Identification Chiral Capillary Electrophoresis

8/12/2019 Identification Chiral Capillary Electrophoresis

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L S V I R J o u r n al o f C h r o m a t o g r a p h y A , 7 3 5 ( 1 9 9 6 ) 7 7 - 1 2 1

JOURN L OFCHROM TOGR PHY

R e v i e w

dentif icat ion o f ch iral dr ug isom ers by capi llary e lectrophoresisSalvatore anali

lstituto di Cromatografia del C.N.R. Area della Ricerca di Roma P.O. Box 10 00016 Monterotondo Scalo Rome Italy

bstrac t

S e p a r a t i o n o f o p t i c a l i s o m e r s o f c o m p o u n d s o f p h a r m a c e u t i c a l i n t e r e st b y c a p il l a ry e l e c t r o p h o r e t i c t e c h n i q u e s i sr e v i e w e d . T h e d i r e c t a n d i n d i re c t s e p a r a t io n m e t h o d , a s w e l l a s t h e m a i n r e s o l u t i o n m e c h a n i s m s a n d t h e p a r a m e t e r si n f l u e n c i n g t h e s t e r e o s e l e c t iv i t y a r e d i s c u s s e d c o n s i d e r i n g c a p i l l a r y z o n e e l e c t r o p h o r e s i s , m i c e l l a r e l e c t r o k in e t i c c h r o m a t o g -r a p h y , i s o t a c h o p h o r e s i s a n d e l e c t r o c h r o m a t o g r a p h y . S e v e r a l c h i ra l s e l e c t o r s h a v e b e e n s u c c e s s f u l l y u s e d i n C E f o r c h i r a ls e p a r a t io n , i n c l u d i n g c y c l o d e x t r i n s a n d t h e ir d e r i v a t i v e s, m o d i f i e d c r o w n - e t h e r s , p r o t e i n s , a n t ib i o t ic s , l i n e a r s a c c h a r i d e s a n dc h i r al s u r f a c t a n ts . O n l y a p p l i c a t i o n s i n t h e p h a r m a c e u t i c a l f i el d w i th t h e m o s t i m p o r t a n t e x p e r i m e n t a l c o n d i t i o n s a r es u m m a r i s e d i n t h e T a b l e s r e p o r t e d i n t h i s p a p er . T h e c h i ra l a n a l y s e s o f d r u g s i n r e a l s a m p l e s l i ke b i o l o g i c a l f l ui d s o rp h a r m a c e u t i c a l f o r m u l a t i o n s a r e a l s o r e p o r t e d .Keywords: R e v i e w s ; E n a n t i o m e r s e p a r a t i o n ; P h a r m a c e u t i c a l a n a l y s i s; C y c l o d e x t r i n s ; D r u g s ; A n t i b i o t ic s ; C r o w n e t h e r s ;M i c e l l e s

C o n t e n t s

1 . In t ro d u c t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 82 . Th e i n d i re c t c h i ra l s e p a ra t i o n me t h o d . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . 7 83 . Th e d i re c t c h i ra l s e p a ra t i o n me t h o d . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . 7 9

3 .1. In c l u s i o n -c o mp l e x a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 8 03 .1.1 . Th e u se o f c y c l o d e x t r i n s o r t h e i r d e r i v a t i v e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . 8 03 .1.2 . M a c ro c y c l i c a n t i b i o t ic s . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . 9 83 .1.3 . C ro w n -e t h e r d e r i v a t i v e s . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . 1 0 2

3 .2. Af f i n i t y e l e c t ro p h o re s i s . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 1 0 33 .3. M i c e l l e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 73 .4. C h i ra l d ru g a n a l y se s b y e l e c t ro c h ro m a t o g ra p h y . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . 1 0 84 . Imp ro v i n g t h e d e t e c t i o n l i mi t i n C E fo r c h i ra l s e p a ra t io n s . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . 1 1 0

5 . C h i ra l i so me r a n a l y s i s i n b i o lo g i c a l f lu i d s a n d p h a rma c e u t i c a l s . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . 1 116 . C o n c l u s i o n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 47 . L i s t o f a b b re v i a t io n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 7Ac k n o w l e d g m e n t s . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 8R e fe re n c e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 8

0 0 2 1 -9 6 7 3 / 9 6 / 3 2 .0 0 1 9 9 6 E l se v i e r Sc i e n c e B .V. Al l r i g h t s re se rv e dSSDI 0 0 2 1 - 9 6 7 3 ( 9 5 ) 0 1 3 2 7 - X


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78 S . F a n a l i / J . C h r o m a t o g r . A 7 3 5 1 9 9 6 ) 7 7 - 1 2 11 I n t r o d u c t i o n

Ma n y c h e mic a l c o mp o u n d s u se d i n p h a r ma c e u -t ica l formula t ions possess ( an) a symmetr ic cen te r ( s )re spons ib le for op t ica l ac t iv i ty tha t can s t ronglyinf luence the i r pha rmacologica l p rope r t ie s . Recent lyi t has been repor ted tha t among 523 na tura l andsemisy nthe t ic drugs 98 .8 a re ch i ra l and 98 .4 ofthem a re so ld a s s ing le enant iomer , whi le in the caseo f 1 3 2 7 sy n th e t i c d r u g s c h i r a l c o mp o u n d s r e p r e se n t39 .8 and only 11 .6 of them a re so ld a s s ing lee n a n t io me r [ 1 ]. E x a m p le s o f d r u g s i n w h ic h o n e o fth e tw o e n a n t io m e r s p o s se s se s a d i f f e r e n t p h a r ma c o -lo g i c a l a c t i v i t y a r e w e l l d o c u me n te d , e . g . , ( - ) - e p i -n e p h r in e a n d ( - ) - t e r b u t a l i n e a r e 1 0 - a n d 4 - t ime smo r e p o t e n t t h a n t h e i r ( + ) - a n t i p o d e , r e sp e c t i v e ly[ 2] , ( - ) - p r o p r a n o lo l i s 1 0 0 - f o ld mo r e p o t e n t t h an( + ) - p r o p r a n o lo l ; i n a d d i t i o n o n e o f t h e tw o e n a n t io -mers of the same drug can even be tox ic ( e .g . ,tha l idomide , ke tamine ) .

T h e d e ma n d f o r a n a ly t i c a l me th o d s w i th h ig hr e so lu t io n p o w e r a n d h ig h e f f i c i e n c y i s r e c e n t lyinc reas ing for , e .g . , ch i ra l d rug pur i ty cont ro l , pha r -ma c o k in e t i c , p h a r ma c o d y n a mic s tu d i e s , e t c .

C h i r a l a n a ly s is i s c u r r e n t l y ca r r i e d o u t e mp lo y in gc h r o ma to g r a p h ic t e c h n iq u e s i n c lu d in g h ig h - p e r f o r m-a n c e l i q u id c h r o ma to g r a p h y ( H P L C ) , t h in - l a y e rc h r o m a t o g r a p h y ( T L C ) a n d g a s c h r o m a t o g r a p h y( G C ) [ 3 - 9 ]

Capi l la ry e lec t rophores is (CE) i s a r ecent power -fu l ana ly t ica l technique wide ly appl ied in d i f fe ren ta reas of r e sea rch , e .g . , pha rmaceut ica l , b io logica l ,e n v i r o n me n ta l , e t c . T h e d i f f e r e n t s e p a r a t i o n mo d e s ,n a me ly c a p i l la r y g e l e l e c t r o p h o r e s is ( C G E ) , mic e l l a re l e ct r o k in e t ic c h r o m a t o g r a p h y ( M E K C ) , i s ot a ch o -p h o r e s i s ( I T P ) , c a p i l l a r y z o n e e l e c t ro p h o r e s i s ( C Z E )a n d th e ma n y c h i r a l s e l e c to r s a v a i l a b l e ma k e C Etechniques a ve ry impor tan t too l for ch i ra l ana lys is inthe pha rmaceut ica l f ie ld . Compared to o the r ana -ly t i c a l t e c h n iq u e s l i k e H P L C , C E c a n o f f e r s e v e r a ladvantages inc luding s impl ic i ty , r ap id i ty of ana lys is ,a u to ma t io n , d i f f e re n t s e p ar a t io n m e c h a n i sms a n d l o wcos t .

The sepa ra t ion of two enant iomers i s a d i f f icu l tta sk in CE because the two ana ly te s possess s imi la rphys ico-chemica l prope r t ie s , un less a ch i ra l envi ron-me n t i s u se d i n o r d e r t o s e l e c t i v e ly mo d i f y t h e i re lec t rophore t ic mobi l i t ie s . The ch i ra l se lec tor i s

u su a l l y a d d e d t o t h e b a c k g r o u n d e l e c t r o ly t e a n d o n lya few /z l o f buf fe r can be used for the ana lys isr e d u c in g t h e c o s t s a n d e n v i r o n me n t p o l l u t i o n a n da l lo w in g i n su c h a w a y th e u se o f e v e n v e r yexpens ive ch i ra l se lec tor s . Most of the work done tod a t e c o n c e r n e d t h e s e p a r a t i o n o f c h i r a l c o mp o u n d s ,i n c lu d in g ma n y d r u g s [ 1 0 - 1 2 ] u s in g t h e d i r e c tsepa ra t ion me thod where d i f fe ren t ch i ra l se lec tor sw e r e a d d e d t o t h e b a c k g r o u n d e l e c t r o ly t e [ 1 3 - 1 6 ] ;even ch i ra l se lec tor s bound to the capi l la ry wa l l [17]or immobi l i sed in to a ge l have been s tud ied [18] .S e v e r a l r e so lu t i o n me c h a n i sms h a v e b e e n d i s c u s se dinc luding inc lus ion-complexa t ion , me ta l -ch i ra l l ig -and complexa t ion , mice l la r so lubi l iza t ion , a f f in i tyand ion-pa i r ing in te rac t ions . Even i f the f ie ld ofch i ra l ana lys is by CE was recent ly inves t iga ted , theto p i c h a s b e e n w id e ly r e v i e w e d [ 1 0 , 1 1 , 1 9 - 3 5 ] .T h e p r e se n t p a p e r r e v i e w s t h e s t a t e o f t h e a r t o fc h i r a l s e p a r a t i o n s p e r f o r me d b y C E t e c h n iq u e sfocus ing the a t ten t ion on the ident i f ica t ion of ch i ra ld r u g i so m e r s c o m p o u n d s . D i f f e r e n t ch i r a l r eso lu t i o nme c h a n i sms a r e c o n s id e r e d e x c lu d in g t h o se t h a twere not appl ied to ch i ra l d rugs sepa ra t ion , e .g . ,l igand-exchange . Fur the rmore , se lec ted appl ica t ionsin pha rmaceut ica l and c l in ica l f ie lds a re a lso d is -cussed .

