CN111487340A

CN111487340A - Method for detecting organic residual solvent in obeticholic acid raw material medicine

Info

Publication number: CN111487340A
Application number: CN202010331561.5A
Authority: CN
Inventors: 付丙月; 王金虎; 张宁; 段崇刚; 孙玲; 王伶; 王帆; 任文杰
Original assignee: Shandong Academy of Pharmaceutical Sciences
Current assignee: Shandong Academy of Pharmaceutical Sciences
Priority date: 2020-04-24
Filing date: 2020-04-24
Publication date: 2020-08-04

Abstract

The invention relates to a method for detecting organic residual solvents in crude drugs of obeticholic acid, which is used for detecting eight organic solvents, namely ethanol, isopropanol, acetonitrile, dichloromethane, n-hexane, ethyl acetate, tetrahydrofuran and toluene by a gas chromatography method through a hydrogen Flame Ionization Detector (FID) in a programmed heating and headspace sampling mode according to an external standard method. The method is simple to operate, high in sensitivity and good in accuracy, and is suitable for detecting residual solvents of seven organic solvents in the obeticholic acid raw material medicine.

Description

Method for detecting organic residual solvent in obeticholic acid raw material medicine

Technical Field

The invention relates to a method for detecting residual solvents in crude drugs of obeticholic acid, which adopts gas chromatography to detect the contents of ethanol, isopropanol, acetonitrile, dichloromethane, n-hexane, ethyl acetate, tetrahydrofuran and toluene in obeticholic acid according to an external standard method and belongs to the field of drug analysis and detection.

Background

In the process of analyzing impurities of the raw material medicines, research on residual solvents is concerned, and research results published by authorities such as the world health organization and the like show that many organic solvents have certain harm to the environment and human bodies. The residue of the organic solvent not only brings about the safety problem of the residual solvent itself, but also is closely related to the quality of the raw material drug. When the residual solvent contained in the medicine is higher than a safe value, the stability of the medicine is influenced, and the human body or the surrounding environment is influenced, so that the pharmacopoeias of various countries require to control the residual quantity of the organic solvent or the volatile impurities used in the production process.

Obeticholic acid (OCA), a farnesoid X receptor agonist, has the following chemical formula:

in 5 months 2014, the U.S. FDA granted the obeticholic acid rapid approval channel approval to Intercept pharmaceutical company. Obeticholic acid indirectly inhibits cytochrome 7A1(CYP7A1) gene expression by activating farnesoid X receptors. Since CYP7A1 is the rate-limiting enzyme of cholic acid biosynthesis, obeticholic acid can inhibit cholic acid synthesis, and can be used for treating primary biliary cirrhosis and non-alcoholic fatty liver disease.

The method accurately measures the content of eight organic residual solvents by adopting a gas chromatography aiming at organic solvents of ethanol, isopropanol, acetonitrile, dichloromethane, n-hexane, ethyl acetate, tetrahydrofuran and toluene used in the synthesis process of the obeticholic acid raw material medicine.

Disclosure of Invention

The invention aims to establish a method for detecting organic residual solvents in crude drugs of obeticholic acid, which is used for quickly and accurately detecting the residual amounts of eight organic solvents, namely ethanol, isopropanol, acetonitrile, dichloromethane, n-hexane, ethyl acetate, tetrahydrofuran and toluene in obeticholic acid by using a gas chromatography. The method has the advantages that: 1. the method is universal, high in practicability and simple to operate; 2. has enough sensitivity, accuracy and good separation degree, and can effectively control impurities.

The invention provides a method for detecting a residual solvent in an obeticholic acid raw material medicine, which comprises the following steps:

(1) respectively placing a proper amount of reference substances of each component to be detected in the same 100ml volumetric flask, adding DMF solvent, diluting to scale, shaking up, and preparing mixed reference substance solutions with 500, 75, 45, 60, 500, 30 and 90 mu g/ml of ethanol, isopropanol, acetonitrile, dichloromethane, n-hexane, ethyl acetate, tetrahydrofuran and toluene respectively;

(2) precisely weighing 50mg of obeticholic acid, placing the obeticholic acid in a 10ml headspace bottle, adding 5ml of DMF, sealing, and shaking up gently to obtain a test solution;

(3) adopting gas chromatography and FID detector, heating, introducing sample via headspace, detecting blank solution, reference solution and sample solution, recording chromatogram, and calculating residual solvent content of ethanol, isopropanol, acetonitrile, dichloromethane, n-hexane, ethyl acetate, tetrahydrofuran and toluene by external standard method.

