DOXORUBICIN

Aristide Vigevani and Martin J . Williamson

I. Description 246
1 . 1 History 246
1.2 Name, Formula, Molecular Weight 246
1.3 Appearance, Color 247
2. Physical Properties 241
2.1 Infrared Spectrum 241
2.2 Nuclear Magnetic Resonance Spectra 247
2.3 Mass Spectra 25 1
2.4 Ultraviolet and Visible Spectrum 253
2.5 Fluorescence Spectra 255
2.6 Circular Dichroism 255
2.7 Optical Rotation 255
2.8 Melting Point 255
2.9 X-ray Diffraction 255
2.10 Differential Scanning Calorimetry 260
2.11 Solubility 260
2.12 Ionization Constant 260
2. I3 Polarography 260
3. Synthesis 260
3.1 Microbiological 260
3.2 Chemical 263
4. Stability 263
5. Metabolism 265
6. Methods of Analysis 261
6.1 Elemental Analysis 267
6.2 Spectrophotometric Analysis 267
6.3 Electrochemical Analysis 267
6.4 Paper Chromatography 267
6.5 Thin Layer Chromatography 268
6.6 Liquid Chromatography 268
7. Determination of Doxorubicin in Biological Fluids 270
8. Analysis of Pharmaceutical Formulations 270
9. Miscellaneous 270
10. Acknowledgments 270
1 1. References 270

CopytighI @ 1980 by Academic Ress, Inc.

Analytical h f i l c s of Drug Substances, 9 245 AIIrighrs Of n - ~ u c t i o nin any form resewed.
ISBN: &12-260809-7
246 ARISTIDE VIGEVANI A N D MARTIN J . WILLIAMSON

1. Description
1.1 History
Doxorubicin is an antineoplastic antibiotic isolated
from a culture of Streptomyces peucetius var. caesius or by
chemical synthesis from daunorubicin. The injectable dosage
form is supplied as the hydrochloride salt in combination
with lactose as a freeze-dried powder.

1.2 Name, Formula, Molecular Weight

Doxorubicin is chemically named (8S-lOS)-lO-
(3-amino-2,3,6-trideoxy-a. -L-e-hexopyranosy1)oxy-

methoxy-5,12-naphthacenedione. (CAS-23214-92-8) .
7,8,9 ,l0-tetrahydro-6,8,1l-trihydroxy-8-hydroxyacetyl-l-
Originally
named (7S:9S)-9-hydroxyacetyl-4-methoxy-7~8,9,lO-tetrahydro-
6,7,9,11-tetrahydroxy-7-~-(2,3,6-trideoxy-3-amino-cl - L - w -
hexopyranosyl)-5,12-naphthacenedione.

NHq H

27H29N01 1 Mw 543.5

Hydrochloride salt (CAS-25316-40-9)

C27H2gNOll.HC1 Mw 580.0
DOXORUBICIN 247

1.3 A p p e a r a n c e , Color
The h y d r o c h l o r i d e s a l t i s a r e d , f r e e - f l o w i n g
c r y s t a l l i n e powder, a n d t h e f r e e z e - d r i e d f o r m u l a t i o n
c o n t a i n i n g lactose i s a r e d cake.

2 Physical Properties
2.1 I n f r a r e d Spectrum
A review of the c a r b o n y l a b s o r p t i o n s o f
a n t i n e o p l a s t i c ( a n t i tumor) a n t h r a c y c l i n e s h a s b e e n
published1. The i n f r a r e d s p e c t r u m o f d o x o r u b i c i n
h y d r o c h l o r i d e recorded from a KBr p e l l e t ( 0 . 4 ) % o n a
Perkin-Elmer model 457 g r a t i n g s p e c t r o p h o t o m e t e r is shown i n
F i g u r e 1. The i n t e r p r e t a t i o n o f t h e m a i n a b s o r p t i o n b a n d s i s
g i v e n i n T a b l e 1.

TABLE 1

I n f r a r e d spectrum o f doxorubicin hydrochloride

~

I R A b s o r p t i o n Band, c m - l A s s i g n men ts

3560-3160 0-H s t r e t c h ( h y d r o g e n bonded)

3160-2300 NH3+ s t r e t c h and OH
s t r e t c h (hydrogen bonded)
1724 C=O (ketone)
1 6 1 3 and 1580 C=O s t r e t c h ( i n t r a hydrogen
bonded q u i n o n e )
1282 C-0-C s t r e t c h ( e t h e r )
1115 C-0 ( t e r t i a r y alcohol)
1071 C-0 (secondary a l c o h o l )
1008 C-0 (primary a l c o h o l )

