CN105092754A

CN105092754A - Method for determining sulfonate genotoxic impurity by using HPLC

Info

Publication number: CN105092754A
Application number: CN201410215531.2A
Authority: CN
Inventors: 严洁; 李轩
Original assignee: Tianjin Hankang Pharmaceutical Biotechnology Co Ltd
Current assignee: Tianjin Hankang Pharmaceutical Biotechnology Co Ltd
Priority date: 2014-05-21
Filing date: 2014-05-21
Publication date: 2015-11-25
Anticipated expiration: 2034-05-21
Also published as: CN105092754B

Abstract

The invention discloses a method for determining sulfonate genotoxic impurity (or suspected genotoxicity) by using HPLC. According to the invention, octadecylsilane chemically bonded silica is taken as a chromatographic column of a stationary phase, a mixed solvent of an organic phase and a buffer is performed with gradient elution to be taken as a mobile phase for direct detection. The detection method has the advantages of high sensitivity, strong specialization, high precision, strong accuracy and convenient operation; and the method has wide applicability and can effectively control the quality of the bulk drugs.

Description

A kind of method utilizing HPLC to measure sulfonic acid esters genotoxicity impurity

Technical field

The invention belongs to Pharmaceutical Analysis field, particularly, relate to one and utilize HPLC(high performance liquid chromatography) measure the method for sulfonic acid esters genotoxicity impurity.

Background technology

In pharmaceutical synthesis, methane-sulforic acid, the sulfonic acid substances such as benzene methanesulfonic acid generate alkyl sulfonic ester as methyl mesylate (MMS) with the lower alcohol of trace in synthetic reaction, ethyl methane sulfonate (EMS), normal-butyl methanesulfonates (NBMS), and aromatic yl sulphonate is as methyl benzene sulfonate (MBS), ethyl benzenesulfonat (EBS), p-toluenesulfonic acid ester (MP-TS), alkylated reaction can be there is in these materials with DNA, thus the inducement of cause cancer may be become, the existence of these latent gene toxic impurities causes the great attention of management organization, be defined in bulk drug the strict control carrying out related substance, European Union specify the limit of genotoxicity impurity be maximum day dosing be 1.5 μ g.

Exist in prior art and use HPLC method to measure sulfonic acid esters impurity, HPLC-UV method is the detection method relatively commonly used, as PR-HPLC method measures MBS, EBS in Amlodipine Besylate Tablet, the method adopts InretsilODS chromatographic column (150mm × 4.6mm, 5 μm), mobile phase is 1% triethylamine (Ph3.0)-acetonitrile, its degree of separation and peak shape are better, but detectability is lower, and when measured object concentration is lower, uv absorption may not be detected, thus impact measures.

At present, the sensitivity of China's conventional medicine determination method is only 10 to 60ppm, the analysis of quantitative and qualitative analysis cannot be carried out at all to genotoxicity impurity, by the continuous adjustment of synthetic route, intermediate and the starting material that may produce genotoxicity impurity can only be avoided.

Applicant finds, be the chromatographic column of Stationary liquid in order to octadecylsilane chemically bonded silica, the mixed solvent of organic phase and acetate buffer is as eluent gradient wash-out, sulfonic acid esters impurity can be detected, and highly sensitive (sensitivity can reach 0.75ppm), can effectively control bulk drug quality, solve genotoxicity cannot qualitative and quantitative analysis detect a difficult problem.

Summary of the invention

The invention provides a kind of method utilizing HPLC to measure sulfonic acid esters genotoxicity impurity, thus realize the control of trace sulfonic acid esters genotoxicity impurity in bulk drug.

Technical scheme of the present invention: a kind of method utilizing HPLC to measure sulfonic acid esters genotoxicity impurity is provided,

Selection octadecylsilane chemically bonded silica is Stationary liquid, using the mixed solvent of organic phase and damping fluid as eluent gradient wash-out.

Detecting step is as follows:

(1) set chromatographic condition: chromatographic column with octadecylsilane chemically bonded silica as Stationary liquid, using the mixed solvent of organic phase and damping fluid as eluent gradient wash-out, column temperature 20 DEG C ~ 30 DEG C, flow velocity is 0.8 ~ 1.2mL/min, and determined wavelength is 260nm ± 10nm;

(2) sample solution is prepared: adopt the mixed liquor of organic solvent or organic solvent and water that detected sample is mixed with sample solution;

(3) compartment analysis: by sample solution 10 μ L, injects high performance liquid chromatograph, completes the mensuration of sulfonic acid esters genotoxicity impurity.

Described organic phase is for being selected from acetonitrile.

Described damping fluid is that acetate rushes liquid.The concentration range of damping fluid is 0.005mol/L ~ 0.05mol/L, preferred 0.01mol/L.

The pH value of described damping fluid is 3.0 ~ 5.1.

