CN105092745B - The method and apparatus for reducing fixedness buffer salt content in LC MS testers - Google Patents

Abstract

The application is related to a kind of method and apparatus of fixedness buffer salt content in reduction LC MS testers.Specifically, the application is related to a kind of method of fixedness buffer salt content in reduction liquid chromatogram eluting fraction in test for LC Mass, and this method comprises the following steps：(1) detectable substance is configured to solution, the solution is injected into liquid chromatograph to be eluted, the object eluting fraction for being ready to use in Mass Spectrometer Method is collected from liquid chromatography eluant；(2) eluting fraction is made to be sufficiently mixed with auxiliary agent；(3) make gained mixed liquor separate suspension and clear liquid by way of filtering, divide and take clear liquid；With optional (4) are concentrated gained clear liquid, are produced.The application method can be effectively reduced the buffer salt content in high performance liquid chromatography eluent, so that the eluent is reduced to mass spectrometric loss when for follow-up mass spectroscopy.The present invention uses simple processing method, it is possible to achieve fixedness buffer salinity is substantially reduced in the LC eluting fractions of LC MS methods, and target substance will not be lost.

Description

The method and apparatus for reducing fixedness buffer salt content in LC-MS testers

Technical field

The application belongs to technical field of analytical chemistry, be related to a kind of associative operation in liquid chromatography-mass spectrometry and its A kind of device used, it is more particularly to a kind of to be used to reduce buffer salt content in liquid chromatography-mass spectrography (LC-MS) method tester Method and a kind of its device used.The application method and apparatus can be effectively reduced in high performance liquid chromatography eluent Buffer salt content so that the eluent is reduced to mass spectrometric loss when for follow-up mass spectroscopy.The present invention Using simple processing method and simple device, it is possible to achieve fixedness buffer salt is dense in the LC eluting fractions of LC-MS methods That spends substantially reduces, and target substance will not be lost.

Background technology

Mass spectral analysis is a kind of analysis method of measurement ion mass-to-charge ratio (mass-charge ratio), and its general principle is to make examination Each component is ionized in an ion source in sample, generates the electrically charged ion of different charge-mass ratios, the effect of accelerated electric field, shape Into ion beam, into mass analyzer.In the mass analyser, electric field and magnetic field is recycled to make the opposite velocity dispersion of generation, They are focused on respectively and mass spectrogram is obtained, so that it is determined that its quality.

Mass-spectrometric technique is a kind of identification technology, and very important effect is played in terms of the identification of organic molecule.It can be fast Molecular weight that is fast and extremely accurately determining large biological molecule, makes proteome research be deep into higher structure from identification of proteins Repercussion study between research and various protein.

With the development of mass-spectrometric technique, the application field of mass-spectrometric technique is also more and more wider.Due to mass spectral analysis have it is sensitive Degree is high, and amount of samples is few, and analyze speed is fast, the advantages of separation and identification are carried out simultaneously, and therefore, mass-spectrometric technique is widely used in Chemistry, chemical industry, environment, the energy is medical, sports medical science, science and technology concerning criminal matters, life science, the every field such as material science.

Mass spectrograph species is various, and different instrument application features are also different, in general, in 300 DEG C or so the samples that can be vaporized Product, can pay the utmost attention to be analyzed with gas chromatography-mass spectrum (GC-MS), because GC-MS uses EI sources, obtained mass spectrum letter Breath is more, can carry out library searching.The separating effect of capillary column might as well.If can not vaporize at 300 DEG C or so, need to use LC-MS is analyzed, now main to obtain molecular weight information, if tandem mass spectrum, can also obtain some structural informations.If biological Macromolecular, it is main to be analyzed using LC-MS and MALDI-TOF, it is main to obtain molecular weight information.For protein example, it can also survey Determine amino acid sequence.Mass spectrometric resolution ratio is an important technology index, high-resolution mass spectrometer can provide compound group into Formula, this is very important for structure determination.When double focusing mass spectrometer, Fourier transform mass spectrometer, flight with reflector Between mass spectrograph etc. all there is resolution capability.

Mass spectrometry has certain requirement to sample.In carrying out the sample of GC-MS analyses and should being organic solution, the aqueous solution Organic matter can not typically determine, extract and separate must be carried out and be changed into organic solution, or use headspace sampling technology.Some compounds Polarity is too strong, is easily decomposed in heating process, for example organic acid compound, can now carry out esterification treatment, acid is changed into Ester carries out GC-MS analyses again, and the structure of acid can be speculated by analysis result.If sample, which can not be vaporized, to be esterified, that is just LC-MS can only be carried out to analyze.It is general using ESI sources for polarity sample, for nonpolar sample, using APCI sources.

Liquid chromatograph-mass spectrometer (liquid Chromatograph Mass Spectrometer), abbreviation LC- MS, is the instrument of liquid chromatogram and mass spectrometry.It combines liquid chromatograph and efficiently separates hot instability and higher boiling chemical combination The separating capacity of thing and mass spectrograph very strong component identification capacity.It is a kind of effective hand for separating the complicated organic mixture of analysis Section.

Chromatograph-mass spectrometer coupling technology is one of analysis method of the present age most important separation and identification.The advantage of chromatogram exists In separation, the separating capacity of chromatogram provides maximally effective selection for mixture separation, but chromatographic process is difficult to obtain structure letter Breath, it reaches the presumption to unknown material structure mainly by with standard specimen contrast；In terms of to the structural analysis of COMPLEX MIXED unknown material Seem weak；For detecting qualitative with a large amount of unknown compounds without ultraviolet absorption compound on conventional UV-detector Analysis also needs to depend on other means.Mass spectrography can provide abundant structural information, with sample amount be used in several spectrum methods again Amount is minimum, but its sample needs preprocessed (purifying, separation), and program is complicated, time-consuming.For a long time, people are to solve this The weakness of two kinds of technologies has developed many technologies, and wherein chromatograph-mass spectrometer coupling technology is the technology of most development and application prospect One of.What application was more at present is gas chromatography-mass spectrum (GC-MS) combination.But GC requires that sample has certain vapour pressure, Only 20% medicine can satisfactorily can be separated without advance chemical treatment with gas-chromatography, it is a variety of in the case of studied Medicine need by appropriate pretreatment and derivatization, could carry out GC-MS analyses to make the sample easily vaporized.And HPLC separates polarity, compound, while LC-MS joins Machine compensate for the deficiency of traditional LC detector, with high separation capacity, high sensitivity, and application is wider and with extremely strong special The features such as attribute, increasingly it is valued by people.According to estimates in known compound about 80% compound be hydrophily it is strong, The low organic matter of volatility, heat-labile compound and large biological molecule, these compounds are widely present in current application and hair In terms of most extensive, the most potential field of exhibition, including biological, medicine, chemical industry and environment, they need to be separated with LC.Therefore, LC and MS combination can solve the insurmountable problems of GC-MS.

Liquid chromatograph-mass spectrometer is the instrument of liquid chromatogram and mass spectrometry.It is effective that it combines liquid chromatograph The separating capacity of heat of dissociation instability and higher-boiling compound and mass spectrograph very strong component identification capacity.It is a kind of separation analysis The effective means of complicated organic mixture.Online key is to be applicable the exploitation of interface, it is necessary to enter ion gun in sample fraction Preceding removal solvent at present, use crawler type heated belt more.

Liquid chromatography mass combined instrument (liquid Chromatograph Mass Spectrometer), abbreviation LC-MS, It is the high end instrument in Analysis of Organic Substances market.Liquid chromatogram (LC) can be effectively by the organic matter in organic matter testing sample Composition is separated, and the analysis that mass spectrum (MS) can be to separated organic matter one by one, obtains organic matter molecular mass, structure is (at certain In the case of a little) and concentration (quantitative analysis) information.It is very simple that powerful electron spray ionisation technology creates LC-MS mass spectrograms It is clean, the characteristics of later data processing is simple.LC-MS is Analysis of Organic Substances laboratory, medicine, Food Inspection room, production process control The essential analysis tool of the departments such as system, quality inspection.

