CN115598238A

CN115598238A - Method for determining content of dextroisomer in levonorgestrel by high performance liquid chromatography

Info

Publication number: CN115598238A
Application number: CN202211154388.1A
Authority: CN
Inventors: 牛犇; 贾志祥; 赵杰; 孙琳琳
Original assignee: Jiangsu Lianhuan Pharmaceutical Co ltd
Current assignee: Jiangsu Lianhuan Pharmaceutical Co ltd
Priority date: 2022-09-19
Filing date: 2022-09-19
Publication date: 2023-01-13

Abstract

The invention discloses a method for determining content of dextroisomer in levonorgestrel by a high performance liquid chromatography, belonging to the technical field of medicines. The invention adopts DAICEL IG chromatographic column, and uses n-heptane-ethanol-trifluoroacetic acid as mobile phase to carry out high performance liquid chromatography to detect the dextroisomer in levonorgestrel, and has the advantages that: the main component and the unknown impurities behind the main component can be well separated; the solvent peak does not interfere with the determination of the principal component; the spectral purity of the destroyed sample under each condition is larger than 990 by peak purity detection, impurities generated by destruction under each condition can be well separated from the main peak, and the specificity of related substance detection is good; meanwhile, when the chromatographic condition parameters are slightly changed, the separation of related substance impurities in the levonorgestrel and the inspection result are not influenced, and the durability is good. The method can be used for quickly, effectively, accurately and reliably separating and detecting the intermediate-to-right isomer in the levonorgestrel, is favorable for improving the product quality of the levonorgestrel and improves the medication safety of patients.

Description

Method for determining content of dextroisomer in levonorgestrel by high performance liquid chromatography

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a method for determining the content of a dextroisomer in levonorgestrel by a high performance liquid chromatography.

Background

Levonorgestrel, the chemical name of which is (-) -13-ethyl-17-hydroxy-18, 19-bis-demethyl-17 alpha-pregn-4-ene-20-alkyne-3-ketone, is a progestational drug widely used at present, the molecular structure of which shows that the chiral carbon configuration of a steroid structure is stable, only 17 chiral carbon atoms can generate dextroisomer, and the activity of the levonorgestrel is 2 times of that of a norethisterone racemate, so that the influence of the isomer on the activity can be seen, and the Chinese pharmacopoeia 2020 edition only has the limit requirement on the rotation degree, and more influencing factors on the rotation degree, so that the detection and separation of the dextroisomer of the levonorgestrel have important significance on the production and storage of raw material drugs and preparations. The structural formula of the levonorgestrel is shown as follows:

disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide a method for determining the content of a dextroisomer in levonorgestrel by a high performance liquid chromatography.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a method for determining the content of dextroisomer in levonorgestrel by a high performance liquid chromatography method comprises the following steps:

(1) Preparing a test solution and a reference solution;

(2) Setting high performance liquid detection conditions;

(3) And respectively injecting the test solution and the control solution into a liquid chromatograph, recording the chromatogram, and calculating the content of the dextroisomer.

Further, in the method, the chromatographic column is DAICEL IG, the length is 250mm, the inner diameter is 4.6mm, and the particle size of the filler is 5 mu m.

Further, the mobile phase consists of n-heptane, ethanol and trifluoroacetic acid, and the volume ratio is 920-940: 60-80: 0.5.

Further, the column temperature of the chromatographic column is 33-37 ℃.

Furthermore, the flow rate of the mobile phase is 0.95-1.05 mL/min.

Further, the detection wavelength of the liquid chromatograph is 237nm.

Further, preparation of a test solution: placing levonorgestrel about 10mg in a 20mL volumetric flask, adding 5mL ethanol for dissolving, adding n-heptane for diluting to constant volume, and mixing uniformly; preparation of control solutions: precisely measuring 1mL of the test solution, placing the test solution into a 100mL measuring flask, diluting the test solution to a scale with a solvent, and shaking up the test solution to be used as a self-control solution.

In the method, the sample amount of the test solution and the sample amount of the control solution are both 10 mu L.

In the method, the area of the dextroisomer peak in the chromatogram of the test solution is not larger than the area of the main peak of the control solution, namely the isomer content is not larger than 1.0 percent.

