Griseofulvin

Analytical Profiles of Drug Substances, 8

GRISEOFULVIN Ecl~1at-dR . Tou1nley 1.

? -.

3. 4. 5. 6.

7.

8 9 I0

Description I . I Name, Formula. Molecular Weight I .2 Appearance, Color. Odor I . 3 Compendia1 References. Other Physical Properties 2.01 Circular Dichroisni 2.02 Nuclear Magnetic Resonance 2.03 Mass Spectrum 2.04 Ultraviolet Spectrum 2.05 Infrared Spectrum 2.06 X-Ray Diffraction 2.07 Fluorescence and Luminescence 2.08 Photolysis 2.09 Optical Rotation 2.10 Melting Range 2.11 Differential Scanning Calorimetry 2.12 Thermogravimetry 2.13 Electrophoretic Properties 2. I4 Solubilitv Production and Synthesis Impurities Stahility Drug Metabolic Products Methods of Analysis 7.01 Identification 7.02 Elemental Analysis 7.03 Spectrophotometric Analysis 7.04 Spectrofluorometric Analysis 7.05 Colorimetric Analysis 7.06 lodometric Analysis 7.07 Turbidimetric Analysis 7.08 Polarographic Analysis. 7.09 Chromatographic Analysis 7.091 Partition Column Chromatography 7.092 Paper Chromatography 7.093 Thin-Layer Chromatography 7.094 Gas chromatography 7.095 High Performance Liquid Chromatography 7.10 Biological Methods of Analysis Identification and Determination in Body Fluids Analysis of Dosage Forms Acknowledgments 219

Copyright 0 1979 by Academic Press. Inc. All rigtits of reproduction in any form reserved. ISBN 0-12-260808-9

EDWARD R. TOWNLEY

220

1.

Description 1.1 Name, Formula, Molecular Weight

Chemical Names (2S-trans)-7-Chloro-2',4,6-trimethoxy-6'-methylspiro [benxofuran-2(3H), 1'-(2) cyclohexene]3,4'-dione 7-chloro-4,6-dimethoxycoumaran-3-one-2-spir0-1'-(2~me thoxy-6'-me thylcyclohex-2'-en-4 '-one)

Generic Names Griseofulvin Trade Names Fulcin; Fulvicin UfF, Fulvicin PIG, Grifulvin; Grisactin; Grisovin, Gris-PEG, Grysio, Lamoryl, Likuden, NeoFulcin, Poncyl; Spirofulvin; Sporostatin. Formula and Molecular Weight

OCH

CH 0 3

c1

1' 7H17 ' 1 ° 6 1.2

CH

3

Molecular Weight

352.77

Appearance, Color, Odor Griseofulvin is a white, odorless, crystalline pow-

der. 1.3

Cornpendial References, Other Griseofulvin is listed in the following compendia: The United States Pharmacopia (1) The British Pharmacoepia (2) The Europeon Pharmacoepia ( 3 ) and the Merck Index (4). A previously published review (5) is a good source for physical and chemical data, production, use, occurrence and biological information.

GRISEOFULVIN

22 I

Physical Properties P h y s i c a l measurements by S c h e r i n g C o r p o r a t i o n are provided f o r Batch Number UGFP-1961. T h i s b a t c h i s chromatographic a l l y p u r e w i t h t h e e x c e p t i o n of 1.1% d e c h l o r o g r i s e o f u l v i n . 2.

2.01

C i r c u l a r Dichroism S p e c t r a The c i r c u l a r d i c h r o i s m s p e c t r a ( F i g u r e 1) w a s obt a i n e d on a 0.0155 mg/ml s o l u t i o n i n methanol w i t h a Cary Model 61 C i r c u l a r Dichroism Spectrophotometer. The f o l l o w i n g molar e l l i p t i c i t y v a l u e s were o b t a i n e d : Table I Molar E l l i pt i c it y

Wavelength nm 345 326 314 294 236 218

PI

+ + + +

shoulder shoulder shoulder peak peak peak

7,520 19,100 23,200 43,700

+110,000

- 97,500

2.02

Nuclear Magnetic Resonance S p e c t r a The p r o t o n NMR spectrum of g r i s e o f u l v i n ( F i g u r e 2) w a s o b t a i n e d i n DMSO-d s o l u t i o n (conc. w/v = 10 mg/0.40 m l ) c o n t a i n i n g TMS as k t e r n a l r e f e r e n c e u t i l i z i n g t h e V a r i a n A s s o c i a t e s CFT-20 S p e c t r o m e t e r o p e r a t i n g a t a frequency of 79.5 MHz. The chemical s h i f t s ( 6 , ppm) are w i t h r e f e r e n c e t o TMS. The e x p e r i m e n t a l c o n d i t i o n s a r e : Sweep w i d t h Pulse width Acquisition t i m e Data t a b l e

= = = =

800 Hz 6.0 1.1 sec ( 25'tip) 5.1 s e c 8K

E D W A R D R. T O W N L E Y

222

1

200

li

FIGURE 1 :

250

300

WAVELENGTH [nm]

350

Circular Dichroism Spectra of Griseofulvin

400

224

EDWARD R. T O W N L E Y

T a b l e I1 Proton

Chemical S h i f t s ( 6 , ppm)

6 '-CH3

0.82

5'-CH 6 '-CH 21

2.3-2.9

m

(3H)

2'-OCH3

3.63

8

(3H)

3.96 4.04

S

4-0CH3 } 6-OCH3

Aromatic

d ( J x 6 . 5 Hz)

(3H)

8

3 '-H 5-€I d= d o u b l e t , s= s i n g l e t , m- m u l t i p l e t . The carbon-13 NMR s p e c t r u m of g r i s e o f u l v i n ( F i g u r e 3 ) w a s o b t a i n e d a t am bi ent t e m p e r a t u r e i n DMSO-d 6 c o n t a i n i n g TMS a s i n t e r n a l r e f e r e n c e u t i l i z i n g V a r i a n A ssoci a t e s XL-100-15 s p e c t r o m e t e r equi pped w i t h F o u r i e r a c c e s s o ries. The s y s t e m was l o c k e d t o t h e d e u t e r i u m r e s o n a n c e f r e quency o f t h e s o l v e n t , and o p e r a t e d a t a f r e q u e n c y of 25.2 MHz f o r carbon-13. The chem i cal s h i f t s are r e p o r t e d ( c , ppm.) from t h e i n t e r n a l s t a n d a r d TMS.

