CN110836935B

CN110836935B - Method for determining 3 genotoxic impurities in suplatast tosilate raw material medicine

Info

Publication number: CN110836935B
Application number: CN201911153738.0A
Authority: CN
Inventors: 杨再香; 吴雪; 牟祥; 卢念红; 雷海燕
Original assignee: Chongqing Liujiang Pharmatech Co ltd
Current assignee: Chongqing Liujiang Pharmatech Co ltd
Priority date: 2019-11-25
Filing date: 2019-11-25
Publication date: 2022-01-28
Anticipated expiration: 2039-11-25
Also published as: CN110836935A

Abstract

The invention discloses a method for determining 3 genotoxic impurities in a raw material medicine of suplatast tosilate. The method disclosed by the invention uses a high performance liquid chromatography-mass spectrometer, and establishes a linear equation by adding a background raw material, so that the time of the method disclosed by the invention is short, 3 impurities are controlled simultaneously, the technical problems that the recovery rate is high, a high-concentration sample pollutes the mass spectrum and the repeatability is influenced are solved, and a reliable method with specificity, sensitivity and accuracy meeting the quality control requirements of the raw material medicines is established.

Description

Method for determining 3 genotoxic impurities in suplatast tosilate raw material medicine

Technical Field

The invention belongs to the field of drug analysis, and particularly relates to a method for determining 3 genotoxic impurities in a suplatast tosilate raw material drug.

Background

Sulfast is a novel antiallergic drug developed by the pharmaceutical industry corporation of Japan Roc in 1995, and exerts an antiallergic effect by inhibiting T cell-induced production of interleukin-4 (IL-4) and interleukin-5 (IL-5), thereby inhibiting infiltration of acidic granulocytes and IgE antibody production. It is mainly used for treating bronchial asthma of children and adults. According to clinical manifestations, the suplatast tosilate can completely or partially replace hormone drugs, and has the characteristics of good curative effect, and safety and convenience in long-term administration.

The initial material used by the existing synthesis process of the raw material medicine of the suplatast tosilate mostly contains genotoxicity warning structures such as aliphatic nitro, monohalogenated alkane, alkyl ester of sulfonic acid and the like. According to ICH M7 (R1), classes 1, 2 and 3 impurities possibly existing in starting materials, intermediates and bulk drugs need to be set below a specific limit, an acceptable limit (TCC) or subjected to bacterial mutation tests.

Disclosure of Invention

The preparation of the bulk drug comprises the synthesis of a key intermediate of the suplatast tosilate, and the preparation process of the intermediate is as follows:

。

an intermediate impurity A possibly remaining in the reaction in the preparation process of the bulk drug: 1-chloro-3- (4-nitrophenoxy) -2-propanol, impurity B of p-nitrophenol for nucleophilic ring opening of compound D: 1, 3-bis (4-nitrophenoxy) -2-propanol, compound E, and impurity a, impurity C, which is a nucleophilic substitution of impurity a: 1- ((1-ethoxy-3- (4-nitrophenoxy) -2-propyl) oxy) -3- (4-nitrophenoxy) -2-propanol, and 3 genotoxic impurities in the bulk drug are reported in pharmacopoeia, import registration standard or related literature.

In order to control the quality of the raw material medicine, the invention provides a method for determining 3 genotoxic impurities in the raw material medicine of the suplatast tosilate, and establishes a method for determining the specificity, sensitivity and accuracy of the 3 genotoxic impurities to meet the quality control requirements of the raw material medicine. The impurity structures to be controlled are respectively as follows:

the method for determining 3 genotoxic impurities in the raw material medicine of the suplatast tosilate comprises the following steps:

1) using a high performance liquid chromatography-mass spectrometer, and setting the following parameters:

a chromatographic column: c18;

a detector: an ultraviolet detector VWD;

the temperature of the sample injection plate is not controlled;

sample introduction volume is 20 muL;

and (3) testing time: 30-60 min;

column temperature: 30-40 ℃;

wavelength: 220-250 nm;

flow rate: 0.2-1.0 ml/min;

mobile phase: the composition comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is 0.005-0.010 mol/L ammonium acetate buffer salt solution or 0.05-0.1 volume percent formic acid aqueous solution, and the mobile phase B is acetonitrile or acetonitrile mixed solution containing 0.05-0.1 volume percent formic acid; performing isocratic or gradient separation;

cutting high-concentration raw material medicines into waste liquid by adopting a two-dimensional chromatograph, and pumping a cleaning mobile phase into the mass spectrum by using a second group of pumps;

mass spectrometer mass separator type: a quadrupole rod;

an ionization mode: electrospray ionization;

capillary voltage: 3000-3500V;

flow rate of drying gas: 12L/min;

atomizing gas pressure 45 psig;

fragmentation voltage: 50-100V;

