Microbial Assay Of Vitamin of B Series

In this slide contains introduction, principle, precautions, solution and assay method for vitamin B series.
Presented by: P. VENKATESH (Department of pharmaceutical analysis),
RIPER, anantapur

1. 1 A Seminar as a part of curricular requirement for I year M. Pharm I semester . Presented by M r P . Venkatesh . (Reg. No. 2 L 8 1 S 7 1 4 ) Under the guidanc e of Dr. K . V i n o d k u m a r , M . p h a r m , P h D . Professor Pharmaceutical Analysis , H e a d o f department - P h a r maceutical analysi s . M I C R OBIAL ASSAY Of V I T A M IN O F B S E R I E S

2. 2 Introduction. Principle. Quick view of B s e r i e s v i t a m i ns . Microorganisms used. Precautions. S o l u t i on s a n d i n o c u l u m preparations . Assay methods. C O N T E N T S

3. 3 Microbial assa y Also Known as microbiological assay. Microbial assay could be a sort of biological assay designed to analyse the compounds that have impact on microorganisms. Hepls to estimate -Con c e n t r a t i o n . -Effency of antibiotics. Introduction

4. 4 PRNCIPLE Vitamins are important growth factors required for growth and multiplication of certain microorganisms. The microorganisms require growth factors in micro or nanograms. The basis of this assay is to measure the ability of test organism to synthetise the factor by utilising the substance being assayed under a proper nutrition conditions . Factor-growth ,nutrition condition-cyanocobalamine.

5. 5 PRNCIPLE Microbial assay of vitamin is based on T est concentration of vitamin is estimate d w i t h k nown concentration of standard. Comparison of growth of microorganisms

6. 6 T h i a m i n e ( B 1 ) : O r g a n i s m : l a c t o b a c i llus v a r i d e s c e n s . D e f i e n c y : memory l o s s , c o n f u s ion hallucination . D a i l y i n t a k e : Q u i c k v i ew of v i tamin B s e r i es Y e a r s M a l e F e m a l e 9 - 1 3 . 9 m g . 9 m g 1 4 - 1 8 1 . 2 m g 1 . m g 1 9 - 5 1 . 2 m g 1 . 1 m g 5 1 a b o v e 1 . 2 m g 1 . 1 m g

7. 7 R i b o f l a v i n ( B 2 ) : O r g a n i s m : l a c t o b acillus case i . D e f i e n c y : s k i n d i s o r d e r s , c h e l o s i s , h a i r l o s s , r e p r o d u ctive p r o b l e ms . D a i l y i n t a k e : Q u i c k v i ew of v i tamin B s e r i es 2 - 5 y e a r s 2 . 1 m g 6 - 1 1 y e a r s 2 . 2 m g 1 2 - 1 9 y e a r s 2 . 3 m g

8. 8 N i a c i n ( B 3 ) : O r g a n i sm : l a c t o bacillus p l a n t a r u m . D e f i ency : p e l l e g r a , d e r m a titis , diarrhea a n d c a n r e s u lt i n d e a th Occurs d u e t o g e n e t i c d i s o r d e r s . D a i l y intake : Q u i c k v i ew of v i tamin B s e r i es A d u l t m e n 1 9 a b o v e 1 6 m g A d u l t w o m e n 1 9 a b o v e 1 4 m g P r e g n ant w o m e n 1 8 m g B r e s t feeding w omen 1 7 m g

9. 9 P a n t o t h e n i c a c i d ( B 5 ) : O r g a n ism : l a c t o bacillus p l a n t a r u m . D e f iency : inherited disorder c a l l e d p a n t o t h e n a t e k i n a s e a s s o c i a ted neuro d e g e neration - c a n 't u s e p a t h o t h e n i c a c i d . D a i l y i n t a ke : Q u i c k v i ew of v i tamin B s e r i es 9 - 1 3 y e a r s 4 m g 1 4 - 1 8 y e a r s 5 m g 1 9 y e a r s a b o v e 5 m g P r e g n ant w o m e n 6 m g

10. 10 P y r o d o x i n ( B 6 ) : O r ganism : s a c c h a r o m y c e s u v a r u m . Defency : s k i n r a s h e s , c r a c k e d l i p s , impaired immune functio n , n e r v e p a i n . D a i l y i n t a k e : Q u i c k v i ew of v i tamin B s e r i es 9 - 1 3 years 1 . m g 1 . m g 1 4 - 1 8 y e a r s 1 . 3 m g 1 . 2 m g 1 9 - 5 y e a r s 1 . 3 m g 1 . 3 m g 5 a b o v e 1 . 7 m g 1 . 5 m g