2 T h e i n d i r e c t c h i r a l s e p a r a t i o n m e t h o dT h e in d i r e c t s e p a r a t i o n me th o d o f e n a n t io me r s i s

b a se d o n a c h e mic a l r e a c t i o n o f t h e tw o a n t i p o d ew i th a c h i r a l c o mp o u n d b e f o r e t h e e l e c t r o p h o r e t i ca n a ly s i s w i th a p r o d u c t io n o f a mix tu r e o f tw od ia s t e r e o i so me r s , a s sh o w n b e lo w :(D,L)-C + (L)-R

a b[ D) -C- - L ) -R] + [ L ) -C- - L ) -R]

c d l )where C is the racemic compound, R the ch i ra lreagent , and D- and L- a re dext ro and levo ro ta tory ,re spec t ive ly . The produc t of the reac t ion leads to thef o r ma t io n o f tw o c o mp o u n d s ( c a n d d ) i n w h ic h Ca n d R a r e b o u n d th r o u g h c o v a l e n t b o n d s a n d t h econf igura t ion of R i s the same in the two de r iva t ives


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s. Fanali / J. Chromatogr. A 735 1996) 77-121 79and thus c and d a re d ia s te reo isomers . The mixturecan be , in pr inc ip le , sepa ra ted by CE us ing an achi ra lb a c k g r o u n d e l e c t r o ly t e b e c a u se t h e c o m p o n e n t s p o s -sess d i f fe ren t phys ico-chemica l prope r t ie s .

T h e i n d i r e c t s e p a r a t i o n me th o d , w id e ly u se d i nH P L C a n d G C , w a s h a r d ly a p p l i e d i n C E . T h i s ma ybe due to the d isadvantages a r i s ing f rom th is me thodin compar ison to the d i rec t sepa ra t ion me thod.A mo n g th e m w e c a n o u t l i n e t h e f o l l o w in g : ( 1 ) t h eme th o d i s t ime - c o n su min g b e c a u se a s a mp le p r e -t r ea tment involv ing a chemica l r eac t ion wi th acha rac te r iza t ion of the chemica l r eac t ion produc ts i smanda tory ; (2) r eac t ing groups on the ana ly te schemica l s t ruc ture a re necessa ry (n i t rogen , hydroxyl ,ca rboxyl ic ) ; (3) one has to use ve ry pure ch i ra lse lec tor s , the presence of an impure ch i ra l se lec torw i l l p r o d u c e tw o c o mp o u n d s i n a d d i t i o n t o t h edias te reo isomers s tud ied which makes the sepa ra t ionmore d i f f icu l t ; (4 ) the two enant iomers should reac twi th the same ra te ; (5) the re sponse of the de tec torf o r t h e tw o d i a s t e r e o i so me r s f o r me d sh o u ld b e t h esame; (6) r eac t ion condi t ions should be appropr ia tein o r d e r t o a v o id s t e r e o t r a n s f o r ma t io n o f e i t h e rch i ra l r eagent , enant iomers or d ia s te reo isomers .

Very of ten i t i s no t easy to r e so lve a l l theo p e r a t i o n a l p r o b l e ms d e sc r ib e d a b o v e . H o w e v e r ,no te tha t the ind i rec t me thod can g ive a va l idso lu t i o n i n t h o se c a se s w h e r e t h e d i r e c t me th o dc a n n o t b e u se d o r w h e n th e d e t e c t a b i l i t y o f a n a ly t e sshould be inc reased .

T h e i n d i r e c t s e p a r a t i o n me th o d w a s ma in ly a p -p l i e d i n C E f o r t h e e n a n t io me r i c r e so lu ti o n o f a min oac ids us ing d i f fe ren t ch i ra l se lec tor s , e .g . , 2 ,3 ,4 ,6-t e t r a - O - a c e ty l - D - g lu c o p y r a n o sy l i so th io c y a n a t e( G I T C ) [ 3 6 ] , ( + ) - O , O ' - d i a c e ty l - L - t a r t a r i c a n h y d r id eo r ( + ) - O , O ' - d ib e n z o y l - L - t a r t a r i c a n h y d r id e [ 3 7 , 3 8 ] ,1- f luoro-2 ,4-d in i t rophenyl-5-L-a lan ine or 1- f luoro-2 ,4-d in i t ro-5-D-a lan ine (L- or o-Mar fey ' s r eagent )[ 3 9 ] . H o w e v e r t h e a p p l i c a b i l i t y o f t h i s me th o d h a sa l so b e e n sh o w n f o r c o mp o u n d s o f p h a r ma c e u t i c a lin te res t by De t te and Watz ig [40 ,41] .

E n a n t io me r s e p a r a t i o n o f a mp h e ta min e , me th a m-phe tami 'ne , ephedr ine , pseudoephedr ine , norephe-d r in e a n d n o r p se u d o e p h e d r in e i n a s in g l e ME K C r u naf te r de r iva t iza t ion wi th 2 ,3 ,4 ,6- te t ra -O-ace ty l - f l -D-g lu c o p y r a n o sy l i so th io c y a n a t e ( G I T C ) w a s d e sc r ib e db y L u r i e [ 4 2 ]. T h e e f f e c t o f o r g a n i c mo d i f i e r t y p eand concent ra t ion , SDS concent ra t ion , cap i l la ry tem-

pera ture and the appl ied vol tage on the re so lu t ionand se lec t iv i ty were s tud ied . The opt imum ex-p e r ime n ta l c o n d i t i o n s f o r t h e e n a n t io me r s e p a r a t i o no f 1 2 p h e n y le th y l a n in e i so me r s w e r e f o u n d to b e a t3 0 C w i th a b a c k g r o u n d e l e c t r o ly t e ( B G E ; h e r e 1 0m M p h o s p h at e + 1 0 m M b o r at e b u f f e r p H 9 ) c o n -t a in in g 10 0 mM S D S a n d 2 0 me th a n o l . T h ea p p l i c a b i l i t y o f t h e o p t imi se d me th o d w a s sh o w na n a ly z in g a f o r e n s i c s a mp le c o n t a in in g 1 R , 2 S - ( - ) -e p h e d r in e a n d 2 S - ( + ) -me th a mp h e ta min e .

One in te res t ing example where the d i rec t r e s -o lu t ion me thod i s d i f f icu l t to apply i s r epresented bythe chira l separation of L- and D-carnit ine . The L-ca rn i t ine enant iomer i s ve ry impor tan t in the me tabo-l i sm of long fa t ty ac ids , whi le the D- isomer p lays atoxic e f fec t on b iochemica l processes , thus the drugshould not contain D-carnit ine . The separation of D-and L-ca rn i t ine has been pe r formed a f te r de r iva t iza -t i o n w i th ( - ) - [ 1 - ( 9 - f l u o r e n y l ) - e th y l ] c h lo r o f o r ma te( F L E C ) i n 5 0 mM p h o sp h a t e b u f f e r p H 2 .6 . T h ea d d i t i o n o f 2 0 mM te t r a b u ty l a mmo n iu m b r o mid eimproved the d ia s te reo isomer re so lu t ion [43] .

Recent ly the ind i rec t ch i ra l sepa ra t ion me thod hasb e e n u se d f o r t h e e n a n t io me r s e p a r a t i o n o f s e v e r a la mp h e ta min e s su c h a s a mp h e ta min e , 4 - h y d r o x y -a mp h e ta min e , 3 , 4 - me th y l e n e d io x y me th a mp h e ta min ea n d 3 , 4 - me th y l e n e d io x y e th a mp h e ta min e a f t e r d e r i -v a t i z at i o n w i th M a r f e y ' s r e a g e n t. T h e B G E w a s 8 0a q u e o u s b u f f e r ( 5 mM b o r a t e p H 9 a n d 1 0 0 mMSD S) and 20 me than ol [44] .

3 T he d i rec t ch i ra l s epa ra t i o n m et ho dThe d i rec t ch i ra l sepa ra t ion me thod i s successfu l ly

a p p l i e d i n C E f o r t h e e n a n t io me r i c s e p a r a t i o n o fs e v e r a l c l a s se s o f c o mp o u n d s , ma in ly , o f p h a r ma -ceut ica l in te res t . The ch i ra l se lec tor can e i the r bea d d e d t o t h e b a c k g r o u n d e l e c t r o ly t e [ 1 3 - 1 5 , 2 2 ] o rbound to the capi l la ry wa l l [17] , o r inc luded in /bound to a ge l ma tr ix [18 ,45] in orde r to a r range as te reose lec t ive envi ronment tha t i s in te rac t ing , dur -i n g t h e e l e c t o p h o r e t i c r u n , w i th t h e tw o e n a n t io me r so n f o r min g l a b i l e d i a s t e r e o i so me r i c c o mp le x e s . T h el a b i l e c o mp le x e s f o r me d a r e mo v in g t o w a r d t h ede tec tor a t a d i f fe ren t ve loc i ty on ly i f they possessd i f fe ren t s tab i l i ty cons tan ts . In th is case re la t ive lyw e a k b o n d s a r e i n v o lv e d i n t h e d i a s t e r e o i so me r


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8 0 S. Fanali / J. Chromatogr. A 735 1996) 77-121

forma t ion process (hydrogen , hydrophobic , 7 r -Tr ,d ip o l e - d ip o l e ) .

The ch i ra l pur i ty of the se lec tor in the d i rec tsepa ra t ion me thod i s no t a s c r i t ica l a f ac tor a s in theindi rec t sepa ra t ion me thod; in f ac t , the impur i ty i si n f lu e n c in g o n ly t h e r e so lu t io n a n d i t h as b e e n sh o w ntha t r e la t ive ly good re su l t s can be obta ined us ing achi ra l se lec tor conta in in g up to 10 of i t s an t ipode[11].

S e v e r a l r e so lu t i o n me c h a n i sms a n d t h e ma in p a -ramete r s in f luenc ing the se lec t iv i ty of the ch i ra lsepa ra t ion wi l l be d iscussed inc luding inc lus ion-complexa t ion , mice l la r and a f f in i ty in te rac t ions3 . 1. l n c l u s i o n c o m p l e x a t i o n

T h e in c lu s io n - c o mp le x a t i o n me c h a n i sm h a s b e e nwide ly appl ied in ana ly t ica l chemis t ry in orde r toimprove the se lec t iv i ty of the sepa ra t ion of seve ra li so me r i c c o mp o u n d s i n c lu d in g g e o me t r i c a l, p o s i t i o n -a l and enant iomer ic . Up to now three c la sses ofc o mp o u n d s , f o r min g i n c lu s io n - c o mp le x e s , h a v e b e e nused in CE, namely cyc lodext r ins (CDs) , te t r aca r -b o x y la t e d c r o w n - e th e r a n d a n t ib io ti c s .3.1 .1 . The use o f cyc lode x t r ins or the i r der i va t i ve s

C D s a r e o l i g o sa c c h a r id e s c o mp o se d b y d i f f e r e n tg lu c o se u n i t s c o n n e c t e d t o e a c h o th e r t h r o u g ha ( 1 , 4 ) - g lu c o s id i c b o n d s a n d t h e y a r e o b t a in e d b yenzymat ic r eac t ion wi th s ta rch . In sp i te of the fac tt h a t C D s f o r me d f r o m 6 t o u p t o 1 2 g lu c o se u n i t shave been i so la ted , on ly those wi th s ix , seven ande ight un i t s a re cu r ren t ly used , te rm ed ce - , /3- andy-CD, re spec t ive ly . The CD's shape i s s imi la r to tha to f a t r u n c a t e d c o n e w i th a r e l a t i v e ly h y d r o p h o b iccavi ty and ab le to hos t ana ly te s , and an hydrophi l l ico u t s id e r e g io n d u e t o t h e p r e se n c e o f h y d r o x y lg r o u p s ( p o s i t i o n 2 , 3 a n d 6 o f g lu c o p y r a n o se ) . T h ethree na t ive cy lodext r ins possess the same depth buth a v e d i f f e r e n t w id th s ( i n c r e a s in g b y t h e n u mb e r o fg lucose uni t s ) .

