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E L S E V I E R Journal of Chrom atography B. 685 (19 96 ) 191 195JOU R N A L OFC H R OMA T OGR A P H Y B :BIOMEDICALAPPLICAIIONS

S h o r t c o m m u n i c a t i o nHigh-performance liquid chromatographic analysis for the

determination of miconazole in human plasma using sol id-phaseextraction

M a r i a K o b y l i f i s k a * , K a m i l a K o b y l i f i s k a , B a r b a r a S o b i kDep ar tmen t ~ f Phar m acology , Phar m aceu t i ca l Res ea r ch Ins t i tu t e . 8 Ryc l' giera S t r ee t . 01- 793 W ar s aw . Po land

Received 15 Decem ber 1995 ; revised 12 February 1 996; accepted 4 M arch 1996

A bs tr ac tA h ig h -p e r fo rm a n c e l i q u id c h ro m a to g ra p h ic m e th o d fo r t h e d e t e rm in a t i o n o f m ic o n a z o l e i n h u m a n p l a sm a i s d e sc r ib e d . A

so l id -p h a se e x t r a c ti o n w a s p e r fo rm e d o n a n o c t a d e c y l (Ct s ) c a r t r i d g e . Mic o n a z o l e w a s e lu t e d w i th m e th a n o l , se p a ra t e d o n are v e r se d -p h a se c o lu m n a n d w a s m e a su re d b y u l t r a v io l e t d e t e c t i o n a t 2 3 0 n m . Th e a b so lu t e e x t r a c t i o n r e c o v e ry f ro m p l a sm asa m p le s w a s 8 5 %. Th e l im i t o f d e t e c t i o n w a s e s t a b l i sh e d a s 5 n g /m l . Th e c o e f f i c i e n t o f v a r i a t i o n o f t h e d e t e rm in a t i o n o fp l a sm a l e v e l s b y t h is m e th o d o v e r t h e s t a n d a rd c u rv e c o n c e n t ra t i o n r a n g e w a s l e ss t h a n 1 0 %, e x c e p t w i th t h e c o n c e n t ra t i o no f 1 0 n g /m l . Th e p l a sm a l e v e l s o f m ic o n a z o l e i n tw e lv e h e a l t h y v o lu n t e e r s g iv e n a 2 5 0 -m g o ra l d o se o f tw o t a b l e t fo rm sw e re d e t e rm in e d b y t h i s m e th o d .K e v w o r d s : M i c o n a z o l e

1 . I n t r o d u c t i o nM i c o n a z o l e [ l - ( 2 , 4 - d i c h l o r o - / 3 - ( ( 2 , 4 - c h l o r o -

b e n z y l ) o x y ) p h e n e t h y l ) i m i d a z o l e ] i s a s y n t h e t i ci m i d a z o l e d e r i v a t i v e w i t h a b r o a d - s p e c t r u m a n t i f u n g -a l a c t i v i t y . I t i s e s t a b l i s h e d a s a u s e f u l d r u g f o r t h et r e a t m e n t o f v a ri o u s s y s t e m i c m y c o s e s , i n c l u d i n gc a n d i d i a s i s , c o c c i d i o i d o m y c o s i s , c r y p t o c o c c o s i s a n dh i s t o p l a s m o s i s 1 1 , 2 ] . I t i s a l s o a c t i v e a g a i n s t G r a m -p o s i t i v e b a c t e r i a [1 ]. S e v e r a l H P L C m e t h o d s h a v eb e e n d e s c r i b e d f o r i t s d e t e r m i n a t i o n i n b i o l o g i c a lf l ui d s [ 3 - 5 ] , b u t n o n e w a s s e n s i t i v e e n o u g h f o rq u a n t i t a t i o n o f t h is d r u g a f t e r o r a l a d m i n i s t r a t i o n .T h e s e m e t h o d s u s e d a l i q u i d - l i q u i d e x t ra c t i o n p r o -c e d u r e a s t h e s a m p l e p r e p a r a t i o n s t e p .*Corresponding author.

