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Fluphenazine Hydrochloride
FLUPHENAZINE HYDROCHLORIDE
Klaus Florey
263
KLAUS FLOREY
CONTENTS DES C R I P T I ON 1.1 N a m e , F o r m u l a , M o l e c u l a r Weight I.2 A p p e a r a n c e , C o l o r , O d o r 2. PHYSICAL P R O P E R T I E S 2 . 1 I n f r a r e d Spectra 2.2 N u c l e a r Magnetic R e s o n a n c e S p e c t r a 2 . 3 U l t r a v i o l e t Spectra 2 . 4 Mass Spectra 2 . 5 pka, p H 2 . 6 Melting R a n g e 2 . 7 Differential Thermal A n a l y s i s 2.8 Thermogravimetric Analysis 2.9 Solubility 2 . 1 0 crystal Properties 3. SYNTHESIS 4 . S T A B I L I T Y - DEGRADATION 5 . DRUG METABOLIC PRODUCTS 6 . METHODS OF ANALYSIS 6.1 Elemental Analysis 6 . 2 Nonaqueous Titration 6 . 3 Polarographic A n a l y s i s 6.4 S p e c t r o f l u o r o m e t r i c A n a l y s i s 6.5 C o l o r i m e t r i c A n a l y s i s 6.6 E l e c t r o p h o r e s i s 6.7 Chromatographic Analysis 6 . 7 1 Paper 6 , 7 2 Thin-Layer 6.73 Column 6 . 7 4 Gas - L i q u i d 7 . COUNTERCURRENT SEPARATION I D E N T I F I C A T I O N AND DETERMINATION 8. I N BODY F L U I D S AND T I S S U E S 9. MISCELLANEOUS 10. REFERENCES
1.
2 64
FLUPHENAZINE H Y D R O C H L O R I D E
1.
DESCRIPTION 1.1 Name Formula, Molecular Weight Fluphenazine Hydrochloride is 4 - { 3 - [ 2 krifluoromethy1)-phenothiazine-10-yll propyl) piperazine ethanol dihydrochloride: also 4-(3-[lo -2(-trifluoromethy1)-phenothiazinylI propy1)-1piperazine-ethanol dihydrochloride; 1- (2-hydroxy ethyl)-4- [2-trifluoromethyl-lO-phenothiaz~nyl) propyl ] piperazine dihydrochloride; 10-(3'-[4"(B-hydroxy ethyl)-1"-piperazinyl] propyl-2-trifluoromethyl-phenothiazine dihydrochloride: S94; SQ 4918.
1.2 Appearance, Color, Odor White to off-white, odorless crystalline powder. PHYSICAL PROPERTIES 2.1 Infrared Spectra The infrared spectra of Squibb House Standard #48101-001, in mineral oil mull and KBr from MeOH, are presented in figures 1 and 2.1 The infrared spectrum of the free base, and a discuss ion of spectra-structure correlation of phenothiazines have been presented. 9 The spectra published by Sammul, et al.4 are in essential agreement with those presented here. 2.
265
FREQUENCY (CM-1)
x
r
t.
5 n
r 0 D rn
<
2
3
4
5
6
7 8 WAVELENGTH (MICRONS1
I0
II
12
13
is
Fig. 1 Infrared Spectrum of fluphenazine hydrochloride in mineral oil mull. Instrument: Perkin-Elmer 621
is
n
r
5 I rn
z B N 2
rn
I
< o n
0 c,
I
r 0
n
0
rn
Fig. 2 Infrared Spectrum of fluphenazine hydrochloride in KBr from methanol. Instrument: Perkin-Elmer 621
KLAUS F L O R E Y
N u c l e a r M a q n e t i c Resonance S p e c t r a The NMR s p e c t r a o f f l u p h e n a z i n e base a n d h y d r o c h l o r i d e a r e p r e s e n t e d i n f i g u r e s 3 and 4 . 5 The s p e c t r u m o f t h e f r e e base i s e s s e n t i a l l y i d e n t i c a l w i t h t h a t p u b l i s h e d p r e v i o u s l y . 3 The f o l lowing a s s i g n m e n t s c a n be made for c h e m i c a l s h i f t o f t h e s p e c t r u m of t h e f r e e base. 6 2.2
a b
6.05 8.09 7.57 6.41 6.93 2.9-3.0
C
d
e f
2.3 corded:
triplet multiplet absorption envelope triplet singlet aromatic
Ultraviolet Spectra The f o l l o w i n g U . V . d a t a h a v e been re1.
i n methanol’
( I n s t r u m e n t Cary 1 5 )
Amax 261 mp E i E m 646
A m a x 310 mp, E1% 69 1cm
268
Fiq. 3 NMR Spectrum of fluphenazine base in deuterochloroform. Inscrument: Varian A-60
-*+
i i
r B C
v)
n
r
0
n
rn
<
L
r a*
-I I
l . . . . , . , . , ~ . , . , , , , , , ~
, . . . . . .
I
7a
.
.
I
.
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A4
I
30
1 1 1 , , 1 , , , , , , , , , 1 , , , , , , , , , ~
I
.(I
.
.
I
.
.
.
I
I
30
10
I
I
I
.
. . I. . . . I 0
' .
.I 0
Fig. 4 NMR Spectrum of fluphenazine hydrochloride in deuterochloroform. Instrument: Varian A-60
M ,i
FLUPHENAZINE HYDROCHLORIDE
2.3
U l t r a v i o l e t S p e c t r a Cont d . 2. i n 95% e t h a n o l 3 Amax 264 Log E 4 . 5 9 Amax 316 Log E 3 . 6 9 Amin 239 Log E 4 . 2 8 Amin 300 Log E 3 . 5 1 3. i n 50% a l c o h o l ( I n s t r u m e n t Beckman
DU)
ElPPm 1c m
Xmax 259 Xmax 305
4.
0.052 0.006 0.013 0.002
Xmin 224 Xmin 280 i n methanol8 Xmax 259 Log
E
4.53
2.4
Mass S p e c t r a The l o w - r e s o l u t i o n m a s s s p e c t r u m o f f l u p h e n a z i n e base i s r e p r e s e n t e d i n f i g u r e 5 . 9 The h i g h - r e s o l u t i o n s p e c t r u m shows t h e f o l l o w i n g f r a g m e n t a t i o n p a t t e r n o f s i d e c h a i n and nucleus’, which i s i n a g r e e m e n t w i t h t h e r e s u l t s o f an independent i n v e s t i g a t i o n . 69
?zyz 307-1H
2 8 0 I157
t
H2 *CH
.+)
-CH2 I
350
27 1
Fig. 5 Low-resolution Mass Spectrum of fluphenazine.
Instrument: MS-9
FLUPHENAZINE HYDROCHLORIDE
Another prominent fragment is t h e follow-
ing:
248
The s i g n i f i c a n t m a s s numbers shown a b o v e c a n be t a b u l a t e d a s f o l l o w s : Mass U n i t s D i f f . C H N 0 S F U n s a t O/E* Found- C a l c u l a t e d 437 2 2 26 3 1 1 3 10 0 0.3 419 2 2 24 3 - 1 3 10 0 -0.7 4 0 6 2 1 2 3 3 - 1 3 10.5 E -1.6 363 19 18 2 - 1 3 10.5 E -0.3 307 16 1 2 1 - 1 3 10 0 -1.1 306 1 6 11 1 - 1 3 1 0 . 5 E -0.3 280 14 9 1 - 1 3 9.5 E -0.1 E 0.5 266 1 3 7 1 - 1 3 9.5 248 14 9 1 - - 3 9.5 E -0.1 1 7 1 9 19 2 1 - - 1.5 E -0.1 E -0.7 1 5 7 8 1 7 2 1 - - 1.5 E -0.1 1 4 3 7 15 2 1 - - 1.5
"0
=
odd e l e c t r o n i o n : E
2.5
pKa;
=
pH
even e l e c t r o n i o n
The a p p a r e n t pKa of f l u p h e n a z i n e d i h y d r o c h l o r i d e i n aqueous a l c o h o l i c s o l u t i o n h a s b e e n d e t e r m i n e d as 8.05 f o r t h e s e c o n d b a s i c g r o u p . F o r t h e f i r s t b a s i c g r o u p , a v a l u e o f 3.90 w a s obt a i n e d . lo I n d e p e n d e n t l y , v a l u e s o f 7 . 8 (estimat e d ) and 3 . 6 0 , r e s p e c t i v e l y , w e r e o b t a i n e d . 7 1 The p H of a n aqueous s o l u t i o n o f t h e d i h y d r o c h l o r i d e i s 2 . 4 ; t h a t o f monohydrochloride is 273
KLAUS FLOREY
5 . 5 . 11 2.6
M e l t i n q Ranqe The f o l l o w i n g m e l t i n g p o i n t s o f t h e hydroc h l o r i d e have been r e p o r t e d : 238.5-239.5O ( R e c r y s t . from abs , e t h a n o l USP M e t h o d p ( R e c r y s c . from abs. e t h a n o l ) l2 2 31-2 33' 224-226OI3, l4 224.5-226O ( R e c r y s t . from m e t h a n o l - e t h e r ) 15 2 3 5-2 3 701 7 l7 231-232OI8 2.7
D i f f e r e n t i a l Thermal A n a l y s i s D i f f e r e n t i a l Thermal A n a l y s i s of S q u i b b House S t a n d a r d #48101-001 showed a n endotherm a t 237OC. ( I n s t r u m e n t : DuPont) 1 Thermoqravimetric A n a l y s i s Thermogravimetric a n a l y s i s o f Squibb House S t a n d a r d #48101-001 gave no w e i g h t l o s s up t o 15Ooc ( I n s t r u m e n t : DuPont) 2.8
2 9
Solubility Fluphenazine hydrochloride is hygroscopic and e x t r e m e l y s o l u b l e i n w a t e r (70% w/v a t 25OC pH 2 . 4 ) . S o l u b i l i t y i n e t h a n o l : 15% w/v a t 25OC. L e s s t h a n 2% (w/v) i n e t h e r , a c e t o n e , benzene, l i g r o i n . 11 I n s o l u b l e i n c h l o r o f o r m and e t h e r . 67 2.10 c r y s t a l P r o p e r t i e s For f o r e n s i c i d e n t i f i c a t i o n , a p i c t u r e o f f l u p h e n a z i n e d i h y d r o c h l o r i d e c r y s t a l s from 95% e t h a n o l h a s been p u b l i s h e d w i t h t h o s e o f o t h e r p h e n o t h i a z i n e s . 19' A n x-ray powder d i f f r a c t i o n p a t t e r n of fluphenazine dihydrochloride is presented i n 274
FLUPHENAZINE HYDROCHLORIDE
table l.20 S l i g h t l y d i f f e r e n t d-values ( 1 8 . 2 ; 9.07; 8 . 7 1 ; 6 . 0 7 ; 5 . 0 3 ; 4 . 8 5 ; 4 . 3 2 ; 4 . 0 2 ; 3. 7 5 ) 7 and ( 1 7 . 2 0 ; 1 1 . 5 0 ; 8 . 4 0 ; 5 . 9 0 ; 5 . 5 0 ; 5 . 4 0 ; 4 . 7 0 ; 4.50; 4 . 2 0 ; 4 . 1 0 ; 4 . 0 0 ; 3 . 9 0 ; 3 . 7 5 ; 3 . 6 0 ; 3 . 3 5 ; 3.20; 3.10; 2.85; 2.80; 2.65; 2.55; 2.45; 2.40; 2 . 2 2 ; 2 . 1 5 ) 77 w e r e a l s o r e p o r t e d . 3 . SYNTHESIS S y n t h e t i c pathways t o f l u p h e n a z i n e ( I ) a r e shown i n f i g u r e 6. The s y n t h e s i s u s u a l l y i n v o l v e s t h e a d d i t i o n of t h e propylpiperazine ethanol s i d e c h a i n t o t h e 3-trifluoromethyl-phenothiazine nuc l e u s (11). The f o l l o w i n g r o u t e s t o ( I ) h a v e b e e n reported: (1)D i r e c t a d d i t i o n o f 4 ( 3 - c h l o r o p r o y l ) -1-pipe r a z i n e e t h a n o l t o (11) t o y i e l d ( I ) .2 P ( 2 ) Formation o f i n t e r m e d i a t e 1 0 ( 3 - c h l o r o p r o p y 1 ) - 3 t r i f l u o r o m e t h y l p h e n o t h i a z i n e (111) und e r a v a r i e t y of c o n d i t i o n s , and c o n d e n s a t i o n o f (111) w i t h p i p e r a z i n e e t h a n o l t o ( I ). l2 16,l7 (3)Condensation of 3(2-trifluoromethyl-10phenothiazinyl) propylpiperazine ( I V ) with ethyle n e o x i d e 1 5 , w i t h @-bromo-ethyl a c e t a t e v i a (VI)1 3 9 14, o r w i t h 2-bromo e t h a n o l . This last method h a s a l s o been used t o p r e p a r e ( I ) , l a b e l e d w i t h 14C i n t h e two e t h a n o l c a r b o n s . 6 ( 4 )R e d u c t i o n o f 4 (f3- ( 1 0 - p h e n o t h i a z i n y l ) t h i o propionyl) piperazine ethanol with lithium aluminum h y d r i d e t o ( I ) .2 2 J
-
4 . STABILITY DEGRADATION F l u p h e n a z i n e h y d r o c h l o r i d e is h y g r o s c o p i c . When m o i s t u r e i s e x c l u d e d , t h e c r y s t a l l i n e m a t e r i a l i s s t a b l e f o r a t l e a s t 6 months a t temperat u r e s as h i g h as 5Ooc, b o t h under a i r and n i t r o gen. The compound i s p h o t o s e n s i t i v e , s i n c e t h e s u r f a c e o f c r y s t a l s exposed t o i n c a n d e s c e n t l i g h t f o r p e r i o d s o f more t h a n 4 weeks d i s c o l o r e d t o 215
KLAUS F L O R E Y
2.10 Crystal Properties
(Continued)
TABLE I X-ray Powder D i f f r a c t i o n p a t - t e r n of S q u i b b House S t a n d a r d #48101-001 d (? * i) Rel. I n t e n s i t y * * 17.65 2 0.05 0.26 0.12 9.80 8.90 0.58 (5) 8.70 0.42 6.02 0:36 5.56 5.46 0.24 0.14 5.36 0.31 5.15 4 . 8 3 _f 0 . 0 2 0.47 4.75 0.59 (4) 4.49 0.71 ( 2 ) 4. 31 0.48 4.23 0.22 4.12 0.12 4.00 0.63 (3) 3.90 1 . 0 0 (1) 3.72 u-x5 3.69 0.47 3.57 0.32 0.21 3.40 0.16 3.27 0.24 3.23 0.42 3.11 3.06 0.19 0.22 3.00 2.93 0.12 2.87 0.15 2.72 0.16 2.67 0.16 2.54 0.14 2.42 0.15 2.34 0.15 2.27 0.14 2.02 0.15 d = (interplanar distance)
m
s i AB n
Q
A-
**
2
1.539 R a d i a t i o n : ka and ka2 c o p p e r 1 Based on h i g h e s t i n t e n s i t y of 1 . 0 0 I n s t r u m e n t : Phillips 276
Curve N O . 357
I
< O m 0 D
I
r 0
a
o rn
Fig. 6
S y n t h e t i c Pathways.
