THE BIOASSAY OF DIGITALIS WITH OBSERVATIONS ON THE pH FACTOR

B y.
vv
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C asim ir T. Ich n io w sk i

T h e sis subm itted, to th e F a c u lty of th e G raduate School

o f the U n iv e r s ity o f M aryland in p a r t i a l
f u l f i l l m e n t o f th e re q u ire m e n ts f o r
th e d eg ree o f D octor o f P h ilo so p h y

19 36
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 Acknowledgement

The a u th o r w ish es to ta k e t h i s o p p o rtu n ity to e x p re ss h i s

s in c e r e th a n k s and g r a t i t u d e to Dr, M arvin R* Thompson f o r h is

in v a lu a b le a d v ic e , c r i t i c i s m and g u id an ce in th e b io a s s a y s tu d ie s

and a n a ly s is and i n t e r p r e t a t i o n o f th e p h y s io lo g ic d a ta .

L ik e w ise , th e a u th o r i s v e ry th a n k fu l and d e ep ly in d e b te d to

D r, E. G. Vanden Bosche who so k in d ly a d v is e d and o f f e r e d c r i t i ­

cism s and s u g g e s tio n s to th e a u th o r in h is s tu d y o f the h y d ro g e n -

io n f a c t o r o f T in c tu re D i g i t a l i s and in the m a th e m atica l i n t e r ­

p r e t a t i o n and a n a ly s is o f c e r t a i n d a ta .
In tr o d u o tio n

F o r a number o f y e a r s l i t e r a t u r e r e p o r t s have shown t h a t v a rio u s

b io a s s a y w o rk ers have d is a g r e e d a s re g a rd s th e b e s t method f o r th e a s s a y

of D ig ita lis .

A problem c o n c e rn in g th e a s s a y m ethods f o r D i g i t a l i s and e v o lv in g

from th e l a b o r a t o r i e s o f the D epartm ent o f Pharm acology o f th e S chool o f

Pharm acy c e n te re d i t s e l f a b o u t th e c a t o r f r o g m ethods because m ost o f

th e p r e p a r a tio n s o f D i g i t a l i s w hich a r e p u t a t th e d is p o s a l o f th e p r a c ­

t i c i n g p h y s ic ia n a r e s ta n d a rd iz e d by e i t h e r th e c a t o r th e f r o g methods*

A review o f th e l i t e r a t u r e shows th a t th e m ost im p o rta n t m ethods u se d in

th e s ta n d a r d iz a tio n o f D i g i t a l i s a r e b a se d on p ro c e d u re s u s in g th e c a t

o r th e f r o g a s th e t e s t o b je c t .

H a tc h e r and Brody (1 ) u s in g th e c a t m ethod found t h a t th e r e s u l t s

w ould be ZVfo too h ig h i f o n ly th e D i g i t a l i s p r e p a r a tio n w ere u sed f o r th e

p e r f u s io n th ro u g h o u t the a s s a y . I n o r d e r to e lim in a te t h i s e r r o r , th e y

m o d ifie d th e p ro c e d u re in such a way t h a t th e a s s a y was te rm in a te d by i n ­

j e c t i n g an o u ab ain s o lu tio n .

H a s k e ll (£) co n clu d ed from h i s s t u d i e s o f th e D i g i t a l i s a s s a y p r o ­

c e d u re s th a t th e o n e -h o u r f r o g method i s s a t i s f a c t o r y and t h a t o u ab ain

s ta n d a r d iz a tio n n u l l i f i e s v a r i a t i o n s in th e f r o g s .

E c k le r (3) b e lie v e s t h a t th e c a t method i s th e m ost c o m p lic a te d and

d i f f i c u l t te c h n iq u e when i t i s a q u e s tio n o f th e c a t , f r o g and g u in e a

p ig m ethods.
 z

E g g le s to n (4) on th e o th e r h a n d , s t a t e s t h a t o f th e th r e e m ethods -

o a t , f r o g and g u in e a p ig - th e o a t method i s th e b e s t.

R ow ntree an d Maoht (5) c laim ed t h a t o f th e two commonly u s e d p ro ­

c e d u re s , c a t and f r o g te c h n iq u e s , th e c a t m ethod i s th e b e t t e r . These

w o rk ers u s e d o n ly a D i g i t a l i s p r e p a r a tio n f o r th e p e r f u s io n .

C olson (6) b e lie v e s t h a t th e c a t method i s b e s t an d , on th e w hole,

g iv e s r e s u l t s w hich a r e low er th a n th o se o b ta in e d by th e f r o g m ethod.

Rowe (7 ) p r e f e r s th e M.L.D* f r o g m ethod to th e c a t and dog m ethods;

and th e c a t , to the dog. However, he b e lie v e s he d id n o t work w ith

enough c a t s .

O’ B rie n and Snyder (8) a ssa y e d T in c tu r e s and F lu i d e x t r a c t s o f D ig­

i t a l i s by th e H a tch e r c a t m ethod, o n e-h o u r an d th e tw e n ty -fo u r h o u r

M.L.D. f r o g methods and th e g u in e a p ig m ethod. These w o rk ers fo u n d th e

c a t a s s a y s to show th e l e a s t d e t e r i o r a t i o n and th e f r o g a s s a y s th e m o st,

i n p r e p a r a tio n s w hich had ag ed f o r two and a h a l f y e a r s .

H a s k e ll, Dowell and T e rry (9) co n clu d ed from t h e i r s tu d y on th e de­

t e r i o r a t i o n o f T in c tu re D i g i t a l i s t h a t th e f r o g and g u in e a p ig methods

show a r a p id d e t e r i o r a t i o n w h ile th e H a tc h e r c a t method shows no a p p a re n t

lo s s in p o te n cy even o v e r a p e r io d a s lo n g a s f i v e y e a r s .

A r e p o r t o f th e A.D.M.A. (10) s t a t e s t h a t fo u r m ethods s u rv iv e d th e

t e s t o f tim e ; (a ) g u in e a p i g , (b) o n e-h o u r f r o g , (c) M.L.D. f r o g and

(d) c a t m ethods. T his r e p o r t a ls o s t a t e s , t h a t th e g r e a t e s t v a r i a t i o n

 3

was o b se rv e d in the com parison o f th e r e s u l t s o b ta in e d by th e c a t find fro g

te c h n iq u e s . F u rth e rm o re , th e r e p o r t r e v e a ls t h a t some c o ll a b o r a t o r s ob­

t a i n e d r e s u l t s w hich w ere in good agreem ent by th e s e m ethods,

H a s k e ll and C ourtney (11) b e lie v e t h a t th e H a tc h e r o a t m ethod e x ­

c e l s th e o th e r m ethods o f a s s a y f o r D i g i t a l i s ,

H a s k e ll (12) s tu d ie d th e e f f e c t o f age on th e p o te n c y o f D i g i t a l i s

by th e o r a l c a t an d th e f r o g m ethods. H is r e s u l t s , by th e s e two m ethods

checked. The H a tch e r m ethod, on th e o th e r h an d , showed h ig h e r r e s u l t s in

aged t i n c t u r e s .

P i t t e n g e r (13) r e p o r tin g a t a m e e tin g o f th e A m erican Drug M anufact­

u r e r s ’ A s s o c ia tio n , s t a t e d t h a t A.D.M.A. members, members o f th e Committee

on B io lo g ic a l A ssay in g o f th e A. Ph. A. and o th e r e x p e r ts found th e g u in e a

p i g and M.L.D. f r o g p ro c e d u re s to y i e l d r e s u l t s t h a t w ere more a c c u r a te

th a n th o s e o b ta in e d by th e o n e-h o u r f r o g o r th e c a t te c h n iq u e . F u r th e r ­

m ore, he s t a t e d t h a t an o b je c tio n to th e g u in e a p ig method was th e c o s t

o f a n im a ls ,

V anderhoff (14) w o rk in g w ith f r e s h and o ld t i n c t u r e s found t h a t th e

f r e s h t i n c t u r e s w ere a b so rb e d more r a p id ly th a n th e o ld o n e s. These t e s t s

w ere c a r r i e d o u t on h e a lth y m e d ic a l s tu d e n ts , c a t s and dogs. The i n t r a ­

venous c a t method showed no d e t e r i o r a t i o n w h ile th e f r o g lymph s a c , o r a l

c a t an d th e human b e in g t e s t s showed lo s s in a b s o r b a b i l i t y - th u s a lo s s

in p o te n c y .
 4

W ible (15) a s s a y e d n in e t i n c t u r e s by th e f r o g and c a t methods* He

fo u n d d is c r e p a n c ie s betw een th e s e two m ethods b u t th e s e v a r i a t i o n s w ere

n o t c o n s is te n t*

T revan, Boock, B um an d G-addum (16) who c a r r i e d o u t a co m p arativ e

stu d y o f b io a s s a y m ethods on two sam ples o f D i g i t a l i s powder r e p o r te d

t h a t th e r e i s a n agreem ent by f r o g , r a b b i t a u r i c l e , g u in e a p ig an d c a t

m ethods p ro v id e d th e sample i s s tr o n g . However, i f th e sam ple be weak,

th e n th e r e i s a s e r io u s d isag ree m e n t among th e r e s u l t s a lth o u g h a l l

m ethods show th e sam ple to be w eak.

Howe (17) s t a t e s t h a t t h e fr o g (.U .S .P .X and M .L.D.) m ethods and

th e c a t m ethod a r e in a p p lic a b le w h ile th e g u in e a p ig m ethod c o u ld be

u s e d b u t th e a n im a ls a r e too e x p e n s iv e .

Swanson and H argreaves (18) u s in g f r o g , c a t an d g u in e a p ig p r o ­

c ed u res fo u n d v a r i a t i o n s w hich w ere not c o n s i s t e n t .

Wokes (19) g iv e s r e s u l t s w h ich show t h a t w ith an in c re a s e in th e

age o f th e p r e p a r a tio n s th e f r o g a s s a y s show a much low er p o te n cy when

compared w ith th e c a t a s s a y r e s u l t s . L ik e w is e , he p o in ts o u t t h a t th e

f r o g p o te n cy d im in is h e s r a p i d l y d u r in g th e f i r s t few months o f s to r a g e

u n t i l th e p o te n c y re a c h e s a l e v e l o f o n e - h a lf to tw o - th ir d s o f i t s o r i ­

g in a l m lu e a t w hich p o in t th e p o te n c y becomes c o n s ta n t f o r s e v e r a l

y ears. The c a t p o te n c y , on th e o th e r h an d , d e c re a s e s much more slo w ly .

In the f r o g m ethods, th e p ro c e d u re a tte m p ts to a c c o u n t f o r th e

 5

a b s o r p tio n w h ile th e o a t m ethod does n o t s in c e th e s o lu ti o n i s i n j e c t e d

intravenously and thus one does away w ith th e a b s o r p tio n f a c t o r .

The 1923 E dinburgh C onference 120) o f th e League o f N a tio n s H e a lth

Committee h e ld th e o p in io n t h a t th e a c t i v i t y o f D i g i t a l i s and i t s a l l i e s

c o u ld be d e te rm in e d w ith re a so n a b le a c c u ra c y e i t h e r by th e l e t h a l dose

m ethod on f r o g s o r l e t h a l dose m ethod on th e c a t w ith slow in tra v e n o u s

in f u s io n and f u r t h e r , th e C onferen ce recommended th e i n v e s t i g a t i o n o f

th e u se of a sam ple o f D i g i t a l i s le a v e s a s a s ta n d a rd f o r D i g i t a l i s .

The s u c c e e d in g C onference h e ld a t Geneva in 1925 (21) recommended

th e fo u r -h o u r f r o g and th e M.L.D, fro g m ethods a s w e ll &s th e Magnus

m o d if ic a tio n o f th e H a tch e r c a t method as s t i f f i c i e n t l y a c c u r a te p ro ­

c e d u re s . F u r th e r th e co n fe ren c e recommended t h a t D i g i t a l i s and i t s

p r e p a r a tio n s be a ss a y e d in term s o f a s ta n d a rd D i g i t a l i s powder*

The r e s u l t s o f a n i n t e r n a t i o n a l stu d y o f th r e e D i g i t a l i s pow ders

a r e r e p o r te d in th e memoranda (22) p re p a re d f o r th e Perm anent Commission

on S ta n d a r d iz a tio n o f B io lo g ic a l P ro d u c ts by K n a ffl-L e n z . The re io o rt

s t a t e s t h a t co n co rd an t r e s u l t s c o u ld be o b ta in e d in d i f f e r e n t c o u n tr ie s

w ith b o th th e c a t te c h n iq u e and t h e f r o g method o f a s s a y . The r e l a t i v e

v a lu e s o b ta in e d how ever w ere n o t a b s o lu t e ly i d e n t i c a l .

F i n a l l y th e F r a n k f u r t C onference (22) h e ld in 1928 d e c id e d to ad o p t

a D i g i t a l i s ' powder a s a r e f e r e n c e s ta n d a rd . T his powder became the

” I n t e r n a t i o n a l D i g i t a l i s Pow der.” The m ethods o f a s s a y a c c e p te d a s

r e l i a b l e and d ependable w ere: th e f r o g method a s recommended a t th e

 6

Geneva C onference o r t h i s f r o g method in i t s o th e r m o d if ic a tio n s and

th e m ethods o f slow in tra v e n o u s in f u s io n in to a mammal: H a tc h e r-

Magnus c a t , K n a ffl-L e n z g u in e a p ig and T iffe n e a u dog te c h n iq u e s .

The 1932 e d i t i o n o f th e B r i t i s h Pharm acopoeia r e q u ir e s t h a t D ig i­

t a l i s and i t s p r e p a r a tio n s he s ta n d a r d iz e d in terras o f th e I n t e r n a t i o n ­

a l S ta n d a rd D i g i t a l i s Powder and t h a t i n th e a c t u a l a s s a y com parison o f

an unknown i s made a g a i n s t th e o f f i c i a l s ta n d a rd p r e p a r a t i o n o f pow dered

D ig ita lis . The f r o g m ethod in v o lv in g th e u se o f a m o r t a l i t y c u rv e , th e

p ro c e d u re s in v o lv in g th e u se o f th e g u in e a p ig o r th e c a t slo w ly p e r ­

fu s e d w ith an in f u s io n o f th e powder o r a s a l i n e d i l u t i o n o f th e t i n c ­

tu r e a r e l i s t e d hy th e B .P. a s te c h n iq u e s t h a t a r e s a t i s f a c t o r y .

The H e a rt Committee (23) o f N.Y. T u b e rc u lo s is and H e a lth A ss o c ia ­

t i o n recommends th a t th e H atcher-B ro d y p ro c e d u re hy u sed in s ta n d a r d iz ­

in g D i g i t a l i s .

C, W. Edmunds (24) in r e p ly in g to t h i s recom m endation o f th e H e art

C om m ittee, s t a t e d t h a t th e R e v isio n Com m ittees o f th e U .S .P . v o te d un ­

an im ously to keep th e f r o g method in 1910, 1920 and 1930. F u rth e rm o re ,

he answ ers t h a t p h a rm a c is ts c la im i t w ould he p r a c t i c a l l y im p o ssib le to

c a r r y o u t c a t t e s t s s in c e th e f ir m s c o u ld n o t o b ta in enough c a ts and

th e n he c o n clu d e s t h a t n o t a s i n g l e m a n u fa c tu re r v o te d to a d o p t th e o a t

p ro c e d u re .
 7

The U .S .P . 1 0 th r e v i s i o n makes m andatory a b io - a s s a y f o r D i g i t a l i s ,

S q u i l l and S tro p h a n th u s an d t h e i r p r e p a r a t io n s . The m ethod o f a s s a y i s

th e o n e-h o u r f r o g p ro c e d u re w ith o u a b ain a s th e s ta n d a rd f o r com p ariso n .

The e le v e n th r e v i s i o n o f th e U .S .P . w hich becomes o f f i c i a l June 1 ,

1936 h a s a s th e o f f i c i a l m ethod f o r the a s s a y o f D i g i t a l i s an d i t s p r e p ­

a r a t i o n s th e o n e-h o u r f r o g te c h n iq u e w ith a s ta n d a rd r e f e r e n c e D i g i t a l i s

powder f o r com parison. F u rth e rm o re , th e U .S .P .X I p o te n c ie s a r e e x ­

p r e s s e d in u n i t s d i r e c t l y r e f e r a b l e to I n t e r n a t i o n a l S ta n d a rd D i g i t a l i s

Powder p o te n c y .

A c o n s id e r a tio n o f th e above l i s t r e v e a ls t h a t th e o f f i c i a l tre n d

o f th o u g h t, a s f a r a s s ta n d a r d iz a tio n o f D i g i t a l i s i s c o n c e rn e d , fa v o rs

and even makes m andatory th e f r o g o r c a t o r g u in e a p i g p ro c e d u re s .

The problem and s tu d y , h e r e i n a f t e r d e s c r ib e d , w ere u n d e rta k e n and

su rv ey ed from a s ta n d p o in t o f p r a c t i c a l a p p l i c a t i o n o f th e f r o g a n d

c a t m ethods to th e r o u ti n e s ta n d a r d iz a tio n and e v a lu a tio n o f D i g i t a l i s

and i t s p r e p a r a t io n s .

P ro c e d u re , A p p aratu s and Technique

1. P r e p a r a tio n o f th e D ilu tio n s

The f r o g m ethods u se d in t h i s stu d y w erei U .S .P .X and m o d ific a ­

tio n s ( 2 5 ) , U .S .P .X I (2 6 ) , and th e C anadian "O verN ight" te c h n iq u e
 8

d e s c r ib e d by Chapman and M o rre ll (2 7 ). The d i l u t i o n s f o r i n j e c t i o n in to

th e f r o g w ere made a c c o rd in g to th e re q u ire m e n ts o f th e p a r t i c u l a r method

u se d . Whenever a g iv e n p r e p a r a t io n was so weak t h a t d e ^ ,l c o h o l iz a tio n

was n e c e s s a iy , th e n th e fo llo w in g p ro c e d u re f o r rem oving th e a lc o h o l was

c a r r i e d o u ts th e r e q u ir e d amount o f th e t i n c t u r e t h a t was n e c e s s a ry f o r

th e d i l u t i o n was p la c e d in a b e a k e r and a c u r r e n t o f a i r p a ss e d o v e r th e

tin c tu re . T his p ro c e d u re was alw ays c a r r i e d o u t a t room te m p e ra tu re .