Liquid chromatography mass combined instrument feature：Liquid chromatogram and mass spectrum connection, can increase extra analysis ability, can It is micro in precise Identification and quantitative picture cell and Tissue lysates, blood, blood plasma, the complex sample matrix such as urine and oral fluid Compound.High performance liquid chromatography mass spectrometer system (ABSciex Eksigent LC/MS and LC/MS/MS) provides some uniquenesses Advantage, including：(1) minimal sample needed for quick analysis and circulation prepares；(2) high sensitivity and combination can analyze multiple chemical combination Thing ability, it might even be possible to across the species of compound；(3) pinpoint accuracy, high-resolution identification and quantified goal analyte.

Although people solve the problem of many analytical chemistry fields by liquid chromatograph mass spectrography, however, carrying out When LC-MS is analyzed, it is the aqueous solution or methanol solution that its sample, which is usually required that, and is especially required in the mobile phase of liquid chromatogram It should not contain or contain nonvolatile salt as few as possible.

But, regrettably, it is used as the mobile phase of liquid chromatogram such as high performance liquid chromatography, it usually needs thereto Add some and do not have the buffer salt of volatilization to obtain excellent chromatographic isolation effect, but these include the buffer salt for not having volatilization Chromatography eluant fraction when for ensuing mass spectroscopy, these fixedness buffer salts be difficult to remove or be difficult to What the object in chromatography eluant fraction was separated.For example, Chinese Pharmacopoeia aceglutamide recorded of page 6 of version two in 2010 When it is relevant material to determine impurity therein, separation analysis, the flowing that the liquid chromatogram is used are carried out by liquid chromatography Potassium dihydrogen phosphate is included in phase, if in the case of determining the wherein ownership or chemical constitution of impurity peaks in the case, Make impurity peaks chromatogram fraction import mass spectrograph to detect it is beneficial, but the biphosphate included in impurity peaks chromatogram fraction Potassium does not have volatility, is unfavorable for mass spectroscopy.If carrying out concentration opinion to the impurity peaks chromatogram fraction using current normal method (method generally dried up using nitrogen), is concentrated although object is impurity, and phosphate therein similarly can be into Concentrate to ratio, and pH change may be caused, influence the stability of composition to be measured, therefore this method for concentration is not drop The proper method of fixedness buffer salinity in low MS determinands.Therefore this method for concentration can not realize reduction MS determinands The purpose of middle fixedness buffer salinity.

Therefore, chemical analysis field technical staff urgently expects have one kind to be effectively reduced liquid chromatography-mass spectrometry The method that fixedness buffer salt content is even removed in tester, and suitable for the device of this method.

The content of the invention

The purpose of the application is that fixedness in liquid chromatography-mass spectrometry tester can be effectively reduced by providing one kind The method that buffer salt content is even removed.It has been unexpectedly discovered that making liquid chromatogram eluting fraction enter according to the application method After row processing, the content of fixedness buffer salt in the liquid chromatogram eluting fraction can be significantly decreased.The application is based on should It was found that and being accomplished.

Therefore, first method of the present invention, which is provided in a kind of test for LC-MS analysis, reduces liquid chromatogram The method of fixedness buffer salt content in eluting fraction, this method comprises the following steps：

(1) detectable substance is configured to solution, the solution is injected into liquid chromatograph to be eluted, from liquid chromatogram elution Liquid collects the object eluting fraction for being ready to use in Mass Spectrometer Method, the eluting fraction is imported the syringe for being pre-filled with auxiliary agent In；

(2) in syringe, the eluting fraction is sufficiently mixed with auxiliary agent, (optional can be with elution in step (1) Fraction extraly adds organic solvent (such as methanol and/or acetonitrile) mixing into syringe together)；

(3) make gained mixed liquor separate suspension and clear liquid by way of filtering, divide and take clear liquid；With, optional

(4) gained clear liquid is concentrated, produced.

The method of any embodiment according to a first aspect of the present invention, wherein the liquid chromatogram is RP-HPLC color Spectrum.

The method of any embodiment according to a first aspect of the present invention, wherein liquid chromatogram elution mobile phase used It is the mixed liquor of aqueous phase and organic phase.In one embodiment, the organic phase is such as, but not limited to：Methanol, acetonitrile, second Alcohol, tetrahydrofuran, isopropanol and combinations thereof；Particularly methanol and acetonitrile.In one embodiment, comprising not in the aqueous phase Volatile buffer salt.

According to the method for present invention any embodiment according to a first aspect of the present invention, wherein the aqueous phase and organic phase Volume ratio can be 5：95~95：5 scope.

The method of any embodiment according to a first aspect of the present invention, wherein the fixedness buffer salt for example but is not limited One or more in phosphate, citrate, acetate, perchlorate.

The method of any embodiment according to a first aspect of the present invention, wherein the alkali root of the fixedness buffer salt is choosing From the alkali root either ammonium root of alkali metal or alkaline-earth metal ions.In one embodiment, the alkali root is selected from：Sodium ion, Potassium ion, magnesium ion, calcium ion, alkali root preferably is sodium ion, potassium ion or ammonium ion.

The method of any embodiment according to a first aspect of the present invention, wherein the fixedness buffer salt includes but do not limited In：Sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, citric acid Sodium, potassium citrate, disodium citrate, citric acid dipotassium, citric acid trisodium, tripotassium citrate, sodium acetate, potassium acetate, ammonium acetate, Sodium perchlorate, potassium hyperchlorate and combinations thereof.

The method of any embodiment according to a first aspect of the present invention, wherein the object is the master in the detectable substance Want composition or non-principal composition (such as impurity).In one embodiment, main component or the non-principal composition is Know material either unknown materials.In chemical analysis field, it is known that material or the unknown materials isolated mesh in liquid chromatogram Mark is looked for after spectral peak or eluting fraction, and the object subsequently enters mass spectrograph detection, can be determined according to Mass Spectrometer Method result The target chemical structure, so as to known substance is made it is accurate confirm, or to the chemical constitution of unknown materials/constitute into Row analysis.

The method of any embodiment according to a first aspect of the present invention, wherein the detectable substance is configured to used in solution Solvent is mobile phase or is selected from following solvent：Water, methanol, acetonitrile, ethanol and combinations thereof.

The method of any embodiment according to a first aspect of the present invention, wherein the auxiliary agent is mineral sulfates.At one In embodiment, the mineral sulfates be selected from magnesium sulfate, sodium sulphate, potassium sulfate, calcium sulfate, its anhydride, its hydrate and It is combined.

The method of any embodiment according to a first aspect of the present invention, wherein the eluting fraction is being mixed with the auxiliary agent When compare 1 with weight：0.01~100 ratio mixing, for example, compare 1 with weight：0.05~10 ratio mixing, such as with weight ratio 1：0.1~5 ratio mixing, for example, compare 1 with weight：0.5~2 ratio mixing.The two weight than regulation be easy realization , the mixed proportion of eluting fraction and auxiliary agent is suitably for example adjusted according to the reduction degree of fixedness buffer salt.

The method of any embodiment according to a first aspect of the present invention, wherein the volume ratio of the aqueous phase and organic phase can be with It is 5：95~95：5 scope.As long as can be realized it has been found that detectable substance and object are generally dissolved in this mobile phase The purpose of reduction fixedness buffer salt wherein used.Also, because the method for first aspect present invention is particularly used for being directed to The LC-MS of a certain detectable substance and/or object is determined, and the method for first aspect present invention is in this LC-MS methods Between increased several simple sample handling procedures.Therefore, it is this to be suitable for well known to a person skilled in the art can very easy be interpreted Detectable substance and/or the LC-MS methods of object analysis are fully able to receive increase several operating procedures of the invention.I.e. it is easy to Infer, the inventive method is suitable for any LC-MS methods for meeting condition determination of the present invention, as long as such as these LC-MS LC In mobile phase used, buffer salt meet the condition of defined, the inventive method is just applied to these LC-MS.Therefore, present invention side Detectable substance and object and its LC and MS methods condition used need not be limited specifically in method, although the present invention exists Hereinafter the inventive method is verified in the method for specific Pharmaceutical Analysis.

