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423 acta farmac utica bonaerense - vol. 24 n 3-a o 2005 C . P r e s e nt i n g a m e d i u m va l u e o f 1 6 1 . 7 C, anda s t a n d a r d d e v i a t i o n o f 0 , 9 3 a n d a R S D o f 0 . 5 8 % . T he s e va l u e s a r e t h e a v e r a g e o f 9 d e t e r - m i n a t i o n s . T h e m e l t i n g r a n g e v a l u e s o b t a i n e d fo r t h e w o r k i n g s t a nd a r d a nd t h e d r u g s a m p l e indicate quite similar result s.Ac c o r d i n g t o t he d a t a o bt a i n e d , t h i s p r o c e - d u r e h a s b e e n f o u n d t o b e p r e c i s e , a c c u r a t e a n d s u it a b l e f o r t h e a n a l ys i s o f c e l e c o x i b .

i b . I t s ho u l d b e no t e d t ha t t he m e t ho d g a v e s i m i l a r a n d f a - vorable result s wit h respect to the low RSD val-u e s . T h e m e lt i n g p o i n t d e t e r m i n e d i n a c a p i l l a r yt u b e i s o n e o f t h e m o s t a c c u r a t e a n d m o r e me a n i n g f u l c r it e r i o n s o f p u r it y. Ult ravio let spectro scopy I n t h e d e v e lo p m e nt o f t h i s m e t ho d , s e v e r a l s o l v e nt s w e r e u s e d , l i k e m e t h a n o l , a c e t o n it r i l e , Figure 2 . T h e u l t r a v i o l e t s p e c t r a o f m e t h a n o l s o l u - t io n s ( 1 0 ( g l 1 ) o f c e l e c o x i b w o r k i n g s t a nd a r d ( u p - p e r l i n e ) a nd t h e s a m p l e ( l o w e r l i n e ) . NaOH (0.1 M) and HCl (0.1 M). The above sol-ve nt s w e r e a l s o u s e d i n c o m b i n a t io n s . T he d e c i - s io n fo r u s i n g a 1 0 0 % m e t ha no l w a s ba s e d o n its sensit ivit y, stabilit y and preparat ion t ime.A s t o c k s o l u t io n o f c e l e c o x i b w a s p r e p a r e d b y d i s s o l v i n g 1 0 m g o f t he s a m p l e a n d t he r e f e r - e n c e m a t e r i a l , i n 2 0 0 m l o f m e t h a no l, t o a c h i e v e a f i n a l c o nc e nt r a t io n o f 5 0 g m l 1 . From this so-lu t io n w a s p r e p a r e d a s o l u t io n c o nt a i n i n g 1 0 g m l - 1 , a n d t h e a b s o r b a n c e w a s m e a s u r e d . T h e s o l v e nt u s e d i n t h e s o l u b i l i z a t io n was a lso used as a b la nk.T h e s p e c t r u m o f c e l e c o x i b i n m e t h a n o l i s s h o w n i n F i g u r e 2 . A m a x i m u m a b s o r p t i o n a t 2 5 2 w a s fo u n d . T h e s p e c t r a o bt a i n e d fo r t h e s a m p l e a nd r e f - e r e n c e m a t e r i a l w a s c o m p a r e d a n d a n a l y z e d , s u g g e s t i n g t ha t t he e xt r a c t io n p r o c e d u r e w a s ad-e q u a t e f o r t h e d o s a g e fo r m t e s t e d ( C e l e b r a

)a n d t h e u l t r a v i o l e t s p e c t r u m c a n b e u s e d f o r id e nt if i c a t io n o f b o t h b u l k s u b s t a nc e a n d t a b l e t s . Nuclear Magnetic Resonance (NMR) T h e N M R s p e c t r o s c o p y i s a p o w e r f u l t e c h- n i q u e fo r t h e c h a r a c t e r i z a t io n of the exact struc-t u r e o f a c o m p o u n d , p r o v i d i n g m u c h m o r e i n f o r m a t i o n a b o u t mo l e c u l a r s t r u c t u r e t h a n a n y other technique. T h e p r o t o n N M R i s t h e m o s t commonly used form of NMR because it s sensit i v it y a n d t he l a r g e a mo u nt o f s t r u c t u r e i n f o r m a - t io n it y i e l d s . T he c o m b i n a t io n o f i n fr a r e d a nd NMR spectroscopy data is often sufficient to det er mine t he st r uct ur e o f a n u nk no w n mo le c u le. T he sp ect r u m s ho w n i n F i g u r e 3 p o s s e s s e s t w o c ha r a c t e r i s t i c s h a r p s i n g l e t p e a k s a t 2 . 3 a n d Figure 3 .NMR spectrum of c e l e c o x i b in DMSO.