2.2 N u c l e a r M a g n e t i c Resonance Spectra

Proton magnetic resonance spectrometry h a s been
e x t e n s i v e l y u s e d a s a f u n d a m e n t a l tool for t h e d e t e r m i n a t i o n
o f t h e s t r u c t u r e o f daunorubicin and r e l a t e d
compounds2r3r4. The lH-NMR s p e c t r u m o f a d r i a m y c i n o n e
p e n t a a c e t a t e i n CDC13 h a s b e e n d e s c r i b e d and t e n t a t i v e l y
assigned5. The lH-NMR s p e c t r u m o f d o x o r u b i c i n
h y d r o c h l o r i d e i n DMSO-d6 s o l u t i o n r e c o r d e d a t 1 0 0 MHz on a
V a r i a n HA-100 s p e c t r o m e t e r a t 8 O o C ( f o r b e t t e r r e s o l u t i o n ) i s
shown i n F i g u r e 2. The i n t e r p r e t a t i o n o f t h e s p e c t r u m is
g i v e n i n T a b l e 2.
I I I
0
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DO XO R UBICIN 249

TABLE 2

lH-NMR d a t a of d o x o r u b i c i n h y d r o c h l o r i d e i n D M s 0 - d ~
s o l u t i o n a t 80°C (TMS as i n t e r n a l r e f e r e n c e ) .

Proton Mu 1t i p l i c i t y a J or WH (Hz)

CH3-5 d 1.15 6.5

H2-2 m 1.77 -
H2-9 m 2.15 -
H2-7 s 2.92 -
H-3 I m 3.31 -
H-4 I bs 3.62 6.0
CH3O S 3.94 -
H-5 dq 4.14 and
H2-14 S 4.57 -
OH-4 bs 4.46 -
H-10 bs 4.90 10.0
H-1 bs 5.25 7.0
H-3 t 7.53 7.0

H-2
H-4
+
3 d 7.79 7.0
-
NH3 bs 7.96
13. 08b
OH-11 two s and
13. 85b

a) s - S i n g l e t ; d = Doublet; t = Triplet;
m = M u l t i p l e t ; b s = Broad s i g n a l ; dq = Double
quartet.
b) A t room t e m p e r a t u r e .
C
.d
u
0
DOXORUBICIN 25 I

The 13C NMR spectra of doxorubicin hydrochloride,

daunorubicin hydrochloride, the corresponding aglycones and
of a-methyl daunosaminide in DMSO-d6 solutions and the
interpretation has been reported6. Figure 3 shows the FT
13C NMR proton noise-decoupled spectrum of doxorubicin
hydrochloride in D20 solution, recorded at 80 MHz on a
Varian CFT-20 NMR spectrometer. Dioxane, which is not shown,
was used as the internal standard. The interpretation of the
spectrum is given in Table 3 .

TABLE 3

13C-NMR data of doxorubicin hydrochloride in D20 solution

values (ppm) are referred to 734s.

Carbon 6 Carbon 6

1 161.0 4a (134.5)
2 (119.2) 5a 111.0
3 137.3 6a (134.0)
4 (120.2) 10a (134.5)
5 (185.9) lla 111.0
6 154.6 12a (120.0)
7 32.8 CH3O 57.2
8 76.6 1' 99.4
9 36.0 2' 28.5
10 69.0 3' 47.7
11 156.2 4' (67.9)
12 (186.1) 5' (67.0)
13 213.9 CH3-5 ' 16.6
14 65.3

Assignments with similar shift values

given in parentheses may be interchanged.

2.3 Mass Spectra

The mass spectrum of doxorubicin hydrochloride
itself cannot be obtained by electron-impact ionization, but
this technique can be used to obtain the spectra of
adriamycinone5 and daunosamine'. A study of
-
N-acetyldaunosamine derivatives has been published8. The
mass spectra of some E-acylated daunorubicin derivatives have
been published9 and also the GC-MS of persilylated aglycone
derivatives of doxorubic in and daunorubic inlo.
 E
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-8
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DOXORUBICIN 253

The i n t a c t molecule can be examined by f i e l d

desorption i o n i z a t i o n mass s p e c t r o m e t r y l l . Figure 4 shows
t h e spectrum obtained on a Varian MAT31lA spectrometer,
equipped w i t h a combined FD/FI/EI source ( e m i t t e r heating
c u r r e n t 2 O m A ) l 2 . Table 4 g i v e s t h e assignments of t h e
major fragmentation peaks.