The method of any embodiment according to a first aspect of the present invention, relative to object eluting fraction obtained by step (1) In fixedness buffer salt for, the remaining rate (%) of the fixedness buffer salt in clear liquid obtained by step (3) reaches 50% Hereinafter, 5~50% degree can be for example reached, for example, can reach 5~40% degree, for example, can reach 5~35% Degree, can for example reach 5~30% degree, for example, can reach 5~25% degree.

The method of any embodiment according to a first aspect of the present invention, relative to object eluting fraction obtained by step (1) In object for, the remaining rate (%) of the object in clear liquid obtained by step (3) reaches more than 70%, for example, can reach 70~100% degree, for example, can reach 70~95% degree, for example, can reach 70~90% degree, for example may be used To reach 75~90% degree.

The method of any embodiment according to a first aspect of the present invention, wherein the volume of organic solvent is to wash in step (2) 0~100 times of de- fraction volume, such as 0~50 times, such as 0~25 times, such as such as 0~10 times, 0~5 times.

The method of any embodiment according to a first aspect of the present invention, wherein the syringe includes：Core bar, sleeve, work Plug, filter, cap bag.

The method of any embodiment according to a first aspect of the present invention, wherein the needle stand of the syringe and entering for filter Liquid mouthful is tightly connected, and the liquid outlet of the filter is sealed by the cap bag.

The method of any embodiment according to a first aspect of the present invention, wherein being pre-filled with the sleeve of the syringe Auxiliary agent.

The method of any embodiment according to a first aspect of the present invention, wherein the auxiliary agent is hermetically present in the injection In the sleeve of device.

The method of any embodiment according to a first aspect of the present invention, wherein the cylinder of the sleeve of the syringe at an upper portion thereof Footpath is bigger than the barrel dliameter of sleeve main body section, to form bulb.Thus when making piston enter bulb core bar pull-up, due to There is space between piston and sleeve, eluting fraction can be caused to import in (inflow) syringe；Similarly, optional supplement adds Plus organic solvent can also be added by this mode.It is other eluting fraction (and optional organic solvent) is imported (inflow) injection Design in device also applies to the present invention's.Because auxiliary agent exists in sleeve interior sealing usually；And using the injection During device, piston is set to enter bulb to core bar pull-up, this will certainly form vacuum in sleeve and hinder core bar to pull up, now Cap bag can be opened to release this vacuum.

In the present invention, when referring to " top " of sleeve of syringe, one end that syringe core rod can be pulled freely is referred to； Comparatively, one end of " bottom " syringe needle of syringe sleeve.

The method of any embodiment according to a first aspect of the present invention, wherein the filter is miillpore filter.At one In embodiment, the miillpore filter is the miillpore filter of miillpore filter, e.g. aperture 0.22um to 1um that aperture is less than 1um. In the practical operation of the present invention, it is found that aperture 0.22um to 1um miillpore filter is all applicable.

The method of any embodiment according to a first aspect of the present invention, wherein making the eluting fraction (and optional organic Solvent) import (inflow) syringe in after, to the main part that core bar makes piston return to sleeve is moved down, so that eluting fraction The mixed liquor of (and optional organic solvent) and auxiliary agent is sealed in sleeve to carry out married operation.To avoid not sufficiently mixed Preceding penetration by liquid filter and flow out, mixing when can with cap bag will seal filter exit seal.Retouched according to the present invention State, these operations are to be very easy to realize.

In view of the auxiliary agent used in the present invention is generally particle or particulate, suspension and clear liquid is set to separate all by way of filtering It is readily obtained the usually said clear liquid of the settled solution of suitable subsequent measurements, i.e. people.Therefore the mode of general filtering all may be used To use, for example, for filtering, general miillpore filter can realize these functions, such as aperture 0.45um or 0.22um miillpore filter can be used.

In view of the target portion obtained by the inventive method processing can be the main component or micro in detectable substance Impurity component, therefore in the case of necessary, in the case of particularly micro impurity, obtained by being handled through the inventive method Clear liquid carries out concentration and is necessary, and follow-up mass spectroscopy is carried out to obtain sufficient concentrations of object.And for target Thing is the situation of main component, because the amount of contained object in clear liquid is larger, is sufficiently used for follow-up mass spectroscopy, therefore should Clear liquid can be without concentration.

Typically, concentration can remove the method (such as by way of nitrogen drying) of solvent volatilizing.Feelings are concentrated herein Under condition, fixedness buffer salt can not be removed effectively in the eluting fraction of mobile phase, these fixednesies buffering Salt can bring immense pressure to follow-up Mass Spectrometer Method, and may cause pH change, influence the stability of composition to be measured. It has been unexpectedly discovered that by means of the invention it is also possible to being effectively reduced fixedness in liquid chromatogram eluting fraction Buffer salt content (concentration in other words).

Further, second aspect of the present invention provides a kind of object in detectable substance and carries out liquid chromatography-mass spectrography The method of analysis, it comprises the following steps：

(1) detectable substance is configured to solution, the solution is injected into liquid chromatograph to be eluted, from liquid chromatogram elution Liquid collects the object eluting fraction for being ready to use in Mass Spectrometer Method, the eluting fraction is imported the syringe for being pre-filled with auxiliary agent In；

(2) in syringe, the eluting fraction is sufficiently mixed with auxiliary agent, (optional can be with elution in step (1) Fraction extraly adds organic solvent (such as methanol and/or acetonitrile) mixing into syringe together)；

(3) make gained mixed liquor separate suspension and clear liquid by way of filtering, divide and take clear liquid；

(4) clear liquid obtained by previous step is taken to be directly used in mass spectroscopy, or be used further to after gained clear liquid is concentrated Mass spectroscopy.

The method of any embodiment according to a second aspect of the present invention, wherein the liquid chromatogram is RP-HPLC color Spectrum.

The method of any embodiment according to a second aspect of the present invention, wherein liquid chromatogram elution mobile phase used It is the mixed liquor of aqueous phase and organic phase.In one embodiment, the organic phase is such as, but not limited to：Methanol, acetonitrile, second Alcohol, tetrahydrofuran, isopropanol and combinations thereof；Particularly methanol and acetonitrile.In one embodiment, comprising not in the aqueous phase Volatile buffer salt.

According to the method for present invention any embodiment according to a second aspect of the present invention, wherein the aqueous phase and organic phase Volume ratio can be 5：95~95：5 scope.

The method of any embodiment according to a second aspect of the present invention, wherein the fixedness buffer salt for example but is not limited One or more in phosphate, citrate, acetate, perchlorate.

The method of any embodiment according to a second aspect of the present invention, wherein the alkali root of the fixedness buffer salt is choosing From the alkali root either ammonium root of alkali metal or alkaline-earth metal ions.In one embodiment, the alkali root is selected from：Sodium ion, Potassium ion, magnesium ion, calcium ion, alkali root preferably is sodium ion, potassium ion or ammonium ion.

The method of any embodiment according to a second aspect of the present invention, wherein the fixedness buffer salt includes but do not limited In：Sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, citric acid Sodium, potassium citrate, disodium citrate, citric acid dipotassium, citric acid trisodium, tripotassium citrate, sodium acetate, potassium acetate, ammonium acetate, Sodium perchlorate, potassium hyperchlorate and combinations thereof.

The method of any embodiment according to a second aspect of the present invention, wherein the object is the master in the detectable substance Want composition or non-principal composition (such as impurity).In one embodiment, main component or the non-principal composition is Know material either unknown materials.In chemical analysis field, it is known that material or the unknown materials isolated mesh in liquid chromatogram Mark is looked for after spectral peak or eluting fraction, and the object subsequently enters mass spectrograph detection, can be determined according to Mass Spectrometer Method result The target chemical structure, so as to known substance is made it is accurate confirm, or to the chemical constitution of unknown materials/constitute into Row analysis.