I n t h e i n c lu s io n - c o mp le x a t i o n me c h a n i sm th ec o mp o u n d f i t s t h e C D c a v i ty w i th t h e w h o le mo le -cu le or wi th i t s hydrophobic pa r t and , thus , the CDtype has a ve ry impor tan t ro le in the sepa ra t ionp r o c e ss . T h e h y d r o p h o b ic i n t e r a c ti o n w i th t h e c a v i tya lone i s no t suf f ic ien t to enab le the sepa ra t ion ofe n a n t io me r c o mp o u n d s ; w e a k b o n d s b e tw e e n su b -

o

CH20H CH2OH CH2OH CH2OHF ig . 1 . S t r u c tu r e o f C D.

s t i t u e n t g r o u p s o n t h e a sy mme t r i c c e n t e r / s o f a n a -ly t e s a n d s e c o n d a r y a n d /o r p r ima r y h y d r o x y l g r o u p sof the CD a re re spons ib le for ch i ra l r ecogni t ion .

F ig . 1 shows the shape of a CD whi le Table 1shows the ma in proper t ie s of the na t ive CDs used inCE.

Due to i t s d imen sions /3-CD is e f fec t ive for thein c lu s io n c o mp le x e s w i th mo le c u l e s c o mp o se d o fo n e o r tw o c o n d e n se d a r o ma t i c g r o u p s , a n d t h emodif ica t ion of hydroxyl groups a t pos i t ion 2 , 3 and6 o f g lu c o p y r a n o se a f t e r c h e mic a l r e a c t i o n s e n o r m-o u s ly e x t e n d s t h e u se o f su c h C D ty p e s i n c h ir a l C Eana lys is . The modif ied CDs can exhib i t ve ry d i f fe r -en t prope r t ie s than the pa ren t ones , which can beuse fu l for improving the se lec t iv i ty in ch i ra l ana ly-ses , e .g . , h ighe r so lubi l i ty , d i f fe ren t conformat ion ,cha rged or cha rgeable groups , e tc . The case ofheptak is - (2 ,6-d i -O-methyl ) - /3-CD can se rve he re a sa n e x a mp le . T h e tw o h y d r o x y l g r o u p s a t p o si t io n 2a n d 6 o f e a c h g lu c o se i s mo d i f i e d w i th me th o x ygroups and as a r e su l t the o l igosacchar ide possessesa deeper cav i ty and an h ighe r so lubi l i ty than /3-CD.

CDs and the i r de r iva t ives have been wide ly ap-p l ied in CE for the enant iomer sepa ra t ion of a la rge

T a b l e 1T h e m a i n p r o p e r ti e s o f n a ti v e C D s s e e R e f . [ 4 6 ] )

C D t y p e

N u m b e r o f g l u c o p y r a n o s e u n i t s 6 7 8M o l e c u l a r m a s s D a ) 9 7 2 1 1 3 5 1 2 9 7I n n e r d i a m e t e r n m ) 0 . 5 7 0 . 7 8 0 . 9 5D e p t h n m ) 0 . 7 8 0 . 7 8 0 . 7 8S o lu b i l i t y i n wa te r g /1 0 0 ml ) 1 4 .5 1 . 85 2 3 . 2[ o~ ]~ + 1 5 0 . 5 + 1 6 2 . 0 + 1 7 7 . 4


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S . Fa n a l i / J . Ch ro m a t o g r . A 7 3 5 1 9 9 6 ) 7 7 - 1 2 1 81n u mb e r o f c o mp o u n d s o f p h a r ma c e u t i c a l i n t e r e s tused a s s tandards wi th the a im to s tudy the pa rame-te r s in f luenc ing the se lec t iv i ty of the sepa ra t ion .

The f i r s t a t tempt to pe r form chi ra l sepa ra t ion bye l e c t ro mig r a t i o n me th o d s w a s d o n e b y S m o lk o v a sg r o u p b y I T P e mp lo y in g d ime th y l a t e d ( d i - O Me - f l -C D ) o r t r ime th y l a t e d - f l - C D ( t r i - O Me - /3 - C D ) a s c h i -ra l se lec tor added to the lead ing e lec t ro ly te [47] .Af te r s tudying d i f fe ren t ope ra t iona l pa ramete r s , e .g . ,C D ty p e a n d c o n c e n t r a t i o n , t h e e n a n t io me r r e so lu -t ion of r acem ic s tandard drugs , inc luding pseudo-e p h e d r in e , n o r p se u d o e p h e d r in e , O - a c e ty lp se u d o -e p h e d r in e a n d p - h y d r o x y n o r p se u d o e p h e d r in e w a sp e r f o r me d . T h i s i s n o t t h e o n ly e x a mp le o f c h i r a ldrug ana lys is by ITP , seve ra l o the r s sepa ra t ionsc a r r i e d o u t b y t h e s a me g r o u p d o c u me n t t h e a p -p l icab i l i ty of ITP to enant iomer sepa ra t ion , e .g . ,ke to t i f en drug and i t s po la r synthes is in te rmedia te sw e r e r e so lv e d i n t h e i r e n a n t io me r s u s in g f l - C D o rd i - O Me - f l - C D [ 4 8 ] . F u r th e r mo r e , a c o u p le d c o lu mnsys tem, in which the two capi l la r ie s conta ined twodif fe ren t CDs, has been d iscussed [49] . Comp ar isono f I T P a n d C Z E f o r e n a n t io me r s e p a r a t i o n o f p h a r -ma c e u t i c a l c o mp o u n d s ( c h lo r a mp h e n ic o l , k e to t i f e n ,ke to t i f en-N-oxide and th ior idaz ine ) was a lso d is -cussed [50] ; the au thors conc luded tha t ITP wasmo r e su i t a b l e t h a n C Z E f o r t h e a n a ly s i s o f min u t ecom pon ents in a la i ge excess o f o the r s bu t CZE ispre fe rab le when h igh e f f ic ienc ies a re r eques ted .T h e o r e t i c a l mo d e l s f o r e n a n t io me r s e p a r a t i o n u s in gITP have a lso been d iscussed [51 ,52] . Unfor tuna te ly ,a f te r these exce l len t r e su l t s ITP was not used forfur the r inves t iga t ions in ch i ra l d rug ana lys is .

CZE has been te s ted for ch i ra l sepa ra t ions ofa min o - a c id d e r iv a t i v e s u sin g l i g a n d e x c h a n g e me c h -anism [53 ,54] or CDs incorpora ted in to a ge l [18] orc h a r g e d C D s [ 1 3 ] . T h e f i r s t a t t e mp t t o u se C Z E f o rth e e n a n t io me r s e p a r a t i o n o f c o mp o u n d s o f p h a r ma -ceut ica l in te res t was d one by us [14] s tudying seve ra lsy mp a th o mim e t i c d ru g s , n a me ly e p h e d r in e , n o r e p h e -dr ine , ep inephr ine , norepinephr ine and i sopro te renol .A mo n g th e C D s s tu d i e d d i - O Me - f l - C D w a s t h e mo s te f fec t ive in ch i ra l sepa ra t ion pe r formed in a shor tc a p i l la r y ( 2 0 c m) c o a t e d w i th l i n e a r p o ly a c r y l a mid e .The inc rease in CD concent ra t ion led to an inc reasein migra t ion t ime and re so lu t ion . The e f fec t o f thec o n c e n t r a t i o n o f C D s o n t h e e n a n t io se l e c t i v i t y w a s

discussed by seve ra l au thors ind ica t ing tha t th ispa ramete r should be ca re fu l ly cont ro l led for thesuccess of ch i ra l d rug sepa ra t ion [15 ,55-57]

As ment ioned above the se lec t ion of the appro-p r i a t e C D i s o f p a r a mo u n t imp o r t a n c e w h e n c h i r a ldrug sepa ra t ions have to be pe r formed. As f i r s tr equi rement for ch i ra l r e so lu t ion us ing CDs, theana ly te s must f i t the cavi ty when forming inc lus ionc o mp le x e s a n d t h u s t h e d ime n s io n o f t h e c h i r a lse lector must be appropria te . Nardi e t a l . [58]sepa ra ted racemic i so lyse rg ic ac id , me luol , te rgur ide ,l i sur ide and n ice rgol ine , e rgot a lka lo id drugs , on lyu s in g y - C D w i th t h e l a r g e s t c a v i t y . T h e e n a n t io me rsepa ra t ion of te rbuta l ine was pe r formed us ing bothf l - C D a n d d i - O Me - f l - C D ; t h e mo d i f i e d C D a l l o w e dto o b t a in a h ig h e r e n a n t io se l e c t i v i t y ( ma x imu mr e so lu t i o n a t 5 mM) th a n t h e p a r e n t o n e ( ma x imu mresolu t ion a t 15 mM). These re su l t s can be expla inedcons ide r ing tha t the modif ied CD possesses a deepe rc a v i ty ( mo r e h y d r o p h o b ic ) t h a n f l - C D a n d mo d i f i e dhydroxyl groups a t pos i t ion 2 and 6 tha t can in-f luence secondary bonds [59] .

T h e mo d i f i c a t i o n o f t h e h y d r o x y l g r o u p s o n t h eC D r ims i s o f p r ima r y imp o r t a n c e i n o r d e r t oimprove the s te reose lec t iv i ty of the sepa ra t ion be -c a u se t h e se g r o u p s a r e f o rmin g s e c o n d a r y b o n d s w i ththe enant iomers . There fore , a t ten t ion should be pa idto the degree of subs t i tu t ion of the CD. This e f fec twas s tud ied b y Va lko e t a l [60] us ing H P- f l -C D a td i f fe ren t degree of subs t i tu t ion for the ch i ra l sepa -ra t ion of seve ra l o rganic ac ids and by Yoshinaga andTanaka [61] us ing d i f fe ren t me thyla ted f l -CD for thechi ra l r e so lu t ion of some dansyl -amino ac ids . Re-cent ly we s tud ied the e f fec t o f d i f fe ren t CD and the i rc o n c e n t r a t io n o n t h e e n a n t io me r s r e so lu t i o n o f so menon-s te ro ida l an t i - in f lammatory drugs (NSAIDs) ,namely fenoprofen , f lu rb iprofen , ke toprofen , ibupro-f e n , i n d o p r o f e n a n d su p r o f e n . A m o n g th e C D s u se d( d i - O Me - f l - C D , t r i - O Me - f l - C D , 6 A - me th y l a min o - f l -C D ) t r i - O Me - , 8 - C D p r o v e d t o b e t h e mo s t e f f e c t i v echi ra l se lec tor a l lowing the base l ine sepa ra t ion o f a llthe enant iomers s tud ied [62] .