TABLE 4

F i e l d desorption mass spectrum of doxorubicin hydrochloride

m/e assignment

544 M+1
543 Molecular ion
414 a d r iamycinone

l+

CH30 0 bH
7, -b isanhyLi0a-L iamycinone

0 OH

336

2.4 U l t r a v i o l e t and V i s i b l e Spectrum

The u l t r a v i o l e t and v i s i b l e spectrum of doxorubicin
hydrochloride i n methanol ( c = l % )is shown i n Figure 513.
The molecular a b s o r p t i v i t i e s a r e given i n Table 5.
 m
=.t
o m
= II
4
U
u
aJ
a
co
m
I 0
V I
a,
U
3
(D m
m 4
m h
DOXORUBICIN 255

TABLE 5

U l t r a v i o l e t a n d V i s i b l e Molecular Absorptivities of
Doxorubicin Hydrochloride i n Methanol

Wavelength E

233 38150
253 255 00
290 8400
471 13050
495 13000
530 7200

2.5 Fluorescence Spectra

T h e f l u o r e s c e n c e spectra of d o x o r u b i c i n
h y d r o c h l o r i d e i n water a n d e t h a n o l a t a p p r o x i m a t e l y 5 ppm,
d e t e r m i n e d u s i n g a P e r k i n - E l m e r MPF-2A s p e c t r o f l u o r i m e t e r ,
a r e shown i n F i g u r e 6.13

2.6 C i r c u l a r Dichroism
T h e c h i r a l c e n t e r s a t C-8 a n d C-10 are r e s p o n s i b l e
f o r t h e C o t t o n e f f e c t s a t 3 4 5 a n d 2 8 5 nm. T h e c i r c u l a r
d i c h r o i s m c u r v e s of m e t h a n o l s o l u t i o n s of d o x o r u b i c i n a n d
d a u n o r u b i c i n h y d r o c h l o r i d e s a n d of a d r i a m y c i n o n e a n d
daunomycinone i n d i o x a n e , d e t e r m i n e d u s i n g a Roussel-Jouan
D i c h r o g r a p h 11, a r e shown i n F i g u r e s 7 a n d 8. T h i s t e c h n i q u e
h a s been used i n t h e deduction o f stereochemical
r e l a t i o n s h i p s i n t h e f i e l d of a n t h r a c y ~ l i n o n e s ~ ~ .

2.7 Optical R o t a t i o n
The o p t i c a l r o t a t i o n C C ] ~ ~of d o x o r u b i c i n
h y d r o c h l o r i d e i n m e t h a n o l ( 0 . 1 ) was d e t e r m i n e d a t 5 8 9 nm
u s i n g a P e r k i n - E l m e r Model 2 4 1 MC polarimeter t o b e +255O.

2.8
Melting Point
D o x o r u b i c i n h y d r o c h l o r i d e melts a t 2 0 5 T w i t h
decomposition.

2.9 X-ray D i f f r a c t i o n
A t t h i s t i m e n o X-ray d i f f r a c t i o n s t u d i e s have been
r e p o r t e d o n d o x o r u b i c i n h y d r o c h l o r i d e or i t s d e r i v a t i v e s .

S i n g l e c r y s t a l X-ray d i f f r a c t i o n of
-
N-bromoacetyldaunorubicin s o l v a t e with acetone15 confirmed
t h e s t r u c t u r e and a b s o l u t e c o n f i g u r a t i o n o f daunorubicin,
which h a d p r e v i o u s l y been d e t e r m i n e d b y c h e m i c a l
s t u d i e s 2 , 3,4. More r e c e n t l y t h e s e r e s u l t s were c o n f i r m e d
b y a n X-ray a n a l y s i s of d a u n o r u b i c i n , a s t h e h y d r o c h l o r i d e
 0
0
ln 4
0 2m
aD c
m
s
4J
0
ln
In c
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0
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d 5
.d
o c
nl '-
I n k
0
0
In
0
a,
d
0
ln
d
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0
d
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(u 0
P
0
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0
a,
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5
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(u
m
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Q)
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256
 70 70

> 60 60
h
ul
5 50 50
I-
z
z 40 40
0
2 30 30
5

L
g 20 20
-
!z
-I 10 10
W
&

0 0
r 1 1 1 1 1 1 1 1 ~ 1

480 520 560 600 640 680 480 520 560 600 640 680
WAVELENGTH (nm)

F i g u r e 6. F l u o r e s c e n c e S p e c t r a of D o x o r u b i c i n H y d r o c h l o r i d e i n Water ( l e f t )
and E t h a n o l ( r i g h t ) . C o n c e n t r a t i o n s a p p r o x i m a t e l y 5 mg/l.
258 ARISTIDE VIGEVANI AND MARTIN J . WILLIAMSON

At2
3t

D AU NORUB ICI N

At2 h(nrn1
3r
I
2

--)--c.---c.

DOXORUBICI N
-1

-2

-3

-4 4
260 2i O 360 3;O 3iO 3 i O 3iO 460
400
A(nmi

Figure 7. Circular Dichroism Curves of Doxorubicin and

Daunorubicin Hydrochlorides in Methanol
DOXORUBICIN 259

A&
27

-2 1
260 O
2; 360 3;O 3iO 3kO 360 460
?dnm 1
A€

-2
260
I 260 360 3iO 3 i O 3kO 360 460
A (nml

Figure 8. Circular Dichroism Curves of Adriamycinone

and Daunomycinone in Dioxane Solution
260 ARISTIDE VIGEVANI A N D MARTIN J . WILLIAMSON

monohydrate p y r i d i n e s a l t 1 6 . Some c o n f o r m a t i o n a l
d i f f e r e n c e s were o b s e r v e d w i t h respect t o t h e N-bromoacetyl
derivative.