The d i l u t i o n s o f th e t i n c t u r e s o r o f th e e x t r a c t s o f D i g i t a l i s u se d

f o r o a t a s s a y s w ere made up w ith 0*9^ NaCl s o lu ti o n and in no in s ta n c e

d id th e d i l u t i o n c o n ta in more th a n \Ofo a lc o h o l by volume.

2. A p p a ra tu s . The equipm ent u se d f o r th e a s s a y s was so c o n s tr u c te d

a s to accommodate 200 f r o g s i n in d iv id u a l com partm ents a t one tim e . A l-

s o , th e a s s a y ta n k was o u t f i t t e d w ith a th e rm o -r e g u la tin g d e v ic e t h a t

m a in ta in e d a s e t te m p e ra tu re r i g i d l y and a c c u r a te ly . A v e ry r i g i d m ain­

te n an c e o f te m p e ra tu re i s n e c e s s a ry s in c e th e r a t e s o f a b s o r p tio n v ary

d i r e c t l y w ith th e te m p e ra tu re ; v id e Baker (2 8 ), S ollm ann, M endenhall and

S tin g e l ( 2 9 ) , R oth (30) an d S m ith and McClosby (3 1 ). The f r o g a p p a r a tu s

u se d in t h i s s tu d y c o n s t i t u t e s a perm anent p a r t o f th e la b o r a to r y e q u ip ­

m ent.

3. P ro c e d u re . The f r o g s r e c e iv e d in any shipm ent w ere p la c e d in a

s to ra g e ta n k an d k e p t th e r e f o r a p e rio d o f a t l e a s t a week b e fo re th e y
 9

w ere u se d i n a n a s s a y , T his r o u ti n e was fo llo w e d i n o r d e r to e lim in a te

th e l e s s r e s i s t a n t ones an d th u s u se th e f r o g s w hich rem ain ed and w ere

more u n ifo rm ly r e s i s t a n t .

On th e day b e fo re th e a s s a y , th e te m p e ra tu re o f th e a s s a y and s t o r ­

age ta n k s was r e g u la te d to 20° 0 , so a s to have th e f r o g s in 20° C. w a te r

f o r a b o u t tw e n ty -fo u r, h o u rs a s i s r e q u ir e d , Then on th e f o llo w in g day

th e a c t u a l a s s a y was c a r r i e d o u t a c c o rd in g to th e p ro c e d u re u se d . A lu e r

tu b e r c u lin 1 c c . s y r in g e , g ra d u a te d in 0 ,0 1 c c . and f i t t e d w ith a b lu n te d

n e e d le p o i n t so a s to p re v e n t p e r f o r a t i o n o f th e s k in was u s e d to make

th e i n j e c t i o n s .

The m o d if ic a tio n s o f th e U .S .P .X p ro c e d u re in v o lv e d o n ly th e tim e

p e r io d ; t h a t i s , in th e tw o-hour and fo u r -h o u r f r o g m ethods th e tim e was

in c re a s e d a s in d ic a te d by th e name o f th e m ethod.

The c a t method t h a t was fo llo w e d was in a l l e s s e n t i a l s t h a t o f

H a tc h e r an d Brody ( 1 ) . The p ro c e d u re d i f f e r e d , how ever, from th e o r i g i ­

n a l method in two r e s p e c t s ; f i r s t , an o u a b ain s o lu tio n was n o t u se d to

k i l l th e c a t - a D i g i t a l i s S o lu tio n a lo n e was u se d th ro u g h o u t th e a s s a y

and seco n d , th e d u r a tio n o f th e a s s a y was l i m it e d to a p e r io d o f from

t h i r t y to f o r t y - f i v e m in u te s . Burn (32) recommends a t h i r t y to f i f t y -

f i v e m in u te p e r io d .

R ecords o f t h i s d ep artm en t show t h a t i t m a tte rs l i t t l e w hat p e rio d

o f tim e i s s e le c te d f o r th e d u ra tio n o f th e a s s a y p ro v id e d t h a t th e p e r io d
 10

o f tim e s e l e c t e d i s m a in ta in e d th ro u g h o u t a l l th e s e r i e s o f a s s a y s

c a r r i e d o u t in any p a r t i c u l a r s tu d y an d f u r t h e r p ro v id e d t h a t a s ta n d ­

a r d o f com parison i s u se d in an i d e n t i c a l m anner, e x p re s s in g th e p o ten o y

in term s o f th e re f e r e n c e s ta n d a r d r a t h e r th a n i n "an im al u n i t s ’*.

B io a ssa v S ta n d a rd s .

The s ta n d a rd s used f o r t h e f r o g a s s a y s w e re: o u a b a in , th e o f f i c i a l

U .S .P .X s ta n d a r d , th e S ta n d a rd D i g i t a l i s Powder o f U .S .P . XI and th e

C anadian S ta n d a rd #428 (85 mgm. e q u iv a le n t t o 100 mgm. I n t e r n a t i o n a l

S tan d ard ) and a second o f f i c i a l sam ple o f C anadian S ta n d a rd (88 mgm.

e q u iv a le n t to 100 mggn. o f I n t e r n a t i o n a l S ta n d a rd ) t h a t was r e le a s e d when

th e su p p ly of S ta n d a rd #428 h a d Been d e p le te d .

The o u a b ain was k e p t in a 1:1000 s to c k s o lu tio n p re p a re d by d i s ­

s o lv in g th e o u a b ain in 70f0 a lc o h o l. Such a s to c k s o lu ti o n was k e p t in

a r e f r i g e r a t o r a t a te m p e ra tu re o f 1-2° C. e x c e p t when m aking th e n e c ­

e s s a r y d i l u t i o n s f o r th e a s s a y . Then the s o lu ti o n was ta k e n from th e

r e f r i g e r a t o r an d b ro u g h t to room te m p e ra tu re b e fo re w ith d ra w in g th e

n e c e s s a ry am ount. These s to c k s o lu tio n s when p re p a re d and k e p t a s d e ­

s c r ib e d above w ere found to be s t a b l e f o r a y e a r o r more a s was ob­

se rv e d by f r e q u e n t and c a r e f u l checks a g a i n s t f r e s h l y p re p a re d o u a b ain

s o lu tio n s .

The s ta n d a rd d i g i t a l i s pow ders, o b ta in e d s e a le d in am puls o f amber

g l a s s , w ere k e p t in a c o n ta in e r in a r e f r i g e r a t o r u n t i l u se d .
 11

The sim p le d e te r m in a tio n o f l e t h a l d o se s o f p r e p a r a tio n s hy th e c a t

method does n o t in d ic a te r e l i a b l y th e p o te n c y o f a g iv e n D i g i t a l i s p ro ­

d u c t. The id e a o f e x p re s s in g r e s u l t s i n term s o f " c a t u n i ts " was ab an ­

doned and i n i t s s te a d a com parison o f unknown D i g i t a l i s p r e p a r a tio n s

was made a g a in s t o u a b ain a n d , in th e more r e c e n t s tu d i e s o f th e r e p o r t ,

a g a i n s t S ta n d a rd D i g i t a l i s Powders*

I n g e n e r a l, th e stu d y in c lu d e s a su rv e y o f s t a b i l i t y d a ta w ith

o b s e r v a tio n s on th e pH f a c t o r , a com parison o f th e o a t m ethod a g a in s t

th e f r o g m ethods o f lo n g and s h o r t p e rio d s o f o b s e r v a tio n and a com­

p a r is o n o f th e more w id e ly u s e d methods o f e x t r a c t i o n o f D i g i t a l i s

Pow ders.

The r e s u l t s o b ta in e d in t h i s i n v e s t i g a t i o n a r e g iv e n in t a b u la r

fo n n , each t a b l e b e in g fo llo w e d by comments a n d d is c u s s io n ,

PART ONE

A. STUDY OP TINCTURE DIGITALIS BY THE ONE, TWO AND POUR HOUR

PROG METHODS AND THE CAT METHOD.

HISTORY OP PREPARATIONS STUDIED

The t i n c t u r e s in t h i s s tu d y w ere o b ta in e d from Dr. J , C. Munch,

Chairman o f th e Committee on Pharm acology and B io assay s o f th e A.Ph.A .

D r. Munch in h i s r e p o r t (33) d e s c r ib e s th e p r e p a r a tio n o f th e s e t i n c t u r e s

a s fo llo w s : "A pp ro x im ately seven pounds o f d r ie d D i g i t a l i s le a v e s w ere

o b ta in e d from th e Pood and Drug A d m in is tra tio n , E l i L i l l y Company,

H.K. M u lfo rd Company, Norwich Pharm acal Company, P a rk e , D avis Company

 12

an d S harp and Dohme. These ground cru d e D i g i t a l i s le a v e s w ere m ixed to

o b ta in a t o t a l o f 18 k i l o s o f cru d e d ru g . T h is was d e f a t t e d w ith p e t ­

roleum e t h e r on F e b ru a ry 3 -4 -5 , 1929 th e n exposed on t r a y s and d r ie d .

A t o t a l o f 1 4 .5 k i l o s o f d e f a t t e d m a t e r ia l was o b ta in e d . T h is was co n ­

v e r te d in to T in c tu re D i g i t a l i s s t r i c t l y fo llo w in g th e m ethod o u tlin e d in

U .S .P .X , e x c e p t t h a t a t o t a l o f 145 l i t e r s was o b ta in e d and no a d j u s t ­

m ents f o r p o te n c y w ere made. T his m a t e r i a l was l a b e l l e d " T in c tu re A".

"To a m easured volume o f 7000 c c . o f T in c tu r e A s u f f i c i e n t 70$ a lc o ­

h o l was added on F e b ru a ry 10, 1929 to make a t o t a l volume o f 10 l i t e r s .

T h is m a te r ia l w hich is 70$ o f T in c tu re A was l a b e l l e d " T in c tu r e B",

T in c tu r e s A and B w ere packaged in one ounce and f o u r ounce f l i n t , amber

and b lu e g l a s s b o t t l e s u s in g th e com m ercial p ro c e d u re . A l l b o t t l e s w ere

th o ro u g h ly w ashed and d r ie d b e fo re u s e ,"

S tu d ie s and o b s e r v a tio n s made on th e s e sam ples d a te from F e b ru a ry ,

1931 so t h a t a t th e b e g in n in g o f th e s e s tu d ie s th e s e sam ples had been

aged f o r two y e a r s .

These sam ples o f th e two t i n c t u r e s w ere l a b e l l e d w ith th e fo llo w in g

P .T . num bers:

Blue A P .T . 23

F lin t A P .T . 24

Amber A P .T . 25

Blue B P .T . 26

F lin t B P .T . 27
Amber B P .T . 28
 13

These b o t t l e s w ere k e p t on a la b o r a to r y t a b l e exposed to d i f f u s e d

l i g h t and to th e c o n d itio n s o f th e room.

The r e s u l t s on th e s e sam ples a r e g iv e n in T able 1,

 14

TABLE 1, C o m p ila tio n o f th e P ro g and Gat A ssay R e s u lts O b ta in ed w ith

P .T . 23, 24, 25, 26, 27 and 28.
.— ^
U .S .P .X O ne-hour P ro g 2 H our-P rog 4 H our-P rog O at Method 1
[P re p -D a te o f O uabain Equiv, D ate o f O uabain D ate o f O uabain D ate of O uabain !
I a r a * A ssay o f 1 oc. MGM A ssay E q u iv , o f A ssay E q u iv .o f A ssay E q u iv . j
tio n 1 c c. MGM lc c . MGM o f 1 cc, i
! MGM :

P . T , 8 /1 4 /3 1 0.0550 2 /2 4 /3 1 0 .0 7 8 /1 5 /3 1 0 .0 5 3 /6 /3 1 0.1057
23 2 /1 0 /3 2 0.0354 8 /1 7 /3 1 0 .0 4 5 4 5 /2 7 /3 1 0.0897
3 /1 5 /3 2 0.0450 2 /9 /3 2 0,0347 3 /1 4 /3 2 0.0363 1 2 /4 /3 1 0 .0 8 8 3
2 /2 7 /3 3 0*0857 3 /1 0 /3 2 0.0438 12/20/32 0.1117

P . T . 2 /2 5 /3 1 0 .0 7 4 /1 7 /3 1 0.0625 4 /1 7 /3 1 0 .0625 6 /1 9 /3 1 0.0889

24 4 /1 8 /3 1 0 .0613 8 /1 7 /3 1 0 .0 5 8 /1 5 /3 1 0 .0 5 8 3 1 2 /9 /3 1 0.0906
8 /1 4 /3 1 0 ,0 5 2 3 2 /9 /3 2 0.0347 3 /1 4 /3 2 0 .0 3 7 5 1 2 /2 3 /3 2 0,0912
2 /1 0 /3 2 0.0344 3 /1 0 /3 2 0 .0 5
3 /1 5 /3 2 0 ,0 4 3
2 /2 7 /3 3 0 .0 8

P .T . 2 /2 5 /3 1 0.0786 4 /2 1 /3 1 0.0635 4 /2 2 /3 1 0.0 4 3 5 5 / 1 /3 1 0.1049

25 4 /1 8 /3 1 0.0625 8 /1 8 /3 1 0 .0 5 9 3 8 /1 5 /3 1 0 .0 5 7 3 12 / 1 0 /3 1 0 .0 9 1 4
8 /1 4 /3 1 0 ,0 5 5 2 /9 /3 2 0*0385 3 /1 4 /3 2 0.0438 12/Q /32 0,0966
2 /1 0 /3 2 0.037 3 /1 0 /3 2 0.0500 5 /1 7 /3 4 0.0648
3 /1 5 /3 2 0.0405 7 /1 2 /3 4 0.0699
2 /2 7 /3 3 0*0857 6 /2 0 /3 5 0.0856
3 /2 9 /3 4 0,0714 4 /1 8 /3 6 0.0891
5 /1 5 /3 4 0.0875
7 /1 2 /3 5 0*0769
3 /2 6 /3 6 0.0769

P .T . 3/19/31 0 .0 5 0 1 0 /24 /3 1 0 .0 3 7 5 1 0 /2 4 /3 1 0.0375 4 /1 0 /3 1 0.0667

26 1 0 /2 3 /3 1 0.033 3 /3 l/3 2 0.0167 4 /2 /3 2 0 .0 2 6 /2 5 /3 1 0.0755
4 / 1 /3 2 0 .0 1 6 1 2 /1 7 /3 1 0.0671
1 2 /2 7 /3 2 0.0691
P .T . 3 /1 9 /3 1 0.0 5 5 3 /2 0 /3 1 0 .045 1 0 /2 4 /3 1 0.0429 7 / l l / 3 1 0.0698
27 1 0 /2 3 /3 1 0 .0 4 1 0 /2 7 /3 1 0.0 3 6 4 4 /2 /3 2 0 .0 1 8 1 2 /1 8 /3 1 0.0746
4 /l/3 2 0.019 3 /3 1 /3 2 0 .0 2 1 2 /2 9 /3 2 0.0722

P .T .4 /1 8 /3 1 0 .0 4 3 /2 0 /3 1 0 .0 5 4 /2 2 /3 1 0 .0 2 5 7 /1 5 /3 1 0.0 6 2 3
28 1 0 /2 3 /3 1 0 .0375 4 /1 8 /3 1 0 .0 5 1 0 /2 4 /3 1 0 .0 4 3 1 2 /2 3 /3 1 0,0535
4 /l/3 2 0 .0 2 1 0 /2 7 /3 1 0 .0 4 4 /2 /3 2 0 .0 2 1 2 /1 6 /3 2 0.0597
3 /2 7 /3 6 0.0556 3 /3 l/3 2 0 .0 2 4 / 2 8 /3 6 0.0587
 15

D is c u s s io n o f T able 1.

In 1931 and 1932 th e p r e p a r a t io n s w ere a s s a y e d by th e U .S .P .X o n e -

hour and th e two and f o u r -h o u r m o d if ic a tio n s o f th e U .S .P .X m ethod and

a ls o by th e c a t m ethod. The c a t method a s a p p lie d in 1931-32 in v o lv e d

th e u se o f a f ix e d a n e s t h e t i c , c h lo re to n e . The c h lo re to n e in 50^ a lc o ­

h o l s o lu tio n was i n j e c t e d i n t r a p e r i t o n e a l l y to induce th e a n e s t h e s ia .

D u rin g in d u c tio n s ta g e th e c a ts w ere a n e s th e ti z e d l i g h t l y w ith e th e r .

The m . l . d . o f o u a b ain d eterm in ed on c a ts a n e s t h e tiz e d , a s j u s t d e s c r ib e d ,

had a v a lu e o f a p p ro x im a te ly 0 .1 mgm. p e r k i l o o f a n im a l. The u se o f

c h lo re to n e o r any f i x e d a n e s t h e t i c , was abandoned a f t e r 1932 and i n i t s

s te a d e t h e r was u s e d a s th e a n e s t h e t i c . The c a t s w ere k e p t l i g h t l y

a n e s th e tiz e d w ith t h i s v o l a t i l e a n e s t h e t i c . T h is m ethod of m a in ta in in g

a r a t h e r r e g u la r d e p th o f a n e s th e s ia was fo u n d to be much more p r a c t i c a l

and c o n v e n ie n t th a n th e p ro c e d u re in v o lv in g th e u se o f a f ix e d a n e s t h e t i c .

G ats a n e s th e tiz e d l i g h t l y w ith e t h e r w ere more s u s c e p tib le to o u a b ain .

A verage m .l.d * s . d eterm in e d o v e r s e v e r a l p e rio d s o f tim e w ere a s fo llo w s ;

0 .0 9 7 , 0.094 and 0.095 mgn. p e r k i l o o f a n im a l, u s in g from f i v e to tw enty

c a ts p e r a s s a y . C ats a n e s th e tiz e d w ith a f ix e d a n e s t h e t i c , c h lo re to n e o r

some b a r b i t u r a t e , w ere l e s s s e n s i t i v e and gave m . l . d . v a lu e s somewhat

d e f i n i t e l y g r e a t e r th a n 0 .1 mgn. p e r k i l o . B auer and Fromherz (34) i n

s tu d y in g th e in flu e n c e o f th e n a r c o ti c on th e m ,l .d . of o u a b ain and

d i g i t a l o i d s found th e m .l.d * o f o u ab ain to be 0 .1 1 4 m g n ., and 0 .0 9 6 mgm,

p e r k i l o o f c a t when th e a n im a ls w ere a n e s th e tiz e d w ith a b a r b i t u r a t e

 16

an d " l i g h t e th e r '1, r e s p e c tiv e ly ,

A c lo s e s c r u tin y o f T a b le 1 r e v e a ls t h a t hy th e c a t m ethod th e r e

was no d e m o n strab le lo s s in p o te n c y in th e t i n c t u r e s . F u rth e rm o re , th e r e

was no d e f i n i t e l y d e m o n strab le d if f e r e n c e in p o te n c y among th e d i f f e r e n t

sam ples o f one s e r i e s , The A t i n c t u r e s how ever w ere d e f i n i t e l y s tro n g e r

th a n th e B sam p les, whose p o te n c y , r e l a t i v e to A on th e b a s is o f th e

d i l u t i o n made, was e s tim a te d q u i t e a c c u r a t e l y .