The method of any embodiment according to a second aspect of the present invention, wherein the detectable substance is configured to used in solution Solvent is mobile phase or is selected from following solvent：Water, methanol, acetonitrile, ethanol and combinations thereof.

The method of any embodiment according to a second aspect of the present invention, wherein the auxiliary agent is mineral sulfates.At one In embodiment, the mineral sulfates be selected from magnesium sulfate, sodium sulphate, potassium sulfate, calcium sulfate, its anhydride, its hydrate and It is combined.

The method of any embodiment according to a second aspect of the present invention, wherein the eluting fraction is being mixed with the auxiliary agent When compare 1 with weight：0.01~100 ratio mixing, for example, compare 1 with weight：0.05~10 ratio mixing, such as with weight ratio 1：0.1~5 ratio mixing, for example, compare 1 with weight：0.5~2 ratio mixing.The two weight than regulation be easy realization , the mixed proportion of eluting fraction and auxiliary agent is suitably for example adjusted according to the reduction degree of fixedness buffer salt.

The method of any embodiment according to a second aspect of the present invention, wherein the volume ratio of the aqueous phase and organic phase can be with It is 5：95~95：5 scope.

The method of any embodiment according to a second aspect of the present invention, relative to object eluting fraction obtained by step (1) In fixedness buffer salt for, the remaining rate (%) of the fixedness buffer salt in clear liquid obtained by step (3) reaches 50% Hereinafter, 5~50% degree can be for example reached, for example, can reach 5~40% degree, for example, can reach 5~35% Degree, can for example reach 5~30% degree, for example, can reach 5~25% degree.

The method of any embodiment according to a second aspect of the present invention, relative to object eluting fraction obtained by step (1) In object for, the remaining rate (%) of the object in clear liquid obtained by step (3) reaches more than 70%, for example, can reach 70~100% degree, for example, can reach 70~95% degree, for example, can reach 70~90% degree, for example may be used To reach 75~90% degree.

The method of any embodiment according to a first aspect of the present invention, wherein the volume of organic solvent is to wash in step (2) 0~100 times of de- fraction volume, such as 0~50 times, such as 0~25 times, such as such as 0~10 times, 0~5 times.

The method of any embodiment according to a second aspect of the present invention, wherein the syringe includes：Core bar, sleeve, work Plug, filter, cap bag.

The method of any embodiment according to a second aspect of the present invention, wherein the needle stand of the syringe and entering for filter Liquid mouthful is tightly connected, and the liquid outlet of the filter is sealed by the cap bag.

The method of any embodiment according to a second aspect of the present invention, wherein being pre-filled with the sleeve of the syringe Auxiliary agent.

The method of any embodiment according to a second aspect of the present invention, wherein the auxiliary agent is hermetically present in the injection In the sleeve of device.

The method of any embodiment according to a second aspect of the present invention, wherein the cylinder of the sleeve of the syringe at an upper portion thereof Footpath is bigger than the barrel dliameter of sleeve main body section, to form bulb.Thus when making piston enter bulb core bar pull-up, due to There is space between piston and sleeve, eluting fraction can be caused to import in (inflow) syringe；Similarly, optional supplement adds Plus organic solvent can also be added by this mode.It is other eluting fraction (and optional organic solvent) is imported (inflow) injection Design in device also applies to the present invention's.Because auxiliary agent exists in sleeve interior sealing usually；And using the injection During device, piston is set to enter bulb to core bar pull-up, this will certainly form vacuum in sleeve and hinder core bar to pull up, now Cap bag can be opened to release this vacuum.

The method of any embodiment according to a second aspect of the present invention, wherein the filter is miillpore filter.At one In embodiment, the miillpore filter is the miillpore filter of miillpore filter, e.g. aperture 0.22um to 1um that aperture is less than 1um. In the practical operation of the present invention, it is found that aperture 0.22um to 1um miillpore filter is all applicable.

The method of any embodiment according to a second aspect of the present invention, wherein making the eluting fraction (and optional organic Solvent) import (inflow) syringe in after, to the main part that core bar makes piston return to sleeve is moved down, so that eluting fraction The mixed liquor of (and optional organic solvent) and auxiliary agent is sealed in sleeve to carry out married operation.To avoid not sufficiently mixed Preceding penetration by liquid filter and flow out, mixing when can with cap bag will seal filter exit seal.Retouched according to the present invention State, these operations are to be very easy to realize.

Further, third aspect present invention, which is provided in a kind of test for LC-MS analysis, reduces liquid phase The method of fixedness buffer salt content in chromatography eluant fraction, or for carrying out liquid phase color to the object in detectable substance The syringe of the method for spectrum-mass spectral analysis, the syringe includes：Core bar, sleeve, piston, filter, cap bag.

The syringe of any embodiment according to a third aspect of the present invention, wherein the LC-MS analysis is tested The method such as first aspect present invention any embodiment of fixedness buffer salt content in middle reduction liquid chromatogram eluting fraction (these embodiments as described above, and are optionally hereby incorporated).

The syringe of any embodiment according to a third aspect of the present invention, wherein the object in detectable substance is carried out The method of LC-MS analysis (these embodiments such as above the as described in second aspect of the present invention any embodiment Two aspects are described, and are optionally hereby incorporated).

The syringe of any embodiment according to a third aspect of the present invention, wherein the needle stand and filter of the syringe Liquid inlet is tightly connected, and the liquid outlet of the filter is sealed by the cap bag.

The syringe of any embodiment according to a third aspect of the present invention, wherein being pre-charged with the sleeve of the syringe There is auxiliary agent.

The syringe of any embodiment according to a third aspect of the present invention, wherein the auxiliary agent is hermetically present in the note In the sleeve of emitter.

The syringe of any embodiment according to a third aspect of the present invention, wherein the sleeve of the syringe is at an upper portion thereof Barrel dliameter is bigger than the barrel dliameter of sleeve main body section, to form bulb.Thus when making piston enter bulb core bar pull-up, by There is space between piston and sleeve, eluting fraction can be caused to import in (inflow) syringe；Similarly, optional supplement The organic solvent of addition can also be added by this mode.It is other eluting fraction (and optional organic solvent) is imported (inflow) note Design in emitter also applies to the present invention's.Because auxiliary agent exists in sleeve interior sealing usually；And using the note During emitter, piston is set to enter bulb to core bar pull-up, this will certainly form vacuum in sleeve and hinder core bar to pull up, this When cap bag can be opened to release this vacuum.

The syringe of any embodiment according to a third aspect of the present invention, wherein the filter is miillpore filter.One In individual embodiment, the miillpore filter is the miillpore filter that aperture is less than 1um, e.g. aperture 0.22um to 1um micropore filter Film.In the practical operation of the present invention, it is found that aperture 0.22um to 1um miillpore filter is all applicable.

The syringe of any embodiment according to a third aspect of the present invention, wherein making the eluting fraction (and optional have Machine solvent) import in (inflows) syringe after, to moving down the main part that core bar makes piston return to sleeve, evaporated so that eluting The mixed liquor of part (and optional organic solvent) and auxiliary agent is sealed in sleeve to carry out married operation.To avoid fully not mixed Close preceding penetration by liquid filter and flow out, can will seal the exit seal of filter with cap bag in mixing.According to the present invention Description, these operations are to be very easy to realize.

Any embodiment of the either side of the application, can be combined with other embodiments, as long as they are not Contradiction occurs.In addition, in any embodiment of the application either side, any technical characteristic goes for other realities The technical characteristic in scheme is applied, as long as they are not in contradiction.

The application is further described below.

All documents recited in the application, their full content is incorporated herein by reference, and if these are literary When offering expressed implication and inconsistent the application, it is defined by the statement of the application.In addition, various terms used in this application and Phrase has well known to a person skilled in the art general sense, nonetheless, the application remain desirable at this to these terms and Phrase is described in more detail and explained that the term and phrase referred to is if any inconsistent with common art-recognized meanings, with the application institute table The implication stated is defined.

