EUROPEAN PHARMACOPOEIA 8.

Etidronate disodium

01/2008:1778 ASSAY
corrected 8.3 Dissolve 0.100 g in 2 mL of anhydrous formic acid R and
dilute to 50 mL with glacial acetic acid R. Titrate with 0.1 M
perchloric acid, determining the end-point potentiometrically
ETIDRONATE DISODIUM
(2.2.20).
1 mL of 0.1 M perchloric acid is equivalent to 12.50 mg
Dinatrii etidronas
of C2H6Na2O7P2.
STORAGE
In an airtight container.

C2H6Na2O7P2
[7414-83-7]

IMPURITIES
Specified impurities : A, B.
Mr 250.0
A. H3PO4 : phosphoric acid,
B. H3PO3 : phosphorous acid.

DEFINITION
Disodium dihydrogen (1-hydroxyethylidene)bisphosphonate.
Content : 98.0 per cent to 102.0 per cent (anhydrous substance).

01/2015:0823

ETOPOSIDE

CHARACTERS
Appearance : white or yellowish, hygroscopic powder.
Solubility : freely soluble in water, practically insoluble in
acetone and in ethanol (96 per cent).

Etoposidum

IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Comparison : etidronate disodium CRS.
The transmittance at about 2000 cm 1 (5 m) is not less
than 40 per cent without compensation.
B. It gives reaction (a) of sodium (2.3.1).
TESTS
pH (2.2.3) : 4.2 to 5.2.
Dissolve 1.0 g in carbon dioxide-free water R and dilute to
100 mL with the same solvent.
Impurities A and B. Liquid chromatography (2.2.29).
Test solution. Dissolve 20.0 mg of the substance to be
examined in water R and dilute to 10.0 mL with the same
solvent.
Reference solution. To 2.0 mL of a 0.3 g/L solution of
phosphoric acid R add 2.0 mL of a 0.25 g/L solution of
phosphorous acid R and dilute to 50.0 mL with water R.
Column :
size : l = 0.15 m, = 4.6 mm ;
stationary phase : anion-exchange resin R (5 m) ;
temperature : 35 C.
Mobile phase : mix 0.2 mL of anhydrous formic acid R and
1000 mL of water R ; adjust to pH 3.5 with an 80 g/L solution
of sodium hydroxide R.
Flow rate : 1.0 mL/min.
Detection : differential refractometer.
Injection : 100 L.
System suitability : reference solution :
resolution : minimum 2.5 between the peaks due to
impurity A and impurity B.
Limits :
impurities A, B : for each impurity, not more than the area
of the corresponding peak in the chromatogram obtained
with the reference solution (0.5 per cent).
Heavy metals (2.4.8) : maximum 20 ppm.
1.0 g complies with test F. Prepare the reference solution using
2 mL of lead standard solution (10 ppm Pb) R.
Water (2.5.32): maximum 5.0 per cent.
Dissolve 50.0 mg in a mixture of equal volumes of anhydrous
acetic acid R and formamide R and dilute to 5.0 mL with the
same mixture of solvents. Use 1.0 mL of the solution.

4306

C29H32O13
[33419-42-0]

Mr 588.6

DEFINITION
(5R,5aR,8aR,9S)-9-[[4,6-O-[(R)-Ethylidene]--Dglucopyranosyl]oxy]-5-(4-hydroxy-3,5-dimethoxyphenyl)5,8,8a,9-tetrahydroisobenzofuro[5,6-f][1,3]benzodioxol6(5aH)-one.
Content : 98.0 per cent to 102.0 per cent (anhydrous substance).
CHARACTERS
Appearance : white or almost white, crystalline powder, slightly
hygroscopic.
Solubility : practically insoluble in water, sparingly soluble
in methanol, slightly soluble in ethanol 96 per cent and in
methylene chloride.
IDENTIFICATION
First identification : A, B.
Second identification : C, D.
A. Specic optical rotation (see Tests).
B. Infrared absorption spectrophotometry (2.2.24).
Comparison : etoposide CRS.
C. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 10 mg of the substance to be
examined in a mixture of 1 volume of methanol R and
9 volumes of methylene chloride R and dilute to 2 mL with
the same mixture of solvents.
Reference solution. Dissolve 10 mg of etoposide CRS in
a mixture of 1 volume of methanol R and 9 volumes of
methylene chloride R and dilute to 2 mL with the same
mixture of solvents.
Plate : silica gel H R as the coating substance.
Mobile phase : water R, glacial acetic acid R, acetone R,
methylene chloride R (1.5:8:20:100 V/V/V/V).
Application : 5 L as bands of 10 mm.
Development : immediately, over 6/7 of the plate.
Drying : in a current of warm air for 5 min.
See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.3

