a semi-automated solid-phase extraction and radio receptor assay for the analysis of scopolamine in...

8/2/2019 A Semi-Automated Solid-phase Extraction and Radio Receptor Assay for the Analysis of Scopolamine in Urine and Plasma

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E L S E V I E R European Journal of Pharmaceutical Sciences, 5 (1997) 3 15 -32 5

I I I I l l l p l i N J I I I I l l l i l . O F

PHARMACEUTICALS C I E N

A s e m i - a u t o m a t e d s o l i d - p h a s e e x t r a c t i o n a n d r a d i o r e c e p t o r a s s a y f o r t h ea n a l y s i s o f s c o p o l a m i n e i n u r i n e a n d p l a s m a

a : ~ aI .J . B o s m a n ' , W . R . D o u m a b, K . E n s a n g , R . A . d e Z e e u w a~Groningen Institute .for Drug Studies (GIDS), U niversity C entre for Pharmacy, Department o f Analytical Ch emis t~ and T oxicology, A . Deusinglaan

l, 9713 AV Groningen, The Netherlands~Universitv Hospital, Department of Pulmonology, Oostersingel 59, 9713 EZ Groningen, The Netherlands

Rece ived 12 October 1995; accepted 23 February 199 7

Abs t r a c tI n a d o u b l e - b l i n d p l a c e b o - c o n t r o l l e d c r o s s - o v e r s t u d y , w e e v a l u a t e d t h e t h e r a p e u t ic e f f i c a c y o f t r a n s d e r m a l s c o p o l a m i n e i n te n p a t i en t s

w i t h r e v e r s i b l e a i rw a y s o b s t r u c t io n . T h e y r e c e i v e d a p a t c h ( S c o p o d e r m T T S ) b e h i n d t h e e a r f o r t h r e e d a y s a n d s a m p l e s o f b l o o d a n du r i n e w e r e t a k e n . A h i g h l y s e n s i t i v e m e t h o d w a s d e v e l o p e d a n d v a l i d a t e d t o m e a s u r e t h e l o w l e v e l s o f f r e e a n d t o ta l s c o p o l a m i n e i n u r in ea n d p l a s m a . T h e p r o c e d u r e c o n s i s t e d o f a s e m i - a u t o m a t e d s o l i d - p h a s e e x t r a c t i o n f o l l o w e d b y t h e a n a ly s i s u s i n g r a d i o r e c e p t o r a s s ay s . T h em e a n p l a s m a c o n c e n t r a t i o n s , t a k e n e v e r y t h ir d p a t c h d a y , o f f r e e an d t o t a l s c o p o l a m i n e w e r e 4 3 . 6 p g / m l a n d 2 2 9 . 0 p g / m l , r e s p e c t i v e l y .I n 2 4 - h u r i n e , c o l l e c t e d e v e r y s e c o n d p a t c h d a y , 6 . 3 t xg o f f r e e s c o p o l a m i n e a n d 8 3 . 4 I . zg t o t a l s c o p o l a m i n e w a s e x c r e t e d . T h i s m e a n s t h a t7 0 % o f th e d e l i v e r e d d o s e ( 1 2 0 ~ g i n 2 4 h ) i s e x c r e t e d i n u r in e . F o r u r i n e s a m p l e s , t h e l i m i t o f d e t e c t io n ( L O D ) o f th e a s s a y i s 5 5 0 p g / m la n d th e li m i t o f q u a n t i t a t io n ( L O Q ) i s 6 1 0 p g / m l . F o r p l a s m a s a m p l e s , t h e L O D i s 1 6 p g / m l a n d t h e L O Q i s 3 8 p g / m l . 1 9 97 E l s e v i e rSc i e n c e B .V.K e y w o r d s : R a d i o r e c e p t o r a s s a y ; S o l i d - p h a s e e x t r a c ti o n ; T r a n s d e r m a l a d m i n i s t ra t i o n ; S c o p o l a m i n e ; P l a s m a ; U r i n e

1 . I n t r o d u c t i o n

S c o p o l a m i n e i s a c o m p e t i t i v e i n h i b i t o r o f t h em u s c a r i n i c r e c e p t o r s o f a c e t y l c h o l i n e . I t i s m a r k e t e da s a t r a n s d e r m a l d r u g d e l i v e r y s y s t e m ( S c o p o d e r m T T S ) f o r t h e p r e v e n t i o n o f n a u s e a a n d v o m i t i n ga s s o c i a t e d w i t h m o t i o n s i c k n e s s . L i k e o t h e r a n t i c h o -l i n e r g i c s , i t c a n a l s o c a u s e r e l a x a t i o n o f th e s m o o t hm u s c l e o f t h e b r o n c h i a n d b r o n c h i o l e s ( C l i s s o l d a n dH e e l , 1 9 8 5 ; D e m e t e r a n d C o r d a s c o , 1 9 8 6 ).

T h e t r a n s d e r m a l t h e r a p e u t i c s y s t e m o f s c o p o l a m i n ei s a t h i n p l a s t e r ( 0 . 2 m m ) , c o m p r i s i n g a r e s e r v o i rw h i c h c o n t a i n s t h e d r u g i n a m i x t u r e o f m i n e r a l o i la n d p o l y i s o b u t y l e n e , h e l d b e t w e e n a i m p e r m e a b l eb a c k i n g l a y e r a n d a r a t e - c o n t r o l l i n g m e m b r a n e . T h e

*Corresponding author. Present address: Faculty of Pharmacy, Depart-ment of Pharmaceutical A nalysis, Sorbonnelaan 16, 3584 CA , Utrecht,The Netherlands. Tel.: +31 30 2537667; fax: +31 30 2537387.0928 -0987/97 /$32 .00 19 97 Elsev ier Science B.V. Al l r igh ts reservedP I I S 0 9 2 8 - 0 9 8 7 ( 9 7 ) 0 0 0 5 8 - 4

u n i t c o n t a i n s 1 . 5 m g o f s c o p o l a m i n e a n d i s d e s i g n e dt o d e l i v e r a to t a l o f 0 .5 m g a t a n c o n s t a n t r a t e o fa p p r o x i m a t e l y 5 I ~ g / h o u r o v e r a 3 - d a y p e r i o d ( C l i s -s o l d a n d H e e l , 1 9 8 5 ) . O n t h e e p i d e r m a l s i d e o f t h em e m b r a n e i s a n a d h e s i v e l a y e r c o n t a i n i n g a p p r o x i -m ate ly 140 p~g as a pr im ing do se to s a tura te ce r t a inb i n d i n g s i t e s w i t h i n t h e s k i n a n d t o a c c e l e r a t ea c h i e v e m e n t o f s t e a d y s t a te b l o o d l e v el s .

T r a n s d e r m a l d r u g d e l i v e r y b y m e a n s o f p a t c h e sm a y o f f e r b en e f it s o v e r c o n v e n t i o n a l d r u g t h e r a p y b yp r o d u c i n g s u s t a i n e d , c o n s t a n t a n d c o n t r o l l e d p l a s m ac o n c e n t r a t i o n s o v e r a p r o l o n g e d p e r i o d o f t i m e . T h i sm a y r e s u l t in a p r o l o n g e d d u r a t i o n o f a c ti o n w h i c hi m p r o v e s p a t i e n t c o m p l i a n c e s i n c e f r e q u e n t i n t a k e o ft h e d r u g i s n o l o n g e r n e c e s s a r y ( G u y a n d H a d g r a f t ,1 9 8 5 ) . F o r t h i s r e a s o n , w e e v a l u a t e d t h e t h e r a p e u t i ce f f i c a c y o f t r a n s d e r m a l s c o p o l a m i n e i n t e n p a t i en t sw i t h r e v e r s i b l e a i r w a y s o b s t r u c t i o n ( D o u m a e t a l . ,


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316 l . J . B o s m a n e t a l . / E u r o p e a n J o u r n a l o f P h a r m a c e u t i c a l S c i e n c es 5 ( 1 9 9 7 ) 3 1 5 - 3 2 5

1997) . The y r e c e ive d a pa t c h ( S c opode r m T T S )be h ind the e a r f o r t h r e e da ys a nd sa m ple s o f b looda nd u r ine w e r e t a ke n . I n o r de r t o c o r r e l a t e t he l e ve l so f s c opo la m ine w i th t he r a pe u t i c e f f i c a c y , a h igh lyse ns i t i ve a na ly t i c a l m e thod w a s ne e de d to m e a su r et h e p l a s m a a n d u r i n ar y c o n c e n t r a ti o n s o f s c o p o l a m i n ein t he se pa t i e n t s . M os t de sc r ibe d m e thods a r e s e ns i -t i ve e nough to m e a su r e t he c onc e n t r a t i ons i n u r ine ,h o w e v e r p l a s m a l e v e l s h a v e r a r e l y b e e n d e t e r m i n e d(Ensing e t a l . , 1988; C l isso ld and Hee l , 1985;S c he u r l e n e t a l ., 1984 ). S inc e s c opo la m ine i s e x t e n -s ive ly m e ta bo l i z e d in g luc u r on id e a nd su lpha t e c on ju -ga t e s, w e d e c ide d to qua n t i t a t e s c opo la m ine as w e l la s t he sum o f s c opo la m ine a nd m e ta bo l i t e s .

The a na ly s i s is pe r f o r m e d by m e a ns o f r a d io re c e p -to r a s sa y ( R R A ) . The p r inc ip l e o f R R A i s ba se d onthe a b i l i t y o f a d r ug to c om pe te w i th a r a d io l a be l l e dl iga nd f o r a spe c i f i c r e c e p to r b ind ing s i t e . The d r ug( sc opo la m ine ) e xe r t s i t s pha r m a c o log ic a l a c t i onth r ough in t e r a c t ion w i th a g ive n r e c e p to r ( m us -c a r in i c ) . The d r ug c a n be a na ly se d qua n t i t a t i ve lyw he n a n a l i quo t o f t he s a m p le i s a dde d to a so lu t iontha t c on ta in s a f i xe d a m oun t o f r e c e p to r a nd a f i xe da m o u n t o f r a d i o l a b e l l e d l i g a n d ([N-methyl-3H ] s c o p o l a m m e m e t h y l c h l o r i d e ) . T h e u n k n o w nq u a n t i t y o f s c o p o l a m i n e i n u r i n e a n d / o r p l a s m a c a nbe c a l c u l a t e d by de t e r m in ing the i nh ib i t i on o f l a -be l l e d l i ga nd b ind ing a nd c om pa r ing th i s t o t hei n h i b i t i o n p r o d u c e d b y k n o w n q u a n t i t i e s o f d r u g i nca l ibra t ion s amp les (~m is te rovf i e t a l ., 1994).