2.10 D i f f e r e n t i a l Scanning Calorimetry

The h e a t i n g c u r v e of d o x o r u b i c i n h y d r o c h l o r i d e
o b t a i n e d w i t h a Perkin-Elmer Model DSC-1B s c a n n i n g
calorimeter a t a t e m p e r a t u r e g r a d i e n t o f 8OC/min. i s shown i n
F i g u r e 917. I t shows a n endotherm, c o r r e s p o n d i n g t o t h e
s o l i d - l i q u i d t r a n s i t i o n a t 202-2O5OCI p a r t i a l l y superimposed
by a n endotherm due t o d e c o m p o s i t i o n which i s a t a maximum a t
26OOC and c o n t i n u e s t o h i g h e r t e m p e r a t u r e s .

2.11 S o l u b i l i t y
Doxorubicin h y d r o c h l o r i d e i s r e a d i l y s o l u b l e i n
water, normal s a l i n e , methanol, a c e t o n i t r i l e and
t e t r a h y d r o f u r a n , b u t o n l y s l i g h t l y s o l u b l e or i n s o l u b l e i n
less polar o r g a n i c s o l v e n t s . The a p p a r e n t p a r t i t i o n
c o e f f i c i e n t (Papp) between 1 - o c t a n o l and T r i s b u f f e r a t pH
7.0 w i t h c o n s t a n t i o n i c s t r e n g t h ( I = 0.1) is 0.52 a t room
t e m p e r a t u r e (22-24OC) a f t e r s h a k i n g f o r 1 5 hours18.

2.12 I o n i z a t i o n C o n s t a n t
A pKa o f 8.22 was d e t e r m i n e d f o r t h e h y d r o c h l o r i d e
w i t h N/20 sodium hydroxide. Solutions of doxorubicin
h y d r o c h l o r i d e show i n d i c a t o r - 1 i k e p r o p e r t i e s I t u r n i n g from
orange-red t o b l u e - v i o l e t a b o u t pH = 913. V a l u e s o f -5.9,
8.2, 10.2, and 13.2 f o r pK1, pK2, pK3 and pK4, d e t e r m i n e d
by s p e c t r o p h o t o m e t r i c methods, have been r e p o r t e d 1 9 .

2.13 m l a r o g r a p h y
Due t o i t s q u i n o i d a l system, d o x o r u b i c i n g i v e s
c h a r a c t e r i s t i c p o l a r o g r a m s a t d i f f e r e n t pH v a l u e s . These
c u r v e s , d e t e r m i n e d u s i n g a Leeds-Northrup Electro-Chemograf
t y p e E p o l a r o g r a p h , are shown i n F i g u r e

3. Synthesis
3.1 M i c r o b i o l o g i c a l
Doxorubicin c a n be o b t a i n e d by a e r o b i c f e r m e n t a t i o n
o f S t r e p t o m y c e s peucetius v a r . c a e s i u s f o l l o w e d by e x t r a c t i o n
w i t h a c i d i c a c e t o n e and p u r i f i c a t i o n by p a r t i t i o n
chromatography o n a column o f cellulose b u f f e r e d a t pH 5.4.
The a n t i b i o t i c is r e c o v e r e d from t h e e l u a t e s i n 1 - b u t a n o l
s a t u r a t e d w i t h pH 5.4 p h o s p h a t e b u f f e r by back e x t r a c t i o n
w i t h d i l u t e a c i d pH 3 , f o l l o w e d by r e - e x t r a c t i o n i n t o
c h l o r o f o r m a t pH 8.6. The c h l o r o f o r m s o l u t i o n i s
c o n c e n t r a t e d and d o x o r u b i c i n c r y s t a l l i z e d a s t h e
h y d r o c h l o r i d e on a d d i t i o n o f a n e q u i v a l e n t o f m e t h a n o l i c
F C
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262 ARISTIDE VIGEVANI AND MARTIN J . WILLIAMSON

4
0.3 0.4 0.5 0.6 0,7 0.8 0.9
1 1.1 1.2 1.3Volt

Figure 10. Polarograms of Doxorubicin i n S o l u t i o n s

of D i f f e r e n t pH Values
DOXORUBICIN 263

hydrogen chloride. Final purification is performed by

crystallization from ethanol or from a methanol/l-propanol
mixture20.