Etoposide

Detection : spray with a mixture of 1 volume of sulfuric

acid R and 9 volumes of ethanol (96 per cent) R and heat at
140 C for 15 min. Cover the plate immediately with a glass
plate of the same size. Examine in daylight.
Results : the principal zone in the chromatogram obtained
with the test solution is similar in position, colour and size
to the principal zone in the chromatogram obtained with
the reference solution.
D. In a test-tube dissolve about 5 mg in 5 mL of glacial acetic
acid R and add 0.05 mL of ferric chloride solution R1. Mix
and cautiously add 2 mL of sulfuric acid R. Avoid mixing
the 2 layers. Allow to stand for about 30 min ; a pink to
reddish-brown ring develops at the interface and the upper
layer is yellow.

Identification of impurities : use the chromatogram supplied

with etoposide for system suitability CRS and the chromatogram
obtained with reference solution (b) to identify the peaks due
to impurities B, C, D, E, N and O.
Relative retention with reference to etoposide (retention
time = about 5 min) : impurity D = about 0.4 ;
impurity E = about 0.8 ; impurity C = about 1.1 ;
impurity B = about 1.2 ; impurity N = about 3.1 ;
impurity O = about 4.2.
System suitability : reference solution (b) :
peak-to-valley ratio : minimum 2.0, where Hp = height
above the baseline of the peak due to impurity C and
Hv = height above the baseline of the lowest point of the
curve separating this peak from the peak due to etoposide ;
and minimum 3.0, where Hp = height above the baseline
TESTS
of the peak due to impurity B and Hv = height above the
baseline of the lowest point of the curve separating this
Appearance of solution. The solution is clear (2.2.1) and not
peak from the peak due to impurity C.
more intensely coloured than reference solution Y6 or BY6
Limits
:
(2.2.2, Method II).
correction factor : for the calculation of content, multiply
Dissolve 0.6 g in a mixture of 1 volume of methanol R and
the peak area of impurity O by 1.7 ;
9 volumes of methylene chloride R and dilute to 20 mL with
the same mixture of solvents.
impurities B, C, D, E, N : for each impurity, not more than
twice the area of the principal peak in the chromatogram
Specic optical rotation (2.2.7) : 114 to 106 (anhydrous
obtained with reference solution (a) (0.2 per cent) ;
substance).
Dissolve 50.0 mg in a mixture of 1 volume of methanol R and impurity O : not more than 1.5 times the area of the
principal peak in the chromatogram obtained with
9 volumes of methylene chloride R and dilute to 10.0 mL with
reference solution (a) (0.15 per cent) ;
the same mixture of solvents.
unspecified impurities : for each impurity, not more than the
Related substances. Liquid chromatography (2.2.29).
area of the principal peak in the chromatogram obtained
Solvent mixture : mobile phase A, mobile phase B (50:50 V/V).
with reference solution (a) (0.10 per cent) ;
total : not more than 10 times the area of the principal peak
Test solution (a). Dissolve 40 mg of the substance to be
in the chromatogram obtained with reference solution (a)
examined in the solvent mixture and dilute to 10.0 mL with
(1.0 per cent) ;
the solvent mixture.
disregard limit : 0.5 times the area of the principal peak in
Test solution (b). Dissolve 50.0 mg of the substance to be
the chromatogram obtained with reference solution (a)
examined in the solvent mixture and dilute to 50.0 mL with
(0.05 per cent) ; disregard any peak due to the solvent.
the solvent mixture.
Heavy metals (2.4.8): maximum 20 ppm.
Reference solution (a). Dilute 1.0 mL of test solution (a) to
100.0 mL with the solvent mixture. Dilute 1.0 mL of this
0.5 g complies with test G. Prepare the reference solution
solution to 10.0 mL with the solvent mixture.
using 1 mL of lead standard solution (10 ppm Pb) R.
Reference solution (b). Dissolve 4 mg of etoposide for system
Water (2.5.12) : maximum 6.0 per cent, determined on 0.250 g.
suitability CRS (containing impurities B, C, D, E, N and O) in
Sulfated ash (2.4.14): maximum 0.1 per cent, determined on
1.0 mL of the solvent mixture.
1.0 g.
Reference solution (c). Dissolve 50.0 mg of etoposide CRS in
ASSAY
the solvent mixture and dilute to 50.0 mL with the solvent
mixture.
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modications.
Column :
Injection : test solution (b) and reference solution (c).
size : l = 0.125 m, = 4.6 mm ;
System suitability :
stationary phase : octadecylsilyl silica gel for
repeatability : maximum relative standard deviation of
chromatography R (5 m) ;
1.0 per cent after 6 injections of reference solution (c).
temperature : 40 C.
Calculate the percentage content of C29H32O13 from the
Mobile phase :
declared content of etoposide CRS.
mobile phase A : anhydrous formic acid R, triethylamine R,
STORAGE
water R (1:1:998 V/V/V) ;
In an airtight container.
mobile phase B : anhydrous formic acid R, triethylamine R,
acetonitrile R (1:1:998 V/V/V) ;
IMPURITIES
Time
Mobile phase A
Mobile phase B
Specified impurities : B, C, D, E, N, O.
(min)
(per cent V/V)
(per cent V/V)
Other detectable impurities (the following substances would,
75
0-7
25
if present at a sufcient level, be detected by one or other of
the tests in the monograph. They are limited by the general
7 - 23
75 27
25 73
acceptance criterion for other/unspecied impurities and/or
by the general monograph Substances for pharmaceutical
Flow rate : 1 mL/min.
use (2034). It is therefore not necessary to identify these
Detection : spectrophotometer at 285 nm.
impurities for demonstration of compliance. See also 5.10.
Injection : 10 L of test solution (a) and reference solutions (a) Control of impurities in substances for pharmaceutical use) : A,
F, G, H, I, J, K, L, M, P, Q, R.
and (b).
General Notices (1) apply to all monographs and other texts