I n p r inc ip l e m e ta bo l i t e s w h ic h pos se s s a b ind inga f f in i t y to t he r e c e p to r , m a y be c ode te r m in e d by ther a d io r e c e p to r a s sa y . H ow e ve r , no in t e r f e r e nc e s o fm e ta bo l i t e s o f s c opo la m ine a r e t o be e xpe c t e d ( C in -t ron and Chen, 1987; Scheur len e t a l . , 1984) . There -f o r e , t he t o t a l a m oun t o f s c opo la m ine ( f r e e p lu sc o n j u g a t e d ) w a s d e t e r m i n e d b y i n c u b a t i o n o f u r i n ea nd p l a sm a sa m ple s w i th g lu su l a se , w h ic h i s aprepara t ion of a 13-glucuronidase and a ry lsu l fa tase . Ino r de r t o e l im ina t e i n t e r f e r e nc e s o f g lu su l a se a ndm a t r ix i n t e r f e re nc e s , a s e m i - a u tom a te d so l id - pha see x t r a c t ion p r oc e du r e w a s r e qu i r e d .

I n t h i s pa pe r , w e de sc r ibe t he de ve lo pm e n t a ndva l ida t ion o f a p r oc e du r e t o a na ly se f r e e ( unc on ju -ga t e d ) a nd to t a l s c opo la m ine . The p r oc e du r e c ons i s t so f a s e m i - a u tom a te d so l id - pha se e x t ra c t ion w i th s i l i c ag e l ( S i) c o l u m n s u s i n g a n A S P E C - s y s t e m ( A u t o m a t i cS a m ple P r e pa r a t ion w i th Ex t r a c t ion C o lum ns ) .S c o p o l a m i n e w a s e l u t e d w i t h d i c h l o r o m e t h a n e a n d

the e x t r a c ts w e r e a na ly se d w i th a r a d io r e c e p to r a s sa y( R R A ) .

2 . M a t e r i a l s a n d m e t h o d s

2.1. Materials[N-methyl-3H]Scopolamine m e t h y l c h l o r i d e

( [3 H ] N M S , 8 0 .4 C i / m m o l ) w a s o b t a i n ed f r o m D uP o n t N E N ( D u P o n t , W i l m i n g t o n , D E , U S A ) .S c o p o l a m i n e h y d r o b r o m i d e t r i h y d r a t e w a s o b t a i n e df r o m M e r c k ( D a r m s t a d t , G e r m a n y ) . G l u s u l a s e w a sob ta ine d f r om C a lb ioc he m ( La Jo l l a , C A , U S A ) .S o d i u m h y d r o x i d e w a s o b t a i n e d f r o m J a n s s e nC h im ic a ( B e e r se , B e lg ium ) . A l l o the r c he m ic a l s a ndso lve n t s w e r e o f a na ly t i c a l g r a de a nd ob ta ine d f r omM e r c k ( D a r m s t a d t , G e r m a n y ) .P o lye th y le ne tube s (4 m l a nd 12 m l ) w e r e ob ta ine df r om G r e ine r ( A lphe n a /d R i jn , The N e the r l a nds ) .T h e G F / B g l a ss f ib r e fi lt er s w e r e f r o m W h a t m a n( M a ids tone , U K ) . R ia lum a w a s u se d a s s c in t i l l a t i onl iqu id , ob t a ine d f r om Lum a c ( O le n , B e lg ium ) , i nc om bina t ion w i th m in i - s c in t i l l a t i on c oun t ing v i a l sf r om P a c ka r d ( G r on inge n , The N e the r l a nds ) . B ondElu t e x t r a c t ion c o lum ns , t ype S i ( s i l i c a ge l ) , be dvo lum e 2 . 8 m l w e r e k ind ly dona te d by V a r i a n ( S a m -p le P r e pa r a t ion P r oduc t s , H a r bo r C i ty , C A , U S A ) .

H e p a r i n i z e d h u m a n p l a s m a w a s o b t a i n e d f r o m t h eloc a l b lood ba nk a nd s to r e d a t - 20 C . C a l f b ra in sw i thou t c e r e be l lum w e r e ob ta ine d f r om the l oc a ls l a u g h t e rh o u s e a n d s t o r ed a t - 8 0 C .2.2. Preparation of solutions

T h e 5 0 m M s o d i u m p h o s p h a t e b u f fe r p H 7 . 4 ( a ss a ybu f f e r ) w a s p r e pa r e d by d i s so lv ing 1 . 38 gN a H e P O 4 . H 2 0 a n d 7 .1 2 g N a 2 H P O 4 . 2 H 2 0 i n 1 l it red is t i l led wa te r .

T h e 5 0 m M s o d i u m p h o s p h a t e b u ff e rs p H 4 . 7 5 o rpH 3 . 75 w e r e p r e pa r e d by the a dd i t i on o f 4 Mhydr oc h lo r i c a c id t o t he a s sa y bu f f e r .

T h e 5 0 m M s o d i u m p h o s p h a t e b u ff e rs p H 1 1.5 o r11 .25 w e r e p r e pa r e d by the a dd i t i on o f 4 M sod iu mhydr ox ide to t he a s sa y bu f f e r .

G lusu la se so lu t ions pH 4 . 75 o r pH 3 . 75 w e r ep r e pa r e d by d i lu t i ng the g lu su l a se s toc k so lu t ion 75t i m e s w i t h 5 0 m M s o d i u m p h o s p h a t e b u f f e r p H 4 . 75o r pH 3 . 75 .


3/11

I . J. B o s m a n e t a l . / E u r o p e a n J o u r n a l o f P h a r m a c e u t i c a l S c i en c e s 5 ( 1 9 9 7 ) 3 1 5 - 3 2 5 31 7

A s c o p o l a m i n e s t o c k s o l u t i o n o f 1 x 1 0 3 M w a sp r e p a r e d i n e t h a n o l a n d s t o r e d a t - 2 0 C .2.3. Preparation of receptor material

C a l f b r a i n s w i t h o u t c e r e b e l l u m w e r e h o m o g e n i z e di n 6 v o l u m e s ( w / v ) i c e c o ld 0 . 3 2 M s u c r os e u s in g aT e f l o n - g l a s s P o t t e r E l v e j e h e m h o m o g e n i z e r ( R W 2 0 ,J a n k e a n d K u n k e l K G , I K A - W e r k , S t a u f e n i . B r e i s -g a u , G e r m a n y ) a t 1 , 2 0 0 r p m . T h e h o m o g e n a t e w a sc e n t r i f u g e d f o r 1 0 m i n a t 1 0 0 0 X g a n d th e r e su l t i n gs u p e r n a ta n t w a s c e n t r i f u g e d f o r 6 0 m i n a t 1 0 0 0 0 0 g( B e c k m a n L 8 - 5 5 u l t r a c e n t r i f u g e , B e c k m a n I n s t r u -m e n t s N e d e r l a n d B . V . , M i j d r e c h t, T h e N e t h e r l a n d s ).T h e p e l l e t w a s r e s u s p e n d e d i n t h e a s s a y b u f f e r a n dc e n t ri f u ge d 3 0 m i n a t 1 0 0 , 0 0 0 g . T h i s w a s h i n g w a sr e p e a t e d a n d t h e f i n a l p e l l e t w a s r e s u s p e n d e d i n 5v o l u m e s ( w / v ) a s s a y b u f f e r . T h e e n t i r e p r o c e d u r ew a s c a r r i e d o u t a t 4 C . T h e o b t a i n e d h o m o g e n a t e w a sf r o z e n w i t h l i q u i d n i t r o g e n a n d l y o p h i l i z e d f o r 2 4 h( H e t o s i c c C D 5 2 - 1 , l y o p h i l i z e r , H e t o , B i r k e r - d , D e n -

mar k) . T he lyophi l i z e d pr e par at ion was s tor e d at- 2 0 C .

T h e o p t i m u m a m o u n t o f l y o p h i l i z e d m a te r ia l fo rt h e r a d io r e c ep t o r a s s a y o f s c o p o l a m i n e i n u r i n e (3 m gt i s su e i n 4 0 0 pA a s s a y b u f f e r ) a n d i n p l a s m a ( 2 m gt i s s u e i n 1 0 0 ~ 1 ) w a s e x p e r i m e n t a l l y d e t e r m i n e d .2.4. Scopolamine analysis

T h e p r o c e d u r e c o n s i s t s o f a s e m i - a u t o m a t e d s o l i d -p h a s e e x t r a c t i o n f o l l o w e d b y t h e a n a l y s i s o fs c o p o l a m i n e ( f r ee a n d t o ta l ) u s i n g r a d i o r e ce p t o ra s s a y s . T o d e t e r m i n e t o t a l s c o p o l a m i n e , t h e u r i n e o rp las ma s amp les we r e pr e tr e a te d w i th g lus u las e (T able1).2.4 .1 . Ins t rumen ta t ion

T h e e x t r a c t i o n w a s p e r f o r m e d w i t h a n A S P E C -s y s t e m ( G i l s o n M e d i c a l E l e c t r o n i c s, V i l l i e r s l e B e l ,F r a n c e ). A s s h o w n i n F i g . 1 , t h e A S P E C - s y s t e mc o n s i s t s o f t hr ee c o m p o n e n t s : A M o d e l 4 0 1 d i lu t o r , a