Exemplary, in process of the present invention is realized, object elution it can be evaporated as obtained by determining step (1) of the present invention (it can be designated as C1 to the concentration of fixedness buffer salt in part in the present invention, represent it is more easily spy with molar concentration It is not that in the case where a variety of buffer salts are applied in combination, such as when using dihydrogen phosphate and disodium hydrogen phosphate simultaneously, can survey Determine the total mol concentration of phosphate anion), then the fixedness buffer salt in clear liquid obtained by measure step (3) of the present invention is dense Spend (it can be designated as C2 in the present invention, and its concentration method for expressing is identical with C1), be then calculated as follows and use present invention side The remaining rate (%) of buffer salt after method processing：

Buffer salt remnants rates (%)=(C2 ÷ C1) × 100%

It has been found that using the inventive method, the remaining rate of the buffer salt can be made to reach less than 50%, such as can be reached 5~50% degree, for example, can reach 5~40% degree, for example, can reach 5~35% degree, for example, can reach To 5~30% degree, for example, it can reach 5~25% degree.It can be seen that, by simple processing method of the invention easily Realize a large amount of removals of buffer salt.

The concentration of fixedness buffer salt in object eluting fraction and clear liquid can use the side of many prior arts Method is determined.For example, typical, acetate, phosphate and citrate can refer to Xu Mingming methods (acetic acid in amino acid injection Salt and phosphatic capillary zone electrophoresis, Chinese Journal of Pharmaceuticals, 2015,46 (1):59) determine.In another example, Typically, perchlorate can refer to Shi Yali methods (trace in chromatography of ions-mass Spectrometry for Determination drinking water and environmental water sample Measure perchlorate, assay office, 2007,26 (4):34) determine.Because the present invention is characterized using C1 and C2 relative change The technology of the present invention effect, as long as therefore C1 and C2 have therebetween relative comparativity, to these specific buffer salts The accuracy of measure need not have extra high requirement.Other assay methods of document report are also that can be applicable completely.

Exemplary, in process of the present invention is realized, object elution it can be evaporated as obtained by determining step (1) of the present invention The concentration (it can be designated as D1 in the present invention, and it easily can be with molar concentration meter) of object in part, is then determined Object in clear liquid obtained by step (3) of the present invention concentration (it can be designated as D2 in the present invention, its concentration method for expressing with D1 is identical), the remaining rate (%) of the buffer salt after being handled using the inventive method is then calculated as follows：

Object remnants rates (%)=(D2 ÷ D1) × 100%

It has been found that using the inventive method, the remaining rate (%) of the object can be made to reach more than 70%, such as may be used To reach 70~100% degree, for example, 70~95% degree can be reached, for example, can reach 70~90% degree, 75~90% degree can for example be reached.

The assay method of target concentration is easily realized according to the condition determination of specific product, this below specific Can readily it recognize in example.

The present invention uses simple processing method, it is possible to achieve fixedness buffer salt in the LC eluting fractions of LC-MS methods Concentration is substantially reduced, and target substance will not be lost.

Brief description of the drawings

Fig. 1 is a kind of typical embodiments of syringe available for the inventive method.

Embodiment

The present invention can be further described by the following examples, however, the scope of the present invention is not limited In following embodiments.One of skill in the art, can be with it is understood that on the premise of without departing substantially from the spirit and scope of the present invention Various change and modification are carried out to the present invention.The present invention carries out general to the material and test method that are arrived used in experiment And/or specific description.Although for realize many materials used in the object of the invention and operating method be it is known in the art that But the present invention is still described in detail as far as possible herein.Following examples further illustrate the present invention, rather than limit this hair It is bright.Any only formal rather than substantial equivalent transformation according to made by present inventive concept is regarded as the present invention Technical scheme category.In following embodiment, the specific mineral sulfates used if not otherwise specified, is used Its anhydride.

First, the embodiment of syringe

The present embodiment combination Fig. 1 is illustrated.

Syringe as shown in Figure 1, it can be used in LC-MS analysis test reducing liquid chromatogram eluting fraction The method of middle fixedness buffer salt content, it can also be used to which LC-MS analysis is carried out to the object in detectable substance Method.The syringe includes：Core bar 1, sleeve 2, piston 3, filter 4, cap bag 5.

In some examples, the liquid inlet of the needle stand 6 and filter 4 of the syringe is tightly connected, the filter Liquid outlet is sealed by the cap bag 5.

In some examples, it is pre-filled with auxiliary agent 11 in the sleeve 2 of the syringe.

In some examples, the auxiliary agent 11 is hermetically present in the sleeve 2 of the syringe.

In some examples, the barrel dliameter of the sleeve 2 of the syringe at an upper portion thereof is bigger than the barrel dliameter of the main part of sleeve 2, To form bulb 21.Thus when making piston 3 enter bulb 21 pull-up of core bar 1, due to being deposited between piston 3 and sleeve 2 In space, eluting fraction can be caused to import in (inflow) syringe；Similarly, the organic solvent of optional supplement addition also may be used Added by this mode.Other designs for making eluting fraction (and optional organic solvent) import in (inflow) syringe Apply to the present invention's.Because auxiliary agent 11 exists in the interior sealing of sleeve 2 usually；And when using the syringe, be to core bar 1 Pull-up makes piston 3 enter bulb 21, and this will certainly form vacuum in sleeve 2 and hinder core bar 1 to pull up, and can now open Cap bag 5 is to release this vacuum.

In some examples, the filter 4 is miillpore filter.In some examples, the miillpore filter is that aperture is less than The miillpore filter of 1um miillpore filter, e.g. aperture 0.22um to 1um.In the practical operation of the present invention, aperture is found 0.22um to 1um miillpore filter is all applicable.

In some examples, wherein making the eluting fraction (and optional organic solvent) import in (inflow) syringe Afterwards, to moving down the main part that core bar 1 makes piston 3 return to sleeve 2 so that eluting fraction (and optional organic solvent) with The mixed liquor of auxiliary agent is sealed in sleeve 2 to carry out married operation.To avoid in not sufficiently mixed preceding penetration by liquid filter and Outflow, the exit seal of filter 4 can will be sealed in mixing with cap bag 5.According to present invention description, these operations are very Easily realize.

2nd, LC-MS analysis is tested, and fixedness buffer salt content in reduction liquid chromatogram eluting fraction Embodiment

Following experiment is carried out underneath with syringe shown in Fig. 1.If not otherwise indicated, filter device therefor is aperture 0.45um miillpore filters.

Embodiment 1：The method for removing buffer salt sodium dihydrogen phosphate in mobile phase

Chinese Pharmacopoeia version two page 130 in 2010 has recorded captopril, and needs to check that impurity Kato therein is general Sharp disulphide.This case expedition removes the effect of buffer salt sodium dihydrogen phosphate in mobile phase.

Lucifuge is operated.Captopril bulk drug is taken, it is accurately weighed, plus flow phased soln and quantify dilution and be made in every 1ml Solution containing about 0.5mg, (faces as need testing solution and uses brand-new)；Captopril Disulfide reference substance separately is taken, it is accurately weighed, Plus methanol dissolves in right amount, then the solution being made in every 1ml containing about 5ug is quantitatively diluted with mobile phase, be used as reference substance solution；Take again Captopril and Captopril Disulfide reference substance, plus methanol dissolve in right amount, be made of flowing phase dilution in every 1ml it is each containing about 0.1mg and 15ug mixed solution, is used as system suitability solution.According to high performance liquid chromatography (Chinese Pharmacopoeia 2010 Two annex VD of version) experiment, using octadecylsilane chemically bonded silica as filler；0.01mol/L sodium dihydrogen phosphates-first Alcohol-acetonitrile (70：25：5) it is mobile phase (with phosphorus acid for adjusting pH value to 3.0)；Detection wavelength is 215nm；40 DEG C of column temperature.Take system Employment and suitability test (E ＆ ST) solution 50ul, injects liquid chromatograph, the separating degree between captopril peak and Captopril Disulfide peak It should be greater than 4.0.Reference substance solution 50ul is taken, liquid chromatograph is injected, detection sensitivity is adjusted, makes Captopril Disulfide color The peak height of spectral peak is about the 50% of full scale；Precision measures need testing solution and each 50ul of reference substance solution again, is injected separately into liquid Chromatography, records chromatogram；If any consistent with Captopril Disulfide retention time in the chromatogram of need testing solution Chromatographic peak, by external standard method with calculated by peak area, must not cross 1.0%.It is possible thereby to determine Captopril Disulfide in bulk drug Content.