I n t h i s w o r k , w e d e s c r i b e a s i m p l e a n d s e n s i t i v eH P L C m e t h o d t o m e a s u re m i c o n a z o le i n hu m a np l a s m a w h i c h r e n d e r s i t a p p l i c a b l e i n b i o a v a i l a b i l i t ys t u d ie s . T h e m e t h o d c o n s i s t s o f a s o l i d - p h a s e e x -t r a c ti o n ( S P E ) p r o c e d u r e a s t h e s a m p l e p r e p a r a t i o ns t e p .

2 . E x p e r i m e n t a l2 . I . C h e m i c a l s

M i c o n a z o l e n i tr a t e w a s s u p p l i e d b y th e P h a r m a c yD e p a r t m e n t o f th e P h a r m a c e u t i c a l R e s e a r c h I n s t i tu t e( W a r s a w , P o l a n d ) . H P L C - g r a d e a c e t o n i t ri l e a n dm e t h a n o l w e r e p u r c h a s e d f r o m J . T . B a k e r ( P h i l l ip -

0378-4347/96/$15.00 199 6 Elsevier Science B.V. All rights reservedP I I S 0 3 7 8 - 4 3 4 7 ( 9 6 ) 0 0 1 2 5 - 9

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192 M. Kobyl#iska et al. / J. Chromatogr. B 685 (1996) 191-195s b u r g , N J , U S A ) . A l l o t h e r r e a g e n t s w e r e o f a n a -l y t i c a l g r a d e .2.2. Chromatograp hic conditions

T h e H P L C s y s te m c o n s is t ed o f a M o d e l L C - 6 Ap u m p , a M o d e l S P D - 6 A V U V - V i s d e t ec t o r o p e r a te da t 2 3 0 n m , a M o d e l C R - 6 A i n t e g r a t o r a n d a M o d e lS I L - 9 A a u t o s a m p l e r ( S h i m a d z u E u r o p a , D u i s b u rg ,G e r m a n y ) . T h e a n a ly t ic a l co l u m n ( 2 5 0 4 m m I .D .,N u c l e o s i l 1 2 0 - 5 C j s ; M a c h e r e y - N a g e l , D i i re n , G e r -m a n y ) w a s p r e c e d e d b y a 3 0 4 m m I . D . N u c l e o s i l1 2 0 - 5 C ~ 8 g u a r d c o l u m n . T h e c o l u m n w a s h e a t e d a t4 0 C u s i n g a M o d e l C T O - 1 0 A o v e n ( S h i m a d z uE u r o p a ). T h e m o b i l e p h a s e c o n s i s t ed o f m e t h a n o l -a c e t o n i t r i l e - 0 . 0 1 M p h o s p h a t e b u f f e r, p H 7 . 0( 3 6 : 3 6 : 2 8 , v / v ) a n d w a s d e l i v e r e d a t a f l o w - r a t e o f2 . 1 m l / m i n . T h e s o l u t i o n w a s f i l t e r e d ( 0 . 4 5 / x mn y l o n m e m b r a n e ) a n d s o n i c a t e d p r i o r t o u s e .2.3. Sample preparati on