Further, in the detection method, a polysiloxane capillary chromatographic column is adopted, the initial temperature is 30-40 ℃, the injection port temperature is 180-230 ℃, the detection temperature is 180-260 ℃, the carrier gas is inert gas, the flow rate is 1.0-4.0 ml/min, and the split ratio is 5-20: 1.

furthermore, in the detection method, the chromatographic column is Agilent DB-624(30m × 0.53mm, 3.0 μm), the initial temperature is preferably 30-50 ℃, the optimal value is 35 ℃, the injection port temperature is preferably 200-210 ℃, the optimal value is 200 ℃, the detection temperature is preferably 190-210 ℃, the optimal value is 200 ℃, and the carrier gas is N₂(ii) a The flow rate is preferably 2.0 ml/min-4.0 ml/min, and the optimal value is 3.0 ml/min; the preferable flow dividing ratio is 5-10: 1, optimal value 5: 1.

compared with the prior art, the invention has the positive effects that:

1. the gas chromatography condition is suitable for detecting the residual organic solvent of the obeticholic acid raw material medicine, and the content of 8 residual solvents such as ethanol, isopropanol, acetonitrile, dichloromethane, normal hexane, ethyl acetate, tetrahydrofuran and toluene can be quickly and accurately detected.

2. The detection method provided by the invention is accurate, simple and convenient to operate, good in reproducibility and high in sensitivity, can fully meet the requirement of determination of the residue of the organic solvent in the crude drug of obeticholic acid, can better control the product quality, and ensures the safety of the drug.

Drawings

FIG. 1 blank solvent gas chromatogram

FIG. 2 gas chromatogram for ethanol system applicability

FIG. 3 gas chromatogram for isopropanol system applicability

FIG. 4 acetonitrile System applicability gas chromatogram

FIG. 5 gas chromatogram for dichloromethane System applicability

FIG. 6 gas chromatogram for n-hexane system applicability

FIG. 7 ethyl acetate system applicability gas chromatogram

FIG. 8 gas chromatogram for tetrahydrofuran System applicability

FIG. 9 gas chromatogram for toluene System applicability

FIG. 10 gas chromatogram of mixing system for ethanol, isopropanol, acetonitrile, dichloromethane, n-hexane, ethyl acetate, tetrahydrofuran, and toluene

FIG. 11 Obeticholic acid sample System applicability gas chromatogram

Detailed Description

The invention will now be further described by way of the following examples, which are not intended to limit the scope of the invention in any way.

Examples

1. Instrument and reagent

The instrument comprises the following steps: agilent 7890A gas chromatograph, detector FID; an Agilent chromatography workstation;

reagent testing: all the reagents are chromatographically pure; obeticholic acid (home made).

2. Chromatographic conditions

Chromatographic column Agilent DB-624(30m × 0.53mm, 3.0 μm);

column temperature: keeping the temperature at 35 ℃ for 6min, increasing the temperature to 200 ℃ at the speed of 30 ℃/min, and keeping the temperature for 6 min;

sample inlet temperature: 200 ℃;

detecting the temperature: 200 ℃;

flow rate: 3 ml/min;

carrier gas: n is a radical of₂；

The split ratio is as follows: 5: 1;

and (3) sample introduction mode: a headspace sampling method;

the balance time is as follows: 20 min;

equilibrium temperature: 80 ℃.

3. Solution preparation

5ml of DMF was precisely taken and placed in a 10ml headspace bottle, sealed, and used as a blank solvent.

Preparing a mixed reference substance solution: accurately weighing appropriate amount of ethanol, isopropanol, acetonitrile, dichloromethane, n-hexane, ethyl acetate, tetrahydrofuran and toluene, placing in a 100ml volumetric flask, adding DMF solvent, diluting to scale, shaking up, and making into mixed reference solution with concentration of ethanol, isopropanol, acetonitrile, dichloromethane, n-hexane, ethyl acetate, tetrahydrofuran and toluene of 500, 75, 45, 60, 500, 30 and 90 μ g/ml respectively.

The test solution is prepared by precisely weighing 50mg of obeticholic acid, placing the obeticholic acid in a 10ml headspace bottle, adding 5m L DMF for dissolution, sealing, and shaking up gently to obtain the test solution.

4. Sample assay

① System suitability test

Precisely measuring 5ml of each of peak location solutions of a blank solvent (DMF), ethanol, isopropanol, acetonitrile, dichloromethane, n-hexane, ethyl acetate, tetrahydrofuran and toluene and a mixed reference solution, and injecting a sample in a headspace.

The blank solvent does not interfere with the organic residual solvent, all components are completely separated, R is more than 1.5, and the retention time and the separation degree of all components are shown in table 1. The original map is shown in FIGS. 1-7.