T a b l e IO v e r v i e w o f t h e a n a l y s i s o f s c o p o l am i n e

U r i n e P l a s m aIncuba t ion w i th g lusu lase *~

I n c u b a t i o n w i t h g l u s u la s e , a d a p t e d m e t h o d

E x t r a c t i o n w i t h t h e A S P E C - s y s t e mC o n d i t i o n i n g

S a m p l e a p p l i c a ti o n

W a s h i n gE l u t i n gE v a p o r a t i o nR e c o n s t i t u t e r e s i d u eR a d i o r e c e p t o r assay

250 t~1 u r i n e7 5 0 ~1 g l u s u l a s e ( p H 4 . 7 5 )i n c u b a t i o n 10 h 37C so lu t ion1 m l p h o s p h a t e b u f f e r ( p H 1 1 .5 )

5 m l m e t h a n o l5 m l w a t e r3 m l d i c h l o r o m e t h a n e2 m l p h o s p h a t e b u f f e r ( p H 7 . 4 )f l o w - r a t e 3 . 0 m l / m i n1000 i l l u r ine *= o r1600 ILl incuba ted sam ple *~f l o w - r a t e 1 . 5 m l / m i n

1 . 5 m l p h o s p h a t e b u f f e rf l o w - r a t e 3 . 0 m l / m i n3 m l d i c h l o r o m e t h a n ef l o w - r a t e 1 . 5 m l / m i nv a c u u m c o n c e n t r a t o r1000 Ix l phosphate bu f fer100 IM of r e c o n s t i t u t e d r e s i d u e400 ~zl r e c e p t o r s u s p e n s i o n5 0 IL l [ ~H ] N - m e t h y l s c o p o l a m i n e

250 IM p lasma750 ix l g lusu lase so lu t ion (pH 3 .75)i n c u b a t i o n 10 h 37CI m l p h o s p h a t e b u f f e r ( p H I 1 , 2 5 )500 Ix l p l asma1500 t~1 g lusu lase so lu t ion (pH 3 .75)incubat ion 10 h 37C2 m l p h o s p h a t e b u f f e r ( p H 1 1 , 2 5 )5 m l m e t h a n o l5 m l w a t e r3 m l d i c h l o r o m e t h a n e2 m l p h o s p h a t e b u f f e r ( p H 7 . 4 )f l o w - r a t e 3 . 0 m l / m i n1500 t .t l pla sm a *-~ or1600 ILl incubated sam ple *~ o r3600 Ixl i ncubated sa mple *~ ( a d a p t e d )f l o w - r a t e 1 . 5 m l / m i n1 .5 ml phosphate bu f ferf l o w - r a t e 3 . 0 m l / m i n3 m l d i c h l o r o m e t h a n ef low-ra t e 1 .5 ml /minv a c u u m c o n c e n t r a t o r100 I~1 phosphate bu f fer100 txl of r e c o n s t i t u t e d r e s i d u e100 ~1 recep to r suspens ion5 0 I M [ 3 H ] N - m e t h y l s c o p o l a m i n e

*1 To d e t e r m i n e t o t a l ( f r e e p l u s c o n j u g a t e d ) s c o p o l a m i n e .* 2 T o d e t e r m i n e f r e e ( u n c o n ju g a t e d ~ s c o p o l a m i n e .


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3 1 8 l . J . B o s m a n e t a l. / E u r o p e a n J o u r n a l o f P h a r m a c e u t i c a l S c i en c e s 5 ( 1 9 9 7 ) 3 1 5 - 3 2 5

F i g . 1 . T h e A S P E C - s y s t e m ( A u t o m a t i c S a m p l e P r e p a r a ti o n w i t h E x -t r a ct i o n C o l u m n s ) , w h i c h c o n s i s t s o f t hr e e m a i n c o m p o n e n t s : A M o d e l4 0 1 d i l u t o r ( A ) , a s a m p l e p r o c e s s o r ( B ) , a n d a s e t o f r a c k s a n d a c c e s s o r i e st o h a n d l e 3 m l S P E - c o l u m n s a n d s o l v e n t s ( C I , C 2 , C 3 ) .

s a m p l e p r o c e s s o r , a n d a s e t o f r a c k s a n d a c c e s s o r i e st o h a n d l e 3 m l S P E - c o l u m n s a n d s o l v e n t s .2 . 4 .2 . I nc uba t ion w i th g lusu lase

T o t a l s c o p o l a m i n e ( f r e e a n d c o n j u g a t e d ) w a s m e a -s u r e d a f t e r i n c u b a t i o n o f t h e u r i n e o r p l a s m a s a m p l e sw i t h g l u s u la s e . T h e b i o l o g ic a l m a t r i ce s w e r e b u f f e r e dw i t h g l u s u l a s e s o l u t i o n i n o r d e r t o o b t a i n a n i n c u b a -t i o n p H b e t w e e n 4 . 5 a n d 6 . 2 .

To 2 50 Ixl ur ine , 7 50 I~1 g lu sulas e so lu t io n pH 4 .75w a s a d d e d a n d v o r t e x m i x e d f o r 5 s e c . T h e m i x t u r ew a s i n c u b a t e d f o r 1 0 h a t 3 7 C u n d e r c o n s t a n t

is h a k in g . T h e r e a c t i o n w a s t e r m i n a t e d b y a d d i n g 1 m lp h o s p h a t e b u f f e r p H 1 1 .5 , t o g e t a f in a l p H o f 7 . 4.F o r p l a s m a s a m p l e s ( 2 5 0 i x l ) , a g l u s u l a s e s o l u t i o n p H3 . 7 5 ( 7 5 0 I ~1 ) w a s u s e d a n d t o t e r m i n a t e t h e i n c u b a -t i o n , p h o s p h a t e b u f f e r p H 1 1 .2 5 ( 1 m l ) w a s a d d e d t og e t a f i na l p H o f 7 . 4. O t h e r w i s e t h e p r o c e d u r e f o rp l a s m a w a s t h e s a m e a s f o r u r i n e . T h e i n c u b a t e ds a m p l e s w e r e v o r t e x e d a n d 1 6 0 0 p J a l iq u o t s w e r eu s e d f o r t h e e x t r a c t i o n .

W h e n t h e i n i t i a l p r o c e d u r e f o r p l a s m a a p p e a r e d t ob e t o o i n s e n s i t i v e t o a n a l y s e t h e a c t u a l l e v e l s , a na d a p t i o n w a s d e v e l o p e d . T o 5 0 0 i l l p l a s m a , 1 5 0 0 t x lg l u s u l a s e s o l u t i o n p H 3 . 7 5 w a s a d d e d a n d v o r t e x e df o r 5 s e c . T h e m i x t u r e w a s i n c u b a t e d f o r 1 0 h a t 3 7 Ca n d t h e r e a c t i o n w a s t e r m i n a t e d b y a d d i n g 2 m lp h o s p h a t e b u f f e r p H 1 1 . 25 , t o g e t a fi n a l p H o f 7 . 4.

T h e i n c u b a t e d s a m p l e w a s v o r t e x e d a n d 3 6 0 0 ~ 1 w a su s e d f o r t h e e x t r a c t i o n .2.4 .3 . Ex trac t ion

B o n d E l u t e x t r a c t i o n c o l u m n s , t y p e S i ( s i l i c a g e l )w e r e s e a l ed w i t h p o l y p r o p y l e n e c a p s a n d i n s t a ll e d o nt h e S P E - r a c k ( F i g . 1 ) . T h e s o l v e n t s a n d s a m p l e s( u r i n e o r p l a s m a ) w e r e p l a c e d i n t h e s o l v e n t p o s i t i o n sa n d s a m p l e r a c k , r e s p e c ti v e l y . P o l y e t h y l e n e t u b e s( 5 5 1 2 m m , 4 m l ) w e r e p l a c e d in t h e c o l l e c ti o nr a ck . T h e e x t r a c t i o n w a s t h e n p e r f o r m e d b y A S P E Ci n a s e q u e n t i a l m o d e a s f o l l o w s .

F i rs t, t h e c o l u m n w a s p r e c o n d i t i o n e d b y s u b -s e q u e n t w a s h i n g s w i t h 5 m l m e t h a n o l , 5 m l w a t e r , 3m l d i c h l o r o m e t h a n e a n d 2 m l a s s a y b u f f e r a t af l o w - r a te o f 3. 0 m l / m i n . T h e u r i n e s a m p l e ( 1 0 0 0 ~ 1) ,p l a s m a s a m p l e ( 1 5 0 0 i l l ) o r t h e i n c u b a t e d s a m p l e( 1 6 0 0 p J o r 3 6 0 0 ~ 1 f o r t h e a d a p t e d p r o c e d u r e ) w a sd i s p e n s e d o n t o t h e c o l u m n a n d p u s h e d t h r o u g h b yp o s i t i v e p r e s s u r e a t a f lo w - r a t e o f 1 . 5 m l / m i n . T op u s h t h e s a m p l e t h r o u g h t h e c o l u m n c o m p l e t e l y , 1 .5m l o f a ir w a s a p p l i ed . A f t e r w a s h i n g t h e c o l u m n w i t h1 .5 m l a s s a y b u f f e r a t a f l o w - r a t e o f 3 .0 m l / m i n , t h ec o l u m n w a s d r i e d b y p a s s i n g t h r o u g h 6 m l o f a i r .S c o p o l a m i n e w a s t h e n e l u t e d i n t o t h e c o l l e c t i o n t u b eb y p a s s i n g t h r o u g h 3 m l o f d i c h l o r o m e t h a n e a t af l o w - r a t e o f 1 .5 m l / m i n a n d 1 .5 m l o f a ir w a s a p p l i e do n t o t h e c o l u m n t o p u s h t h e d i c h l o r o m e t h a n e th r o u g ht h e c o l u m n c o m p l e t e l y .