The f r o g m ethods r e v e a le d , on th e o th e r h an d , a t f i r s t a g ra d u a l

drop in p o te n c y in term s o f o u ab ain b e g in n in g w ith th e 1931 a ss a y s and

c o n tin u in g th ro u g h 1932, A su rv e y o f th e r e s u l t s by th e f r o g m ethods

showed t h a t th e s e p ro c e d u re s gave good ag reem en t betw een th e r e l a t i v e

p o te n c ie s o f th e A and B t i n c t u r e s b u t th e s e p o te n c ie s w ere v e ry much

low er th a n th o s e shown by th e c a t a s s a y s .

F u rth e rm o re , a com parison o f th e f r o g m eth o d s, one a g a i n s t th e

o t h e r , d is c lo s e d th e f a c t t h a t by th e fo u r -h o u r te c h n iq u e a much b e t t e r

and c l e a r e r end p o in t co u ld be o b serv ed and a l s o th e a b s o r p tio n from th e

lymph s a c , on th e w h o le, was good. The a b s o r p tio n was much b e t t e r th a n

co u ld be o b se rv e d in c a r r y in g o u t th e one and two h o u r- fro g m ethods.

The a b s o r p tio n in th e f r o g s u se d in th e s h o r t e r p e rio d s o f a s s a y was i r ­

r e g u la r a-nrt i n c o n s i s t e n t ; sometimes good a n d a t o th e r tim e s p o o r. The

end p o i n t , in f r o g s , o f th e one and tw o-hour te c h n iq u e s w as, on th e whole,

l e s s c l e a r and d e f i n i t e th a n in th e case o f th e fo u r hour p ro c e d u re .

 17

F u r th e r s tu d ie s on th e p o te n cy o f th e s e p r e p a r a tio n s w ere li m it e d

to o b s e r v a tio n s on P .T . 25 because th e r e l a t i v e p o te n c ie s o f th e s e

sam ples w ere d e f i n i t e l y e s ta b lis h e d a n d th e r e was no p u rp o se in r e p e a t ­

in g t e s t s on s i m i l a r p r e p a r a t io n s . Theo n e -h o u r te c h n iq u e o f the

U .S .P .X was fo llo w e d in th e s e l a t t e r a s s a y s s in c e i t was th e o f f i c i a l

p ro c e d u re and o f f i c i a l o p in io n p o in te d to th e r e t e n t i o n o f th e o n e -h o u r

te c h n iq u e in th e U .S .P .X I. I n th e f i n a l s e t o f a s s a y s o b s e r v a tio n s on

P .T . 28 w ere in c lu d e d to see w h e th er th e 100:70 r a t i o o f p o te n cy s t i l l

h e ld .

The e s tim a tio n s o f p o te n cy c a r r i e d o u t b e g in n in g w ith 1933 showed

r a t h e r rem arkable and i n t e r e s t i n g r e s u l t s . The p o te n c y o f P .T . 25 had

r i s e n from a l e v e l o f a p p ro x im a te ly 50$ U .S .P .X p o te n cy to a p o te n cy o f

s l i g h t l y more th a n 100$. A ssays in 1934 showed p o te n c ie s o f 86$ and

105$ o f U .S .P .X re q u ire m e n ts . C at p o te n c ie s f o r th e p e rio d in 1934

w ere a t a l e v e l o f 79$ and 84$ in term s o f o u a b a in ; a r a t h e r good a g re e ­

m ent betw een th e o f f i c i a l method and th e c a t m ethod. In 1935, th e f r o g

m ethod showed a p o te n cy o f 92$, w h ile th e c a t te c h n iq u e gave a r e s u l t

o f 102$; a g a in a good agreem ent. The 1936 a ssa y s d is c lo s e d th e f a c t

t h a t P .T . 25, by th e f r o g te c h n iq u e , was 93$ an d by th e c a t te ch n iq u e

107$, P .T . 28 was 67$ by th e f r o g m ethod and 71$ by th e c a t a s s a y .

T his r a t h e r p e c u l i a r b e h a v io r, t h a t i s , a g ra d u a l f a l l in p o ten cy

and th e n an in c re a s e in p h y s io lo g ic a c t i v i t y was n o t th e f in d i n g o f

t h i s la b o r a to r y a lo n e . Dr. Munch, commenting on th e b io a s s a y f o r D ig i-

 18

t a l i s "before th e Pharm acology S e c tio n a t th e j o i n t c o n v e n tio n o f th e

A m erican M edical and C anadian M ed ical A s s o c ia tio n s h e ld in A t l a n t i c C ity

i n th e s p r in g o f 1935 s t a t e d t h a t th e sam ples s u b m itte d f o r c o ll a b o r a t i v e

s tu d y by th e A, Ph. A* w ere showing a rem ark ab le and p e c u l i a r a c t i v i t y -

th e sam ples w hich in p re v io u s y e a rs had been d e t e r i o r a t i n g w ere now g a in ­

in g p o te n c y .

An e x p la n a tio n o f f e r e d by t h i s la b o r a to r y f o r su ch a b e h a v io r i s th e

f a c t t h a t th e f r o g s u s c e p t i b i l i t y to D i g i t a l i s and o u a b ain i s f a r from

p a r a l l e l from a q u a n t i t a t i v e s ta n d p o in t. I n 1931 and 1932 th e m ,s .d . f o r

o u a b ain ra n g e d from 0,00025 mgm. to 0,00005 mgm. p e r o f f r o g w hereas

i n 1933, 1934, 1935 and 1936 th e v a lu e s w ere 0,0006 mgm,, 0*0007 mgm.,

0,0005 to 0 ,0006 mgm. an d 0,0005 mgn. p e r gm. f r o g , r e s p e c t iv e l y . T hus,

in th e y e a r s 1931-32 th e f r o g s w ere v e ry s e n s itiv e to o u a b ain r e q u ir in g

a p p ro x im a te ly from 2 - 1 3 tim es l e s s o u ab ain p e r g?n, o f f r o g th a n in th e

su b se q u e n t y e a rs 1933-36. The m ,s ,d . o f P .T . 25, t r a c e d in an an alo g o u s

m anner, ra n g e d from 0 ,0 0 4 5 c c. to 0 ,0015 c c . p e r gn. o f a n im al in 1931-

32 to 0.007 c c . , 0,008 c c , , 0,0065 c c . , and 0.0065 c c . p e r gm. o f f r o g

f o r 1933, 1934, 1935 a n d 1936 r e s p e c ti v e ly . From th e above d is c u s s io n

i t w i l l be s e e n , in a g e n e r a l way, t h a t th e f r o g changes i n s e n s i t i v i t y

to o u a b ain were much g r e a t e r th an to D i g i t a l i s . Thus o v e r a p e rio d o f

f i v e y e a r s th e range o f m .s .d . f o r o u ab ain was much g r e a t e r th a n f o r any

g iv e n t i n c t u r e .
 19

T able I I i s in c lu d e d a s a supplem ent to T a b le I and i t shows th e

s ta n d a r d d e v ia t io n from th e a v e ra g e f o r th e c a t a s s a y s shown i n T ab le

I. The C a lc u la tio n s w ere made a c c o rd in g to Burn ( 3 5 ) .

TABLE I I

P re p a ra - M .L .D .of S td .D e v ia ­ O uabain e q u i­ S td . D ev ia­

t i o n No. D ate P re p . t i o n from v a le n t of t i o n from
co. E r. th e Mean P rp p . MGM th e Mean

23 3 /6 /3 1 1 .0 5 ± 0 .0 4 0.1112 ± 0.0162
5 /2 7 /3 1 1 .2 4 ± 0 .1 0 0.1112 ±0.0162
1 2 /4 /3 1 1 .2 0 ±0 .0 6 0.106 ± 0 ,0 1 9 2
12/ 2 0 /3 2 1*02 ± 0 .0 6 0.114 ±0,0168
24 6 / I 9 / 3I 1.2 5 ± 0 .0 9 0.1112 ±0.0162
1 2 /9 /3 1 1 .1 7 ±0.07 0 .106 ± 0.0192
1 2 /2 3 /3 2 1 .2 5 ±0,06 0.1 1 4 ±0.0168
25 5 /1 /3 1 1 ,0 6 ± 0.0 3 0.1112 ±0.0162
I 2/ I O / 3 I 1.1 6 ±0.10 0 .1 0 6 ± 0.0192
1 2 /8 /3 2 1 .1 8 ±0.09 0 .1 1 4 ±0.0168
5 / I 7 /3 4 1 .5 0 iO .16 0.09719 ± 0 .0 2 2 3
7 /1 2 /3 4 1. 39 ±0.10 0.09719 ±0.0223
6 /2 0 /3 5 1 .1 0 ±0.07 0 .0 9 4 3 ±0.0052
4 /1 8 /3 6 1 .1 0 ±0.08 0,0981 ±0.0137
26 4 /1 0 /3 1 1,67 ± 0 ,1 0 0 .1 1 1 2 *0.0162
6 /2 5 /3 1 1.47 ± 0 .0 9 0.1112 ±0.0162
12 /1 7 /3 1 1 .5 8 ± 0 .1 1 0.106 ±0.0192
12/ 2 7 /3 2 1 .6 5 ±0.09 0 .1 1 4 ±0.0168
27 7 /ll/3 1 1.59 ± 0.12 0.1112 ±0,0162
1 2 /1 8 /3 1 1.4 2 ±0. 06 0 ,1 0 6 ±0.0192
1 2 /2 9 /3 2 1.58 ±0.21 0.114 ±0.0168
28 7 /l5 /3 1 1 .7 8 ± 0.1 4 0.1112 ± 0 .0 1 6 2
1 2 /2 3 /3 1 1 .9 8 ±0.08 0.106 ±0.0192
1 2 /1 6 /3 2 1.9 1 ±0.08 0 .1 1 4 ±0.0168
4 /2 8 /3 6 1. 67 ±0.08 0.0981 ±0,0137
 20

PART ftVO

THE HYDROGEN ION CONCENTRATION STUDY OP TINCTURE DIGITALIS

I t was d e c id e d , a lth o u g h n o t o r i g i n a l l y p la n n e d , to u n d e rta k e a

pH s tu d y o f th e T in c tu r e s o f D i g i t a l i s d is c u s s e d in P a r t One. The p u r ­

pose o f su ch an u n d e rta k in g was to o b serv e w h e th er o r n o t th e r e was any

r e l a t i o n s h i p betw een th e p o te n c y and th e pH o f th e s e t i n c t u r e s as th e y

aged.

The r e g u l a r p ro c e d u re s t h a t a r e fo llo w e d in th e d e te rm in a t ion o f

pH v a lu e s o f s o lu tio n s r e q u ir e a p p ro x im a te ly 15 c c . o f th e m a te r ia l f o r

a s in g le e x p e rim e n t. S in ce check r e s u l t s a r e u s u a l l y c a r r i e d o u t, i t

w ould n e c e s s a ry to u se 30 cc# o f th e m a te r ia l a t one tim e. Inasmuch

a s th e w ork on th e s e t i n c t u r e s was to be c a r r i e d o u t o v e r a f a i r l y lo n g

p e r io d o f tim e and would r e q u ir e t h a t th e pH d e te n n in a tio n s be c a r r i e d

o u t e v e ry th r e e o r fo u r m onths, i t was found t h a t 30 cc# w ould be too

la r g e a volume to use a t one tim e . As a consequence an a c c u r a te m icro ­

m ethod had to be found. Such a m ethod was f i r s t s u g g e s te d by B iilm ann (36).

T his m ethod in v o lv e d t h e fo llo w in g system s

* •• •
I ^uinhydrone , # S a t* d . huinhydrone
* #• •
, KC1 0*09 M KOI Unknown . Pt
• ** •
. HOI 0#01 M .* • S o lu tio n
• •• •

I f c a p i l l a r y tu b e s a r e u sed in s te a d o f th e o rd in a ry e le c tr o d e v e s s e l s ,
 Ga/Vanometer

Regu/afin? S t'd Cell

Rheostat

V-02
 Separatory Funnel
acting os reserve***-
for s a t'd RGL sol.

To 2-way sw itch

Electrode vessel
— conlo/mng unknown
solution in tip

Thermometer —
Plug a t ■
absorbent co tto n

A gor plu g s o f d
with KCI

Beaker contain ins 5of% ^cfrng 95

tt?e referen ce haff c e ll
 21

i t i s p o s s ib le to d e te rm in e th e pH o f s o l u t i o n s u s in g o n ly a few d ro p s

o f th e m a t e r i a l i n q u e s tio n . T h e re fo re su ch a m ethod w ould be econom i­

c a l when one ta k e s in to c o n s id e r a tio n th e m a t e r i a l b e in g t e s t e d .

I n th e f i r s t s e r i e s o f d e te r m in a tio n s , a p l a i n s a l t b rid g e w ith o u t

a r e s e r v o i r f o r th e s a t u r a t e d KC1 s o l u t i o n was u s e d (s e e diagram # 2 ).

The en d d ip p in g in to th e s o l u t i o n o f th e r e f e r e n c e h a l f - c e l l was p lu g g ed

i n w ith a b s o r b e n t c o tto n p r e v io u s ly soaked in s a t u r a t e d KOI s o l u t i o n .

The " a g a r s a f e ty - tu b e " was n o t u s e d a t t h a t tim e .

I n th e sec o n d and su b se q u e n t s e r i e s o f d e te r m in a tio n s , th e a r r a n g e ­

m ent (a n im provem ent o v e r t h a t j u s t d e s c rib e d above) o f th e two h a l f - c e l l s ,

a s shown i n diagram # 2 , was u s e d .

M a t e r i a l . E quipm ent an d P ro c e d u re .

1. S ta n d a rd W eston C e ll (E p p ley L abs, )•

2. S tu d e n t P o te n tio m e te r (L eeds and N o rth ru p ).

3. Lamp G alv an o m eter (Leeds an d N o rth ru p )*

4 . Two-way s w itc h .

5. Three d ry c e l l s to s u p p ly c u r r e n t to th e p o te n tio m e te r.

6. Two d ry c e l l s f o r c u r r e n t to g a lv a n o m e te r,

7. A b u f f e r s o l u t i o n 0 .0 1 M.HSC1
0 .0 9 M.KC1
a s th e s o l u t i o n f o r th e r e f e r e n c e h a l f - c e l l ,

8. A gar g e l s a t u r a t e d w ith KC1.

The e le c tr o d e v e s s e l w hich i s to h o ld th e unknown s o l u t i o n i s th e

 22

one recommended "by C u lle n and B iilm an n (37) f o r s m a ll q u a n t i t i e s o f f l u i d .

B rig h t s t r i p s o f p la tin u m w ire #21 gauge (B-S s c a le ) s e a le d in to

o r d in a r y f i n e g la s s tu b in g w ere u s e d a s th e e l e c tr o d e s . These e le c tr o d e s

a r e c o n ta c te d by means o f m ercury p la c e d in to th e s e g l a s s tu b e s . The

e le c tr o d e to be d ip p e d in to th e unknown s o l u t i o n i s f i r s t m o iste n e d w ith

some o f th e unknown m a te r ia l and th e n d ip p e d in to some s o l i d quinhy&rone

so a s to make a few c r y s t a l s a d h e r e . The e l e c t r o d e , th u s p r e p a r e d , i s

p la c e d in to th e c a p i l l a r y e le c tr o d e v e s s e l c o n ta in in g some o f th e unknown

s o l u t i o n w hich h a s been drawn in to th e c a p i l l a r y .

A g r e a t many in c o n v e n ie n c e s r e s u l t e d in u s in g th e s a l t b rid g e f o r th e

f i r s t s e r i e s o f pH d e te r m in a tio n s . F i r s t , th e d i f f u s i o n o f th e KOI s o lu ­

t i o n from th e b r id g e th ro u g h t h e c o tto n p lu g in to th e s o lu tio n o f th e

r e f e r e n c e h a l f - c e l l made i t im p e ra tiv e to change th e b u f f e r s o l u tio n q u ite

f r e q u e n tly . S e c o n d ly , th e re was no p r a c t i c a l means f o r c le a n in g t h a t

p o r ti o n o f th e b rid g e in to w hich some o f th e unknown s o lu tio n had d if f u s e d

from th e c a p i l l a r y . T h ird ly , th e maximum number o f d e te rm in a tio n s t h a t

co u ld be c a r r i e d o u t w ith any d e g re e o f a c c u ra c y was l im it e d to two o r

th re e . So t h a t , a l l in a l l , th e p ro c e d u re u sed in th e f i r s t s e r i e s o f

d e te rm in a tio n s was v ery in c o n v e n ie n t an d consumed to o much tim e . Because

o f th e s e d is a d v a n ta g e s , the a rran g em en t shown i n diagram #2 was devised*

Any s o l u t i o n t h a t may d i f f u s e from th e b rid g e i s cau g h t in th e " a g a r

s a fe ty -tu b e " . The r a t e o f d i f f u s i o n th ro u g h th e a g a r p lu g i s n e g a tiv e

 23

o r p r a c t i c a l l y n e g l i g i b l e when o b s e r v a tio n s w ere made on a d if f u s i o n

t e s t o f from f o u r to f i f t e e n h o u rs . A f t e r each d e te r m in a tio n th e c u rv e d

p o r t i o n o f th e s a l t b r id g e i s e a s i l y f lu s h e d by tu r n in g on th e s t o p ­

cock o f th e s e p a r a to r y fu n n e l*

The s a l t b rid g e shown in diag ram #2 was c o n s tr u c te d o f o rd in a r y

s o f t g l a s s tu b in g (0*5 cm* e x t e r n a l d ia m e te r ) . The b rid g e was made

5*5 cm* i n h e i g h t and 5*4 cm. lo n g . The c u rv e d p o r t i o n m easu red 2 .7 cm.

i n h e ig h t.

