CN111413417A

CN111413417A - Detection method for sodium levofolinate raw material and related substances in preparation thereof

Info

Publication number: CN111413417A
Application number: CN201910014478.2A
Authority: CN
Inventors: 雷勇胜; 康江鹏; 魏巍; 潘毅
Original assignee: Tianjin Institute of Pharmaceutical Research Co Ltd
Current assignee: Tianjin Institute of Pharmaceutical Research Co Ltd
Priority date: 2019-01-07
Filing date: 2019-01-07
Publication date: 2020-07-14

Abstract

The invention provides a detection method of sodium levofolinate raw material and related substances in a preparation thereof, which is carried out by adopting a high performance liquid chromatography, wherein the chromatographic conditions are as follows: the chromatographic column is an octadecylsilane chemically bonded silica chromatographic column; mobile phase a is a salt solution; the pH value of the salt solution is 6.0-9.0; the mobile phase B is one or a mixture of acetonitrile, methanol and ethanol; the salt solution is a salt solution containing an ion-pair reagent; adopting a gradient elution method, wherein the flow rate of a mobile phase is 0.2-1.2ml/min, the column temperature of a chromatographic column is 35-45 ℃, and the detection wavelength is 200-300 nm; the detection method is simple and convenient to operate, high in separation degree, strong in specificity of the detection method, and accurate and reliable in detection result.

Description

Detection method for sodium levofolinate raw material and related substances in preparation thereof

Technical Field

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a detection method and application of sodium levofolinate as a raw material and related substances in a preparation thereof.

Background

Sodium levofolinate (English name: Disodium L evofolinate) has the chemical name (2S) -2- [ [4- [ [ [ (6S) -2-amino-5-formyl-1, 4,5,6,7, 8-hexahydro-4-oxo-6-dishedPyridyl radical]Methyl radical]Amino group]Benzoyl radical]Amino group]-glutaric acid disodium salt, molecular formula C₂₀H₂₁Na₂N₇O₇Molecular weight 517.40, CAS accession number:

284461-73-0. The structure is shown in formula I.

Sodium levofolinate is approved to be marketed in the United kingdom in 3.19.2008, is marketed in more than 20 countries such as Germany, France, Belgium and the like at present, and the levofolinate is used as an auxiliary therapeutic drug for tumor chemotherapy and is combined with 5-FU for treating cytotoxicity to reduce the toxic and side effects of the chemotherapeutic drug and improve the life quality of tumor patients, so that the sodium levofolinate has remarkable clinical significance and is in the first-line medication status at present. Levofolinic acid is also the classical drug for rescue therapy of high doses of methotrexate. The safety and effectiveness of levofolinic acid therapy has been well-verified clinically. Levofolinic acid is a biologically active levorotatory form of the racemic leucovorin, which is involved in various metabolic processes including purine synthesis, pyrimidine nucleotide synthesis and amino acid metabolism. Levofolinic acid is frequently used to reduce toxicity and neutralize the effects of folic acid antagonists, such as methotrexate. The levofolinic acid and the folic acid antagonist share a membrane transport carrier, competitively enter cells, and stimulate the folic acid antagonist to flow out of the cells. The cells are protected from the folate antagonist by filling the reduced folate pool. Levofolinic acid does not need to be reduced by dihydrofolate reductase. It therefore acts as a source of pre-reduction of tetrahydrofolate. It is therefore able to bypass the folate antagonism of dihydrofolate reductase and provide a source of various co-enzymes of folate. The biochemical mechanism of the combination of sodium levofolinate and FU is: fluorouracil is capable of inhibiting DNA synthesis by binding to thymidylate synthase. The combination of sodium levofolinate and fluorouracil results in the formation of a stable triple composition comprising thymidylate synthase, deoxyuridine 5-fluoromonophosphate and 5, 10-methylenetetrahydrofolate. This increases the inhibitory effect of DNA biosynthesis, resulting in the extended block of thymidylate synthase.