KLAUS F L O R E Y
light tan. I n aqueous s o l u t i o n , f l u p h e n a z i n e hyd r o c h l o r i d e d e g r a d e s when e x p o s e d t o l i g h t . S o l u t i o n s a t 0.01% i n e t h a n o l , w a t e r , 0.1g H C 1 and 0 . 5 N H2SO4 w e r e k e p t i n t h e d a r k o r e x p o s e d t o f l u o r e s c e n t c e i l i n g l i g h t a t room t e m p e r a t u r e (see s e c t i o n 2 . 3 ) . A f t e r 1 7 days i n t h e d a r k , a l l sol u t i o n s r e t a i n e d t h e i r U.V. absorbance i n t h e Only t h e 0 . 5 g H2S04 s o l u t i o n 255-265 m p r e g i o n . showed a s l i g h t d e c l i n e i n a b s o r b a n c e . In the light-exposed samples, only t h e e t h a n o l i c solut i o n r e m a i n e d unchanged, w h i l e i n t h e r e m a i n i n g s o l u t i o n s d i s c o l o r a t i o n and t h e a p p e a r a n c e o f m u l t i p l e b a n d s a t 240 and 255 mw w e r e n o t e d . 6 4 Exposing a 0 . 0 5 % f l u p h e n a z i n e h y d r o c h l o r i d e i n 0.1g H C 1 s o l u t i o n i n p y r e x g l a s s t o s u n l i g h t c a u s e d a 40% d r o p o f a b s o r b a n c e i n 2 h o u r s . 2 3 A s t u d y o f t h e pH dependency o f t h e l i g h t i n s t a b i l i t y showed a s i m i l a r d e c l i n e i n a b s o r b a n c y from p H 0 . 3 t o 9, w h e r e a s above pH 11 t h e d e g r a d a t i o n w a s r e t a r d e d . 2 4 Again, it w a s found t h a t s o l v e n t s s u c h as m e t h a n o l , e t h a n o l , dimethylformami d e , e t h y l e n e g l y c o l , and e t h e r p r e v e n t o r r e t a r d degradation. The l i g h t - c a t a l y z e d d e g r a d a t i o n i s n o t prev e n t e d b y e x c l u s i o n o f oxygen25 and p r o c e e d s t h r o u g h f o r m a t i o n of t h e f r e e r a d i c a l ( c f 2 6 ) . The r a d i c a l c a n a l s o be formed b y a n o x i d i z i n g a g e n t , s u c h as c e r i c ammonium s u l f a t e . 26 I n phenothiazines g e n e r a l l y , t h e semiquinone r a d i c a l u n d e r g o e s d i s p r o p o r t i o n a t i o n . The p r o d u c t s o f o x i d a t i o n ar e t h e s u l f o x i d e o r t h e 3 ( o r 7 ) -hydroxy-derivatives. However, f o r m a t i o n of t h e l a t t e r is s e v e r e l y i n h i b i t e d i n 10-substituted p h e n o t h i a z i n e s . 2 6 The s e c o n d - o r d e r d e c a y r a t e c o n s t a n t of t h e s e m i q u i n o n e r a d i c a l of f l u p h e n a z i n e w a s f o u n d t o be 1100 (l/mole/min) , as meas u r e d by e l e c t r o n s p i n r e s o n a n c e . 26 The s u l f o x i d e c o n t e n t o f s e v e r a l commercial b a t c h e s o f f l u p h e n a z i n e w a s d e t e r m i n e d t o be a r o u n d 0 . 1 % b y 218
FLUPHENAZINE H Y DROCH L O R I DE
a s p e c t r o f l u o r o m e t r i c method ( e x c i t a t i o n wavel e n g t h 350 m@; f l u o r e s c e n c e w a v e l e n g t h 400 mp; see a l s o s e c t i o n 6 . 4 ) .27 The s u l f o x i d e c o n t e n t c a n a l s o be measured b y a t h i n - l a y e r method (see a l s o s e c t i o n 6.72).2 8 So f a r , t h e r e i s no e v i d e n c e f o r t h e d e g r a d a t i v e f o r m a t i o n of 7 - h y d r o x y f l u p h e n a z i ~ which h a s been i d e n t i f i e d as a m e t a b o l i c p r o d u c t (see s e c t i o n 5 ) . 5 . DRUG METABOLISM Acetylf luphenazine ( V I , Figure 6 ) was hydrolyzed e n z y m a t i c a l l y t o f l u p h e n a z i n e ( I ) i n v i t r o b y homogenates o f s m a l l i n t e s t i n a l mucosa, l i v e r , and b r a i n from e i t h e r t h e r a t o r t h e monkey.29 ( S e e a l s o s e c t i o n 7 ) . When f l u p h e n a z i n e w a s i n c u b a t e d w i t h a " m i c r o s o m a l and s o l u b l e " f r a c t i o n o f r a t l i v e r s , h y d r o x y l a t i o n on t h e p h e n o t h i a z i n e r i n g was o b s e r v e d . 3 0 The s u l f o x i d e w a s n o t det e c t e d i n t h e e x t r a c t , and it seems u n l i k e l y t h a t b i o l o g i c a l sulfoxidation precedes hydroxylation i n t h e s e s y s t e m s . 3 0 The b i o l o g i c a l d i s p o s i t i o n and m e t a b o l i c f a t e of fluphenazine-14C i n t h e dog and r h e s u s monkey w a s s t u d i e d . 7 6 When dogs w e r e t r e a t e d w i t h f l u p h e n a z i n e - 1 4 C , 7-hydroxyfluphenazine ( V I I , F i g u r e 6) w a s i from t h e u r i n e a n d i d e n t i f i e d by s y n t h e s i s . human b l o o d and u r i n e s m a l l amounts o f f l u p h e n a z i n e and i t s s u l f o x i d e , b o t h i n f r e e and c o n j u g a t e d form w e r e i d e n t i f i e d . 7 9 T i s s u e d i s t r i b u t i o n and e x c r e t i o n w e r e s t u d i e d i n t h e r a b b i t . 73 6. METHODS OF ANALYSIS 6.1 E l e m e n t a l A n a l y s i s % Found Element % Calc. R e f . 11 12 15
Wa:f
C H
51.76 5.53
51.72 5.62 8.04
0
8.23 3.13 6.28
F N
S
c1
11.17
13.89
-
-
6.05 13.66
51.73 5.86
-
8.00
-
-
-
51.68 5.42
-
-
-
KLAUS F L O R E Y
6 . 2 Nonaqueous T i t r a t i o n Nonaqueous t i t r a t i o n i n g l a c i a l a c e t i c a c i d w i t h 0.01g p e r c h l o r i c a c i d and Q u i n a l d i n e Red o r c r y s t a l v i o l e t i n d i c a t o r g a v e a n e u t r a l i z a t i o n e q u i v a l e n t t o 259 ( c a l c . 2 5 5 . 2 2 ) . 32
6 . 3 Polarographic Analysis The h a l f - w a v e p o t e n t i a l i n a l c o h o l i c s u l f u r i c a c i d s o l u t i o n w a s f o u n d t o be 0 . 7 1 4 ( v o l t v s . normal c a l o m e l e l e c t r o d e ) , c o m p a r a b l e t o t h a t o f o t h e r p h e n o t h i a z i n e s .8 6 . 4 Spectrofluorometric Analysis Fluphenazine hydrochloride, l i k e o t h e r phenothiazine drugs, e x h i b i t s fluorescence. This phenomenon w a s f i r s t e x p l o r e d i n 0 . 2 g s u l f u r i c a c i d . 33 The f l u o r e s c e n c e p e a k w a s f o u n d i n t h e r a n g e o f 450 t o 475 mp, s h i f t i n g t o s l i g h t l y l o w e r w a v e l e n g t h upon o x i d a t i o n w i t h p o t a s s i u m permanganate. A s l i t t l e as 0 . 0 1 t o 0 . 0 5 1-14 o f t h e p u r e s u b s t a n c e per m l . o f 0 . 2 g s u l f u r i c a c i d c o u l d be d e t e c t e d . Due t o i n t e r f e r e n c e by o t h e r substances, t h e s e n s i t i v i t y i n biological f l u i d s was l o w e r ( a b o u t 0 . 8 pg/ml f l u i d ) . T h i s method i s s u p e r i o r t o d e t e r m i n a t i o n b y U . V . , where about 4 p.g/ml a r e r e q u i r e d . I n 50% a c e t i c a c i d , t h e f o l l o w i n g d a t a w e r e o b t a i n e d , w i t h o u t o r w i t h o x i d a t i o n w i t h 30% hydrogen peroxide34:
A c t i v a t i o n maximum,m~ F l u o r e s c e n c e maximum,my Unoxidized Oxidized Unoxidized Ox i d i z e d Ref 34 Ref 35
330
-
345 3 50
460
-
380 410
I n t h e u n o x i d i z e d form, a s h a r p i n c r e a s e i n f l u o r e s c e n c e i n t e n s i t y o c c u r r e d above p H 7; i n t h e o x i d i z e d form, i n t e n s i t y w a s a b o u t e q u a l from 280
FLUPHENAZINE HYDROCHLORIDE
p H 2 t o p H 1 2 and f e l l p r e c i p i t o u s l y a t p H 1 2 . I n t h e o x i d i z e d form, as l i t t l e as 0 . 5 wg/ml c o u l d be d e t e r m i n e d q u a n t i t a t i v e l y . F l u p h e n a z i n e w a s r e l a t i v e l y l e s s f l u o r e s c e n t t h a n some o t h e r p h e n o thiazines. In concentrated s u l f u r i c acid, t h e a c t i v a t i o n maximum w a s found a t 325 m p and t h e f l u o r e s c e n c e maximum a t 500 mp. 36 The i n s t r u m e n t u s e d i n a l l i n v e s t i g a t i o n s w a s t h e Aminco-Bowman S p e c t r o f l u o r o m e t e r .