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

the high performance liquid chromatography can quickly, effectively, accurately and reliably separate and detect the dextroisomer in the levonorgestrel bulk drug, the main component and the unknown impurities behind the levonorgestrel bulk drug can be well separated under the chromatographic condition of the high performance liquid chromatography, the separation degree is 4.3, the blank solvent has no micro-peak inversion interference, and the solvent peak does not interfere the determination of the main component; the spectral purity of the destroyed sample under each condition is larger than 990 by peak purity detection, impurities generated by destruction under each condition can be well separated from the main peak, and the specificity of related substance detection is good; meanwhile, when the chromatographic condition parameters are slightly changed, the separation of related substance impurities in the levonorgestrel and the inspection result are not influenced, and the durability is good. The method can be used for quickly, effectively, accurately and reliably separating and detecting the intermediate-to-right isomer in the levonorgestrel, is favorable for improving the product quality of the levonorgestrel and improves the medication safety of patients.

Drawings

FIG. 1 is a test chromatogram under chromatographic conditions according to the invention;

FIG. 2 is a levonorgestrel nondestructive profile;

FIG. 3 is a levonorgestrel-photodisruption profile;

FIG. 4 is a levonorgestrel-hyperthermia disruption profile;

FIG. 5 is a levonorgestrel-pyrolysis destruction profile;

fig. 6 is a standard curve chart, wherein fig. 6a is a standard curve of levonorgestrel dextroisomer, and fig. 6b is a standard curve chart of levonorgestrel.

Detailed Description

To make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described below with reference to specific embodiments. In the following examples, unless otherwise specified, all technical means used are conventional means well known to those skilled in the art.

Example 1

A method for determining the content of a dextroisomer in levonorgestrel by adopting a high performance liquid chromatography method specifically comprises the following steps:

1) Preparing a reference substance solution, a test substance solution and a mixed solution, wherein the test substance solution is prepared by the following steps: placing levonorgestrel about 10mg in a 20mL volumetric flask, adding 5mL ethanol for dissolving, adding n-heptane for diluting to constant volume, and mixing uniformly; preparation of control solution: precisely measuring 1mL of the test solution, placing the test solution into a 100mL measuring flask, diluting the test solution to a scale with a solvent, and shaking the test solution uniformly to serve as a self-contrast solution; the test effect of the method of the invention is tested by preparing mixed solution and injecting, and the mixed solution is prepared as follows: taking a proper amount of levonorgestrel and norgestrel racemate, and dissolving and diluting the levonorgestrel and norgestrel racemate by using a solvent to prepare a mixed solution containing 0.5mg/mL of levonorgestrel and 5 mu g/mL of levonorgestrel dextroisomer; the solvent in the reference solution and the mixed solution is composed of n-heptane-ethanol, and the ratio is 3: 1.

2) Setting high performance liquid detection conditions; detection conditions are as follows: the chromatographic column is celluloid DAICEL IG with specification of 25cm × 0.46cm and 5 μm, the mobile phase is composed of n-heptane-ethanol-trifluoroacetic acid, and the volume ratio of n-heptane to ethanol to trifluoroacetic acid is 930:70:0.5; the column temperature is 35 ℃, and the flow rate is 1.0mL/min; the detection wavelength of the high performance liquid chromatograph detector is 237nm.

3) Taking 10 mu L of the self-contrast solution, injecting into a liquid chromatograph, adjusting the detection sensitivity to make the peak height of the main component peak about 10-20% of the full range, precisely measuring 10 mu L of the mixed solution, injecting into the liquid chromatograph, and recording the chromatogram (figure 1). As can be seen from FIG. 1, the main component and the unknown impurities behind the main component can be well separated under the chromatographic conditions, the separation degree is 4.3, and the method is applicable because the blank solvent has no micro-peak-inversion interference.

Example 2

The detection method of example 1 was verified in several ways, including system applicability, destructive testing, limit of quantitation, limit of detection, linear relationship, precision, accuracy, and solution stability, and is described in detail below.

1) System suitability determination:

taking a proper amount of norgestrel and levonorgestrel reference substances, precisely weighing, adding ethanol, respectively dissolving and diluting to prepare storage solutions containing about 1mg/mL of norgestrel (containing about 0.5mg/mL of levonorgestrel and dextroisomer) and 0.5mg/mL of levonorgestrel. Appropriate amounts of the norgestrel and levonorgestrel stock solutions are transferred and added with a solvent to respectively prepare positioning solutions containing 20 mu g/mL of norgestrel (containing about 10 mu g/mL of each of levonorgestrel and dextroisomer) and 10 mu g of levonorgestrel.