4307

Etoposide

EUROPEAN PHARMACOPOEIA 8.3

F. (5R,5aR,8aR,9S)-9-[[4,6-O-[(R)-ethylidene]--Dglucopyranosyl]oxy]-5-[4-[(phenoxyacetyl)oxy]3,5-dimethoxyphenyl]-5,8,8a,9-tetrahydroisobenzofuro[5,6-f][1,3]benzodioxol-6(5aH)-one
(4-phenoxyacetyletoposide),
A. (5R,5aR,8aR,9S)-5-[4-[[(benzyloxy)carbonyl]oxy]-3,5dimethoxyphenyl]-9-[[4,6-O-[(R)-ethylidene]--Dglucopyranosyl]oxy]-5,8,8a,9-tetrahydroisobenzofuro[5,6-f][1,3]benzodioxol-6(5aH)-one (4-carbobenzoyloxyethylidene-lignan P),

B. (5R,5aS,8aR,9S)-9-[[4,6-O-[(R)-ethylidene]--Dglucopyranosyl]oxy]-5-(4-hydroxy-3,5-dimethoxyphenyl)5,8,8a,9-tetrahydroisobenzofuro[5,6-f][1,3]benzodioxol6(5aH)-one (picroethylidene-lignan P ; cis-etoposide),

C. (5R,5aR,8aR,9S)-9-[[4,6-O-[(R)-ethylidene]--Dglucopyranosyl]oxy]-5-(4-hydroxy-3,5-dimethoxyphenyl)5,8,8a,9-tetrahydroisobenzofuro[5,6-f][1,3]benzodioxol6(5aH)-one (-etoposide),

D. (5R,5aR,8aR,9S)-9-(-D-glucopyranosyloxy)-5-(4-hydroxy3,5-dimethoxyphenyl)-5,8,8a,9-tetrahydroisobenzofuro[5,6-f][1,3]benzodioxol-6(5aH)-one (lignan P),

E. (5R,5aR,8aR,9S)-9-hydroxy-5-(4-hydroxy-3,5-dimethoxyphenyl)-5,8,8a,9-tetrahydroisobenzofuro[5,6-f][1,3]benzodioxol-6(5aH)-one (4-desmethylepipodophyllotoxin),

