The assay method of R- succinum love song Ge Lieting optical puritiesTechnical field
The present invention relates to a kind of dipeptidyl peptidase-IV（DPP-Ⅳ）Inhibitor is R- succinum love song Ge Lieting optical puritiesDetection method.
Background technology
Succinum love song Ge Lieting（Trelagliptin Succinate）It is a kind of dipeptidyl peptidase-IV（DPP-Ⅳ）SuppressAgent, its chemical entitled 2- ((6- ((3R) -3- amino-piperidino) -3,4- dihydro -3- methyl -2,4- dioxo -1 (2H)-phoneticPiperidinyl) methyl) -4- fluorobenzonitrile succinates.Its main component is song Ge Lieting.Have in the compound structure one it is chiral inThe heart, but it is real have pharmacological action for R configurations, its structural formula is shown in formula 1：
Formula 1：Succinum love song Ge Lieting structural formulas
Succinum love song Ge Lieting is by the dipeptidyl peptidase-IV of Japanese force field pharmacy exploitation（DPP-Ⅳ）Inhibitor, by selectingProperty, persistence suppress DPP-IV, so as to control blood sugar level.Incretin（GLP-Ⅰ）With glucose dependency pancreotropic hormonePolypeptide（GIP）Play an important role in blood glucose regulation, and DPP-IV is to cause incretin（GLP-Ⅰ）And PortugalGrape sugar dependency pancreotropic hormone polypeptide（GIP）Inactivation.By suppressing DPP-IV, it becomes possible to increase blood sugar level dependency islets of langerhansElement secretion, so that reach the purpose of control blood glucose.Succinum love song Ge Lieting is can to control blood glucose by improving body self-abilityThe new type Ⅱdiabetes mellitus medicine of level, as single medicine, is taken weekly.Its mechanism of action is unique, low with not producingBlood glucose, do not cause increased weight, and the unique advantage such as Small side effects, cause the incidence rate of gastrointestinal side effect also very low.In March, 2015 obtains Japanese MHLW（MHLW）Listing approval, be mainly used in treating type Ⅱdiabetes mellitus, toleration is goodIt is good.
The synthesis of R- succinum love song Ge Lieting is generated with the reaction of 2- cyano group -5- fluorine bromobenzyl with 3- methyl -6- chlorouracils2- ((chloro- 3,4- dihydros -3- methyl -2 of 6-, -1 (2H)-pyrimidine radicals of 4- dioxo) methyl) -4- fluorobenzonitriles, 2- ((6- chloro- 3,- 1 (2H)-pyrimidine radicals of 4- dihydro -3- methyl -2,4- dioxo) methyl) -4- fluorobenzonitriles again with the double hydrochloric acid of R-3- amino piperidinesReactant salt generates R- song Ge Lieting, the R- succinum love song Ge Lieting that R- songs Ge Lieting is obtained into salt with succinic acid again.
With the development of chiral liquid phase separation techniques, high performance liquid chromatography has become and can accurately detect material opticsThe reliable method of purity.But there is not been reported so far for the chiral separation method of R- succinum love song Ge Lieting.Trace it to its cause, oneIt is that the chromatographic behavior of enantiomer impurity compares similar with principal agent behavior, it is necessary to could be by the two from specific chirality paddingIt is completely separated, needs to carry out substantial amounts of chiral column screening experiment；Two be can be with chiral chromatogram column packing due to its chiral centreThe active force for producing hydrogen bond, dipole-dipole or space behavior is less, and the separation efficiency of conventional chiral chromatographic column is relatively low, it is impossible to realNow efficiently separate.In order to effectively control product quality, a kind of fast and accurately detection method of exigence.
The purpose of the present invention：In order to solve relatively low this problem of separation efficiency, we have developed using addition diethylamineThe method that the chiral chromatography of mobile phase determines R- succinum love song Ge Lietingting optical purities.The method is using in mobile phaseAddition diethylamine, adjusts mobile phase pH, and the steric configuration of stable chirality padding changes chirality padding and R- succinum love songs lattice are arrangedThe bonding action of spit of fland and S- succinum love song Ge Lieting, so as to improve R- succinum love song Ge Lieting and S- succinum love songs Ge Lieting itBetween separation efficiency, realize carries out the measure of optical purity with chiral chromatography, innovatively solves R- succinic acid firstBent Ge Lieting optical purities determine this technical barrier.