424 PRIMO F.T. & FR EHLICH P.E. 3 . 3 p p m t ha t b e lo n g t o t he m e t h y l a nd s u l fo n- a m i d e p r o t o ns o f c e l e c o x i b . T h e s p e c t r u m a l s o r e v e a l s p e a k s fr o m 7 . 3 t o 7 . 9 , w h i c h i s d u e t o t he p r o t o n s o f a r o m a t ic g r o u p s . T h e c ha r a c t e r is - t i c s p e a k s f r o m t h e w o r k i n g s t a n d a r d a g r e e w e l l w it h t ho s e o b s e r v e d i n t he s a m p le s , c o n s i d e r i n g t h e f a c t t h a t a c o ns t a nt s h i ft i s o b s e r v e d i n a l lp e a k s 11, 12 . T h e c h a r a c t e r i s t i c p e a k s i n t he r a n g e o f 2 . 0 - 4 . 0 p p m c a n b e u s e d fo r i d e n t i f i c a t io n o f e xt r a c t e d s a m p l e a n d w o r k i ng s t a nd a r d . Infrared Spectro sco py T h e i n f r a r e d s p e c t r o s c o p y i s o ne o f t h e mo s t c o m m o n l y u s e d t e c h n i q u e fo r t h e i d e n t i f i c a t io no f c o m p o u n d s , t h i s f a c t i s t r u e be c a u s e it s s p e c - t r a i s a l mo s t i n s e n s it i v e b y t h e p r e s e n c e o f i m - p u r it i e s in lo w c o n c e n t r a t io n s . T h e i n f r a r e d s p e c t r u m m e t h o d c a n b e u s e d f o r mo l e c u l e s m u c h a s a f i n g e r p r i nt . I f t h e i n- f r a r e d s p e c t r a o f t w o s u b s t a n c e s a r e c o m p a r e d a n d m a t c h p e a k fo r p e a k , it i s p o s s i b l e t o s a yt h a t t h e y a r e t h e s a m e c o m p o u n d 12 . Thismethodology is also important to d e t e r m i n e s t r u c t u r a l i n fo r m a t io n a b o u t t he mo l e c u l e . T h e i n f r a r e d a b s o r p t i o n s p e c t r u m w e r e o b - t ained b y mix ing 1.5 m g o f t h e f i n e l y g r o u n d e x- t r a c t e d s a m p l e o r t he w o r k i ng s t a nd a r d , w i t h 1 5 0 m g o f p o w e r e d p o t a s s i u m b r o m i d e ( p r e v i o u s l y d r i e d ) , a nd t he n p r e s s e d t h i s m i xt u r e u nd e r h i g hpressure, in order to obtain a KBr disk. This KBrd i s k w a s t h e n i n s e r t e d i nt o a ho ld e r i n t h e s p e c - t r o m e t e r . T r it u r a t e 1 . 5 m g o f t h e s u bs t a n c e t o be e x a m i n e d w it h 1 5 0 m g o f f in e l y p o w d e r e d a n d d r i e d p o t a s s i u m b r o m i d e . T h e s e q u a nt it i e s w e r e sufficient to give a KBr disc o f 13-mm diametera n d a s p e c t r u m o f s u it a b l e i n t e n s it y. C a r e f u l l y t hes u b s t a n c e a n d t h e p o t a s s i u m b r o m i d e w e r e g r i n d e d . T h e m i x t u r e w a s t h e n , s p r e a d i t u n i - fo r m l y i n