Precisely weighing a proper amount of levonorgestrel in a 20mL measuring flask, adding a proper amount of norgestrel stock solution in the same measuring flask, dissolving with 5mL of ethanol, and diluting with n-heptane to obtain a mixed solution containing dextroisomer about 5 mu g/mL and levonorgestrel about 0.5mg/mL, which is used as a system applicability solution.

Respectively injecting 10 mu L of each of the blank solvent, the system applicability solution and each of the impurity positioning solutions into a high performance liquid chromatograph, recording a chromatogram, and inspecting the separation condition of each component, wherein the result is shown in table 1, and the result can be known from table 1 that the peak emergence sequence of the mixed solution is that dextroisomer, levonorgestrel, dextroisomer and other impurities can be effectively separated under the detection wavelength of 237nm, and the solvent peak does not interfere with the measurement of the levonorgestrel and the dextroisomer.

TABLE 1 impurity location and degree of separation

Time to peak	Levonorgestrel dextroisomer	Levonorgestrel
			Positioning solution (minutes)	11.712	15.881
SST (minutes)	11.713	15.538
			Degree of separation	7.6	4.3

2) Destructive test

Preparing a test solution: because the isomer is detected by using a chiral column (normal phase), acid, alkali and oxidation destruction tests are not carried out.

Solution before destruction: and (3) taking about 10mg of levonorgestrel, placing the levonorgestrel into a 20mL volumetric flask, adding 5mL of ethanol for dissolving, adding n-heptane for diluting to a constant volume, and uniformly mixing.

Light irradiation to destroy the solution: and (3) taking about 10mg of levonorgestrel, placing the levonorgestrel into a 20mL volumetric flask, adding 5mL of ethanol for dissolving, adding n-heptane for diluting to a constant volume, and uniformly mixing. The solution is used as a light damage solution after being placed under the irradiation of ultraviolet rays with the wavelength of 254nm and 365nm for damage for 20 minutes.

High temperature destruction of the solution: placing about 10mg of levonorgestrel in a 20mL volumetric flask, destroying at high temperature for 4h, taking out, cooling at room temperature, adding 5mL of ethanol for dissolving, adding n-heptane for constant volume, and mixing to obtain high-temperature destruction solution.

Pyrolysis destruction solution: placing levonorgestrel about 10mg in a 20mL volumetric flask, adding 5mL of ethanol for dissolving, adding a small amount of n-heptane, destroying at high temperature for 4h, taking out, cooling at room temperature, adding 5mL of ethanol for dissolving, adding n-heptane for constant volume, and mixing to obtain pyrolysis destruction solution.

Injecting 10 μ L of each sample solution into chromatograph, recording chromatogram (figure 2-5), and analyzing impurity change. And (3) measuring the sample solution under various damage conditions by adopting a diode array to measure the peak purity or the spectral similarity. The results are shown in table 2, and it can be seen from table 2 that, under each destruction condition, the degradation product generated by the sample can be effectively separated from the main peak, the degradation product does not interfere the determination of the dextroisomer, the reduction amount of the main peak destruction and the reduction of the content thereof should be basically consistent, the purity of the main peak is not less than 990, and the material is conserved.

Table 2 material balance test results

3) Limit of quantitation and limit of detection tests

And taking norgestrel reference stock solution, adding a solvent to dilute the norgestrel reference stock solution into 1mL of solution containing 1 mu g of norgestrel (containing levonorgestrel and about 0.5 mu g of dextroisomer respectively), and measuring the quantitative limit (S/N is more than or equal to 10) and the detection limit (S/N is more than or equal to 3) by using a dilution method. The results are shown in table 3, and it can be seen from table 3 that the quantitative limit and the detection limit of levonorgestrel and its dextroisomer meet the requirement of isomer inspection under the condition of the chromatogram.

TABLE 3 quantitation and detection limits

4) Linear relation

The norgestrel control stock solution with the concentration of 1mg/mL is diluted by adding a solvent to prepare a solution containing 20 mug of norgestrel (containing about 10 mug of each of levonorgestrel and dextroisomer) per 1mL, and the solution is used as a linear stock solution. Linear solutions were prepared as in table 4.

TABLE 4 Linear solution formulation

Precisely measuring 10 μ L of the solution, injecting into high performance liquid chromatograph, recording chromatogram (FIG. 6), measuring peak area, and performing linear regression with peak area A as ordinate and concentration C as abscissa. The results are given in Table 5 below.

