CN105974033A - Method for detecting Tipracil or relevant hydrochloride matter thereof through high performance liquid chromatography - Google Patents
Method for detecting Tipracil or relevant hydrochloride matter thereof through high performance liquid chromatography Download PDFInfo
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- CN105974033A CN105974033A CN201610597277.6A CN201610597277A CN105974033A CN 105974033 A CN105974033 A CN 105974033A CN 201610597277 A CN201610597277 A CN 201610597277A CN 105974033 A CN105974033 A CN 105974033A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N2030/027—Liquid chromatography
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Abstract
The invention provides a method for detecting Tipracil or relevant hydrochloride matter thereof through high performance liquid chromatography. The method comprises the steps that a relevant matter Tipracil or hydrochloride thereof containing sample is prepared into a detection solution with diluents, octadecylsilane chemically bonded silica gel is adopted as a fixed phase, an ion-pairing agent containing inorganic salt buffer solution-organic phase is adopted as a mobile phase, gradient elution is carried out, and an HPLC spectrogram is recorded. Compared with the prior art, the octadecylsilane chemically bonded silica gel is adopted as a chromatographic column of filler, the ion-pairing agent containing inorganic salt buffer solution system-organic phase is adopted as the mobile phase, impurities possibly existing in Tipracil or hydrochloride thereof can be effectively separated and quantitatively determined, therefore, the quality of Tipracil or a hydrochloride product thereof is effectively controlled, high specificity is achieved, high sensitivity is achieved, and good accuracy is achieved.
Description
Technical field
The invention belongs to analysis of compounds technical field, particularly relate to a kind of utilization high performance liquid chromatography detection
The method that Tipracil or its hydrochlorate have related substance.
Background technology
Tipracil or its pharmaceutically acceptable salt are as the effective ingredient of therapeutic agent for inflammatory bowel disease, permissible
The effectively therapeutic agent for inflammatory bowel disease including ulcerative colitis and Crohn disease of safety is provided.
Tipracil hydrochlorate (TPI) can combine with nucleoside analog FTD (α, α, α-trifluorothymidine), wherein,
Trifluorothymidine is a kind of ucleosides anticarcinogen, and the synthesis of interference cancerous cell DNA, Tipracil is thymidine phosphates
Change enzyme inhibitor, the degraded of trifluorothymidine can be reduced, though a combination thereof be a kind of the most also
Stable can the medical composition (TAS-102) of oral administration, type or recurrence can not be excised for treatment
Type advanced colorectal cancer.
On March 24th, 2014, the TAS-102 of roc Pharmaceutical obtains Japan's MHLW approval, JIUYUE 22 in 2015
Day, obtain FDA approval listing.The chemical entitled 5-of Tipracil hydrochlorate (TPI) chloro-6-(2-imino group pyrroles
Alkane-1-base) methyl-2,4 (1H, 3H)-hybar X hydrochlorates；No. CAS is 183204-72-0；Point
Minor is C9H11ClN4O2·HCl；Structural formula is as follows:
During synthesizing this compound, the intermediate of key is had to take to follow-up owing to reaction is incomplete
Reaction in, reaction of formation by-product；There is the byproduct of reaction introduced owing to initiation material purity is the highest；
There is the catabolite produced by medicine auto-degradation.More than belong to this medicine has related substance, needs to carry out
Strict quality control, its 8 impurity structural formulas comprised are shown in Table 1.
The structural formula of impurity in table 1Tipracil or its hydrochlorate
For in Tipracil hydrochlorate synthetic route introduce impurity and catabolite, crude drug need into
The quality control that row is strict.Therefore, it is achieved the separation of potential impurity all to Tipracil hydrochlorate, exist
The process control of Tipracil hydrochlorate synthesis technique, and the quality control of crude drug is respectively provided with extremely important
Meaning.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is that providing a kind of uses high performance liquid chromatography
The method that detection Tipracil or its hydrochlorate have related substance, the method can be deposited in Tipracil or its hydrochlorate
Impurity be effectively separated and quantitative determine, thus effectively control Tipracil or its hydrochlorate product
Quality, specificity is strong, and highly sensitive, accuracy is good.