-

Sweep w i d t h Pulse width Acquisition t i m e Acquisition delay

= 5500 Hz

15 P sec (66'

= 1.6 sec = 0.20 sec

tip)

T a b l e 111 Carbon

Chemical S h i f t (6,)

1' 2' 3'

4'

5' 6' 6' CH 4' ocd, 2' OCH3

90.11 170.22 104.60 195.45 39.49 35.52 13.77 57.54 56.97

Carbon 6 OCH3 3 3a 4 5 6 7 7a

Chemical S h i f t ( 56.52 191.12 104 04 157.59 91.27 164.44 95.24 168.57

6,)

I

I

1

I

I

I 1

I

I 1

,

I

I

m I

A

I

I

I

I

I

I

1

I

I

I

I

I

I

I

#

I

I

I %

m I

FIGURE 3:

I

I

I

I

I

I

1,

1

I "

' I '

I

I

I

I

I

,

I

Carbon 1 3 , Nuclear Magnetic Resonance Spectra of Griseofulvin i n DMSO d6

I

I

I

,

*,

~

226

EDWARD R. T O W N L E Y

The spectrum is in substantial agreement with the data reported by Wenkert et. al, (6). However, on the basis the proton coupled carbon 13 N M R spectra, the assignments for 6 and 7ci are reversed [Brambilla (16)] from those previously reported. The new assignments are based on long range H-C-0-C couplings. 2.03

Mass Spectrum The medium resolution, electron-impact mass spectrum of griseofulvin (Figure 4) was run on a Varian-Mat CH-5 Mass Spectrometer. Instrumental conditions were; Electron Energy 70eV; Source Temperature 25OoC; Sample Probe Temperature 14OoC. The fragmentation ions, given below, are consistent with the griseofulvin structure. Table IV Mass (amu)

Ions

Losses

352 337 321 310 284

CH30 CH3 CH CO Me6=CHCO

OCH

l+

215

138

I+

-t

k

i-

U

a

al

[I)

vl

[I)

9

.. U

F

s8

EDWARD R. TOWNLEY

228

I+ 123

2.04

U l t r a v i o l e t Spectrum The u l t r a v i o l e t spegtrum of g r i s e o f u l v i n i n anhyd r o u s methanol s o l u t i o n a t 25 C gave t h e f o l l o w i n g absorpt i v i t y values Wavelength maximum i s a t 324 nm; a = 15.5 Wavelength maximum i s a t 291 nm; a = 68.3 Wavelength maximum i s a t 235 nm; a = 64.0 The u l t r a v i o l e t spectrum i s shown i n F i g u r e 5.

2.05

I n f r a r e d Spectrum The i n f r a r e d spectrum of g r i s e o f u l v i n , o b t a i n e d as a m i n e r a l o i l m u l l , was r u n on a Perkin-Elmer Model 180 g r a t i n g Important a b s o r p t i o n assignments are I R spectrophotometer. given i n Table V. The spectrum i s g i v e n i n F i g u r e 6. Table V

-1

Wavenumber (cm

1

1703 ( s ) 1658 ( s ) 1615, 1597, 1580 ( s ) 1501 (m) 1220, 1210 ( 8 )

Assignment C=O s t r e t c h ; benzofuranone r i n g carbonyl C-0 s t r e t c h ; cyclohexenone c a r b o n y l C=C s t r e t c h , a r o m a t i c and c y c l i c unsaturation C=C s t r e t c h , a r o m a t i c C-0 s t r e t c h , a r y l methoxyl

I nt e n s i t y s

m

- strong -

medium

2.06

X-ray D i f f r a c t i o n The X-ray d i f f r a c t i o n p a t t e r n of g r i s e o f u l v i n w a s o b t a i n e d on a P h i l l i p s ADP-3500 X-ray D i f f r a c t o m e t e r u s i n g Cu K, r a d i a t i o n (1.5405A0) and N i f i l t e r . The d a t a is g i v e n i n Table VI.

GRISEOFULVIN

2.06

229

X-Ray Powder D i f f r a c t i o n P a t t e r n of G r i s e o f u l v i n Table V I

20 4 009 4.117 10.679 13.123 13.844 14.497 16.422 17.669 19.194 19.625 20.184 21.562 21.986 22.462 23.765 24.067 24.295 25.780 26.567 28.418 29.835 31.104 31.185 31.385 32.624 32.674 34.852 34.914 35.896 36.142 36 202 36.290 37.077 37.270 38.500 38.569 38.659

d(Ao)* 22.038 21.460 8.284 6.746 6.397 6.110 5.398 5.020 4.624 4.523 4.399 4.121 4.043 3.958 3.744 3.698 3.663 3.456 3.355 3.141 2.995 2.875 2.868 2.850 2.745 2.741 2.574 2.570 2.502 2.485 2.481 2.475 2.425 2.413 2.338 2.334 2.329

