Vous êtes sur la page 1sur 2





04/2015:2765 stationary phase : base-deactivated end-capped octadecylsilyl

silica gel for chromatography R (3 m) ;
temperature : 30 C.
Mobile phase :
mobile phase A : 0.1 per cent V/V solution of phosphoric
acid R ;
mobile phase B : phosphoric acid R, acetonitrile R,
methanol R (0.1:40:60 V/V/V) ;
0 - 25

Mobile phase A
(per cent V/V)

Mobile phase B
(per cent V/V)

25 - 32

54 40

46 60

32 - 45



Mr 414.5

Flow rate : 1.0 mL/min.

Detection : spectrophotometer at 240 nm.
Injection : 20 L of test solution (a) and reference solutions (a),
9,11-Epoxy-7-(methoxycarbonyl)-3-oxo-17-pregn-4-ene- (b) and (c).
Identification of impurities : use the chromatogram
Content : 97.5 per cent to 102.0 per cent (anhydrous substance). supplied with eplerenone for system suitability CRS and the
chromatogram obtained with reference solution (a) to identify
the peaks due to impurities A and D ; use the chromatogram
Appearance : white, almost white or slightly yellow, crystalline supplied with eplerenone for peak identification CRS and the
chromatogram obtained with reference solution (b) to identify
Solubility : slightly soluble in water, freely soluble in methylene the peak due to impurity B.
Relative retention with reference to eplerenone (retention
chloride, slightly soluble in ethanol (96 per cent).
time = about 9 min) : impurity D = about 0.71 ;
It shows polymorphism (5.9).
impurity A = about 0.74 ; impurity B = about 1.2.
System suitability : reference solution (a) :
peak-to-valley ratio : minimum 5.0, where Hp = height
Infrared absorption spectrophotometry (2.2.24).
above the baseline of the peak due to impurity D and
Comparison : eplerenone CRS.
Hv = height above the baseline of the lowest point of the
If the spectra obtained in the solid state show differences,
curve separating this peak from the peak due to impurity A.
dissolve the substance to be examined and the reference
substance separately in methanol R, evaporate to dryness and Calculation of percentage contents :
for each impurity, use the concentration of eplerenone in
record new spectra using the residues.
reference solution (c).
Limits :
Specic optical rotation (2.2.7) : 16.0 to 14.0.
impurities A, B : for each impurity, maximum 0.3 per cent ;
Dissolve 0.250 g in acetonitrile R and dilute to 25.0 mL with
unspecified impurities : for each impurity, maximum
the same solvent.
0.10 per cent ;
Related substances. Liquid chromatography (2.2.29).
total : maximum 0.6 per cent ;
Solvent mixture : acetonitrile R, methanol R, water R
reporting threshold : 0.05 per cent.
(25:25:50 V/V/V).
Water (2.5.12) : maximum 0.5 per cent, determined on 1.000 g.
Test solution (a). Dissolve 25.0 mg of the substance to be
Sulfated ash (2.4.14): maximum 0.1 per cent, determined on
examined in the solvent mixture and dilute to 50.0 mL with
1.0 g.
the solvent mixture.
Test solution (b). Dilute 1.0 mL of test solution (a) to 10.0 mL ASSAY
with the solvent mixture.
Liquid chromatography (2.2.29) as described in the test for
Reference solution (a). Dissolve 5 mg of eplerenone for system related substances with the following modication.
suitability CRS (containing impurities A and D) in the solvent
Injection : test solution (b) and reference solution (d).
mixture and dilute to 10.0 mL with the solvent mixture.
Calculate the percentage content of C24H30O6 taking into
Reference solution (b). Dissolve 5 mg of eplerenone for peak
account the assigned content of eplerenone CRS.
identification CRS (containing impurity B) in the solvent
mixture and dilute to 10.0 mL with the solvent mixture.
Reference solution (c). Dilute 1.0 mL of test solution (a) to
Specified impurities : A, B.
100.0 mL with the solvent mixture. Dilute 1.0 mL of this
solution to 10.0 mL with the solvent mixture.
Other detectable impurities (the following substances would,
Reference solution (d). Dissolve 25.0 mg of eplerenone CRS in if present at a sufcient level, be detected by one or other of
the tests in the monograph. They are limited by the general
the solvent mixture and dilute to 50.0 mL with the solvent
acceptance criterion for other/unspecied impurities and/or
mixture. Dilute 1.0 mL of the solution to 10.0 mL with the
by the general monograph Substances for pharmaceutical use
solvent mixture.
(2034). It is therefore not necessary to identify these impurities
Column :
for demonstration of compliance. See also 5.10. Control of
impurities in substances for pharmaceutical use) : C, D, E, F, G.
size : l = 0.15 m, = 4.6 mm ;
General Notices (1) apply to all monographs and other texts