S m a l l d r o p l e t s o f w a t e r i n t h e d i c h l o r o m e t h a n ef r a c ti o n w e r e r e m o v e d m a n u a l l y b y m e a n s o f a p ip e t.D i c h l o r o m e t h a n e w a s e v a p o r a t e d t o d r y n e s s w i th i n4 5 m i n , u s i n g a v a c u u m c o n c e n t r a t o r , a t 1 5 0 0 r p ma n d 4 0 C ( B e u n d e R o n d e , A b c o u d e , T h e N e t h e r -l a n d s) . 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 in 1 0 0 0 p J( u r i n e s a m p l e s ) o r 1 0 0 p J ( p l a s m a s a m p l e s ) a s s a yb u f f e r b y v o r t e x m i x i n g f o r 5 s A f t e r s t a n d i n g f o r 1h o u r , t h e t u b e w a s v o r t e x e d a g a i n f o r 5 s . T od u p l i c a t e p o l y e t h y l e n e t u b e s ( 1 2 m l ) , 1 0 0 p J o f th eu r i n e e x t r a c t w a s p i p e t t e d a n d u s e d f o r t h er a d i o r e c e p t o r a s s ay . H o w e v e r , t h e e n t i r e r e c o n s t i tu t e dp l a s m a s a m p l e ( 1 0 0 ~ 1 ) h a d t o b e u s e d i n t h er a d i o r e c e p t o r a s s a y w h i c h m e a n t t h a t f o r d u p l i c a t ea n a l y s e s t w o e x t r a c t i o n s w e r e n e c e s s a r y .2 . 4 .4 . R ad iore c e p tor as say ( RRA)

T o e a c h p o l y e t h y l e n e tu b e ( 1 2 m l ) w i t h t h e 1 0 0 IL le x t r a c t i o n s a m p l e , 4 0 0 ~ 1 r e c e p t o r s u s p e n s i o n ( f o ru r i n e s a m p l e s ) o r 1 0 0 I, l r e c e p t o r s u s p e n s i o n ( f o rp l a s m a s a m p l e s ) w a s a d d e d . T h e t u b e s w e r e v o r t e x e d


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a n d i n c u b a t e d d u r i n g 6 0 m i n a t 0 C b e f o r e 5 0 t x l o f[ 3 H ] N M S s o l u ti o n ( 4 X 1 0 - 9 M ) w a s a d d e d . T h et u b e s w e r e m i x e d a g a i n a n d i n c u b a t e d f o r a n o t h e rh o u r a t 0 C . A f t e r t h e a d d i t i o n o f 4 m l i c e c o l d a s s a yb u f f e r , th e s a m p l e s w e r e i m m e d i a t e l y f il t er e d t h r o u g hW h a t m a n G F / B g l a ss f ib r e fi lt e rs u n d e r v a c u u m u s i n ga f il t ra t i o n a p p a r a t u s ( 4 8 S , U n i v e r s i t y C e n t r e f o rP h a r m a c y , G r o n i n g e n , T h e N e t h e r l a n d s ) . T h e t u b e sw e r e r i n s e d t w i c e w i t h 4 m l i c e c o l d b u f f e r , w h i c hwas a l so f i l t e red . The to ta l f i l t ra t ion and r ins ingp r o c e s s , t a k i n g p l a c e i n a p p r o x i m a t e l y 1 5 s , w a sc a r r i e d o u t o n e a c h t u b e i n t u r n . T h e f i l t e r s w e r et r a n s f e r r e d i n t o m i n i - s c i n t i l l a t i o n v i a l s a n d d i s p e r s e di n 3 . 5 m l s c i n t i l l a t i o n c o c k t a i l b y s h a k i n g f o r 1 2 0m i n . T h e v i al s w e r e c o u n t e d f o r 4 0 0 0 0 c o u n t s o r 5m i n i n a li q u i d s c i n t i ll a t i o n c o u n t e r , w h i c h e v e r c a m ef i r s t ( M i n a x i , P a c k a r d , G r o n i n g e n , T h e N e t h e r l a n d s ) .F i f t y ~ zl o f t h e u s e d [ 3 H ] N M S s o l u t i o n w e r e a d d e d t o2 m i n i - s c i n t i l l a t i o n v i a l s a n d c o u n t e d a s w e l l .

o n t h e a s s a y . T h e a d a p t e d p r o c e d u r e f o r t o t a ls c o p o l a m i n e i n p l a s m a w a s v a l i d a te d w i t h t h e q u a l i t yc o n t r o l o f 1 . 2 5 x 10 9 M .

T h e o b t a i n e d b i n d i n g v a l u e s ( B q ) o f t h e q u a li tyc o n t r o l s a m p l e s w e r e i n t r o d u c e d i n t h e f i t t e d c a l i -b r a t i o n c u r v e s a n d t h e c o n c e n t r a t i o n s i n u r i n e o rp l a s m a w e r e c a l c u l a t e d .

F o r u r i n e s a m p l e s , i n t e r - d a y d a t a ( b e t w e e n d a y s )w e r e a s s e s s e d b y m e a s u r i n g t h e q u a l i t y c o n t r o ls a m p l e s i n d u p l i c a t e o n d i f f e r e n t d a y s ; i n t r a - d a y d a t a( w i t h in d a y ) w e r e d e t e r m i n e d b y d u p l i c a te m e a s u r e -m e n t s o f t h e q u a l i t y c o n t r o l s a m p l e s a f t e r 5 s e p a r a t ee x t r a c t i o n s o n t h e s a m e d a y .

T h e q u a l i t y c o n t r o l s a m p l e s i n p l a s m a w e r e a n a -l y s e d i n d u p l i c a t e o n t e n d i f f e r e n t d a y s . B e c a u s e f o rp l a s m a s a m p l e s d u p l i c a t e a n a l y s e s m e a n s d u p l i c a t ee x t r a c t i o n s , i n t r a - a n d i n t e r - d a y d a t a w e r e c a l c u l a t e df r o m t h e s a m e s a m p l e s u s i n g o n e - w a y a n a l y s i s o fv a r i a n c e .

2.5. Calibration curve 2. 7. Patient samplesF r o m t h e s c o p o l a m i n e s t o c k s o l u t i o n , a p p r o p r i a t e

d i lu t i o n s w e r e m a d e f r e s h l y o n t h e d a y o f a n a l y s is i nb l a n k u r i n e t o p r o v i d e c o n c e n t r a t i o n s r a n g i n g f r o m5 x 1 0 - 1 M t o 2 X 1 0 - 7 M . A f t e r e x t r a c t i o n , t h i sr e s u l t e d i n f i n a l a s s a y c o n c e n t r a t i o n r a n g i n g f r o m9 . 0 9 x 1 0 ~ M t o 3 . 6 4 X 1 0 8 M . I n b l a n k p l a s m a ,t h e c o n c e n t r a t i o n s w e r e r a n g i n g f r o m 1 X 1 0 -~ 1 M t o5 X 1 0 9 M g i v i n g f in a l as s a y c o n c e n t r a t io n s o f 6 X1 0 ~ M t o 3 X 1 0 8 M . T h e s a m p l e s o f t h e ca l i-b r a t i o n c u r v e s w e r e a n a l y s e d i n d u p l i c a t e . T h e c u r v e sw e r e f i t t e d w i t h t h e L i g a n d c u r v e f i t t i n g p r o g r a m t oc a l c u l a t e t h e p a r a m e t e r s w h i c h c h a r a c t e r i z e t h e c a l i -b r a t i o n c u r v e s ( M u n s o n a n d R o d b a r d , 1 9 8 0 ) .2.6. Quali b, control

T h e a s s a y o f u r i n e w a s v a l i d a t e d w i t h q u a l i t yc o n t r o l s a m p l e s w h i c h w e r e p r e p a r e d i n b l a n k u r i n e ,at 5X 10 -9 M, l X l 0 8 M a n d 5 x 1 0 - 8 M , re sp e c-t i v e l y . I n b l a n k p l a s m a , q u a l i t y c o n t r o l s a m p l e s w e r ep r e p a r e d w i t h c o n c e n t r a t io n s o f 1 . 2 5 x 1 0 - ~ M , 3 x1 0 - ~ o M a n d 1 . 2 5 x 10 -9 M, r e s p e c t i v e l y . T h e q u a l i t yc o n t r o l s a m p l e s w e r e s t o r e d a t - 2 0 C . O n t h e d a y o fa n a l y s i s , t h e q u a l i t y c o n t r o l s a m p l e s w e r e e x t r a c t e da n d a n a l y s e d i n d u p l i c a t e .

I n c u b a t e d q u a l i t y c o n t r o l s a m p l e s ( 5 x 1 0 - 8 M a n d1 . 2 5 x 1 0 - 9 M i n u r i n e a n d p l a s m a , r e s p e c t i v e l y )w e r e c o d e t e r m i n e d t o s t u d y t h e i n f l u e n c e o f g l u s u l a se

U r i n a r y a n d p l a s m a l e v e ls o f f r e e a n d t ot als c o p o l a m i n e w e r e d e t e r m i n e d i n t e n p a t i e n t s w i t hr e v e r s i b l e a i r w a y s o b s t r u c t i o n ( D o u m a e t a l . , 1 9 9 7 ) .T h e p a t ie n t s r e c e i v e d a s c o p o l a m i n e p a t c h( S c o p o d e r m T T S ) o r p l a c e b o b e h i n d t h e e a r f o r 7 2h . T h e p a t c h e s ( p l a c e b o a n d S c o p o d e r m T T S ) w e r ek i n d l y p r o v i d e d b y C i b a G e i g y , A r n h e m , T h e N e t h e r -l a n d s . E v e r y s e c o n d p a t c h d a y u r i n e w a s c o l l e c t e dd u r i n g 2 4 h a n d e v e r y t h i r d p a t c h d a y a t 9 . 0 0 a . m .b l o o d w a s t a k e n f r o m t h e p a t i e n t s . P l a s m a w a so b t a i n e d b y c e n t r i f u g a t i o n o f th e b l o o d s a m p l e( c o l l e c t e d i n a t u b e c o n t a i n i n g h e p a r i n ) a t 5 , 0 0 0 r p mf o r 1 1 m i n ( M e g a f u g e 1 . 0 , H e r a e u s S e p a t e c h , G e r -m a n y ) . T h e u r i n e a n d p l a s m a s a m p l e s w e r e s t o r e d a t- 2 0 C u n t i l a n a l y s i s .