I/I'** 18 14 46 48 8 59 100 7 30 16 24 34 23 45 72 10 13 26 87 57 29 20 19 24 14 14 12 11 18 14 15 14 14 13 18 18 19

*d ( i n t e r p l a n a r d i s t a n c e ) = n / 2 s i n 0 = r e l a t i v e i n t e n s i t y (based on t h e h i g h e s t i n t e n s i t y of 100)

**Ill'

230

EDWARD R. T O W N L E Y

WAVELENGTH [nm] FIGURE 5:

U l t r a v i o l e t Spectrum of G r i s e o f u l v i n Obtained i n Anhydrous Methanol Solvent

2.5

4600

FIGURE 6:

3

3500

4

3000

2500

WAVELENGTH 5 6

2000

MICRONS 8 9 10

7

1700 1400 FREQUENCY (CM’)

1100

12 14

18 22

800

500

Infrared Spectrum of Griseofulvin Obtained a s a Mineral O i l Mull.

3550

205

EDWARD R. TOWNLEY

232

2.07

Fluorescence and Luminescence Griseof u l v i n e x h i b i t s b o t h f l u o r e s c e n c e and luminescence. A r e p o r t by Neely e t al., ( 7 ) g i v e s c o r r e c t e d f l u o r e s c e n c e e x c i t a t i o n (max. 295 nm) and e m i s s i o n (max. 420 nm) s p e c t r a , v a l u e s f o r quantum e f f i c i e n c y of f l u o r e s c e n c e (0.108) c a l c u l a t e d f l u o r e s c e n c e l i f e t i m e (0.663 n s e c ) and phosphorescence decay t i m e (0.11 s e c . ) . The f l u o r e s c e n c e e x c i t a t i o n and emission s p e c t r a a r e g i v e n i n F i g u r e 7. 2.08

Photolysis There i s no change i n t h e t h i n l a y e r chromatogram (single spot) o r i n the fluorescence o r u l t r a v i o l e t spectra a f t e r i r r a d i a t i o n i n methanol w i t h a xenon lamp f o r 20 hours. It is t h e r e f o r e concluded t h a t t h e r e is no s i g n i f i c a n t photod e g r a d a t i o n of g r i s e o f u l v i n under r e a s o n a b l e c o n d i t i o n s of l i g h t exposure ( 7 ) . 2.09

O p t i c a l Rotation

Griseofulvin e x h i b i t s the following o p t i c a l r o t a t i o n when d i s s o l v e d i n t h e s e s o l v e n t s . Table V I I

[a]

k7'=

+370

4

Dimethylformamide

[a]:60=

+358

17

Dioxane

[a]:60=

+302

17

S a t u r a t e d chloroform Acetone

2.10

M e l t i n g Range The D i f f e r e n t i a l Thermal Gravimetry c u r v e ( F i g u r e 9 ) demonstrates t h a t t h e g r i s e o f u l v i n m e l t i n g p o i n t t a k e s p l a c e with decomposition. The m e l t i n g range of g r i s e o f u l v i n from s e v e r a l s o u r c e s i s g i v e n i n Table VIII. Table V I I I Me1t i n g Range 0 C

-

218 t o 224 2 20 218 217-224

Reference 3 4 17 51

GRISEOFULVIN

233

8C 7c

60

50 >

I-

w 40 k

z -

30

20 10

0 200 FIGURE 7:

400 WAVELENGTH

300

500

Corrected Fluorescence and Emission Spectra of Griseofulvin: la, Excitation Spectrum with Emission at 420 nm; l b , Emission Spectrum with Excitation at 295 nm.

EDWARD R. TOWNLEY

234

2.11

D i f f e r e n t i a l Scanning C a l o r i m e t r y F i g u r e 8 shows t h e DSC thermogram of g r i s e o f u l v i n o b t a i n e d w i t h a DuPont Model 900 Thermal Analyzer. A single s h a r p m e l t i n g endokherm o c c u r s f o r t h i s s u b s t a n c e w i t h o n s e t t e m p e r a t u r e a t 216 C.

2.12

Thermogravimetry F i g u r e 9 shows t h e TG thermogram of g r i s e o f u l v i n o b t a i n e d w i t h a DuPont Model 950 Thermogravimetric Analyzer. The thermogram shows no weight l o s s from ambient t o about 2OO0C followed by weight loss due t o s u b l i m a t i o n . 2.13

Electrophoretic Properties Zeta p o t e n t i a l s of d i s p e r s e d g r i s e o f u l v i n have been s t u d i e d b o t h a l o n e , and i n t h e p r e s e n c e of s u r f a c e - a c t i v e a g e n t s , t h e l a t t e r a t a c o n t r o l l e d pH ( 8 ) . A M o b i l i t y / Z e t a P o t e n t i a l pH p l o t of = +25mV g r i s e o f u l v i n , shows a p o s i t i v e c h a r g e a t pH 1 . 5 which r a p i d l y d e c r e a s e s t o z e r o a t pH 2 . 4 . There i s t h e n r e v e r s a l of c h a r g e followed by a n i n c r e a s e o v e r t h e pH r a n g e 2 . 4 t o 7 . 0 . The z e t a p o t e n t i a l a t t h e l a t t e r pH i s -45 mV. The p o t e n t i a l t h e n s t a y s c o n s t a n t o v e r t h e pH range 7 t o 10.