TABLE 1 measurement results of retention time and resolution of each component

② precision test

The mixed control solution was injected into the headspace continuously for 6 times with RSD within 5%, see table 2.

Table 2 precision test results (n ═ 6)

③ Linear test

Precisely measuring the mixed reference stock solutions 0.1 ml, 0.5 ml, 1.0 ml, 2.0ml, 5.0 ml and 10ml, respectively placing in 50ml volumetric flasks, adding DMF to dilute to scale, shaking up, and making into a series of linear mixed reference solutions with different concentrations. And (3) introducing samples in a headspace, continuously introducing samples of each concentration solution for 2 times, performing linear regression by taking the peak area of a reference substance as a vertical coordinate and the concentrations of the reference substance solutions of ethanol, isopropanol, acetonitrile, dichloromethane, normal hexane, ethyl acetate, tetrahydrofuran and toluene as a horizontal coordinate, and obtaining the measurement result shown in table 3.

TABLE 3 results of the Linear test

④ recovery test

Precisely weighing 9 parts of obeticholic acid in total of 100mg, respectively adding 5ml of ethanol, isopropanol, acetonitrile, dichloromethane, n-hexane, ethyl acetate, tetrahydrofuran and toluene mixed reference substance stock solutions, adding DMF (dimethyl formamide) to dilute to a scale, shaking up to obtain sample solutions with recovery rates, respectively sampling 5ml of headspace, recording a chromatogram, and calculating the recovery rate according to the following formula. The recovery rates that could be saved were between 90% and 110% (100% + -10%), and the results are shown in Table 4.

TABLE 4 results of recovery test

⑤ detection line and quantitative limit

Calculating a detection line by using the signal-to-noise ratio S/N ═ 5, calculating a quantification limit by using the signal-to-noise ratio S/N ═ 10, sequentially and respectively diluting ethanol, isopropanol, acetonitrile, dichloromethane, normal hexane, ethyl acetate, tetrahydrofuran and toluene mixed reference substance stock solution, taking 5ml of headspace sample injection, recording a chromatogram, and obtaining the measurement results of the detection line and the quantification limit in a table 5.

TABLE 5 measurement results of each component detection line and quantitative limit

⑥ sample testing

Precisely measuring 5ml of each of a test solution and a reference solution under a '3 solution preparation' phase, introducing a sample in a headspace, recording a chromatogram, and calculating according to an external standard method by using a peak area, wherein the ethanol content is not more than 0.5%, the isopropanol content is not more than 0.5%, the acetonitrile content is not more than 0.041%, the dichloromethane content is not more than 0.06%, the n-hexane content is not more than 0.029%, the tetrahydrofuran content is not more than 0.5%, the ethyl acetate content is not more than 0.5%, and the toluene content is not more than 0.089%.

The organic residual solvent of the sample is detected according to the method, wherein the organic residual solvent comprises 0% of ethanol, 0% of isopropanol, 0% of acetonitrile, 0% of dichloromethane, 0% of n-hexane, 0% of tetrahydrofuran, 0% of ethyl acetate and 0% of toluene.

Claims

1. A method for detecting residual solvents in crude drugs of obeticholic acid is characterized in that a detection method combining a gas chromatography and an external standard method is adopted, and the amounts of 8 organic residual solvents, namely ethanol, isopropanol, acetonitrile, dichloromethane, n-hexane, ethyl acetate, tetrahydrofuran and toluene, are simultaneously measured.

2. The detection method according to claim 1, wherein the chromatographic column used in the detection method is a polysiloxane capillary chromatographic column.

3. The detection method according to claim 1, wherein the detection method employs a programmed temperature rise.

4. The method of claim 1, wherein the detection method is initiated at a temperature of 20 ℃ to 50 ℃.

5. The detection method according to claim 1, wherein the injection port temperature is 180 ℃ to 230 ℃.

6. The method according to claim 1, wherein the detection temperature is 180 to 260 ℃.

7. The detection method according to claim 1, wherein the carrier gas is an inert gas and has a flow rate of 1.0 to 4.0 ml/min.

8. The detection method according to claim 1, wherein the flow split ratio is 5-20: 1.

9. the detection method according to claim 1, wherein headspace sampling is employed.

10. The detection method according to claim 1, wherein the sample detection step of the detection method is as follows:

the method comprises the following steps: preparing a reference substance solution of the residual solvent to be detected;

step two: preparing a test solution;

step three: detecting blank solution, reference solution and sample solution by gas chromatography, recording chromatogram, and calculating residual content of ethanol, isopropanol, acetonitrile, dichloromethane, n-hexane, ethyl acetate, tetrahydrofuran and toluene by external standard method.