-
6.5 Colorimetric Analysis Color T e s t s f o r Identification The g e n e r a l method o f Ryan37 t o q u a n t i t a t e unoxidized phenothiazine d e r i v a t i v e s by c o l o r complex f o r m a t i o n w i t h p a l l a d i u m c h l o r i d e ( 5 0 0 550 mw m a x i m a ) h a s a l s o b e e n a d o p t e d f o r f l u p h e n a z i n e h y d r o c h l o r i d e ( s e e N . F . X I I 1 ) and c a n b e u s e d f o r s t a b i l i t y measurements. Like o t h e r phenothiazines, fluphenazine h y d r o c h l o r i d e reacts w i t h c e r i c s u l f a t e , f i r s t b y forming a red-colored semiquinone f r e e r a d i c a l (420 ml), f o l l o w e d by f o r m a t i o n o f t h e c o l o r l e s s s u l f o x i d e d e r i v a t i v e . T h i s r e a c t i o n h a s b e e n made t h e basis o f a p h o t o m e t r i c t i t r a t i o n . 3 8 The f o l l o w i n g c o l o r t e s t s h a v e been reported. ( F o r a d d i t i o n a l t e s t s , see a l s o s e c t i o n s 6 . 7 1 and 6 . 7 2 ) . FOR COLOR TESTS SEE TABLE I1
6 . 6 E l e c t r o p h o r e s is E l e c t r o p h o r e s i s w a s c a r r i e d o u t on f l u p h e n a z i n e h y d r o c h l o r i d e and s e v e r a l o t h e r phenot h i a z i n e t r a n q u i l i z e r s i n var io u s b u f f e r s prop o s e d by Werrum. 41 A Gordon-Misco a p p a r a t u s , Whatman 3MM paper 30 c m i n w i d t h , and a p o t e n t i a l d i f f e r e n c e of 500 t o 800 v w e r e u s e d . Detection o f s p o t s w a s c a r r i e d o u t w i t h a 40% s u l f u r i c a c i d
zx 1
TABLE I1 Reaqent 0.001N M e r c u r i c n i t r a t e i n h y d r o c h l o r i c a c i d 0.5% aq . ammonium m o l y b d a t e 0.5% aq. ammonium v a n a d a t e 3 7% f o r m a l d e h y d e i n c o n c . s u l f u r i c a c i d (1:20) 0.5% aq. s e l e n i o u s a c i d 1% sodium t u n g s t a t e 0.5% titanium dioxide i n conc.sulfuric acid Fuming n i t r i c a c i d , t h e n e t h a n o l i c p o t a s s i u m hydroxide (3%) 10% aq. sucrose Conc. s u l f u r i c a c i d p-dimethylaminobenzaldehyde 10% i n g l a c i a l acetic acid 1% c o b a l t a c e t a t e : 10% i s o p r o p y l a m i n e i n a c e t o n e 10% aq. c h l o r a m i n e T 1% aq. palladium c h l o r i d e a c i d i f i e d w i t h hydrochloric acid N i t r i c acid Conc. s u l f u r i c a c i d , 6% u r a n i u m n i t r a t e i n 95% e t h a n o l ( 3 : 1 7 ) 0 . 5 % ammonium v a n a d a t e i n c o n c . s u l f u r i c a c i d 4% a q . s i l v e r n i t r a t e s o l u t i o n a c i d i f i e d w i t h 0 . 1 N sulfuric acid 2% Zmmonium f e r r o u s s u l f a t e i n c o n c . sulfuric acid
as.
32
N
Color pink-violet blue-pink-violet grey-yellow 1i g h t red grey-green purple orange
R ef -
39 40 40 40 40 40 40
yellow-green grey-yellow l i g h t brown
40 40
5
l i g h t brown light blue l i g h t yellow
40 18 18
<
orange orange-brown
18 18
l i g h t orange y e l l o w brown
18 18
c r e a m wh it e
18
-
40
75
x
5
FLUPHENAZINE HYDROCHLORIDE
spray (orange color) or fluorescence under W light.42 For fluphenazine hydrochloride, anodic migration (cm) after 40 to 60 minutes was: pH :
Migration:
3.3 6.8
4.7 3.7
6.0
4.3
7.2 4.5
8.0 2.7
9.3 1.2
6.7 Chromatoqraphic Analysis 6.71 Paper Chromato-ra hic Analysis The following chromatographic systems have been reported: Solvent Systems: 1% Sodium formate 1% Sodium formate 90/n-Propanol 10 1% Sodium formate 90/1N Ammonia 10 1g Sodium formate 97/Formic acid 3 1% Sodium acetate 1% Sodium acetate 90/n-Propanol 10 10% Sodium chloride 92/n-Propanol 8 Formamide + Ammonium formate/Benzene Formamide + Ammonium formate/Cyclohexane-benzene (9:1) Petroleum (b.p. 195-220°) /EthanolAmmonia-water (60:2: 38)
Rf Ref 0.23 42 0.32 42 0.19 42 0.42 42 0.17 42 0.48 42 0.45 42 0.71 43 0.22 43 0.59 43
Ref color Detection systems : 40% H2S04 spray orange 42,43 Fluorescence under W light bluish yellow 42,43 orange 43 Dragendorff-reagent Cerium sulfate in 2g sulfuric acid red 43 1% gold (111) chloride red 43 1% dimethylaminobenzaldehyde in ethano1:chloroform (95:5)red 43 A quantitative paper chromatographic method62 can be used to measure the purity of '83
K L A U S FLOREY
fluphenazine, following published procedures63. The paper is impregnated with castor oil ( U S P ) in ether. Developing solvent is methanol-water 85:15. Rf values of fluphenazine base approx. 0.79, fluphenazine hydrochloride 0.83. The zones are eluted from the chromatogram with 95% ethanol and the absorbances o f the eluates are determined at 261 m p in a spectrophotometer. 6.72
3
The following thin-layer chromatographic systems have been reported: FOR ABOVE SEE TABLE I11
6.73 Column (Ion-exchanqe) Chromatographic Analysis~ Ion-exchange analysis of fluphenazine hydrochloride and other phenothiazine tranquilizers on a column of Dowex sulfuric acid resin 50W-X8 was possible, but not too practical for analysis of tablets.56 6.74 Gas-Liquid Chromatoqraphic Analysis Fluphenazine was not eluted after 90 minutes at 270°, using a 210 SE-30 silicone polymer on a 80-100 mesh Gas-chrom S diatomaceous earth column57; with a 3% SE-30 on 80-100 mesh Gas-chrom Q at 25Ooc, it eluted in 4.9 minutes.28 Fluphenazine was also well separated from other tranquilizers on a 120 mesh silanized Gas-chrom P column coated with 1% Hi-Eff-8B (cyclohexanedimethanol succinate) at a temperature of 22Ooc and a retention time of 5 minutes.58 The identification of fluphenazine and other phenothiazine tranquilizers by gas chromatography of their pyrolysis products has also been reported.59
284
TABLE I11 Ads orbent Silica Gel 11
I 11 I1
I1 I' 1' 11
N
m vI
I1
Cellulose Silica Gel 11
11 11 I1
11 11
So1vent Svs tem tert. Butyl alcohol 90/1g Ammonia 10 n-Propanol 88/1g Ammonia 1 2
Ether, sat. with water 70% Methanol 85% n-Propanol n-Butanol sat. with 1s Ammonia Benzene-Dioxane-aq. Ammonia ( 6 0 : 3 5 : 5 ) Ethanol-Acetic acid-water ( 5 0 :30: 2 0 ) Methanol-butanol ( 6 0 :4 0 ) Cyclohexane-Diethylamine (9: 1) 5% aq. Ammonium sulfate sat. with Isobutanol Cyclohexane-methanol ( 5 0 : 5 0 and 2 5 : 7 5 ) Chloroform-acetone ( 5 0 : 5 0 ) Chloroform-methanol ( 5 0 : 5 0 ) chloroform-methanol (90: 10) Chloroform-acetone-methanol ( 5 0 : 2 5 : 2 5 ) Chloroform-acetone-methanol ( 2 5 : 5 0 : 2 5 ) Chloroform-acetone-methanol-ammonia ( 5 0 : 25: 25: 1)
Acetone Acetone Acetone-ammonia (100:1)
Rf 0.37 0.56 0.09 0.68 0.27 0.57 0.34 0.58 0.68 0.05
Re f 42 11
11 11 It 11
n
5211
C
r -o
1
rn 11
53
z B N z rn
1
0.03 0.36 0.03 0.59 0.44 0.42 0 , 32
54 44 44 51,44,50 45 44 44
0.54 0.06
44 44 45 44,51
0.16 0.33
0
n
0
c
1
r
0
rn
TABLE I11 Cont'd.