The invention provides a kind of utilization high performance liquid chromatography detection Tipracil or its hydrochlorate has related substance
Method, including:
Tipracil containing related substance or its hydrochloride sample diluent preparing are become detection solution, with ten
Eight alkyl silane keys and silica gel are fixing phase, use the inorganic salt buffer-organic facies containing ion-pairing agent
Mutually and carry out gradient elution for flowing, record HPLC spectrogram.
Preferably, the Tipracil containing related substance in described detection solution or the concentration of its hydrochloride sample are
0.001～1.0mg/ml.
Preferably, the inorganic salt in described inorganic salt buffer is potassium dihydrogen phosphate, dipotassium hydrogen phosphate, phosphorus
Acid dihydride sodium, disodium hydrogen phosphate, ammonium dihydrogen phosphate, diammonium phosphate and one or more in ammonium acetate；
Described organic facies is methanol and/or acetonitrile.
Preferably, described ion-pairing agent is sodium heptanesulfonate and/or perfluorooctane sulfonate.
Preferably, in described inorganic salt buffer, the concentration of inorganic salt is 0.002～0.05mol/L；Described nothing
The pH value range of machine salt buffer is 2.5～6.0.
Preferably, the concentration of reagent is by the described inorganic salt buffer intermediate ion containing ion-pairing agent
1.0～1.2g/L.
Preferably, the flow velocity of described flowing phase is 0.8～1.2ml/min.
Preferably, the column temperature of described fixing phase is 25 DEG C～45 DEG C.
Preferably, in described gradient elution, the inorganic salt buffer containing ion-pairing agent is mobile phase A,
Organic facies is Mobile phase B, and with volume percent, elution program is: 0～30min, 90%～98%A；
30～45min, 50%～70%A；45～60min, 90%～98%A.
Preferably, described HPLC spectrogram is the HPLC spectrogram at detection wavelength 210～282nm record.
The invention provides a kind of utilization high performance liquid chromatography detection Tipracil or its hydrochlorate has related substance
Method, including: the Tipracil containing related substance or its hydrochloride sample diluent preparing is become detection
Solution, with octadecylsilane key and silica gel for fixing phase, uses the inorganic salt containing ion-pairing agent to delay
Rush liquid-organic facies and mutually and carry out gradient elution for flowing, record HPLC spectrogram.Compared with prior art,
The present invention is the chromatographic column as filler with octadecylsilane key and silica gel, inorganic with containing ion-pairing agent
Salt buffer system-organic facies is flowing phase, can be to Tipracil or its hydrochlorate impurity that may be present
It is effectively separated and quantitative determines, thus effectively control Tipracil or the quality of its hydrochlorate product, specially
Attribute is strong, and highly sensitive, accuracy is good.
Accompanying drawing explanation
Fig. 1 is the chromatogram of the embodiment of the present invention 1 empty solvent；
Fig. 2 is the chromatogram of mixed solution in the embodiment of the present invention 1；
Fig. 3 is the chromatogram of Tipracil hydrochloride, crude in the embodiment of the present invention 2；
Fig. 4 is the chromatogram not destroying sample in the embodiment of the present invention 3；
Fig. 5 is the chromatogram that in the embodiment of the present invention 3, acid destroys sample；
Fig. 6 is the chromatogram that in the embodiment of the present invention 3, alkali destroys sample；
Fig. 7 is the chromatogram of Oxidative demage sample in the embodiment of the present invention 3；
Fig. 8 is the chromatogram of high temperature sample in the embodiment of the present invention 3；
Fig. 9 is the chromatogram that in the embodiment of the present invention 3, illumination destroys sample；
Figure 10 is the chromatogram of Tipracil hydrochloride, crude in the embodiment of the present invention 4.
Detailed description of the invention
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Describe, it is clear that described embodiment is only a part of embodiment of the present invention rather than all wholely
Embodiment.Based on the embodiment in the present invention, those of ordinary skill in the art are not making creativeness
The every other embodiment obtained under work premise, broadly falls into the scope of protection of the invention.