2.14

Solubility The f o l l g w i n g d a t a are g i v e n f o r t h e s o l u b i l i t y of g r i s e o f u l v i n a t 25 C ; a c e t o n e 30 g/L, carbon t e t r a c h l o r i d e 2 g/L, d i c h l o r o e t h a n e 80 g/L, dimethylacetamide, 40 g/L, dioxane 30 g/L, e t h y l e t h e r 0 . 7 g/L, h e p t a n e 0.3 g/L, methanol 0 . 4 g/L; m i n e r a l o i l (0.1 g/L; propylene g l y c o l 2 g/L; Span 80 0.2 g/L, Tween 80, 7 g/L water 0 . 2 g/L ( 1 7 ) .

3.

P r o d u c t i o n and S y n t h e s i s Griseof u l v i n is b i o s y n t h e t i c a l l y manufactured by e l a b o r a t i o n w i t h P e n i c i l l i u m griseofulvum and r e l a t e d s t r a i n s of Penicillia. The b i o s y n t h e s i s h a s been t h e s u b j e c t of numerous chemical and b i o l o g i c a l s t u d i e s , t h e l a t e s t of which is g i v e n by Harris, e t . a l . ( 9 ) F i g u r e 10. Other proposed b i o s y n t h e t i c pathways a r e d i s c u s s e d . G r i s e o f u l v i n was f i r s t i s o l a t e d i n 1938 by Oxford ( 1 0 ) (1939); i t s t o t a l s y n t h e s i s was accomplished i n 1960 and f o l l o w i n g y e a r s i n s e v e r a l l a b o r a t o r i e s ( B r o s s i e t a l . , 1960 ( 1 1 ) Grove, 1963; ( 1 2 ) Mutant s t r a i n s of P. patulum are used f o r t h e commercial p r o d u c t i o n of t h e a n t i b i o t i c by f e r mentation (9).

et. al.,

4.

Impurities Some f e r m e n t e r b r o t h i m p u r i t i e s have been l i s t e d by Holbrook, B a i l e y and B a i l e y (13) and r e p e a t e d i n a d e s c r i p t i o n

FIGURE 8:

Differential Scanning Calorimetry Curve of Griseofulvin

20

0

\ 50

100 T. 'C

FIGURE 9 :

200 250 300 350 150 (CORRECTED FOR CHROMEL ALUMEL THERMOCOUPLES)

Thermogravimetry Curve of Griseofulvin

400

450

500

237

GRISEOFULVIN

acetate

-

0

0

0

0

-

CHJO

a

0

0

OH

-

FIGURE 10:

0

I

CHJ

OH H,C

a

I

CHJ

A Biosynthetic Route f o r G r i s e o f ulvin.

EDWARD R. TOWNLEY

238

of e f f i c i e n t l i q u i d chromatographic s e p a r a t i o n systems by B a i l e y and B r i t t a i n ( 1 4 ) and i n a g a s chromatographic s e p a r a t i o n system by Margosis ( 1 5 ) . S t r u c t u r e s are i n F i g u r e 11. The common i m p u r i t y found i n commercial b a t c h e s of g r i s e o f u l v i n is d e c h l o r o g r i s e o f u l v i n ( 1 4 , 1 5 ) which a p p e a r s t o b e i n t h e range of 0.5 t o 3 . 5 % .

5.

Stability G r i s e o f u l v i n is a s t a b l e drug s u b s t a n c e . After 1 2 y e a r s s t o r a g e a t room t e m p e r a t u r e no decomposition was d e t e c t e d by d i f f e r e n t i a t i n g LC methods (16). There is no p h o t o d e g r a d a t i o n under r e a s o n a b l e c o n d i t i o n s of l i g h t exposure ( 7 ) . G r i s e o f u l v i n is c o n v e r t e d t o g r i s e o f u l v i c a c i d under a c i d i c c o n d i t i o n s . 6.

Drug Metabolic P r o d u c t s The major human m e t a b o l i t e of g r i s e o f u l v i n i s 6-demethylg r i s e o f u l v i n and i t s g l u c u r o n i d e ( 1 7 , l B ) which a c c o u n t f o r about 65% of t h e i n t r a v e n o u s d o s e ( 1 9 ) and 3 5 t o 65% of t h e o r a l dose ( 2 0 , 2 1 ) . The 6 - d e m e t h y l g r i s e o f u l v i n is a l s o t h e major m e t a b o l i t e i n dogs ( 2 2 ) and r a b b i t s ( 2 3 ) w h i l e b o t h 4d e m e t h y l g r i s e o f u l v i n and 6 - d e m e t h y l g r i s e o f u l v i n are major m e t a b o l i t e s i n r a t s ( 2 4 ) and mice ( 2 5 ) . These m e t a b o l i t e s can b e determined by g a s l i q u i d chromatography v i a i s o p r o poxyl d e r i v a t i v e s ( 1 8 ) o r t r i m e t h y l s i l y l e t h e r d e r i v a t i v e s ( 2 6 , 2 7 ) . The 6-demethylgriseofulvin h a s been measured i n u r i n e by h i g h performance l i q u i d chromatography ( 2 8 ) and u l t r a v i o l e t spectrophotometry ( 1 9 ) . Only t r a c e amounts of g r i s e o f u l v i n are found i n t h e u r i n e ( 2 8 ) .

7.

Methods of A n a l y s i s

7.01

Identification A wine r e d c o l o r is produced when about 5 mg of g r i s e o f u l v i n are d i s s o l v e d i n 1 m l of s u l f u r i c a c i d w i t h about 5 mg of powdered potassium dichromate ( 2 9 ) .