At present, in the synthesis process of sodium levofolinate raw materials, the number of the starting materials, the process and the degraded known impurities which may be contained in the final raw material product is 7, and the starting materials, the process and the degraded known impurities are respectively as follows: p-aminobenzoyl glutamic acid (impurity A), 5, 10-diformyltetrahydrofolic acid (impurity B), folic acid (impurity C), 10-formylfolic acid (impurity D), 5-formyltetrahydropteroic acid (impurity E), 10-formyldihydrofolic acid (impurity F) and dihydrofolic acid (impurity G), and the chemical structure is as follows:

the invention relates to a detection method of sodium levofolinate raw materials and preparation related substances thereof, which mainly aims at effectively detecting and separating the seven known impurities of sodium levofolinate. Wherein folic acid (impurity C) is used as a starting material; 5, 10-diformyltetrahydrofolic acid (impurity B), 10-formylfolic acid (impurity D) and 5-formyltetrahydropteroic acid (impurity E) are process impurities; 4-aminobenzoylglutamic acid (impurity A), 10-formyldihydrofolic acid (impurity F) and dihydrofolic acid (impurity G) are degradation products. The detection method provided by the invention can be used for effectively detecting one or more of the seven impurities.

Disclosure of Invention

The invention aims to provide a method for separating and measuring A, B, C, D, E, F, G7 impurities in a sodium levofolinate raw material and a preparation thereof by using a high performance liquid chromatography, which can be used for the preparation process of sodium levofolinate and the quality control of a final product. The method is carried out by adopting a high performance liquid chromatography, and comprises the following steps: (1) preparing a sodium levofolinate sample solution, wherein the sodium levofolinate sample solution comprises a test sample solution (2), injecting the sample solution obtained in the step (1) into a high performance liquid chromatograph, performing gradient elution by using a mobile phase A and a mobile phase B as mobile phases, and recording a chromatogram.

The preparation method of the test solution comprises the following steps: precisely weighing sodium levofolinate raw materials or preparation samples, adding a diluent to dissolve and dilute the sodium levofolinate raw materials or preparation samples until the concentration of the sodium levofolinate is 0.2mg/ml-1mg/ml, and obtaining a test solution.

The chromatographic conditions were as follows: the chromatographic column is an octadecylsilane chemically bonded silica chromatographic column, the mobile phase A is a salt solution, the mobile phase B is one or two of acetonitrile, methanol and ethanol, and the solvent is a salt solution or a mixed solution of the salt solution and one or two of methanol, acetonitrile, ethanol and water.

Preferably, the number of theoretical plates of the column is not less than 5000 in terms of sodium levofolinate.

Preferably, the pH value range of the salt solution is 6.0-9.0, and the preferred pH value range is 7.0-8.0.

Preferably, the salt solution is selected from a solution of disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium dihydrogen phosphate, diamine hydrogen phosphate, ammonium formate or ammonium acetate; the salt solution contains an ion pair reagent, wherein the ion pair reagent is tetrabutylammonium hydroxide, hexadecyltrimethylammonium bromide, tetrabutylammonium bromide, sodium heptanesulfonate, sodium octane sulfonate, sodium hexanesulfonate or sodium dodecyl sulfonate.

Preferably, the salt-containing solution is disodium hydrogen phosphate, the concentration is 0.005-0.02 mol/L, and the optimal concentration is about 0.01 mol/L.

Preferably, the ion-pair agent is tetrabutylammonium hydroxide, and the concentration is 0.5-3.0 ml/L, and the preferable concentration is about 1 ml/L.

Preferably, the mobile phase A salt solution is prepared by taking 25 percent tetrabutylammonium hydroxide 4m L and 3.6g disodium hydrogen phosphate dodecahydrate, adding 1000m L into the mixture to dissolve the tetrabutylammonium hydroxide and the disodium hydrogen phosphate, stirring the mixture for 10min, and adjusting the pH value of the mixture to 7.5 by using 40 percent phosphoric acid, wherein the mobile phase B is methanol, and the gradient is set as follows by taking the volume of the mobile phase as 100 percent:

the detection method provided by the invention is characterized in that the chromatographic column is selected from ZORBAX Eclipse Plus C8, ZORBAX Eclipse XDB-phenyl column, ZORBAX Eclipse XDB-CN, ZORBAX Eclipse Plus C18; ZORBAX Eclipse Plus C18 is preferred.

According to the detection method provided by the invention, the flow rate of the mobile phase is 0.2-1.2 ml/min; preferably 0.5-1 ml/min.