A f t e r e a c h d e te r m in a tio n th e e le c tr o d e an d e le c tr o d e v e s s e l w ere

rem oved and c le a n e d a s f o llo w s : th e e le c tr o d e was d ip p e d i n to a

(HgSO^+'CrOg) m ix tu r e , rem oved and w ashed w ith d i s t i l l e d w a te r , a lc o h o l

a n d e t h e r a n d th e n d r ie d i n a c u r r e n t o f a i r . The same p ro c e d u re was

f o llo w e d w ith th e e l e c t r o d e v e s s e l w ith t h i s a d d i t i o n a l s t e p : a fte r

d r y in g th e c a p i l l a r y tu b e , th e c a p i l l a r y was r i n s e d w ith a p o r ti o n o f

th e unknown s o l u t i o n , th e n some o f th e unknown m a t e r i a l was drawn up

i n t o th e c a p i l l a r y ; th e n th e e l e c t r o d e , p re p a re d w ith unknown s o l u t i o n

a n d q u in h y d ro n e , a s d e s c r ib e d p r e v io u s l y , was p la c e d in to th e e le c tr o d e

v e s s e l.

The f o llo w in g was th e m ethod em ployed f o r p r e p a r in g th e a g a r g e l

and -the a g a r s a f e t y - t u b e s : 2 Grms. o f pow dered a g a r ( J . T. Baker* s

U. S . P . X b ran d ) w ere ru b b e d to a t h i n p a s t e in a g l a s s m o rta r w ith

25 - 30 c c . o f th e s a t u r a t e d KOI s o l u t i o n . In th e m eanw hile, 75-70 c c .

 24

o f s a t u r a t e d KC1 s o l u t i o n , in a b e a k e r, w ere b e in g h e a te d to a b o il*

A t t h i s p o i n t th e t h i n p a s te was p o u red in to th e b e a k e r w ith c o n s ta n t

s tirrin g . T h is m ix tu re was th e n h e a te d to a n i n c i p i e n t b o i l ( d i s ­

t i l l e d w a te r i n sm a ll am ounts sh o u ld be added to re p la c e th e w a te r

w hich h a s b e en d riv e n o f f ) an d th e n th e flam e was removed. I n o rd e r

to p re p a re th e ”a g a r s a f e ty - tu b e s ” a sm a ll p o r ti o n o f th e h o t a g a r

m ix tu re was p o u re d in to a s m a ll b e a k e r u n t i l a l a y e r 1 cm* deep was

o b ta in e d . W hile th e a g a r m ix tu re was s t i l l h o t , th e d e s ir e d number

o f tu b e s (th o ro u g h ly c le a n e d and d rie d ) was i n s e r t e d in to the b e a k e r

and a llo w e d to s ta n d u n t i l th e a g a r m ix tu re g e ll e d com pletely*

The o r d in a r y p o te n tio m e tr io s e t- u p ( s e e diagram #1} was u se d in

th e s e d e te r m in a tio n s , th e o n ly d if f e r e n c e b e in g t h a t th e c a p i l l a r y

tu b e was u s e d a s th e c o n ta in e r f o r th e s o lu tio n o f th e h a l f - c e l l con­

ta i n i n g th e unknown m a t e r i a l . The s o lu ti o n o f th e re f e r e n c e h a l f - c e l l

was made by d is s o lv in g a sm a ll clump o f q u in hydrone in th e HC1-KC1

s o lu tio n .

F o r th e c a l c u l a t i o n o f pH v a lu e s th e fo llo w in g fo rm u la (36) was

u se d :

pH - 2 .0 2 9 + f t ________
0.00019847"
in w hich i t i s th e o b s e rv e d p o t e n t i a l and
-y i s th e a b s o lu te te m p e ra tu re ,

if i s p o s i t i v e i f th e pH y 2*029 and n e g a tiv e i f f 2*029.

No c o r r e c t io n was made f o r th e e r r o r cau sed by th e a lc o h o l ic c o n -

 25

t e n t ( i . e . in th e c a se o f th e g a l e n i c a ls ) o f th e p re p a ra tio n * R alph B*

Sm ith (38) b e l i e v e s , s in c e i t i s in c o n v e n ie n t to r e p o r t r e s u l t s i n te n n s

o f e .m .f . (w hich i s in r e a l i t y w hat i s d e te rm in e d j i t i s custom ary to

c a l c u l a t e pH u s in g th e o r d in a r y c o n s ta n ts f o r w a te r. La M otte w o rk ers

s t a t e d ( p r i v a t e com m unication) t h a t in th e c a se o f su ch p r e p a r a tio n s as

g a l e n i c a l s , c a l c u l a t i o n s o f pH w ere made n o t c o r r e c t i n g f o r th e e r r o r

t h a t th e a l c o h o l i c c o n te n t p lay s*

W ith th e a rran g em en t u s e d , e q u ilib r iu m , in th e system , i s re a c h e d

in s ta n ta n e o u s ly and th e p o t e n t i a l i s c o n s ta n t f o r a b o u t th r e e m in u tes

w ith aqueous s o lu tio n s ( b u f f e r s ) and even lo n g e r th a n t h i s p e rio d o f

tim e w ith p r e p a r a t io n s su ch a s T in c tu re D i g i t a l i s . A f te r t h i s , th e r e i s

a d r i f t in p o t e n t i a l due to d i f f u s i o n changes t h a t s t a r t to a r i s e .

B iilm an n (36) g iv e s r e s u l t s w hich show t h a t th e use o f th e c a p i l l a r y v e s ­

s e l a s th e e le c tr o d e v e s s e l com pares v e ry f a v o ra b ly w ith th e u se o f th e

o r d in a r y e le c tr o d e v e s s e l s em ployed. I n o r d e r to ch eck BiiltQann, s view ,

th e s e t- u p u s e d was t e s t e d a g a i n s t a s e r i e s o f b u f f e r s o f known pH v a lu es.

F o r r i n s i n g an d p r e s e r v in g th e e le c tr o d e s , B iilm ann (36) s t a t e s

t h a t i t s u f f i c e s o r d i n a r i l y to wash w ith HC1 and th e n w ith d i s t i l l e d

w a te r , f i n a l l y to p la c e them in d i s t i l l e d w a te r o r d i l u t e HC1 o r in

th e d ry s t a t e p r e s e r v e d a g a i n s t d u s t. Even th e n the p la tin u m e le c tr o d e

(h e s t a t e s ) w i l l undergo changes so t h a t i t becomes n e c e s s a ry to u se a

warm m ix tu re o f % S 0 4 ( c o n c .) -f'GrOs. Then th e e le c tr o d e i s w ashed w ith

d i s t i l l e d w a te r and h e a te d to re d n e ss in a n a lc o h o l fla m e . In h e a tin g

 26

th e e l e c t r o d e , one m ust be c a r e f u l to a v o id f i s s u r i n g th e g la s s tu b e

s in c e m ercury may le a k o u t an d c o v e r p a r t o f th e p la tin u m w ire and th u s

g iv e r i s e to f a l s e p o t e n t i a l s . As s t a t e d ab o v e, th e e le c tr o d e s u sed in

th e s e e x p e rim e n ts w ere c le a n e d in HgSO^-f-CrOrj m ix tu r e ; th e h e a ti n g i n an

a lc o h o l flam e was o m itte d . When th e e le c tr o d e s w ere n o t u se d th e y were

k e p t immersed in c o ld “ c le a n in g s o l u t i o n " .

The e le c tr o d e s when p re p a re d s h o u ld be checked a g a i n s t a s o lu t io n

o f known pH. I f th e m easurem ents check th e pH o f the s o lu tio n th e n one

may p ro c e e d to u se th e e le c t r o d e s . I f th e m easurem ents o b ta in e d show

th e r e s u l t s to be in a c c u r a te , th e n a new s e t o f e le c tr o d e s m ust be p r e ­

p a re d .

Some r e s u l t s u s in g e le c tr o d e v e s s e ls whose c a p i l l a r i e s w ere o n e -

h a l f th e s iz e recommended by O u lle n an d B iilm an n , w ere o b ta in e d in th e

c o u rse o f th e e x p e rim e n ts u se d to ch eck th e p o te n tio m e tr ic s e t- u p w ith

s ta n d a r d b u f f e r s o l u t i o n s .

The a c c u ra c y o f th e s e t n i p , from day to d a y , was c o n t r o l l e d by th e

u se o f a f r e s h l y p re p a re d 0*05 M. p o ta ssiu m a c id p h th a la te s o lu ti o n b e ­

f o r e c a r r y in g o u t th e pH d e te r m in a tio n s o f th e t i n c t u r e s on any g iv e n

day. I n th e c a s e o f th e f i r s t s e r i e s o f v a lu e s o b ta in e d , how ever, an

HOI s o lu tio n (pH 1 .3 ) was u se d to ch eck the sy stem ; t h e r e a f t e r , th e

p h th a la te s o l u t i o n was u sed .
 27

E x p e rim e n ta l

TAB2E A

B u ffe r S o lu tio n s pH R e s u lts U sin g

PH 10 mm, c a p i l l a r y 20 mm. c a p i l l a r y

5 .3 ( p h th a la te ) 5 .5 9 5 .8 2

5*59 5 .8 1

5 .6 6

6*0 ( p h th a la te ) 5 .7 6 6 .0 1

5 .8 0 6 .0 2

5 .8 3

6*2 ( p h th a la te ) 5 .9 4

5 .9 3

5.98

5*8 (p h o sp h a te) 5 .5 8 5 .8 2

5 .6 4 5 .8 1

6 .2 (p h o sp h a te) 5 .9 5

6 .0 4

2 .8 ( p h th a la te ) 2 .6 3 2 .8 2

2.66 2 .8 0

3 .8 ( p h th a la te ) 3.67 3.78

3.70 3.79

5 .8 (LaM otte) 5 .8 0

5.8 2

4 .6 3 (S ta n d a rd a c e t a t e ) 4 .6 2

4 .6 3
 28

TABLE B

P .T . D ate S e r ie s D ate S e r ie s D ate S e r ie s D ate S e r ie s

No. #1
pH
n #3 #4
PH pH pH

23 8 /2 1 /3 1 5 . 39 1 /3 0 /3 2 5 .6 2 4 /2 9 /3 2 5 .6 6 2 /9 /3 3 5 .1 8

24 n 5 .6 6 11 5 .7 5 it
5 .8 4 n 5 .7 5

25 ii
5*69 11 5 .6 3 11 5 .7 1 11 5 .5 5

26 8 /2 2 /3 1 5 .2 2 n 5 .5 0 it 5 .5 4 ti 5 .0 3

27 ii
5 .6 5 ti
5 .5 8 n 5.7 7 11 5 .7 3

28 it 5 .7 4 it
5 .5 9 n 5 .7 1 11 5.5 9

TABLE C

P .T . No. D ate pH U. S. P . X
% P o ten cy

302 2 /1 0 /3 3 5.5 1 70

317 « 5 .4 8 106

303 11
5 .4 5 110

306 ti 5 .0 2 120

284 11 5.0 9 100

294 tt 5 .5 9 175

309 n 5.49 105

110 it 5 .1 2 140
 29

D is c u s s io n o f th e H ydrogen-Ion C o n c e n tra tio n S tudy

The b u f f e r s u s e d w ere p re p a re d a s d i r e c t e d in C la rk * s "The D e te r­

m in a tio n o f Hydrogen Io n s" 2nd. E d i tio n p . 99-109. The b u f f e r s p re p a re d

w ere checked ( o o l o r i m e t r i c a l l y ) a g a in s t a f r e s h l y p re p a re d 5 .8 b u f f e r

o b ta in e d from La M otte C hem ical P ro d u c ts Company. The same p h th a la te

s o l u t i o n (0*2 M*) was u s e d to make a l l o f th e p h t h a l a te b u f f e r s an d th e

same p h o sp h a te s o l u t i o n ( 0 .2 M .) was u se d to p re p a re a l l o f th e p h o s­

p h a te b u f f e r s . The p h t h a l a t e an d p h o sp h ate b u f f e r s o f pH 5*8, pH 6 .0

an d pH 6*2 w ere checked w ith b ro m c re so l p u rp le s ta n d a rd s (La M o tte) and

th e 6*0 a n d 6*2 b u f f e r s o f b o th s e t s w ere checked w ith bromthymol b lu e

s ta n d a r d s (La M o tte) in o rd e r to ch eck th e c o n s is te n c y o f th e b u f f e r s .

I n a l l in s ta n c e s th e r e s u l t s a g re e d . Prom th e above s ta te m e n ts i t may

b e s a i d t h a t v a lu e s a s s ig n e d to th e b u f f e r s w ere q u ite c o r r e c t .

The f i r s t s e r i e s o f d e te r m in a tio n s on the b u f f e r s was c a r r i e d o u t

l/l5 /3 2 - l/l6 /3 2 . E le c tr o d e v e s s e ls w ith th e s h o r t c a p i l l a r y t i p w ere

u se d i n th e f i r s t s e r i e s o f d e te r m in a tio n s . The e le c tr o d e v e s s e ls r e c ­

ommended by C u lle n an d B iilm ann (37) w ere u s e d in th e second s e r i e s o f

r e s u l t s o b ta in e d in 1 /2 9 /3 2 . Prom an in s p e c tio n o f th e r e s u l t s in

T able A, one may c o n clu d e t h a t th e v e s s e l , a s recommended by th e two

above m en tio n ed w o rk e rs m ust be u s e d .

A g r e a t d e a l o f em phasis can n o t be p la c e d on th e f i r s t s e r i e s o f

d e te r m in a tio n s on t i n c t u r e s (P .T . 23 - 28 in c lu s iv e ) s in c e th e s e d e t e r ­

m in a tio n s w ere c a r r i e d o u t w h ile u s in g th e f i r s t type o f s a l t b rid g e .

 30

However, th e r e s u l t s w ere q u ite a c c u r a t e , s in c e i t was p o s s ib le to check

th e pH o f an HD1 s o l u t i o n (pH 1 .3 ) w ith t h a t s e t- u p .

I n th e su b se q u e n t s e r i e s o f r e s u l t s , th e d e te rm in a tio n s w ere a c ­

c u r a te to w ith in th e e x p e rim e n ta l e r r o r o f th e quirihydrone e l e c t r o d e .

R e s u lts o f s e r i e s $3 showed, on th e w h o le, a d e c re a s e i n a c i d i t y when

com pared w ith s e r i e s # 2, w h ile d e te rm in a tio n s o f th e #4 g roup showed an

in c r e a s e i n a c i d i t y when compared w ith s e r i e s #3, The d if f e r e n c e s in

pH, among th e v a r io u s p r e p a r a tio n s w ere so s m a ll w h ile th e f l u c t u a t i o n s

in p o te n c y w ere so g r e a t a n d n o t c o n s i s t e n t w ith th e pH c h an g e s, t h a t i t

was c o n clu d e d t h a t th e r e was no r e l a t i o n s h i p betw een p o te n cy and pH,

t h a t i s , w here th e p o in t u n d e r c o n s id e r a tio n i s th e pH o f a t i n c t u r e p r e ­

p a re d by th e o r d in a r y p ro c e d u re s and w ith o u t th e a d d iti o n o f a c id o r a l ­

k a l i to e i t h e r th e e x t r a c t i n g menstruum o r th e f i n i s h e d p ro d u c t.

The o b s e r v a tio n s r e p o r te d in Table C a r e i n t e r e s t i n g and f u r t h e r the

c o n te n tio n t h a t th e r e i s no r e l a t i o n s h i p betw een pH and p o te n cy . A ll o f

th e p r e p a r a t io n s in t h i s ta b le w ere t i n c t u r e s e x c e p t P ,T # 110 and P .T .

284; th e s e l a t t e r two w ere f l u i d e x t r a c t s o f D i g i t a l i s . P .T . 317 was P.T ,

302 f o r t i f i e d by r e - p e r c o l a t i n g more d ru g w ith P .T . 302. The pH v a lu e s *

o f th e s e p r e p a r a tio n s and t h e i r p o te n c ie s show no r e l a t i o n s h i p .

Haag and J a r e t t (39) and V/okes (40) b e lie v e th e r e i s no r e l a t i o n s h i p

betw een p o te n c y and pH. Wokes made no a tte m p t to r e g u la te th e pH o f h is

tin c tu re s ; an d J a r e t t o b ta in e d r e s u l t s on sam ples o f T in c tu re D ig i­

t a l i s w hich w ere o b ta in e d from p h arm acies th ro u g h o u t V ir g in ia . P o s te r

 31

an d Van Dyke (41) s t a t e t h a t d e t e r i o r a t i o n o f t i n c t u r e s c o u ld n o t be

c o r r e l a t e d w ith an in c r e a s e in pH*

As a r e s u l t o f th e s tu d y d is c u s s e d above i t was d e c id e d t h a t f u r ­

t h e r i n v e s t i g a t i o n o f th e pH f a c t o r o f T in c tu r e o f D i g i t a l i s w ould be

im p r a c tic a l and u n n e c e s s a ry .

The 1934 r e p o r t o f th e A.D.M.A* (42) g iv e s some v e ry i n t e r e s t i n g

in fo r m a tio n p e r t a i n i n g to th e h y d ro g e n -io n f a c t o r . Four c h e m ists r e ­

p o r ti n g pH v a lu e s f o r two sam ples o f T in c tu r e D i g i t a l i s g iv e th e f o llo w ­

in g v a lu e s f o r one t i n c t u r e j (a ) 5 .5 8 5 and 5 .5 , (b ) 5 .6 0 , (c ) 5 .3 and

(d ) 5 .3 , V alues f o r th e second t i n c t u r e a r e : (a) 4 .4 8 , lb ) 4 .3 , (c)

4*5 and (d) u n s a t i s f a c t o r y re a d in g s ra n g in g from 4*609 to 4*915. These

i n v e s t i g a t o r s u s e d th e quinhydro n e e le c tr o d e . One wo rice r who checked

h i s a p p a r a tu s w ith a 0 .0 5 M. p o ta ssiu m a c id p h th a la te s o lu tio n o b ta in e d

good r e s u l t s f o r t h i s b u f f e r . Two o th e r s , o f th e s e w o rk e rs, checked

t h e i r s e t s w ith b u f f e r s ; one m e re ly r e p o r ts t h a t he checked h i s s e t and

th e o th e r g iv e s r e s u l t s of 6 .6 and 6 .6 f o r a b u f f e r whose c o m p o sitio n

was n o t e x p re s s e d . In th e c ase o f th e f i r s t t i n c t u r e th e d if f e r e n c e

betw een th e h ig h and th e low v a lu e s f o r t h a t p r e p a r a tio n , was 0 .3 o f a

pH u n i t w h ile in th e c a se o f th e second t i n c t u r e th e d if f e r e n c e was 0*2

o f a pH u n i t , when o n ly th e s a t i s f a c t o r y r e s u l t s a r e c o n sid e re d . Thus

th e s e in d iv id u a l r e s u l t s prove t h a t c h e m ists d e te rm in in g th e pH o f th e

same t i n c t u r e s a r e n o t a b le to o b ta in th e c o n s is te n c y t h a t i s u s u a lly
 32

a c c o rd e d th e q u in h y d ro n e m ethod f o r aqueous s o lu tio n s . I t may be s a i d ,

th e r e f o r e , t h a t d if f e r e n c e s o f s e v e r a l te n th s o f a pH u n i t , when ob ­

t a i n e d w ith p r e p a r a t io n s su ch a s T in c tu r e D i g i t a l i s , a r e n o t v e ry s i g n i ­

f i c a n t when a tte m p tin g to c o r r e l a t e th e p o te n cy and pH o f a g iv e n p re p ­

a ra tio n .
 33

P a r t Three

D e te r i o r a t i o n D ata on P o u r Sam ples o f T in c tu re D i g i t a l i s .