T w e l v e B a k e r b o n d S P E o c t a d e c y l C 1 8 c o l u m n s ( 3m l , 2 0 0 m g ) ( J . T . B a k e r ) w e r e p l a c e d o n t h e B a k e rS P E - 1 2 G v a c u u m e x t r a c t i o n m a n i f o l d a n d w e r ep r e w a s h e d w i t h 2 m l o f m e t h a n o l f o l l o w e d b y 5 m lo f w a t e r a n d 2 m l o f 0 . 2 M b o r a t e b u f f e r , p H 1 0.T h e n , p l a s m a s a m p l e s ( 1 m l ) e a c h m i x e d w i t h 1 m lo f 0 . 2 M b o r a t e b u f f e r , p H 1 0, w e r e l o a d e d o n t h ec o l u m n s a n d d r a w n t h r o u g h b y m e a n s o f th e v a c u u mm a n i f o l d . T h e f l o w - r a t e w a s k e p t c o n s t a n t ( < 1 m l /m i n ) . T h e s o l i d p h a s e s w e r e w a s h e d a g a i n w i t h 2 m lo f w a te r , 2 m l o f m e t h a n o l - w a t e r ( 3 0 : 7 0 , v / v ) , 1 m lo f a c e t o n i t r i le - 0 . 2 M b o r a t e b u f fe r p H 1 0 ( 7 0 : 3 0 ,v / v ) , 1 m l o f a c e t o n i t r il e a n d 1 m l o f n - h e x a n e , a t af l o w -r a te o f 5 m l / m i n . T h e n , t h e S P E c o l u m n s w e r ed r i ed u n d e r v a c u u m f o r 1 0 m i n . M i c o n a z o l e w a ss l o w l y e lu t e d b y 1 m l o f m e t h a n o l ( < 1 m l / m i n ) .T h e e l u e n t w a s e v a p o r a t e d t o d r y n e s s u n d e r a n a i rs t r e a m a t 5 0 C . T h e r e s i d u e w a s r e c o n s t i t u t e d i n 1 0 0/ x l o f a c e t o n i t r i le a n d a n a l i q u o t o f th i s s o l u t i o n ( 6 0# 1 ) w a s i n j e c te d o n t o t h e H P L C s y s t e m f o r a n a ly s i s.T h e e x p e r i m e n t s w e r e c a r r i e d o u t i n d u p l i c a t e .2.4. Drug solutions

A s t o c k s o l u t i o n w a s p r e p a r e d b y d i s s o l v i n gm i c o n a z o l e n i tr a te i n m e t h a n o l ( 1 m g / m l c a l c u la t e da s f r e e b a s e ) a n d w a s s t o r e d a t 4 C .

2.5. Ca libration and recoveryC a l i b r a t i o n w a s p e r f o r m e d b y a d d i n g k n o w n

a m o u n t s o f m i c o n a z o l e t o b l a n k h u m a n p l a s m a t oy i e l d c o n c e n t r a t i o n s o v e r a r a n g e o f 1 0 - 1 0 0 0 n g / m l .T h e s e s t a n d a r d s w e r e t h a n e x t r a c t e d a c c o r d i n g t o t h ep r o c e d u r e d e s c r i b e d i n S e c t i o n 2 . 3 .

T h e a b s o l u t e r e c o v e r y o f m i c o n a z o l e w a s c a l c u -l a t e d b y c o m p a r i n g t h e p e a k a r e a o b t a i n e d f r o me x t r a c t s o f s p i k e d p l a s m a s a m p l e s a n d t h e p e a k a r e ao b t a i n e d f r o m d i r e c t in j e c ti o n o f k n o w n a m o u n t s o fs t a n d a r d s o l u t i o n s o f m i c o n a z o l e .

T h e p r e c i s i o n o f a n a l y s i s w a s a s s e s s e d b y s ixr e p l i c a te a n a l y s e s o f h u m a n p l a s m a s p i k e d w i t hm i c o n a z o l e t o g i v e c o n c e n t r a ti o n s o f 5 0, 3 0 0 a n d1 0 0 0 n g / m l , a n d t h e n t h e w i t h i n - d a y v a r i a t i o n s w e r ec a l c u l a t e d . B e t w e e n - d a y v a r i a t i o n s w e r e a l s o c a l c u -l a t e d fo r t h e a b o v e c o n c e n t r a t i o n s ( T a b l e l ) .2.6. Plasma samples