7.02

Elemental A n a l y s i s A n a l y s i s of g r i s e o f u l v i n , w a s determined f o r c a r b o n , hydrogen, and c h l o r i n e . The carbon, and hydrogen a n a l y s i s w a s performed on a P e r k i n . E l m e r Model 240 i n s t r u m e n t . A n a l y s i s f o r c h l o r i n e w a s performed by combustion of t h e sample and c o u l o m e t r i c t i t r a t i o n u s i n g a n American I n s t r u m e n t Co. Chloride T i t r a t o r . The r e s u l t s from t h e e l e m e n t a l a n a l y s i s are l i s t e d i n

Table I X .

239

GRISEOFULVIN

0 I1

C

0 0

CH03

0

CH 3

-- 0

Dechlorgr i seo fulvin

@i0

CHO

\

c1

0

-

cii 3 0

=c

c1

CII 3

Dihydrogr iseofulvin

CH 3

Dehydrogriseofulvin

C H 30

c1

CH 3

Tetrahydrogriseofulvin CH30

C1

CtI 3

OCR 3

Griseofulvic Acid

c1 Isogriseofulvin FIGURE 11:

Impurities Found in the Fermenter Broth

EDWARD R. TOWNLEY

240

Table I X Elemental A n a l y s i s of G r i s e o f u l v i n : Element C H

c1

Batch UGFP-1961

% Theory

% Found

57.88 4.86 10.05

57.97 4.84 9.92

7.03

Spectrophotometric Analysis Q u a n t i t a t i v e u l t r a v i o l e t a n a l y s i s of g r i s e o f u l v i n may b e performed by comparison t o a R e f e r e n c e S t a n d a r d . The u l t r a v i o l e t absorbance i s d e s c r i b e d i n S e c t i o n 2.04 and F i g u r e 5. 7.04

Spectrofluorometric Analysis Griseof u l v i n e x h i b i t s f l u o r e s c e n t p r o p e r t i e s which have been u t i l i z e d f o r h i g h l y s e n s i t i v e a n a l y s e s i n blood and serum (30-33) s k i n and sweat ( 3 4 ) . Riegelman (32) h a s combined TLC s e p a r a t i o n w i t h a f l u o r i m e t r i c d e n s i t o m e t e r r e a d o u t t o g i v e a h i g h l y s p e c i f i c and s e n s i t i v e g r i s e o f u l v i n d e t e r mination i n plasma. Other a n a l y s e s are commonly performed i n e i t h e r 1%aqueous e t h a n o l ( 3 0 ) , a c t i v a t i o n maxima 295 and 335 nm, f l u o r e s c e n c e maxima a t 450 nm o r anhydrous methanol (31) a c t i v a t i o n maxima unchanged a t 295 and 335 nm, f l u o r e s c e n c e maxima 420 nm. Values are u n c o r r e c t e d . Other a p p l i c a t i o n s t o t h e a n a l y s i s of b u l k d r u g s , dosage forms o r as a d e t e c t i o n method f o r high performance l i q u i d chromatography are f e a s i ble. 7.05

Colorimetric Analysis A c o l o r i m e t r i c a s s a y of g r i s e o f u l v i n , based on t h e yellow-orange c o l o r (Xmax=420 nm) which d e v e l o p s when g r i s e o f u l v i n is h e a t e d w i t h i s o n i c o t i n i c a c i d h y d r a z i d e i n a l k a l i n e medium h a s been d e s c r i b e d by Unterman (35) and t h e mechanism i n v e s t i g a t e d by Unterman and Duca (36). 7.06

Iodometric Analysis Iodometric a n a l y s i s has been a p p l i e d t o t h e d e t e r mination of g r i s e o f u l v i n i n s t a g e s of t h e manufacturing proc e s s (37). The mycelium i s e x t r a c t e d w i t h chloroform and t h e a n a l y s i s c a r r i e d out i n a l c o h o l i c solution. The 0.01N i o d i n e s o l u t i o n is s t a n d a r d i z e d w i t h g r i s e o f u l v i c a c i d . 7.07

Turbidimetric Analysis A t u r b i d i m e t r i c a s s a y f o r potency e v a l u a t i o n of g r i s e o f u l v i n h a s been r e p o r t e d (38). The drug i s d i s s o l v e d i n

GRISEOFULVIN

24 1

e t h y l e n e g l y c o l monomethyl e t h e r (niethyl c e l l o s o l v e ) . Polym e r i z a t i o n i s induced w i t h g l y c e r o l and guanosine-5'-triphosp h a t e (GTP). 7.08

Polarographic Analysis A s t u d y d i r e c t e d toward a comparison of t h e reduct i o n p o t e n t i a l s f o r g r i s e o f u l v i n homologs and a n a l o g s s u g g e s t s t h a t polarography i s a method of g r i s e o f u l v i n i d e n t i f i c a t i o n ( 3 9 ) . In e t h a n o l i c s o l u t i o n w i t h 0 . 2 M K C 1 s u p p o r t i n g elect r o l y t e , g r i s e o f u l v i n shows two p o l a r o g r a p h i c waves w i t h h a l f wave p o t e n t i a l s a t about -1.58 V and -1.84 V. T h i s system h a s been a p p l i e d w i t h good r e s u l t s t o t h e d e t e r m i n a t i o n of f i n i s h ed p r o d u c t s i n c l u d i n g t a b l e t s . The a c c u r a c y , p r e c i s i o n and s e l e c t i v i t y of t h e method w a s compared w i t h t h e i o d o m e t r i c method ( 4 0 ) . I s o g r i s e o f u l v i n and g r i s e o f u l v i c a c i d do n o t interfere. (41,42) 7.09

Chromatographic Analyses 7.091

P a r t i t i o n Column Chromatography G r i s e o f u l v i n maybe s e p a r a t e d from o b s e r v e d s t r u c t u r a l l y s i m i l a r i m p u r i t i e s i n t h e f e r m e n t e r b r o t h by means of p a r t i t i o n column chromatography ( 1 3 ) . A C e l i t e c o l umn packing and s o l v e n t system c o n s i s t i n g of methano1:water: hexane:chloroform ( 8 : 2 : 9 : 1 ) was used. Tetrahydrogriseofulvin, dihydrogriseofulvin, i s o g r i s e o f u l v i n , dechlorogriseofulvin are s e p a r a t e d from g r i s e o f u l v i n . The s t r u c t u r e s f o r t h e s e compounds have been g i v e n i n S e c t i o n 5. 7.092 T a b l e X.