T h e s a m e p r o c e d u r e a s d e s c r i b e d f o r t h e q u a l i t yc o n t r o l s a m p l e s w a s p e r f o r m e d t o c a l c u l a t e t h eu n k n o w n a m o u n t o f f r e e a n d t o ta l s c o p o l a m i n e i nu r i n e a n d p l a s m a o f t h e p a t i e n t s .

3 . R e s u l t s a n d d i s c u s s i o n

3.1. Scopolamine analysis: solid-phase extractionand radioreceptor assay3.1.1. Radioreceptor assay

F o r t h e a n a l y s i s w e u s e d a r a d i o r e c e p t o r a ss a y


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( R R A ) , b a s e d o n t h e c o m p e t i t i o n b e t w e e n a r a d i o -l a b e l l e d l i g a n d ( [ 3 H ] N M S ) a n d u n l a b e l l e d d r u g( s c o p o l a m i n e ) f o r b i n d i n g t o m u s c a r i n i c r e c e p t o r s ,b e c a u s e t h e y a r e h i g h l y s e n s i t i v e a n d s e l e c t i v e .

B e c a u s e o f th e p o t e n c y o f s c o p o l a m i n e , t h e c o n -c e n t r a t i o n s o f t h e d r u g i n p l a s m a a n d u r i n e w i l l b ev e r y l o w a n d d i f f i c u l t t o d e t e c t . T h e r e f o r e , t h es e n s i t i v i t y o f t h e r e c e p t o r a s s a y s w a s e n h a n c e d b ya p p l y i n g a p r e - i n c u b a t i o n s t e p o f t h e d r u g w i t h t h er e c e p t o r p r e p a r a t i o n a t 0 C b e f o r e t h e r a d i o l a b e l l e dl i g a n d w a s a d d e d ( S m i s t e r o v f i e t a l. , 1 9 9 4 ) . T h i sr e s u l t e d i n a g a i n i n s e n s i t i v i t y o f a f a c t o r 2 a sc o m p a r e d w i t h e q u i l i b r i u m c o n d i t i o n s a t 3 7 C . H o w -e v e r , t h i s g a i n w a s m u c h s m a l l e r t h a n a p r e v i o u s l yd e s c r i b e d g a i n o f a f a c t o r 6 . 7 u s i n g [ 3 H ] d e x e t i m i d ea s ra d i o l a b e l l ed l i g a n d ( E n s i n g a n d d e Z e e u w , 1 9 8 4 ).P r o b a b l y , t h e g a i n i n s e n s i t i v it y is n o t o n l y d e p e n d e n to n t h e c o m p e t i t i v e d r u g b u t d e p e n d s a l s o o n t h er a d i o l a b e l le d d r u g .

B e s i d e s a h i g h s e n s i t i v i t y , a n o t h e r a d v a n t a g e o fR R A i s t h e s e le c t i v it y t o w a r d s c o m p o u n d s w h i c hp r o d u c e a p h a r m a c o l o g i c a l e f f e c t v ia a n i n t e r a c t io nw i t h r e c e p t o r s . T h i s m e a n s t h a t i n p r i n c i p l e m e t a b o -l it e s m a y b e c o d e t e r m i n e d w h e n t h e y h a v e a n a f f i n it yt o w a r d s t h e r e c e p t o r . I n c a s e o f s c o p o l a m i n e , n oi n t e r f e r e n c e s o f m e t a b o l i t e s a r e t o b e e x p e c t e d . T h eg l u c u r o n i d e a n d s u l p h a t e c o n j u g a t e s o f s c o p o l a m i n ew i l l h a v e n o a f f in i t y t o w a r d s t h e m u s c a r i n i c r e c e p t o rb e c a u s e o f t h e i r p h y s i c o - c h e m i c a l p r o p e r ti e s . S c h e u r -l e n e t a l . ( 1 9 8 4 ) s h o w e d t h a t s c o p o l i n e a n d s c o p i n e ,p o s s i b l e d e g r a d a t i o n p r o d u c t s o f s c o p o l a m i n e , w i l lh a v e n o i n t e r f e r e n c e w i t h t h e a s s a y . T h i s w a s a l s oo b s e r v e d i n a p r e v i o u s s t u d y , t es t in g t h e d i s p l a c e m e n to f b i n d i n g o f r a d i o l a b e l l e d [ 3 H ] d e x e t i m i d e t o m u s -c a r i n i c r e c e p t o r s . T h e v e r y l o w a f f i n i t y c o n s t a n t s o fs c o p o l i n e ( 4 . 1 10 6 M - ~ a n d a p o s c o p o l a m i n e ( 3. 7 1 0 3 M - l ) , c o m p a r e d w i t h 1 . 7 1 0 9 M - x f o rs c o p o l a m i n e , s h o w e d t h a t s c o p o l i n e a n d a p o s -c o p o l a m i n e w i l l n o t i n t e r f e r e w i t h t h e a s s a y o fs c o p o l a m i n e .

I t h a s b e e n s h o w n b e f o r e t h a t d i r e c t r a d i o r e c e p t o ra s s a y s o f s c o p o l a m i n e i n u r i n e c o u l d b e p e r f o r m e db e c a u s e o f t h e r e l a t i v e l y h i g h u r i n e c o n c e n t r a t i o n sa f t e r t h e r a p e u t i c d o s i n g ( E n s i n g e t a l. , 1 9 8 8 ) . H o w -e v e r , u s i n g t h i s d i r e c t a s s a y , c a l i b r a t i o n c u r v e s h a d t ob e p r e p a r e d f o r e a c h p a t i e n t i n h i s / h e r o w n b l a n ku r i n e t o c o m p e n s a t e f o r t h e i n h i b i t io n o f b i n d i n gc a u s e d b y b l a n k u r i n e . M o r e o v e r , t h e a d d i t i o n o fg l u s u l a s e t o u r i n e s a m p l e s , a d d e d t o d e t e r m i n e t h e

t o t a l a m o u n t o f s c o p o l a m i n e , a l s o y i e l d e d s o m ei n h i b i t i o n o f b i n d i n g . A l t h o u g h th i s in h i b i t i o n s e e m e dh i g h l y r e p r o d u c i b l e , e x t r a c a l i b r a t i o n c u r v e s ( t ow h i c h g l u s u l a s e h a d b e e n a d d e d ) h a d t o b e p r e p a r e di n e a c h p a t i e n t ' s b l a n k u r i n e .

F o r p l a s m a , a s a m p l e c l e a n - u p a n d c o n c e n t r a t i o ns t e p w a s c o n s i d e r e d u n a v o i d a b l e b e c a u s e p l a s m ap r o t e i n s d i s t u r b t h e s e n s i t i v i t y a n d a c c u r a c y o f t h ea s s a y , a n d b e c a u s e d r u g c o n c e n t r a t i o n s in p l a s m a a r em u c h l o w e r t h a n i n u r i n e .3.1 .2 . Sol id-ph ase ex trac t ion

S o l i d - p h a s e e x t r a c t i o n ( S P E ) w a s a p p l i e d a s a ne f f e c t i v e s a m p l e p r e - t r e a t m e n t t o e l i m i n a t e m a t r i xi n t e r f e r e n c e s a n d t h e i n t e r f e r e n c e s o f g l u s u l a s e , a n dt o c o n c e n t r a t e p l a s m a s a m p l e s . T h e d e v e l o p e d e x -t r a ct i o n p r o c e d u r e w a s b a s e d o n t h e d e s c r i b e d s a m p l ep r e p a r a ti o n o f N - 0 4 3 7 i n p l a s m a a n d u r i n e w i t hS i - c o l u m n s ( S w a r t e t a l . , 1 9 9 0 ) , a n d e a r l i e r i n v e s -t i g a t i o n s a b o u t t h e e x t r a c t i o n o f s c o p o l a m i n e f r o mur ine .

T h e s e m a n u a l p r o c e d u r e s w e r e a d a p t e d a n d a u t o -m a t e d u s i n g a G i l s o n a u t o m a t e d e x t r a c t i o n s y s t e mA S P E C ( F i g . 1 ) . D i f f e r e n t f l o w - r a t e s , a i r v o l u m e sa n d e l u t i n g v o l u m e s w e r e t e s t e d t o i m p r o v e t h ee x t r a c t io n p r o c e d u r e . A d d i t i o n a ll y , th e A S P E C o f f e rst w o p o s s i b i l i t i e s t o c a r r y o u t e x t r a c t i o n s : A b a t c hm o d e a n d a s e q u e n t i a l m o d e . I n t h e s e q u e n t i a l m o d e ,t h e A S P E C a c c o m p l i s h e s t h e c o m p l e t e e x t r a c t i o np r o c e s s o f t h e f i r s t s a m p l e , a n d t h e n c o n t i n u e s w i t ht h e n e x t s a m p l e , w h e r e a s i n t h e b a t c h m o d e a l lS P E - c o l u m n s a r e t r e a t e d b a t c h w i s e . T h e s e q u e n t i a lm o d e w a s u s e d b e c a u s e h i g h e r r e c o v e r i e s a n d r e -p r o d u c i b i l i t i e s c a n b e o b t a i n e d c o m p a r e d w i t h t h eb a t c h m o d e ( C h e n e t a l . , 1 9 9 3 ) .3.2 . Val idat ion o f the assay

R e p r e s e n t a t i v e c a l i b r a t i o n c u r v e s o f s a m p l e s , p r e -p a r e d f r e s h l y o n t h e d a y o f a n a l y s is i n b l a n k u r i n e o rb l a n k p l a s m a , a r e s h o w n i n F i g . 2 . T h e p a r a m e t e r sw h i c h c h a r a c t e r i s e t h e c a l i b r a t i o n c u r v e s a r e g i v e n i nT a b l e 2 . T h e r e l a t i v e l y s m a l l v a r i a t i o n s i n t h e c a l c u -l a t e d a f f i n i t y c o n s t a n t s o f s c o p o l a m i n e ( K 2 1 ) i n d i c a t et h e h i g h r e p r o d u c i b i l i ty o f t h e a s s a y u n d e r n o n -e q u i l i b r i u m c o n d i t i o n s . F r o m t h e f i t t e d c a l i b r a t i o nc u r v e s , t h e I C 5 o v a l u e w a s c a l c u l a t e d , w h i c h i sd e f i n e d a s t h e c o n c e n t r a t i o n o f l i g a n d ( s c o p o l a m i n e )w h i c h d i s p l a c e s 5 0 % o f th e b o u n d r a d i o l i g a n d


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l . J . Bosman et a l . / European Journal o f Pharmaceut ica l Sciences 5 (19971 315-325 321

Z

om

500

40 0

30 0

20 0

100

\o - - . - o ........ -#.. ~

"'\,, Q

i 0 - I 0 1 0 - 9 i 0 - 8 1 0 - 7F in a l a s say co ne sco p o l am in e (M)

Fig. 2. Calibration curves for t h e i n h i b i t i o n of [~H]N-methylscopolamine([3H]NMS ) binding by scopolamine, m = urine; = plas ma.