Adsorbent Silica Gel I1
I1 I1 I1
I1
11 11 I1
1 3
II
m
I1
rn
'1 11 '1
11 '1
*
*
* ** **
Silica Ge1,Plain Chromagram,Plain
solvent System Acetone-ammonia (95: 5 ) Acetone-methanol (50 :50) Acetone-methanol-ammonia (50 :50 :1) Methanol Methanol-ammonia ( 98 :2 ) Methanol-ammonia (98: 2 ) Chloroform-methanol-ammonia ( 8 0 : 20 : 1) Chloroform-ethanol-ammonia (80: 2 0 : 1) Acetone-benzene-ammonia (30:70:5) Butanol-pyridine-water (1:2: 1) Cyclohexane-benzene-diethylamine (75:15:10) Cyclohexane-benzene-diethylamine (75: 1 5 : 20) Methanol Acetone Benzene-ethanol-ammonia (95: 15: 5 ) Methanol 95% ethanol Chloroform + cyclohexane-diethylamine (50:40: 1 0 ) Chloroform + cyclohexane-diethylamine
Rf Ref 0.54 0.38 0.56 0.34 0.54 0.67 0.68 0.76
44
0.52
47
0.06
48
0.18 0.60
45 48
--
45 30 23 46
0.25
0.30 0.15
45 48
0.24
49
0.06
0.48
TABLE I11 C o n t d A d s o rp t i o n
Chromagram + fluor indicator Silica G e l It
*
** ***
S o l v e n t System
Rf Ref -
c h l o r o f o r m + cyclohexane-diethylamine C h l o r o f o r m - h e x a n e (1:1) 15% aq.ammonium a c e t a t e - m e t h a n o l (20:80)
p r e p a r e d w i t h 0 . 1 M KOH p r e p a r e d w i t h 0.1M NaHS03 I n t h i s s y s t e m , f l u p h e n a z i n e - s u l f o x i d e h a d a n Rf
0.40 0.48
49 50
0.75
28***
of 0.59 2
m
The f o l l o w i n g d e t e c t i o n s y s t e m s h a v e b e e n u s e d : System F l u o r e s c e n c e u n d e r U.V. lamp 40% s u l f u r i c a c i d s p r a y 50% a q . s u l f u r i c a c i d i n e t h a n o l ( 4 : l ) Potassium iodoplatinate 5% f e r r i c c h l o r i d e , 20% p e r c h l o r i c a c i d , 50% n i t r i c a c i d (5:45:50) ( F P N ; F o r r e s t r e a g e n t ) Iodine vapors 1%p o t a s s i u m p e r m a n g a n a t e Dragendorff r eag en t
I
Color Reaction
-
<
0
Ref
42,44,51,30,28,50
orange 42,46,47 orange-red47,52 violet 52,54,44,51,49,28 (flesh or (pale pink 44,45,51,53,54,48 brown 54,44,48 51 brown 51,48
a
0 0
I
6 z0 rn
D e t e c t i o n systems used Cont' d. System 5% p-dimethylaminobenzaldehyde i n 1 8 s s u l f u r i c acid Furf u r a l reagent 1%c e r i u m s u l f a t e i n 2 g s u l f u r i c a c i d 1%g o l d (111) - c h l o r i d e Folin-Ciocalteau reagent 1%ammonium v a n a d a t e i n 10 m l . conc. sulfuric acid 5% cinnamic aldehyde and 5% H C 1 i n e t h a n o l p-benzoquinone i n d i c h l o r o e t h a n e
color
R e a c tion
pink o r orange cameo red red cameo flesh cameo
-
Ref 45,47,51 51,45 47
47 45 45,48 45 55
n
r
0
n
rn
<
F LUPH E NA 2 I N E HY D R OCH LOR I DE
countercurrent Separation F l u p h e n a z i n e w a s s e p a r a t e d from a c e t y l f l u phenazine ( V I , f i g u r e 6) by countercurrent d i s t r i b u t i o n (see a l s o s e c t i o n 5 ) a c r o s s 25 t u b e s i n a s o l v e n t s y s t e m o f 0.1M a c e t a t e b u f f e r , pH 3 . 8 2 , and 1 . 5 % i s o a m y l a l c o h o l i n h e p t a n e . 2 9 7.
I d e n t i f i c a t i o n and D e t e r m i n a t i o n i n Body F l u i d s and T i s s u e s Many o f t h e r e f e r e n c e s c i t e d i n p r e v i o u s sect i o n s c o n c e r n methods d e v i s e d t o i d e n t i f y a n d d e t e r m i n e f l u p h e n a z i n e h y d r o c h l o r i d e and o t h e r phenothiazines f o r pharmacological, toxicological, and f o r e n s i c p u r p o s e s ( c f . 6 0 ) . T h e s e methods c a n be c l a s s i f i e d as f o l l o w s : 8.
43,45,55,39,18,40,74,75 Color r e a c t i o n s 61,43,45,51,74,79 S e p a r a t i o n schemes 33,34,35,36,68 Spectrof luorometry 42 Electrophoresis 43,43 Paper c h r o m a t o g r a p h y (28,42-49, 51-54, 6 5 , 7 4 , T h i n - l a y e r chromatog(78,79 raphy Gas - 1 i q u i d chromat og28,57,58 raphy 65 So 1u b i 1i t y X-ray d i f f r a c t i o n b a n d s 7 M i c r o c r y s t a l l i n e iden19,72 t if i c a tion Miscellaneous The a b s o r p t i o n o f f l u p h e n a z i n e d i h y d r o c h l o r i d e and o t h e r p h e n o t h i a z i n e d e r i v a t i v e s b y s o l i d a d s o r b a n t s , s u c h a s k a o l i n , t a l c and c h a r c o a l , h a s b e e n s t u d i e d b y S o r b y e t a l . i0 The v o l u m e t r i c d e t e r m i n a t i o n o f f l u p h e n a z i n e by s t o i c h i o m e t r i c combinat i o n w i t h a n i o n i c s u r f a c e - a c t i v e a g e n t s , s u c h as sodium l a u r y l s u l f a t e and sodium d i o c t y l s u l f o s u c c i n a t e , h a s b e e n 9.
28Y
KLAUS F L O R E Y
described.66 The surface tension-lowering activity of fluphenazine and other tranquilizers h a s been studied.70 Ion-pair extraction has been described.71 REF ER ENC ES (1) G.A.Brewer, Squibb Institute, personal communication. (2) W.E Thompson, R.J.Warren, J.B.Eisdorfer and J. E.Zarembo, J.Pharm.S c i . 3 , 1819 (1965). (3) R.J.Warren, J.B.Eisdorfer,W.E ,Thompson and J.E.Zarembo, J.Pharm.Sci .55, 144 (1966). (4) O.R.Sammu1, W.L.Brannon and A.L.Hayden, J.Ass .Cffic.Anal.Chem.47, 918 (1964). (5) A.I.Cohen and G.M.Jennings, Squibb Institute, personal communication. (6) H.L.Yale, A.I.Cohen and F.Sowinski, J.Med. Chem.5, 347 (1963). (7) P.Rajeswaran and P.L.Kirk, Bull.Narcotics, 14, 19 (1962), C.A.57, 7390h (1962). (8) FKabasakalian and J.McGlotten, Anal.Chem.2, 431 (1959). (9) A. I.Cohen, Squibb Institute, personal communication. (10)D.L.Sorby, E.M.Plein and J.D.Benmaman, J. Pharm. Sci. 55, 785 (1966). (ll)H.Cords, Squibb Institute, personal communicat ion. (12)H.L.Yale and F.Sowinski, J.Amer.Chem.Soc. 82, 2039 (1960). (13)Smith, Kline and French Laboratories, Brit. Patent 829, 246 (1960), c. A. 54, 174281.1 (1960), (14)G.E.Ullyot, U.S.Patent 3,058,979 (1962), C.A. 58, 68419 (1963).
2Y0
F LUPHENAZINE HYDROCHLORIDE
( 1 5 )E . I . Anderson, G . B . B e l l i n z o n a , P . N . C r a i g , G . E . J a f f e , K . P . Janeway, C . K a i s e r , B . M . L e s t e r , E . J . N i k a w i t z , A.M. P a v l o f f , H . E . R e i f f and C . L . Z i r k l e , A r z n e i m i t t e l - F o r s c h u n g -91 2 937 (1962). ( 1 6 ) S c h e r i c o L t d . , B r i t i s h P a t e n t 833,474 (1960) C . A . 5 4 , 21,144h ( 1 9 6 0 1 . ( 1 7 )H . L . Y a l e , F . A . S o w i n s k i and J . B e r n s t e i n , U . S . P a t e n t 3 , 2 2 7 , 7 0 8 ( 1 9 6 6 ). (18)D.K.Yung and M . P e r n a r o w s k i , J . P h a r m . S c i . 52, 365 ( 1 9 6 3 ) . ( 1 9) P . R a j e s w a r a n and P . L K i r k , B u l l . N a r c o t ics , U.N.Dept. S o c i a l A f f a i r s l3, 2 1 (19611, C . A . 57, 7390h ( 1 9 6 2 ) . ( 2 0 )J . Q . O c h s and N . H . Coy, S q u i b b I n s t i t u t e , p e r s o n a l communication. (21)Michio Nakanishi, J a p a n . P a t e n t 18,512 ( 1 9 6 6 ) , C . A . 66, 3 7 , 9 3 5 ~ ( 1 9 6 7 ) . ( 2 2 ) M . Izumi and M . N a k a n i s h i , J a p a n . P a t e n t 4537 ( 1 9 6 2 ) , C . A . 58, 10,209h ( 1 9 6 3 ) . ( 2 3 )J . E . F a i r b r o t h e r , S q u i b b I n s t i t . u t e , p e r s o n a l communication. ( 2 4 )H . Kadin, S q u i b b I n s t i t u t e , p e r s o n a l communic a tion. ( 2 5 )H . Kadin, S q u i b b I n s t i t u t e , p e r s o n a l communic a tion. ( 2 6 ) T . ~ . T o z e rand L . D.Tuck, J . Pharm. S c i . 5 4 , 1169 (1964). ( 2 7 )J .E . F a i r b r o t h e r , Squibb I n s t i t u t e , p e r s o n a l communication. ( 2 8 ) Z . Kofoed, C . F a b i e r k i e w i c z and G . H . W . L u c a s , J . C h r o m a t o g r . 23, 410 ( 1 9 6 6 ) . ( 2 9 ) C . I . S m i t h and J . C . Burke, Toxic01 . Appl . P h a r m a c o l . 2, 553 ( 1 9 6 0 ) . ( 3 0 ) A . E . R o b i n s o n , J . P h a r m . P h a r m a c . l8, 1 9 ( 1 9 6 6 ) . ( 3 1 )J . D r e y f u s s and A . I . Cohen, J . Pharm. S c i . -960 826 ( 1 9 7 1 ) . ( 3 2 )J . A l i c i n o , S q u i h b I n s t i t u t e , p e r s o n a l communication.
.
79 I
KLAUS F L O R E Y
(33)T.J.Mellinger and C.E.Keeler, Anal.Chem. 35, 554 (1963). (34)J.B.Ragland and V.J.Kinross-Wright, Anal. Chem. 36, 1356 (1964). (35)S.L.Tompsett, Acta Pharmacol.Toxico1. 2 6 , 298 (1968). (36)E.A.Martin, Can. J. Chem. 44, 1783 (1966). (37)J.A.Ryan, J.Pharm.Sci. 48, 240 (1959). (38)S.P.Agarwal and M.I.Blake, J.Pharm.Sci. 48, 1011 (1969). (39)F.M.Forrest, J.S.Forrest and A.S.Mason, Amer. J.Psychiat. 116, 549 (1959). (40)P.Rajeswaran and P.L.Kirk, Bull. on Narcotics 13, No. 3 , 1 (1961), C.A. 56, 11705b (1962). (41)L.N.Werrum, H.T.Gordon and W.J.Thornburg, J. Chromatogr. 2, 125 (1960). (42)T.J.Mellinger and C. E.Keeler, J.Pharm.Sci.51, 1169 (1962). (43)K.Macek and J.Vecerkova, Pharmazie 2 0 , 605 (1965). (44)A.NoirfaliseY J.Chromatogr. l9, 68 (1965). (45)I.Zingales, J. Chromatogr. 3 l , 405 (1967). (46)T.Fuwa, T.Kido and H.Tanaka, Yakuzaigaku 25, 138 (1965), C. A. 64, 6405f (1966). (47)Y.Kiroshita, H.Koike, K.Iwashima and S.Oriyama, Nagayu Shiritzu 12, 33 (1964), C.A. 64, 19324h (1966). (48)W.W.Fike, Anal. Chem. 3 8 , 1697 (1966). (49)P.Schweda, Anal.Chem. 2, 1019 (1967), C.A. 67, 50918j (1967). (50)F.Dondzila, Squibb Institute, personal communication, (51)A.Noirfalise, J.Chromatogr. 20, 61 (1965). (52)J.Cochin and J.W.Daly, J.Pharmaco1.Exp.Ther. 139, 154 (1963). (53)J.Sunshine, Amer. J.Clin. Pathol. 40, 576 (1963). (54)A.Noirfalise and M.H.Grosjean, J.Chromatogr. 16, 236 (1964). 292
FLUPHENAZINE H Y D R O C H L O R I D E
25 (1968), C.A. (55)J.Jeunier, Ann. Pharm.Fr. 26, 68, 117170a (1968). (56)J.W.Steele, Can.Pharm.J.Sci.Sect. 97, 91 (1964), C.A. 60, 15681d (1964). (57)M.W.Anders and G . J.Mannering, J.Chromatogr. 7, 258 (1962). (58)N.C.Cain and P.L.Kirk, Microchem. J. l2, 256 (1967). (59)C.R.Fontan, N.C.Cain and P.L.Kirk, Mikrochim. 1chnoanal.Acta 1964, 326. (GO)J.Sunshine, Handbook of Analytical Toxicology. The Chemical Rubber Co. (1969). (Gl)S.L.Tompsett, Acta Pharmacol.Toxico1. 2% 303 (1968). (62)H.R.Roberts, Squibb Institute, personal communication. (63)H.R.Roberts and K.Florey, J.Pharm.Sci. 5 l , 794 (1962). (64)R.B. Stasiak and N.H.Coy, Squibb Institute, personal communication. (65)J.L.Kiger and J.G.Kiger, Ann.Pharm.Franc. 23, 489 (1965), C.A. 6 4 , 6407e (1966). (66)L.A.Araya Ro jas, An.Fac.Quim.Farm.Univ.Chile 15, 101(1963), C.A. 62, 1 2 9 7 6 ~(1965). (67)LJacobson, Squibb Institute, personal communication. M.A.Mahju and R.P.Maicke1, Biochem.Pharmaco1. (68 18, 2701 (1969). (69 J.N.T.Gilbert and B.J.Millard, 0rg.Mass. Spectrom. 2, 17 (1969). (70 P.M.Seeman and H.S.Bialy, Biochem.Pharmaco1. 12, 1181 (1963). (71)B.A.Persson, Acta Pharm.Suec. 2, 335 (1968). (72)C.N.Andres,J.Ass .Offic.Anal.Chem. 51, 1020 (1968) and ibid. 53, 834 (1970). (73)H.Weissenstein, E.Feller and G.Schelle, Arzneimittel-Forschung l8, 685 (1968). (74)M.Pinkasova, Cesk.Farm. l7, 181 (1968).