I n t h i s o b s e r v a tio n f o u r sam ples w ere a ss a y e d by th r e e methods and

t h e i r S e e p in g q u a l i t i e s fo llo w e d . Three sam ples P .T . 843, P .T . 844 and

P.T# 845 w ere sam ples p re p a re d f o r com m ercial d i s t r i b u t i o n ■while th e

T in c tu re P , was n o t. The m ethods o f p r e p a r a t io n o f th e f i r s t th re e

sam ples w ere n o t a v a i l a b l e . T in c tu re P was p re p a re d by c o l l e c t i n g

1000 c c . o f p e r c o la te from 100 gjns. o f C anadian D i g i t a l i s L eaf, The

U. S. P . X te c h n iq u e was u se d in p r e p a r in g t h i s l a t t e r t i n c t u r e .

P .T . 845 was l a b e l l e d f o u r tim e s t i n c t u r e s tr e n g th w h ile th e l a b e l s

o f P .T . 843 and P .T . 844 b o re no s p e c ia l s ta te m e n ts o r e x p la n a tio n s .

The m ethods fo llo w e d in a s s a y in g th e s e sam ples w e re: (1) U .S .P .X ,

(E) C anadian O v e rn ig h t and (3) th e c a t te c h n iq u e s . The C anadian powder

used i n t h i s p a r t i c u l a r s tu d y had th e f o llo w in g r e l a t i o n to th e I n t e r ­

n a t i o n a l S ta n d a rd : 1*0 Gm. I n t e r n a t i o n a l S ta n d a rd was e q u iv a le n t to

0 .8 8 Gm. C anadian S ta n d a rd Powder, The r e s u l t s o f th e o b s e r v a tio n s a r e

re p o rte d in T able 3. T ab le 4 sup p lem en ts th e r e s u l t s in T a b le 3 in t h a t

i t g iv e s th e s ta n d a rd d e v ia t io n o f th e mean f o r th e c a t a s s a y s .
 T able 3. D e te r i o r a ti o n D ata

; 1
Id e n ­ F re s h P r e p a r a tio n s A f t e r 6 Months Affine
t i t y U .S .P .X O.-M. C anadian — Gat U .S .P .X C.-M. C anadian C at
lOf M ethod O v e rn ig h t M ethod M ethod O v e rn ig h t Method
jPrep.
j O uabain C an .S td . O uabain C an.Std* O uabain O uabain C an .S td . O uabain C a n .S td . Ouabain
| E q u iv . Pow d.Equiv, E q u iv . Pow d,Equiv. E q u iv . E q u iv . Pow d.Equiv. E q u iv . E q u iv . E q u iv .
• o f 1 cc. o f 1 c c . o f 1 cc. o f 1 c c . of 1 oc. o f l e e . o f 1 oc. o f 1 c c . of l e e . o f 1 CCS
MGMS. MGMS • MOMS. MGMS. MGMS. MGMS. MGMS. M G-M S. MOMS. MGMS.

Tr.B
843 .1 0 9 100.7 .0481 103.9 .1117 .1 0 0 7 3 .7 .0 4 3 8 8 .5 .0 9 9 0

Tr.A
.100 120.6 .0576 92.6 .0996 .100 8 5 .5 .0498 8 4 .3 .0 9 4 2 |
844

T r.
Cone •
512.7 .5514 .417 351 .2 3 9 4 6 1 .4 .5165 ;
845 a al 44
it 537.6 .256
\
T r. * Not a s ­
1 2 0 .3 .0870 96.7 .1041 .1 ** 6 6 .6 .0454 86 .8 .0971 j
F sayed

* P r e p a r a tio n 4 .5 months o ld when f i r s t assayed*

** 1 3 .5 months o ld .

52
 35

T able 4
F I R S T ASSAYS

P re p ­ M .L .D .of S td . D evi­ O uabain S td . D evia­ M.L.D. o f S td . D evia­

a r a ­ P r e p .e e . a tio n of M.L.D. t i o n o f the Can. S td , tio n o f th e
tio n p e r Kg* th e Mean MGMS.per Mean POwd.MGrMS. Mean
Kg. p e r Kg.

T r. B
P .T .
843 0 .8 4 tO . 063 0.09429 ±0.005 87*67 ±5*57
T r. A
P .T .
844 0.9 5 ± 0 .0 4 0,09429 ±0.005 87.67 ± 5 .5 7
T r.
Cone.
P .T .
845 0.171 + 0.008 0.09429 ±0* 005 87.67 ± 5 . 57
T r. F 0 .9 1 ± 0 .0 4 0.09429 ±0.005 87,67 ±5,57

s s c o N D A S S AYS

P re p ­ M .L.D .of StcL. D evia­- O uabain S td . D evia­ M .L.D .of S td . D evia­

a ra ­ P re p . co. tio n of M.L.D. tio n o f th e C an .S td , t i o n o f th e
tio n p e r Kg. th e Mean m m . per Mean Powd.MGMS. Mean
Kg. p e r Kg.

T r. B
P .T .
843 0.9 9 1 0 .0 8 0.09805 ±0.0137 87,67 ± 5,57

T r. A
P .T .
844 1 .0 4 ± 0.09 0,09805 ±0.0137 87*67 ± 5 ,5 7

Tr. Cone.
P .T .
845 0 .1 9 0 ± 0.001 0.09805 ±0.0137 87,67 ± 5 .5 7

T r. F 1 .0 1 ±0.09 0.09805 ±0.0137 87.67 ±5.57

 36

D is c u s s io n .

The p o te n c y r e l a t i o n s h i p s e x i s t i n g among P .T . 843, P .T . 844 and

P.T* 845 w i l l he c o n s id e re d f i r s t . The c o n c e n tra te was fo u n d to he fo u r

tim e s th e p o te n c y o f A and B hy th e U. S, P . X p ro c e d u re . The c a t r e ­

s u l t s f o r A and B a r e in agreem ent w ith th e one^hour p o te n c ie s h u t the

o a t p o te n c y o f the c o n c e n tr a te was 1*25 tim e s g r e a t e r th a n th e U .S.P .X

f r o g p o te n c y and was 5*5 and 5 tim es th e p o te n cy o f A and B r e s p e c tiv e ly

when th e c a t p o te n c ie s a re r e f e r r e d to ouabain a s th e s ta n d a rd . The

r a t i o o f th e p o te n c ie s hy th e c a t method in term s o f the C anadian S ta n d ­

a rd w ere th e same a s w ith th e c a t o u a b ain v a lu e s . T h e re fo re , in th e

c ase o f th e c o n c e n tr a te , the c a t method re v e a le d a d e f i n i t e l y g r e a t e r

p o ten cy th a n th e o n e -h o u r f r o g p ro c e d u re . The Canadian o v e rn ig h t te c h ­

n iq u e showed t h a t th e c o n c e n tra te was 5 .3 tim es s tr o n g e r th an B and 4 .4

tim es s tr o n g e r th a n A when the r e s u l t s a r e r e f e r r e d to th e C anadian

S ta n d a rd pow der. On th e o th e r h an d , a lth o u g h th e o v e rn ig h t o uabain

v a lu e s in d ic a te d th e p o te n c ie s o f th e sam ples to he in th e same r a t i o a s

t h a t o b ta in e d w ith th e C anadian S ta n d a rd Powder, th e y d id n o t ag ree w ith

th e U. S. P . X v a lu e s . The o v e rn ig h t o u ab ain p o te n c ie s w ere from 50 -

60$ o f th e U. 5 . P . o u a b ain values*

These p r e p a r a t io n s , a f t e r s ta n d in g s i x months on the la b o ra to ry

ta b le and u n d e r o r d in a r y room c o n d itio n s , d id n o t show a lo s s o f p o ten cy

by the U. S. P. X p ro c e d u re . The c a t te ch n iq u e showed a s l i g h t drop in

 37

p o te n c y b u t t h i s l o s s i s i n s i g n i f i c a n t when one c o n s id e rs th e m arg in o f

e r r o r a cc o rd e d the procedure* By th e C anadian te c h n iq u e A and B d e t e r ­

i o r a t e d to 71$ and 72$ o f t h e i r o r i g i n a l r e s p e c tiv e p o te n c ie s * th e con­

c e n t r a t e a ss a y e d 60$ o f i t s o r i g i n a l p o te n c y .

T in c tu r e P c o u ld n o t be compared as f u l l y a s th e j u s t p r e v io u s ly

d is c u s s e d p r e p a r a t io n s s in c e o n e -h o u r r e s u l t s on P were n o t o b ta in e d

when f i r s t a ss a y e d because th e a v a il a b le s to c k o f fro g s had been d e ­

p l e t e d in o b ta in in g o v e rn ig h t r e s u l t s . A com parison o f the o v e rn ig h t

and c a t m eth o d s, how ever, r e v e a ls a g a in t h a t d e t e r i o r a t i o n by th e c a t

method i s i n s i g n i f i c a n t w h ile th e o v e rn ig h t method s e t s th e p o te n cy o f

P a t 59$ o f i t s p re v io u s p o te n c y ,

A r a t h e r s i g n i f i c a n t p o in t in th e above d e t e r i o r a t i o n s tu d y i s th e

f a c t t h a t the dro p in p h y s io lo g ic a c t i v i t y f o r A, B and th e c o n c e n tra te

ranged from 65$ - 72$ by th e C anadian te ch n iq u e and fu rth e rm o re th e con­

c e n t r a t e was d e t e r i o r a t i n g a s r a p id ly as th e o rd in a ry t i n c t u r e .
 38

PART 4

THE RELATIVE MERITS OF PROCESSES OF EXTRACTION OF DIGITALIS

POWDERS BY MACERATION, PERCOLATION, AND SOXHIET EXTRACTION.

A. RESIDUAL ACTIVITY STUDIES

I n th e c o u rse o f an a s s a y o f a D i g i t a l i s p r e p a r a tio n f o r C anadian

d i s t r i b u t i o n , i t was fo u n d t h a t t h i s p re p a r a tio n was e q u iv a le n t to a

g r e a t e r amount o f C anadian S ta n d a rd D i g i t a l i s when th i s com m ercial

sample was compared w ith a tw o-h o u r a b s o lu te a lc o h o l e x t r a c t o f th e Cana­

d ia n S ta n d a rd p re p a re d a c c o rd in g to th e r e g u la r tw o-hour C anadian te c h ­

n iq u e (43) w h ile t h i s same sample h ad a low er p o ten cy in term s o f a two-

h o u r 95tfo a lc o h o l e x t r a c t . T h is o b se rv e d d if f e r e n c e in p o ten cy i n i t i a t e d

an i n v e s t i g a t i o n o f th e C anadian p ro c e d u re o f e x t r a c t i o n o f D i g i t a l i s

pow ders. Thus i t became th e o b je c t o f t h i s stu d y to d eterm in e w h eth er

o r n o t the C anadian te c h n iq u e , i f r i g i d l y fo llo w e d , was f u l l y e f f i c i e n t

in e x t r a c t i n g th e t o t a l a c t i v i t y o f a g iv e n D i g i t a l i s powder. T his i n ­

fo rm a tio n would p ro v e to be v a lu a b le to b io - a s s a y i s t s s in c e m a n u fa ctu rers

who d i s t r i b u t e D i g i t a l i s in Canada s ta n d a rd iz e th e s e p ro d u c ts a c c o rd in g

to th e C anadian p ro c e d u re . F u rth e rm o re , i f th e C anadian tech n iq u e d id

n o t y i e l d a c o m p lete e x t r a c t i o n , th e n the a c tu a l p o ten cy o f g a l e n i c a l s ,

and p o s s ib ly a ls o th e crude d ru g w ould be w eaker in term s o f th e a b s o lu te

p o te n cy o f th e s ta n d a rd .

The a c t i v i t y o f th e v a rio u s e x t r a c t i v e s p re p a re d was fo llo w e d by u sin g

th e r e g u la r C anadian O v e rn ig h t m ethod of a ss a y (2 7 ). The o n ly d e v ia tio n ,

 39

and. t h a t "by n e c e s s i t y , from t h i s te c h n iq u e was th e number o f fr o g s used

in e s tim a tin g th e p o te n c y o f th e m a jo r ity o f th e r e s i d u a l e x t r a c t s .

The number o f f r o g s u s e d p e r dose o f p r e p a r a t io n i s in d i c a te d in Table 5.

T able 5 i s a c o m p ila tio n o f th e ’’r e s i d u a l a c t i v i t y ” d a ta .

The re a s o n f o r u s in g l e s s th a n tw e n ty -fiv e fro g s was t h a t th e

r e s i d u a l e x t r a c t was o b ta in e d from th e r e - e x t r a c t i o n o f amounts o f

sam ples t h a t a r e o r d i n a r i l y u sed in th e C anadian m ethod. In th e se c a se s

th e amount o f th e C anadian S ta n d a rd o r o f a n o th e r sample o f D i g i t a l i s i s

a p p ro x im a te ly o n e - h a lf o f one gram. Thus th e volume c o n ta in in g th e r e s ­

id u a l a c t i v i t y i n d e te c ta b le am ounts was a p p ro x im a te ly 5-10 c c . T here­

f o r e a la r g e number o f fr o g s c o u ld n o t be in je c te d when one c o n s id e rs

t h a t o r i e n t i n g a s s a y s had to be c a r r i e d o u t. The sam ples e x tr a c te d on.

4-15-36 w eighed a p p ro x im a te ly th re e grams o r s i x tim es th e u s u a l w eig h t

o f a sample u se d in o rd in a ry r o u ti n e a s s a y . The p urpose o f u s in g a

l a r g e r q u a n tity w as: (1) to o b ta in a volume o f r e s id u a l e x t r a c t t h a t

w ould p ro v id e a q u a n tity s u f f i c i e n t l y g r e a t enough to tak e c a re o f twen­

t y - f i v e fr o g s and (Z) to reduce an y p o s s ib le e r r o r r e s u l t i n g in lo s s e s

due to t r a n s f e r o f s m a lle r volumes o f e x t r a c t . The same menstruum th a t

was u sed to e x t r a c t th e powder a t f i r s t was a ls o u sed f o r the r e —

e x tr a c ­

t io n p r o c e s s .

The r e s i d u a l a c t i v i t y o f any g iv e n sample was e s tim a te d by com paring

th e a c t i v i t y o f the f i r s t e x t r a c t and th e r e - e x t r a c t e d f r a c t i o n w ith

o u a b ain . I n t h i s way th e t o t a l a c t i v i t y o f any powder c o u ld be computed.

 40

O uabain le n d s i t s e l f a d m ira b ly f o r su ch a com parison s in c e i t i s a d e f i ­

n i t e s t a b l e ch em ical e n t i t y .

I n a n o th e r p h a se o f t h i s s tu d y , th e a c t i v i t y o f e x t r a c t s o b ta in e d

w ith m e n stru a o th e r th a n a b s o lu te a lc o h o l was com pared. These r e s u l t s

a r e r e p o r te d in T able 6 and w i l l be d is c u s s e d a t le n g th l a t e r .

D is c u s s io n

I n t h i s s tu d y , th e o u a b ain e q u iv a le n ts o f the f i r s t e x t r a c t and

a ls o o f th e r e s id u a l e x t r a c t w ere alw ays d eterm in ed . The sum o f th e

ou ab ain e q u iv a le n ts o f th e two t o t a l e x t r a c ts in d ic a t e s th e t o t a l p h y s io ­

lo g ic a c t i v i t y o f an y g iv e n sam ple. The fo llo w in g example i s g iv en to

i l l u s t r a t e how th e r e s i d u a l a c t i v i t y was c a lc u la te d . Exam ple: 0 .0 2 cc.

p e r gm. f r o g o f an aqueous d i l u t i o n o f an e x tr a c t o f powder P .T . 733

cau sed 50^ m o r t a l i t y w hich c o rre sp o n d s to a dose number o f 4*0 ( c f . Chap­

man and M o rre ll (27) ) w h ile 0.00035 mgm. o f o u ab ain caused lOfo m o r ta lit y

c o rre sp o n d in g to a dose number of 4*22. T h e re fo re 0*02 c c . i s e q u iv a ­

le n t to :

x 0.00035 s 0.000332 mgm. o u ab ain ,

4 .2 2

S in ce th e t o t a l volume o f t h i s d i l u t i o n was 20 c c . , th e o u ab ain e q u iv ­

a l e n t o f th e t o t a l sample o f t h i s aqueous d i l u t i o n , w hich r e p r e s e n ts th e

 t o t a l amount o f the sam ple o f powder by th e f i r s t e x t r a c t i o n , becomes:

0.000332 X20 , = 0 , 332 mgm.

0.02
The aqueous d i l u t i o n o f th e r e s id u a l e x t r a c t o r r e - e x tr a c t e d p o rtio n in

a dose o f 0 .0 2 5 c c . p e r gm. o f f r o g cau sed a m o r t a lit y o f 40% c o r r e s ­

ponding to a dose number o f 3*9 w h ile 0 ,00035 mgjn* o f o u a b ain produced

70$ m o r t a l i t y . Thus 0 .0 2 5 c c . o f th e aqueous d i l u t e d r e - e x t r a c t i v e is

e q u iv a le n t to :

— X 0.00035 = 0.000323 mgm. o uabain

4 .2 2
The o u a b a in e q u iv a le n t o f th e t o t a l r e s i d u a l e x tr a c t becomes:

0,000323 X 9 - 0.11628 mgn. o u a b ain , sin c e

0.025
th e t o t a l volume r e p r e s e n tin g th e r e s id u a l e x t r a c t was 9 c c .