a s u it a b l e d i e , a nd s u b m it it i n va c u u m t o a p r e s s u r e o f a bo u t 8 0 0 M P a . T h e w o r k i n g s t a n d a r d a n d t h e e x t r a c t e d samp le were e x a m i n e d b y t h e s a m e p r o c e d u r e a n d r e c o r d t he s p e c t r a be t w e e n 4 0 0 0 cm -1 and670 cm 1 u nd er t he s a me o per at io na l co nd it io ns 13 .T h e t r a n s m i s s io n m i n i m u m ( a b s o r p t io n m a x - i m u m ) i n t h e s p e c t r u m o bt a ine d w it h t he s u b- st a nc e t o be e xa mine d co r r e spo nd s in

p o s i t i o n a n d r e l a t i v e s i z e t o t h o s e i n t h e s p e c t r u m o b - t a i n e d w it h t h e r e f e r e nc e s u b s t a n c e . U s i n g t h e p o s i t i o n s o f t h e b a n d s o f t h e w o r k i ng s t a n d a r d a s r e f e r e nc e , t he p o s it io n s o f t h e s i g n i f i c a n t b a n d s i n t h e s p e c t r u m o f t h e samp le to be examined and t he reference spec-t r u m s h o u l d c o r r e s p o n d t o t h e w a v e n u m b e r s c a l e . T h e r e l a t i v e s i z e s o f t h e b a nd s a r e c o n c o r - d a n t i n b o t h s p e c t r a . T h e t w o s p e c t ra w e r e compared and the major observed bands andtheir assignments for celecoxib a r e f o u n d i n Table 1.T h e w o r k i n g s t a n d a r d a n d t h e s a m p l e s wereanalyzed and co mpared peak for peak, t hrought heir c h a r a c t e r i s t i c s s p e c t r a l b a n d s . T h e s p e c - trum itself is shown in Figure 4. Thin- layer chro matography O n e o f t he m o s t e f f e c t i v e s c r e e n i n g m e t ho d s is the thin-layer chromatography (TLC), which ist h e s i m p l e s t o f a l l t h e w i d e l y u s e d c h r o m a t o - g r a p h i c m e t h o d s t o p e r fo r m. I n t he d e t e r m i n a - t i o n o f t h i s m e t h o d , d i f f e r e n t c h r o m a t o g r a p h y s ys t e m s w e r e t e s t e d a nd a na l yz e d a c c o r d i n g t o t he c l a s s i f i c a t io n p r o p o s e d b y M o f f a t 13 , w h i c h d i v i d e t h e d r u g s i n t hr e e c a t e g o r ie s : a c i d , ba s i c a n d n e u t r a l s based on t he ir po larit y and acidcharacterist ics. T his procedure is important in Band Frequency (cm 1 ) A s s i g n m e n t 1 1 5 0 1 3 5 0 S = O s t r e t c h i n g ( s u l fo n a m i d e group)1 5 5 0 1 6 0 0 N H s t r e t c h i n g 3 3 0 0 3 5 0 0 N H 2 st ret ching Table 1 . I n fr a r e d f r e q u e n c y a s s i g n m e nt s o f c e l e c o x i b . Figure 4 .The infrareds p e c t r u m o f t he w o r k i n g s t a nd a r d ( u p p e r l i n e ) a n d t h e s a m p l e ( lo w e r l i n e ) .