Erythropoietin concentrated solution


corrected 8.4

A. 3-oxo-17-pregn-4-ene-7,9:21,17-dicarbolactone,

Erythropoietini solutio concentrata

Mr approx. 30 600
B. 11,12-epoxy-7-(methoxycarbonyl)-3-oxo-17-pregn4-ene-21,17-carbolactone,

C. 7-(methoxycarbonyl)-3-oxo-17-pregna-4,9(11)-diene21,17-carbolactone,

D. (2R)-9,11-epoxy-3,5-dioxo-4,5-dihydro-3Hspiro[androst-4-ene-17,2-furan]-7-carboxylic acid,

E. 9,11-epoxy-7-(methoxycarbonyl)-3-oxo-17-pregn-4ene-21,17-carbolactone,

F. 9,11-epoxy-17-hydroxy-7-(methoxycarbonyl)-3-oxo17-pregn-4-ene-21-carboxylic acid,

G. 7-(methoxycarbonyl)-3-oxo-17-pregn-4-ene-21,17carbolactone.


Erythropoietin concentrated solution is a solution
containing a family of closely-related glycoproteins which
are indistinguishable from the naturally occurring human
erythropoietin (urinary erythropoietin) in terms of amino acid
sequence (165 amino acids) and average glycosylation pattern,
at a concentration of 0.5-10 mg/mL. It may also contain buffer
salts and other excipients. It has a potency of not less than
100 000 IU/mg of active substance determined using the
conditions described under Assay and in the test for protein.
Erythropoietin is produced in rodent cells in vitro by a method
based on recombinant DNA technology.
Prior to batch release, the following tests are carried out on
each batch of the nal product, unless exemption has been
granted by the competent authority.
Host cell-derived proteins : the limit is approved by the
competent authority.
Host cell- and vector-derived DNA : the limit is approved
by the competent authority.
Appearance : clear or slightly turbid, colourless solution.
A. It gives the appropriate response when examined using the
conditions described under Assay.
B. Capillary zone electrophoresis (2.2.47).
Test solution. Dilute the preparation to be examined
with water R to obtain a concentration of 1 mg/mL.
Desalt 0.25 mL of the solution by passage through a
micro-concentrator cartridge provided with a membrane
with a molecular mass cut-off of not more than 10 000 Da.
Add 0.2 mL of water R to the sample and desalt again.
Repeat the desalting procedure once more. Dilute the
sample with water R, determine its protein concentration
as described under Tests and adjust to a concentration of
approximately 1 mg/mL with water R.
Reference solution. Dissolve the contents of a vial of
erythropoietin for physicochemical tests CRS in 0.10 mL of
water R. Proceed with desalting as described for the test
Capillary :
material : uncoated fused silica ;
size : effective length = about 100 cm, = 50 m.
Temperature : 35 C.
CZE buffer concentrate (0.1 M sodium chloride, 0.1 M
tricine, 0.1 M sodium acetate). Dissolve 0.584 g of sodium
chloride R, 1.792 g of tricine R and 0.820 g of anhydrous
sodium acetate R in water R and dilute to 100.0 mL with
the same solvent.
1 M putrescine solution. Dissolve 0.882 g of putrescine R in
10 mL of water R. Distribute in 0.5 mL aliquots.
See the information section on general monographs (cover pages)