T h e m e t h o d o f a n a ly s i s w a s a p p l i ed t o th e p l a s m af r o m t w e l v e h e a l t h y n o n - s m o k i n g v o l u n t e e r s ( o n em a l e a n d e l e v e n f e m a l e s ) , a g e d 2 2 - 4 0 y e a r s( m e a n _ + S .D . = 2 9 .8 + _ _ 6 . 0 ) w h o p a r t i c i p a t e d i n p h a r -m a c o k i n e t i c s tu d i e s o f m i c o n a z o l e . T h e i r a v e r a g ew e i g h t a n d h e i g h t ( m e a n _ + S . D . ) w e r e 5 7 . 5 - - + 6 . 7 k ga n d 1 6 1 . 8 + 5 . 8 c m , r e s p e c t i v e l y . T h e p a r t i c i p a n t sw e r e c o n f i r m e d t o b e i n g o o d h e a l t h b y p h y s i c a l

Table 1Within- and between-day precision of miconazole in humanplasmaC o n c e n t r a t i o n C o n c e n t r a t i o n C.V.added determined (%)(ng ml) (mean -+S.D.) (ng/ ml )Within-day" (n=6)50 43.395_ 2.618 6.03300 318.076_+ 3.407 1.071000 1000.462 -+24.965 2.50

Between-dayh (n=6)50 42.13 8- 3.072 7.29300 316.692-+ 3.827 1.211000 997.265 -+25.095 2.52" Within-day assay variance was calculated from six differentassay values obtained on a single day of an alysis for 1-ml aliquotsof spiked plasma standards.b Between-day assa y variance was calculated from the assayvalues obtained on six different day s of analysis for l-ml aliquotsof spiked plasma standards.

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M . K o b y l i rl s k a e t a l . / J. C h r o m a t o g r . B 6 8 5 ( 1 0 9 6 ' 1 9 1 - 1 9 5 19 3

e x a min a t i o n a n d l a b o r a to r y t e s t i n g . T h e s t u d y w a sp e r f o r me d a c c o r d in g o f t h e e th i c a l g u id e l i n e s o f t h er e v i s e d D e c l a r a t i o n o f H e l s in k i . A l l s u b j e c t s g a v ew r i t t e n i n f o r me d c o n s e n t a n d t h e s t u d y p r o to c o l w a sa p p r o v e d b y t h e C o mi t t e e o n M e d ic a l E th i c s o f th eW a r s a w M e d i c a l A c a d e m y . T h e s u b j e c t r e c e i v e dMik o n a z o l t a b l . ( P h a r ma c e u t i c a l R e s e a r c h I n s t i t u t e ,W a r s a w , P o l a n d ) a n d D a k ta r i n t a b l . ( J a n s s e n ,C o p e n h a g e n , D e n ma r k ) , 1 2 5 mg o r a l l y i n a 2 5 0 - r a gd o s e ( tw o t a b l e t s) i n a c r o s s - o v e r d e s ig n s t u d y w i tho n e w e e k w a s h - o u t p e r i o d . B lo o d s a mp le s w e r et a k e n f r o m a f o r e a r m v e in i n to h e p a r in i z e d t u b e simmedia te ly be fore ( t ime ze ro) and a t 0 .5 , 1 , 1 .5 , 2 ,4 , 6 a n d 8 h a f t e r d r u g a d min i s t r a t i o n . T h e p l a s maw a s s e p a r a t e d b y c e n t r i f u g a t i o n a n d s t o r e d a t - 2 0 Cunt i l d rug ana lys is .2. 7. An ah,sis of da ta

P la s ma c o n c e n t r a t i o n s a n d t ime d a t a w e r e a n a -l y s e d b y t h e o n e - c o m p a r t m e n t m o d e l a c c o r d i n g t ot h e P h a r m / P C S p r o g r a m [ 6] . T h e m a x i m u m p l a s m ac o n c e n t r a t i o n s ( C . . . ) a n d t h e t ime t o p e a k ( TmaX) fo rmic o n a z o l e w e r e d i r e c t l y o b t a in e d f r o m th e e x -p e r ime n ta l d a t a . T h e a r e a u n d e r t h e c o n c e n t r a t i o nv e r s u s t ime c u r v e s ( A U C o _ , ) w a s c a l c u l a t e d u s in gthe l inea r t r apezoida l ru le . The apparent te rmina l r a tec o n s t a n t k ~ w a s c o m p u t e d b y l o g - l i n e a r r e g r e s s i o no v e r t h e l a s t d a t a p o in t s o f t h e c o n c e n t r a t i o n v e r s u st ime curve . The va lue of k~] was then used toe x t r a p o l a t e t h e A U C o , v a lu e s u n t i l i n f i n i t y ( t o t a lAUC) . For s ta t i s t ica l ana lys is , the t -pa i r S tudent te s twas used wi th p- -