P a p e r Chromatography A p a p e r chromatography system i s g i v e n i n

(43) Table X

S o l v e n t System Benzene :Cyclohexane; Methanol :Water (5:5:6:4) Glacial a c e t i c a c i d , 0.5%, w a s added t o t h e o r g a n i c phase of t h e s o l v e n t after equilibration. 7.093 i n T a b l e XI.

Paper

Detection

Reference

Whatman No. 1

uv

43

Thin Layer Chromatography Thin l a y e r chromatographic systems are g i v e n The d e t e c t i o n method w a s U.V.

EDWARD R. T O W N L E Y

242

Table XI Adsorbent

Tr

Reference

Methanol :n-bu tanol; 95% ethano1:conc. ammonium hydroxide (4:1:2:1, by ~01.)

Silica Gel

0.64

44

Ch1oroform:isopropanol (3:1, by vole)

Silica Gel

0.86

44

n-butano1:formic acid: Silica Gel water (77:10:13 by vol.)

0.86

44

n-Butanol:95% ethanol conc. ammonium hydroxide:water (4:1:2:1 by vole)

Silica Gel

0.64

44

Ch1oroform:acetone (93:7 by vole)

Silica Gel

0.65

45

Ch1oroform:methanol (lot1 by vole)

Silica Gel

0.82

46

-

47

Solvent System

Ch1oroform:acetic acid Silica Gel diethyl ether (17:1:3) Methanol :benzene (2:98 by vol.)

Silica Gel

0.50

48

Ethyl acetate

Silica Gel

0.50

17

7.094

Gas Chromatopraphy Successful griseofulvin analyses by gas chromatography are reported for simulated samples (49) fermentation extracts (45) and pharmaceutical bulk and dosage forms (50). Margosis (15) describes the application of gas chromatography to the purity determination of griseofulvin. Separation from the related compounds; dehydrogriseofulvin, isogriseofulvin and dechlorogriseofulvin was demonstrated. In a subsequent collaborative study (50) the accuracy and precision of the method was established. Although griseofulvin is thermally stable, the high GLC temperatures require precautions similar to those taken for steroids when preparing columns, column supports and associated equipment (45).

GRISEOFULVIN

243

The s p e c i f i c and s e n s i t i v e GLC d e t e r m i n a t i o n of g r i s e o f u l v i n i n body f l u i d s and t i s s u e s such as s k i n , sweat, u r i n e and plasma w i t h e l e c t r o n c a p t u r e d e t e c t i o n h a s been used by s e v e r a l i n v e s t i g a t o r s (34,51,52). Table XII.

Gas chromatographic c o n d i t i o n s are g i v e n in Table XI1

Carrier Gas

Column

150 cm x 4 mm I.D.; Ushaped s t a i n l e s s s t e e l t u b i n g , 1.5% QF-1 on Anakrom ABS 3 f t x 4 mm I.D.; coiled g l a s s tubing, 3% OV-101 on Gas Chrom Q

N2

He2

Column Temp.

Internal Standard diphenylphthalate

230'

245'

5 f t x 4 mm I.D.; g l a s s 10% Methane column, 3% OV-17 on 90% Argon Chromosorb W. o r Gas or Chrom Q.

Reference

tetraphenylcyclopentadienone

27OoC

diazepam

45,49

15

34,50, 51,52

N2

3 f t x 4 mm I.D.; glass coiled. % OV-17 on Gas Chrom. Q

He 2

225'

tetraphenyl15 cyc l o p e n t adienone

High Performance L i q u i d Chromatography G r i s e o f u l v i n can b e r e a d i l y chromatographed on e i t h e r normal o r r e v e r s e p h a s e columns. S e p a r a t i o n of dec h l o r o g r i s e o f u l v i n , t h e most common s y n t h e t i c i m p u r i t y i s accomplished on C / 1 8 , CN (17) o r ETH columns. (15) I s o g r i s e o f u l v i n , i s s e p a r a t e d on e i t h e r CN (17) o r ETH columns (15).

7.095

L i q u i d chromatography c o n d i t i o n s are g i v e n i n T a b l e XIII.

EDWARD R. T O W N L E Y

244

Table XI11 Column PBondapak C/18 ( o c t y l d e c y l c h e m i c a l l y bonded to silica)

Reverse phase

mondapak C/18 ( o c t y l d e c y l c h e m i c a l l y bonded to silica)

II

Zorbax CN (cyanop r o p y l c h e m i c a l l y bonded to silica)

11

Mobile Phase

Internal Standard

Refer-

Methanol: water 3:2

n-butyl p-hydroxy benzoate

16

45% a c e t o - d i a z e p a n

53

ence

nitrile in 45mM KH PO4 pn = 3.6

Permaphase ETH Normal (C-7 e t h e r c h e m i c a l l y phase bonded t o p e l l i c u l a r 30-50 mesh g l a s s packing)

Methanol: water 3:2 5% c h l o r o form i n hexane

m-phenyl phenol

16

14

7.10

B i o l o g i c a l Methods o r A n a l y s i s M i c r o b i o l o g i c a l p r o c e d u r e s have been developed f o r a s s a y of g r i s e o f u l v i n and a p p l i e d t o t h e a n a l y s i s of b u l k d r u g s and dosage forms ( 1 ) . The c y l i n d e r p l a t e a g a r d i f f u s i o n method is t h e o f f i c i a l m i c r o b i o l o g i c a l method of d e t e r m i n a t i o n ( 5 5 ) . Microsporum gypseum (ATCC 14683) i s t h e t e s t organism. 8.