Then, according to the above method, then precision measures need testing solution 50ul, injects liquid chromatograph, Kato is intercepted respectively The eluting fraction (being designated as fraction A herein) of Puli's chromatographic peak part and the elution of Captopril Disulfide chromatographic peak part evaporate Part (being designated as fraction B herein).For fraction A, determine captopril concentration (being designated as D1) therein and phosphate radical therein is dense Spend (being designated as C1).For the eluting fraction of Captopril Disulfide chromatographic peak part, Kato therein also can be similarly determined Puli's disulphide concentration and phosphate concentration therein.

Then, respectively, by above-mentioned fraction A or fraction B, handled according to present invention below method and step：

(1) detectable substance is configured to solution, the solution is injected into liquid chromatograph to be eluted, from liquid chromatogram elution Liquid collects the object eluting fraction for being ready to use in Mass Spectrometer Method, imports the eluting fraction and is pre-filled with the syringe of auxiliary agent (having completed this step above, gained fraction is fraction A or fraction B respectively)；

(2) in syringe, the eluting fraction and auxiliary agent (magnesium sulfate, eluting fraction and auxiliary agent weight ratio about 1 are made：1) It is sufficiently mixed；

(3) make gained mixed liquor separate suspension and clear liquid by way of filtering, divide and take clear liquid；Determine in clear liquid Captopril concentration or Captopril Disulfide concentration (being designated as D2), and the phosphate concentration (being designated as C2) in clear liquid；

(4) captopril clear liquid obtained by previous step is taken to be directly used in mass spectroscopy, to determine and if captopril It coincide；Mass spectroscopy is used further to after gained Captopril Disulfide clear liquid is carried out into nitrogen drying concentration (to 1/10 volume), To determine if to coincide with Captopril Disulfide.(in the case that when determining MS, sample size is not enough, can be with repeat step (1) to obtain many parts of eluting fractions and merge it, similarly hereinafter).

Embodiment 2：The method for removing buffer salt sodium dihydrogen phosphate in mobile phase

According to embodiment 1, the fraction A wherein obtained or fraction B is handled according to present invention below method and step：

(1) detectable substance is configured to solution, the solution is injected into liquid chromatograph to be eluted, from liquid chromatogram elution Liquid collects the object eluting fraction for being ready to use in Mass Spectrometer Method, imports the eluting fraction and is pre-filled with the syringe of auxiliary agent (having completed this step above, gained fraction is fraction A or fraction B respectively)；

(2) in syringe, the eluting fraction and auxiliary agent (sodium sulphate, eluting fraction and auxiliary agent weight ratio about 1 are made： 0.5) it is sufficiently mixed；

(3) gained mixed liquor is separated suspension and clear liquid by filtering the mode of (miillpore filter, 0.8um), divide and take Clear liquid；Determine the captopril concentration or Captopril Disulfide concentration (being designated as D2) in clear liquid, and the phosphoric acid in clear liquid Root concentration (is designated as C2)；

(4) captopril clear liquid obtained by previous step is taken to be directly used in mass spectroscopy, to determine and if captopril It coincide；Mass spectroscopy is used further to after gained Captopril Disulfide clear liquid is carried out into nitrogen drying concentration (to 1/5 volume), with Determine if to coincide with Captopril Disulfide.

Embodiment 3：The method for removing buffer salt sodium dihydrogen phosphate in mobile phase

According to embodiment 1, the fraction A wherein obtained or fraction B is handled according to present invention below method and step：

(1) detectable substance is configured to solution, the solution is injected into liquid chromatograph to be eluted, from liquid chromatogram elution Liquid collects the object eluting fraction for being ready to use in Mass Spectrometer Method, imports the eluting fraction and is pre-filled with the syringe of auxiliary agent (having completed this step above, gained fraction is fraction A or fraction B respectively)；

(2) in syringe, the eluting fraction and auxiliary agent (calcium sulfate, eluting fraction and auxiliary agent weight ratio about 1 are made：2) Be sufficiently mixed (in step (1) together with eluting fraction in advance into syringe plus acetonitrile (its volume is the 25 of eluting fraction Times))；

(3) gained mixed liquor is separated suspension and clear liquid by filtering the mode of (miillpore filter, 0.22um), divide and take Clear liquid；Determine the captopril concentration or Captopril Disulfide concentration (being designated as D2) in clear liquid, and the phosphoric acid in clear liquid Root concentration (is designated as C2)；

(4) captopril clear liquid obtained by previous step is taken to be directly used in mass spectroscopy, to determine and if captopril It coincide；Mass spectroscopy is used further to after gained Captopril Disulfide clear liquid is carried out into nitrogen drying concentration (to 1/5 volume), with Determine if to coincide with Captopril Disulfide.

Embodiment 4：The method for removing buffer salt sodium dihydrogen phosphate in mobile phase

According to embodiment 1, the fraction A wherein obtained or fraction B is handled according to present invention below method and step：

(1) detectable substance is configured to solution, the solution is injected into liquid chromatograph to be eluted, from liquid chromatogram elution Liquid collects the object eluting fraction for being ready to use in Mass Spectrometer Method, imports the eluting fraction and is pre-filled with the syringe of auxiliary agent (having completed this step above, gained fraction is fraction A or fraction B respectively)；

(2) in syringe, the eluting fraction and auxiliary agent (potassium sulfate, eluting fraction and auxiliary agent weight ratio about 1 are made：1) Be sufficiently mixed (in step (1) together with eluting fraction in advance into syringe plus acetonitrile (its volume is the 10 of eluting fraction Times))；

(3) gained mixed liquor is separated suspension and clear liquid by filtering the mode of (miillpore filter, 0.45um), divide and take Clear liquid；Determine the captopril concentration or Captopril Disulfide concentration (being designated as D2) in clear liquid, and the phosphoric acid in clear liquid Root concentration (is designated as C2)；

(4) captopril clear liquid obtained by previous step is taken to be directly used in mass spectroscopy, to determine and if captopril It coincide；Mass spectroscopy is used further to after gained Captopril Disulfide clear liquid is carried out into nitrogen drying concentration (to 1/5 volume), with Determine if to coincide with Captopril Disulfide.

Embodiment 5：The method for removing buffer salt sodium dihydrogen phosphate in mobile phase

According to embodiment 1, the fraction A wherein obtained or fraction B is handled according to present invention below method and step：

(1) detectable substance is configured to solution, the solution is injected into liquid chromatograph to be eluted, from liquid chromatogram elution Liquid collects the object eluting fraction for being ready to use in Mass Spectrometer Method, imports the eluting fraction and is pre-filled with the syringe of auxiliary agent (having completed this step above, gained fraction is fraction A or fraction B respectively)；

(2) in syringe, making the eluting fraction, (calcium sulphate dihydrate, eluting fraction and auxiliary agent weight ratio are about with auxiliary agent 1：0.5) it is sufficiently mixed；

(3) gained mixed liquor is separated suspension and clear liquid by filtering the mode of (miillpore filter, 0.22um), divide and take Clear liquid；Determine the captopril concentration or Captopril Disulfide concentration (being designated as D2) in clear liquid, and the phosphoric acid in clear liquid Root concentration (is designated as C2)；

(4) captopril clear liquid obtained by previous step is taken to be directly used in mass spectroscopy, to determine and if captopril It coincide；Mass spectroscopy is used further to after gained Captopril Disulfide clear liquid is carried out into nitrogen drying concentration (to 1/10 volume), To determine if to coincide with Captopril Disulfide.