The content of the invention
At present, prepared by succinum love song Ge Lieting flow processs as shown in formula 2.
The technological process of 2 succinum love song Ge Lieting of formula
Technical scheme：
A kind of assay method of R- succinums love song Ge Lieting optical purities, it is characterised in that
Chromatographic condition：Chromatographic system is high performance liquid chromatograph, and chromatographic column filler is amylose-three（3,5- xylyl ammoniaCarbamate）, i.e. AD-H chiral chromatographic columns（250mm×4.6mm,5μm）；Mobile phase A is normal hexane, and Mobile phase B is diethylamineEthanol solution, and the volume ratio of the ethanol solution of described normal hexane and 0.1% diethylamine be 55:45~65:35, diethylamineConcentration is 0.05% ~ 0.15%, and mobile phase is in 0.8 ~ 1.2mL min-1, Detection wavelength：278nm.
In technical solution of the present invention in the ethanol solution of diethylamine, the effect of diethylamine is modifying agent, is typically incorporated within positiveMobile phase in, main effect is to adjust mobile phase pH, and the steric configuration of stable chirality padding changes chirality padding to R, SThe bonding action of enantiomer.
In the mobile phase in the ethanol solution of diethylamine, the concentration of diethylamine is 0.1%.
In the mobile phase, the volume ratio of the ethanol solution of normal hexane and 0.1% diethylamine is 60:40.
The detection method of the optical purity of the present invention, it is characterised in that containing following steps：
Take R- succinum love song Ge Lieting reference substances, S- succinum love song Ge Lieting reference substances each appropriate, be respectively placed in 25ml measuring bottles,Respectively plus anhydrous alcohol solution and quantitatively dilution make respectively be about 0.5mg ml-1 solution, as R- succinum love song Ge LietingReference substance stock solution；
（1）R, S- succinum love song Ge Lieting reference substance solution：Precision measures each 0.25ml of above-mentioned stock solution, puts same 100ml amountsIn bottle, plus dehydrated alcohol is diluted to scale, shakes up, obtains final product, as system suitability test solution；
（2）S- succinum love song Ge Lieting reference substance solution：Precision measures above-mentioned S- succinums love song Ge Lieting reference substance stock solutions0.25ml, is placed in 100ml measuring bottles, plus dehydrated alcohol is diluted to scale, shakes up, and obtains final product, used as S- succinums love song Ge Lieting pairSolution is positioned according to product；
（3）R- succinum love song Ge Lieting reference substance solution：Precision measures above-mentioned R- succinums love song Ge Lieting reference substance stock solutions0.25ml, is placed in 100ml measuring bottles, plus dehydrated alcohol is diluted to scale, shakes up, and obtains final product, used as R- succinums love song Ge Lieting pairSolution is positioned according to product；
（4）R- succinum love song Ge Lieting need testing solutions：Precision weighs R- succinum love song Ge Lieting test samples in right amount, plus anhydrous secondQuantitatively solution of every 1ml containing about 0.5mg is made in dilution to alcohol, used as need testing solution；
（5）Precision measures need testing solution 0.5ml, puts in 200ml measuring bottles, plus dehydrated alcohol is diluted to scale, shakes up, used as rightAccording to solution；
（6）20 l of contrast solution injection chromatograph of liquid is taken, detection sensitivity is adjusted, makes the peak height of main constituent chromatographic peak about fullThe 10%～20% of range.Precision measures need testing solution and each 20 l of contrast solution, is injected separately into chromatograph of liquid, records chromatographFigure to 2 times of main constituent peak retention time, by areas of peak normalization method calculating optical purity；
Chromatographic condition：With CHIRALPAK AD-H chiral columns as chromatographic column；With normal hexane-dehydrated alcohol（60：40）（Containing 0.1% 2Ethamine）For mobile phase；Detection wavelength is 278nm；Column temperature is 25 DEG C；Flow velocity is 1.0mL per minute, 20 L of sample size.