3.2 Chemical
Doxorubicin can be obtained2l by reacting
daunorubicin hydrochloride in a methanol/dioxane solvent
mixture with a chloroform solution of bromine, forming
14-bromodaunorubicin. This is then hydrolyzed with an
aqueous methanolic solution of sodium hydroxide under a
nitrogen atmosphere. After dilution with water, the solution
is extracted with chloroform and the organic extracts dried
over anhydrous sodium sulfate, concentrated, treated with
hydrogen chloride in anhydrous methanol, and then diluted
with ethyl ether. The precipitate formed is doxorubicin
hydrochloride, which is purified by crystallization from a
mixture of methanol and 1-propanol. The above reaction
pathway can be summarized as shown in Figure ll, in which the
anthraquinone moiety is not shown.

4. Stability
Doxorubicin hydrochloride is very stable in the solid
state. It has been stored for years at room temperature
without any loss in potency or indications of degradation.
The lyophilized powder of doxorubicin hydrochloride with
lactose is also stable, if dry and stored in well closed
containers at room temperature13. The active drug
substance has also been found to be stable for three months
at 60°C, and for three months in light of 500 ft. candles of
illumination at room temperature. The lyophilized
formulation is stable mder similar lighting conditions, and
at 6OoC if the moisture content in the sealed vial is less
than 1.0%.

The effect of pH values and buffer concentrations on the

stability of aqueous solutions of doxorubicin hydrochloride
has been determined by spectrophotometric and chromatographic
methods. Doxorubicin is stable in acidic solutions in the pH
range 3.0 to 6.5, but decomposes at increasing rates as the
pH is increased from 6.5 to 12. Decomposition in aqueous
solution gives complex mixtures of pigmented compounds with a
wide range of chromatographic polarities. Apart from the
isolation of adriamycinone from dilute acid solutions13 the
identification of the components of these mixtures has not
been accomplished.
F i g u r e 11. S y n t h e t i c Pathway for Doxorubicin
DOXORUBICIN 265

5. Metabolism
The two major metabolic transformations of doxorubicin in
laboratory animals and in man are:

a) The reduction of the side chain carbonyl group to a

secondary alcohol, giving 13-dihydrodoxorubicin
(adriamycinol) .
b) The reductive cleavage of the daunosamine moiety with
the formation of 10-deoxyadriamycinone.

The first reaction is catalyzed by an enzyme named

"daunorubicin reductase," an aldo-keto reductase of a very
ubiquitous nature. The reductive splitting of the benzylic
glycosidic bond is, on the contrary, rather unique as no
other examples of enzyme catalysis of this otherwise
chemically very facile reaction have been described1. The
aglycone-like compounds thus formed are then further
metabolized by other typical reactions such as 2-methylation
and conjugat ion22* 23.

Doxorubicin and its metabolites extracted from the urine

of patients treated with the drug were separated by
chromatography on columns of silicic acid. The following
compounds were isolated (in order of increasing polarity)
(see Figure 1 2 ) : 13-dihydroadriamycinone (3), lO-deoxy-13-
dihydroadriamycinone(4), l-demethyl-lO-deoxy-13-
dihydroadriamycinone(5), doxorubicin(1) ,
13-dihydrodoxorubicin(2), l-demethyl-lO-deoxy-13-
d ihydr oadr iamyc inone-1-2-s u1fate( 6 ) , 1-demethy1-10-deoxy-13 -
dihydroadriamycinone-l-~-R-D-glucuronide(7). A total of 60%
of the fluorescence in the urine was due to metabolites and
the remainder was unchanged drug. As the recovery of
doxorubicin fluorescence in bile and urine from a patient was
about 60% of the administered dose, the authors pointed out
the possibility of the presence of non-fluorescent
metabolite^^^. The above mentioned metabolites were also
detected in the plasma of patients under doxorubicin
treatment25.

Protein binding studies using the ultracentrifugation

method suggested that doxorubicin is bound to rabbit and
human plasma proteins to an extent of 50%26, but a
re-examination of the original Scatchard plot data changed
this value to 90%27. Other studies using equilibrium
dialysis have suggested complex binding relationships that
need further investigation2*.
 &
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0 0-
0
I I N
0
I
0-
0
X I I 0
X .rl
u
3
It
\
8-&-
0\ I
0
0- p
0 -0
IOU2
0
0-Ul
I
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0 0
I 0
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I
DOXORUBICIN 261

The main biochemical effects of doxorubicin are concerned

with nucleic acid synthesis. The binding of this drug to DNA
is considered responsible for the interference with template
DNA function29. The DNA-doxorubicin binding constant has
been determined to be approximately 2 x lo6 M-l (30).

6. Methods of Analysis
6.1 Elemental Analysis
The elemental analysis of doxorubicin hydrochloride
(Farmitalia reference standard batch GDA 1) is as follows:

% Theory % Found

C 55.91 56.08
H 5.22 5.33
N 2.41 2.16
c1 6.11 5.85

6.2 Spectrophotometric Analysis

The visible absorption maximum at 495 nm (El% = 223)
lcm
can be used for the quantitation of doxorubicin in dosage
forms.