Both na t ive and de r iva t i sed CDs have been in-ves t iga ted by Al t r ia e t a l . [15] for ch i ra l sepa ra t iono f p h a r ma c e u t i c a l c o mp o u n d s b y C Z E . P i c u me te r o land c lenbute ro l , two f l - agonis t ic drugs were re so lvedin the i r enant iom ers and the e f fec t o f seve ra l pa rame-te r s , e .g . , pH and ion ic s t r ength of the BGE, CD type


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82 S . F a n a l i / J . C h r o m a t o g r . A 7 3 5 1 9 9 6 ) 7 7 - 1 2 1a n d c o n c e n t r a ti o n , c a p i l la r y t e mp e r a tu r e o n e n a n t io -se lec t iv i ty were inves t iga ted . The capi l la ry tempera -ture can s t rongly a f fec t the re so lu t ion (gene ra l ly alower tempera ture inc reases r e so lu t ion) a s a lso d is -cussed by Schutzne r and Fana l i [63] s tudying thec h i r a l s e p a r a t i o n o f so me sy mp a th o mime t i c d r u g s ,n a me ly n o r e p in e p h r in e , e p in e p h r in e a n d i so -p r o t e r e n o l . I n c r e a s in g t h e c o lu mn t e mp e r a tu r ec a u se d a d e c r e a se o f b o th mig r a t i o n t ime a n dr e so lu ti o n . T h e d e c r e a se o f e n a n t io se l e c t i v it y c a n b ea sc r ib e d t o t h e sh o r t e r t ime sp e n t b y t h e a n a ly t e s i n toth e C D c a v i ty a n d t o a mo d i f i c a t i o n o f t h e r mo -d y n a m ic p a r a me te rs . T h e e f f e c t o f t h e p H o n t h ee n a n t io me r s e p a r a t i o n w a s s tu d i e d b y P a lma r sd o t t i ra n d E d h o lm [ 5 5 ] a n d t h e r e so lu t i o n o f t e r b u t a l i n ed e c r e a se d b y i n c r e a s in g t h e p H ( 2 . 5 - 8 ) w h e n d i -O Me - f l - C D w a s u se d .E n a n t io se l e c t i v i t y , e f f i c i e n c y a n d r e so lu t i o n c a na l so b e i n f lu e n c e d mo d i f y in g o th e r p a r a me te r s su c has the organic addi t ive a s shown by us for these p a r a ti o n o f p r o p r a n o lo l . 3 0 ( v /v ) me th a n o l w a sth e a d d i t i v e c o n t a in e d i n p h o sp h a t e b u f f e r - u r e a a n d4 0 m M o f / 3 - C D [ 59 ]. R e c e n t ly s i m il a r e f f e ct w asnot iced for the ch i ra l sepa ra t ion of r acemic f lurb i -p r o f e n ( s e e F ig . 2 ) ; h e r e t h e a n a ly t e sh o w e d p o o rr e so lu t i o n a t 5 mM o f t r i - O Me - f l - C D ( v e r y g o o dr e so lu t i o n w a s o b t a in e d a t 3 0 mM o f C D w i th o u to r g a n i c a d d i t iv e ) b u t t h e a d d i t i o n o f m e th a n o l t o t h eB G E ( M E S a t p H 5 ) c a u se d a n i n c re a se o f r e so lu t io n[ 6 2 ] . T h e e f f e c t o f o r g a n i c mo d i f i e r o n e n a n t io me r sreso lu t ion us ing CDs as ch i ra l se lec tor was theore t -i c a l l y d i s c u s se d b y W r e n [ 6 4 ] , w h o sh o w e d th a tw h e n th e C D c o n c e n t r a t i o n w a s a t o r b e lo w th eo p t imu m v a lu e ( ma x imu m r e so lu t i o n ) , t h e a d d i t i o no f o r g a n i c m o d i f i e r c a u se d a r e d u c t io n o f r e so lu t io nd u e t o t h e c h a n g e o f s t a b i li t y c o n s ta n t s o f th e tw oc o mp le x C D - e n a n t io me r s .

Ma n ip u l a t i o n o f t h e B G E f o r imp r o v in g e n a n t io -se lec t iv i ty and e f f ic iency of ch i ra l d rug sepa ra t ionsc a n a l so b e d o n e u s in g a lk y lh y d r o x y a lk y l c e l l u lo sed e r iv a t i v e s a d d e d t o t h e B G E c o n ta in in g C D s , a ssh o w n f o r d r u g s l i k e t h r e o - c h lo r a mp h e n ic o l a n dk e to t i f e n [ 5 0 ]; h y d r o x y e th y l c e l l u lo se w a s a d d e d t oth e B G E c o n ta in in g d i - O Me - f l - C D . S imi l a r e f f e c tw a s r e c o r d e d b y B e ld e r a n d S c h o mb u r g [ 6 5 ] f o r t h ea n a ly s i s o f t o c a in id e a n a lo g u e s u s in g p o ly v in y la l c o h o l ( P V A ) . T h e p o ly me r u se d r e d u c e d t h e e l e c -t ro-osmot ic f low, a s we l l a s the adsorp t ion on the

W

. ___ ~ . . .

mira

rainFig. 2. Electropherograms of the enantiomer separation of racemicflurbiprofen using background electrolytes at pH 5 containing 5mM of tr i -OMe-fl-CD and different concentrat ions of methanol(0-4 0 ). A pparatus, Biofocus 3000 (reprinted with permissionfrom Ref. [62]).

c a p i l l a r y w a l l c a u s in g a n imp r o v e me n t i n e n a n t io -se lec t iv i ty . Recent ly i t has been shown tha t thea d d i t io n o f sh o r t c h a in t e t r a a lk y l a mm o n iu m c a t io n s( T A A ) to t h e c h i r a l B G E c a n s t r o n g ly a f f e c t t h ese lec t iv i ty of the enant iomer sepa ra t ion of seve ra lr a c e mic d r u g s [ 6 6 ] . T MA a n d T B A w e r e a d d e d t oth e B G E a t p H 2 .5 c o n t a in in g d i f fe r e n t C D s ( f l - C D ,d i - O M e - f l - C D , t r i - O M e - f l - C D a n d H P - f l - C D ) f o rth e e n a n t io me r s e p a r a t i o n o f 2 2 c o mp o u n d s o fp h a r ma c e u t i c a l i n te r es t . T h e u se o f T A A c a u se d t h ereve rs ion of the e lec t ro-osmot ic f low, a gene ra l lyinc rease of e i the r migra t ion t ime and s te reose lec t iv i -t y w a s r e c o r d e d . C o mp a r e d t o T MA , T B A p r o v id e da d i f fe ren t se lec t iv i ty inc reas ing the so lubi l i ty off l - C D a n d l o w e r c o n d u c t iv i t y , h o w e v e r i n so mee x te n t a r e d u c t io n o f r e so lu t io n w a s r e c o r d e d d u e t oth e c o mp e t i t i o n o f t h i s c a t i o n f o r t h e C D c a v i ty . T oi l lus t ra te th is, F ig . 3 shows the e f fec t o f TM A andT B A o n th e e n a n t io me r s e p a r a t i o n o f t r imip r a min e[66].


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S. Fanali / J. Chromatogr. A 735 1996) 77-121 83

a )

I I1 0 t i m e ~ , ,. ..~ t .,, ;-~ g O

t i m e r a i n )

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i I6 7

t i m e r a i n )Fig. 3 . E f fec t o f TMA and TBA on the enan t iomer separa tion o ftr imipramine. B ackgro und electrolytes: 2 0 mM H P-/3-CD + a) 50m M s o d i u m p h o s p h at e p H 2 . 5; b ) 1 0 0 m M T B A - p h o s p h a t e p H2.5; c) 50 mM TM A pH 2.5 modified from Ref. [66])

R e v e r s a l o f t h e e l e c t r o - o s mo t i c f lo w w a s a l s o u s e dby S ta lbe rg e t a l . [67] for improving the enant iomersepa ra t ion of seve ra l loca l anaes the t ic drugs , wi tht e t r a p r o p y l a m m o n i u m ( T P A ) a n d T B A b e i n g a d d e dto t h e B G E a t p H 3 c o n t a in in g d i - O Me - /3 - C D

R e c e n t l y u n c h a r g e d / 3 - C D p o ly me r h a s b e e ns tudied for ch i ra l d rug sepa ra t ion in CE. The poly-me r c o n t a in s s e v e r a l / 3 - C D u n i t s b o u n d t o e a c h o th e ra f te r r eac t ion wi th ep ich loroydr in and , thus , in -c lu s io n - c o mp le x a t i o n i n t e r a c t i o n a lo n e c a n n o t e x -p la in the s te reose lec t iv i ty .

Nish i e t a l . [68] sepa ra ted seve ra l t r ime toquinola n a lo g u e s , b r o n c h o d i l a to r d r u g s, u s in g 7 o f / 3 - C Dp o ly me r w i th a B G E a t p H 6 . 5 . R e c e n t l y w ein v e s t i g at e d t h e e f f e c t o f t h e u n c h a r g e d /3 - C D p o ly -

me r o n t h e e n a n t i o me r s e p a r a t i o n o f a l a r g e n u mb e rof drugs , inc luding /3-b locke r s , ce - and f l - an tago-n is t s , and anaes the t ic s [69 ,70] . Inc reas ing the con-c e n t r a t i o n o f c h i r a l p o ly me r a g e n e r a l imp r o v e me n to f t h e e n a n t i o m e r r e s o lu ti o n w a s r e c o r d e d w h i l e t h eaddi t ion of organic addi t ive caused a dec rease inr e s o lu ti o n , e x c e p t f o r p r o p a n o lo l a n d e p h e d r in e . T h esame ch i ra l se lec tor was employed by Nish i e t a l .[71] for the ch i ra l pur i ty cont ro l o f seve ra l d rugss u c h a s t r ime to q u in o l , d e n o p a min e a n d t r ime p id iu m.T h e C E me th o d w a s s h o w n to b e v e r y r e p r o d u c ib l eand use fu l for quant i ta t ive ch i ra l ana lys is . To i l lus -t r a te th is , F ig . 4 shows the e lec t ropherogram of thee n a n t i o me r s e p a r a t i o n o f t r ime to q u in o l a n d d e -n o p a min e w h e r e 0 . 2 a n d 0 . 5 o f min o r i s o me rwas de tec ted .

Many imidazole de r iva t ives a re cur ren t ly used a sant imy cot ic s , an t ineoplas t ic agents , an t iep i le t ic s , e tc .,and apa r t f rom th is some imidazole and t r iazo lede r iva t ives have been sepa ra ted in the i r enant iomersb y C E [ 5 6 , 7 2 - 7 4 ] . R e c e n t l y , e n a n t i o me r s e p a r a t i o no f p h a r ma c e u t i c a l c o mp o u n d s c o n t a in in g imid a z o l emo ie ty w a s s y s t e ma t i c a l l y s t u d i e d b y C E u s in gdi f fe ren t CDs [75] . Na t ive ( a - , /3 - and 3 , -CD) andd e r iv a t i s e d C D s ( H P - /3 - C D ) w e r e t e s t e d f o r t h echi ra l r e so lu t ion of r acemic b i fonazole , o rn idazole ,e c o n a z o l e , mic o n a z o l e , e n i l c o n a z o l e , k e to c o n a z o l e ,me to m id a t e a n d l o f e x id in e . / 3 - , y - o r H P - /3 - C D w e r ea b l e t o r e s o lv e mo s t o f th e s t u d i e d c o mp o u n d s . T h echi ra l sepa ra t ion was inf luenced not on ly by the CDtype but a l so by the ch i ra l se lec tor concent ra t ion andb y t h e B G E ' s p H a n d t h e o r g a n i c a d d i t i v e . E n i l -c o n a z o l e c o u ld b e r e s o lv e d i n i t s e n a n t i o me r s a t p Hlo w e r th a n 6 - 7 u s in g u n c h a rg e d C D s , t h e o p t imu mexper imenta l condi t ions were found a t pH 3 wi th 2 .5m M / 3 - C D a n d 1 0 m e t h an o l .