2) solution preparation

Diluting the solvent: an aqueous acetonitrile solution, preferably 30-80 volume%, more preferably, 50 volume% aqueous acetonitrile solution;

test solution preparation: preparing a test solution by using acetonitrile and a diluting solvent, wherein the concentration of the test solution is 10 mg/ml;

impurity reference solution: preparing an impurity control solution by using acetonitrile and a diluting solvent, wherein the concentration of the impurity control solution is 0.05 mug/ml;

adding standard solution into the test sample: preparing a sample adding solution by using acetonitrile and a diluting solvent, wherein the concentration of a sample raw material medicine in the sample adding solution is 2-10 mg/ml, preferably 10mg/ml, and the concentration of impurity A, B, C is 0.05 microgram/ml;

linear solution: from the lower limit of quantification to the limit concentration level of 200%, acetonitrile and a diluting solvent are respectively prepared in a sample solution of 2-10 mg/ml; the basis for quantification is: area or area ratio to internal standard;

3) calculating the formula:

C=a×A+b

X = C×S×1000/m

in the formula: x: content (ppm) of analyte in sample

A: peak area ratio of object to be measured and internal standard substance in chromatogram of test solution

a: slope of linear regression curve

b: linear regression curve and Y-axis intercept

m: sample weighing of the test sample, mg

S: dilution factor of test sample

C: and (4) the concentration (mug/ml) of the to-be-detected object in the chromatogram of the test solution.

In embodiments of the present invention, the quantitative ion of the impurity A, B, C may be specifically set according to the mobile phase.

In an embodiment of the invention, the safety limits of the impurities are each 5 ppm; the quantitative limit is required to be at least 50% of the safety margin, i.e. ≦ 2.5 ppm.

In the above embodiment, preferably, the liquid chromatography conditions of step 1), preferably shimadzu Inert Sustain C18, 2.1mm × 150mm 3 μm, test time: 30min, and the column temperature is 40 ℃; flow rate: 0.300 ml/min; mobile phase: the mobile phase A is 0.1 volume percent formic acid aqueous solution, and the mobile phase B is acetonitrile mixed solution containing 0.1 volume percent formic acid; and (5) gradient separation.

In the above embodiment, preferably, the mass spectrometry conditions, preferably single quadrupole mass spectrometry, capillary voltage: 3000V; fragmentation voltage: 70V; flow rate of drying gas: 12L/min; atomizing gas pressure 45 psig; fragmentation voltage: 100V. In a mobile phase system containing 0.1 volume percent of formic acid, A, B, C quantitative molecular ion peaks m/z are preferably 276, 379 and 481.

In the above embodiment, preferably, the two-dimensional chromatography in step 1) comprises a chromatographic separation system with only additional valve switching, and two or more liquid phase pumps in combination.

In the above-mentioned implementationIn one embodiment, the addition form of the quantitative ion comprises H⁺、Na⁺、NH₄ ⁺、H^-、HCOO^-、CH₃COO^-。

In the above embodiment, preferably, the diluting solvent of step 2) is a 50 vol% acetonitrile aqueous solution;

wherein, the preparation of the impurity reference solution is as follows: taking the suplatast tosilate raw material medicine and about 10mg of impurity A, B, C, precisely weighing, respectively placing in 10ml measuring bottles, adding acetonitrile for dissolving, fixing the volume to a scale, and shaking up to obtain 1mg/ml stock solution of 4 substances; respectively and precisely transferring 0.05ml of impurity A, B, C stock solution into a 10ml measuring flask, adding a diluting solvent for dissolving, fixing the volume to a scale, and shaking up to obtain 5 microgram/ml of impurity mother solution; simultaneously transferring 50 mul of impurity A, B, C into the same 5ml measuring flask, adding a diluting solvent for dissolving, fixing the volume to a scale, and shaking up to prepare control solutions containing 0.05 mug/ml of impurity A, B, C;

the preparation of the sample adding standard solution comprises the following steps: taking 50mg of the raw material medicine, precisely weighing, placing in a 5ml measuring flask, simultaneously transferring 50 mul of the impurity mother liquor, adding a diluting solvent to dissolve, fixing the volume to a scale, and shaking up to prepare a solution containing 10mg/ml of the raw material medicine and 0.05 mug/ml of impurity A, B, C respectively.

In the above embodiment, preferably, the linear solution of step 2): ranging from the lower limit of quantitation to the 200% limit concentration level, each prepared in 10mg/ml sample solution.