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1 9 4

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o.o 2.0 4.0 6.0 s.o looTL'ne (h)F i g . 2 . M e a n p l a s m a l e v e l s o f m i c o n a z o l e i n t w e l v e h e a l t h yv o l u n t e e r s f o l l o w i n g a s i n g l e o r a l d o s e o f 250 m g M i k o n a z o l t a b l.o r D a k t a r i n t a b l .

a n a ly z in g 1 - ml a l i q u o t s o f s p ik e d p l a s ma s a mp le sc o n t a in in g 5 0 , 3 0 0 a n d 1 0 0 0 n g /m l o f mic o n a z o l eon s ix d i f f e ren t days . In bo th cases , the coe f f ic ien t o fv a r i a t i o n w a s < 1 0 % a t a l l c o n c e n t r a t i o n s i n v e s t i -g a t ed . T h e a b s o l ut e r e c o v e r y o f m i c o n a z o l e f r o m t h eh u m a n p l a s m a u s i n g t h i s m e t h o d w a s 8 5 . 8 2 + 4 . 8 3 % .

T h e a p p l i c a b i l i t y o f t h e a s s a y p r o c e d u r e i s i l l u s -t r a t e d i n F ig . 2 , w h ic h s h o w s t h e a v e r a g e p l a s mac o n c e n t r a t i o n - t i m e c u r v e s o f m i c o n a z o l e a f t e r a d -min i s t r a t i o n o f Mik o n a z o l t a b l . a n d D a k ta r i n t a b l . t otw e lv e v o lu n t e e r s . T a b l e 2 s u mma r i z e s s o me o f t h eme a n p h a r ma c o k in e t i c p a r a me te r s a n d t h e i r s t a n d a r de r r o r s . N o s i g n i f i c a n t d i f f e r e n c e s w e r e o b s e r v e db e tw e e n t h e s e p a r a me te r s .