TABLE 5 Linear relationship test results

The results of known impurity correction factor calculations are shown in table 6:

K _{levonorgestrel} : slope of standard curve of levonorgestrel

K _{Levonorgestrel dextroisomer} : levonorgestrel dextroisomer slope

Table 6 known impurity correction factors

Composition (I)	Slope of	Correction factor
			Levonorgestrel	29.3311	-
Levonorgestrel dextroisomer	29.3369	1.00

As can be seen from tables 5 and 6, each known impurity showed good linearity (r > 0.999) with its peak area in the respective concentration range, and the levonorgestrel dextroisomer correction factor was 1.00, which was calculated by the self-control method.

5) Precision of sample introduction

And taking the norgestrel control stock solution with the concentration of 1mg/mL, adding a solvent to dilute the solution to prepare 1mL of a solution containing 10 mu g of norgestrel (containing about 5 mu g of each of levonorgestrel and dextroisomer), taking 10 mu L of the solution, injecting the solution into a liquid chromatograph, continuously injecting samples for 6 times, and recording the peak area. The results are shown in Table 7, and it can be seen from Table 7 that the peak areas RSD% of the levonorgestrel and dextroisomer are less than or equal to 2.0% and the injection precision is good when 6 needles are continuously injected.

TABLE 7 sample introduction precision results

Precision of sample introduction	Levonorgestrel dextroisomer	A Levonorgestrel
			1	157.104	157.032
2	157.032	156.538
			3	156.881	156.825
4	156.892	156.631
			5	157.240	157.024
6	157.136	156.788
			RSD(％)	0.09％	0.13％

6) Solution stability test

The solution stability test was performed on the control solution and the test solution, respectively.

(1) Stability of the control solution:

taking a proper amount of norgestrel reference stock solution with the concentration of 1mg/mL, adding a solvent to dilute the stock solution to prepare 1mL of solution containing 10 mu g of norgestrel (containing about 5 mu g of each of levonorgestrel and dextroisomer), injecting samples at different times respectively, and inspecting the stability in the day. The results are shown in Table 8, and it can be seen from Table 8 that the levonorgestrel and dextroisomer control had good solution stability (RSD. Ltoreq.2.0%) over 24 hours.

Table 8 impurity control solution stability results

(2) And (3) stability of the test solution:

time (hours)	Peak area _Dextroisomer	Peak area _{Levonorgestrel}
			0	157.104	157.032
4	156.395	156.164
			7	156.100	155.746
9	156.053	155.623
			11	156.239	156.415
14	156.035	155.724
			16	156.056	155.625
21	158.047	157.477
			24	160.095	159.886
RSD％	0.87％	0.88％

Taking the product to prepare a test solution containing 0.5mg/mL of levonorgestrel according to a proposed method, injecting samples at different times respectively, calculating the content of impurities according to an area normalization method, and inspecting the daily stability of the impurities. The results are shown in Table 9, and it can be seen from Table 9 that the isomer content of the test article is 0.016% -0.017% within 24 hours, no obvious change is caused, and the stability is good.

TABLE 9 test article solution stability results

Time (hours)	Content of dextrorotatory isomer/%)
		0	0.017
4	0.016
		7	0.016
9	0.016
		11	0.017
14	0.016
		16	0.017
21	0.017
		24	0.017

7) Repeatability test

Placing a levonorgestrel test sample in a 20mL volumetric flask, adding 5mL ethanol for dissolving, adding n-heptane for diluting to a scale, and preparing a solution containing 0.5mg per mL as a test sample solution; 1mL to 100mL of the test solution is removed and diluted to the scale with the solvent to serve as a self-control solution.

The measurement was repeated 6 parts. The results are shown in Table 10, and it can be seen from Table 10 that the absolute value of the difference between the content of the dextroisomer and the mean value of the test results of 6 repetitive samples is less than or equal to 10.0% of the limit, and the repeatability is good.

TABLE 10 repeatability results

8) Accuracy test (sample recovery rate)

The norgestrel control stock solution with the concentration of 1mg/mL is diluted by adding a solvent to prepare a solution containing 200 mug of norgestrel (containing about 100 mug of each of levonorgestrel and dextrorotatory isomer) in every 1mL, and the solution is used as a recovery stock solution.