( [ 3 H ] N M S ) . T h e m e a n I C s0 v a l u e s i n p l a s m a w e r es i g n i f i c a n t l y h i g h e r i n c o m p a r i s o n w i t h u r i n e ( t w os a m p l e t t e s t, p < 0 . 0 5 ) . T h e d i f f e r e n c e s i n f it t edp a r a m e t e r s b e t w e e n t h e c u r v e s p r e p a r e d i n u r i n e o rp l a s m a c a n b e e x p l a i n e d b y d i f f e r e n c e s i n r e c e p t o rc o n c e n t r a t i o n s a n d c o n c e n t r a t i o n s o f th e r a d i o l a b e l le dd r u g i n t h e a s s a y . T h e a s s a y i n p l a s m a d i f f e r e d f r o mt h e a s s a y i n u r i n e b e c a u s e h i g h e r s e n s i t i v i t i e s w e r en e e d e d t o a n a l y s e t h e l o w l e v e l s o f s c o p o l a m i n e i np l a s m a . T h e r e f o r e , t h e i n c u b a t i o n v o l u m e o f t h ea s s a y i n p l a s m a w a s a d a p t e d to a v o l u m e o f 2 5 0 ~ 1 ,b e c a u s e t h e s m a l l e r t h e v o l u m e u s e d , t h e g r e a t e r t h es e n s i t iv i t y ( C r e v a t - P i s a n o e t al ., 1 9 8 6 ) .

W i t h t h e t h u s o b t a i n e d c a l i b r a t i o n c u r v e s , t h es a m p l e s u s e d f o r t h e f i t t i n g w e r e b a c k - c a l c u l a t e d t ot he i r or i g i na l c onc e nt r a t i ons i n ur i ne or p l as m a , andt h e a c c u r a c y a n d p r e c i s i o n d a t a w e r e e s t a b l i s h e d( T a b l e 3 ) . A s e x p e c t e d , t h e p r e c i s i o n b e c a m e w o r s e

T a b l e 2T h e c a l c u l a t e d p a r a m e t e r s w h i c h c h a r a c t e r i z e t h e c a l i b r a t i o n c u r v e s (n = 11).K,, i s the appare nt affinity constan t of [3H]NMS (M '); K_~, i s the apparentaffinity constant of scopolamine (M -,); R, i s t h e r e c e p t o r concentration (M); N t i s p e r c e n t a g e t h e n o n - s p e c i f i c a l l y b o u n d [3H]NMS: MSQ is the means u m o f s q u a r e s ; I C ~ o i s t h e concentration of scopolamine w h i c h d i s p l a c e s 50% of [3H]NMS (M).P a r a m e t e r g K I (x 10 q) K2~ (x 1 0 9 ) R, (X 10 -9) N~(%) MSQ (X 10 ~3) ICs~l x 10 ")Urine (n = 11 )Mean 3.81 5.75 1.35 1.90 0.90 1.89S.D. 0.97 0.79 0.26 0.23 0.61 0.28CN. 25.6 13.7 19.[) 12.0 68.1 14.8P la sma (n = 101mean 1.37 3.64 2.15 2.37 2.21 2.5 IS.D. 0.33 0.74 0.38 (1.71 1.94 0.3(IC.V. 23.7 20.2 17.8 30.2 88.0 11.8

T a b l e 3Accuracy and precis ion of the back-calculated concent rations of t h e s p i k e d calibration samples in urine (n = 11) and in pla sma ( n= 10)A. Added cone in urine 2x 10 -~ M 5X l0 -'~ M lX l0 -8 M 2Xl 0 8 M 5x l0 ~ M l 10 -7 M(Log final assay conc) (-9.44) (-9.04) (-8.74) (8 .4 4) (-8.04) (-7.74)mean 1.83 4.94 1.01 2.04 4.81 *~ 0.97sd 0. l 8 0.34 0.03 0.03 0.17 0.03ace 91.7 98.8 100.5 I 01.8 96.2 96.6cv 9.7 6.8 2.8 1.6 3.6 3.3B. Added conc in plasma 5x10 ,, M 1x10 -t M 2x10 -'~ M 5x10 "M Ix l0 "M 2x10 '~ M(Log final assay conc) (-9.52) (-9.22) (-8.92) (-8.52) (-8.22) (-7.92)Mean 5.22 0.90 2.07 4.95 1 .02 1 .97S.D. 0.99 0.12 0.12 0.16 0.03 0.12ACC 104.3 89.6 103.3 98.9 101.7 98.2C.V. 18.9 13.7 6.0 3.1 3.1 5.8*1 n=10


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322 l . J . Bosman et al . / European Journal of Pharmaceutical Sciences 5 ( 1 9 9 7) 3 1 5 - 3 2 5

3 5

3 0 ' \2 5

2 0

;=o 1 5

10. 0

0 i I I1 0 - 1 0 1 0 - 9 1 0 - 8 1 0 - 7F i n a l a s s a y c o n e s c o p o l a m i n e (M)

Fig. 3. P r e c i s i o n d a t a o f t h e b a c k - c a l c u l a t e d c o n c e n t r a t i o n s o f t h e s p i k e dcal ib rat io n sam p les . == = u r in e; = p l a s m a .

at the low and h igh end o f the curves (Fig . 3 ) . Forplasma, the prec i s ion exceeds the 15% coeff ic ient o fvariation (cv) at plasma concentrations lower than5 x 1 0 -1~ M . For ur ine , prec i s ion data were be low15% at urine concentrations higher than 2x 1 0 - 9 M .The h igher cv va lues in p lasma in comparison wi th

urine can be explained by the difference in ex-perinaental set-up. In case of plasma samples , dupli-cate analyses means duplicate extraction whereas forurine samples only one extraction is needed toperform duplicate measurements .

The method was val idated with quality controlsamples without glusulase at three different con-centrations (Table 4). The results show that the assayprovides good accuracy, which should be within_+ 15% o f the actual value, and ex cel len t pr ecis ion,which should not exceed 15% cv (Shah et a l . , 1992) .Only the lowest quality control prepared in plasmadeviates m ore than 15% o f the actual value. Ho w -ever, at the l imit of quantitation (LO Q), the accept-able accuracy and precis ion l imits are set at 20%.This means that for plasma the LOQ is close to1.25x 10 -] M, which corresponds to 38 pg /ml forplasma samples o f 1 .5 ml .The LOQ should be obta ined independent ly fromthe cal ibration curve which means that at least f ivesamples independent from the samples o f the ca l i -bration curve should be determined (Shah et a l . ,1992). Therefore, extra quality control samples wereprepared in blank urine at 2x 1 0 - 9 M. The calculatedintra-day precis ion and accuracy, determined byduplicate analyses of f ive samples on the same day,

T a b l e 4A c c u r a c y a n d p r e c i s i o n o f q u a l i t y c o n t r o l s a m p l e s p r e p a r ed i n u ri n e ( A ) a n d p l a s m a (B)Q u a l i t y c o n t r o l s a m p l e s W i t h o u t g l u s u l a s e W i t h g l u s u l a s eAA d d e d c o n c i n u r i n e 5X 10 -9 M 11 0 -~ M 5X10 s(Log f i n a l a s s a y c o n c ) (-9.04) (-8.74) (-8.04)

M 510 s M(-8.74)

I n t e r - d a y (n=9)Mean 4.95 0.96S.D. 0.35 0.07ACC 99.0 96.2C.V. 7.1 7.5Intra-day (n=5)M e a n 4.97 0.99S.D. 0.13 0.04ACC 99.4 99.0C.V. 2.7 4.1BA d d e d c o n e i n p l a s m a 1.25x10 -'~ M 3x 10 -'~ M(Log f i n a l a s s a y c o n c ) (-9.12) (-8.74)

4.610.41

92.28.94.650.19

93.04.1

1.25 X 10 -'~ M(-8.12)

4.970.33

99.56.65.330.05

1 0 6 . 51.0

1.2 5X10 '~ M *~(-9.OO)

M e a n 1.23 2.70 1.31 1.29I n t e r - d a y S.D. 0.21 0.21 0.10 0.24Intra-day S.D. 0.08 0.14 0.04 0. ! 8ACC 98.4 90.1 104.7 103.1Inter-day C.V. 17.1 7.7 7.4 13.9Intra-day C.V. 6.4 5.1 2.9 10.3*1 n=9


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l . J . B o s m a n e t a l. / E u r o p e a n J o u r n a l o f P h a r m a c e u t i c a l S c i e n c e s 5 ( 1 9 9 7 ) 3 1 5 - 3 2 5 323

w e r e 8 . 3 % a n d 1 1 3 . 3 % , r e s p e c t i v e l y . T h e i n t e r - d a yd a t a , o b t a i n e d b y d u p l i c a t e a n a l y s i s o n f i v e d i f f e r e n td a y s , s h o w t h a t a u r i n e c o n c e n t r a t i o n o f 2 10 -9 M isn e a r t h e L O Q : t h e p r e c is i o n w a s 2 5 . 6% a n d t h ea c c u r a c y 8 5 . 2 % . H e n c e , t h e L O Q c a n b e e s t im a t e d t ob e a b o u t 6 1 0 p g / m l f o r u r i n e s a m p l e s o f 1 m l .