KLAUS FLOREY
(75)B.Gothelf and A.G.Karczmar, 1nt.J.Neuropharmacol. 2, 95 (1963). (76)J. Dreyfuss, J. J.Ross, Jr. and E.C.Schreiber, J.Pharm.Sci. 60, 821 (1971). (77)A. deLeenheer and A. Heyndrickx, J.Ass. O f f ic. Anal.Chem. 54, 625 and 6 3 3 (1971). (78)C . Korczak-Fabierkiewicz, G. C imbura, J. Chromatogr. 53, 413 (1970). (79)A.Viala, J.P.Cano and A.Philippe, Ann.Pharm. Franc. 27, 511 (1969).
294
Klaus Florey
263
KLAUS FLOREY
CONTENTS DES C R I P T I ON 1.1 N a m e , F o r m u l a , M o l e c u l a r Weight I.2 A p p e a r a n c e , C o l o r , O d o r 2. PHYSICAL P R O P E R T I E S 2 . 1 I n f r a r e d Spectra 2.2 N u c l e a r Magnetic R e s o n a n c e S p e c t r a 2 . 3 U l t r a v i o l e t Spectra 2 . 4 Mass Spectra 2 . 5 pka, p H 2 . 6 Melting R a n g e 2 . 7 Differential Thermal A n a l y s i s 2.8 Thermogravimetric Analysis 2.9 Solubility 2 . 1 0 crystal Properties 3. SYNTHESIS 4 . S T A B I L I T Y - DEGRADATION 5 . DRUG METABOLIC PRODUCTS 6 . METHODS OF ANALYSIS 6.1 Elemental Analysis 6 . 2 Nonaqueous Titration 6 . 3 Polarographic A n a l y s i s 6.4 S p e c t r o f l u o r o m e t r i c A n a l y s i s 6.5 C o l o r i m e t r i c A n a l y s i s 6.6 E l e c t r o p h o r e s i s 6.7 Chromatographic Analysis 6 . 7 1 Paper 6 , 7 2 Thin-Layer 6.73 Column 6 . 7 4 Gas - L i q u i d 7 . COUNTERCURRENT SEPARATION I D E N T I F I C A T I O N AND DETERMINATION 8. I N BODY F L U I D S AND T I S S U E S 9. MISCELLANEOUS 10. REFERENCES
1.
2 64
FLUPHENAZINE H Y D R O C H L O R I D E
1.
DESCRIPTION 1.1 Name Formula, Molecular Weight Fluphenazine Hydrochloride is 4 - { 3 - [ 2 krifluoromethy1)-phenothiazine-10-yll propyl) piperazine ethanol dihydrochloride: also 4-(3-[lo -2(-trifluoromethy1)-phenothiazinylI propy1)-1piperazine-ethanol dihydrochloride; 1- (2-hydroxy ethyl)-4- [2-trifluoromethyl-lO-phenothiaz~nyl) propyl ] piperazine dihydrochloride; 10-(3'-[4"(B-hydroxy ethyl)-1"-piperazinyl] propyl-2-trifluoromethyl-phenothiazine dihydrochloride: S94; SQ 4918.
1.2 Appearance, Color, Odor White to off-white, odorless crystalline powder. PHYSICAL PROPERTIES 2.1 Infrared Spectra The infrared spectra of Squibb House Standard #48101-001, in mineral oil mull and KBr from MeOH, are presented in figures 1 and 2.1 The infrared spectrum of the free base, and a discuss ion of spectra-structure correlation of phenothiazines have been presented. 9 The spectra published by Sammul, et al.4 are in essential agreement with those presented here. 2.
265
FREQUENCY (CM-1)
x
r
t.
5 n
r 0 D rn
<
2
3
4
5
6
7 8 WAVELENGTH (MICRONS1
I0
II
12
13
is
Fig. 1 Infrared Spectrum of fluphenazine hydrochloride in mineral oil mull. Instrument: Perkin-Elmer 621
is
n
r
5 I rn
z B N 2
rn
I
< o n
0 c,
I
r 0
n
0
rn
Fig. 2 Infrared Spectrum of fluphenazine hydrochloride in KBr from methanol. Instrument: Perkin-Elmer 621
KLAUS F L O R E Y
N u c l e a r M a q n e t i c Resonance S p e c t r a The NMR s p e c t r a o f f l u p h e n a z i n e base a n d h y d r o c h l o r i d e a r e p r e s e n t e d i n f i g u r e s 3 and 4 . 5 The s p e c t r u m o f t h e f r e e base i s e s s e n t i a l l y i d e n t i c a l w i t h t h a t p u b l i s h e d p r e v i o u s l y . 3 The f o l lowing a s s i g n m e n t s c a n be made for c h e m i c a l s h i f t o f t h e s p e c t r u m of t h e f r e e base. 6 2.2
a b
6.05 8.09 7.57 6.41 6.93 2.9-3.0
C
d
e f
2.3 corded:
triplet multiplet absorption envelope triplet singlet aromatic
Ultraviolet Spectra The f o l l o w i n g U . V . d a t a h a v e been re1.
i n methanol’
( I n s t r u m e n t Cary 1 5 )
Amax 261 mp E i E m 646
A m a x 310 mp, E1% 69 1cm
268
Fiq. 3 NMR Spectrum of fluphenazine base in deuterochloroform. Inscrument: Varian A-60
-*+
i i
r B C
v)
n
r
0
n
rn
<
L
r a*
-I I
l . . . . , . , . , ~ . , . , , , , , , ~
, . . . . . .
I
7a
.
.
I
.
I
A4
I
30
1 1 1 , , 1 , , , , , , , , , 1 , , , , , , , , , ~
I
.(I
.
.
I
.
.
.
I
I
30
10
I
I
I
.
. . I. . . . I 0
' .
.I 0
Fig. 4 NMR Spectrum of fluphenazine hydrochloride in deuterochloroform. Instrument: Varian A-60
M ,i
FLUPHENAZINE HYDROCHLORIDE
2.3
U l t r a v i o l e t S p e c t r a Cont d . 2. i n 95% e t h a n o l 3 Amax 264 Log E 4 . 5 9 Amax 316 Log E 3 . 6 9 Amin 239 Log E 4 . 2 8 Amin 300 Log E 3 . 5 1 3. i n 50% a l c o h o l ( I n s t r u m e n t Beckman
DU)
ElPPm 1c m
Xmax 259 Xmax 305
4.
0.052 0.006 0.013 0.002
Xmin 224 Xmin 280 i n methanol8 Xmax 259 Log
E
4.53
2.4
Mass S p e c t r a The l o w - r e s o l u t i o n m a s s s p e c t r u m o f f l u p h e n a z i n e base i s r e p r e s e n t e d i n f i g u r e 5 . 9 The h i g h - r e s o l u t i o n s p e c t r u m shows t h e f o l l o w i n g f r a g m e n t a t i o n p a t t e r n o f s i d e c h a i n and nucleus’, which i s i n a g r e e m e n t w i t h t h e r e s u l t s o f an independent i n v e s t i g a t i o n . 69
?zyz 307-1H
2 8 0 I157
t
H2 *CH
.+)
-CH2 I
350
27 1
Fig. 5 Low-resolution Mass Spectrum of fluphenazine.