The tr u e o r a b s o lu te a c t i v i t y of t h i s p a r t i c u l a r sample o f powdered

D i g i t a l i s becomes th e e q u iv a le n t o f 0 ,3 3 2 0*11628 - 0.44828 mgm, o f

o uab ain and th e % r e s i d u a l a c t i v i t y i s r e p re s e n te d b y :

0*11§|Q . x 100 2 25* 9$

0.44828
The p o te n c y o f the r e s i d u a l e x t r a c t o f th e C anadian powder o f

1-15-36 e x tr a c te d wi th 95$ a lc o h o l i s re p o rte d in Table 5 as l e s s th an

9.9 $ and a ls o t h a t o n ly two fr o g s had been used in t h i s e s tim a tio n o f

p o te n cy . The o r i e n t i n g t e s t s t h a t w ere c a r r ie d out to lo c a te th e f i n a l

dose f o r i n j e c t i o n had to bo re p e a te d s e v e r a l tim es and w ith co m p letely

n e g a tiv e r e s u l t s . A t t h i s p o in t in th e l a s t t o s t (on two fro g s) a l l o f

th e r e s i d u a l e x t r a c t had been consumed and s t i l l th e o r i e n t in g a ssa y

 4J

showed, n e g a tiv e r e s u l t s . The p o te n c y , th e r e f o r e , was c a lc u la te d on th e

has i s o f th e a ssu m p tio n t h a t had th e l a s t dose o f th e aqueous d i l u t i o n

o f th e r e s i d u a l e x t r a c t p ro d u ced d e a th s th e n th e r e s i d u a l a c t i v i t y would

have heen a p p ro x im a te ly 9 .9 $ .

A gain in th e case o f Powder #432 d a te d 7 /1 6 /3 5 th e r e s id u a l p o te n ­

cy i s re c o rd e d a s b e in g g r e a t e r th a n 2 0 .2 $ . In t h i s in s ta n c e , th e o r i ­

e n tin g a s s a y in d ic a te d a c e r t a i n dose p e r gjn. o f f r o g as th e l.D . 50.

T his dose upon i n j e c t i o n produced a m o r t a l i t y o f 100$. The t e s t co u ld

n o t be re p e a te d s in c e a l l o f th e e x t r a c t had been consumed. T his m axi­

mal r e a c t i o n in d ic a te d a r e s i d u a l p o te n c y o f a t l e a s t 20. 2$ o f the t o t ­

a l ; th e r e f o r e th e re c o rd e d p o te n cy f o r t h i s sample was g iv e n a s g r e a te r

th a n 2 0 .3 $ .

A c a re fu l stu d y o f T able 5 re v e a ls t h a t : (1) 10 - 25$ o f th e a c ti v ­

i t y rem ain s in th e powders e x tr a c te d w ith a b s o lu te a lc o h o l f o r twoh o u rs ,

(2) a f iv e - h o u r e x t r a c t i o n w ith a b s o lu te a lc o h o l and a tw o-hour e x tr a c ­

t i o n w ith 95$ a lc o h o l y i e l d e x t r a c t s o f the same p o ten cy and 13) th e

r e s id u a l a c t i v i t y a f t e r a f iv e - h o u r a b s o lu te and tw o-hour 95$ a lc o h o l

e x tr a c ti o n s i s l e s s th an 10$ o f th e t o t a l p o ten cy o f th e powder.

The p e r io d o f tim e t h a t was fo llo w e d in th e r e - e x t r a c t io n p ro c e ss e s

v a r ie d a s i s in d ic a te d in T able 5. The p e rio d o f time s e le c te d a t f i r s t

f o r r e —e x t r a c t i o n was s e t a t f i v e h o u rs s in c e l i t e r a t u r e r e p o r ts in d ic a te

t h a t s i x to e ig h t h o u r 3 g iv e com plete e x tr a c t i o n s . The 1932 E d i tio n of

th e B r i t i s h Pharm acopoeia (44) s p e c if ie s a s i x —

ho u r e x tr a c ti o n w ith ab­
 4,

s o lu te a lc o h o l in a c o n tin u o u s e x t r a c t i o n a p p a ra tu s . P o s te r and Van Dyke

(41) s t a t e e ig h t h o u rs c o n tin u o u s e x t r a c ti o n w ith a b s o lu te a lc o h o l g iv e s

a com plete e x t r a c t i o n , how ever th e y p o in t o u t t h a t fo u r-h o u r e x t r a c t s

w ere n o t s i g n i f i c a n t l y l e s s p o te n t th an th e e ig h t- h o u r e x t r a c t .

L a te r i t was deemed a d v is a b le to o b serv e th e in flu e n c e o f p e rio d s

o f tim e l e s s a n d g r e a t e r th a n th e f iv e - h o u r r e - e x t r a c t i o n p e rio d on th e

r e s id u a l a c t i v i t y o f th e D i g i t a l i s pow ders. The d a ta com piled in Table

5 in d ic a te t h a t r e - e x t r a c t i o n o f a l e a s t f o u r h o u rs i s n e c e s s a ry in

o rd e r to e s tim a te th e a c t i v i t y o f th e s e r e s id u e s . F u rth erm o re , th e r e ­

s u l t s show t h a t th e a c t i v i t y d e te n n in e d f o r th e r e s id u a l e x t r a c t s o f the

powders e x tr a c te d f o r f iv e h o u rs w ith a b s o lu te a lc o h o l and th o se e x ­

t r a c t e d f o r two h o u rs w ith 9Sfo a lc o h o l , i s o f such a m agnitude a s to be

i n s i g n i f i c a n t from a s ta n d p o in t o f b io -a s s q y s ta n d a r d iz a tio n and th u s

t h i s in d ic a te s t h a t f o r p r a c t i c a l purposes o f r o u tin e s ta n d a r d iz a tio n by

the C anadian O v e rn ig h t te c h n iq u e , a f iv e - h o u r a b s o lu te a lc o h o l e x tr a c t

and a tw o-hour 9d$ a lc o h o l e x t r a c t may be c o n sid e re d a s r e p r e s e n tin g the

f u l l a c t i v i t y o f th e powder.
 TABIE 5. Data on R esid u a l E x tr a c tiv e s

Id e n tity O uabain E q u iv , O uabain Equiv, D u ra tio n A b so lu te $ o f A bso- Number o f

of o f T o ta l E x t , , o f T o ta l R esi- of R e s i- O uabain E quiv. l u t e O ua- P ro g s u sed
P r e p a r a tio n a f te r 1 s t, d u a l E x t. d u a l E x t1n . o f the D ig i- b a in Equiv, in th e Re­
E x t1n , MGM, MGM, in h o u rs t a l i s Powd. R em aining si dual te s t
MGM* a f t e r 1 s t.
E x t r a c tio n

1 2 h r.a b s .e x t. 0. 3320 0.11628 0.44828 25*9 10

pow d,P ,T ,733
o f 3-22-35
2 2 h r .a b s .e x t. 0,36613 0.09030 0 .4 5 6 4 3 19.8
Can. Powd. o f
3-22-35
0. 33330 0.09263 0.42593 21.7 10
3 2 h r.a b s .e x t.
Can. Powd. o f
4 -2 6 -3 5 10
4 '2 h r . a b s . e x t . 0.42625 N eg ativ e 2. 5
Can.Powd. o f
5 -23-35 10
0.40000 0.10137 6 ,7 5 0.50137 20.2
5 2 h r .a b s .e x t.
Can.Powd. o f
6 -2 7 -3 5 0.65250 2 0 .3
0.52035 0.13215 8 .5
6 2 h r .a b s .e x t.^
Powd. :t 4=32 of
7-16-35 0.46144 16*2
0.38675 0.07469 8 .5
*? 2 h r .a b s .e x t .
Can.Powa. o f
7-16-35 6 .5 0.31342 14*1
a 2 h r .a b s .e x t . 0.26917 0.04425
Can.Powd. o f
10-15-35 0.53769 7 .6 10
0 .04092 12
9 5 h r .a b s .e x t . 0.49677
Can,Powd. o f
1 -7 -3 6
0.07381 13 0.43281 1 7 .5
10 2 h r . a b s . e x t . 0.35900
Can. Powd. o f
1-15-36 0.47763 9 .9
0 .04725 13
11 2 h r . 95/o a l e . 0.43038
e x t. Can.Powd.
o f 1-15-36 2.33685 11.7 25
12 2 h r . a b s . e x t . 2 .0 6 305 0.27380
Can. Powd. o f
4 -1 5 -3 6 ^ 2.551852 6 .7 25
13 2 h r . 9&;o a l e . 2.381652 0.17020
e x t . Can. Powd.
o f 4 -1 5 -3 6 3.521531 18.1 25
14 2 h r . a b s . e x t . 2.885883 0.6355648
U .S .P .X I Powd.
o f 4 -15-36 3.619272 6.6 25
15 2 h r . 95fo a l e . 3. 380872 0 .23840
e x t. U .S .P .X I
if*
Powd. o f 4-15-36
 As a r e s u l t o f th e s t u d i e s r e p o r te d in T able 5 , in w hich th e optimum

a c c u ra c y o f th e m ethod u sed c o u ld n o t be r e a l i z e d b ecau se a s u f f i c i e n t

number o f f r o g s was n o t u se d in m ost o f the in s ta n c e s , i t was d e c id e d

to compare th e p o te n c ie s o f D i g i t a l i s e x t r a c t s o b ta in e d by ( l | th e

U. S. P. XI p ro c e s s f o r T in c tu r e D i g i t a l i s (slo w p e r c o l a t i o n ) , (2)

m a c e ra tio n a s p r e s c r i b e d f o r th e U, S. P. XI S ta n d a rd D i g i t a l i s w ith

th e s in g le e x c e p tio n t h a t 10 cc* o f menstrumm w ere u se d f o r e a c h gm.

o f powder ta k e n and (3) S o x h le t e x t r a c t i o n w ith 95% and a b s o lu te a l c o ­

h o l f o r the p e r io d s o f tim e i n d ic a te d in T able 6 w h ich c o n ta in s th e

in fo rm a tio n a n d d a ta on th e s e v a r io u s e x t r a c t s . The S o x h le t e x tra c ­

t i o n p ro c e s s was c a r r i e d o u t fo llo w in g v e ry c l o s e l y the C anadian

te c h n iq u e w ith s p e c i a l a t t e n t i o n b e in g g iv e n to th e c a r e f u l c o n tr o l o f

th e r a t e o f b o i l i n g . The p o te n c y d e te r m in a tio n s w ere made by fo llo w ­

in g th e C anadian O v e rn ig h t m ethod and u s in g 25f r o g s f o r th e sam ple

and 25 f o r th e s ta n d a r d in each f i n a l a s s a y .

A f te r a c a r e f u l e x a m in a tio n o f Table 6 , one comes to th e c o n c lu ­

s io n t h a t th e tw o-hour p e r io d o f e x tr a c ti o n w ith a b s o lu te a lc o h o l in a

S o x h le t a p p a ra tu s does n o t p e rm it the r e a l i z a t i o n o f a com plete ex ­

tra c tio n . On the o th e r h a n d , tw o -h o u r e x tr a c ti o n w ith 95% a lc o h o l ,

fiv e —
hour e x t r a c t i o n w ith a b s o lu te a lc o h o l- in a S o x h le t a p p a r a tu s ,

m a c e ra tio n and p e r c o la ti o n y i e l d a p p ro x im a te ly 20% s tr o n g e r e x tr a c ts *

An e x c e p tio n to t h i s was n o te d in th e case of the C anadian S ta n d a rd

Powder e x t r a c t s o b ta in e d by p e r c o l a t i o n and m a c e ra tio n , in which c ase

 46

th e p e r c o l a t e was e q u a l in p o te n c y to th e tw o -h o u r a b s o lu te a lc o h o l

e x t r a c t w h ile th e m a ce ra te was 12jo s tr o n g e r . These r e s u l t s in d i c a te

t h a t i f e x t r a c t i o n in a S o x h le t a p p a ra tu s is to be fo llo w e d in p r e p a r ­

in g an e x t r a c t f o r a s s a y by th e C anadian m ethod, a f iv e - h o u r e x t r a c t i o n

w ith a b s o lu te a lc o h o l o r a tw o-h o u r e x t r a c t i o n w ith 9&jo a lc o h o l w i l l

g iv e a more com plete e x t r a c t i o n th a n th e p r e s e n t C anadian p ro c e d u re .

However, i t m ust be p o in te d o u t t h a t a lth o u g h th e C an ad ian s p e c i f i c a ­

t io n s do n o t p e rm it a com plete e x t r a c t i o n , th e te c h n iq u e does p e rm it

a n a s s a y i s t r i g i d l y a d h e rin g to th e p r e s c r i b e d s te p s o f the m ethod to

o b ta in a c o n s ta n tly u n ifo rm e x t r a c t . By t h i s l a s t s ta te m e n t, i t i s

m eant t h a t the r e l a t i v e p o te n cy o f a g iv e n powder o f D i g i t a l i s com pared

w ith th e C anadian s ta n d a rd w i l l be in d ic a te d a c c u r a t e l y s in c e th e two

powders w i l l be e x t r a c t e d to th e same d e g re e . T h is c o n d itio n w i l l n o t

h o ld , on th e o t h e r h an d , when i t i s a q u e s tio n o f s ta n d a r d iz in g a t i n c ­

t u r e of D i g i t a l i s p re p a re d f o r in s ta n c e , f o r com m ercial d i s t r i b u t i o n ,

s in c e th e e x t r a c t i o n f a c t o r in t h i s c ase i s e lim in a te d . However, i t is

p o s s ib le to o b ta in check a s s a y s on a t i n c t u r e o r f l u i d e x t r a c t in term s

of th e C anadian S ta n d a rd Powder b u t th e p o te n cy found f o r a t i n c t u r e w i l l

be g r e a t e r th a n th e a c t u a l p o te n cy o f t h a t g a l e n i c a l . A t i n c t u r e s ta n d ­

a r d iz e d by th e p r e s e n t C anadian te c h n iq u e p a s s e s in to th e tra d e a c t u a l l y

w eaker th a n i s in d ic a te d by th e a s s a y . The tr u e o r a b s o lu te e q u iv a le n t

i n mgms. o f D i g i t a l i s S ta n d a rd o f su ch a t i n c t u r e may be e x p re ss e d by
 47

th e fo llo w in g fo rm u las

® = A. X ^ wil*°k

T s The a b s o lu te v a lu e in mgms. o f s ta n d a rd

A s The a p p a re n t m lu e in mgms. o f s ta n d a rd a s found

by th e a s s a y

x s f r a c t i o n r e p r e s e n tin g th e r e s id u a l a c t i v i t y
y
 48

TABIE 6. C om parisons o f E x t r a c tio n Methods

Id e n tity D ate E q u iv a le n t o f 100 mgms. f0

of of Can. S td . Powder. E q u iv a le n t
P r e p a r a tio n A ssay 2 Hour A bs. E x t. o f 2 Hour Abs*
M IS . E x t. Can.Powder

Gan. Powd.
2 Hr. A bs. E x t. 1-7 -3 6 100 100

Can. Powd.
5 Hr. A bs. E x t. 1-7-36 84-. 11 118.9

Can. Powd.
2 Hr. 95/5 E x t. 1 -15-36 84 119

Can. Powd.
2 Hr. A bs. E x t. 1 -15-36 100 100

U. S. P . XI
S td , Powd.
2 H r. A bs. E x t. 4 -15-36 7 0 .7 141.4

U. S. P . XI
S td . Powd.
2 H r. 9Sjl E x t. 4-15-36 60 167

Can. Powd.
2 H r. Abs. E x t. 4-15-36 100 100

Can. Powd.
2 H r. 9£^b E x t. 4-15-36 8 3 .1 1 2 0 .3

U. S. P . XI
S td . Powd.
M acerate 4 -1 6 -3 6 55*6 179.9

U. S. P . XI
“ S td . Powd.
P e r c o la te 4 -1 6 -3 6 58 .1 172.1

Can. Powd.
M acerate 4 -1 6 -3 6 8 9 .4 111.9

Can. Powd.
P e r c o la te 4 -1 6 -3 6 101 99
 49

B. STUDY OF THE DETERIORATION OF THE AQUEOUS DILUTIONS OF THE SOXHXET EXTRACTS

The C anadian te c h n iq u e r e q u ir e s t h a t th e e x t r a c t i o n and d i l u t i o n o f

th e e x t r a c t sh o u ld be made w ith in tw e n ty -fo u r h o u rs o f th e b e g in n in g o f

in je c tio n s . I t was d e c id e d to o b ta in d a ta on th e p o te n c y o f th e aqueous

d i l u t i o n s a s th e y a g ed . These aqueous d i l u t i o n s , aged f o r p e rio d s o f

tim e ra n g in g from one weex to seven and o n e -h a lf m onths, were a ssa y e d

a g a in s t f r e s h l y p re p a re d e x t r a c t s o f th e C anadian s ta n d a rd . The a g in g

aqueous d i l u t i o n s o f th e e x t r a c t s w ere Icept in g la s s c o n ta in e r s w e ll

s to p p e re d in a r e f r i g e r a t o r a t a te m p e ra tu re of 4° C. The r e s u l t s o f

t h i s s tu d y a r e g iv e n in two t a b l e s , T ab le 7 and T able 8*

 50

IABIE 7. D e te r io r a tio n D ata

Id e n tity Age o f E q u iv a le n t o f 100 M&M. b

of D ilu te d o f F re s h 2 h r . A bs. A le , E q u iv a le n t of
P r e p a r a tio n E x tra c t E x t, o f Gan. S td . Powd. F re s h 2 h r.
MGM. Abs, Ext*

Gan. Powd*
2 h r . Abs. E x t. F re s h 100 100

Gan* Powd*
5 h r . Abs. E x t. F re s h 84*11 1 1 8 .9

Can. Powd.
2 h r . Abs. E x t. 7 .5 Months 113*63 88

Gan. Powd.
2 h r . Abs. E x t. 5 .5 Months 1 2 9 .6 3 77

* Powd. #432
2 h r . Abs. E x t. 5 .7 5 Months 179*37 4 7 .7

Can. Powd.
2 h r . Abs. E x t. 5 .7 5 Months 138.88 72

Can. Powd.
2 h r . Abs. E x t. 4. 5 Months 140.85 71

* 85*6 mgm, of Powder #432 w ere e q u iv a le n t to 100 mgr., o f C anadian

S ta n d a rd Powder when f r e s h l y p re p a re d .
 51

TABLE 8. D e te r io r a ti o n D ata

Id e n tity Age o f E q u iv a le n t o f 100 MGM.

of
i
D ilu te d o f P re s h E h r . Abs. E q u iv a le n t o f
P r e p a r a tio n E x tra c t A le . E x t. o f Can. S td . P re s h E h r .
Powder Abs. E x t,
M IS .