T h e L O Q m u s t b e d i f f e r e n t i a te d f r o m t h e l i m i t o fd e t e c t i o n ( L O D ) w h i c h f o r R R A c a n b e d e f in e d ast h e c o n c e n t r a t i o n o f a n a l y t e a t w h i c h t h e f r a c t i o nb o u n d l a b e l i s s i g n i f i c a n t l y s m a l l e r t h a n t h e f r a c t i o nb o u n d l a b e l i n t h e a b s e n c e o f a n a l y t e ( ~ m i s t e r o v f i e ta l ., 1 9 9 4 ) . T h i s w a s d e t e r m i n e d e x p e r i m e n t a l l y b yp e r f o r m i n g r e p l ic a t e m e a s u r e m e n t s o f t h e z e r o s t a n-d a r d ( a m o u n t o f b o u n d [ S H ] N M S i n a b s e n c e o fs c o p o l a m i n e ) . F r o m t h e s e d a t a t h e s t a n d a rd d e v i a t i o n( s d ) w a s c a l c u l a t e d a n d t h e L O D w a s f o u n d a t t h ei n t e r s e c t i o n o f t h e l i n e r e p r e s e n t i n g t h e m e a n z e r os t a n d a r d m i n u s 2 s d w i t h t h e c o m p o s i t e c a l i b r a t i o nc u r v e . F o r u r i n e s a m p l es , t h e L O D o f t h e a s sa y w a s5 5 0 p g / m l a n d f o r p l a s m a s a m p l e s 1 6 p g / m l .

I n c u b a t e d q u a l i t y c o n t r o l s a m p l e s w e r e c o d e t e r -m i n e d t o s t u d y t h e i n f l u e n c e o f g l u s u l a s e o n t h ea s s a y . T h e q u a l i t y c o n t r o l d a t a f o r p l a s m a w e r eo b t a i n e d w i t h t h e i n i t i a l p r o c e d u r e . T h e p r e s e n t e dd a t a in T a b l e 3 s h o w t h a t th e i n c u b a t e d s a m p l e s m e e tt h e a b o v e m e n t i o n e d c r i t e r i a ( a c c u r a c y _ + 1 5 % , C . V .- - - 1 5 % ) . T h i s m e a n s t h a t a s i n g l e c a l i b r a t i o n c u r v ec a n b e p r e p a r e d t o m e a s u r e b o t h f r e e a n d t o t a ls c o p o l a m i n e i n u r i n e o r p l a s m a . H o w e v e r , t h e l e v e l so f to t al s c o p o l a m i n e i n p l a sm a s a m p l e s o f t h ep a t i e n t s w e r e n o t d e t e c t a b l e w i t h t h e i n i t i a l p r o -c e d u r e . T h e r e f o r e , th e v o l u m e o f t h e p la s m a s a m p l e sw a s i n c r e a s e d a n d t h e a d a p t e d p r o c e d u r e w a s v a l-i d a t e d w i t h a q u a l i t y c o n t r o l s a m p l e o f 1 . 2 5 x 1 0 9 Ms c o p o l a m i n e . T h e m e a s u r e d a c c u r a c y w a s 1 1 0 .7 %( _ +0 .4 % ) a n d t h e a d a p t e d p r o c e d u r e w a s f o u n d t o b en o t s t a t i st i c a ll y d i f f e r e n t f r o m t h e i n i t ia l p r o c e d u r e( t w o s a m p l e t t e s t , p < 0 . 0 5 ) .

3.3. Patient samples

3.3.1. UrineA f t e r a p p l i ca t io n o f S c o p o d e r m T T S t o t h e

p o s t a u r i c u l a r a r e a o f t h e p a t i e n t s , w e f o u n d t h a t a na v e r a g e o f 6 .3 f xg ( r a n g e 3 . 3 - 9 . 3 p , g) o f f r e es c o p o l a m i n e a n d a n a v e r a g e o f 8 3 .4 lx g ( r a n g e 4 8 . 0 -1 3 4. 5 I xg ) o f t o t a l s c o p o l a m i n e w a s e x c r e t e d i n 2 4 - hu r i n e ( T a b l e 5 ) . T h e m e a s u r e d u r i n e c o n c e n t r a t i o n s

( f r e e a n d t o t a l ) w e r e a l l h i g h e r t h a n t h e l o w e s tq u a l i t y c o n t r o l o f 5 X 1 0 - 9 M ( 1 . 5 n g / m l ) . I n t h ep l a c e b o p h a s e n o s c o p o l a m i n e w a s d e t e c t e d .

T h e o b s e r v e d v a r i a t i o n s i n s c o p o l a m i n e e x c r e t i o nc o r r e s p o n d s w i t h t h e f o u r t o s i x - f o l d v a r i a t i o n s i nu r i n e e x c r e t i o n r a t e s r e p o r t e d b e f o r e ( P a r r o t t , 1 9 8 9 ) .D u r i n g s t e a d y - s t a t e c o n d i t i o n s a p p r o x i m a t e l y 1 2 0 t xgo f s c o p o l a m i n e i s d e l i v e r e d t o t h e p a t i e n t i n 2 4 h . W ef o u n d t h a t o f t h i s d o s e , a b o u t 6 5 % i s e x c r e t e d i n 2 4 - hu r i n e a s g l u c u r o n i d e a n d s u l p h a t e c o n j u g a t e s a n do n l y 5 % o f t h e d o se i s e x c r e t e d u n c h a n g e d . T h e s er e s u l t s a r e i n l i n e w i t h e a r l i e r s t u d i e s i n w h i c h l e s st h a n 1 0% o f t h e a d m i n i s t e r e d d o s e w a s e x c r e t e du n c h a n g e d ( C l i s s o l d a n d H e e l , 1 9 8 5 ; S c h e u r l e n e t a l .,1 9 84 ) . H o w e v e r , w e f o u n d 7 0 % o f to t al s c o p o l a m i n ee x c r e t e d i n 2 4 - h u r i n e , w h e r e a s S c h e u r l e n e t a l .( 1 9 8 4 ) f o u n d o n l y 3 4 % o f t h e d r u g i n t h e u r i n ew h i c h m a y b e d u e t o t h e i n c u b a t i o n p r o c e d u r e .3.3.2. Plasma

U p t il l n o w , p l a s m a c o n c e n t r a t io n s o f s c o p o l a m i n eh a v e r a r e l y b e e n r e p o r t e d b e c a u s e a s e n s it iv e m e t h o df o r th e m e a s u r e m e n t o f s u c h l o w l e v e ls w a s l a c k in g( C l i s s o l d a n d H e e l , 1 9 8 5 ) . W e m e a s u r e d a v e r a g ep l a s m a c o n c e n t r a t i o n s o f f r ee a n d t o t a l s c o p o l a m i n eo f 4 3 . 6 p g / m l ( r a n g e 1 6 . 8 - 6 5 . 4 p g / m l ) a n d 22 9 . 0p g / m l ( r a n g e 1 6 5 . 6 - 3 7 7 . 2 p g / m l ) r e s p e c t i v e l y( T a b l e 5 ) . T h r e e o f t en m e a s u r e d c o n c e n t r a t i o n s o ff r e e s c o p o l a m i n e w e r e b e l o w t h e L O Q b u t a b o v e t h eL O D . W i t h t h e a d a p t e d p r o c e d u r e , t h e p l a s m a c o n -c e n t r a t i o n s o f to t a l s c o p o l a m i n e w e r e a l l a b o v e t h eL O Q o f 7 . 5 1 0 - ' ~ M i n t h e a s s ay , w h i c h c o r r e -s p o n d s w i t h 1 2 4 p g / m l i n p l a s m a u s e d f o r t h e t o ta ls c o p o l a m i n e d e t e r m i n a t i o n . I n t h e p l a c e b o p h a s e n os c o p o l a m i n e w a s d e t e c t e d .

M u i r a n d M e t c a l f e ( 1 9 8 3 ) r e p o r t e d a m e a n p e a kp l a s m a c o n c e n t r a t i o n f o r f r e e s c o p o l a m i n e o f 0 . 4 2 l 0 9 M ( 1 2 7 p g / m l ) i n 4 v o l u n t e e r s a f t e r t r a n s d e r -m a l a d m i n i s tr a t io n o f a s c o p o l a m i n e p a t c h w i t h ap r i m i n g d o s e o f 2 0 0 j xg a n d a re l e a s e r a t e o f 1 0p o g /h o u r. H o w e v e r , a l th o u g h t h e d o s e d e l i v e r e d w a st w i c e a s h i g h a s i n o u r s t u d y , t h e d r u g w a s d e t e c t a b l ei n o n l y 4 o f 1 2 v o l u n t e e r s . R e c e n t l y , p l a s m a c o n -c e n t r a t i o n s i n a g r o u p o f 8 s u b j e c t s w e r e d e t e r m i n e db y a m e t h o d w h i c h c o n s i s t i n g o f a p r e p a r a t i v ee x t r a c t i o n s t e p w i t h C ~ s c o l u m n s , f o l l o w e d b y a na n a l y t i c a l q u a n t i t a t i o n s t e p w i t h a m u s c a r i n i cr a d i o r e c e p t o r a s s a y ( C i n t r o n a n d C h e n , 1 9 8 7 ). T h em e a n f r e e p l a s m a c o n c e n t ra t i o n w a s 1 34 p g / m l


10/11

324 l.J. B o s m a n e t al. / European Journal of Pharmaceutical Sciences 5 (1997) 315-325

T a b l e 5F r e e a n d t o ta l s c o p o l a m i n e i n 2 4 - h u r i n e a n d i n p l a s m a o f I 0 p a t i e n t s .P a t i e n t F r e e P h a s e 1 F r e e P h a s e 2 T o t a l P h a s e 1 T o t a l P h a s e 2A. F r e e a n d t o t a l s c o p o l a m i n e (tzg) in 24-h urine of 10 patients

6789

10MeanS.D.C.V.n=10MeanS.D.C.V.