According to the detection method provided by the invention, the temperature of the chromatographic column is 35-45 ℃; preferably 38 to 42 ℃.

The detection method provided by the invention is characterized in that the detector is an ultraviolet detector, a differential detector, an evaporative light scattering detector and a diode array detector; preferably an ultraviolet detector and a diode array detector.

According to the detection method provided by the invention, the detection wavelength is 200-300 nm; preferably 220-290 nm; more preferably 280 nm.

The gradient elution setup is preferably as follows:

time (minutes)	Mobile phase A (%)	Mobile phase B (%)
			0	83	17
25	83	17
			35	75	25
45	70	30
			51	70	30
52	83	17
			75	83	17

The invention also provides application of the detection method in detection of the sodium levofolinate preparation.

The method for detecting the impurities in the sodium levofolinate provided by the invention adopts a high performance liquid chromatography to realize the rapid and accurate determination of 7 impurities, has higher sensitivity and specificity, is simple and convenient to operate, has the separation degree meeting the standard (namely, the separation degree of a main peak and the separation degree of the impurities are both more than 1.50) (the ratio of the retention time difference of two adjacent peaks to the average peak width is also called resolution, and represents the separation degree of the two adjacent peaks, the larger R is, the better R is, the two peaks are partially overlapped, when R is 1.0, the separation degree can reach 98 percent, when R is 1.5, the separation degree can reach 99.7 percent, when R is 1.5, the R is generally used as the mark that the two adjacent components are completely separated, when R is 1, the separation is called 4 sigma separation, the two peaks are basically separated, the area of the exposed peak is 95.4 percent, the area of the inner side group is overlapped by about 2 percent, when R is 1.5, the area of the exposed peak is called 6 separation, the bare peak area was 99.7%. R.gtoreq.1.5 is referred to as complete separation. The Chinese pharmacopoeia stipulates that R is more than 1.5. The separation degree calculation formula: and R is 2(tR2-tR1)/(W1+ W2)), can be used for quality control of sodium levofolinate, lays a foundation for research and development and quality detection of the compound, and has practical significance.

Drawings

FIG. 1 is a liquid chromatogram of a test solution in example 3;

FIG. 2 is a liquid chromatogram of a test sample solution for forced acid degradation in example 3;

FIG. 3 is a liquid chromatogram of a test sample solution for alkali forced degradation in example 3;

FIG. 4 is a liquid chromatogram of a high temperature forced degradation test sample solution in example 3;

FIG. 5 is a liquid chromatogram of a test sample solution for oxidative forced degradation in example 3;

FIG. 6 is a liquid chromatogram of a solution of the test sample in example 3, which was subjected to forced degradation by illumination;

FIG. 7 is a liquid chromatogram of the system adaptation solution of example 3;

FIG. 8 is a liquid chromatogram of the system adaptation solution of example 4;

FIG. 9 is a liquid chromatogram of the system adaptation solution of example 5;

FIG. 10 is a liquid chromatogram of the test solution in example 5.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.

Example 1

Selection of solvent and concentration of test solution:

the solvent for dissolving the sample is selected from water, acetonitrile, ethanol and a mobile phase, wherein the solubility of water to the sample is the best.

We used the mobile phase as the solvent of the sample, respectively prepared 0.5mg/m L, 0.6mg/m L and 0.7mg/m L, 0.7mg/m L is more suitable for the detection of impurities in the sample, and we finally selected 0.7mg/m L as the concentration of the test solution.

Example 2

Selection of salt solution in mobile phase:

liquid chromatography is commonly used with a wide variety of salt solutions, such as: disodium hydrogenphosphate, sodium dihydrogenphosphate, potassium dihydrogenphosphate, dipotassium hydrogenphosphate, diammonium hydrogenphosphate, ammonium formate, or ammonium acetate, and the like. The ion pair reagent is selected from tetrabutylammonium hydroxide, hexadecyltrimethylammonium bromide, tetrabutylammonium bromide, sodium heptane sulfonate, sodium octane sulfonate, sodium hexane sulfonate or sodium dodecyl sulfonate.

