EUROPEAN PHARMACOPOEIA 5.

0 Doxorubicin hydrochloride

mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with

the mobile phase.
The chromatographic procedure may be carried out using :
— a stainless steel column 0.12 m long and 4 mm in internal
diameter packed with spherical octylsilyl silica gel for
chromatography R (5 µm) with a specific surface area of
220 m2/g and a pore size of 80 nm,
— as mobile phase at a flow rate of 1 ml/min a mixture of C. (E)-3-(dibenzo[b,e]oxepin-11(6H)-ylidene)-N-
30 volumes of methanol R and 70 volumes of a 30 g/l methylpropan-1-amine,
solution of sodium dihydrogen phosphate R, previously
adjusted to pH 2.5 with phosphoric acid R,
— as detector a spectrophotometer set at 254 nm,
maintaining the temperature of the column at 50 °C.
Inject 20 µl of the test solution. Adjust the sensitivity of the
system so that the height of the principal peak is at least
50 per cent of the full scale of the recorder. The test is not
valid unless the resolution between the first peak (E-isomer) D. (Z)-3-(dibenzo[b,e]oxepin-11(6H)-ylidene)-N,N-
and the second peak (Z-isomer) is at least 1.5. dimethylpropan-1-amine.
The ratio of the area of the peak due to the E-isomer to the
area of the peak due to the Z-isomer is 4.4 to 6.7. 01/2005:0714
Heavy metals (2.4.8). 1.0 g complies with limit test D
(20 ppm). Prepare the standard using 2 ml of lead standard DOXORUBICIN HYDROCHLORIDE
solution (10 ppm Pb) R.
Loss on drying (2.2.32). Not more than 0.5 per cent, Doxorubicini hydrochloridum
determined on 1.000 g by drying in an oven at 100-105 °C.
Sulphated ash (2.4.14). Not more than 0.1 per cent,
determined on 1.0 g.

ASSAY
Dissolve 0.250 g in a mixture of 5 ml of anhydrous acetic
acid R and 35 ml of acetic anhydride R. Titrate with 0.1 M
perchloric acid until the colour changes from blue to green,
using 0.2 ml of crystal violet solution R as indicator.
1 ml of 0.1 M perchloric acid is equivalent to 31.58 mg of
C19H22ClNO.
STORAGE
Protected from light.
IMPURITIES
A. dibenzo[b,e]oxepin-11(6H)-one,
B. (11RS)-11-[3-(dimethylamino)propyl]-6,11-
dihydrodibenzo[b,e]oxepin-11-ol,
C27H30ClNO11 Mr 580.0
DEFINITION
(8S,10S)-10-[(3-Amino-2,3,6-trideoxy-α-L-lyxo-
hexopyranosyl)oxy]-6,8,11-trihydroxy-8-(hydroxyacetyl)-
1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione
hydrochloride.
Substance produced by certain strains of Streptomyces
coeruleorubidus or Streptomyces peucetiusor obtained by
any other means.
Content : 98.0 per cent to 102.0 per cent (anhydrous,
solvent-free substance).
CHARACTERS
Appearance : orange-red, crystalline powder, hygroscopic.
Solubility : soluble in water, slightly soluble in methanol.
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Comparison : doxorubicin hydrochloride CRS.
B. Dissolve about 10 mg in 0.5 ml of nitric acid R, add
0.5 ml of water R and heat over a flame for 2 min. Allow
to cool and add 0.5 ml of silver nitrate solution R1. A
white precipitate is formed.
TESTS
pH (2.2.3) : 4.0 to 5.5.
Dissolve 50 mg in carbon dioxide-free water R and dilute to
10 ml with the same solvent.