Described mobile phase is the mixed solvent gradient elution of organic phase and damping fluid, and during gradient elution 0 ~ 5min, the ratio of damping fluid, organic phase is volume ratio 90%:10%; During 5 ~ 14min, damping fluid volume ratio is reduced to 80%, and organic phase volume ratio increases to 20%; 14 ~ 15min, buffer ratio is that volume ratio is reduced to 10%, and the ratio of organic phase is volume ratio when increasing to 90%:15 ~ 25min, and the ratio of damping fluid, organic phase is that volume ratio 10%:90% is constant; During 25 ~ 30min, damping fluid volume ratio increases to 90%, and organic phase volume ratio is reduced to 10%; During 30 ~ 35min, the ratio of damping fluid, organic phase is that volume ratio 90%:10% is constant.

Described testing conditions: column temperature 20 DEG C ~ 30 DEG C, preferably 25 DEG C; Flow velocity is 0.8 ~ 1.2mL/min, preferably

1.0mL/min。Determined wavelength is 260nm ± 10nm, preferred 260nm.

Detection method of the present invention, can realize according to following methods:

(1) get detected sample appropriate, dissolve with the mixed solvent of acetonitrile or it and water, be mixed with the sample solution of suitable concn.

(2) get sulfonic acid esters impurity appropriate, dissolve with the mixed solvent of acetonitrile or it and water, be mixed with the reference substance solution of suitable concn.

(3) arranging flow rate of mobile phase is 0.8 ~ 1.2mL/min, and the flow velocity of mobile phase is preferably 1.0mL/min; Detect

Wavelength 260nm; Chromatogram column temperature is 20 DEG C ~ 30 DEG C, is preferably 25 DEG C.

(4) get respectively (1), 2) sample solution and reference substance solution 10 μ L, inject high performance liquid chromatograph, complete the assay of sulfonic acid esters toxic impurities.

Technique effect of the present invention: by utilizing HPLC, carry out trace detection analysis to genotoxicity impurity in bulk drug (or doubtful genotoxicity) sulfonic acid esters toxic impurities, sensitivity can reach 0.75ppm.

Embodiment

Be described below in detail embodiments of the invention, it should be noted that embodiment described below is exemplary, only

For explaining the present invention, and limitation of the present invention can not be interpreted as.In addition, if do not clearly not stated, adopted in the following embodiments all reagent are, and market can be buied, or can according to text or the synthesis of known method, for the reaction conditions do not listed, be also that those skilled in the art easily obtain.

Embodiment 1

Instrument: Agilent1260 high performance liquid chromatograph, 1260 UV-detector

Chromatographic column: octadecylsilane chemically bonded silica is the chromatographic column (250 × 4.6mm, 5 μm) of filling agent;

The ammonium acetate buffer (pH5.1) of mobile phase A: 0.01mol/L

Mobile phase B: acetonitrile

Gradient elution sees the following form;

Flow velocity: 1.0mL/min

Determined wavelength: 260nm

Column temperature: 25 DEG C

Sampling volume: 10 μ L

Thinning agent: acetonitrile

Test procedure:

Need testing solution: take Amlodipine Besylate Tablet sample 1.0014g and put 10mL measuring bottle, dissolve with thinning agent and be diluted to scale, shaking up.

Reference substance solution: precision takes P-TOLUENE SULFO ACID 99's methyl esters reference substance 7.48mg and puts in 100mL measuring bottle, is diluted to scale by water-soluble solution, shakes up, as stock solution A.Precision measures 1.0mL stock solution A and puts in 100mL measuring bottle, is diluted to scale, shakes up with thinning agent, and as stock solution B, precision measures 1.0mL stock solution B and puts in 10mL measuring bottle, is diluted to scale, shakes up with thinning agent, in contrast product solution (0.75ppm).

Get reference substance, need testing solution respectively, under above-mentioned chromatographic condition, carry out efficient liquid phase chromatographic analysis, record chromatogram, the retention time methyl benzene sulfonate chromatographic peak that to be 6.282min chromatographic peak be in figure, adopts area normalization method to calculate impurity content.

Conclusion: the methyl benzene sulfonate in Amlodipine Besylate Tablet sample is less than 0.75ppm.

Embodiment 2

Instrument: Agilent1260 high performance liquid chromatograph, 1260 UV-detector

The ammonium acetate buffer (pH3.0) of mobile phase A: 0.01mol/L

Mobile phase B: acetonitrile

Gradient elution sees the following form;

Flow velocity: 1.0mL/min

Determined wavelength: 250nm

Column temperature: 20 DEG C

Sampling volume: 10 μ L

Thinning agent: acetonitrile

Test procedure:

Need testing solution: take methane-sulforic acid T-3811 crude product 1.0014g and put 10mL measuring bottle, dissolve with thinning agent and be diluted to scale, shaking up.