The concentration of the salt solution is selected, the concentration of the salt solution is considered, the concentration is determined to be 0.005-0.02 mol/L and is an appropriate concentration range, and the optimal concentration is 0.01 mol/L.

Example 3

1) Instrumentation and test conditions

Waters 1525 liquid chromatograph manufactured by Wauter corporation, autosampler, PDA detector, column: ZORBAX Eclipse Plus C18(5.0 μm, 4.6 × 250mm), detection wavelength: 280mn, column temperature: 40 ℃; linear gradient elution was performed with methanol as phase B using disodium hydrogenphosphate buffer (25% tetrabutylammonium hydroxide 4m L and 3.6g disodium hydrogenphosphate dodecahydrate, water 1000m L was added for dissolution, stirring was performed for 10min, pH was adjusted to 7.5 with 40% phosphoric acid) as phase A, methanol as phase B, according to the following table:

2) Experiment step 1. preparation of solution:

preparing impurity stock solution, namely precisely weighing a proper amount of impurity reference substances, and dissolving the impurity reference substances by using 0.025 mol/L sodium carbonate solution to obtain mixed impurity stock solution with impurities of 0.7mg/m L respectively.

The system adaptability solution is prepared by precisely weighing appropriate amount of reference substance of levofolinic acid, adding appropriate amount of mixed impurities, dissolving in water, and diluting to obtain mixed solution containing levofolinic acid 0.7mg per 1m L and impurities 7.0 μ g respectively as system adaptability solution.

The test solution is prepared by dissolving appropriate amount of sodium levofolinate as raw material in water under ultrasonic treatment, and diluting to obtain solution containing 0.7mg of levofolinate per 1m of L.

Precisely weighing about 30.60mg of the sodium levofolinate sample, putting the sodium levofolinate sample into a 10ml measuring flask, adding water to dissolve and dilute the sodium levofolinate sample to scale, transferring 2.5ml of the sodium levofolinate sample into the 10ml measuring flask, adding 0.1 mol/L hydrochloric acid 0.5ml of the sodium levofolinate sample, neutralizing the sodium levofolinate sample to be neutral after 15min, and diluting the sodium levofolinate sample to scale with water to obtain the acid forced degradation test sample solution.

The method comprises the steps of precisely weighing about 30.60mg of sodium levofolinate to be tested, adding 8ml of 0.1 mol/L sodium hydroxide solution for dissolution, transferring 2ml of the sodium levofolinate to a 10ml measuring flask, placing the measuring flask in a water bath at 90 ℃ for 1h, taking out the measuring flask, neutralizing the measuring flask to be neutral by using 0.1 mol/L hydrochloric acid, diluting the measuring flask to a scale by using water, and using the measuring flask as a test sample solution for forced degradation by using alkali.

Precisely weighing about 30.60mg of the sodium levofolinate sample, putting the sodium levofolinate sample into a 10ml measuring flask, adding water to dissolve the sodium levofolinate sample, and diluting the sodium levofolinate sample to a scale; 2.5ml of the suspension was transferred into a 10ml measuring flask and diluted to the mark with water. And (4) heating the mixture in a water bath at 90 ℃ for 16h, taking out the mixture, and cooling the mixture to room temperature. As a high temperature forced degradation test sample solution.

The sodium levofolinate sample of about 15.30mg is precisely weighed and placed in a 5ml measuring flask, dissolved by water and diluted to the scale. Transferring 2.5ml from the sample into a 10ml measuring flask, adding 0.4ml of 3% hydrogen peroxide solution, heating in a water bath at 90 deg.C, taking out after 15min, cooling, and diluting with water to the scale. As an oxidative forced degradation test sample solution.

Precisely weighing about 30.60mg of the sodium levofolinate sample into a 10ml measuring flask, transferring 2.5ml of the sodium levofolinate sample into the 10ml measuring flask, adding water to dilute the sodium levofolinate sample to a scale, standing the sodium levofolinate sample in the sunlight for 3 hours, taking the sodium levofolinate sample out, and diluting the sodium levofolinate sample to the scale with water. The test sample solution was forcibly degraded as light.