11. 11 F o l i c a c i d ( B 9 ) : O r g a n i sm : l a c t o bacillus case i . D e f i ency : m e g a l o b l a s t i c a n e m i a . D a i l y intake : Q u i c k v i ew of v i tamin B s e r i es A d u l t 4 m c g P r egnant w o m e n 1 m c g

12. 12 B i o t i n ( H ) : O r g a nism : l a c t o bacillus p l a n t a r um . D e f i ency : r a r e i n h e rited disorder w h e r e b o d y i s n o t a b l e t o use b i o t i n , d e f i e n c y c a u s e d b y m u t a t ion . D a i l y intake : Q u i c k v i ew of v i tamin B s e r i es Y e a r s M a l e F e m a l e 4 - 8 1 2 m c g 1 2 m c g 9 - 1 3 2 m c g 2 m c g 1 4 - 1 8 2 5 m c g 2 5 m c g 1 9 a b o v e 3 m c g 3 m c g

13. 13 Q u i c k v i ew of v i tamin B s e r i es S t i c k m o d e l s t r u c tur e . S k e l e t a l m o d e l s t r ucture

14. 14 Also known as vitamin B 1 2 . It is a water soluble vitamin. Structure is similar to that of heme where iron is replaced with cobalt as center of molecule. Q u i c k v i ew of c y a nocobalamine

15. 15 Main sources of cyanocobalamine are liver,eggs,milk,meat and fish. D aily intake of cyanocobalamine Q u i c k v i ew of c y a nocobalamine 9 - 1 3 y e a r s 1 . 8 m c g / d a y 1 4 above y e a r s 2 . 4 m c g / d a y P r e g nant w o m e n 2 . 6 m c g / d a y B r e s t feeding w o m e n 2 . 8 m c g / d a y

16. 16 Defiency causes pernicious anemia. Helps in maintaining healthy nervous system. Aids in production of DNA and RNA. Help to keep red blood cells healthy. Q u i c k v i ew of c y a nocobalamine

17. 17 Q u i c k v i ew of v i tamin B s e r i es

18. 18 M I C R O O RGANISM U S E D microorganism u s e d h e r e a r e L a c t o b a c i l l u s l e i c h m a n n i T e s t o r g a n i s m s e l e c t ed must b e c a p a b l e o f u t i l i z i ng f r e e c y a n o cobalamine . G r a m n e g ative b a c t e r i a . N o n - p a t h o g e n i c . E a s y t o c u l ture a n d e a s i l y a v a i l a b l e . I s o l a ted f r o m - M i l k - C h e e s e a n d d a i r y p r o d u c t s .

19. 19 G r e a t c a r e m u s t b e t a k e n t o a v o i d c o n t a m ination o f p reparations . A l l g l a s s w a r e s m u s t b e f r e e f r o m c h e m i cals a n d m i c r o b e s . G l a s s w a r e m u s t b e h e a t e d a t 2 5 ° C f o r at least 1 h o u r b e f o re u s e . T h e w h o l e e x p i r e m e n t m u s t be c a r r i e d u n d e r a s c e p t i c conditions . A s c e p t i c c onditions - l a m i n o r a i r f l o w c o n d i tions P R E C A U T I O NS

20. 20 S t a n d a r d B 1 2 s t o c k solution . S t a n d a r d B 1 2 s o l u tion . T e s t s o l u t i o n t o b e a s s a y e d . B a s a l m e d i u m s t o c k solution . Suspension m e d i u m . Preparation o f i n o c u l u m . S O L U T I O N S , I N O C U L U M P R E P E R A T I O N

21. 21 S t a n d a rd B 1 2 S t o c k s o l u tion : A c c u r a t e l y w e i g h ed a m o u nt o f cy a nocobalamin e r e f e r e n c e s t a n d a r d A d d ed 2 5 % ethanol - m a k e u p t o 1 m l R e s u l t i n g S o l u t ion 1 . m i c r o g r ams o f cy a nocobalamin e p e r m l Continuation . . . . . . . . . .