The fluorescence properties of doxorubicin can be used

for the determination of total anthracycline at low
concentrations31.

6.3 Electrochemical Analysis

ChronopotentiometricJ1, cyclic voltammetr ic32
and p~larographic~~r33 assays of doxorubicin hydrochloride
have been reported. These techniques determine the total
anthracycline content.

6.4 Paper Chromatography

Doxorubicin can be separated from the aglycone,
adriamycinone, by paper chromatography using either of the
following two systemsl3.

A) 1-butanol saturated with pH 5.4 M/15 phosphate buffer.

B) 1-propanol/ethyl acetate/water, 7/1/2 by volume.

The Rf values for doxorubicin and adriamycinone are

0.1 and 0.3, and 0.25 and 0.65 for systems A and B
respectively .
268 ARISTIDE VIGEVANI AND MARTIN J. WILLIAMSON

6.5 Thin Layer Chromatography

Thin l a y e r c h r o m a t o g r a p h i c s y s t e m s f o r d o x o r u b i c i n
are g i v e n i n T a b l e 6.

TABLE 6

Thin l a y e r c h r o m a t o g r a p h i c s y s t e m s f o r d o x o r u b i c i n .

Adsorbent S o l v e n t System -
Rf Reference

Silica gel Methylene c h l o r i d e /

me t h a n o l / w a t e r (100/20/2) 0.17 13

Silica g e l 1-butanol/acetic acid/water

(4/1/5) 0.33 13

Silica gel Chloroform/95% e t h a n o l /

trifluoroacetic acid
(75/20/5) 0.23 34

Silica gel Chloroform/me t h a n o l / a c e t i c

acid (93/5/2, p l a t e d r i e d ,
t h e n 76/20/4) 0.2 35

Silica gel Ch loroform/me thanol/wa ter

sprayed with (140/60/10)
phosphate
buffer
(pH = 7.0) 0.3 36

Polyamide/ l-Butanol/2-propanol/isopropyl
cellulose e t h e r / a c e t i c acid/wa ter
(35/6/6/9/44) 0.3 37

6.6 L i q u i d Chromatography
L i q u i d c h r o m a t o g r a p h i c s y s t e m s for d o x o r u b i c i n
h y d r o c h l o r i d e a r e g i v e n i n T a b l e 7.
DOXORUBICIN 269

TABLE 7

Liquid chromatographic systems for doxorubicin

Stationary Mobile Approximate Doxorubicin Ref.

Phase Phase Capacity Factor (k’)

Silica 2-pr opanol/i sopropyl

(5 micron) e ther/0 .125 M acetate
buffer pH 4.5 (65/30/5) 10 38

Silica 2-propanol/0.5 sodium

(5 micron) acetate buffer pH 4.5
(96.2/3.8) 4 39

Silica Methylene chloride/

(5 micron) methanol/25% ammonia/
water (90/9/0.1/0.8) 4 40

Cyanopropylsilica Chloroform/methanol/
(10 micron) acetic acid/water
(79.8/14.1/4.7/14 ) 3 41

Cyanopropylsilica Ch lor oform/me thanol/

(10 micron) water (96/5/1) - 42

Corasi1-pheny 1 Linear gradient, 16%

(37-75 micron) acetonitrile/in water
to 20% acetonitrile/
80% pH 4 formate buffer 10 43

Coras i1-pheny1 Linear gradient 0 to 40%

(37-50 micron) acetonitrile in pH 4.0
ammonium formate buffer 10 44

Octadecyl-silica Me thanol/aqueous solution

(10 micron) of PIC B-7 (heptane-
sulfonic acid) (50/50) 14 45

Oc tadecyl-s ilica Acetonitrile/aqueous

(10 micron) phosphoric acid pH 2,
(31/69) 3 46

Octadecyl-silica Me thanol/water/acetic
(10 micron) acid (66/33.2/0.8) 1.4 47
Octyl-silica Acetonitrile/10’2 M.
(10 micron) aq. phosphoric acid
(40/60) 2 48
210 ARISTIDE VIGEVANI AND MARTIN J . WILLIAMSON

7. Determination of Doxorubicin in Biological Fluids

Total anthracycline compounds in biological fluids can be
determined by fluorimetric method^^^,^^. Radioimmunoassay
procedures have also been reported50. The emphasis is now
on the separate determination of metabolites and intact drug
in biological fluids. One such method coupled liquid
chromatography followed by RIA^^ but it was rather
time-consuming. TLC followed by fluorescence scanning has
been reported35 and used for disposition prediction^^^.