The invention provides a kind of utilization high performance liquid chromatography detection Tipracil or its hydrochlorate has related substance
Method, including: by the sample of the Tipracil containing related substance or its hydrochlorate with diluent preparing become inspection
Surveying solution, with octadecylsilane key and silica gel for fixing phase, employing contains the inorganic salt of ion-pairing agent
Buffer-organic facies mutually and carries out gradient elution for flowing, records HPLC spectrogram.
Wherein, the diluent that described diluent is well known to those skilled in the art, there is no special limit
System, the present invention is preferably water, inorganic salt buffer, the mixed solution of acetonitrile and inorganic salt buffer,
Methanol and the mixed solution of inorganic salt buffer, the aqueous solution of acetonitrile or the aqueous solution of methanol；Such as diluent
For inorganic salt buffer or the mixed solution containing inorganic salt buffer, inorganic salt buffer therein is preferred
For the inorganic salt buffer mutually consistent with flowing；The mixed solution of described acetonitrile and inorganic salt buffer and
Methanol is preferably more than with the maximum volume concentration of methanol in the mixed solution of inorganic salt buffer or acetonitrile
40%；In the present invention, most preferably, using water as diluent.
Tipracil containing related substance or its hydrochloride sample diluent preparing are become detection solution；Described
The Tipracil containing related substance in detection solution or the concentration of its hydrochloride sample are preferably 0.001～1.0
Mg/ml, more preferably 0.01～1.0mg/L, be further preferably 0.1～1.0mg/L.
Detection solution high performance liquid chromatography being detected, its sample size is preferably 10～20 μ l.
In the present invention, described high performance liquid chromatography is with octadecylsilane key and silica gel for fixing phase, preferably
For CNW Athena C18-WP or YMC-Pack ODS-A；Its specification is preferably 4.6 × 150mm,
3μm。
Inorganic salt buffer-the organic facies containing ion-pairing agent is used mutually and to carry out gradient elution for flowing；
The ion-pairing agent that described ion-pairing agent is well known to those skilled in the art, there is no special restriction,
The present invention is preferably sodium heptanesulfonate and/or perfluorooctane sulfonate, more preferably sodium heptanesulfonate；Described contain
The inorganic salt buffer intermediate ion having ion-pairing agent is preferably 1.0～1.2g/L to the concentration of reagent, more excellent
Elect 1.1g/L as；What the inorganic salt in described inorganic salt buffer was well known to those skilled in the art can conduct
The inorganic salt of buffer reagent, there is no special restriction, is preferably potassium dihydrogen phosphate, phosphorus in the present invention
In acid hydrogen dipotassium, sodium dihydrogen phosphate, disodium hydrogen phosphate, ammonium dihydrogen phosphate, diammonium phosphate and ammonium acetate
One or more, more preferably potassium dihydrogen phosphate；In described inorganic salt buffer, the concentration of inorganic salt is excellent
Electing 0.002～0.05mol/L as, more preferably 0.005～0.03mol/L, be further preferably 0.01～0.02mol/L,
Most preferably 0.01mol/L；The pH value of described inorganic salt buffer is preferably 2.5～6.0, more preferably
3～5, it is further preferably 4～5, most preferably 4.6；Organic facies in described flowing mutually be preferably methanol and/or
Acetonitrile, more preferably acetonitrile.
The present invention uses the buffer salt system adding ion pair as flowing phase, solves Tipracil or its hydrochloric acid
Salt and have the separation determination problem of related substance, ensures that the quality of Tipracil or its hydrochlorate can
Control.
In the present invention, the flow velocity of described flowing phase is preferably 0.8～1.2ml/min, more preferably 0.9～1.1
Ml/min, is further preferably 1.0ml/min；The column temperature of described fixing phase is preferably 25 DEG C～45 DEG C, more preferably
It is 30 DEG C～40 DEG C, is further preferably 35 DEG C～40 DEG C, most preferably 40 DEG C.
