圆二色性检测在SFC中的应用在没有峰分辨率的情况下测定对映体比率

对映体比例的测定对于表征和研究各对映体的药理作用至关重要。圆二色性检测器（CD）测量左右圆偏振光的差分吸收。尽管对映体对紫外线的吸收是相等的，但它们对左右圆偏振光的吸收不同。测量对映体之间这种差异吸收的能力将允许在不解析峰的情况下测定对映体比率，从而消除了实现对映体分离的耗时过程。测定这些药物的对映体纯度是必要的，因为它们是许多商业上可获得的药物之一，作为外消旋和对映体纯形式的完全不同的药物提供。为了满足FDA对手性药物生产的要求，有必要确认正在生产的手性药物的正确形式，并且必须分析对映体纯度。The Use of Circular Dichroism Detection in SFC to Determine Enantiomeric Ratios without Peak Resolution DJ Tognarelli l ， J ohn Burchelll ， Satoe l ij i ma2， Yoshiro Kondo2， Masao Bounoshita2， Yasuyo Sato2， Miki Kuwaj i ma2.1JASCO Incorporated ， 28600 Mary's Court， Easton， MD 21601；2JASCO Corporation， 2967-5Ishikawa -machi ， Hachioji ， Tokyo 192-8537 J apan.e-mail： dtog n are lli @j as c oin c.co m Introduction The determinat i on of enantiomeric r atios is imperat i ve i n characterizing and studying the pharmacological effects of each enantiomer. The FDA requires individual testing of chiral drug molecules as significant differences in behavior between enantiomers in the body can occur as history has shown. Some enantiomers just differ in potency， but others can be inhibitive to each other and even some detrimental . In the 1950s one enantiomer of thal i domide was used against morning sickness ， however， the other enantiomer caused birth defects1. Tuberculosis is t reated with a variety of medications including the ethambutol (S，S)-(+) enantiomer， but the (R，R)-(-) enantiomer causes blindness2. Currently， the time-consuming task of achieving baseline resolution of enantiomers i s required for determining t he enantiomeric pur i ty. This typically requires SFC screening employing many columns and solvent combinations under a variety separation conditions.The resulting conditions are applicable to t he separat i on of t hose speci f ic enantiomers， while other r acemic mixtures require a new solvent-column screen. The circular dichroism detector (CD) measures the differential absorption of right and left circularly polarized light . Although enantiomers wi l l absorb UV light equally， t hey wil l absorb right and left circular l y polarized light differently. The ability to measure this differential absorption between the enantiomers will allow the determination of the enantiomeric ratio without resolving the peaks， thereby eliminating the t ime-consuming process of achieving enantiomeric separation. The determinat i on of the enantiomer i c purity of these drugs i s imperative since they are among many commercially available drugs t hat are offered as completely di f ferent drugs in the racemic and enantiomeric pure forms . To meet the FDA requirements for chiral drug production i t is necessary to conf i rm the correct form of the chiral drug i s being produced and the enantiomeric purity must be analyzed. Methods and Materials Test compounds F l avanone， Trans Stilbene Oxide， (S)-(+)-Ketoprofen， Ketoprofen，Sulfamethazine were all purchased f rom Sigma-Aldrich (St. Louis， MO， USA). The columns used were obtained from Phenomenex and Chiral Technologies. The JASCO SFC consisted of a CO2 pump， co-solven t pump， autosampler， column oven ， UV detector， circular dichroism detector， Advion CMS-L， and back pressure regulator controlled under ChromNAV 2 software. Results The separation of TSO is shown in figure 1. At 254nm the top chromatogram shows the separation of the 2 enantiomers and the bottom shows the differential， equal and opposite CD absorption of the enantiomer at 254nm. Figure 1. Chr o m at o g ram o f TSO . Top 254n m UV， B a t to m 254nm CD. The individual f lavanone enantiomers in figure 2 show the CD absorption spectrum of each.They show equal and opposite differential absorption at 254nm， 310nm， 337nm and 370nm. Figu r e 2. T h e in d ivid u al CD spe ct ra o f ea c h e na i n tiome r of l lav a none The enant i omer i c separation of flavanone at 254nm and at 340nm i s shown in f i gure 3. In this example the enantiomeric ratio can be determined to be 50：50 using either UV or CD.When the enantiomers co-elute， the UV shows the peak， however the equal and opposite CD absorption of the enantiomers cancel their signals as shown in t he left chromatograms in figure 4. The (s)-(+)-ketoprofen i s shown in the right chromatograms and demonstrate the CD signal from the pure enantiomer. F i g u re 4. The c hromatog ra m o f r ac e mic k etop r o fe n at 225n m (l e f t ) an d (S)(+)-ketop r ofen (r ight ). The co-elution of enantiomers would inhibit t he ability to determine the enantiomeric excessive using UV， MS， or most detectors. However， t he CD detector allows for determination of the enantiomeric ratio without resolution as shown in figure 5. The chromatogram shows the overlay of ketoprofen 90%， 96%， 97% 98% and 99% S. The cal i bration curve shows a correlation of 0.99. Fig ur e 5. Ch r omato gr am an d ca l i b r at i on curv e of Ketop r oten 90% S t o 99% S . The parallel 2 column screen of a mixture of flavanone， ketoprofen and sulfamethazine is shown i n f i gure 6. The top TIC shows t he detection of all 3 compounds from both columns as the run in parallel， but recombine before the MS. The 2nd and 3rd chromatograms are from column 1 through the CD detector at 310nm. The 4t h chromatograms shows the 2nd column at 254nm. The CD detector shows the abi l ity to identify the enantiomers dur i ng an achiral separation. Figu re 6. Par a lle l s tac ked c h r o m at o gr a m s of f l avan one . Th e top t ra c e i s the T I C fr om t he MS aft er bot h c olu m n s r ecombin e d . T he 2an d 3c h rometogr a ms .UV an d CD sign al， ar e t r om co l u m n 1. T h e 4m ch r oma t o gr am i s f r om t h e 2 par a l l e l c o l u m n goi n g t o a UV de t e ct or . Conclusions E na nt i omeric ex c ess can on l y be d e te rmi ned by U V a f ter basel i ne r esol u tio n o f t he enan tio me r s i s ac hi eved， w hich can be ti m e c o ns um i ng i n screeni n g co lum ns a nd so l ve n ts.T h e CD de t ecto r p rovid e s the u n iq ue ability t o d e t e rmi n e tha t enantiom er ic excess wi th out t he n eed fo r c hr omatog r a phi c separat i o n of the enantiomers . 1. C . Bl ak emore an d S. J ennet t， Th e Oxfor d C o m pa nion to the Body. Oxford Univers i t y Press . 2001. 2.N. Chha b ra ，M. Aseri a n d D . Padmana b han . A r eview o f dr u g i someri s m and it s signi f icance . Int . J. Appl. Basic Med.Res. 2013.3 (1).16-18.