The most recently published methods have used

rever sed-phase 1 iquid ~ h r o m a t o g r a p h y 5s2
~ or normal phase
liquid ~ h r o m a t o g r a p h y ~with
~ , ~ ~fluorescence detection.
These methods have been applied to tissue distribution
studies5l. Similar methodss4 used for daunorubicin and
metabolites should also be applicable.

8. Analysis of Pharmaceutical Formulations

The identification and/or determination of doxorubicin
hydrochloride in Adriamycin involves the use of visible
spectrophotometry, thin layer chromatography followed by
spectrophotometry or microbiological agar diffusion55.
However, the recently published liquid chromatographic
procedure46 is replacing the above physical methods.

9. Miscellaneous
Ph a rmace u t ica 1 preparations of doxor u b i c in hy dr och lor ide ,
trade-marked Adriamycin, have been patented20.

10. Acknowledgments
Acknowledgment is made to Drs. F. Arcamone and S. Penco
of Farmitalia-Carlo Erba SPA. and Drs. G. Davis, W. Hausmann
and J. Short of Adria Inc., for their useful advise during
the preparation of the manuscript.

11. References - Literature covered until March, 1979.

F. Arcamone, in "Topics in Antibiotics Chemistry,"
-
P. Sammes Ed., Vol. 2, Ellis Horwood Publ.,
Chichester, 1978, pp. 102-239, and references therein.

F. Arcamone, G. Franceschi, P. Orezzi, S. Penco, and

R. Mondelli, Tetrahedron Letters, 3349 (1968).

F. Arcamone, G. Cassinelli, G. Franceschi, P. Orezzi,

and R. Mndelli, Tetrahedron Letters, 3353 (1968).

F. Arcamone, G. Cassinelli, G. Franceschi,

R. Mondelli, P. Orezzi, and S. Penco, Gazz. Chim.
Ital., 100, 949 (1970), and references therein.
DOXORUBICIN 27 1

F. Arcamone, G. F r a n c e s c h i , S. Penco, and A. S e l v a ,

T e t r a h e d r o n Letters, 1007 (1969).

A. Arnone, G. Fronza, R. M o n d e l l i , and A. V i g e v a n i ,

T e t r a h e d r o n Letters, 3349 (1976).

B. G i o i a , F a r m i t a l i a Research L a b o r a t o r i e s , P r i v a t e
Communication (1972).

A V i g e v a n i , B. Gioia, and G. C a s s i n e l l i , C a r b o h y d r a t e
as., 32, 321 (1974).

P. P. Roller, M. S u t p h i n , and A. A. A s z a l o s , Biomed.

Mass Spectrom., 3, 166 (1976).

K. K. Chan, and E. Watson, J. Pharm. Sci., 67,1748

(1978).

K. H. Maurer, U. Rapp, K. Chan, and W. Sadee,

Communication p r e s e n t e d a t t h e "Mario Negri 2nd
I n t e r n a t i o n a l Symposium on Mass S p e c t r o m e t r y i n
B i o c h e m i s t r y and Medicine", M i l a n , 24-26 J u n e , 1974.

B. Gioia, F a r m i t a l i a Research L a b o r a t o r i e s , P r i v a t e
Communication (1978).

F. Arcamone, G. C a s s i n e l l i , G. F r a n c e s c h i , S. Penco,
C. P o l , S. R e d a e l l i , a n d A. S e l v a , " I n t e r n a t i o n a l
Symposium o n Adriamycin," S. K . C a r t e r , A. D i Marco,
M. Ghione, I. H. K r a k o f f , and G. Mathe Eds.,
S p r i n g e r - V e r l a g , B e r l i n , 1972, pp. 1-22.

H. Brockmann, H. Brockmann Jr., and J. Niemeyer,

T e t r a h e d r o n Letters, 4719 ( 1 9 6 8 ) .

R. A n g i u l i , E. F o r e s t i , L. Riva d i S a n s e r v e r i n o ,
N. W. Isaacs, 0. Kennard, W. D. S. Motherwell,
D. L. Wampler, and F. Arcamone, Nature. New Biology,
-234, 78 (1978)

S. N e i d l e and G. T a y l o r , Biochim. Biophys. Acta.,

-
479, 450 (1977).

E. P e l l a , Carlo Erba Research Laboratories, P r i v a t e

Communication (1978).

G. G o n f a l o n i e r i and G. Vasconi, Carlo Erba R e s e a r c h

L a b o r a t o r i e s , P r i v a t e Communication, (1975).
212 ARISTIDE VIGEVANI AND MARTIN J . WILLIAMSON

19) R. J. S t u r g e o n , and S. S. Schulman, J. Pharm. Sci.,

-
66, 958 (1977).

20) F. Arcamone, G. C a s s i n e l l i , G. F a n t i n i , A. G r e i n ,
P. O r e z z i , C. p o l , and C. Spalla, B i o t e c h n o l .
Bioeng., &, 1 1 0 1 (1969).