The stock solutions of the above recovery rates were precisely aspirated and diluted to prepare 40%, 100% and 200% solutions in an amount of 3 parts per concentration as shown in Table 11. And (3) sucking 10 mu L of each sample, injecting a sample, recording the area of the peak of the dextroisomer, and calculating the recovery rate and RSD of the dextroisomer. The results are shown in Table 11, and it is understood from Table 12 that the recovery rates of the dextroisomer and the average recovery rate are in the range of 101.0% to 104.3%, the RSD% of each recovery rate is 1.1%, and the recovery rate is good.

TABLE 11 recovery ratio solution preparation

TABLE 12 recovery results

9) Intermediate precision

And the same operator performs isomer content inspection on the same batch of samples at different times, by different operators and by different instruments according to a repeatability test method. The results are shown in Table 13, and it is clear from Table 13 that the isomer testing method of this product is excellent in intermediate precision.

TABLE 13 results of intermediate precision test

Name of experiment	Experimenters	Instrument numbering	Isomer content (%)
				Repeatability of	Experimenter A	CA15A23008	0.018％
At different times	Experimenter A	CA15A23008	0.017％
				Different instruments	Experimenter A	CA15A23008	0.017％
Different persons	Experimenter B	CA15A23003	0.018％

10 Durability test

The durability of the levonorgestrel isomer method is mainly verified from three aspects of different proportions, different flow rates and different column temperatures of a mobile phase.

The experiment is carried out according to the conditions in the table 14, the results are shown in the table 15, and the table 15 shows that the levonorgestrel dextroisomer and the levonorgestrel can be effectively separated when the mobile phase proportion, the flow rate and the column temperature slightly change and the chromatographic columns of the same type are replaced, the detection result is not obviously influenced, the requirements are met, and the chromatographic conditions have good durability.

TABLE 14 durability conditions

Parameter(s)	Specified parameters	Extent of variation
			Flow phase ratio	N-heptane-ethanol-trifluoroacetic acid (930: 70: 0.5)	±1％
Column temperature
		35℃	Column temperature + -2 deg.C
Flow rate of flow				1.0mL/min	Flow rate. + -. 0.05mL/min

TABLE 15 durability results

Claims

1. A method for determining the content of dextroisomer in levonorgestrel by a high performance liquid chromatography method is characterized by comprising the following steps:

(1) Preparing a test solution and a reference solution;

(2) Setting high performance liquid detection conditions;

(3) And respectively taking the test solution and the control solution, injecting the test solution and the control solution into a liquid chromatograph, recording a chromatogram, and calculating the content of the dextroisomer.

2. The method for determining the content of dextroisomer in levonorgestrel according to claim 2, wherein the chromatographic column is DAICEL IG with length of 250mm, inner diameter of 4.6mm and filler diameter of 5 μm.

3. The method for determining the content of dextroisomer in levonorgestrel according to claim 2, wherein the mobile phase comprises n-heptane, ethanol and trifluoroacetic acid at a volume ratio of 920-940: 60-80: 0.5.

4. The method for determining the content of dextroisomer in levonorgestrel according to claim 2, wherein the column temperature of the chromatographic column is 33-37 ℃.

5. The method for determining the content of dextroisomer in levonorgestrel according to claim 2, wherein the flow rate of the mobile phase is 0.95-1.05 mL/min.

6. The method for determining the content of dextroisomer in levonorgestrel according to claim 2, wherein the detection wavelength of the liquid chromatograph is 237nm.

7. The method for determining the content of dextroisomer in levonorgestrel according to claim 1, wherein the preparation of the test solution comprises the following steps: taking about 10mg of levonorgestrel, placing the levonorgestrel into a 20mL volumetric flask, adding 5mL of ethanol for dissolving, adding n-heptane for diluting to a constant volume, and uniformly mixing; preparation of control solutions: precisely measuring 1mL of the test solution, placing the test solution into a 100mL measuring flask, diluting the test solution to a scale with a solvent, and shaking up the test solution to be used as a self-control solution.

8. The method for determining the content of dextroisomer in levonorgestrel according to claim 1, wherein the sample volume of the test solution and the sample volume of the control solution are 10 μ L.

9. The method for determining the content of dextroisomer in levonorgestrel according to claim 1, wherein the area of the peak of dextroisomer in the chromatogram of the test solution is not larger than the area of the main peak of the control solution.