The present invention uses gradient elution, the inorganic salt buffering in described gradient elution, containing ion-pairing agent
Liquid is mobile phase A, and organic facies is Mobile phase B, and with volume percent, elution program is preferably: 0～30
Min, 90%～98%A；30～45min, 50%～70%A；45～60min, 90%～98%A；More excellent
Elect 0～30min, 92%～96%A as；30～45min, 55%～65%A；46～60min, 92%～96%A；
It is further preferably 0～30min, 92%A；30～45min, 60%A；46～60min, 92%A or 0～30min,
96%A；30～45min, 60%A；46～60min, 96%A.
After gradient elution, record HPLC spectrogram, preferably record HPLC at detection wavelength 210～282nm
Spectrogram.
More specifically, the present invention can realize in accordance with the following methods:
High performance liquid chromatograph: Shimadzu LC-20AT, PDA detector
Chromatographic column: CNW Athena C18-WP, 4.6 × 150mm, 3 μm
Mobile phase A: (1.36g potassium dihydrogen phosphate, 1.1g sodium heptanesulfonate add water phosphate buffer
1000ml)
Mobile phase B: acetonitrile
Detection wavelength: 210nm
Flow velocity: 1.0ml/min
Sample size: 10 μ l
Column temperature: 40 DEG C
Gradient program is shown in Table 2.
Table 2 gradient elution program
Diluent: water
More specifically, the present invention can realize in accordance with the following methods:
Chromatographic column: YMC-Pack ODS-A, 4.6 × 150mm, 3 μm
Column temperature: 40 DEG C
Flow velocity: 1.0ml/min
Sample size: 10 μ l
Detection wavelength: 210nm
Mobile phase A: (1.36g potassium dihydrogen phosphate, 1.1g sodium heptanesulfonate add water phosphate buffer
1000ml)
Mobile phase B: acetonitrile
Gradient elution degree is shown in Table 3.
Table 3 chromatographic column is gradient elution program table during YMC-Pack ODS-A
Diluent: water
In the present invention, described " Tipracil or its hydrochlorate " is in the case of the most clearly distinguishing, permissible
It is the composite of Tipracil, sterling, crude drug, the composite of Tipracil hydrochlorate, sterling, raw material
Medicines etc. are using Tipracil as the product of main constituent.The assay method of the present invention is all applicable to the matter of the said goods
Amount controls, and the assay method having related substance of these products is also protected within the scope of the present invention.
For existing document to Tipracil or its hydrochloride Starting material and the disappearance of preparation impurity identification research thereof,
The present invention by process trial, force Degrading experiment Tipracil hydrochloride impurities to be enriched with, separates and carry
Pure, identify 8 main known impurities, and impurity has been carried out ownership of tracing to the source, including Tipracil salt
The initiation material of hydrochlorate and catalyst (SM1, SM2, DBU), synthetic intermediate (TPI-1), Tipracil
Hydrochlorate synthesising by-product (impurity A, impurity B) and degradation impurity (impurity C, impurity D).As
Contrast, separates each known impurities by isocratic elution, but between TPI-1 and SM2, separating degree is undesirable.
The present invention uses linear gradient elution method, through to flowing phase composition and the optimal screening of ratio, determining analysis method.
Carry out specificity by Chinese Pharmacopoeia the 4th general rule 0512 (high performance liquid chromatography) of version in 2015 simultaneously
Checking, result shows that the method can have by impurity that may be present all to Tipracil or its hydrochlorate
Effect separates and quantitative determines, and has related substance research to provide reliable impurity spectrum for Tipracil or its hydrochlorate
Reference, has the most progressive bigger effect and actual application value.
From above technical scheme, the present invention is the chromatographic column as filler with octadecylsilane key and silica gel,
With the inorganic salt buffer system-organic facies containing ion-pairing agent for flowing phase, can to Tipracil or
Its hydrochlorate impurity that may be present is effectively separated and quantitative determines, thus effectively control Tipracil or
The quality of its hydrochlorate product, specificity is strong, and highly sensitive, accuracy is good.