Instrument: MS-9
FLUPHENAZINE HYDROCHLORIDE
Another prominent fragment is t h e follow-
ing:
248
The s i g n i f i c a n t m a s s numbers shown a b o v e c a n be t a b u l a t e d a s f o l l o w s : Mass U n i t s D i f f . C H N 0 S F U n s a t O/E* Found- C a l c u l a t e d 437 2 2 26 3 1 1 3 10 0 0.3 419 2 2 24 3 - 1 3 10 0 -0.7 4 0 6 2 1 2 3 3 - 1 3 10.5 E -1.6 363 19 18 2 - 1 3 10.5 E -0.3 307 16 1 2 1 - 1 3 10 0 -1.1 306 1 6 11 1 - 1 3 1 0 . 5 E -0.3 280 14 9 1 - 1 3 9.5 E -0.1 E 0.5 266 1 3 7 1 - 1 3 9.5 248 14 9 1 - - 3 9.5 E -0.1 1 7 1 9 19 2 1 - - 1.5 E -0.1 E -0.7 1 5 7 8 1 7 2 1 - - 1.5 E -0.1 1 4 3 7 15 2 1 - - 1.5
"0
=
odd e l e c t r o n i o n : E
2.5
pKa;
=
pH
even e l e c t r o n i o n
The a p p a r e n t pKa of f l u p h e n a z i n e d i h y d r o c h l o r i d e i n aqueous a l c o h o l i c s o l u t i o n h a s b e e n d e t e r m i n e d as 8.05 f o r t h e s e c o n d b a s i c g r o u p . F o r t h e f i r s t b a s i c g r o u p , a v a l u e o f 3.90 w a s obt a i n e d . lo I n d e p e n d e n t l y , v a l u e s o f 7 . 8 (estimat e d ) and 3 . 6 0 , r e s p e c t i v e l y , w e r e o b t a i n e d . 7 1 The p H of a n aqueous s o l u t i o n o f t h e d i h y d r o c h l o r i d e i s 2 . 4 ; t h a t o f monohydrochloride is 273
KLAUS FLOREY
5 . 5 . 11 2.6
M e l t i n q Ranqe The f o l l o w i n g m e l t i n g p o i n t s o f t h e hydroc h l o r i d e have been r e p o r t e d : 238.5-239.5O ( R e c r y s t . from abs , e t h a n o l USP M e t h o d p ( R e c r y s c . from abs. e t h a n o l ) l2 2 31-2 33' 224-226OI3, l4 224.5-226O ( R e c r y s t . from m e t h a n o l - e t h e r ) 15 2 3 5-2 3 701 7 l7 231-232OI8 2.7
D i f f e r e n t i a l Thermal A n a l y s i s D i f f e r e n t i a l Thermal A n a l y s i s of S q u i b b House S t a n d a r d #48101-001 showed a n endotherm a t 237OC. ( I n s t r u m e n t : DuPont) 1 Thermoqravimetric A n a l y s i s Thermogravimetric a n a l y s i s o f Squibb House S t a n d a r d #48101-001 gave no w e i g h t l o s s up t o 15Ooc ( I n s t r u m e n t : DuPont) 2.8
2 9
Solubility Fluphenazine hydrochloride is hygroscopic and e x t r e m e l y s o l u b l e i n w a t e r (70% w/v a t 25OC pH 2 . 4 ) . S o l u b i l i t y i n e t h a n o l : 15% w/v a t 25OC. L e s s t h a n 2% (w/v) i n e t h e r , a c e t o n e , benzene, l i g r o i n . 11 I n s o l u b l e i n c h l o r o f o r m and e t h e r . 67 2.10 c r y s t a l P r o p e r t i e s For f o r e n s i c i d e n t i f i c a t i o n , a p i c t u r e o f f l u p h e n a z i n e d i h y d r o c h l o r i d e c r y s t a l s from 95% e t h a n o l h a s been p u b l i s h e d w i t h t h o s e o f o t h e r p h e n o t h i a z i n e s . 19' A n x-ray powder d i f f r a c t i o n p a t t e r n of fluphenazine dihydrochloride is presented i n 274
FLUPHENAZINE HYDROCHLORIDE
table l.20 S l i g h t l y d i f f e r e n t d-values ( 1 8 . 2 ; 9.07; 8 . 7 1 ; 6 . 0 7 ; 5 . 0 3 ; 4 . 8 5 ; 4 . 3 2 ; 4 . 0 2 ; 3. 7 5 ) 7 and ( 1 7 . 2 0 ; 1 1 . 5 0 ; 8 . 4 0 ; 5 . 9 0 ; 5 . 5 0 ; 5 . 4 0 ; 4 . 7 0 ; 4.50; 4 . 2 0 ; 4 . 1 0 ; 4 . 0 0 ; 3 . 9 0 ; 3 . 7 5 ; 3 . 6 0 ; 3 . 3 5 ; 3.20; 3.10; 2.85; 2.80; 2.65; 2.55; 2.45; 2.40; 2 . 2 2 ; 2 . 1 5 ) 77 w e r e a l s o r e p o r t e d . 3 . SYNTHESIS S y n t h e t i c pathways t o f l u p h e n a z i n e ( I ) a r e shown i n f i g u r e 6. The s y n t h e s i s u s u a l l y i n v o l v e s t h e a d d i t i o n of t h e propylpiperazine ethanol s i d e c h a i n t o t h e 3-trifluoromethyl-phenothiazine nuc l e u s (11). The f o l l o w i n g r o u t e s t o ( I ) h a v e b e e n reported: (1)D i r e c t a d d i t i o n o f 4 ( 3 - c h l o r o p r o y l ) -1-pipe r a z i n e e t h a n o l t o (11) t o y i e l d ( I ) .2 P ( 2 ) Formation o f i n t e r m e d i a t e 1 0 ( 3 - c h l o r o p r o p y 1 ) - 3 t r i f l u o r o m e t h y l p h e n o t h i a z i n e (111) und e r a v a r i e t y of c o n d i t i o n s , and c o n d e n s a t i o n o f (111) w i t h p i p e r a z i n e e t h a n o l t o ( I ). l2 16,l7 (3)Condensation of 3(2-trifluoromethyl-10phenothiazinyl) propylpiperazine ( I V ) with ethyle n e o x i d e 1 5 , w i t h @-bromo-ethyl a c e t a t e v i a (VI)1 3 9 14, o r w i t h 2-bromo e t h a n o l . This last method h a s a l s o been used t o p r e p a r e ( I ) , l a b e l e d w i t h 14C i n t h e two e t h a n o l c a r b o n s . 6 ( 4 )R e d u c t i o n o f 4 (f3- ( 1 0 - p h e n o t h i a z i n y l ) t h i o propionyl) piperazine ethanol with lithium aluminum h y d r i d e t o ( I ) .2 2 J
-
4 . STABILITY DEGRADATION F l u p h e n a z i n e h y d r o c h l o r i d e is h y g r o s c o p i c . When m o i s t u r e i s e x c l u d e d , t h e c r y s t a l l i n e m a t e r i a l i s s t a b l e f o r a t l e a s t 6 months a t temperat u r e s as h i g h as 5Ooc, b o t h under a i r and n i t r o gen. The compound i s p h o t o s e n s i t i v e , s i n c e t h e s u r f a c e o f c r y s t a l s exposed t o i n c a n d e s c e n t l i g h t f o r p e r i o d s o f more t h a n 4 weeks d i s c o l o r e d t o 215
KLAUS F L O R E Y
2.10 Crystal Properties
(Continued)
TABLE I X-ray Powder D i f f r a c t i o n p a t - t e r n of S q u i b b House S t a n d a r d #48101-001 d (? * i) Rel. I n t e n s i t y * * 17.65 2 0.05 0.26 0.12 9.80 8.90 0.58 (5) 8.70 0.42 6.02 0:36 5.56 5.46 0.24 0.14 5.36 0.31 5.15 4 . 8 3 _f 0 . 0 2 0.47 4.75 0.59 (4) 4.49 0.71 ( 2 ) 4. 31 0.48 4.23 0.22 4.12 0.12 4.00 0.63 (3) 3.90 1 . 0 0 (1) 3.72 u-x5 3.69 0.47 3.57 0.32 0.21 3.40 0.16 3.27 0.24 3.23 0.42 3.11 3.06 0.19 0.22 3.00 2.93 0.12 2.87 0.15 2.72 0.16 2.67 0.16 2.54 0.14 2.42 0.15 2.34 0.15 2.27 0.14 2.02 0.15 d = (interplanar distance)
m
s i AB n
Q
A-
**
2
1.539 R a d i a t i o n : ka and ka2 c o p p e r 1 Based on h i g h e s t i n t e n s i t y of 1 . 0 0 I n s t r u m e n t : Phillips 276
Curve N O . 357
I
< O m 0 D
I
r 0
a
o rn
Fig. 6
S y n t h e t i c Pathways.
KLAUS F L O R E Y
light tan. I n aqueous s o l u t i o n , f l u p h e n a z i n e hyd r o c h l o r i d e d e g r a d e s when e x p o s e d t o l i g h t . S o l u t i o n s a t 0.01% i n e t h a n o l , w a t e r , 0.1g H C 1 and 0 . 5 N H2SO4 w e r e k e p t i n t h e d a r k o r e x p o s e d t o f l u o r e s c e n t c e i l i n g l i g h t a t room t e m p e r a t u r e (see s e c t i o n 2 . 3 ) . A f t e r 1 7 days i n t h e d a r k , a l l sol u t i o n s r e t a i n e d t h e i r U.V. absorbance i n t h e Only t h e 0 . 5 g H2S04 s o l u t i o n 255-265 m p r e g i o n . showed a s l i g h t d e c l i n e i n a b s o r b a n c e . In the light-exposed samples, only t h e e t h a n o l i c solut i o n r e m a i n e d unchanged, w h i l e i n t h e r e m a i n i n g s o l u t i o n s d i s c o l o r a t i o n and t h e a p p e a r a n c e o f m u l t i p l e b a n d s a t 240 and 255 mw w e r e n o t e d . 6 4 Exposing a 0 . 0 5 % f l u p h e n a z i n e h y d r o c h l o r i d e i n 0.1g H C 1 s o l u t i o n i n p y r e x g l a s s t o s u n l i g h t c a u s e d a 40% d r o p o f a b s o r b a n c e i n 2 h o u r s . 2 3 A s t u d y o f t h e pH dependency o f t h e l i g h t i n s t a b i l i t y showed a s i m i l a r d e c l i n e i n a b s o r b a n c y from p H 0 . 3 t o 9, w h e r e a s above pH 11 t h e d e g r a d a t i o n w a s r e t a r d e d . 2 4 Again, it w a s found t h a t s o l v e n t s s u c h as m e t h a n o l , e t h a n o l , dimethylformami d e , e t h y l e n e g l y c o l , and e t h e r p r e v e n t o r r e t a r d degradation. The l i g h t - c a t a l y z e d d e g r a d a t i o n i s n o t prev e n t e d b y e x c l u s i o n o f oxygen25 and p r o c e e d s t h r o u g h f o r m a t i o n of t h e f r e e r a d i c a l ( c f 2 6 ) . The r a d i c a l c a n a l s o be formed b y a n o x i d i z i n g a g e n t , s u c h as c e r i c ammonium s u l f a t e . 26 I n phenothiazines g e n e r a l l y , t h e semiquinone r a d i c a l u n d e r g o e s d i s p r o p o r t i o n a t i o n . The p r o d u c t s o f o x i d a t i o n ar e t h e s u l f o x i d e o r t h e 3 ( o r 7 ) -hydroxy-derivatives. However, f o r m a t i o n of t h e l a t t e r is s e v e r e l y i n h i b i t e d i n 10-substituted p h e n o t h i a z i n e s . 2 6 The s e c o n d - o r d e r d e c a y r a t e c o n s t a n t of t h e s e m i q u i n o n e r a d i c a l of f l u p h e n a z i n e w a s f o u n d t o be 1100 (l/mole/min) , as meas u r e d by e l e c t r o n s p i n r e s o n a n c e . 26 The s u l f o x i d e c o n t e n t o f s e v e r a l commercial b a t c h e s o f f l u p h e n a z i n e w a s d e t e r m i n e d t o be a r o u n d 0 . 1 % b y 218
FLUPHENAZINE H Y DROCH L O R I DE
a s p e c t r o f l u o r o m e t r i c method ( e x c i t a t i o n wavel e n g t h 350 m@; f l u o r e s c e n c e w a v e l e n g t h 400 mp; see a l s o s e c t i o n 6 . 4 ) .27 The s u l f o x i d e c o n t e n t c a n a l s o be measured b y a t h i n - l a y e r method (see a l s o s e c t i o n 6.72).2 8 So f a r , t h e r e i s no e v i d e n c e f o r t h e d e g r a d a t i v e f o r m a t i o n of 7 - h y d r o x y f l u p h e n a z i ~ which h a s been i d e n t i f i e d as a m e t a b o l i c p r o d u c t (see s e c t i o n 5 ) . 5 . DRUG METABOLISM Acetylf luphenazine ( V I , Figure 6 ) was hydrolyzed e n z y m a t i c a l l y t o f l u p h e n a z i n e ( I ) i n v i t r o b y homogenates o f s m a l l i n t e s t i n a l mucosa, l i v e r , and b r a i n from e i t h e r t h e r a t o r t h e monkey.29 ( S e e a l s o s e c t i o n 7 ) . When f l u p h e n a z i n e w a s i n c u b a t e d w i t h a " m i c r o s o m a l and s o l u b l e " f r a c t i o n o f r a t l i v e r s , h y d r o x y l a t i o n on t h e p h e n o t h i a z i n e r i n g was o b s e r v e d . 3 0 The s u l f o x i d e w a s n o t det e c t e d i n t h e e x t r a c t , and it seems u n l i k e l y t h a t b i o l o g i c a l sulfoxidation precedes hydroxylation i n t h e s e s y s t e m s . 3 0 The b i o l o g i c a l d i s p o s i t i o n and m e t a b o l i c f a t e of fluphenazine-14C i n t h e dog and r h e s u s monkey w a s s t u d i e d . 7 6 When dogs w e r e t r e a t e d w i t h f l u p h e n a z i n e - 1 4 C , 7-hydroxyfluphenazine ( V I I , F i g u r e 6) w a s i from t h e u r i n e a n d i d e n t i f i e d by s y n t h e s i s . human b l o o d and u r i n e s m a l l amounts o f f l u p h e n a z i n e and i t s s u l f o x i d e , b o t h i n f r e e and c o n j u g a t e d form w e r e i d e n t i f i e d . 7 9 T i s s u e d i s t r i b u t i o n and e x c r e t i o n w e r e s t u d i e d i n t h e r a b b i t . 73 6. METHODS OF ANALYSIS 6.1 E l e m e n t a l A n a l y s i s % Found Element % Calc. R e f . 11 12 15