425 acta farmac utica bonaerense - vol. 24 n 3-a o 2005 o r d e r t o i nc r e a s e t h e i n fo r m a t io n o bt a i n e d o n l y b y c h a ng i n g t h e mo b i l e p h a s e , a nd t hu s r e s u lt - ing in a significant change in select ivit y.T he s e c h r o ma t o g r a p h i c s ys t e m s c o n s i s t e d o f m i xt u r e s o f t he fo l l o w i n g s o l v e n t s : c h lo r o fo r m- a c e t o n i t r i l e , e t h y l a c e t a t e , a c e t o n e a n d m e t h a - no l, i n d i f f e r e n t c o nc e nt r a t io n s . N e v e r t h e l e s s , a l l t h e m o b i l e p h a s e s t e s t e d w e r e n o t a d e q u a t e f o r t he p r o p e r i d e nt i f i c a t io n o f t h e d r u g . W it h t heo b j e c t i v e t o d e v e l o p m o r e r e l i a b l e c h r o m a t o - g r a p h i c met ho d, a mo d if ic at io n in t he s yst e mt est e d w as pr o po se d, in o r der t o i m p r o v e t h e c h r o m a t o g r a p h i c r e s o lu t io n . T h e mo b i l e p h a s e used c o n s i s t e d o f c h lo r o fo r m- e t h y l a c e t a t e - e t he r ( 1 0 : 5 : 1 v/ v / v) . T h e s ys t e m w a s c h o s e n d u e t o it s sensit ivit y, simplicit y and efficacy.T h e p r e f e r e n c e t o u s e c o m m e r c i a l l y p r e - p a r e d s i l i c a g e l p l a t e s w a s d u e t o it s d u r a b i l i t ya n d ho mo g e n e it y o f t h e a b s o r b e nt la ye r . A s a m p l e s o l u t io n o f t h e w o r k i n g s t a nd a r d a nd t he d r u g s a m p l e ( 5 0 ( g m l 1 ) w e r e s p o t t e d o nt o a s i l i c a g e l p l a t e w it h a m i c r o p i p e t t e a nd t h e c h r o m a t o g r a m w a s d e v e l o p e d b y p l a c i n g t he p l a t e i n a t a nk c o n t a i n i n g t he mo b i l e p h a s e . F o l l o w i n g d e v e l o p m e n t t h e i n d i v i d u a l s o l u t e s p o t s w e r e i d e nt i f i e d u nd e r U V l a mp ( 2 5 4 a n d 3 6 5 n m ) . T h e s p o t s i n t h e d r u g s a m p l e a n d t he w o r k i n g s t a n d a r d p r e s e n t e d s i m i l a r R f v a l u e s . T he R f v a l u e o bt a i n e d fo r d r u g s a m p l e a n d t h e w o r k i n g s t a n d a r d w e r e 0 . 4 5 a n d f o r r o f e c o x i b 0.32. The picture of the TLC plate is shown inFigure 5.c a t io n o f c e l e c o x i b . B e i n g a r e l a t i v e l y n e w d r u g , t h e r e a r e no t m a n y p u b l i c a t io ns c o nc e r n i n g it s analysis, and so far it is not included in any offi-c i a l c o d e . S o m e o f t he m e t ho d o lo g i e s a r e s i m p l e , a n d h a v e b e e n f o u n d t o b e a c c u r a t e , p r e c i s e a n d suitable, giving, by this way, reliable results.H e nc e , t he m e t h o d s e va l u a t e d i n t h i s s t u d ya r e a p p l i c a b l e fo r t he i d e nt i f i c a t io n o f c e l e c o x i b, i n r o u t i n e q u a l i t y c o n t r o l l a b o r a t o r i e s , f o r t h e ident ificat ion of this drug. Acknowledgements . T h e a u t ho r s w i s h t o t ha nk t h e m a n a g e m e nt o f D r . R e d d y s Laboratories Ltd. for theirs u p p o r t i n p r o v i d i n g t h e r e q u ir e d w o r k i n g s t a n d a r d s f o r o u r r e s e a r c h , a n d D r . G r a c e G o s m a n n f o r h e r help in the NMR spectra.

REFERENCES 1. Bombardier, C.; L. Laine, A. Reicin, D. Shapiro,R. Burgos-Vargas, B. Davis, R. Day, M.B. Fe-rraz, C.J