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 G r i s e o f u l v i n h a s u s u a l l y been determined i n body f l u i d s and t i s s u e s by s p e c t r o f l u o r i m e t r i c (30-34) o r g a s chromatograp h i c methods (45,46,48). More r e c e n t l y g r i s e o f u l v i n h a s been determined i n plasma by h i g h performance l i q u i d chromatography (53,541. A n a l y s i s of Dosage forms Usual dosage forms of g r i s e o f u l v i n are c a p s u l e s , t a b l e t s and b o l u s e s . These may b e p r e p a r e d f o r a n a l y s i s by s i m p l e l i q u i d s o l i d e x t r a c t i o n of drug s u b s t a n c e . Margosis (15,50) used chloroform as an e x t r a c t i n g s o l v e n t w i t h g e n t l e h e a t . A compendia procedure d e s c r i b e s t h e e x t r a c t i o n of g r i s e o f u l v i n from t a b l e t s w i t h b o i l i n g a l c o h o l . The a n a l y s i s f o r e x t r a c t e d drug s u b s t a n c e has been performed by s e v e r a l methods. Most common is a simple u l t r a v i o l e t a n a l y s i s ( 2 , 3 , 5 5 ) . Polarography u t i l i z i n g t h e system g i v e n i n S e c t i o n 7.08 h a s been used (39). Thin-layer, g a s and l i q u i d chromagraphy may a l s o be 9.

G R I S EO F U LV I N

245

used u t i l i z i n g systems d e s c r i b e d i n S e c t i o n s 7.093, 7.094, and 7.095 r e s p e c t i v e l y . These l a t t e r methods are v a l u a b l e because of t h e i r s p e c i f i c i t y . I n t h e United S t a t e s , g r i s e o f u l v i n drug s u b s t a n c e and dosage forms must conform t o t h e r e g u l a t i o n s of t h e F e d e r a l Food and Drug A d m i n i s t r a t i o n concerning a n t i b i o t i c d r u g s (55, 5 6 ) . M i c r o b i o l o g i c a l a s s a y r e s u l t s o b t a i n e d by a n a l y t i c a l methods d e s c r i b e d i n t h e s e compendia a r e c o n c l u s i v e . 10.

Acknowledgements The a u t h o r wishes t o thank D r . M. D. Yudis and D r . H. Suprenant f o r t h e i r encouragement f o r t h i s work and t o acknowledge t h e v a l u a b l e a s s i s t a n c e of members of t h e S c h e r i n g C o r p o r a t i o n , P h y s i c a l Organic Research S e c t i o n : D r . R. B r a m b i l l a , M r . P. B a r t n e r , M r . C. Eckhart and M r . R. F o e s t e r f o r t h e a c q u i s i t i o n and i n t e r p r e t a t i o n of t h e p h y s i c a l d a t a , and t o t h e S c h e r i n g l i b r a r y s t a f f p a r t i c u l a r l y M s . J . Nocka, f o r t h e l i t e r a t u r e s e a r c h e s and S c h e r i n g General O f f i c e S t a f f f o r t h e c a r e f u l t y p i n g of t h i s monograph.

EDWARD R. T O W N L E Y

246

References 1.

U.S.

Pharmacopeial Convention Inc. (1970). The U.S. Pharmacopeia, 19th rev., Easton, Pa. ,Mack p. 673.

2.

British Pharmacopeia Commission (1973) British Pharmacopeia, London, HMSO, p. 221.

3.

Council of Europe (1971) Europeon Pharmacopoeia, Europeon Treaty Series, 50, 2, Paris, Maisonnmeuve, p. 234.

4. Stecher, P.G., ed (1976). Rahway, N.J.,

5. 6. 7. 8.

The Merck Index, 9th ed., Merck & CO., 4392.

International Agency for Research on Cancer, 10, 153 (1976). WHO; Geneva, Switzerland. S.G. Levine, R.E. Hicks, H.E. Gottlieb and E. Wenkert,

J. Org. Chem., 40, 2540 (1975).

W.C. Neely and J.R. McDuffie, J. Assoc. Off. Anal. Chem.,

55, 1300 (1972).

J.B. byes, J. Pharm. Pharmacol., 29, 163 (1977).

9. C.M. Harris, J.S. Roberson and T.M. Harris, J. Am. Chem. SOC., 98, 5380 (1976). 10. A.E. Oxford, H. Raistrick and P. Simonart, Biochem. J . , 33, 240 (1939). 11.

A. Brossi, M. Baumann, M. Gerecke, E. Kyburz, Helv. Chim. Acta, 43, 1444 (1960).

12.

J.F. Grove, Q. Rev., l7, 1, (1963).

13.

A. Holbrook, F. Bailey and Greta M. Bailey, J. Pharm. and Pharmacol. Suppl., 15,274T (1963).

14

F. Bailey and P.N. Brittain, J. Chromatogr., 83, 431 (1973).

15.

M. Margosis, J. Chromatogr., 7 0 , 73 (1972).

16.

Schering Research Data.

17. M.J. Barnes and B. Boothroyd, Biochem. J . , 78, 41 (1961).

GRISEOFULVIN

247

18.