1 3.3 69.12 9.0 134.53 3.7 48.04 9.3 72.15 7.9 114.3

4.0 59.07.5 108.55.1 73.9

6.7 86.76.6 68.3

7.4 5.6 107.8 67.21 .6 2.4 25.2 1 3 . 0

22.1 42.1 23.4 19.4

6.3 83.52.2 27.3

34.8 32.7

B. Free and total scopolamine (pg/m!) in plasma of 10 patients

6789

10M e a nS.D.C.V.n=10MeanS.D.C.V.

1 16.8 204.12 57.0 377.23 44.5 216.44 65.4 235.65 29.3 198.2

39.6 236.734.0 165.660.8 243.2

40.0 200.148.6 213.1

45.3 42.5 246.1 217.715.9 15.8 93.0 15.535.2 37.1 37.8 7.1

43.6 229.015.0 56.934.4 24.8

P h a s e 1 : P a t i e n t s fi rs t r e c e i v e d s c o p o l a m i n e , t h e n p l a c e b o .P h a s e 2 : P a t i e n t s f i r s t r e c e i v e d p l a c e b o , t h e n s c o p o l a m i n e .A l l u r i n e a n d p l a s m a s a m p l e s f r o m p a t i e n ts t h a t r e ce i v e d p l a c e b o s h o w e d n o d e t e c t a b l e s c o p o l a m i n e c o n c e n t r at i o n s .

(range 82 -239 pg /ml) which i s three t imes h igher asthe mean concentra t ion we measured. I f we assum ethat doubling of the dose results in two times higherconcentrations, our results are comparable with themean plasma concentration reported by Muir andMetca l fe (1983) , tak ing into account that they wereunable to detect scopo lamine in 8 o f 12 vo lunteers .The method we descr ibed o f fers the advantage o fassay ing both free and to ta l scopo lamine which g ivesextra information on the bioavailabil i ty and kineticso f the drug .

4 . C o n c l u s i o nWe developed and va l ida ted a method which

cons i s ts o f a semi-automated so l id-phase ex tract ionfo l lowed by the ana lys i s o f free and to ta lscopo lamin e with radioreceptor assay (RR A). A l-though the concentrations after transdermal drugdelivery of scopolamine are very low, the assay issufficiently sensitive to measure these levels . Forurine samples o f 1 ml , the l imi t o f detect ion (L OD )of the assay i s 550 pg /m l and the l imi t o f quanti ta -


11/11

I .J . B o s m a n e t a l. / E u r o p e a n J o u r n a l o f P h a r m a c e u t i ca l S c i e n c e s 5 ( 1 9 9 7 ) 3 1 5 - 3 2 5 32 5

t i o n (L O Q ) i s 6 1 0 p g / m l . Fo r p l a s m a s a m p l e s o f 1 . 5m l , th e L O D i s 1 6 p g / m l a n d th e L O Q i s 3 8 p g / m l .

R e f e r e n c e sC h e n , X . H . , F r a n k e , J . E , E n s i n g , K . , W i j s b e e k , J. , d e Z e e u w , R . A . , 1 9 93 .S e m i - a u t o m a t e d s o l i d - p h a s e e x t r a c t io n p r o c e d u r e f o r d r u g s c r e e n i n g i n

b i o l o g i c a l f l u i d s u s i n g t h e A S P E C s y s t e m i n c o m b i n a t i o n w i t h C l e a nS c r e e n D A U c o l u m n s . J . C h r o m a t o g r . 6 1 3 , 2 8 9 - 2 9 4 .

C i n t r o n , N . M . , C h e n , Y .M . , 1 9 87 . A s e n s i t iv e r a d i o r e c e p t o r a s s a y f o rd e t e r m i n i n g s c o p o l a m i n e c o n c e n t r a t i o n s i n p l a s m a a n d u r i n e . J .P h a r m . S c i . 7 6 , ( 4 ) 3 2 8 - 3 3 2 .

C l i s s o l d , S . E , H e e l , R . C . , 1 9 85 . T r a n s d e r m a l h y o s c i n e ( s c o p o l a m i n e ) . Ap r e l i m i n a r y r e v i e w o f i t s p h a r m a c o d y n a m i c p r o p e r t i e s a n d t h e r a p e u t i ce f f i c a c y . D r u g s 2 9 , 1 8 9 - 2 0 7 .

C r e v a t - P i s a n o , E , H a r i t o n , C . , R o l l a n d , E H . , C a n o , J . E , 1 9 86 . F u n d a m e n -t a l s o f r a d i o r e c e p t o r a s s a y s . J . P h a r m . B i o m e d . A n a l . 4 , ( 6 ) 6 9 7 - 7 1 6 .

D e m e t e r , S . L . , C o r d a s c o , E . M . , 1 9 86 . T r a n s d e r m a l s c o p o l a m i n e i n t h et r e a t m e n t o f a s t h m a : a p r e l i m i n a r y r e p o r t . J . A s t h m a 2 3 , ( 4 ) 2 0 3 - 2 0 6 .

D o u m a , W . R . , B o s m a n , I . J. , R u t g e r s , S . R . , E n s i n g , K . , d e Z e e u w , R . A . ,K o ~ t e r , G . H . , a n d P o s t m a , D . S . ( 1 9 9 7 ) E f f e c t s o f t r a n s d e r m a ls c o p o l a m i n e o n p u l m o n a r y f u n c t i o n , s y m p t o m s a n d b r o n c h i a l h y p e r -r e s p o n s i v e n e s s to m e t h a c h o l i n e . E u r. J . P h a r m . S c i . 5 , 3 2 6 - 3 3 4 .

E n s i n g , K . , d e Z e e u w , R . A . , 1 9 84 , P r e - i n c u b a t i o n a n d l o w t e m p e r a t u r e sin quan t i t a t ive rad io re cep to r as says . Anal . Le t t . 17 , 1647-165 8 .

Ens ing , K . , i n ' t Hou t , W.G. , Halm a, E , Kl inkers , H .M .J . , Ens ing , G .J . , deZ e e u w , R . A . , 1 9 88 . D e v e l o p m e n t a n d a p p l i c a t io n o f a r a d i o r e c e p t o ra s s a y f o r sc o p o l a m i n e . A r z n e i m . F o r s c h . ( D r u g R e s . ) 3 8 , ( 1 ) 1 0 6 - 1 1 1 .

G u y , R . H . a n d H a d g r a f t , J . ( 1 9 8 5 ) T r a n s d e r m a l d r u g d e l i v e r y : t h e g r o u n dr u l e s a r e e m e r g i n g . P h a r m a c y I n t e r n a t i o n a l , M a y , 1 1 2 - 1 1 6 .

M u i r , C . , M e t c a l f e , R . , 1 9 8 3. A c o m p a r i s o n o f p l a s m a l e v e l s o f h y o s c i n eaf t e r o ra l and t ransderm al admin i s t ra t ion . J. Pharm. Biom . Anal . 1 ,3 6 3 - 3 6 7 .

Munson , P .J . , Rodbard , D . , 1980 . L igand : a versa t i l e computer i zeda p p r o a c h f o r c h a r a c t e r i z a t i o n o f l i g a n d - b i n d i n g s y s t e m s . A n a l . B i o -c h e m . 1 0 7 , 2 2 0 - 2 3 9 .

P a r r o tt , A . C . , 1 9 89 . T r a n s d e r m a l s c o p o l a m i n e : a r e v i e w o f i ts e f f e c t su p o n m o t i o n s i c k n e s s , p s y c h o l o g i c a l p e r f o r m a n c e , a n d p h y s i o l o g i c a lf u n c t i o n i n g . A v i a t . S p a c e . E n v i r o n . M e d . 6 0 , ( 1 ) 1 - 9 .

S c h e u r l e n , M . , B i t t i g e r , H . , A m m a n n , B . , 1 9 8 4 . S i m p l e r a d i o l i g a n db i n d i n g a s s a y f o r t h e d e t e r m i n a t i o n o f u r i n a ry s c o p o l a m i n e . J . P h a r m .Sci . 73 ,14) 561-563 .

Shah , V .E , Midha , K .K. , D igh e , S . , McG i lveray , l . J . , Skel ly , J .P . , Yacob i ,A . , L a y l o f f , T . , V i s w a n a t h a n , C . T . , C o o k , C . E . , M c D o w a l l , R . D . ,Pi t tman , K .A. , Specto r , S . , 1992 . Analy t i ca l methods va l ida t ion :b i o a v a i la b i l it y , b i o e q u i v a l e n c e , a n d p h a r m a c o k i n e t i c s t u d ie s . J . P h a r m .S c i . 8 1 , ( 3 ) 3 0 9 - 3 1 2 .

Smis terovzi , J . , Ens ing , K . , de Zee uw, R .A. , 1994 . Me thodo lo g ica l aspec t so f q u a n t i t at i v e r e c e p t o r a ss a y s . J. P h a r m . B i o m e d . A n a l . 1 2 ,( 6 ) 7 2 3 -745.

S w a r t , E J . , D r e n t h , B . F . H . , d e Z e e u w , R . A . , 1 9 90 . R e v e r s e d - p h a s e l iq u i dc h r o m a t o g r a p h i c m e t h o d w i t h a m p e r o m e t r i c d e t e c t i o n f o r t h e d e -t e r m i n a t i o n o f t h e d o p a m i n e a g o n i s t 2 - ( N - p r o p y l - N - 2 -t h i e n y l e t h y la m i n o ) - 5 - h y d r o x y t e t r a l i n ( N - 0 4 3 7 ) i n h u m a n p l a s m a a n dur ine . J . Chromatogr . , Bio ta . App l . 528 , 464-472 .

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