Reference substance solution: precision takes methyl mesylate reference substance 37.42mg and puts in 100mL measuring bottle, molten with thinning agent

Separate and be diluted to scale, shaking up, as stock solution A.Precision measures 1.0mL stock solution A and puts in 100mL measuring bottle, is diluted to scale, shakes up with thinning agent, and as stock solution B, precision measures 1.0mL stock solution B and puts in 100mL measuring bottle, is diluted to scale, shakes up with thinning agent, in contrast product solution (3.75ppm).

Get reference substance, need testing solution respectively, under above-mentioned chromatographic condition, carry out efficient liquid phase chromatographic analysis, record chromatogram, in figure, retention time is 4.545min chromatographic peak is methyl mesylate chromatographic peak, adopts area normalization method to calculate impurity content.

Conclusion: this method is applicable to the content of the methyl mesylate detecting variable concentrations scope in methane-sulforic acid T-3811 sample.

Reference examples 1

Adopt the genotoxicity impurity of GC-FID method P-TOLUENE SULFO ACID 99 Amlodipine of the prior art to carry out detection to analyze, further checking the present invention adopt accuracy and the sensitivity of method.

The preparation of need testing solution: get Amlodipine Besylate Tablet appropriate, accurately weighed, first dissolve by little water, then by acetone diluted and constant volume makes 20mg/ml, as need testing solution; Get methyl benzene sulfonate reference substance appropriate, accurately weighed, also dilute with acetone solution and make 0.05mg/ml solution, product solution in contrast; Get above-mentioned need testing solution and reference substance solution headspace sampling in accordance with the law respectively, record chromatogram, with peak area external standard method result of calculation.

Chromatographic condition:

Injection port: shunting, temperature 250 DEG C;

Chromatographic column: polyglycol is that Stationary liquid or polarity are close;

Column temperature: temperature programme 150 DEG C keep 4 minutes then with 20 DEG C per minute be warmed up to 240 DEG C keep 5 minutes;

Column flow rate: 2.0ml/min;

Detecting device: flame ionization ditector (FID), temperature 280 DEG C;

Split sampling: split ratio 10:1;

Sample introduction pattern: headspace sampling;

Head-space sampler: furnace temperature 150 DEG C insulation 10 minutes, pin temperature 150 DEG C, sample size 1ml.

Define methyl benzene sulfonate according to the appearance time of reference substance solution and need testing solution to exist, and conform to the result that the present invention measures according to the content going out the methyl benzene sulfonate in peak area determination test sample of methyl benzene sulfonate.

Claims

1. utilize HPLC to measure a method for sulfonic acid esters genotoxicity impurity, it is characterized in that:

(1) chromatographic condition: chromatographic column take octadecylsilane chemically bonded silica as Stationary liquid, using the mixed solvent of organic phase and damping fluid as eluent gradient wash-out, column temperature 20 DEG C ~ 30 DEG C, flow velocity is 0.8 ~ 1.2mL/min, and determined wavelength is 260nm ± 10nm;

(2) preparation of sample solution: adopt the mixed liquor of organic solvent or organic solvent and water that detected sample is mixed with sample solution;

(3) compartment analysis: by sample solution 10 μ L, injects high performance liquid chromatograph, completes the mensuration of sulfonic acid esters toxic impurities.

2. method according to claim 1, is characterized in that: described mobile phase is the mixed solvent gradient elution of organic phase and damping fluid, and during gradient elution 0 ~ 5min, the ratio of damping fluid, organic phase is volume ratio 90%:10%; During 5 ~ 14min, damping fluid volume ratio is reduced to 80%, and organic phase volume ratio increases to 20%; 14 ~ 15min, buffer ratio is that volume ratio is reduced to 10%, and the ratio of organic phase is that volume ratio increases to 90%; During 15 ~ 25min, the ratio of damping fluid, organic phase is that volume ratio 10%:90% is constant; During 25 ~ 30min, damping fluid volume ratio increases to 90%, and organic phase volume ratio is reduced to 10%; During 30 ~ 35min, the ratio of damping fluid, organic phase is that volume ratio 90%:10% is constant.

3. method according to claim 1 and 2, is characterized in that: described organic phase is acetonitrile.

4. method according to claim 1 and 2, is characterized in that: described damping fluid is for being selected from acetate buffer.

5. method according to claim 4, is characterized in that: the concentration range of described damping fluid is 0.005mol/L ~ 0.05mol/L, preferred 0.01mol/L.

6. method according to claim 4, is characterized in that: the pH value of described damping fluid is 3.0 ~ 5.1.

7. method according to claim 1, is characterized in that: the flow velocity of mobile phase is 1.0mL/min.

8. method according to claim 1, is characterized in that: column temperature is 25 DEG C.

9. method according to claim 1, is characterized in that: determined wavelength is 260nm.