First respectively to R, S- succinums love song Ge Lieting reference substance solution is entered with S- succinum love song Ge Lieting reference substance solutionSample, carries out system suitability analysis, then carries out optical purity measure to R- succinum love song Ge Lieting need testing solutions, return by areaOne changes the optical purity that method calculates sample.
System suitability test：R, S- succinum love song Ge Lieting has reached baseline separation, and separating degree is more than 2.0, sees explanationBook accompanying drawing 3, with R- succinum love song Ge Lieting, S- succinum love song Ge Lieting reference substance chromatograms（Figure of description 4 and 5）Contrast,Determine that RT=14.113 is S configurations, RT=15.773 is R configurations.
The useful achievement of the present invention：
1 adds diethylamine in mobile phase, adjusts mobile phase pH, and the steric configuration of stable chirality padding changes chirality padding pairThe bonding action of R, S- succinum love song Ge Lieting, so that improve separation efficiency.
Baseline separation is reached between R in 2 analytes, S- song Ge Lieting, workable, analysis method specificity, spiritSensitivity and accuracy are high, can for ensure medicine safely, effectively, the strong foundation of quality controllable offer.
3 main peak peak types are good, and before and after main peak, impurity peaks are good with main peak separating degree, and the system suitability of chromatographic condition is goodIt is good.
The average recovery rate of 4 the method is 1.75% for 101.52%, RSD, fully meets requirement.
In technical solution of the present invention, the R- succinums love song Ge Lieting reference substances and S- succinum love song Ge Lieting reference substancesConfirmation：
Chromatographic condition：Chromatographic system is high performance liquid chromatograph, and chromatographic column filler is octadecylsilane chemically bonded silica（250mm×4.6mm,5μm）, flow velocity：1.0ml min-1, column temperature：35 DEG C, Detection wavelength：278nm, 20 L of sample size, mobile phase：With secondNitrile is mobile phase A, the potassium dihydrogen phosphate of 0.01mol L-1（PH value is adjusted to 6.2 with saturation potassium hydroxide solution）- acetonitrile（90:10）For Mobile phase B, pressing list procedure carries out gradient elution；
-1-1| Time (minute) | Mobile phase A (%) | Mobile phase B（%） |
| 0 | 1 | 99 |
| 30 | 20 | 80 |
| 55 | 85 | 15 |
| 55.5 | 1 | 99 |
| 60 | 1 | 99 |
Purity testing：Precision weighs R- succinums love song Ge Lieting reference substances with S- succinum love song Ge Lieting reference substances in right amount respectively,Plus acetonitrile quantitatively dilutes and makes solution of every 1ml containing about 0.5mg, sample detection, by areas of peak normalization method calculated purity, purityShould be greater than 99.0%；
Description of the drawings：
Fig. 1：RS- succinum love song Ge Lieting purity testing chromatograms.
Fig. 2：RS- succinum love song Ge Lieting chromatographic peak spectrograms.
Fig. 3：RS- succinum love song Ge Lieting optical purities determine chromatogram.
Fig. 4：S- succinum love song Ge Lieting optical purities determine chromatogram.
Fig. 5：R- succinum love song Ge Lieting optical purities determine chromatogram.
Fig. 6：R- succinum love song Ge Lieting samples（PTG141202 batch test samples）Optical purity determines chromatogram.
Caption：EP/JP represents European Pharmacopoeia and Japanese Pharmacopoeia high performance liquid chromatography separating degree computational methods.
Specific embodiment：
Once embodiment is used for illustrating the present invention, but not as limitation of the present invention.
The measure of the optical purity of 1 R- succinum love song Ge Lieting samples of embodiment
Instrument and reagent：High performance liquid chromatograph（HIT；Thermo Fisher Scientific Inc.）；AD-H chirality colorsSpectrum post（250mm×4.6mm,5μm；Daicel medicine chiral technology company limited）；Thermostat water bath（The emerging instrument of Community of Jin Tan County sectionFactory）；BT-25S electronic balances（Switzerland, Mei Tele companies）.DL-1000D Ultrasound Instrument（The letter Instrument Ltd. in Shanghai）.