21) F. Arcamone, G. F r a n c e s c h i , and S. Penco, U.S. Patent

3,803,124 (Apr. 9, 1974).

22) M. A. A s b e l l , R. Schwartzbach, F. J. B u l l o c k , and

D. W. Yesair, J. Pharmacol. Exp. Ther., 182, 63
(1972).

23) F. J. B u l l o c k , R. J. B r u n i , M. A. A s b e l l , J.
Pharmacol. Exp. Ther., 182, 70 (1972).

24) S. Takanashi and N. R. Bachur, Drug Metabolism and

D i s p o s i t i o n , Q, 79 (1976).

25) R. S. Benjamin, C. E. Riggs, Jr., and N. R. Bachur,

Cancer Res., 37, 1416 (1977).

26) P. A. H a r r i s and J. F. Gross, Cancer Chemother, Rep.,

-
59, 819 (1975).

27) K. K. Chan, J. L. Cohen, J. F. Gross, K.

J. H i m e l s t e i n , J. R. Bateman, Y. Tsu-Lee, and
A. S. Marlis, Cancer Treatment Reports, 62, 1161
(1978).

28) M.Menozzi, F a r m i t a l i a Research L a b o r a t o r i e s , P r i v a t e

Communication (1978).

29) A. DiMarco and F. Arcamone, Arzneim.-Fbrsch. (Drug

R e s e a r c h ) , 25, 368 (1975) and r e f e r e n c e s c i t e d
t h e r ein.

30) S. R. Byrn and G. D. Dolch, J. Pharm. S c i . , 67, 688

(1978) and r e f e r e n c e s c i t e d .

31) L. Dusonchet, N. Gebbia, and F. G e r b a s s i , Pharm. Res.

Commun., 2, 55 (1971).

32) G. M. Rao, J. W. LOwn, and J. A. Plambeck, J.

Electrochem. Soc., 125, 534 (1978).

33) L. A. S t e r n s o n and G. Thomas, A n a l . Letters, 10, 99

(1977).
DO XO RUBICIN 273

G. W. C l a r k , Adria Laboratories , P r i v a t e
Communication, (1979).

E. Watson and K. K. Chan, Cancer Treatment R e p o r t s ,

-
60, 1611 (1976)

Federal Register 41, 14184, A p r i l 2, 1976 S e c t i o n

436.315.

Federal Register 41, 14184, A p r i l 2, 1976 S e c t i o n

436.314.

F a r m i t a l i a SpA., J u n e 1975, P r i v a t e Communication.

H . G. B a r t h and A. Z. COnner, J. Chromatogr., 131,

375 (1977).

R. Hulhoven and J. P. Desager, J. Chromatogr., 125,

369 (1976).

P. A. Harris, Proc. Amer. A s s o c . Cancer R e s e a r c h , 2,

131 (1976).

V. P. M a r s h a l l , E. A. R e i s e n d e r , and P. F. W i l l e y .
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J. J. Langone, H v a n Vunakis, and N. Bachur, Biochem.

Med., 2,283 (1975).

M. I s r a e l , W. J. Pegg, P. M. W i l k i n s o n and
M. B. G a r n i c k , " B i o c h e m i c a l / B i o l o g i c a l A p p l i c a t i o n s
of L i q u i d Chromatography," G. L. Hawk, e d i t o r , Marcel
D e k k e r Inc., N.Y. 1978, Chap. 22.

Waters P h a r m a c e u t i c a l A p p l i c a t i o n Note #312.

Federal Register 43, 44836, September 29, 1978,

S e c t i o n 436.322.

L. M a l s p e i s , Ohio S t a t e U n i v e r s i t y , P e r s o n a l
Communication, (1978).

S. Eksborg, J. Chromatogr., 149, 225 (1978).

N. R. Bachur, A. L. Moore, J. G. B e r n s t e i n a n d A.
L i u , Cancer Chemotherap. Rep., 54, 89 (1970).

H.van Vunakis, J. J. Langone, L. J. R i c e b e r g and L.

Levine, Cancer R e s e a r c h , 34, 2546 (1976).
274 ARISTIDE VIGEVANI A N D MARTIN J . WILLIAMSON

51) M. Israel, W. J. Pegg., P. M. Wilkinson and M. B.

Garnick, J. Liquid Chromatogr., 1,795 (1978).

52) R. Baurain, D. Deprez-DeCampeneere and A. Trouet,

Analytical Biochemistry, 99, 112 (1979).

53) J. H. Peters and J. F. Murray Jr., J. Liquid

Chromatogr., 2, 45 (1979)

54) S. Eksborg, H. Ehrsson, B. Anderson and M. Beran, J.

Chromatogr., 153, 211 (1978).

55) Federal Register 41, 14184, April 2, 1976, Section

450.24.