4308

G. (5R,5aR,8aR,9S)-5-[4-[[(benzyloxy)carbonyl]oxy]3,5-dimethoxyphenyl]-9-[[4,6-O-[(R)-ethylidene]2,3-di-O-formyl--D-glucopyranosyl]oxy]-5,8,8a,9tetrahydroisobenzofuro[5,6-f][1,3]benzodioxol-6(5aH)one (4-carbobenzoyloxydiformylethylidene-lignan P),

H. (5R,5aR,8aR,9S)-9-ethoxy-5-(4-hydroxy-3,5dimethoxyphenyl)-5,8,8a,9-tetrahydroisobenzofuro-[5,6f][1,3]benzodioxol-6(5aH)-one (4-O-desmethyl-1-Oethylepipodophyllotoxin),

I. (5R,5aR,8aR,9S)-9-[[4,6-O-[(R)-ethylidene]--Dglucopyranosyl]oxy]-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9tetrahydroisobenzofuro[5,6-f][1,3]benzodioxol-6(5aH)one (4-O-methylethylidene-lignan P),

J. (5R,5aR,8aR,9S)-5-(4-hydroxy-3,5-dimethoxyphenyl)9-methoxy-5,8,8a,9-tetrahydroisobenzofuro-[5,6f][1,3]benzodioxol-6(5aH)-one (4-O-desmethyl-1-Omethylepipodophyllotoxin),
See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.3

Etoposide

K. 9,9-oxybis[(5R,5aR,8aR,9S)-5-(4-hydroxy-3,5dimethoxyphenyl)-5,8,8a,9-tetrahydroisobenzofuro[5,6-f][1,3]benzodioxol-6(5aH)-one] (di-4-Odesmethylepipodophyllotoxin),

O. (5R,5aR,8aR,9S)-9-[[2,3-bis-O-(dichloroacetyl)-4,6-O-[(S)ethylidene]--L-glucopyranosyl]oxy]-5-(4-hydroxy-3,5dimethoxyphenyl)-5,8,8a,9-tetrahydroisobenzofuro[5,6f][1,3]benzodioxol-6(5aH)-one,

L. (5R,5aR,8aR,9R)-9-hydroxy-5-(4-hydroxy-3,5-dimethoxyphenyl)-5,8,8a,9-tetrahydroisobenzofuro[5,6-f][1,3]benzodioxol-6(5aH)-one (4-O-desmethylpodophyllotoxin),

P. 9-hydroxy-5-(4-hydroxy-3,5-dimethoxyphenyl)isobenzofuro[5,6-f][1,3]benzodioxol-6(8H)-one,

M. (5R,5aR,8aR,9R)-9-hydroxy-5-(3,4,5-trimethoxyphenyl)5,8,8a,9-tetrahydroisobenzofuro[5,6-f][1,3]benzodioxol6(5aH)-one (podophyllotoxin),

Q. 5-(4-hydroxy-3,5-dimethoxyphenyl)isobenzofuro[5,6f][1,3]benzodioxol-6(8H)-one,

N. (5R,5aR,8aR,9S)-9-[[4,6-O-[(R)-ethylidene]-D-glucopyranosyl]oxy]-5-[4-[[(5R,5aR,8aR,9S)5-(4-hydroxy-3,5-dimethoxyphenyl)-6-oxo5,5a,6,8,8a,9-hexahydroisobenzofuro[5,6f][1,3]benzodioxol-9-yl]oxy]3,5-dimethoxyphenyl]5,8,8a,9-tetrahydroisobenzofuro-[5,6-f][1,3]benzodioxol6(5aH)-one.

R. (5R,5aR,8aR,9S)-9-[[4,6-O-[(R)-ethylidene]--Dglucopyranosyl]oxy]-5-[4-[[(5R,5aR,8aR,9R)-5-(4hydroxy-3,5-dimethoxyphenyl)-6-oxo-5,5a,6,8,8a,9hexahydroisobenzofuro[5,6-f][1,3]benzodioxol9-yl]oxy]-3,5-dimethoxyphenyl]-5,8,8a,9tetrahydroisobenzofuro[5,6-f][1,3]benzodioxol6(5aH)-one.

General Notices (1) apply to all monographs and other texts

4309