Wa:f
C H
51.76 5.53
51.72 5.62 8.04
0
8.23 3.13 6.28
F N
S
c1
11.17
13.89
-
-
6.05 13.66
51.73 5.86
-
8.00
-
-
-
51.68 5.42
-
-
-
KLAUS F L O R E Y
6 . 2 Nonaqueous T i t r a t i o n Nonaqueous t i t r a t i o n i n g l a c i a l a c e t i c a c i d w i t h 0.01g p e r c h l o r i c a c i d and Q u i n a l d i n e Red o r c r y s t a l v i o l e t i n d i c a t o r g a v e a n e u t r a l i z a t i o n e q u i v a l e n t t o 259 ( c a l c . 2 5 5 . 2 2 ) . 32
6 . 3 Polarographic Analysis The h a l f - w a v e p o t e n t i a l i n a l c o h o l i c s u l f u r i c a c i d s o l u t i o n w a s f o u n d t o be 0 . 7 1 4 ( v o l t v s . normal c a l o m e l e l e c t r o d e ) , c o m p a r a b l e t o t h a t o f o t h e r p h e n o t h i a z i n e s .8 6 . 4 Spectrofluorometric Analysis Fluphenazine hydrochloride, l i k e o t h e r phenothiazine drugs, e x h i b i t s fluorescence. This phenomenon w a s f i r s t e x p l o r e d i n 0 . 2 g s u l f u r i c a c i d . 33 The f l u o r e s c e n c e p e a k w a s f o u n d i n t h e r a n g e o f 450 t o 475 mp, s h i f t i n g t o s l i g h t l y l o w e r w a v e l e n g t h upon o x i d a t i o n w i t h p o t a s s i u m permanganate. A s l i t t l e as 0 . 0 1 t o 0 . 0 5 1-14 o f t h e p u r e s u b s t a n c e per m l . o f 0 . 2 g s u l f u r i c a c i d c o u l d be d e t e c t e d . Due t o i n t e r f e r e n c e by o t h e r substances, t h e s e n s i t i v i t y i n biological f l u i d s was l o w e r ( a b o u t 0 . 8 pg/ml f l u i d ) . T h i s method i s s u p e r i o r t o d e t e r m i n a t i o n b y U . V . , where about 4 p.g/ml a r e r e q u i r e d . I n 50% a c e t i c a c i d , t h e f o l l o w i n g d a t a w e r e o b t a i n e d , w i t h o u t o r w i t h o x i d a t i o n w i t h 30% hydrogen peroxide34:
A c t i v a t i o n maximum,m~ F l u o r e s c e n c e maximum,my Unoxidized Oxidized Unoxidized Ox i d i z e d Ref 34 Ref 35
330
-
345 3 50
460
-
380 410
I n t h e u n o x i d i z e d form, a s h a r p i n c r e a s e i n f l u o r e s c e n c e i n t e n s i t y o c c u r r e d above p H 7; i n t h e o x i d i z e d form, i n t e n s i t y w a s a b o u t e q u a l from 280
FLUPHENAZINE HYDROCHLORIDE
p H 2 t o p H 1 2 and f e l l p r e c i p i t o u s l y a t p H 1 2 . I n t h e o x i d i z e d form, as l i t t l e as 0 . 5 wg/ml c o u l d be d e t e r m i n e d q u a n t i t a t i v e l y . F l u p h e n a z i n e w a s r e l a t i v e l y l e s s f l u o r e s c e n t t h a n some o t h e r p h e n o thiazines. In concentrated s u l f u r i c acid, t h e a c t i v a t i o n maximum w a s found a t 325 m p and t h e f l u o r e s c e n c e maximum a t 500 mp. 36 The i n s t r u m e n t u s e d i n a l l i n v e s t i g a t i o n s w a s t h e Aminco-Bowman S p e c t r o f l u o r o m e t e r .
-
6.5 Colorimetric Analysis Color T e s t s f o r Identification The g e n e r a l method o f Ryan37 t o q u a n t i t a t e unoxidized phenothiazine d e r i v a t i v e s by c o l o r complex f o r m a t i o n w i t h p a l l a d i u m c h l o r i d e ( 5 0 0 550 mw m a x i m a ) h a s a l s o b e e n a d o p t e d f o r f l u p h e n a z i n e h y d r o c h l o r i d e ( s e e N . F . X I I 1 ) and c a n b e u s e d f o r s t a b i l i t y measurements. Like o t h e r phenothiazines, fluphenazine h y d r o c h l o r i d e reacts w i t h c e r i c s u l f a t e , f i r s t b y forming a red-colored semiquinone f r e e r a d i c a l (420 ml), f o l l o w e d by f o r m a t i o n o f t h e c o l o r l e s s s u l f o x i d e d e r i v a t i v e . T h i s r e a c t i o n h a s b e e n made t h e basis o f a p h o t o m e t r i c t i t r a t i o n . 3 8 The f o l l o w i n g c o l o r t e s t s h a v e been reported. ( F o r a d d i t i o n a l t e s t s , see a l s o s e c t i o n s 6 . 7 1 and 6 . 7 2 ) . FOR COLOR TESTS SEE TABLE I1
6 . 6 E l e c t r o p h o r e s is E l e c t r o p h o r e s i s w a s c a r r i e d o u t on f l u p h e n a z i n e h y d r o c h l o r i d e and s e v e r a l o t h e r phenot h i a z i n e t r a n q u i l i z e r s i n var io u s b u f f e r s prop o s e d by Werrum. 41 A Gordon-Misco a p p a r a t u s , Whatman 3MM paper 30 c m i n w i d t h , and a p o t e n t i a l d i f f e r e n c e of 500 t o 800 v w e r e u s e d . Detection o f s p o t s w a s c a r r i e d o u t w i t h a 40% s u l f u r i c a c i d
zx 1
TABLE I1 Reaqent 0.001N M e r c u r i c n i t r a t e i n h y d r o c h l o r i c a c i d 0.5% aq . ammonium m o l y b d a t e 0.5% aq. ammonium v a n a d a t e 3 7% f o r m a l d e h y d e i n c o n c . s u l f u r i c a c i d (1:20) 0.5% aq. s e l e n i o u s a c i d 1% sodium t u n g s t a t e 0.5% titanium dioxide i n conc.sulfuric acid Fuming n i t r i c a c i d , t h e n e t h a n o l i c p o t a s s i u m hydroxide (3%) 10% aq. sucrose Conc. s u l f u r i c a c i d p-dimethylaminobenzaldehyde 10% i n g l a c i a l acetic acid 1% c o b a l t a c e t a t e : 10% i s o p r o p y l a m i n e i n a c e t o n e 10% aq. c h l o r a m i n e T 1% aq. palladium c h l o r i d e a c i d i f i e d w i t h hydrochloric acid N i t r i c acid Conc. s u l f u r i c a c i d , 6% u r a n i u m n i t r a t e i n 95% e t h a n o l ( 3 : 1 7 ) 0 . 5 % ammonium v a n a d a t e i n c o n c . s u l f u r i c a c i d 4% a q . s i l v e r n i t r a t e s o l u t i o n a c i d i f i e d w i t h 0 . 1 N sulfuric acid 2% Zmmonium f e r r o u s s u l f a t e i n c o n c . sulfuric acid
as.
32
N
Color pink-violet blue-pink-violet grey-yellow 1i g h t red grey-green purple orange
R ef -
39 40 40 40 40 40 40
yellow-green grey-yellow l i g h t brown
40 40
5
l i g h t brown light blue l i g h t yellow
40 18 18
<
orange orange-brown
18 18
l i g h t orange y e l l o w brown
18 18
c r e a m wh it e
18
-
40
75
x
5
FLUPHENAZINE HYDROCHLORIDE
spray (orange color) or fluorescence under W light.42 For fluphenazine hydrochloride, anodic migration (cm) after 40 to 60 minutes was: pH :
Migration:
3.3 6.8
4.7 3.7
6.0
4.3
7.2 4.5
8.0 2.7
9.3 1.2
6.7 Chromatoqraphic Analysis 6.71 Paper Chromato-ra hic Analysis The following chromatographic systems have been reported: Solvent Systems: 1% Sodium formate 1% Sodium formate 90/n-Propanol 10 1% Sodium formate 90/1N Ammonia 10 1g Sodium formate 97/Formic acid 3 1% Sodium acetate 1% Sodium acetate 90/n-Propanol 10 10% Sodium chloride 92/n-Propanol 8 Formamide + Ammonium formate/Benzene Formamide + Ammonium formate/Cyclohexane-benzene (9:1) Petroleum (b.p. 195-220°) /EthanolAmmonia-water (60:2: 38)
Rf Ref 0.23 42 0.32 42 0.19 42 0.42 42 0.17 42 0.48 42 0.45 42 0.71 43 0.22 43 0.59 43
Ref color Detection systems : 40% H2S04 spray orange 42,43 Fluorescence under W light bluish yellow 42,43 orange 43 Dragendorff-reagent Cerium sulfate in 2g sulfuric acid red 43 1% gold (111) chloride red 43 1% dimethylaminobenzaldehyde in ethano1:chloroform (95:5)red 43 A quantitative paper chromatographic method62 can be used to measure the purity of '83
K L A U S FLOREY
fluphenazine, following published procedures63. The paper is impregnated with castor oil ( U S P ) in ether. Developing solvent is methanol-water 85:15. Rf values of fluphenazine base approx. 0.79, fluphenazine hydrochloride 0.83. The zones are eluted from the chromatogram with 95% ethanol and the absorbances o f the eluates are determined at 261 m p in a spectrophotometer. 6.72
3
The following thin-layer chromatographic systems have been reported: FOR ABOVE SEE TABLE I11
6.73 Column (Ion-exchanqe) Chromatographic Analysis~ Ion-exchange analysis of fluphenazine hydrochloride and other phenothiazine tranquilizers on a column of Dowex sulfuric acid resin 50W-X8 was possible, but not too practical for analysis of tablets.56 6.74 Gas-Liquid Chromatoqraphic Analysis Fluphenazine was not eluted after 90 minutes at 270°, using a 210 SE-30 silicone polymer on a 80-100 mesh Gas-chrom S diatomaceous earth column57; with a 3% SE-30 on 80-100 mesh Gas-chrom Q at 25Ooc, it eluted in 4.9 minutes.28 Fluphenazine was also well separated from other tranquilizers on a 120 mesh silanized Gas-chrom P column coated with 1% Hi-Eff-8B (cyclohexanedimethanol succinate) at a temperature of 22Ooc and a retention time of 5 minutes.58 The identification of fluphenazine and other phenothiazine tranquilizers by gas chromatography of their pyrolysis products has also been reported.59
284
TABLE I11 Ads orbent Silica Gel 11
I 11 I1
I1 I' 1' 11
N
m vI
I1
Cellulose Silica Gel 11
11 11 I1
11 11
So1vent Svs tem tert. Butyl alcohol 90/1g Ammonia 10 n-Propanol 88/1g Ammonia 1 2
Ether, sat. with water 70% Methanol 85% n-Propanol n-Butanol sat. with 1s Ammonia Benzene-Dioxane-aq. Ammonia ( 6 0 : 3 5 : 5 ) Ethanol-Acetic acid-water ( 5 0 :30: 2 0 ) Methanol-butanol ( 6 0 :4 0 ) Cyclohexane-Diethylamine (9: 1) 5% aq. Ammonium sulfate sat. with Isobutanol Cyclohexane-methanol ( 5 0 : 5 0 and 2 5 : 7 5 ) Chloroform-acetone ( 5 0 : 5 0 ) Chloroform-methanol ( 5 0 : 5 0 ) chloroform-methanol (90: 10) Chloroform-acetone-methanol ( 5 0 : 2 5 : 2 5 ) Chloroform-acetone-methanol ( 2 5 : 5 0 : 2 5 ) Chloroform-acetone-methanol-ammonia ( 5 0 : 25: 25: 1)
Acetone Acetone Acetone-ammonia (100:1)
Rf 0.37 0.56 0.09 0.68 0.27 0.57 0.34 0.58 0.68 0.05
Re f 42 11
11 11 It 11
n
5211
C
r -o
1
rn 11
53
z B N z rn
1
0.03 0.36 0.03 0.59 0.44 0.42 0 , 32
54 44 44 51,44,50 45 44 44
0.54 0.06
44 44 45 44,51
0.16 0.33
0
n
0
c
1
r
0
rn
TABLE I11 Cont'd.