As ment ioned be fore , to a ce ta in ex ten t i t could bea d v a n t a g e o u s t o d e r i v a t i z e t h e tw o e n a n t i o me r sbe fore the CE ana lys is us ing an achi ra l r eagent wi ththe a im to e i the r improve the de tec tab i l i ty or toin t roduce groups tha t can eas i ly in te rac t wi th thechi ra l se lec tor present in the e lec t rophore t ic sepa -ra t ion sys tem. This in te res t ing approach has beenshown for the sepa ra t ion of D- and L-ca rn i t ine tha tdue , to the chemica l s t ruc ture , were not sepa ra tedf r o m e a c h o th e r w i th o u t d e r i v a ti z a ti o n . H e r e t h e tw oenant iomers a re in jec ted for the CE ana lys is a f te rde r iva t iza t ion wi th an achi ra l r eagent , 9 - f luorenyl -


8/45

84 S . F a n a l i / J . C h r o m a t o g r . A 7 3 5 1 9 9 6 ) 7 7 - 1 2 1

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t i m e m i n )Fig. 4. Electropherogramof the enantiomer separation of trimetoquinol and denop amine using uncharged/3-C D polym erand di-OMe-/3-CD.Background electrolytes: (a) 25 mM phosphate buffer pH 2.7 and 5 %/3-C D p olymer 20 kV applied voltage); (b) 25 mM phosphate bufferpH 2.7, 2 M urea and 20 mM di-OMe-fl-CD (15 kV applied voltage); capillary, 57(50) cm 75 /zm I.D. (modified from Ref. [71])

m e t h y l c h l o r o f o r m a t e . T h e u s e o f p h o s p h a t e b u f f e r a tp H 2 . 6 c o n t a i n i n g T B A B r a n d y - C D a l l o w e d t h ee n a n t i o m e r s r e s o l u t i o n o f c a m i t i n e d e r i v a t i v e s [ 4 3 ]

O p t i m i z a t i o n s t u d i e s h a v e b e e n i n v e s t i g a t e d d e -v e l o p i n g a t h e o r e t i c a l m o d e l w h e r e t h e e l e c t r o p h o -r e t i c m o b i l i t y w a s c o r r e l a t e d t o t h e C D c o n c e n -t r a t i o n . A n o p t i m u m C D c o n c e n t r a t i o n w a s f o u n d f o rp r o p r a n o l o l u s i n g d i - O M e - f l - C D a s c h i r a l s e l e c t o r[ 7 6 ] . T h e s a m e m o d e l w a s a p p l i e d f o r t h e e n a n t i o -m e r s e p a r a t i o n o f o x p r e n o l o l a n d m e t o p r o l o l [ 7 7 ] ,for prac to lo l , ephedr ine and a t enolo l [64] . Penn e t a l .c a l c u l a t e d t h e s t a b i l i t y c o n s t a n t s o f t i o c o n a z o l ee n a n t i o m e r s w i t h H P - f l - C D a n d t h e o p t i m u m r e s -o l u t i o n e x p e r i m e n t a l c o n d i t i o n s w e r e p r e d i c t e d . T h ee l e c t ro p h o r e t i c m o b i l i t y w e r e c o r r e c t e d f o r t h e e f f e c to f t h e c h a n g e o f v i s c o s i t y d u e t o t h e i n c r e a s e o f t h eC D c o n c e n t r a t i o n . A m o r e c o m p l e x t h e o r e t i c a l

m o d e l s t u d y i n g t h e e f f e c t o f p H a n d c o n c e n t r a t i o n o ff l - C D o n c h i r a l s e l e c t i v i t y f o r f e n o p r o f e n a n d i b u -p r o f e n w a s d i s c u s se d b y V i g h ' s g r o u p [ 78 ]. T h es a m e g r o u p u s e d H P - f l - C D i n C E f o r t h e s t u d y o ft h e e f f e c t o f p H a n d C D c o n c e n t r a t i o n o n p e a kr e s o l u t i o n o f e n a n t i o m e r s o f a t r o p i n e ( d e s i o n o s e l e c -t i v e s e p a r a t i o n ) , c h l o r o a m p h e t a m i n e ( i o n o s e l e c t i v es e p a r a ti o n ) a n d p r o p r a n o l o l ( d u o s e l e c t iv e s e p a r at i o n ).P e a k r e s o l u t i o n s u r f a c e s w e r e c a l c u l a t e d a n d u s e d f o rthe ana lys i s of the poss ib i l i ty of ch i ra l s epara t ions[79] . The theore t i ca l approach was a l so used forw e a k a c i d s l i k e n a p r o x e n a n d f e n o p r o f e n c o n c l u d i n gtha t the succes s of the ch i ra l s epara t ion was pr imar i -l y i n fl u e n c e d b y t h e p H o f th e B G E a n d s e c o n d a r i l yby the concent ra t ion of the ch i ra l s e l ec tor [80] .

O t h e r m a n i p u l a t io n s o f t h e B G E f o r i m p r o v i n g t h es e l e c t i v i t y o f t h e e n a n t i o m e r s e p a r a t i o n i n c l u d e t h e


9/45

S . F a n a l i / J . C h r o m a t o g r . A 7 3 5 1 9 9 6 ) 7 7 - 1 2 1 8

addi t ion of ach i ra l sur fac tan ts to the BGE conta in ingC D s . T h i s me th o d p r o v e d t o b e e f f e c t i v e ma in ly f o rt h e r e s o lu t i o n o f u n c h a r g e d c o mp o u n d s .

T h e e l e c t r o p h o r e t i c s y s t e m i s c o mp o s e d o f i o n i csur fac tan ts forming mice l le s (SDS is the most used)a n d a n a q u e o u s p h a s e ( b u f f e r c o mb in e d w i th C D s ) .T h e u n c h a r g e d a n a ly t e s a r e mo v in g w i th t h e e l ec t r o -osmot ic f low and a r epa r t i t ion equi l ib r ium is e s tab-l i shed be tween the mice l la r -aqueous phases caus inga d i f f e ren t ve loc i ty of ana ly te s . The ch i ra l sepa ra t ionis due to the CD tha t wi l l cause a lower ing of themig r a t i o n t ime s o f t h e tw o e n a n t i o me r s d e p e n d in gf r o m th e C D - a n a ly t e c o mp le x a t i o n .

S e v e r a l p a r a me te rs c a n b e mo d i f i e d f o r imp r o v in gthe s te reose lec t iv i ty whe n CD -M EK C is used , e .g . ,s u r f a c t a n t t y p e a n d c o n c e n t r a t i o n , C D ty p e a n dconcent ra t ion , buf fe r type , ion ic s t r ength , pH andorganic addi t ive .

C D - M E K C h a s b e e n w i d e l y e m p l o y e d f o r t h eenant iomer sepa ra t ion of de r iva t i sed amino ac ids .D a n s y l - a min o a c id s h a v e b e e n r e s o lv e d u s in g S D Sand y-CD [81] or a mix ture of y- and /3-CD, whi lef o r n a p h th a l e n e - 2 , 3 - d i c a r b o x a ld e h y d e ( C B I - a min oa c id s ) y - C D /S D S p r o v e d t o b e e f f e c t i v e [ 8 2 , 8 3 ] .T h e a p p l i c a b i l i t y o f C D - ME K C f o r t h e c h i r a l s e p a -r a ti o n o f p h a r ma c e u t i c a l co m p o u n d s h a s b e e n s h o w nby Nish i e t a l . [84] , who showed the sepa ra t ion ofth iopenta l and pentoba rb i ta l in the i r enant iomers a tp H 9 i n t h e p r e s e n c e o f S D S a n d y - C D ; t h ee n a n t i o s e l e c ti v i t y w a s im p r o v e d b y a d d in g l - m e th o x y -ace t ic (1-men) or d-camphor -10-su l fonic ac ids (d-c a m) t o t h e B G E . T h e e n a n t i o me r s e p a r a t i o n o fp h a r ma c e u t i c a l c o mp o u n d s b y C D - ME K C in c lu d e scyc le tan ine [85] , d in iconazol and uniconazol [56 ,86] ,th iazole de r iva t ives [87] , hexobarb i ta l , mephobarb i -ta l , secobarb i ta l , g lu the t imide , g lu te th imide ana -logues and fadro zole [88] . Dif fe ren t CDs in combi-n a t io n w i th S D S h a v e b e e n t e s te d f o r t h e e n a n t i o me rs e p a r a t i o n o f me p h e n y to in , p h e n y to in a n d t h e i r 4 -h y d r o x y d e r iv a t i v e s a n d t h e u se o f / 3 - C D a l l o w e d t h ecomple te r e so lu t ion of the s tud ied compounds [89] .R e c e n t l y mo d i f i e d c h a r g e d a n d n o n - c h a r g e d / 3 - C Dh a v e b e e n u s e d i n mic e l l a r s y s t e m a t p H 9 . 5 c o n -ta in ing e i the r SDS or STDC for the ch i ra l r e so lu t ionof seve ra l r acemic compounds inc luding /3-agonis t s ,/3 -an tagonis t s , phenyle thylamine s t imulants and theant idepressant d ic lofens ine [90] .

T h e c o mb in a t i o n o f /3 - C D a n d S T D C w a s a l s o

used for the ch i ra l sepa ra t ion of mephenyto in andh y d r o x y m e p h e n y to in [9 1 ], a s w e l l a s f o r t h e s e p a -r a ti o n o f b a c lo f e n a n d i t s a min o p h o s p h o r i c a n a lo g u e s[911.

A c o mp a r i s o n o f t h e d i f f e r e n t C E mo d e s , n a me lyC Z E a n d ME K C h a s b e e n s h o w n b y A n ig b o g u e t a l .[92] for the enant iomer sepa ra t ion of amino-g lu te th imide , a drug used for the t r ea tment ofadrenocor t ica l tumours . At pH 9 , where the ana ly te sa r e n eu t ra l , th e c o mb in a t i o n o f / 3 - C D a n d C M- j3 - C Din t h e p r e s e n c e o f 5 0 Me O H e n a b l e d th e b a s e -l i n esepa ra t ion . Good re su l t s were a l so obta ined a t pH 3us ing CZ E; c~-, a s we l l a s y -CD a l lowed the ch i ra lr e so lu t ion .

R e c e n t l y e n a n t i o me r s e p a r at i o n o f o x a mn iq u in e b yC E w a s c o mp a r e d w i th t h e r e s u l t s o b t a in e d u s in gHPLC [93] . The ch i ra l s ta t iona ry phase (Cyc lobond Iand I I ) used in HPLC did not a l low the enant iomerreso lu t ion of the s tud ied compound, whi le a ch i ra l -AGP pro te in s ta t iona ry phase a l lowed to ob ta insuccessfu l ch i ra l sepa ra t ion , bu t the r e su l t s weres t rongly inf luenced by sma l l changes of pH (0 .2u n i t s ) . / 3 - C D a d d e d t o t h e B G E a t p H 1 2 s h o w e d t h a tCE can g ive be t te r r e su l t s than HPLC; in f ac t , goodresu l ts were obta ined , even i f the pH was chan ged 1pH unit . In a recent study Piperaki e t a l . [94]inves t iga ted the enant iomer sepa ra t ion of f luoxe t inea n d n o r f l u o x e t in e u sin g H P L C a n d C E . T h e b in d in gc o n s t an t s w e r e me a s u r e d w i th b o th t e c h n iq u e s u n d e rthe same exper imenta l condi t ions , g iv ing goodagreement be tween the da ta ob ta ined . B inding con-s tan ts were inf luenced by the CD type , ion ic addi -t ives and organic so lvents . I t was conc luded tha t CEcan be used advantageous ly in orde r to pred ic t theuse fu l o rganic so lvent for HPLC.