2. Specificity test and methodology verification:

2.1 Special Property test (forced degradation test)

Precisely measuring the sample solution and 20 μ l of the forced degradation test sample solution, injecting into a liquid chromatograph, injecting sample according to a set method, and recording chromatogram. And calculating the content of each impurity in the test solution before and after the damage and the content of the main component in the test solution by a normalization method.

And calculating the content of each impurity in the test solution before and after the damage and the content of the main component in the test solution by a normalization method. The results are shown in Table 1-Table 2, and the drawings are shown in FIG. 1-FIG. 6.

And (3) material balance calculation:

recovery (%) - (A)_{After destruction}/C_{After destruction})÷(A_{Before breaking}C_{Before destruction})]×100％

In the formula: c_{After destruction}Concentration of destructive sample

A_{After destruction}Sum of peak areas of total impurities and main peak in chromatogram of destroyed sample

C_{Before destruction}Concentration of the sample before disruption

A_{Before destruction}Sum of peak areas of total impurities and main peak in chromatogram of sample before destruction

TABLE 1 sodium levofolinate forced degradation test Material balance results

TABLE 2 sodium levofolinate forced degradation test results

2.2 System Adaptation solution assay results (n ═ 6)

Taking the adaptive solution of the system, and sequentially and continuously injecting samples for determination, wherein the relative retention time of impurities is listed in table 3, and the test results are listed in table 4. The chromatogram is shown in figure 7.

Table 3 list of relative retention times of impurities

Serial number	Name of impurity	Retention time (min)	Relative retention time
				1	Impurity A	15.712	0.52
2	Impurity B	23.792	0.78
				3	Impurity C	50.003	1.64
4	Impurity D	47.841	1.57
				5	Impurity E	8.634	0.28
6	Impurity F	21.361	0.70
				7	Impurity G	33.801	1.11
8	Sodium levofolinate	30.508	1.00

TABLE 4 systematic adaptation solution determination results and precision tests

2.3 detection Limit

Detection limit solution: accurately weighing appropriate amounts of impurity A, impurity C, impurity D, impurity E, impurity F, impurity B, impurity G and folinic acid respectively, adding water to gradually dilute until S/N is 3, thus obtaining a detection limit solution, and preparing 2 parts in parallel.

The determination method comprises the following steps: each 20. mu.l of the detection limit solution was measured precisely and measured under the set chromatographic conditions. The results are shown in Table 5.

TABLE 5 detection Limit measurement results

2.4 solution stability test

Respectively taking the sample solution for 0h, 2.5h, 7.5h, 12.5h, 17.5h and 25h, and carrying out sample injection and determination. The RSD% of the peak area was calculated as shown in table 6.

Table 6 results of solution stability test (n ═ 5)

Example 4

1) Instrumentation and test conditions

Waters 1525 liquid chromatograph manufactured by Wauter corporation, autosampler, PDA detector, column: ZORBAX Eclipse Plus C18(5.0 μm, 4.6 × 250mm), detection wavelength: 280mn, column temperature: 40 ℃; linear gradient elution was performed with methanol as phase B using disodium hydrogenphosphate buffer (25% tetrabutylammonium hydroxide 4m L and 9.0g disodium hydrogenphosphate dodecahydrate, water 1000m L was added for dissolution, stirring was performed for 10min, pH was adjusted to 7.5 with 40% phosphoric acid) as phase A, methanol as phase B, according to the following table:

2) Experimental procedure

Preparing a solution:

the system adaptability solution is a mixed solution which is prepared by accurately weighing a proper amount of a levofolinic acid reference substance, removing a proper amount of the levofolinic acid reference substance from mixed impurities, adding water to dissolve the levofolinic acid reference substance and diluting the levofolinic acid reference substance into the mixed impurities, wherein each 1m L contains 0.7mg of levofolinic acid and 7.0 mu G of impurities G respectively, and the mixed solution is used as the system adaptability solution.

3) Results of the experiment

The separation degree of the impurity G peak and the main peak cannot reach baseline separation, the separation degree is less than 1.2, and a system adaptability solution chromatogram is shown in figure 8.

Example 5

1) Instrumentation and test conditions

time of day(minutes)	Mobile phase A (%)	Mobile phase B (%)
			0	83	17
25	83	17
			35	75	25
45	70	30
			51	70	30
52	83	17
			75	83	17

2) Experimental procedure

Preparing a solution:

And (3) taking a proper amount of the sodium levofolinate injection, adding water, and ultrasonically diluting to obtain a solution containing 0.7mg of levofolinate in each 1m of L, wherein the solution is used as a test solution.