Embodiment 6：The method for removing buffer salt sodium dihydrogen phosphate in mobile phase

According to embodiment 1, the fraction A wherein obtained or fraction B is handled according to present invention below method and step：

(1) detectable substance is configured to solution, the solution is injected into liquid chromatograph to be eluted, from liquid chromatogram elution Liquid collects the object eluting fraction for being ready to use in Mass Spectrometer Method, imports the eluting fraction and is pre-filled with the syringe of auxiliary agent (having completed this step above, gained fraction is fraction A or fraction B respectively)；

(2) in syringe, the eluting fraction and auxiliary agent (magnesium sulfate, eluting fraction and auxiliary agent weight ratio about 1 are made：2) Be sufficiently mixed (in step (1) together with eluting fraction in advance into syringe plus methanol (its volume is the 50 of eluting fraction Times))；

(3) gained mixed liquor is separated suspension and clear liquid by filtering the mode of (miillpore filter, 0.22um), divide and take Clear liquid；Determine the captopril concentration or Captopril Disulfide concentration (being designated as D2) in clear liquid, and the phosphoric acid in clear liquid Root concentration (is designated as C2)；

(4) captopril clear liquid obtained by previous step is taken to be directly used in mass spectroscopy, to determine and if captopril It coincide；Mass spectroscopy is used further to after gained Captopril Disulfide clear liquid is carried out into nitrogen drying concentration (to 1/10 volume), To determine if to coincide with Captopril Disulfide.

Embodiment 7：The method for removing buffer salt sodium dihydrogen phosphate in mobile phase

According to embodiment 1, the fraction A wherein obtained or fraction B is handled according to present invention below method and step：

(1) detectable substance is configured to solution, the solution is injected into liquid chromatograph to be eluted, from liquid chromatogram elution Liquid collects the object eluting fraction for being ready to use in Mass Spectrometer Method, imports the eluting fraction and is pre-filled with the syringe of auxiliary agent (having completed this step above, gained fraction is fraction A or fraction B respectively)；

(2) in syringe, the eluting fraction and auxiliary agent (sodium sulphate, eluting fraction and auxiliary agent weight ratio about 1 are made： 0.5) it is sufficiently mixed；

(3) gained mixed liquor is separated suspension and clear liquid by filtering the mode of (miillpore filter, 0.22um), divide and take Clear liquid；Determine the captopril concentration or Captopril Disulfide concentration (being designated as D2) in clear liquid, and the phosphoric acid in clear liquid Root concentration (is designated as C2)；

(4) captopril clear liquid obtained by previous step is taken to be directly used in mass spectroscopy, to determine and if captopril It coincide；Mass spectroscopy is used further to after gained Captopril Disulfide clear liquid is carried out into nitrogen drying concentration (to 1/10 volume), To determine if to coincide with Captopril Disulfide.

As a result：Above example 1-7, through mass spectroscopy fraction A and fraction B, mass spectral results in each experiment respectively with card Top's profit and Captopril Disulfide coincide；In each experiment, buffer salt remnants rates are for example implemented in the range of 6~28% The remaining rate of the buffer salt of example 1 is 19%；Captopril object remnants rates are in the range of 75%~85%, such as embodiment 2 The remaining rate of captopril object be 83%；Captopril Disulfide object remnants rates are in 73%~87% scope Interior, the remaining rate of Captopril Disulfide object of such as embodiment 3 is 82%.It is clear obtained by above example 1-7 steps (3) Liquid after measured, wherein essentially free of sulfate radical.In the experiment of supplement, according to above example 1-7 method, it is different only It is the aluminum sulfate that sulfate therein is changed to equivalent；As a result the remaining rate of display buffer salt is all higher than 93%.

Embodiment 8：The method for removing mixing buffer salt potassium dihydrogen phosphate and disodium hydrogen phosphate in mobile phase

Chinese Pharmacopoeia version two page 179 in 2010 has recorded the side that Cefodizime Sodium bulk drug content is determined using HPLC methods Method, this case expedition removes the effect that buffer salt is mixed in mobile phase.

It is filler with octadecylsilane chemically bonded silica；With phosphate buffer (take potassium dihydrogen phosphate 0.87g with it is anhydrous Disodium hydrogen phosphate 0.22g, is dissolved in water and is diluted to 1000ml, shake up)-acetonitrile (920：80) it is mobile phase；Detection wavelength is 262nm.Reference substance solution 10ml, plus 0.1mol/L hydrochloric acid solution 1ml are taken, room temperature is placed 24 hours, then adds 0.1mol/L hydrogen-oxygens Change sodium solution 1ml, shake up, take 20ul to inject liquid chromatograph, cephalo crop peak and forward and backward adjacent degradation impurity peak separation Degree should be not less than 3.0 and 4.0 respectively.Determination method：Take this product appropriate, accurately weighed, being dissolved in water and quantifying dilution is made every 1ml In solution containing about Cefodizime 0.1mg, shake up, precision measures 20ul injection liquid chromatographs, records chromatogram；Separately take cephalo Ground piperazine reference substance is appropriate, is measured in the same method.By external standard method with calculated by peak area test sample C20H20N6O7S4 content.Thus method The content of Cefodizime C20H20N6O7S4 in bulk drug test sample can be determined.

Then, according to the above method, then precision measures need testing solution 20ul, injects liquid chromatograph, intercepts Cefodizime The eluting fraction (being designated as fraction A herein) of chromatographic peak part.Determine Cefodizime concentration (being designated as D1) in fraction A and its In phosphate concentration (being designated as C1).

Then, the operation by above-mentioned fraction A respectively according to step (1) to (4) in embodiment 1-7 is handled.

As a result：Through mass spectroscopy, mass spectral results coincide with Cefodizime；Buffer salt remnants rates are in the range of 7~27%, mesh The remaining rate of thing is marked in the range of 72%~89%.

Embodiment 9：The method for removing mixing buffer salt acetate and perchlorate in mobile phase

Chinese Pharmacopoeia version two page 107 in 2010 has recorded the side that lavo-ofloxacin bulk drug content is determined using HPLC methods Method, this case expedition removes the effect of mixing buffer salt acetate and perchlorate in mobile phase.

It is filler with octadecylsilane chemically bonded silica；Ammonium acetate 4.0g and height (are taken with ammonium acetate sodium perchlorate solution Sodium chlorate 7.0g, the 1300ml that adds water makes dissolving, with phosphorus acid for adjusting pH value to 2.2)-acetonitrile (85：15) it is mobile phase；Detection wavelength For 294nm.Weigh lavo-ofloxacin reference substance, Ciprofloxacin reference substance and impurity E reference substance each appropriate, plus 0.1mol/L hydrochloric acid Solution, which dissolves and diluted, is made mixed solution in every 1ml containing about lavo-ofloxacin 0.12mg, Ciprofloxacin and each 6ug of impurity E, Take 10ul to inject liquid chromatograph, record chromatogram, the retention time at lavo-ofloxacin peak is about 15 minutes, lavo-ofloxacin peak It should be respectively greater than 2.0 and 2.5 with the separating degree at impurity E peak and lavo-ofloxacin peak and Ciprofloxacin peak.Take lavo-ofloxacin former Expect medicine about 60mg, it is accurately weighed, put in 50ml measuring bottles, plus 0.1mol/L hydrochloric acid solutions dissolve and are quantitatively diluted to scale, shake up, Precision measures 5ml, puts in 50ml measuring bottles, is diluted to scale with 0.1mol/L hydrochloric acid solutions, shakes up, and precision measures 10ul injection liquid Chromatography, records chromatogram；It is another to take lavo-ofloxacin reference substance appropriate, it is measured in the same method, it is left with calculated by peak area by external standard method Ofloxacin content, is produced.Thus method can determine the content of lavo-ofloxacin in bulk drug test sample.

Then, according to the above method, then precision measures need testing solution 20ul, injects liquid chromatograph, intercepts levofloxacin The eluting fraction (being designated as fraction A herein) of star valley point.Determine levofloxacin concentration (being designated as D1) in fraction A and its In acetate concentration (being designated as C1) and perchlorate's concentration (being designated as C2).

Then, the operation by above-mentioned fraction A respectively according to step (1) to (4) in embodiment 1~4 is handled.