T r ime p id iu m, t r ime to q u in o l a n d d e n o p a min e e n -ant iomers were successfu l ly sepa ra ted us ing CE andH P L C w i th t h e d i r e c t s e p a r a t i o n me th o d . C E p r o v e dto be use fu l for qua l i ty cont ro l o f ch i ra l d rugs and i twas poss ib le to de tec t a s l i tt l e a s 0 .1 of inac t iveenant iomer us ing CE [71] .

Recent ly , ion- spray mass spec t rometry coupledw i th C E w a s u s e d f o r mo n i to r i n g t h e e n a n t i o me rsepa ra t ion of te rbuta l ine and ephedr ine . The sys temwas ab le to de tec t bo th f r ee enant iomers and enant io-me r - C D c o mp le x e s f o r t e r b u t a l i n e . T h e me th o d w a sshown to be su i tab le for the ana lys is of te rbuta l ineenant iomers sp iked in to a ur ine sample and the MS


10/45

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2mMNHO2mMsoumeaae

pH71mMSD2Mue1MeO

a3mMCS

1mMppepH2515Mue

a3mMCS

1mMTsHO4pH2901

MH00mMHAa1mMCS

5mMppepH33a13mMCS

9(2mMTsHO4pH24

a5mMCS)1MeO

2mMTsccadpH35

01Ha1mMCS

[9 [6 [6 [111

[1 [9]

I1 [5 [6 [1 [1 [1 [9 [6 [8 [1 [1 [7 [9 [5

~q

Con

op8

-~


12/45

Deasmnakn

mae(amamn

mhmamnpogamamn

Dhodndvv

Dmhn

Dnc

e

E

n

1mMbaebepH861mM

SD1%Aa2mMCS

5mMppepH33a3mMCS

2mMNHO1mMccad

pH4a1mMCS

5mMsoumeaaeppc

adpH22a3mMCS

ppebepH25455

MeO08%a61mMCS

ppepH3(102mMT

a6mMCS

5mMppepH25a

1mmCS

98%(2mMTsHPO4pH24

a5mMCS)12%MeO

2mMppepH272Mue

a2mMCS

2mMTsppe01%o05%

HMCa6mMCS

C

HC

1mMccadTspH6

[1

a03%CS

C

HyC

2mMppepH6a05%CS

[1

C

HC

5mMppepH33a

[7

3gmlCS

C

HC

1mMppcadehamn

[9

pH3a3mMCS

CMEK

yCodOMeC

1mMbaepH91mMSD

[58

2Muea5mMCS

C

dOMeC

l0mMTsppepH24

[1

a2mMCS

C

dOMeC

5mMppepH33a3mMCS

[7

Te2(cn

Cm

Cy

C

B

Ree

Copamn

CMEK

fC

pH35Mue5mMSD

[1

a1mMCS

C

fC

1mMppepH251%MeO

[1

15Muea3mMCS

C

HCofC

1mMppcadehamn

[9

pH3a3o1mMCS

C

fCdOMeC

5mMTMAppepH25a

[6

tOMeC

2mMCS

HC

Ccean

CMEK

yC

[8

Ceeo

C

HC

[7

C

fC

[1

C

HC

[1

.~

C

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[1

C

HC

[6

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/3Cpym

[6

~

Cn

C

dOMeC

[9

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mn

C

dOMeC

[711

C

dOMeC

[1


13/45

E

n

Enn

Een

Eoa

Foe

Fuamn

meaFuamin

ooFuamin

Fdmamhdvv

Foe

C C C C C C C C C C C C C C C C C C C CMEK

C C C CMEK

C

fC dOMeC

dOMeC

dOMeC

dOMeCo

HC

dOMeC

fCdOMeC

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dOMeC

dOMeC

dOMeC

dOMeC

dOMeCM-C

fC fCdOMeC

HC

fCpym

HC

dOMeC

M-3CoTC

fCdOMeC

dOMeC

tOMeC

TC fCTD

CD

1mMTppepH25

a2mMCS

5mMLppepH25

a5mMCS

3mMTsppepH25

a4g1CS

2mMppepH251mMT

01HCa1mMCS

1mMppcadehamn

pH3a3mMCS

5mMppepH25a1mMCS

5mMTMAppepH25

a2mMCS

2mMppepH25a2mMCS

1mMTsppepH24

a2mMCS

1mMppepH25a2mMCS

5mMppepH25a2mMCS

1mMNHO4pH251MeO

a3mMCS

2mMTsHO4pH23

a9mMCS

1mMppeTpH25

a2mMCS

5mMTMAppepH25

a2mMCS

5mMppepH25

a1mmCS

5mMppepH33a3mMCS

2mMppepH32Mue

2mMbaeppepH7

a1mMCS

2mMNHO2mMsoumeaae

pH71mMSD2Mue1MeO

a3mMCS

1mMppcadehamn

pH33MeOa1mMCS

1mMppcadTspH25

a4mMCS

1mMppcadTspH25

a3mMCS

3mMNHO1mMHBpH72

a2mMfC

mMTD

2mMMEpH4502H

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[1 [6 [1 [1 [9 [5 [6 [1 [1 [1 [6 [1 11

]1 [6 [6 [7 ill

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Conop9

.q g


14/45

Te2(cn

Cm

Cy

C

B

Ree

C

HC

2mMMEpH4402%H

[8

a2mMCS

C

fC

6mMMEpH4602%H

[1

a1mMCS

C

tOMeC

1mMMEpH5a3mMCS

[6

Fun

CMEK

tOMeC

1mMTspH2701%MHCA

[1

a1mMCS

Fubpoe

C

tOMeC

1mMMEpH5a3mMCS

[6

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C

HC

1mMppcadehamn

[9

pH3a2mMCS

Guehmd

C

dOMeC

5mMppepH25a1mMCS

[8

H

ba

C

oCfC

5mMppe1mMbae

[1

.~

dOMeC

pH9a1mMCS

C

fCdOMeC

1mMTsbcadpH832mM

[1

H~C

EDA0513%CS

CMEK

fCdOMeC

2mMNHO2mMsoumeaae

[8

.~

pH71mMSD2Mue1%MeO

a3mMCS

Hmon

C

fC

3mMppebepH62

[1

02%Ha1mMCS

Iboe

C

fC

2mMMEpH4502%H

[71

~

a1mMCS

o

C

tOMeC

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C

HC

5mMppepH33a3mMCS

[7

,~

Imdedvv

C

fCoTC

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[7

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e

a2mMfo5mM~C

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loednmomde

imdedvv

C

HC

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.[7

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eec

eoedn

a2mMCS

momdemc

e

Inoe

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Ioen

Iooee

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yC

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dOMeCo

HC

C

dOMeC

C

dOMeC

C

fC

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dOMeC

C

dOMeC

C

fCdOMeC

tOMeCHC

1mMMEpH5a3mMCS

[6

01MppebepH25a3mMCS

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1mMppcadehamn

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pH3a1o3mMCS

1mMTsppepH24a1mMCS

[1

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[6

1mMppeTpH25a2mMCS

[1

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[9

a2mMCS

5mMppepH33a3mMCS

[7

5mMTMAppepH25a2mMCS

[6


15/45

Kamn

Kooe

Koeasnemae

Lsud

Ledn

Meon

Meu

Me

on4hom

on

Me

ba

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Meooo

Meuhn

Mehmamn

Meooo

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Nmhe

n

Nmhp

n

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C C C C CMEK

CMEK

CMEK

C C C C C C C C C C C C

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f-C yC HC

HC

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n-C

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HC

dOMeC

dOMeC

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dOMeC

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5mMppepH25a5mMCS

5mMppepH25a2mMCS

5mMppepH33a3mMCS

5mMppepH25a2mmCS

1mMMEpH5a3mMCS

L5mMsoumaaepH5502H

a1mMCST1mMfaan

2mMTsccadpH3500H

a1mMCS

01MppebepH25

a3mMCS

5mMppepH33a3mMCS

5mMppepH33a3mMCS

01MppebepH25

a3mMCS

3mMNHO1mMbcadpH72

5mMTD

a2mMCS

1mMppe6mMbaepH91

1mMSD12po

a5mMCS

2mMNHO2mMsoumeaae

pH71mMSD2Mue1MeO

a3mMCS

1mMTsHPO4pH2901MH

00mMHAa1mMCS

5mMppepH33a3mMCS

4mMLppepH3a3mMCS

9(2mMTsHO4pH24

a5mMCS)1MeO

98(2mMTsHO4pH24

a5mMCS)12MeO

5mMTMAppepH25

a2mMCS

5mMppepH33a3mMCS

5mMppepH33a3mMCS

3mMTsppepH25

a4gCS

pH25HCT

2mMppepH251mMT

a1mMCS

2mMppepH251mM

T

a1mMCSHT

2mMppepH251mMT

a1mMCS

[6 [6 [7 [6 [6 [48]

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16/45

Te2(cn

Cm

Cy

C

B

Ree

No

C

HC

5mMsoumppepH257mM

[9

TMAa2mMCS

C

HC

5mMTMAppepH25

[6

a2mMCS

No

C

HC

2mMMETpH504%pymc

[1

ava1mMCS

C

HC

2mMMEpH4802%H

[8

a5mMCS

Nom

C

HC

5mMppepH33a3mMCS

[7

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C

yC

01MppebepH25

[5

a3mMCS

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C

H3C

5mMppepH33a3mMCS

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n

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1mMTsppepH24

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5mMppepH33a3mMCS

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3mMTsppepH25

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~

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9%(2mMTsHO4pH24

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a5mMCS)1%MeO

~

C

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[6

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C

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[1

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C

dOMeC

5mMppepH25a2mMCS

[6

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fC

1mMppeTpH25

[1

a2mMCS

C

H3C

3mMTsccadpH255mM

[11

1CH4a2mMCS

C

dOMeCHC

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[6

a2mMCS

C

fCpym

5mMppepH25

[6

a1mmCS

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H3C

5mMppepH33a3mMCS

[7

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n

IT

fC

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[4

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C

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9%(2mMTsHOpH24

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a5mMCS)1%MeO

Nvam

C

tOMeC

1mMppcadehamn

[9

pH3a2mMCS

C

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6mMppepH25a6mMCS

[1


17/45

Oayp

n

Oomn

Omnqn

Ommn

Oeo

Poba

pHop

n

Pamn(mhdmheh

amamnmhdmhmha

amamnmhamamn

amamne

nenn

p

n

Pamn(mhdmhmh

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mhdmhhamamn)

Pen

Pcmeo

Pno

Paoo

IT C C C C C C C C C CMEK

IT C CMEK

C C C C CMEK

C C C

f-C dOMeC

HCfC HC

dOMeC

dOMeC

dOMeCHC

fCpym

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)-C fC HC

H3C

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OMeC

dOMeC

dOMeC

dOMeC

fCdOMeC

nCdOMeC

L5mMsoumaaepH54

a1mMCST1mMfaan

5mMppebepH25

a2mMCS

5mMppepH33a3mMCS

5mMNHO4pH1a2mMCS

1mMppcadehamn

pH3a1mMCS

5mMsoumppepH257mM

TMAa2mMCS

5mMLppepH3a3mMCS

5mMTMAppepH25

a2mMCS

5mMppepH25

a1mmCS

5mMppe1mMbaepH9

a0mMCS

2mMppebaepH95mMSD

a3mMCS6mMm

o4mMdcm

L5mMsoumaae02HpH

54a1mMCST0mMfaan

1mMccad12mMNHO4

pH25a1mMCS

[4 [6 [7 [9 [9 [9 [7 [6 [6 [1 [8 [4 [1

5mMbaepH955po

o

5mMSDa6o1mMCS

5mMppepH33a3mMCS

2mMNHO1mMccad

pH4a1mMCS

2mMbaeppcadpH23

a3mMCS

5mMsoumppepH257mM

TMAa2mMCS

1mMTsHO4pH2901MH

00mMHAa0mMCS

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pH3a1mMCS

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5mMppepH25a4mMCS

[9 [7 [1 [11

[9 [1 [9 [6 [6

Conop9


18/45

Te2(cn

Cm

Cy

C

B

Ree

Pmn

C

dOMeC

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[1

24mMCS

C

TM-C

2mMppepH272Mue

[1

a2mMCS

C

yC

2mMppebaepH27

[7

a2mMCS

IT

fC

L1mMsoumaaepH55a

[1

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C

TC

pH3

[1]