P l a s ma l e v e l o f mic o n a z o l e a f t e r o r a l a d min i s t r a -

J. Chromatogr. B 685 (1996) 191-1 95

t i o n i s v e r y l o w a n d w a s e s t ima t e d u s in g a mi -c r o b io lo g i c a l me th o d o n ly [ 1 ] . T h e p r o b l e m in v o lv e din t h e d e t e r min a t i o n o f mic o n a z o l e f r o m p l a s ma i st h e f a c t t h a t c l e a r s e p a r a t i o n o f mic o n a z o l e f r o mp la s ma c o mp o n e n t s i s e s s e n t i a l f o r o p t ima l q u a n t i t a -t i o n . O th e r H P L C me th o d s u s e d l i q u id - l i q u id e x -t r a c t io n o r a c e to n i t ri l e d e p r o t e in i z a t i o n a s t h e s a mp lep r e p a r a t i o n s t e p s [ 2 - 4 ] . T h o s e m e t h o d s h a v e as e n s it i v it y o f 0 . 1 - 1 / z g / m l o f p la s m a , w h i c h i s n ote n o u g h t o m e a s u r e p l a s m a l e v el o f m i c o n a z o l e a f t ero r a l a d min i s t r a t i o n . T h i s l o w s e n s i t i v i t y r e s u l t e df r o m d e t e c t i o n a t 2 5 4 n m. A t 2 5 4 n m, c o n t r i b u t i o n st o t he c h r o m a t o g r a m f r o m p l a s m a c o n t a m i n a n t s w e r em i n i m i z e d b u t t h e a b s o r b a n c e o f m i c o n a z o l e w a slo w e r . W e m o n i to r e d t h e c o lu mn e lu e n t a t 2 3 0 n m,t h e p e a k a b s o r b a n c e w a v e l e n g t h o f m i c o n a z o l e . T h ec o lu mn w a s ma in t a in e d a t 4 0 C to min imiz e b a n db r o a d e n in g . A o n e - c o mp a r tme n t mo d e l w a s f i t t e d t oth e o b s e r v e d p l a s ma c o n c e n t r a t i o n s , a n d d i f f e r e df r o m a t h r e e - c o m p a r t m e n t o p e n m o d e l f o r p l a s m ac o n c e n t r a t i o n s o b t a in e d a f t e r i n t r a v e n o u s i n f u s io n o fmic o n a z o l e ( 5 2 2 mg ) o v e r 1 5 min [ 8 ] . I n o u r s t u d y ,t h e p h a r ma c o k in e t i c p a r a m e te r s k ~ a n d t~ /2 ( T a b l e2) a re s imi la r to k~2 and t~ /2 ca lcu la ted for a f a s tc o m p a r tm e n t b y L e w i e t al . [8 ] (0 . 3 9 0 h ~ a n d 2 . 0 8 0h , r e s p e c t i v e ly ) . U n f o r tu n a t e ly , o u r me th o d s t i l l h a sto o l i t t l e s e n s i t i v i t y t o me a s u r e t h e p l a s ma l e v e l o fmic o n a z o l e f o r l o n g e r t h a n 8 h a f t e r o r a l a d min i s t r a -t i o n a n d w e c o u ld c a l c u l a t e p h a r ma c o k in e t i c p a r a me -t e r s f o r f a s t c o mp a r tme n t o n ly .

I n s u mma r y , t h e d e s c r i b e d me th o d f o r t h e d e -t e r m i n a t i o n o f m i c o n a z o l e i n h u m a n p l a s m a i ss e n s i t i v e a n d r e p r o d u c ib l e . T h i s me th o d w o u ld a l l o wp h a r ma c o k in e t i c s t u d i e s f o r mic o n a z o l e a f t e r o r a la d min i s t r a t i o n t o b e c o n d u c t e d . T h e b io e q u iv a l e n c eo f Mik o n a z o l t a b l . 1 2 5 mg w a s f o u n d t o b e n o t

T a b l e 2P h a r m a c o k i n e t i c p a r a m e t e r s o f m i c o n a z o l e i n t w e l v e h e a l t h y v o l u n t e e r s a f t e r a s i n g l e 2 5 0 - m g o r a l d o s e o f M i k o n a z o l t a b l . a n d D a k t a r i nt ab l .P h a r m a c o k i n e t i c M i k o n a z o l D a k t a r i np a r a m e t e r ( m e a n + - S .D . , n = 1 2 ) ( m e a n _ + S . D . , n = 1 2 )k ~ ( h ~) 0 . 3 9 7 + _ 0 .1 0 5 0 . 4 3 8 + 0 . 0 9 8k a ( h ' ) 0 . 8 7 2 _ + 0 . 2 9 4 0 . 7 2 1 _ + 0 . 1 2 6t , 2 ( h ) 1 . 8 11 + 0 . 4 0 4 1 . 6 4 7 _ + 0 . 3 2 5T . . . ( h ) 1 . 7 3 9 _ + 0 . 1 9 6 1 . 8 0 7 + 0 . 2 7 4C m , x ( n g m l ~ ) 2 0 5 . 9 8 5 _ + 1 2 5 . 5 6 7 1 8 7 . 2 3 3 _ + 1 1 9 . 1 8 0A U C o_ .~ ( n g h m l - ~ 9 6 7 . 5 7 4 -+ 6 0 6 . 2 4 7 8 3 7 . 4 5 7 _ + 4 5 7 . 7 0 9

7/27/2019 Journal of Chro

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