3) Results of the experiment

Taking the system adaptability solution and the sample solution, and sequentially and continuously feeding sample for determination, wherein the system adaptability solution chromatogram is shown in figure 9, and the sample solution chromatogram is shown in figure 10.

Each impurity peak was well separated from the main peak.

Claims

1. A detection method for sodium levofolinate and related substances in a preparation thereof is characterized in that the method is a high performance liquid chromatography method,

the chromatographic conditions were as follows:

the chromatographic column is an octadecylsilane chemically bonded silica chromatographic column;

mobile phase a is a salt solution; the pH value of the salt solution is 6.0-9.0;

the mobile phase B is one or a mixture of acetonitrile, methanol and ethanol;

the salt solution solvent is a salt solution containing an ion pair reagent; adopting a gradient elution method, wherein the flow rate of a mobile phase is 0.2-1.2ml/min, the column temperature of a chromatographic column is 35-45 ℃, and the detection wavelength is 200-300 nm;

the procedure for gradient elution was:

time (minutes) Mobile phase A (%) Mobile phase B (%) 0 78～88 22～12 20～30 78～88 22～12 30～40 70～80 30～20 40～50 65～75 35～25 46～56 65～75 35～25 47～57 78～88 22～12 70～80 78～88 22～12

Wherein, the related substances of the sodium levofolinate comprise: one or more of sodium levofolinate-related substance impurities A, B, C, D, E, F, G, wherein the sodium levofolinate-related substance impurities A are p-aminobenzoylglutamic acid; the related substance impurity B of the sodium levofolinate is 5, 10-diformyltetrahydrofolic acid; the related substance impurity C of the sodium levofolinate is folic acid; the related substance impurity D of the sodium levofolinate is 10-formylfolic acid; the related substance impurity E of the sodium levofolinate is 5-formyl tetrahydropteroic acid; the related substance impurity F of the sodium levofolinate is 10-formyldihydrofolic acid; the related substance impurity G of the sodium levofolinate is dihydrofolic acid;

2. the detection method according to any one of claims 1, characterized in that: the pH value of the salt solution is 7.0-8.0.

3. The detection method according to any one of claims 1 to 2, characterized in that: the salt solution is selected from the solution of disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, ammonium formate or ammonium acetate; the ion pair reagent is selected from tetrabutylammonium hydroxide, hexadecyltrimethylammonium bromide, tetrabutylammonium bromide, sodium heptane sulfonate, sodium octane sulfonate, sodium hexane sulfonate or sodium dodecyl sulfonate.

4. The detection method according to any one of claims 1 to 3, wherein the salt solution is disodium hydrogen phosphate having a concentration of 0.005 to 0.02 mol/L, preferably 0.01 mol/L, and the ion pair reagent is tetrabutylammonium hydroxide having a concentration of 0.5 to 3.0 ml/L, preferably about 1.0 ml/L.

5. The detection method according to any one of claims 1 to 4, characterized in that: the gradient elution procedure is as follows:

。

6. The detection method according to any one of claims 1 to 5, wherein the chromatography column is selected from the group consisting of ZORBAX Eclipse Plus C8, ZORBAX Eclipse XDB-phenyl column, ZORBAX Eclipse XDB-CN, ZORBAX Eclipse Plus C18; ZORBAX Eclipse Plus C18 is preferred.

7. The detection method according to any one of claims 1 to 6, wherein the flow rate of the mobile phase is 0.5 to 1 ml/min; the temperature of the chromatographic column is 38-42 ℃.

8. The detection method according to any one of claims 1 to 7, wherein the detector is selected from the group consisting of an ultraviolet detector, a differential detector, an evaporative light scattering detector, a diode array detector; preferably an ultraviolet detector or a diode array detector.

9. The detection method according to any one of claims 1 to 8, wherein the detection wavelength is 220-290 nm; preferably 280 nm.

10. Use of the assay according to any one of claims 1 to 9 for the detection of substances associated with sodium levofolinate raw materials or preparations.