C

fC

01MppebepH25ue

[5

MeOa4mMCS

C

HCoHC

2mMTsppepH24

[9

a2mMCS

C

dOMe3C

4mMLppepH3MeOoA

[71

a3mMCS

C

/3CdOMeC

5o1mMppebepH25

[91

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ToTMAa2mMCS

CMEK

fC

4mMbaepH933mMSD

[1

a1mMCS

C

tOMeC

1mMppcadehamn

[9

pH3a1mMCS

'~

C

HC

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[1

04%pymava1mMCS

C

HC

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[1

C

fCdOMeC

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[6

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a2mMCS

.q

C

fCpym

5mMppepH25a2mMCS

[6

~

C

HCdOMeC

5mMKPO4pH3MeOa2mMCS

[1

23dOdayC

IT

dOMeC

[4

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fC

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[1

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[6

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fC

[1

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aCDI1CH4

[1

Pomhn

Poao

Poao

Poaoadvv

P

n

Pmuhn

Qnd

L5mMsoumaaepH5402%

Ha1mMCST1mMfaan

1mMTppepH25

a2mMCS

2mMppepH251mMT

a1mMCS

5mMTMAppepH25

a2mMCS

9%(2mMTsHO4pH24

a5mMCS)1%MeO

5mMppepH25a3mMCS

1mMTsccadpH222mM1CH4

a2mMCS


19/45

Sbam

Sbamareaeme

SDEA4(novdu

S

ba

Soon(5Has

3amnk6cbmd1234

teaobeareae

Nakao(GT

Sao

SoeS

n

Tban

Tgd

Toa

Todn

Tmdum

C C C C C CMEK

CMEK

C C C C C C C C C C CMEK

IT IT C C CMEK

dOMeC

H3C

dOMe3C

TC o-C

OMeaC

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tOMeC

dOMe3C

fCdOMC

HCofC

H3C

fCpym

fCdOMe3C

H3CyC TC yC yC TC yC yC

pH33ppepH3(=02mMT

a6mMCS

5mMNHO1mMccadpH25

a1mMCS

1mMbaebepH1a2mMCS

5mMppe1mMbaepH9

a1mMCS

2mMNHPO2mMsoumeaae

pH71mMSD2Mue1MeO

a3mMCS

3mMNHPO41mMbcadpH70

a2mMC5mMSD

5mMppepH33a3mMCS

1mMppcadehamn

pH3a1mMCS

1mMMEpH5a3mMCS

5mMppepH33a3mMCS

1mMppebepH25

a1mMo5mMoCS

1mMppcadehamn

pH3a1mMCS

ppepH3(102mMT

a6mMCS

5mMppepH25

a1mmCS

5mMppepH2516a

6

a52mMCS

01MppebepH25

a3mMCS

2mMppebaepH9

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a3mMCS

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pH55a5mMCST0mM

amnocad

2mMTsHO4pH25a5mMCS

2mMppebaepH27

a2mMCS

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5mMDAa2mMCS

[7 [6 [1 [1 [1 [88 [1 [7 [9 [6 [7 [5 [9 [6 [6 [55

1

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[84]

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20/45

Te2(cn

Cm

Cy

C

B

Ree

Tndao

C

yC

Tmon

C

dOMC

C

dOMC

Tmonao

C

fCpym

C

fCofCpym

Tmpamn

C

HC

Uce

CMK

yC

Vam

CMK

tOMC

Waan

Zc

C

tOMCofC

C

tOMC

C

tOMCHC

C

dOM~C

C

HBC

4mMsoumppepH300PV

[6

a5mMCS

2mMppe2MuepH25

[1

a14mMCS

2mMppepH27a5CS

[7

2mMppepH272Mue

[1

a1CS

2mMppepH65(2Mue

[7

a4mMfCo3~C

pym2765

5mMTMppepH25

[6

a2mMCS

1mMbaepH91mMSD2M

[5

ue1soaa5mMCS

2mMTsppepH2701M

[1

00mMHA2ehegya

1mMCS

1mMppcaddehamn

[9

pH3a2o1mMCS

6mMppepH25a6mMCS

[1

5mMTMppepH25a2mMCS

[6

1mMppepH832MO

[1

a4mMCS

5mMppepH33a3mMCS

[7

t~

e -4


21/45

S . F a n a l i / J . C h r o m a t o g r . A 7 3 5 1 9 9 6 ) 7 7 - 1 2 1 97d e t e c t i o n p r o v id e d a 1 0 0 0 - f o ld imp r o v e me n t i ns ig n a l - t o - n o i s e r a t i o c o mp a r e d t o t h e U V d e t e c t i o n[95] . To our knowledge , th is i s the f i r s t example onu s in g C E - M S f o r th e a n a ly s is o f e n a n t i o me r s o fpha rmaceut ica l in te res t and we a re convinced tha tth is i s an in te res t ing way to ident i fy ch i ra l d rugisomers .

T h e e n a n t i o me r s e p a r a t i o n s p e r f o r me d b y C Eusing unch arged CD s as ch i ra l se lec tor inc luded ala rge number of drugs ( an t idepressants , an-t ipsychot ic s , hypnot ic s , ba rb i tura te s , anaes the t ic s ,adrene rg ic , b ronchodi la tor s , non-s te ro ida l an t i - in -f lammator ie s , an t i - a s thmat ic s , an t i -h is taminics , an t i -coagulan ts , an t i - funga l drugs , f l -b locke r s , ca lc ium-channe l b locke r s , an t i -hyper tens ion drugs , an t i - a r -rhythmics , an t i - chol ine rg ics , an t i - cance r drugs , an t i -ma la r ia drugs , an t i -bac te r ia l d rugs and a romataseinhibitor) are l is ted in Table 2.

A mo n g th e l a r g e f a mi ly o f C D s , t h o s e p o s s e s s in gc h a r g e d / c h a r g e a b l e g r o u p s p r o v e d t o b e v e r y e f f e c -t i v e f o r th e e n a n t i o m e r s e p a r at i o n o f a w id e r a n g e o fa n a ly t e s , i n c lu d in g c o mp o u n d s o f p h a r ma c e u t i c a linterest .

T h e mo d i f i c a t i o n o f n a t i v e C D s b y i n t r o d u c in gchargeable groups , e .g . , me thylamino , su l fa te , ca r -b o x y l a t e , s u l f o b u ty l , a l l o w s t h e u s e o f a c h a r g e d /cha rgeable ch i ra l se lec tor wi th d i f f e ren t prope r t ie st h a n t h e p a r e n t C D . T h e p r e s e n c e o f t h e a b o v e -ment ioned subs t i tuent groups inc reases the so lubi l i tyof the CD, a l lows the ana lys is of uncharged ana ly te sand in t roduce o the r in te rac t ions , e .g . , e lec t ros ta t ic .T h e mo d i f i e d C D s , u n d e r t h e i n f l u e n c e o f t h e a p p l i e de lec t r ic f ie ld , move wi th the i r own mobi l i ty , and thecha rge of the ch i ra l se lec tor can p lay a ve ry im-por tan t ro le in the sepa ra t ion mechanism.

Cons ide r ing the s imples t theore t ica l approachd e s c r ib e d b y W r e n s g r o u p [ 7 6 ,7 7 , 1 4 0 ] f o r e n a n t i o -mer sepa ra t ion us ing CDs as ch i ra l se lec tor s , thed i f f e r e n c e i n mo b i l i t y o f t h e tw o e n a n t i o me r s i si n f l u e n c e d b y t h e d i f f e r e n c e i n mo b i l i t y o f f r e ea n a ly t e ( / x f) a n d c o mp le x e d c o mp o u n d ( /X c ). T h e u s eo f c h a r g e d C D s i s c a u s in g t h e i n c r e a s e o f t h e s epa ramete r s and , thus , the inc rease of r e so lu t ion .

M o n o ( 6 - f l - a m i n o e t h y l a m i n o - 6 - d e o x y ) - f l - C D ( C -D e n ) a n d 2 - c a r b o x y m e th y l - f l - C D h av e b e e n e m-p lo y e d i n C E f o r t h e f i r s t t ime b y T e r a b e a n dco-w orke r s [ 13 ,141 ] for the en ant iom er sepa ra t ion ofseve ra l dansyl -amino ac ids , and la te r on Nard i e t a l .

u s e d p o s i t i v e ly c h a r g e d f l - C D d e r iv a t i v e s , n a me ly6 g - me th y l a min o , a n d 6 A a n d 6 D - d im e th y l a min o - f lC D f o r t h e e n a n t i o me r s e p a r a t i o n o f s o me h y d r o x yac id de r iva t ives . Also , seve ra l g roups inves t iga tedd i f f e r e n t mo d i f i e d C D w i th c h a r g e d / c h a r g e a b l egroups for the ch i ra l CE sepa ra t ion of pha rmaceu-t i c a l c o mp o u n d s [ 1 6 , 7 5 , 9 9 , 1 0 2 , 1 1 4 , 1 1 7 , 1 4 2 - 1 4 7 ] .

In a ve ry in te res t ing s tudy Schmit t and Enge lha rd tu s e d c a r b o x y me th y t a t e d - f l - C D , f o c u s in g o n t h e im-por tance of the CD cha rge on the ch i ra l r ecogni t ion ,w h ic h c a n s t r o n g ly b e i n f l u e n c e d b y t h e p H o f t h eB G E . T h e c a r b o x y l i c g r o u p o f t h e C D w a s n o tcha rged a t pH lower than 4 and thus the ch i ra lse lec tor behav ed as a quas i s ta t iona ry phase , wh i le a th ig h e r p H ( > 5 ) i t h a d i ts o w n m o b i l i t y a l l o w in g th echi ra l sepa ra t ion of uncharged ana ly te s of pha rma-ceut ica l in te res t , such a s hexobarb i ta l and ox-azol id inone [16]

A d d i t i o n a l i n f o rma t io n o n t h e u s e o f c h a r g e d C D sh a s b e e n o b t a in e d a n d d i s c u s s e d b y o u r g r o u p u s in ga n e g a t i v e ly c h a r g e d f l - C D p o ly me r [ 1 4 3 ] . T h echi ra l se lec tor possesses ve ry h igh so lubi l i ty and canb e u s e d e i t h e r i n a c h a r g e d o r u n c h a r g e d mo d es e l e c t i n g t h e a p p r o p r i a t e p H o f t h e B G E . T h ep o ly me r w a s s u c c e s s f u l u s e d f o r t h e e n a n t i o me rs e p a r a t i o n o f b a s i c c o mp o u n d s o f p h a r ma c e u t i c a lin te res t such a s te rbuta l ine , p ropranolo l , ep inephr ine ,n o r e p h e d r in e a n d n o r p h e n y l e p h r in e . T e rb u t a l i n e a n dp r o p r a n o lo l s e e me d t o s h o w th e h ig h e s t c o mp le x -a t i o n w i th t h e C D p o ly me r t h a t a t p H > 4 c a u s e d a nin v e r s io n o f mo b i l i t y o f t h e tw o a n a ly t e s . T h eme n t io n e d e f f e c t c a n b e u s e d a d v a n t a g e o u s ly f o rr e v e r sin g t h e m ig r a t io n o r d e r o f tw o e n a n t i o me r s a n dth u s imp r o v e t h e q u a n t i t a t i o n w h e n t h e min o r c o m-ponent i s migra t ing behind the ma jor one . Toi l lus t r a te th is F ig . 5 shows the inve r s ion of themigra t ion orde r of propranolo l .