In the above embodiment, preferably, the quantification in step 2) is based on preferably: no internal standard was added.

The beneficial results of the invention are:

the method provided by the invention can simultaneously control 3 impurities in a short time, overcomes the technical problems of high recovery rate, high-concentration sample pollution on mass spectrum and influence on repeatability, and provides a method with specificity, sensitivity and accuracy meeting the current guiding principle for quality control of the mesolast bulk drug.

Drawings

FIG. 1 shows a blank sample profile;

FIG. 2 shows a profile of a specific solution;

FIG. 3 shows a quantitative lower limit solution map;

FIG. 4 shows a blank sample pattern for an ammonium acetate mobile phase system;

FIG. 5 shows a spectrum of a specific solution in an ammonium acetate mobile phase system;

FIG. 6 shows a lower quantitative lower limit solution diagram of an ammonium acetate mobile phase system.

Detailed Description

Embodiments of the present invention are described below by way of examples of the present invention.

Example 1 specificity and sensitivity in an acidic mobile phase System

(1) Preparation of sample solution

Diluting the solvent: 50% by volume aqueous acetonitrile solution

A special solution:

taking the suplatast tosilate raw material medicine and about 10mg of impurity A, B, C, precisely weighing, respectively placing in 10ml measuring bottles, adding acetonitrile for dissolving, fixing the volume to a scale, and shaking up to obtain 1mg/ml stock solution of 4 substances; and respectively precisely transferring 0.05ml of impurity A, B, C stock solution into a 10ml measuring flask, adding a diluting solvent for dissolving, fixing the volume to a scale, and shaking up to obtain 5 microgram/ml of impurity mother solution. Taking 50mg of the raw material medicine, precisely weighing, placing the raw material medicine in a 5ml measuring flask, simultaneously transferring 50 mu l of impurity mother liquor, adding a solvent to dissolve the raw material medicine, fixing the volume to a scale, and uniformly shaking to prepare solutions containing 10mg/ml of the raw material medicine and 0.05 mu g/ml of impurity A, B, C respectively as special solutions.

Quantitative lower limit solution: meanwhile, 50 mul of impurity A, B, C mother liquor is transferred into the same 5ml measuring flask, a diluting solvent is added for dissolution, the volume is determined to be a scale, and the solution is shaken up to prepare a control solution (5 ppm) containing 0.05 mug/ml of impurity A, B, C. Precisely transferring 0.4ml of the control solution, precisely adding 1.6ml of a diluting solvent, and uniformly mixing to obtain a quantitative lower limit solution (1 ppm).

Respectively adding 20 mul of blank solution, special solution and quantitative lower limit solution, and detecting conditions of HPLC-MS are as follows:

an octadecyl silica gel chromatographic column (150 mm multiplied by 2.1mm, 3 mu m) takes an aqueous solution containing 0.1% by volume of formic acid as a mobile phase A, acetonitrile containing 0.1% by volume of formic acid as a mobile phase B, and the flow rate is 0.3000 ml/min; the column temperature was 40 ℃, the sample size was 20 μ l, and elution was performed according to the following gradient program:

single quadrupole mass spectrometry, capillary voltage: 3000V; fragmentation voltage: 70V; flow rate of drying gas: 12L/min; atomizing gas pressure 45 psig; the m/z of the quantitative molecular ion peak of the impurity A, B, C is preferably 276, 379 and 481.

The other set of pumps for cleaning mass spectrum, mobile phases A1 and B1 are the same as A, B respectively, and the flow rate is 0.3000 ml/min, and the proportion is 50 percent respectively. The valve switching times and positions were: 0min, B site; 10mim, A-position; 22min, B site. Wherein the A site represents the entering of the effluent of the chromatographic column into the mass spectrum and the B site represents the entering of the waste liquid.

The blank sample has no interference at the analyte peak position of each channel (see fig. 1, the peak time of the impurity A, B, C is about 11.5min, 16.2min and 18.4min respectively), and 276, 379 and 481 in the special sample are separated from the adjacent peaks. Wherein impurity C in channel 481 has 2 chiral centers and the peak has a split, calculated as the sum of areas (see fig. 2); the lower limit of quantitation (25% of the limit concentration level) sensitivity was satisfactory with signal-to-noise ratios of 9.3, 15.9, 55.5 for 276, 379, 481, respectively (see fig. 3).