Can, Powd,
E h r . Aba. E x t. F re s h 100 100

Can, Powd,
E h r . 95$ E x t. P re s h 84 119

Can. Powd,
E h r . Abs. E x t. 1 Week 1 1 1 ,4 8 9 .8

Can. Powd.
5 h r , A bs. E x t. 1 Week 98*43 10E

D is c u s sio n

Prom a su rv e y o f T a b le s 7 and. 8 , i t i s e v id e n t t h a t w ith in a week

th e aqueous d i l u t i o n s o f the D i g i t a l i s e x t r a c t s show a d e c re a se in p o te n ­

cy even when th e s e d i l u t i o n s a r e k e p t a t th e te m p e ra tu re o f th e r e f r i g e r a ­

to r, A ltho ugh a c t u a l b io lo g ic t e s t s do n o t show a v e ry a p p r e c ia b le d e t e r i ­

o r a tio n in th e s e w e ek -o ld aqueous d i l u t i o n s , th e t e s t s i n d ic a te t h a t th e r e

i s evidence o f l o s s in p o te n c y . Prom th e d a ta on hand, i t a ls o a p p e a rs

t h a t th e d e t e r i o r a t i o n r a t e re a c h e s a n e q u ilib riu m l e v e l a t a p p ro x im a te ly

70$ o f th e o r i g i n a l p o te n c y a f t e r a g in g f o u r and a h a l f m onths. T h e re fo re ,

 52

th e re q u ire m e n ts s e t f o r t h "by the C anadian method p e r ta in in g to th e im­

m e d ia te u se o f th e aqueous d i l u t i o n s o f the D i g i t a l i s e x tr a c ts a r e p e r ­

fe c tly ra tio n a l.

0. COMPARISON OF EXTRACTION TECHNIQUES BY THE U. S. P . XI AND CANADIAN

OVERNIGHT METHODS OF ASSAY

I n t h i s p a r t i c u l a r phase o f th e stu d y o f th e r e l a t i v e p o te n c ie s o f

D i g i t a l i s e x t r a c t s o b ta in e d by m a c e ra tio n and p e r c o la tio n (a s d e sc rib e d

in p a r t A) and S o x h le t e x t r a c t i o n , i t was d e cid e d to pay s p e c ia l a t t e n ­

tio n to the p h y s io lo g ic a c t i v i t y o f th e se e x t r a c t s in term s o f U. S. P . X

and U. S. P. XI re q u ire m e n ts . The s ta n d a rd s f o r com parison in th is

s e r i e s o f o b s e r v a tio n s w ere o u a b ain and the m acerate of the U. S. P. XI

S ta n d a rd Powder (1 c c . o f m a cerate b e in g e q u iv a le n t to 100 mgm. o f t h i s

powder)* T a b le 9 g iv e s the r e s u l t s o f t h i s in v e s tig a ti o n . P otency

e s tim a tio n s by th e C anadian O v e rn ig h t Method a r e a ls o in c lu d e d in t h i s

ta b le f o r th e sak e o f com paring th e o n e-h o u r method a g a in s t the o v er­

n ig h t te c h n iq u e . R e s u lts by th e C anadian te ch n iq u e a r e lik e w is e r e f e r ­

a b le d i r e c t l y to the m a ce ra te o f th e U* S. P. XI D i g i t a l i s S ta n d a rd ,
 53

TABIE 9 . C o m p a riso n o f E x t r a c t i o n M e th o d s

U .S .P .X U .S .P .X I C. -M. C a n a d ia n Me th o d C a t M eth o d
P re p a r a tio n M eth o d O u a b a in
M e th o d U .S .P .X I O u a b a in
O u a b a in U .S .P .X I S t d . Powd. E q u iv a le n t E q u iv a le n t
E q u iv .o f S t d . Powd. E q u iv . o f of 1 cc. o f 1 cc.
1 o c. MGM,, E q u iv .o f 1 c c . MGM. MGM. MGM.
1 oc.MGM

M a c e r a te o f
U .S .P .X I Powd. 0 .1 6 7 100 100 0 ,1 2 3 2

P e rc o la te o f
U .S .P .X I Pow d. 0 .1 6 7 100 95 0 .1 1 6 9

M a c e r a te o f
C an. S t d . Powd. 0 .1 4 2 8 8 5 .7 62 0 .0 7 6 1

P e rc o la te o f
C an. S t d . Powd. 0 .1 4 2 8 8 5 .7 55 0 .0 6 7 3

2 h r . 95$ A lc .E x t.
U .S .P .X I Powd. 0 .1 6 7 100 92 0 .1 1 3 3

2 h r . A b s. A le .
E x t . U .S .P .X I
Powd. 0 .1 2 5 75 78 0 .0 9 6 1

2 h r . 95$ A l c .E x t .
C an. S t d . Pow d. 0 .1 2 5 75 67 0 .0 8 1 9

2 h r . A h s. A le .
E x t. C an. S t d .
Powd. 0 .1 0 0 -0 .1 2 5 6 0 -7 5 55 0 .0 6 8 2

0 .1 0 0 60 37 0 .0 4 3 0 0 .0 9 9
P .I . 843 T r . B

0 .1 0 0 60 43 0 .0 4 9 8 0 .0 9 4 2
P .T . 844 T r. A

P .T , 845 T r .
0 .3 3 3 -0 .5 0 0 200 -300 195 0 .2 3 8 5 0 .5 1 6 5
C one.

4 2 .9 2 7 .5 0 .0 3 2 1 0 .0 8 9 1
P .T . 25 0 .0 7 1 4

3 3 .3 19 0 .0 2 2 1 0 .0 5 8 7
P .T . 28 0 .0 5 5 5

3 3 .3 0 .0 3 8 8 0 .0 9 7 1
T r. P 0 .1 0 0 60

M a c e r a te o f
Pow d. #432 0 .1 3 7 5 75

P e rc o la te o f
Powd. #432 0 .1 3 7 5 75

M a c e r a te o f 0 .0 9 1 7 -
Powd. A ■>.110 5 0 -6 0

P e rc o la te o f
Powd. A 0 .1 0 0 5 4 .5
M a c e r a te o f
P o w d .P .T . 417 0 .1 0 5 3 6 3 .2

P e rc o la te of
Powd. P .T . 417 0 .1 2 6 7 6 6 .7

M a c e r a te o f
Powd. P .T . 632 0 .1 2 5 75

P e rc o la te o f
Powd. P .T . 632 0 .1 2 5 75

M a c e r a te o f 0 .1 5 0 -
Powd. P .T . 747 0 .1 6 7 7 5 -1 0 0

P e rc o la te o f
Powd. P .T . 747 0 .1 1 8 7 0 .6

M a c e r a te o f
Powd. M inn. P o l . 0 .1 6 7 100

P e rc o la te of
Powd. M in n . P o l . 0 .1 5 4 9 2 .3
 54

D is c u s s io n

O b se rv a tio n of* th e s e t e s t s r e v e a ls t h a t the m a c e ra tio n te c h n iq u e

Of the U* S . P. XI ( 2 6 ) , tw o-ho u r e x t r a c t i o n in a S o x h le t a p p a r a tu s

w ith 9dfo a lc o h o l and slow p e r c o l a t i o n y i e l d e x t r a c t s , in g e n e r a l, o f

th e same p o te n cy . However, i t m ust be p o in te d o u t t h a t i t a p p e a rs

t h a t th e m a c e ra te i s a t r i f l e s tr o n g e r th a n th e p e r c o l a t e when th e

p o te n c ie s o f th e se two ty p e s o f e x t r a c t s a r e c o n s id e re d in th e l i g h t

o f b o th th e o n e -h o u r and C anadian o v e rn ig h t te c h n iq u e s .

I n s e v e r a l in s ta n c e s th e o n e -h o u r te c h n iq u e d id n o t y i e l d c l e a r

c u t r e s u l t s s in c e a ra n g e o f p o te n c ie s , r a t h e r th a n any one s e t p o t ­

en cy , was the b e s t t h a t c o u ld be o b ta in e d . The C an ad ian te c h n iq u e

e lim in a te s th e p o s s i b i l i t y o f any p e r s o n a l i n t e r p r e t a t i o n , a f a c t o r

w hich does a r i s e and w i l l p e r s i s t a s lo n g as th e s o - c a l l e d ’♦minimum

s y s t o l i c s t a n d s t i l l ’* i s the end p o in t in the o n e -h o u r te c h n iq u e . In

th e case of c o m p a ra tiv e ly s tr o n g and f r e s h p r e p a r a t i o n s , th e end

p o in ts as a r u l e a r e q u ite d e f i n i t e and e a s i l y d is c e r n i b l e . On th e

o th e r hand i f weak sam ples o r aged sam ples o f t i n c t u r e s be a s s a y e d ,

th e n th e en d r e a c t i o n i s n o t so c l e a r s in c e sto p p e d h e a r t s (ev en a f t e r

'*m assive” d o ses) a r e r e a d i l y s tim u la te d and w i l l b e a t th en s to p and

b e a t a g a in , F u rth e n u o re w ith ag ed sam ples th e a b s o r p tio n i s e r r a t i c

and t h i s f a c t o r a g a in c o m p lic a te s th e p i c t u r e . I s th e p o o r a b so rp ­

t i o n due to th e in h e r e n t p r o p e r t i e s o f an ag ed sample o r o f a weak

 55

sam ple which, h a s to be c o n c e n tra te d , b e f o r e a s u i t a b l e d i l u t i o n can be

made, o r i s th e p o o r a b s o r p tio n c au se d by th e f a i l u r e o f c i r c u l a t i o n

a f t e r th e f r o g h as a b so rb e d enough to cau se h e a r t sto p p a g e ?

A com parison o f th e r e s u l t s o b ta in e d by th e C anadian te c h n iq u e

and th o s e o b ta in e d by th e U, S. P . XI o n e -h o u r m ethod shows t h a t th e

r e s u l t s by th e fo rm e r m ethod a r e on th e w hole lo w er th a n th e p o te n c ie s

shown by th e U* S. P . te c h n iq u e . C om parison o f th e o u a b a in e q u iv a l e n ts

by th e o v e r n ig h t and o n e -h o u r m ethods r e v e a l s t h a t th e f r o g i s much

more s u s c e p tib le to o u a b ain by th e o v e r n ig h t m ethod and a s a consequence

o f t h i s v a r i a t i o n in s u s c e p t i b i l i t y , a d is c u s s io n o f p o te n c ie s in term s

o f o u ab ain e q u iv a le n ts i s r u le d o u t.

A t t h i s p o in t i t i s w o rth w h ile to in c lu d e a b r i e f summary o f th e

c o n c lu s io n s and recom m endations made by th e Subcom m ittee on D i g i t a l i s

Of the A. D. M. A. a s r e p o r te d in th e p ro c e e d in g s (45) o f t h i s a s s o c ia ­

tio n . Among t h e i r recom m endations a r e two t h a t a r e q u ite p e r t i n e n t ;

H i th e a b an d o n in g o f o u a b ain a s a re f e r e n c e s ta n d a rd f o r D i g i t a l i s an d

(2) th e re p la c e m e n t o f th e Ll.S.D. o n e -h o u r f r o g te c h n iq u e by an M.L.D.

te c h n iq u e o f s i x h o u rs o r o v e r n ig h t, so a s to a llo w s u f f i c i e n t tim e f o r

a com plete a b s o r p tio n and p h y s io lo g ic e f f e c t . T his p a r t i c u l a r A.D.1I.A#

r e p o r t (45) i s b a se d on a c o lla b o r a t iv e stu d y c a r r i e d o u t by o p e r a to r s

in tw elv e l a b o r a t o r i e s . Two sam ples o f T in c tu r e D i g i t a l i s w ere used in

t h i s stu d y .
In 1 9 3 5 , t h i s same Subcom m ittee r e p o r te d (46) the c o lla b o r a t iv e
 56

e f f o r t s o f te n l a b o r a t o r i e s on two sam ples o f T in c tu r e D i g i t a l i s . One

t i n c t u r e , sam ple B, was p re p a re d by d i l u t i n g a p o r ti o n o f th e sam ple

l a b e l l e d nA ,f to tw o—th i r d s th e p o te n c y o f A# The t e s t s w ere c a r r i e d o u t

by the o n e -h o u r M .S.D. and th e o v e rn ig h t M .L.D. f r o g m ethods w ith Cana­

d ia n S ta n d a rd D i g i t a l i s Powder #428 a s a r e f e r e n c e s ta n d a r d . The p o t ­

e n c ie s of th e t i n c t u r e s a ssa y e d in t h i s c o l l a b o r a t i v e stu d y w ere r e p o r te d

in term s of I n t e r n a t i o n a l S ta n d a rd Powder p o te n c y . The r e s u l t s o b ta in e d

in t h i s s tu d y w ere much more u n ifo rm th a n in th e p re v io u s y e a r when o u a­

b a in was u sed a s a re f e r e n c e s ta n d a r d . The a v e ra g e r e s u l t s o f th e te n

la b o r a to r ie s by th e o n e -h o u r m ethod was 94.6$ f o r A an d 76.3$ f o r B and

by th e o v e rn ig h t m ethod 97.6$ f o r A and 69.8$ f o r B. Thus, by th e o n e -

hour p ro c e d u re B was 80.7$ o f A and by th e o v e rn ig h t m ethod B was 70,5$

o f A. The o n e -h o u r r e s u l t s te n d to show f o r B a g r e a t e r p o te n c y th a n i s

t h e o r e t i c a l l y p o s s i b l e , i f th e m ethod o f p r e p a r in g B i s k e p t in m ind. T his

com m ittee in a n a ly z in g the com piled d a ta o f .the te n l a b o r a t o r i e s r e v e a ls

t h a t th e r e w ere from fo u r to s i x d i f f e r e n t c o n c lu s io n s t h a t c o u ld be

m a th e m a tic a lly c a l c u l a t e d an d t h a t i t was im p o ssib le to say w hich o f two

o r th r e e o f th e more l i k e l y i n t e r p r e t a t i o n s w a s a c t u a l l y c o r r e c t . T his

com m ittee c i t e s a s an exam ple t h a t th e d a ta o b ta in e d in t e s t i n g T in c tu re

A by th e o n e -h o u r m ethod gave i n t e r p r e t a t i o n s ra n g in g from 77$ to 100$.

The p o s s i b i l i t y of i n t e r p r e t a t i o n o f r e s u l t s o b ta in e d by th e C anadian

technique i s n e g l i b l e i f n o t n e g a ti v e ; on the o th e r hand such a c o n d itio n

does n o t p r e v a i l i f th e o n e -h o u r te c h n iq u e as o u tlin e d by e i t h e r U .S .P .X
 57

o r U .S .P .X I i s fo llo w e d . The c o ll a b o r a t i v e work c a r r i e d o u t u n d e r

A.D.M.A. a u s p ic e s p o in ts to th e a d o p tio n o f an o v e rn ig h t te c h n iq u e s in c e

such a p ro c e d u re g iv a s c l e a r c u t r e s u l t s t h a t a re d e v o id o f th e d i f f i ­

c u l t y o f i n t e r p r e t a t i o n o f an end p o i n t , r e a c t i o n .

I t i s i n t e r e s t i n g from a c o m p a ra tiv e s ta n d p o in t to s e e how th e r e ­

s u l t s o f t h i s l a b o r a to r y on th e s e two sam ples a r e r e l a t e d to th e r e s u l t s

r e p o r te d by th e D i g i t a l i s Subcom m ittee;

ONE HOUR PROG METHOD

P r e p a r a tio n A.D.M.A. R e s u lt o f th e Pharm acology la b , o f th e

Ave. R e s u lt School o f Pharmacy

T r. A 94.6fb lOVfo

T r. B 76 . 3 f o 86f0

OVERNIGHT FROG METHOD

T r. A 97 .& ?o 10 9fo

Tr. B 6 9 ,8 %

The r e s u l t s of t h i s la b o r a to r y a re in concordance w ith the g e n e ra l

f in d in g s o f th e A.D.M.A. r e p o r t . By the o n e-h o u r method 3 i s QS% of A

and by th e o v e r n ig h t te c h n iq u e B i s 6 8 .8 yb o f A. Thus by the o v e rn ig h t

 58

p ro c e d u re t h i s la b o r a to r y was a b le to e s tim a te a c c u r a t e ly th e t h e o r e t i ­

c a l p o te n c y o f B.