General Notices (1) apply to all monographs and other texts 1481
Doxycycline hyclate EUROPEAN PHARMACOPOEIA 5.0

Related substances. Liquid chromatography (2.2.29). IMPURITIES

Prepare the solutions immediately before use.
A. daunorubicin,
Test solution (a). Dissolve 50.0 mg of the substance to be
examined in the mobile phase and dilute to 50.0 ml with the
mobile phase.
Test solution (b). Dilute 10.0 ml of test solution (a) to
100.0 ml with the mobile phase.
Reference solution (a). Dissolve 10.0 mg of doxorubicin
hydrochloride CRS and 10 mg of epirubicin
hydrochloride CRS in the mobile phase and dilute to 50.0 ml
with the mobile phase. Dilute 10.0 ml of the solution to
100.0 ml with the mobile phase.
Reference solution (b). Dilute 5.0 ml of reference solution (a)
to 20.0 ml with the mobile phase. B. R = OCH3 : (8S,10S)-10[(3-amino-2,3,6-trideoxy-α-L-lyxo-
Reference solution (c). Dissolve 50.0 mg of doxorubicin hexopyranosyl)oxy]-8-(2-bromo-1,1-dimethoxyethyl)-6,8,
hydrochloride CRS in the mobile phase and dilute to 50.0 ml 11-trihydroxy-1-methoxy-7,8,9,10-tetrahydrotetracene-5,
with the mobile phase. Dilute 10.0 ml of the solution to 12-dione,
100.0 ml with the mobile phase.
C. R + R = O : (8S,10S)-10[(3-amino-2,3,6-trideoxy-α-L-lyxo-
Column : hexopyranosyl)oxy]-8-(bromoacetyl)-6,8,11-trihydroxy-1-
— size : l = 0.25 m, Ø = 4.0 mm, methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione,
— stationary phase: end-capped octadecylsilyl silica gel
for chromatography R (5 µm).
Mobile phase : mix equal volumes of acetonitrile R and a
solution containing 2.88 g/l of sodium laurilsulfate R and
2.25 g/l of phosphoric acid R.
Flow rate : 1 ml/min.
Detection : spectrophotometer at 254 nm.
Injection : 5 µl ; inject test solution (a) and reference D. (8S,10S)-6,8,10,11-tetrahydroxy-8-(hydroxyacetyl)-
solutions (a) and (b). 1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione
(doxorubicin aglycone, doxorubicinone).
Run time : 3.5 times the retention time of doxorubicin.
Retention time : doxorubicin = about 8 min.
System suitability : reference solution (a) : 01/2005:0272
— resolution : minimum of 2.0 between the peaks due to
doxorubicin and to epirubicin. DOXYCYCLINE HYCLATE
Limits :
— any impurity : not more than the area of the peak
Doxycyclini hyclas
corresponding to doxorubicin in the chromatogram
obtained with reference solution (b) (0.5 per cent),
— disregard limit : 0.1 times the area of the peak
corresponding to doxorubicin in the chromatogram
obtained with reference solution (b) (0.05 per cent).
Ethanol (2.4.24, System B) : maximum 1.0 per cent.
Water (2.5.12) : maximum 4.0 per cent, determined on
0.100 g.
Bacterial endotoxins (2.6.14) : less than 2.2 IU/mg, if
intended for use in the manufacture of parenteral dosage (C22H25ClN2O8),1/2C2H6O,1/2H2O Mr 512.9
forms without a further appropriate procedure for the
removal of bacterial endotoxins. DEFINITION
Hydrochloride hemiethanol hemihydrate of
ASSAY
(4S,4aR,5S,5aR,6R,12aS)-4-(dimethylamino)-3,5,10,
Liquid chromatography (2.2.29) as described in the test for 12,12a-pentahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,
related substances. 12a-octahydrotetracene-2-carboxamide.
Injection : test solution (b) and reference solution (c). Substance obtained from oxytetracycline or metacycline or
Calculate the percentage content of C27H30ClNO11. by any other means.
Content : 95.0 per cent to 102.0 per cent of C22H25ClN2O8
STORAGE (anhydrous and ethanol-free substance).
In an airtight container. If the substance is sterile, store in a
sterile, airtight, tamper-proof container. CHARACTERS
Appearance : yellow, crystalline powder, hygroscopic.
LABELLING Solubility : freely soluble in water and in methanol, sparingly
The label states, where applicable, that the substance is free soluble in ethanol (96 per cent). It dissolves in solutions of
from bacterial endotoxins. alkali hydroxides and carbonates.

1482 See the information section on general monographs (cover pages)