Example 2 specificity and sensitivity in ammonium acetate mobile phase System

Respectively carrying out 20 mul of the hollow white solution, the special solution and the quantitative lower limit solution in the embodiment 2, wherein the detection conditions of HPLC-MS are as follows:

an octadecyl silica gel chromatographic column (250 mM multiplied by 4.6mM, 5 mu m) takes an aqueous solution containing 5mM ammonium acetate as a mobile phase A and acetonitrile as a mobile phase B, and the flow rate is 1.000 ml/min; the column temperature was 40 ℃, the sample size was 20 μ l, and elution was performed according to the following gradient program:

single quadrupole mass spectrometry, capillary voltage: 3000V; fragmentation voltage: 70V; flow rate of drying gas: 12L/min; atomizing gas pressure 45 psig; the m/z of the quantitative molecular ion peak of the impurity A, B, C is preferably 290, 393, 459. The other group of pumps for cleaning the mass spectrum has mobile phases A1 and B1 which are respectively the same as A, B, and the flow rate is 0.4000 ml/min, and the proportion respectively accounts for 50%. The valve switching times and positions were: 0min, B site; 20mim, position a; 50min, B site. Wherein the A site represents the entering of the effluent of the chromatographic column into the mass spectrum and the B site represents the entering of the waste liquid. The blank sample has no interference at the analyte peak position of each channel (the peak time of the impurity A, B, C is about 27.3min, 32.5min and 35.7min respectively) (see figure 4), and the separation of 290, 393 and 459 from the adjacent peaks in the special sample meets the requirement (see figure 5); the lower limit of quantitation (25% of the limit concentration level) sensitivity was satisfactory with signal-to-noise ratios of 290, 393, 459 of 20.7, 35.0, 17.5, respectively (see fig. 6).

Example 3 measurement of recovery and establishment of a Linear equation by adding a standard to a sample of 10mg/ml of crude drug

Preparing a linear solution: weighing about 100mg of the raw material medicine into a 10ml measuring flask, and adding linear mother liquor with impurity A, B, C5 of 5 mug/ml to prepare linear solution (1 ppm) with limit concentration of 25% by 20 mul respectively. Then linear solutions with the limiting concentrations of 40%, 50%, 80%, 100%, 150% and 200% are prepared in sequence according to the method. Preparing an accurate solution: to a sample solution of 10mg/ml of the drug substance, A, B, C impurities with limited concentrations of 50%, 100%, and 150% were added, and 3 parts were prepared in parallel as an accuracy solution. The linear fitting coefficients of impurity A, B, C are: 0.9966, 1.0000, 0.9999; the recovery rates of impurity a at 3 concentration levels were: 116.9%, 102.2%, 98.5% (see table 1); the recovery rates of impurity B at 3 concentration levels were: 102.6%, 103.1%, 99.8% (see table 2); the recovery rates of impurity C at 3 concentration levels were: 110.8%, 110.9% and 103.4%. All fall between 75% and 120% recommended by the pharmacopoeia 9101 guidelines.

Table 1: table for calculating recovery rate of impurity A

Table 2: table for calculating recovery rate of impurity B

Table 3: table for calculating recovery rate of impurity C

Comparative example 1 establishment of Linear equation without addition of background drug substance and determination of recovery

Preparing a linear solution: to the same 10ml measuring flask, 20 μ l each of linear mother liquors of A, B, C5 μ g/ml of impurities was added to prepare a linear solution (1 ppm) of a limit concentration of 25%. In this way, linear solutions with limiting concentrations of 40%, 50%, 80%, 100%, 150%, 200% were prepared in this order. The solution was serially diluted with a linear mother liquor of A, B, C5 mug/ml impurity to a linear solution of limiting concentrations 25%, 40%, 50%, 80%, 100%, 150%. Preparing an accurate solution: to a sample solution of 10mg/ml of the drug substance, A, B, C impurities with limited concentrations of 50%, 100%, and 150% were added, and 3 parts were prepared in parallel as an accuracy solution. The linear fitting coefficients of impurity A, B, C are: 0.9965, 0.9990, 0.9907; the recovery rates of impurity a at 3 concentration levels were: 79.1%, 106.9%; the recovery rates of impurity B at 3 concentration levels were: 96.3%, 141.0%, 213.7%; the recovery rates of impurity C at 3 concentration levels were: 75.6%, 151.7%, 197.0%. The linear fit coefficient met the requirements, but the recovery rate did not fall far between 75% and 120% recommended by pharmacopoeia 9101 guidelines.