Adsorbent Silica Gel I1
I1 I1 I1
I1
11 11 I1
1 3
II
m
I1
rn
'1 11 '1
11 '1
*
*
* ** **
Silica Ge1,Plain Chromagram,Plain
solvent System Acetone-ammonia (95: 5 ) Acetone-methanol (50 :50) Acetone-methanol-ammonia (50 :50 :1) Methanol Methanol-ammonia ( 98 :2 ) Methanol-ammonia (98: 2 ) Chloroform-methanol-ammonia ( 8 0 : 20 : 1) Chloroform-ethanol-ammonia (80: 2 0 : 1) Acetone-benzene-ammonia (30:70:5) Butanol-pyridine-water (1:2: 1) Cyclohexane-benzene-diethylamine (75:15:10) Cyclohexane-benzene-diethylamine (75: 1 5 : 20) Methanol Acetone Benzene-ethanol-ammonia (95: 15: 5 ) Methanol 95% ethanol Chloroform + cyclohexane-diethylamine (50:40: 1 0 ) Chloroform + cyclohexane-diethylamine
Rf Ref 0.54 0.38 0.56 0.34 0.54 0.67 0.68 0.76
44
0.52
47
0.06
48
0.18 0.60
45 48
--
45 30 23 46
0.25
0.30 0.15
45 48
0.24
49
0.06
0.48
TABLE I11 C o n t d A d s o rp t i o n
Chromagram + fluor indicator Silica G e l It
*
** ***
S o l v e n t System
Rf Ref -
c h l o r o f o r m + cyclohexane-diethylamine C h l o r o f o r m - h e x a n e (1:1) 15% aq.ammonium a c e t a t e - m e t h a n o l (20:80)
p r e p a r e d w i t h 0 . 1 M KOH p r e p a r e d w i t h 0.1M NaHS03 I n t h i s s y s t e m , f l u p h e n a z i n e - s u l f o x i d e h a d a n Rf
0.40 0.48
49 50
0.75
28***
of 0.59 2
m
The f o l l o w i n g d e t e c t i o n s y s t e m s h a v e b e e n u s e d : System F l u o r e s c e n c e u n d e r U.V. lamp 40% s u l f u r i c a c i d s p r a y 50% a q . s u l f u r i c a c i d i n e t h a n o l ( 4 : l ) Potassium iodoplatinate 5% f e r r i c c h l o r i d e , 20% p e r c h l o r i c a c i d , 50% n i t r i c a c i d (5:45:50) ( F P N ; F o r r e s t r e a g e n t ) Iodine vapors 1%p o t a s s i u m p e r m a n g a n a t e Dragendorff r eag en t
I
Color Reaction
-
<
0
Ref
42,44,51,30,28,50
orange 42,46,47 orange-red47,52 violet 52,54,44,51,49,28 (flesh or (pale pink 44,45,51,53,54,48 brown 54,44,48 51 brown 51,48
a
0 0
I
6 z0 rn
D e t e c t i o n systems used Cont' d. System 5% p-dimethylaminobenzaldehyde i n 1 8 s s u l f u r i c acid Furf u r a l reagent 1%c e r i u m s u l f a t e i n 2 g s u l f u r i c a c i d 1%g o l d (111) - c h l o r i d e Folin-Ciocalteau reagent 1%ammonium v a n a d a t e i n 10 m l . conc. sulfuric acid 5% cinnamic aldehyde and 5% H C 1 i n e t h a n o l p-benzoquinone i n d i c h l o r o e t h a n e
color
R e a c tion
pink o r orange cameo red red cameo flesh cameo
-
Ref 45,47,51 51,45 47
47 45 45,48 45 55
n
r
0
n
rn
<
F LUPH E NA 2 I N E HY D R OCH LOR I DE
countercurrent Separation F l u p h e n a z i n e w a s s e p a r a t e d from a c e t y l f l u phenazine ( V I , f i g u r e 6) by countercurrent d i s t r i b u t i o n (see a l s o s e c t i o n 5 ) a c r o s s 25 t u b e s i n a s o l v e n t s y s t e m o f 0.1M a c e t a t e b u f f e r , pH 3 . 8 2 , and 1 . 5 % i s o a m y l a l c o h o l i n h e p t a n e . 2 9 7.
I d e n t i f i c a t i o n and D e t e r m i n a t i o n i n Body F l u i d s and T i s s u e s Many o f t h e r e f e r e n c e s c i t e d i n p r e v i o u s sect i o n s c o n c e r n methods d e v i s e d t o i d e n t i f y a n d d e t e r m i n e f l u p h e n a z i n e h y d r o c h l o r i d e and o t h e r phenothiazines f o r pharmacological, toxicological, and f o r e n s i c p u r p o s e s ( c f . 6 0 ) . T h e s e methods c a n be c l a s s i f i e d as f o l l o w s : 8.
43,45,55,39,18,40,74,75 Color r e a c t i o n s 61,43,45,51,74,79 S e p a r a t i o n schemes 33,34,35,36,68 Spectrof luorometry 42 Electrophoresis 43,43 Paper c h r o m a t o g r a p h y (28,42-49, 51-54, 6 5 , 7 4 , T h i n - l a y e r chromatog(78,79 raphy Gas - 1 i q u i d chromat og28,57,58 raphy 65 So 1u b i 1i t y X-ray d i f f r a c t i o n b a n d s 7 M i c r o c r y s t a l l i n e iden19,72 t if i c a tion Miscellaneous The a b s o r p t i o n o f f l u p h e n a z i n e d i h y d r o c h l o r i d e and o t h e r p h e n o t h i a z i n e d e r i v a t i v e s b y s o l i d a d s o r b a n t s , s u c h a s k a o l i n , t a l c and c h a r c o a l , h a s b e e n s t u d i e d b y S o r b y e t a l . i0 The v o l u m e t r i c d e t e r m i n a t i o n o f f l u p h e n a z i n e by s t o i c h i o m e t r i c combinat i o n w i t h a n i o n i c s u r f a c e - a c t i v e a g e n t s , s u c h as sodium l a u r y l s u l f a t e and sodium d i o c t y l s u l f o s u c c i n a t e , h a s b e e n 9.
28Y
KLAUS F L O R E Y
described.66 The surface tension-lowering activity of fluphenazine and other tranquilizers h a s been studied.70 Ion-pair extraction has been described.71 REF ER ENC ES (1) G.A.Brewer, Squibb Institute, personal communication. (2) W.E Thompson, R.J.Warren, J.B.Eisdorfer and J. E.Zarembo, J.Pharm.S c i . 3 , 1819 (1965). (3) R.J.Warren, J.B.Eisdorfer,W.E ,Thompson and J.E.Zarembo, J.Pharm.Sci .55, 144 (1966). (4) O.R.Sammu1, W.L.Brannon and A.L.Hayden, J.Ass .Cffic.Anal.Chem.47, 918 (1964). (5) A.I.Cohen and G.M.Jennings, Squibb Institute, personal communication. (6) H.L.Yale, A.I.Cohen and F.Sowinski, J.Med. Chem.5, 347 (1963). (7) P.Rajeswaran and P.L.Kirk, Bull.Narcotics, 14, 19 (1962), C.A.57, 7390h (1962). (8) FKabasakalian and J.McGlotten, Anal.Chem.2, 431 (1959). (9) A. I.Cohen, Squibb Institute, personal communication. (10)D.L.Sorby, E.M.Plein and J.D.Benmaman, J. Pharm. Sci. 55, 785 (1966). (ll)H.Cords, Squibb Institute, personal communicat ion. (12)H.L.Yale and F.Sowinski, J.Amer.Chem.Soc. 82, 2039 (1960). (13)Smith, Kline and French Laboratories, Brit. Patent 829, 246 (1960), c. A. 54, 174281.1 (1960), (14)G.E.Ullyot, U.S.Patent 3,058,979 (1962), C.A. 58, 68419 (1963).
2Y0
F LUPHENAZINE HYDROCHLORIDE
( 1 5 )E . I . Anderson, G . B . B e l l i n z o n a , P . N . C r a i g , G . E . J a f f e , K . P . Janeway, C . K a i s e r , B . M . L e s t e r , E . J . N i k a w i t z , A.M. P a v l o f f , H . E . R e i f f and C . L . Z i r k l e , A r z n e i m i t t e l - F o r s c h u n g -91 2 937 (1962). ( 1 6 ) S c h e r i c o L t d . , B r i t i s h P a t e n t 833,474 (1960) C . A . 5 4 , 21,144h ( 1 9 6 0 1 . ( 1 7 )H . L . Y a l e , F . A . S o w i n s k i and J . B e r n s t e i n , U . S . P a t e n t 3 , 2 2 7 , 7 0 8 ( 1 9 6 6 ). (18)D.K.Yung and M . P e r n a r o w s k i , J . P h a r m . S c i . 52, 365 ( 1 9 6 3 ) . ( 1 9) P . R a j e s w a r a n and P . L K i r k , B u l l . N a r c o t ics , U.N.Dept. S o c i a l A f f a i r s l3, 2 1 (19611, C . A . 57, 7390h ( 1 9 6 2 ) . ( 2 0 )J . Q . O c h s and N . H . Coy, S q u i b b I n s t i t u t e , p e r s o n a l communication. (21)Michio Nakanishi, J a p a n . P a t e n t 18,512 ( 1 9 6 6 ) , C . A . 66, 3 7 , 9 3 5 ~ ( 1 9 6 7 ) . ( 2 2 ) M . Izumi and M . N a k a n i s h i , J a p a n . P a t e n t 4537 ( 1 9 6 2 ) , C . A . 58, 10,209h ( 1 9 6 3 ) . ( 2 3 )J . E . F a i r b r o t h e r , S q u i b b I n s t i t . u t e , p e r s o n a l communication. ( 2 4 )H . Kadin, S q u i b b I n s t i t u t e , p e r s o n a l communic a tion. ( 2 5 )H . Kadin, S q u i b b I n s t i t u t e , p e r s o n a l communic a tion. ( 2 6 ) T . ~ . T o z e rand L . D.Tuck, J . Pharm. S c i . 5 4 , 1169 (1964). ( 2 7 )J .E . F a i r b r o t h e r , Squibb I n s t i t u t e , p e r s o n a l communication. ( 2 8 ) Z . Kofoed, C . F a b i e r k i e w i c z and G . H . W . L u c a s , J . C h r o m a t o g r . 23, 410 ( 1 9 6 6 ) . ( 2 9 ) C . I . S m i t h and J . C . Burke, Toxic01 . Appl . P h a r m a c o l . 2, 553 ( 1 9 6 0 ) . ( 3 0 ) A . E . R o b i n s o n , J . P h a r m . P h a r m a c . l8, 1 9 ( 1 9 6 6 ) . ( 3 1 )J . D r e y f u s s and A . I . Cohen, J . Pharm. S c i . -960 826 ( 1 9 7 1 ) . ( 3 2 )J . A l i c i n o , S q u i h b I n s t i t u t e , p e r s o n a l communication.
.
79 I
KLAUS F L O R E Y
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