For the present invention furtherly, a kind of use high-efficient liquid below in conjunction with what the present invention was provided by embodiment
Phase chromatograph detection Tipracil or its hydrochlorate have the method for related substance to be described in detail.
Reagent used in following example is commercially available.
Embodiment 1
Instrument: Shimadzu LC-20AT high performance liquid chromatograph and work station thereof, PDA detector
Chromatographic column: CNW Athena C18-WP, 4.6 × 150mm, 3 μm
Mobile phase A: (1.36g potassium dihydrogen phosphate, 1.1g sodium heptanesulfonate add water phosphate buffer
1000ml)
Mobile phase B: acetonitrile
Detection wavelength: 210nm
Flow velocity: 1.0mL/min
Sample size: 10 μ L
Column temperature: 40 DEG C
Gradient elution program is shown in Table 4.
Table 4 embodiment 1 gradient elution program
Diluent: water
Take Tipracil hydrochlorate, impurity A, impurity B, impurity C, SM1, SM2, TPI-1 respectively
Appropriate with DBU, add diluent and make the hydrochlorate containing Tipracil and be about 1mg/mL, remaining each material list
Body is about the mixed solution of 1 μ g/mL.
Take blank solvent i.e. diluent and mixed solution, be measured by above-mentioned chromatographic condition, record chromatograph
Figure.
Typical color spectrogram is shown in Fig. 1, Fig. 2.
In Fig. 2 retention time be the chromatographic peak of 10.897min be Tipracil hydrochlorate chromatographic peak, remaining is each
Peak is the chromatographic peak of 7 impurity of Tipracil hydrochlorate, in figure each chromatographic peak be followed successively by SM1, impurity C,
Impurity B, SM2, TPI-1, Tipracil hydrochlorate, impurity A, DBU.As can be seen from Figure, empty
White solvent not interference measurement, introduces impurity, non-sample impurity for DUB between impurity A and DBU；Tipracil
Hydrochlorate all can reach good separation with its each impurity, meets the requirement of Chinese Pharmacopoeia.
Embodiment 2
Instrument: Shimadzu LC-20AT high performance liquid chromatograph and work station thereof, PDA detector
Chromatographic column: CNW Athena C18-WP, 4.6 × 150mm, 3 μm
Mobile phase A: (1.36g potassium dihydrogen phosphate, 1.1g sodium heptanesulfonate add water phosphate buffer
1000ml)
Mobile phase B: acetonitrile
Detection wavelength: 210nm
Flow velocity: 1.0mL/min
Sample size: 10 μ L
Column temperature: 40 DEG C
Gradient elution program is shown in Table 5.
Table 5 embodiment 2 gradient elution program
Diluent: water
Take Tipracil hydrochloride, crude, add diluent and make the hydrochlorate containing Tipracil and be about 1mg/mL's
Crude product solution.
Take Tipracil hydrochloride, crude solution, be measured by above-mentioned chromatographic condition, record chromatogram.
Typical color spectrogram is shown in Fig. 3.
In Fig. 3, retention time be the chromatographic peak of 10.844min be Tipracil hydrochlorate chromatographic peak, 7.266
Min be impurity C, 9.309min be SM2,9.845min be intermediate 1,16.129min be impurity
A, other be unknown impuritie.Tipracil hydrochlorate all can reach good separation with its each impurity, meets
The requirement of Chinese Pharmacopoeia.
Embodiment 3
Instrument: Shimadzu LC-20AT high performance liquid chromatograph and work station thereof, PDA detector
Chromatographic column: CNW Athena C18-WP, 4.6 × 150mm, 3 μm
Mobile phase A: (1.36g potassium dihydrogen phosphate, 1.1g sodium heptanesulfonate add water phosphate buffer
1000ml)
Mobile phase B: acetonitrile
Detection wavelength: 210nm
Flow velocity: 1.0mL/min
Sample size: 10 μ L
Column temperature: 40 DEG C
Gradient elution program is shown in Table 6.
