the mobile phase B is methanol or/and acetonitrile; the elution procedure was followed with a gradient from 0 to 50 minutes as follows:

。

2. the method of claim 1, wherein: the content of ammonium formate in the buffer solution is 0.02 mol/L-0.08 mol/L.

3. The method of claim 1, wherein: the content of ammonium formate in the buffer solution is 0.04 mol/L-0.06 mol/L.

4. The method of claim 1, wherein: the pH is selected from 2.8-3.2.

5. The method according to claim 1 or 4, characterized in that: trifluoroacetic acid was used to adjust the pH.

6. The method of claim 1, wherein: the column temperature is 35-45 ℃.

7. The method of claim 1, wherein: the flow rate of the mobile phase is 0.8-1.2ml/min.

8. The method of claim 1, wherein: detection wavelength: 210-400 nm.

9. The method of claim 1, wherein: the related substances include compounds of the following structures:

。

10. a method for simultaneously detecting two compounds, comprising: the two compounds have the structural formula:

the detection is carried out by adopting high performance liquid chromatography, which comprises the following steps:

a chromatographic column: octadecylsilane chemically bonded silica gel column

Mobile phase: the method comprises the following steps of (1) preparing a mobile phase A and a mobile phase B, wherein the mobile phase A is an ammonium formate buffer solution, and the pH value of the mobile phase A is adjusted to 2.5-3.5 by trifluoroacetic acid; the mobile phase B is methanol or/and acetonitrile; the elution procedure was followed with a gradient of 0-50 min:

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