C. Lin, J. Margat, R. Chang, J. McGlotten and S . Symchowicz, J. Pharmacol. Exp. Ther., 187, 415 (1973).

19.

M. Rowland and S. Riegelman, J. Pharm. Sci., 62, 2030 (1973)

20.

W.L.

*

Chiou and S . Riegelman, J. Pharm. Sci.,

(1971).

21.

1376

C.C. Lin and S . Symchowicz, Drug Metabolism Reviews, 4 ( 1 ) 75,

22.

60,

(1975).

P.A. Harris and S . Riegelman, J. Pharm. Sci., 58, 93, 1969

23

S. Symchowicz, M. S . Staub and K. K. Wong, Biochem. Pharmacol., l6, 2405 (1967).

24.

S . Symchowicz and K. K. Wong, Biochem. Pharmacol., 1595 (1966).

25.

C. Lin, R. Chang, J. Magat and S . Symchowicz, J. Pharm. and Pharmacol., 24, 9 1 1 (1972).

26.

A. Zlatkis, "Advances in Gas Cromatography 1967," Proceedings of the Fourth International Symposium held in New York, N.Y., April 3-6, 1967. Preston Technical Abstracts Co., Evanston, Ill. 1967, p. 129.

27.

P. Kabasakalian, M. Katz, B. Rosenkrantz and E. Townley, J. Pharm. Sci., 59, 595 (1970).

28.

15,

E. Papp, K. Magyar and H. J. Schwartz, J. Pharm. Sci.,

65, 441 (1976).

29

H. Auterhoff and M. Kliem, Arch der Pharmazie, 309, 326 (1976)

30

C. Bedford, K.J. Child and E.G. Tomich, Nature, 2,364 (1959)

31.

M. Kraml, J. Dubuc and D. Dvornik, J. Pharm. Sci., 5 4 , 655 (1965).

32.

L.J. Fisher and S . Riegelman, J. Chromatogr.,.u, 268 (1966)

EDWARD R. TOWNLEY

248

19, ( 6 )

33.

K. E i s e n b r a n d t , Pharmazie,

34.

V. P Shah, S. Riegelman and W. L. E p s t e i n , J. Pharm. S c i . , 6 l , 634 ( 1 9 7 2 ) .

35.

H.W.

36

H.W. Unterman and A l . Duca, Rev. Roum Chim. (1971).

37.

H.W. Unterman and A l . Duca,. Chim. Anal. 196 ( 1 9 7 2 ) .

38

J. Hoebeke and G. Van V i j e n , L i f e S c i . , l7, 5 9 1 ( 1 9 7 5 ) .

39.

H.W. Unterman and A l . Duca, Isr. J. Chem., l2, ( 5 ) 985 (1974).

40.

J. Kadar-Pauncz, Acta Pharm Hung., 3 5 , 297 ( 1 9 6 3 ) .

41.

H.W. Unterman and A l . Duca, Rev. Chim. ( B u c h a r e s t ) ( 1 9 7 1 ) ; 2, 188 ( 1 9 7 2 ) .

42

H.W. Unterman, and A l . Duca, Chim. Anal. 1 ( 2 ) , 97 ( 1 9 7 1 ) .

43.

S. Symchowicz and K.K.

Unterman, Rev. Chim. ( B u c u r e s t i )

1595 ( 1 9 6 6 ) .

W.A. Creasey, K.G. Bensch and S.E. Pharmacol., 20, 1579 ( 1 9 7 1 ) .

45.

R.J. Cole, J . W . K i r k s e y and C.E. b i o l . 9 l9, 1 0 6 , ( 1 9 7 0 ) .

46.

C. L i n , R. Chang, C . Casmer, and S. Metab. Dispos., 1,6 1 1 ( 1 9 7 3 ) .

47.

2 . Durackova, V.

48

H.J.

141 ( 1 9 7 6 ) .

16,( 5 )

286 ( 1 9 6 5 ) .

16,1077

(Bucharest) 2 ( 3 )

1,97

(Bucharest)

Wong, Biochem. Pharmacol, l5,

44.

49.

406 ( 1 9 6 4 ) .

Malawista, Biochem.

Holaday, Appl. MicroSymchowicz, Drug

B e t i n a , P. Nemec, J. Chromatog.,

116,

I s s a q , E.W. Barr, T. Wei, C. Meyers, A. A s z a l o s , J. Chromatog., 133, 291 ( 1 9 7 7 ) .

S. I g u c h i , M. Yamamoto and T. Goromaru, J. Chromatogr.,

24,

182 ( 1 9 6 6 ) .

GRISEOFULVIN

249

64,

50.

M. M a r g o s i s , J. Pharm. S c i . ,

51.

H . J . S c h w a r t z , B.A. Waldman, and V. Madrid, J. Pharm. S c i . , 65, 370 (1976).

52.

V.P.

Shah, S . Riegelman and W.L.

1020 ( 1 9 7 5 ) .

E p s t e i n , J. Pharm. S c i . ,

6 1 , 634 (1972).

155, 206

53.

L.P. Hackett and L . J . (1978).

54.

R.L. N a t i o n , G.W. Peng, V. Smith and W.L. Pharm. S c i . , 67, 805, 1978.

55.

US Code of F e d e r a l R e g u l a t i o n s (1976) Food and DruRs, T i t l e 21, p a r t 436.105, Washington, D.C., US Government

D u s i , J. Chromatog.,

Chiou, J.

printing office. 56.

E.M. Oden, G.H. Wagman, and M . J . W e i n s t e i n , A n a l y t i c a l M i c r o b i o l o g y , Vol. 11, Academic P r e s s , 1972, p 385.