22. 22 S t a n d ard B 1 2 s o lution : F u r t h e r d i l u t i on o f s t o c k solution m a d e a s f o l l o ws . 1 m l s t o c k s o l u t i on - 9 9 m l p u r i f ied w a t e r . ( 1 m l - 1 n g ) A d d 1 m l a b o v e s o l u t ion 1 9 9 m l p u r i f ied water . ( 1 m l - . 5 n g ) T e s t s o l u tion to be assayed : A c c u r a t e a m o unt of t e s t solution i s d i s s o l ved i n water , d i l u te H C L o r N A O H s o l u tion i s a d d e d t o a d j u s t p h - 6 . . Continuation . . . . . . . . . .

23. 23 B a s a l m e d i um stock solution : Continuation . . . . . . . . . .

24. 24 Suspension m e d i u m : P r e p a r e 1 m l s o l u t ion b y m i x i n g e q u a l v o l ume o f b a s a l m e d i u m stock solution a n d d i s t i l l ed w a t e r . P r e p aration o f i n o c ulum : T r a n s f e r a l o o p f u l l o f l a c t o b a c i l l u s l i e c h m a n n i f r o m r e c e nt s u b c u l t u r e i n t o t w o t u b e s e a c h c o n t a i n i n g 1 m l o f s t e r i l e c u l t u r e m e d i u m . I n c u b a t e t h e t u b e f o r 1 8 t o 2 4 h o u r s a t 3 7 d e g r e e s c e ntigrade . Continuation . . . . . . . . . .

25. 25 C e n t r i f uge m a k e a suspension o f c e l l that s e t t l e d o w n u s i n g 1 m l o f s t e r i l e s u s p n t i o n m e d i u m . C e n t r i fuge a g a i n a n d a g a i n s u s p e n d t h e c e l l i n 1 m l o f s t e r ile m e d i u m . R e p e a t t h i s a t l e a s t 3 t i m e s t o a v o i d c o n t a m i nation . Aseptically t r a n s f e r 1 m l o f suspension m e d i u m i n 1 m l o f s t e r i l e suspension medium . T h i s s u s p e nsion i s u s e d a s i n o c u lum . Continuation . . . . . . . . . .

26. 26 A s s a y i s p e r f o rme d b y t w o m e t h o d s T i t r i m e t r i c m e t h o d . T u r b i d i m e t r i c m e t h o d . T i t r i m e t r i c m e t h o d : C l e a n t h e t e s t t u b e a n d a d d , . 5 , 1 . , 2 . , 2 . 5 , 3 . , 3 . 5 , 4 . , 4 . 5 a n d 5 . m l o f s t a n d a r d c y a n o c o b a l a mine s o l u tion . T o e a c h t u b e a d d 5 m l o f b a s a l medium s o l u t ion . A S S A Y M E T H O D S

27. 27 V o l u m e o f e a c h i s a d j u s t e d w i t h 1 m l d i s t i l l e d w a t e r . I n a n o t h e r 4 t e s t t u b e s a d d 1 , 2 , 3 , 4 m l o f t e s t s o l ution w h i c h i s t o b e a s s a y e d . V o l u m e of e a c h i s a d j u sted w i t h 1 m l o f d i s t i l l ed water . S t e r i l i z e a l l t h e t e s t t u b e s i n a u t o c l a v e a t 1 2 1 ° C f o r 1 5 m i n u tes . C o o l t h e t e s t tube t o r o o m t e m p e r a t u r e . I n o c u l a t e a d r o p inoculum p r e p a r e d . Continuation . . . . . . . .

28. 28 I n c u b a t e t h e t e s t t u b e f o r 6 4 t o 7 2 h o u r s a t a t e m p e r a ture r a n g e o f 3 t o 3 7 d e g r ee s centigrade . A f t e r i n c u b a t i o n c o n t e n t o f e a c h t e s t t u b e i s t i t r a ted w i t h . 5 N N a O H u s i n g b r o m o t h y m a l b l u e a s a n indicator u n t i l g r e e n c o l o u r a p p e ar s . R e c o r d a l l t r i t a n t r e a d i n g c l e a r l y . Continuation . . . . . . . .

29. 29 T u r b i d i m e t r i c method : A l s o p e r f o rmed by Turbidimetric m e t h o d . B y u s i ng un i n o c u lated m e d i a a s b l a n k a n d a d j u s t i n g transmittance a t 6 4 n m . T r a n s m i t t a n c e values a r e n o t ed . P l o t a g r a p h b y c o n s i d e r i n g transmittance v e r s e s s t a n d a r d c y anocobalamine solution . Continuation . . . . . . . .