Table 6 embodiment 3 gradient elution program
Diluent: water
Do not destroy: take Tipracil hydrochlorate 10mg, put in 10ml measuring bottle, add diluent and dissolve and determine
Hold to scale, as not destroying sample.
Acid destroys: takes Tipracil hydrochlorate 10mg, puts in 10ml measuring bottle, adds 0.1mol/L hydrochloric acid
Solution 2ml, puts 60 DEG C of water-baths 30 minutes, adds 0.1mol/L sodium hydroxide solution 2ml and neutralizes, stands
To room temperature, add diluted to scale, shake up, destroy sample as acid.
Alkali destroys: takes Tipracil hydrochlorate 10mg, puts in 10ml measuring bottle, adds 0.1mol/L hydrogen-oxygen
Change sodium solution 2ml, put 60 DEG C of water-baths 2 minutes, add 0.1mol/L hydrochloric acid solution 2ml and neutralize, stand
To room temperature, add diluted to scale, shake up, destroy sample as alkali.
Oxidative demage: take Tipracil hydrochlorate 10mg, put in 10ml measuring bottle, add 3% hydrogen peroxide
Solution 2ml, puts 60 DEG C of water-baths 2 hours, stands to room temperature, adds diluted to scale, shakes up,
As Oxidative demage sample.
High temperature: take Tipracil hydrochlorate 10mg, put in 10ml measuring bottle, under the conditions of putting 60 DEG C 6
Hour, stand to room temperature, add diluent and dissolve and be settled to scale, as high temperature sample.
Illumination destroys: takes Tipracil hydrochlorate 10mg, puts in 10ml measuring bottle, puts 5000 ± 500l ux
Under the conditions of 10 days, add diluent and dissolve and be settled to scale, as illumination destroy sample.
Take above-mentioned solution respectively, be measured by above-mentioned chromatographic condition, record chromatogram.
Typical color spectrogram is shown in Fig. 4 to Fig. 9, and wherein Fig. 4 is the chromatogram not destroying sample；Fig. 5 is that acid is broken
The chromatogram of bad sample；Fig. 6 is the chromatogram that alkali destroys sample；Fig. 7 is the chromatograph of Oxidative demage sample
Figure, wherein in Fig. 7, B figure is the partial enlarged drawing of A figure；Fig. 8 is the chromatogram of high temperature sample；
Fig. 9 is the chromatogram that illumination destroys sample.
In Fig. 4 retention time be the chromatographic peak of 11.345min be Tipracil hydrochlorate chromatographic peak, each peak divides
Good from degree.
In Fig. 5 retention time be the chromatographic peak of 11.312min be Tipracil hydrochlorate chromatographic peak, newly-increased main
Wanting two degradation impurity, retention time is respectively 5.563min, 7.632min, and wherein 7.632min is
Impurity C.Each peak separating degree is good.
In Fig. 6 retention time be the chromatographic peak of 11.323min be Tipracil hydrochlorate chromatographic peak, newly-increased two
Individual principal degradation impurity, retention time is respectively 5.824min, 7.647min, and wherein 7.647min is
Impurity C.Each peak separating degree is good.
In Fig. 7 retention time be the chromatographic peak of 11.343min be Tipracil hydrochlorate chromatographic peak, newly-increased three
Individual principal degradation impurity, retention time is respectively 6.619min, 7.115min, 7.651min, and wherein 7.115
Min is impurity D, and 7.651min is impurity C.Each major impurity peak separating degree is good.
Without the most newly-increased impurity in Fig. 8 and Fig. 9.Each peak separating degree is good.
Fig. 1 to Fig. 9 shows, the method for the present invention is easy, quick, accuracy is high, can clearly by
Tipracil hydrochlorate is each known with it or unknown impuritie good separation, thus effectively control Tipracil or
The quality of its hydrochlorate product.