Normal hexane（Chromatographically pure, Tianjin Biao Shiqi Science and Technology Ltd.s）；Dehydrated alcohol（Chromatographically pure, Tianjin scholar young tiger are strangeScience and Technology Ltd.）；Diethylamine（Analysis is pure, and Tianjin is extensively into chemical reagent company limited）；R- succinum love song Ge Lieting reference substances（Purity 99.9%, drug research institute of Di Sha Pharmaceuticaies group）；S- succinum love song Ge Lieting reference substances（Purity 99.0%, enlightening are huskyDrug research institute of Pharmaceutical group）；R- succinum love song Ge Lieting samples（PTG141202 batches, purity 99.0%, enlightening sand Pharmaceutical collectionDrug research institute of group）.
We have carried out the screening of mobile phase chromatographic condition by many experiments：
The selection of 1.1 mobile phases
Succinum love song Ge Lieting is the larger compound of polarity, therefore selects normal hexane-dehydrated alcohol system to be mobile phase.Mobile phaseProportioning investigate and find, the appropriate ratio for increasing normal hexane is conducive to improving separating degree, but normal hexane large percentage can causeRetention time is long, is unfavorable for analysis detection.Add diethylamine in mobile phase, main peak peak type can be made to tend to spike, post effect increases,Number of theoretical plate reaches requirement.By to diethylamine addition（0.05%、0.1%、0.15%）System optimization show that 1.0 %'s addsDosage makes separation efficiency optimal, thus selects 0.1% diethylamine.Flow velocity finds in investigating that appropriate reduction flow velocity can also be carriedHigh separating efficiency, but analysis time can be caused long, cause unnecessary waste.By to flow velocity（0.8 mL·min-1、1.0 mL·min-1、1.2mL·min-1）System optimization show that the flow conditions of 1.0 mL min-1 make separation efficiency mostIt is good, thus flow velocity selects 1.0 mL min-1.
The selection of 1.2 ultraviolet wavelengths：Uv-absorption maximum wavelengths of the succinum love song Ge Lieting in mobile phase is 278nm.Due to succinum love song Ge Lieting it is basically identical with the physicochemical property of its enantiomer, uv-absorption maximum wavelength be also278 nm, thus 278 nm are selected as the Detection wavelength of enantiomer.
The selection of 1.3 sample concentrations：Relatively concentration is respectively 0.3mg mL-1、0.5mg·mL-1、1.0mg·mL-1'sThe chromatogram of sample, experimental result show, 0.3mg mL-1Sample peak height it is relatively low, the less enantiomer of content is not examinedGo out；1.0mg·mL-1Sample concentration it is too big, chromatographic column has the sign of overload；0.5mg·mL-1The sample of concentration in peak height andRequirement can be met in terms of peak area, it is thus determined that 0.5mg mL-1For detecting the concentration of sample.
1.4 ruggedness：Ruggedness investigation is carried out by the change to mobile phase ratio, flow velocity and column temperature to method, has been tiedFruit finds, when proportion of ethanol is between 45%-35%, flow velocity occurs minor variations between 0.8-1.2 mL min-1, to separatingEfficiency does not affect, and enantiomer can be detected, and its content does not have significant changes.Room temperature is changed between 17 ~ 23 DEG CWhen, retention time is slightly affected, separation efficiency is not affected.
As described in technical scheme, detection method detects the optical purity of R- succinum love song Ge Lieting.
Sample determination（PTG141202 batch test samples）
It is appropriate that precision weighs PTG141202 batches test sample, plus dehydrated alcohol quantitatively dilution to make every 1ml molten containing about 0.5mgLiquid, used as need testing solution；Precision measures need testing solution 0.5ml, puts in 200ml measuring bottles, plus dehydrated alcohol is diluted to scale,Shake up, as contrast solution；20 l of contrast solution injection chromatograph of liquid is taken, detection sensitivity is adjusted, is made main constituent chromatographic peakPeak height be about full scale 10%～20%.Precision measures need testing solution and each 20 l of contrast solution, is injected separately into liquid phase colorSpectrometer, 2 times of record chromatogram to main constituent peak retention time, by areas of peak normalization method calculating optical purity（See accompanying drawing 6）；
Although the above-mentioned accompanying drawing that combines is described to the specific embodiment of the present invention, not to the scope of the present inventionLimit, skilled artisan should be understood that on the basis of technical scheme those skilled in the art are notThe various modifications made by needing to pay creative work or deformation are still within protection scope of the present invention.