Finally, it is noted that the above-mentioned embodiments illustrate rather than limit the invention, and that, while the invention has been described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. The method for determining 3 genotoxic impurities in the raw material medicine of the suplatast tosilate comprises the following specific structures:

a chromatographic column: c18; a detector: an ultraviolet detector VWD; the temperature of the sample injection plate is not controlled; the injection volume is 20 mul; and (3) testing time: 30-60 min; column temperature: 30-40 parts of; wavelength of ° c: 220-250 nm; flow rate: 0.2-1.0 ml/min; mobile phase: the composite material consists of a mobile phase A and a mobile phase B, wherein the mobile phase A is 0.005-0.010 mol/L ammonium acetate buffer salt solution or 0.05-0.1 volume percent formic acid aqueous solution, the mobile phase B is acetonitrile or acetonitrile mixed solution containing 0.05-0.1 volume percent formic acid, and isocratic or gradient separation is carried out; cutting high-concentration raw material medicines into waste liquid by adopting a two-dimensional chromatograph, and simultaneously pumping a cleaning mobile phase into the mass spectrum by a second group of pumps;

mass spectrometer mass separator type: a quadrupole rod; an ionization mode: electrospray ionization; capillary voltage: 3000-3500V; flow rate of drying gas: 12L/min; atomizing gas pressure 45 psig; fragmentation voltage: 50-100V;

2) solution preparation method

Diluting the solvent: acetonitrile water solution;

impurity reference solution: preparing an impurity reference solution by using acetonitrile and a diluting solvent, wherein the concentration of the impurity reference solution is 0.05 mu g/ml;

adding standard solution into the test sample: preparing a sample adding standard solution by using acetonitrile and a diluting solvent, wherein the concentration of a sample raw material medicine in the sample adding standard solution is 10mg/ml, and the concentration of impurities is 0.05 mu g/ml;

3) calculating the formula:

C＝a×A+b

X＝C×S×1000/m

in the formula: x: the content of the analyte in the sample is ppm

a: slope of linear regression curve

b: linear regression curve and Y-axis intercept

m: sample weighing of the test sample, mg

S: dilution factor of test sample

C: the concentration of the analyte in the chromatogram of the test solution is μ g/ml.

2. The method for detecting 3 genotoxic impurities in the case of mesolast as claimed in claim 1, wherein the immobilization of the chromatographic column in step 1) is an octadecyl silica gel chromatographic column, the particle size is 3-5 μm, the length is 150-250 mm, and the inner diameter is 2.1-4.6 mm.

3. The method of determining 3 genotoxic impurities in a suplatast tosilate crude drug according to claim 1, wherein the safety limits for the impurities are all 5 ppm; the quantitative limit is required to be at least 50% of the safety margin, i.e. ≦ 2.5 ppm.

4. The method of determining 3 genotoxic impurities in a mesolast drug substance as claimed in claim 1, wherein the liquid chromatography conditions of step 1), preferably shimadzurt sustatin C18, 2.1mm x 150mm 3 μm, test time: 30min, and the column temperature is 40 ℃; flow rate: 0.300 ml/min; mobile phase: the mobile phase A is 0.1 volume percent formic acid aqueous solution, and the mobile phase B is acetonitrile mixed solution containing 0.1 volume percent formic acid; and (5) gradient separation.

5. The method for determining 3 genotoxic impurities in a case drug of suplatast tosilate according to claim 1, wherein the mass spectrometric conditions, preferably single quadrupole mass spectrometry, capillary voltage: 3000V; fragmentation voltage: 70V; flow rate of drying gas: 12L/min; atomizing gas pressure 45 psig; fragmentation voltage: 100V; in a mobile phase system containing 0.1 volume percent of formic acid, A, B, C quantitative molecular ion peaks m/z are preferably 276, 379 and 481.

6. The method for detecting 3 genotoxic impurities in the case of the mesolast drug substance as claimed in claim 1, wherein the two-dimensional chromatographic technique of step 1) comprises a chromatographic separation system combining two or more liquid pumps with only additional valve switching.

7. The method for determining 3 genotoxic impurities in a suplatast tosilate crude drug according to claim 1, wherein the addition form of the quantification ions of impurities A, B and C of step 1) comprises H⁺、Na⁺、NH₄ ⁺、H^-、HCOO^-、CH₃COO^-。

8. The method for detecting 3 genotoxic impurities in the bulk drug of suplatast tosilate according to claim 1, wherein the volume of acetonitrile in the dilution solvent in step 2) is 30-80%.

9. The method for detecting 3 genotoxic impurities in the mesolast bulk drug according to claim 1, wherein the linear solution in step 2) is prepared in a solution with a constant concentration of the bulk drug, and the concentration is 2-10 mg/ml.

10. The method for determining 3 genotoxic impurities in a case of mesolast as claimed in claim 1, wherein the quantification in step 2) is based on the peak area or peak area ratio.