Embodiment 4
Instrument: Shimadzu LC-6AD high performance liquid chromatograph and work station thereof, UV-detector
Chromatographic column: YMC-Pack ODS-A, 4.6 × 150mm, 3 μm
Column temperature: 40 DEG C
Flow velocity: 1.0ml/min
Sample size: 10 μ l
Detection wavelength: 210nm
Mobile phase A: (1.36g potassium dihydrogen phosphate, 1.1g sodium heptanesulfonate add water phosphate buffer
1000ml)
Mobile phase B: acetonitrile
Gradient elution program is shown in Table 7.
Table 7 embodiment 4 gradient elution program
Diluent: water
Take Tipracil hydrochloride, crude, add diluent and make the hydrochlorate containing Tipracil and be about 1mg/mL's
Crude product solution.
Take Tipracil hydrochloride, crude solution, be measured by above-mentioned chromatographic condition, record chromatogram.
Typical color spectrogram is shown in Figure 10, and wherein in Figure 10, B figure is the partial enlarged drawing of A figure.
In Figure 10, retention time be the chromatographic peak of 13.378min be Tipracil hydrochlorate chromatographic peak, 9.636
Min be impurity C, 11.451min be impurity B, 11.676min be intermediate 1,12.346min be SM2,
17.756min be impurity A, other be unknown impuritie.Tipracil hydrochlorate impurity each with it all can reach
Good separation, meets the requirement of Chinese Pharmacopoeia.
Claims (10)
1. use the method that high performance liquid chromatography detection Tipracil or its hydrochlorate have related substance, its
It is characterised by, including:
Tipracil containing related substance or its hydrochloride sample diluent preparing are become detection solution, with ten
Eight alkyl silane keys and silica gel are fixing phase, use the inorganic salt buffer-organic facies containing ion-pairing agent
Mutually and carry out gradient elution for flowing, record HPLC spectrogram.
Method the most according to claim 1, it is characterised in that containing relevant thing in described detection solution
The concentration of the Tipracil of matter or its hydrochloride sample is 0.001～1.0mg/ml.
Method the most according to claim 1, it is characterised in that the nothing in described inorganic salt buffer
Machine salt be potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, ammonium dihydrogen phosphate,
Diammonium phosphate and one or more in ammonium acetate；Described organic facies is methanol and/or acetonitrile.
Method the most according to claim 1, it is characterised in that described ion-pairing agent is heptane sulphur
Acid sodium and/or perfluorooctane sulfonate.
Method the most according to claim 1, it is characterised in that inorganic in described inorganic salt buffer
The concentration of salt is 0.002～0.05mol/L；The pH value range of described inorganic salt buffer is 2.5～6.0.
Method the most according to claim 1, it is characterised in that the described nothing containing ion-pairing agent
Machine salt buffer intermediate ion is 1.0～1.2g/L to the concentration of reagent.
Method the most according to claim 1, it is characterised in that the flow velocity of described flowing phase is 0.8～1.2
ml/min。
Method the most according to claim 1, it is characterised in that the column temperature of described fixing phase is 25 DEG C
～45 DEG C.
Method the most according to claim 1, it is characterised in that in described gradient elution, containing from
Son is mobile phase A to the inorganic salt buffer of reagent, and organic facies is Mobile phase B, with volume percent,
Elution program is: 0～30min, 90%～98%A；30～45min, 50%～70%A；45～60min,
90%～98%A.
Method the most according to claim 1, it is characterised in that described HPLC spectrogram is in inspection
Survey the HPLC spectrogram of wavelength 210～282nm record.
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Citations (1)
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|---|---|---|---|---|
| CN106018573A (en) * | 2016-03-16 | 2016-10-12 | 江苏悦兴药业有限公司 | Method for detecting purity of tipiracil hydrochloride in TAS-102 |
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2016
- 2016-07-26 CN CN201610597277.6A patent/CN105974033A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106018573A (en) * | 2016-03-16 | 2016-10-12 | 江苏悦兴药业有限公司 | Method for detecting purity of tipiracil hydrochloride in TAS-102 |
Non-Patent Citations (1)
| Title |
|---|
| 陆丹慧等: "盐酸替哌拉索杂质结构鉴定", 《药学与临床研究》 * |
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