30. 30 Continuation . . . . . . . . T r a n s m i ttance V a l u e Standard cyanocobalamine S o lution

31. 31 P r inciple : V i t a m i n B 1 2 i s e x t r a c t e d f o r m s a m p l e u s i n g S o d i u m a c e t a t e b u f f e r i n p r e s e n c e o f s o d i um c y a n i d e . a t 1 d e g r ees centigrade E x t r a c t a r e p u r i f i ed a n d c o n c e n t r a ted with I m m u n o a f f i n i t y c o l u m n . H P L C m e t h o d o f analysis

32. 32 S a m p l e preparation : I f t h e s a m p l e i s i n p o w d e r f o r m w e i g h 2 5 g o f s a m p l e a n d t a k e n i n a b e a k e r . A d d 2 g o f s a m p l e a n d suspension i s h o m o g e n i zed w i t h p o l y t r o n . E x t r a c t i o n w e i g h 6 g r a m s of above s o l utio n a n d t a k e n i n 2 5 m l f l a t b o t t o m a m b e r g l a s s f l a s k . A d d 1 m l s o d i um cyanide s o l u tion . I f s a m p l e contain s t a r c h a d d . 5 g o f a l p h a a m y l a s e . H P L C m e t h o d o f analysis

33. 33 M i x thoroughly a n d i n c u b a te f o r 1 5 m i n u t e s . A d d 2 5 m l o f s o dium a c e t a t e s o l u tion a n d p l a c e t h e f l a s k i n b o i l i n g w a t e r b a t h . Q u a n t i t a tively transfer t h e c o n t e n t of t h e f l a s k i n t o 1 m l a m b e r e d v o l u m e t r ic f l a s k . F i l t e r t h e c o ntent a n d p a s s t h r o ugh i m m u n o a f f i n i t y c o l u m n A n a l ysis : M a k e a n inj e ction o f s t a ndard a n d t e s t s o l u tion . M e a s u re Chromatographic p e a k responce . H P L C m e t h o d o f analysis

34. 34 C h r o m a tographic c onditions : F l o w r a t e . 4 m l / m i n I n j e c tion v o l u m e 5 m i c r o l i t e r s D e t e c t o r U V D e t e c tion w a v e l e n g th 3 6 1 n m M o b i l e p h a s e A - 1 m l w a t e r + T F A 2 5 m l B - 1 m l a c e t o n y t r i l + T F A 2 5 m l E l u t i o n G r a d i ent H P L C m e t h o d o f analysis

35. 35 I d e n t i f i c a tion : I d e n t i f y B 1 2 p e a k i n c h r o m a t o g r a m o f t e s t s o l u tion c o m p a ring with that r e t e ntion time a n d U V s p e c t r u m o f corresponding peak o f o b t a i ned from s t a n d a r d . Q u a n t i f ication : H P L C m e t h o d o f analysis

36. 36 W h e r e A = Response o f p e a k o b t a i n e d f o r t h e s a m p l e . I = Intercept o f calibration c u r v e . S = s l o p e o f c a l i b ration curve . V = v o l u m e o f t e s t s o l u t ion i n m l . V 2 = V o l u m e i n w h i ch a l i q u i t e o f s a m p l e S o l u t ion f r o m i m m u n o a f f i n i t y cleanup . m = w e i g h t o f t e s t portion i n g r a m s . V 1 = V o l u m e o f a l i q u o t o f s a mple s o l u t ion l o a d e d i n t o affinity c o l u m n . H P L C m e t h o d o f analysis

37. 37 B i s h o n i k a p i l , k a t a r i a Mahesh , M i c r o b i al a s s a y o f v i t a m i n B , international r e s u r c h j o n o u r a l o f p h a r macy , 2 1 2 , 3 ( 2 ) , p . n o - 7 4 - 8 1 . G a r r a t , D . C . , T h e q u a n t i t a tive a n a l ysis o f d r u g s 2 n d edition . I n d i a , v a l l a b h p r a k a s h a n , 2 1 , p . n o - 8 1 3 - 8 2 5 . J a i n , N . K . . Pharmaceutical m i c r o b i o l o g y , 2 n d edition . I n d i a M a c m i l l a n l t d , N e w Delhi . p . n o - 3 7 - 1 7 . E s t h e r Campos G i m e n e z . , I m p r o ved A O A O f i r s t action 2 1 1 . 8 f o r t h e a n a l y sis o f v i tamin B 1 2 , J o u r n a l o f A O A C i n t e r n a tional , 2 1 4 , v o l . 3 7 n o - 5 , p . n o - 3 , 4 , 5 . R e f e r e nces

38. 38 T h a n k y o u