Some d a ta o b ta in e d w ith c a t a s s a y s a r e in c lu d e d in T ab le 9 f o r th e

sake o f a rev iew and li m i t e d com parison o f th e t h r e e w id e ly u sed te c h ­

n iq u e s f o r D i g i t a l i s S ta n d a r d iz a tio n , These c a t a s s a y s w ere p r e v io u s ly

d is c u s s e d in th e d e t e r i o r a t i o n s tu d i e s and th e r e f o r e no comments on t h i s

p a r t i c u l a r group o f a s s a y s w i l l be made,

D. A COMPARISON OP THE POTENCY OP DIGITALIS IN TERMS OP THE U. S. P . X

AND U. S. P. XI REQUIREMENTS

T h is s tu d y was u n d e rta k e n w ith th e view o f d e te rm in in g th e r e l a t i v e

p o te n c ie s when a ssa y ed (1) in term s cf th e U, S. P . X re q u ire m e n ts and

(E) in terras o f U. S, P , XI re q u ire m e n ts . The r e s u l t s o f t h i s i n v e s t i ­

g a tio n a r e r e p o r te d in Table 10,

The re fe re n c e s ta n d a rd f o r com parison w ere o u a b ain (s u p p lie d by th e

Pood and Drug A d m in is tra tio n o f th e U, S. D epartm ent o f A g r ic u ltu r e ) and

th e TJ . 3. P . XI S ta n d a rd D i g i t a l i s Powder (s u p p lie d by th e Board o f

T r u s te e s o f th e U, S. P, C o n v en tio n ). T his U. S. P . R eferen ce D i g i t a l i s

Powder i s 1 ,3 4 tim es a s s tro n g as t h e MU, 3 . P, D i g i t a l i s U n it" w hich in

tu r n i s e q u iv a le n t to 100 mgms, o f I n te r n a tio n a l S ta n d a rd D i g i t a l i s

s in c e 7 4 ,5 mgms* o f th e U, 3. P# XI R eferen c e Powder a r e e q u iv a le n t to

one U. S . P . D i g i t a l i s U n it.
 59

TABLE.10. C o m p a riso n o f P o t e n c i e s o f D i g i t a l i s E x t r a c t s in
Terms o f U. S. P . X an d U. 3 . P . XI R e q u ire m e n ts

P re p a ra tio n d R a t io o f U .S .P .X
p p
U .S .P .X P o te n c y U .S .P .X I P o te n c y P o te n c y to
U .S .P .X I P o te n c y

M a c e ra te
U .S .P .X I Powd. 200 134 1 .4 9

P e rc o la te
U .S .P .X I Powd. 200 134 1 .4 9

M a c e ra te
C a n .S td . Powd. 172 115 1 .5 0

P e rc o la te
C an. S t d . Powd. 172 115 1 .5 0

2 h r . 9dfo E x t .
U .S .P .X I Powd, 200 134 1 .4 9

Z h r . A b s. E x t .
U .S .P . XI Powd. 151 101 1 .5 0

2 h r . 95/b E x t .
C an. Powd. 151 101 1 .5 0

2 h r , A b s. E x t .
1 2 1 -1 5 1 80-101 1 .4 8 (a v e ra g e )
Can, Powd.

121 80 1 .5 1
P .T . 843

121 80 1 .5 1
P .T . 844

4 0 1 -6 0 2 26 8 -4 0 2 1 .5 0 (a v e ra g e )
P .T . 845

86 57 1 .5 1
P .T . 25

67 45 1 .4 9
P .T . 28

121 80 1 .5 1
T r. F

M a c e r a te 1 .6 4
Powd. #432 166 101

P e rc o la te 1 .6 4
Powd. #432 166 101

P e rc o la te
Powd. A 121 73 .
1 66

M a c e r a te
M inn. P o l . 200 134 1 .4 9

P e rc o la te
M inn. P o l . 186 124 1 .5 0

M a c e r a te
P . T . 417 127 85 1 .4 9

P e rc o la te
P .T . 417 152 89 1 .7 1

M a c e r a te
P .T . 632 151 101 1 .5 0

P e rc o la te
P .T . 632 151 101 1 .5 0

M a c e r a te
P .T . 747 1 8 1 -2 0 0 1 0 1 -1 3 4 1 ,6 2 ( a v e r a g e )

P e rc o la te
P .T . 747 142 95 1 .4 9
 60

A s u rv e y o f T able 10 p o in ts to th e f a c t t h a t th e U. S. P . XI

p o te n c y re q u ire m e n ts a re d e f i n i t e l y g r e a t e r th a n th o se o f th e U. S.' P . X.

By a c t u a l a s s a y th e a v e ra g e o f tw e n ty -s ix t e s t s r e v e a le d t h a t th e U. S. P. XI

p o te n c y i s 1 .5 3 2 tim e s a s g r e a t a s th e U. 3. P. X re q u ire m e n ts .

The C anadian S ta n d a rd D i g i t a l i s Powder u se d in t h i s p a r t i c u l a r stu d y

h ad th e fo llo w in g r e l a t i o n s h i p : 88 mgms. o f C anadian S ta n d a rd a re

e q u iv a le n t to 100 mgms. o f I n t e r n a t i o n a l S ta n d a rd . Thus t h i s sam ple o f

C anadian Powder i s 1.136 tim e s a s s tr o n g a s th e I n t e r n a t i o n a l S ta n d a rd ,

The p o te n c y o f t h i s C anadian S ta n d a rd r e f e r r e d to th e I n t e r n a t i o n a l

Powder th ro u g h a com parison w ith th e U. S. P. XI R eferen c e Powder and

a s d e te rm in e d by th e U. S . P . XI p ro c e d u re , was found to be 119$ of

I n t e r n a t i o n a l Powder p o te n c y .

C om parison o f the U. S. P . XI D i g i t a l i s S ta n d a rd Powder was made

on f o u r d i f f e r e n t o c c a s io n s . The fo llo w in g t a b l e summ arizes th e s e t e s t s ;

D ate M .5.D. o f O uabain M.S.D. Of U .S .P . XI S td .

MGMS. p e r Gm. P ro " Powder Msms. p e r Gm. Pros:
3 -26-36 0.00050 0.300

3-31-36 0.00055 0 .3 2 5

4-^-3& 0.00050 0.300

4 -7 -3 6 0.00050 0.300

I n th r e e o f th e t e s t s 100 mgms, o f the D i g i t a l i s S ta n d a rd Powder

w ere e q u iv a l e n t to 0,167 mgms. o f o u ab ain o r 200^> U. S. P. X p o te n cy

b u t o n ly 134$ in term s o f U. S. P. XI re q u ire m e n ts . In th e o th e r t e s t

 61

th e o u a b a in e q u iv a le n t o f 100 mgms. o f t h i s D i g i t a l i s v/as s l i g i t l y

g r e a t e r - 0,1 6 9 mgms.

G e n e ra l D is c u s s io n

As a r e s u l t o f th e e x p e rie n c e s o b ta in e d in c a r r y in g o u t th e f r o g

and c a t t e s t s m en tio n ed in t h i s s tu d y , a c e r t a i n ro u tin e p ro c e d u re f o r th e

b io a s s a y c o n tr o l o f D i g i t a l i s was d ev elo p ed . A t f i r s t , a p re lim in a ry

a s s a y i s c a r r i e d o u t on two o r th re e c a ts g iv in g a p p ro x im a te ly s im il a r

r e s u l t s in o r d e r to l o c a t e th e ap p ro x im ate p o ten cy o f any g iv e n D i g i t a l i s

sam ple. F o r a com plete a s s a y , i . e , a c a t a s s a y , a t l e a s t f i v e c a ts a r e

u se d . The c a t m, l . d , r e s u l t s a r e alw ays c a l c u l a te d and e x p re sse d in

term s o f a s u i t a b l e r e f e r e n c e s ta n d a rd . Then th e a s s a y i s c a r r i e d o u t

on f r o g s . The p u rp o se o f such a p r e lim in a r y c a t a s s a y i s tw o -fo ld :

(1) t h i s p ro c e d u re e n a b le s an a s s a y i s t to s e l e c t d o ses f o r i n j e c t i o n in ­

to f r o g s w ith o u t th e p r e lim in a r y o r i e n t a t i o n t e s t s w ith fr o g s and [Z]

th e c a t r e s u l t s s e rv e to check o r c o n tr o l th e f r o g a s s a y s in c e c a t r e ­

s u l t s o b ta in e d w ith th e te c h n iq u e a s a p p lie d in t h i s la b o r a to r y a r e in

r a t h e r good ag reem en t w ith f r o g r e s u l t s . The c a t te ch n iq u e g iv in g r e ­

s u l t s q u ite com parable to f r o g r e s u l t s i s t h a t method w hich in v o lv e s

a n e s t h e t i z i n g th e c a t l i g h t l y w ith e th e r .

An o u t l i n e o f t h i s c a t te c h n iq u e may be d e s c rib e d a s fo llo w s ; th e

fe m o ra l v e in o f th e c a t i s exposed and c a n n u liz e d . The s tr e n g th o f th e

p e r f u s in g f l u i d i s a d ju s t e d from a p r e lim in a r y a s s a y such, t h a t th e

m .l .d . p e r k i l o i s c o n ta in e d in te n c c , The d i g i t a l i s d i l u t i o n i s

i n j e c t e d in to th e a n im a l a t th e r a t e o f one c c , p e r m in u te u n t i l s tro n g

symptoms o f D i g i t a l i s b ra d y c a rd ia a r e m a n if e s t. T his r a t e o f p e r f u s io n

m a in ta in e d th ro u g h o u t th e b ra d y c a rd ia c s ta g e o f symptoms i s slow ed to a

r a t e o f t h r e e - q u a r t e r s o f a c c . w ith th e f i r s t ap p ea ra n ce o f a ta c h y ­

c a r d ia . As th e ta c h y c a rd ia c symptoms d ev elo p and in c re a s e to a p ro ­

nounced r a p id r a t e , th e r a t e o f flo w i s checked s t i l l more u n t i l the

flo w i s now one c c . e v e ry two to th r e e m in u tes n e a r th e te rm in a tio n o f

th e e x p e rim e n t. W ith th e f i r s t p ro n ounced s ig n s o f c a r d ia c f l u t t e r the

p e r f u s io n i s sto p p e d c o m p le te ly . The h e a r t w i l l th e n f l u t t e r to a s to p

w ith in a s h o r t tim e o f th e f i r s t f i b r i l l a t i o n .

The o v e rn ig h t f r o g te c h n iq u e i s to be p r e f e r r e d to th e o n e-h o u r

f r o g m ethod b e ca u se th e o v e rn ig h t p ro c e d u re a llo w s s u f f i c i e n t tim e f o r

com plete a b s o r p tio n , a f a c t o r o f param ount im portance in o b ta in in g con­

s i s t e n t l y good r e s u l t s , and th u s f u l l p h y s io lo g ic e f f e c t . A t th e same

tim e t h i s m ethod e lim in a te s the p e r s o n a l e q u a tio n in th e i n t e r p r e t a t i o n

o f th e end p o in t e f f e c t s as i s o f te n r e q u ir e d in th e o n e-h o u r te c h n iq u e .

The abandonm ent o f o u a b ain as a re fe re n c e s ta n d a rd f o r D i g i t a l i s

s ta n d a r d i z a t i o n removes an o b je c t io n to th e U, d. P . X p ro c e d u re f o r

D i g i t a l i s s ta n d a r d iz a tio n s in c e D i g i t a l i s and o u a b ain a r e n o t i d e n t i c a l

from a s ta n d p o in t o f a b s o r p tio n and f r o g s u s c e p t i b i l i t y .

The m ost p r a c t i c a l m ethod, a m ethod t h a t sh o u ld g iv e more uniform

 63

r e s u l t s , f o r th e p r e p a r a t io n o f a D i g i t a l i s e x t r a c t o f tlie re fe re n c e

s ta n d a r d i s th e m a c e ra tio n te c h n iq u e a s d e s c rib e d f o r th e R eferen ce

D i g i t a l i s Powder o f t h e U. S. P . XI (2 6 ], The m a c e ra tio n te c h n iq u e

r e q u ir e s v e ry l i t t l e o f th e a s s a y i s t 's tim e . The v a r ia b le s in c id e n t

to a p e r c o l a t i o n p ro c e d u re a re e lim in a te d i f a m a cerate be p re p a re d f o r

u se in th e a s s a y . B e rry and D avis (47) com paring th e r e l a t i v e m e r its

o f m a c e ra tio n and p e r c o l a t i o n f o r th e p r e p a r a tio n o f T in c tu r e o f D ig i­

t a l i s found t h a t m a c e ra tio n was j u s t a s e f f e c t i v e a s p e r c o la ti o n .

 64

Summary and C o n c lu sio n s

1* The c a t m ethod a s a p p lie d to th e a s s a y o f t i n c t u r e o f D i g i t a l i s

showed v e ry l i t t l e l o s s in p o te n cy o v e r a p e r io d o f a p p ro x im a te ly f i v e

y e a r s when the m ethod in v o lv e s e x p r e s s io n o f th e p o te n c y in te r n s o f a

r e f e r e n c e stan d a rd *

2. The o n e-h o u r f r o g m ethod o f th e U. S, P . Z w ith o u a b ain a s th e

s ta n d a r d re v e a le d p e c u l i a r r e s u l t s in a d e t e r i o r a t i o n s tu d y co n d u cted

o v e r a p e r io d o f a p p ro x im a te ly f iv e y e a r s . A c o n s i s t e n t d e c re a se in

p o te n c y o f th e t i n c t u r e s fo llo w e d by an in c re a s e in p o te n cy was o b serv ed .

3. When th e d e t e r i o r a t i o n o f sam ples o f T in c tu re o f D i g i t a l i s was f o l ­

lowed by th e U. S. P. X, C anadian O v e rn ig h t f r o g an d th e c a t m ethods,

th e C an ad ian te c h n iq u e was th e o n ly method to r e v e a l an a p p re c ia b le lo s s

in p o te n c y o f t i n c t u r e s aged f o r a p p ro x im a te ly s i x m onths. The c a t m eth­

od showed a lo s s t h a t was n o t v e ry s i g n i f i c a n t .

4. The u n s u i t a b i l i t y o f o u a b ain a s a re fe re n c e s ta n d a rd f o r D i g i t a l i s

i s co n firm e d .

5. No r e l a t i o n s h i p betw een th e p o te n cy and pH o f t i n c t u r e s c o u ld be

o b se rv e d .

6* An a c c u r a te method f o r th e d e te rn iin a tio n o f pH o f sm all amounts o f

f l u i d i s d e s c r ib e d .
 65

7* The C anadian e x t r a c t i o n te c h n iq u e d o ss n o t e x h a u st th e a c t i v i t y o f

a g iv e n D i g i t a l i s pow der. The a c t i v i t y re m a in in g in th e sample r e p r e s e n ts

a p p ro x im a te ly 20[o o f th e t o t a l a c t i v i t y o f th e pow der.

8. M a c e ra tio n , p e r c o l a t i o n and a m o d ifie d C anadian te c h n iq u e o f S o x h le t

e x t r a c t i o n o f f i v e h o u rs w ith a b s o lu te a lc o h o l o r two h o u rs w ith 95/b a l c o ­

h o l y i e l d e x t r a c t s o f a p p ro x im a te ly e q u a l p o te n c y .

9. The m a c e ra tio n te c h n iq u e a s d e s c r ib e d in th e U. S. P . XI f o r th e e x ­

t r a c t i o n o f th e D i g i t a l i s R e fe re n c e Powder an d in v o lv in g f r e q u e n t a g i t a ­

t i o n was fo u n d to be th e m ost s a t i s f a c t o r y e x t r a c t i o n p ro c e s s for- u se in

ro u tin e b io a s s a y s t a n d a r d iz a tio n t e s t s o f D i g i t a l i s .

10* The p o te n c y re q u ire m e n ts f o r T in c tu r e D i g i t a l i s U. S. P . XI a r e 1*53

tim e s g r e a t e r th a n th e U. 5 . P. X r e q u i s i t e s , b a se d on th e r e l a t i v e s u s ­

c e p t i b i l i t i e s o f f r o g s to o u ab ain and D i g i t a l i s in th e s p r in g o f 1936*

11* The o v e r a i ^ i t te c h n iq u e o f th e C anadian m ethod i s th e b e s t f r o g p r o ­

ced u re f o r ro u tin e s ta n d a r d iz a tio n o f D i g i t a l i s p ro d u c ts .

12. A r o u ti n e m ethod f o r th e la b o r a to r y c o n tr o l o f D i g i t a l i s p r e p a r a tio n s

i s o u tlin e d *

13* A lth o u g h th e U. 3. P . XI and C anadian o f f i c i a l T in c tu re s of D i g i t a l i s

a r e t h e o r e t i c a l l y e q u iv a le n t to 100 mgms. o f I n t e r n a t i o n a l S ta n d a rd D ig i­

t a l i s Pow der, the U. 3 . P. XI t i n c t u r e i s a p p ro x im a te ly 2<$ s tr o n g e r th an

 66

th e C an ad ian t i n c t u r e when th e U. S. P , t i n c t u r e i s e v a lu a te d and com­

p a re d by b o th th e C anadian m ethod o f a s s a y a n d th e p r e s c r i b e d U, S. P. XI

te c h n iq u e .
 67

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1 . H a tc h e r and B rody, Am. J . Pharm. 82 (1 9 1 0 ), 360.

2. H a s k e ll, Am. J , Pharm. 83 (1 9 1 1 ), 201,

3. E c k le r , J .A .P h .A ., 1 (1 9 1 2 ), 715,

4 . E g g le s to n , Am. J , Pharm. 85 (1 9 1 3 ), 99,

5. R ow ntree a n d M a o h t, J.A .M .A . 66 (1 9 1 6 ), 870.

6. O o lson, J .A .P h .A . 7 (1 9 1 8 ), 13*

7. Rowe, J .A .P h .A ,, 8 (1 9 1 9 ), 900,

8. O 'B rie n and S n y d er, J .A .P h .A ., 8 (1 9 1 9 ),9 1 4 .

9. H a s k e ll, Dowell a n d T e rry , J .A .P h .A ., 11 (1 9 2 2 ), 918

10. A.D.M.A. P ro c . (1 9 2 3 ), 204.

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24. C. W. Edmunds - J.A .M .A ., 102 (1 9 3 4 ), 1246.

25. U. S. Pharm acopoeia - 1 0 th R e v ., p. 394,

26. U .S. P harm acopoeia - 1 1 th Rev. , p . 397.

27. Chapman and M o r r e ll, J . P harm aco l. Exp. T her. , 46 (1 9 3 2 ), 229.

28. B a k e r, Am. J. P h a rm ,, 84 (1 9 1 2 ), 247.

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31. Sm ith an d M cClosky, U .S . Pub. H e a lth R ep o rt #52 (1 9 2 5 ),

32. B um - “Methods o f B io lo g ic a l Assay**, O xford U niv. P re s s 1928, p . 16*

33. R e p o rt o f th e Committee on Pharm acology and B io assay s o f th e A. Ph. A.

J u l y 2 9 , 1931,

34. B au er and Froraherz - A rch. Exp. P a th . Pharm akol. , 172 (1 9 3 3 ), 693.

35. B um "M ethods o f B io lo g ic a l A ssay ", O xford U niv. P re s s 1928, p . 117.

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37. C u lle n a n d B iilm a n n , J . B io l. Chein., 64 (1 9 2 5 ), 727.

38. S m ith , J.A .P h .A , 17 (1 9 2 8 ), 241.

39. Haag and J a r e t t , J .A .P h .A , 20 (1 9 3 1 ), 471.

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42. A.D.M.A. P r o c ., (1934), 334.

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Dr. C. W. Chapman.

44. B r i t i s h Pharm acopoeia, 1932 Ed, p. 621.

 69

45. A.D.M.A. P r o c ., (1 9 3 4 ), 302.

46. A.D.M.A. P r o c . ,1 1 9 3 5 ), 292.

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