As a result：Through mass spectroscopy, mass spectral results coincide with lavo-ofloxacin；Acetate remnants rates are in 11~29% scopes Interior, perchlorate remnants rates are in the range of 7~24%, and object remnants rates are in the range of 73%~88%.

Embodiment 10：The method for removing mixing buffer salt acetate and perchlorate in mobile phase

Chinese Pharmacopoeia version two page 221 in 2010 has recorded the side that Sparfloxacin bulk drug content is determined using HPLC methods Method, this case expedition removes the effect of buffer salt citrate in mobile phase.

It is filler with octadecylsilane chemically bonded silica；Citric acid 2.104g and Chinese holly (are weighed with sodium citrate buffer Rafter acid sodium 2.941g, adds water to 500ml, and pH value is adjusted to 2.4)-acetonitrile (70 with 70% perchloric acid solution：30) it is mobile phase； Detection wavelength is 298nm.Take Sparfloxacin reference substance appropriate, plus flowing phased soln and dilute that every 1ml is made is molten containing about 0.1mg Liquid, irradiates 20 hours under 4500lx illumination, as system suitability solution, measures 10ul note people's liquid chromatographs, Chromatogram is recorded, Sparfloxacin peak retention time is about 7 minutes, and retention time is about at 0.9 corresponding thereto at Sparfloxacin peak The separating degree of impurity peaks should meet the requirements.

Sparfloxacin bulk drug about 50mg is taken, it is accurately weighed, put in 100ml measuring bottles, plus appropriate methanol, shake well makes molten Solution, with methanol dilution to scale, shakes up, precision measures 2ml, puts in 25ml measuring bottles, scale is diluted to mobile phase, shake up, essence It is close to measure 20ul note people's liquid chromatographs, record chromatogram；It is another to take Sparfloxacin reference substance appropriate, it is measured in the same method, by external standard method With calculated by peak area, produce.Thus method can determine the content of Sparfloxacin in bulk drug test sample.

Then, according to the above method, then precision measures need testing solution 20ul, injects liquid chromatograph, intercepts Sparfloxacin The eluting fraction (being designated as fraction A herein) of valley point.Determine Sparfloxacin concentration (being designated as D1) in fraction A and therein Citric acid radical concentration (is designated as C1).

Then, the operation by above-mentioned fraction A respectively according to step (1) to (4) in embodiment 1~4 is handled.

As a result：Through mass spectroscopy, mass spectral results coincide with Sparfloxacin；Citrate remnants rate in the range of 8~26%, Object remnants rates are in the range of 72%~89%.

Experiment of the invention by foregoing exemplary, using simple processing method, it is possible to achieve the LC elutions of LC-MS methods Fixedness buffer salinity is substantially reduced in fraction, and target substance will not be lost.This is determined to MS later Extremely beneficial.

The application is described in detail by the various aspects above to the application, but the application is not limited to this states These specific embodiments, spirit and scope should be defined by appended claims.

Claims (9)

1. a kind of captopril for RPLC-mass spectral analysis test bag impurity containing Captopril Disulfide The method of fixedness buffer salt content in liquid chromatogram eluting fraction is reduced during bulk drug, this method comprises the following steps：

(1) detectable substance is configured to solution, the solution is injected into liquid chromatograph to be eluted, received from liquid chromatography eluant Collection is ready to use in the object eluting fraction of Mass Spectrometer Method, imports the eluting fraction and is pre-filled with the syringe of auxiliary agent；Institute State eluting fraction and import the operation that following (1a) to (1d) is specifically included before syringe：

(1a) lucifuge is operated；Captopril bulk drug is taken, it is accurately weighed, plus flow phased soln and quantify dilution and be made in every 1ml Solution containing 0.5mg, as need testing solution, the need testing solution, which faces, uses brand-new；Captopril Disulfide reference substance separately is taken, It is accurately weighed, plus methanol dissolves in right amount, then quantitatively diluted with mobile phase the solution containing 5g in every 1ml is made, it is molten as reference substance Liquid；Take captopril and Captopril Disulfide reference substance, plus methanol to dissolve in right amount again, every 1ml is made with flowing phase dilution In each mixed solution containing 0.1mg and 15 μ g, be used as system suitability solution；

(1b) is tested according to the specification of the Chinese Pharmacopoeia two contained high performance liquid chromatographies of annex VD of version in 2010, with octadecane Base silane bonded silica gel is filler；Using volume ratio as 70：25：5 0.01mol/L sodium dihydrogen phosphates-methanol-acetonitrile is Mobile phase, mobile phase phosphorus acid for adjusting pH value to 3.0；Detection wavelength is 215nm；40 °C of column temperature；Take system suitability The μ l of solution 50, inject liquid chromatograph, and the separating degree between captopril peak and Captopril Disulfide peak should be greater than 4.0；

(1c) takes the μ l of reference substance solution 50, injects liquid chromatograph, adjusts detection sensitivity, makes Captopril Disulfide chromatogram The peak height at peak is the 50% of full scale；Precision measures need testing solution and each 50 μ l of reference substance solution again, is injected separately into liquid chromatogram Instrument, records chromatogram；If any the chromatographic peak consistent with Captopril Disulfide retention time in the chromatogram of need testing solution, By external standard method with the content of Captopril Disulfide in calculated by peak area bulk drug；

(1d) precision measures the μ l of need testing solution 50 again, injects liquid chromatograph, captopril chromatographic peak part is intercepted respectively It is designated as fraction A eluting fraction and the eluting fraction for being designated as fraction B of Captopril Disulfide chromatographic peak part；For evaporating Part A, determining captopril concentration therein, it is designated as D1 and phosphate concentration therein it is designated as C1；For captopril two The eluting fraction of sulfide chromatographic peak part, similarly determines Captopril Disulfide concentration therein and phosphoric acid therein Root concentration；

(2) in syringe, the eluting fraction is made to be sufficiently mixed with auxiliary agent；

(3) make gained mixed liquor separate suspension and clear liquid by way of filtering, divide and take clear liquid；Determine the Kato in clear liquid Puli or the concentration for being designated as D2 of Captopril Disulfide, and C2 phosphate concentration is designated as in clear liquid；With,

(4) captopril clear liquid obtained by taking step (3) is directly used in mass spectroscopy, to determine if to coincide with captopril； Gained Captopril Disulfide clear liquid is carried out to be used further to mass spectroscopy after nitrogen drying is concentrated into 1/10 volume, to determine it Whether it is coincide with Captopril Disulfide,

Wherein,

The auxiliary agent is magnesium sulfate, and eluting fraction is 1 with auxiliary agent weight ratio：1；

Clear liquid obtained by step (3) after measured, wherein essentially free of sulfate radical.

2. method according to claim 1, the auxiliary agent is sodium sulphate, eluting fraction is 1 with auxiliary agent weight ratio：0.5.

3. method according to claim 1, the syringe includes：Core bar, sleeve, piston, filter, cap bag；The injection The needle stand of device and the liquid inlet of filter are tightly connected, and the liquid outlet of the filter is sealed by the cap bag；The auxiliary agent is close Feud is present in the sleeve of the syringe, and the filter is the miillpore filter in 0.22 μm to 1 μm of aperture.

4. method according to claim 3, barrel dliameter of the barrel dliameter than sleeve main body section of the sleeve of the syringe at an upper portion thereof Greatly, to form bulb.

5. method according to claim 1, the auxiliary agent is calcium sulfate, eluting fraction is 1 with auxiliary agent weight ratio：2.

6. method according to claim 1, the auxiliary agent is potassium sulfate, eluting fraction is 1 with auxiliary agent weight ratio：1.

7. method according to claim 1, the auxiliary agent is calcium sulphate dihydrate, eluting fraction is 1 with auxiliary agent weight ratio：0.5.

8. method according to claim 1, the auxiliary agent is magnesium sulfate, eluting fraction is 1 with auxiliary agent weight ratio：2.

9. method according to claim 1, the auxiliary agent is sodium sulphate, eluting fraction is 1 with auxiliary agent weight ratio：0.5.