R e c e n t l y a n e w c h a r g e d f l - C D d e r iv a t i v e ( 4 - s u l -f o b u ty l - f l -C D , S B E - f l - C D ) h a s b e e n s y n th e s i s ed a n dwide ly s tud ied in CE for ch i ra l sepa ra t ions . F ig . 6s h o w s t h e c h e mic a l s t r u c tu re o f t h e S B E - f l - C D . T h enew CD possesses 4 su l fonic groups bound a tp o s i t io n 6 o f 4 g lu c o p y r a n o s e o f t h e C D th r o u g h t h ebuty l cha in . The ch i ra l se lec tor i s nega t ive ly cha rgeda t any pH and thus th is f ea ture broadens i t s use inC E . C E a n a ly s i s o f S B E - f l - C D u s in g i n d i r e c t U Vde tec t ion revea led tha t the ch i ra l se lec tor i s anhe te rogeneous mixture [148] .


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9 8 S . Fa n a li / J . Ch ro ma to g r . A 7 3 5 (1 9 9 6 ) 7 7 -1 2 1

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0 m i s k ~ o ~ l i m ( m i n ) I kF i g . 5 . I n v e r s i o n o f m i g r a t i o n o r d e r o f t h e t w o s e p a r a t e d e n a n t i o -m e r s o f p r o p r a n o l o i . C a p i l l a r y 1 7 c m 2 5 / z m I . D . c o a t e d ) ;b a c k g r o u n d e l e c t r o ly t e , 5 0 m M a c e t a t e b u f f e r p H 4 . 5 + a ) 1 0m g / m l o f n e g a ti v e c h i r a l p o l y m e r ; b ) re v e r s e d p o l a r it y a n o d ed i r e c t i o n ) , 2 0 m g / m l o f n e g a t i v e c h i r a l p o l y m e r ; a p p l i e d v o l t a g e1 2 k V r e p r i n t e d w i t h p e r m i s s i o n f r o m R e f . [ 1 4 3 ] ) .

T h e a p p l i c a b i l i t y o f SBE - f l - CD i n CE f o r t h ee n a n t i o me r s e p a r a t io n o f p h a r ma c e u t i c a l c o mp o u n d swas sh own b y Det t e e t a l . [117] fo r the separa t ion o fsome ephedr ine a lka lo ids ephedr ine , no rephedr ine ,me t h y l e p h e d r i n e , p s e u d o e p h e d r i n e a n dme t h y l p s e u d o e p h e d r in e ) u s i n g a BG E a t p H 1 0,c lose to the pKa o f th e s t u d i e d c o mp o u n d s . T h e s a me

s o s o s oF i g . 6 . S t r u ct u r e o f t h e S B E - ~ - C D .

ch i ra l s e l ec to r a t a concen t ra t ion range 1 .5 -5 raM )was also used by Tai t et al . [145] as a chiral selectora t pH 2 .5 fo r the enan t iome r separa t ion o f ephedr ine ,pseudoephedr ine , ad rena l ine , no radrena l ine andDOPA.

Bas ic pharma ceu t i ca l com pound s c l enbu tero l ,d imeth inde ne , e t i l e f r ine , ima fen , i soprena l ine , l o fex-id ine , mef loqu ine , metomida te and mianser ine) werereso lved in the i r enan t iomers a t pH 3 .1 us ing con-c e n t ra t io n s o f SBE - f l - CD a s l o w a s < 4 0 / . tM. T h ech i ra l s e l ec to r was a l so t es t ed in a coun ter -cu r ren tf low where the ch i ra l s e l ec to r was in j ec t ed fo r on ly1.8 s high pressure) on catho de as pre-r inse whi lethe ana ly te d imeth indene) was in j ec t ed on thea n o d e . Fu r t h e r mo r e t h e n e g a t i v e l y c h a r g e d CD w a ssuccess fu l ly used fo r the enan t iomer separa t ion o ftha l idomide , a neu t ra l d rug moving wi th the eo f andin te rac t ing wi th SBE-f l -CD.

A su l fa t ed - f l -CD has be en t es t ed as ch i ra l s e l ec to rf o r t h e e n a n t i o me r s e p a r a t i o n o f u n c h a r g e d c o m-pounds , such as phensux imide and indapamine , us inga BGE a t pH 6-8 [149] .

Rec en t ly we ha ve inves t iga ted the e f fec t o f severa lparameters such as CD concen t ra t ion , pH of theBGE, s t ruc tu re o f the ana ly tes on the enan t iomersepara t ion o f severa l bas i c and ac id ic compounds o fpharmaceu t i ca l i n t e res t us ing SBE-f l -CD [147] .Racemic warfar in , acenocoumaro l , t e rbu ta l ine , bup i -vaca ine , p rometha z ine were success fu l ly separa t ed inthe i r enan t iomers . To i l lu s t ra te th i s F ig . 7 show s thee n a n t i o me r s e p a r a t i o n o f r a c e mi c c o mp o u n d s o fp h a r ma c e u t i c a l i n t e r e s t e mp l o y i n g SBE - f l - CD a schiral selector .

Tab le 3 summar izes the i somer separa t ion o fch i ra l d rugs by CE us ing charged CDs as ch i ra lselectors .3 1 2 Macrocyclic antibiotics

The use o f the macrocyc l i c an t ib io t i cs van-c o my c i n , r i f a my c i n B a n d r i s to c e t in A w a s r e c e n t l yin t roduced fo r ch ira l s epara t ions in CE b y A rms t ronget a l . [150-152] . Tab le 4a and b show the com-pounds o f pharmaceu t i ca l i n t e res t reso lved in the i renan t iomers us ing macrocyc l i c an t ib io t i cs as ch i ra lselector .Di f fe ren t bas i c compounds o f pharmaceu t i ca l i n -t e res t i nc lud ing ad renerg ic , vasocon t r i c to rs , b ron-chod i l a to rs , vasod il a to rs, f l -ad ren erg ic b lockers w ere


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F i g . 7 . E l e c t r o p h e r o g r a m s o f t h e e n a n t i o m e r s e p a r a t io n o f ra c e m i c c o m p o u n d s o f p h a r m a c e u t i c a l i n t e r e s t w h e n a s u l f o b u t y l a t e d - f l - C D i su s e d a s c h i r a l s e le c to r . B a c k g r o u n d e l e c t ro l y t e 5 0 m M p h o s p h a t e b u f f e r p H 6 c o n t a in i n g S B E - / 3 - C D A , B a n d C , 6 m g / m l ; D , 1 0 m g / m l ) ;c a p i l l a ry 5 0 4 5 . 5 ) c m 5 0 / z m I . D .; a p p l i e d v o l t a g e 1 5 k V r e p r i n t e d w i t h p e r m i s s i o n f r o m R e f . [ 1 4 7 ] ) .

r e so lv e d i n t h e i r e n a n t io me r s u s in g r i f a my c in B a sc h i r a l s e l e c to r d i s so lv e d i n a n o r g a n i c - a q u e o u sbuf fe r due to i t s low so lubi l i ty in wa te r . Due to thes t ro n g a b so r p t i o n a t r e l a t i v e ly sh o r t U V w a v e l e n g th so f t h e c h i r a l c o mp o u n d in d i r e c t U V d e t e c t i o n w a se mp lo y e d . A s c a n b e s e e n i n F ig . 8 r i f a my c in Bp o sse s se s o n e c a r b o x y l i c g r o u p t h a t w a s d i s so c i a t e da t the ope ra t ing pH (pH 7) and thus the ch i ra lc o mp o u n d w a s mo v in g a s n e g a t i v e ly c h a r g e d i n t h eo p p o s i t e d i r e c t io n o f t h e a n a ly se d b a s ic c o m p o u n d s .D i f f e r e n t t y p e s o f i n t e r a c t i o n s w e r e i n v o lv e d i n t h echi ra l r ecogni t ion process , namely cha rge -cha rge ,h y d r o g e n - b o n d in g a n d h y d r o p h o b ic - in c lu s io n .

R i f a my c in B p r o v e d t o b e a g o o d c h i r a l s e l e c to rfor amino a lcohols and i t s ch i ra l enant iose lec t iv i tyw a s s t r o n g ly i n f lu e n c e d b y t h e c h e mic a l s t r u c tu re o fa n a ly t e s ( t h e p o s i t i o n o f t h e h y d r o x y g r o u p , t h eamine type ) . In f ac t , when the OH group was a t thep o s i t i o n o f t h e a r o ma t i c r i n g o r a s e c o n d a r y a min ogroup was present the ch i ra l r ecogni t ion was en-h a n c e d . F u r th e r mo r e , t h e p r e se n c e o f mo r e t h a n o n ea r o ma t i c r i n g i n t h e a n a ly t e ' s s t r u c tu r e r e d u c e d t h eposs ib i l i ty of enant iomer ic r e so lu t ion , ind ica t ing theimpor tance of the inc lus ion in te rac t ion .

A mo n g o th e r o p e r a t i o n a l p a r a me te r s t h e o r g a n i cmo d i f i e r t y p e a n d c o n c e n t r a t i o n , t h e B G E ty p e a n di ts ion ic s t r ength , the concent ra t ion of r i f amyc ins t rongly inf luenced the ch i ra l r ecogni t ion of thes tu d ie d b a s ic c o mp o u n d s . T h e o p t im u m e x p e r ime n ta lc o n d i t i o n s w e r e f o u n d u s in g 0.1 M p h o sp h a t e b u f f e ra t p H 7 (6 0 ) , 2 -p r o p a n o l (4 0 ) a n d 2 5 mM o fr i f a my c in B .

V a n c o m y c in a n d r i s t o c e ti n a r e ma c r o c y c l i c g ly c o -pept ide an t ib io t ic s tha t showed no mobi l i ty a t a pHo f a b o u t 7 ; a t l o w e r a n d h ig h e r p H th e y a r e p o s i t i v e -ly a n d n e g a t iv e ly c h a r g e d , r e sp e c t i v e ly . T h e tw ochira l se lec tor s have been used for the ch i ra l sepa -r a t io n o f mo r e t h a n 1 0 0 c o mp o u n d s , i n c lu d in g so meof the pha rmaceut ica ls l i s ted in Table 4b . Ther e so lu t i o n o f n a p r o x e n i n c r e a se d b y i n c r e a s in g t h ec o n c e n t r a t i o n o f v a n c o my c in , w h i l e i n c r e a s in g t h epH reduced the enant iose lec t iv i ty [150] . A s imi la re f f e c t w a s r e c o r d e d f o r k e to p r o f e n w h e n

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