EUROPEAN PHARMACOPOEIA 8.

Alprostadil

04/2015:1488 System A
Column :

ALPROSTADIL
Alprostadilum

size : l = 0.25 m, = 4.0 mm ;

stationary phase : base-deactivated octylsilyl silica gel for
chromatography R (4 m) with a pore size of 6 nm ;
temperature : 35 C.
Mobile phase :
mobile phase A : dissolve 3.9 g of sodium dihydrogen
phosphate R in water R and dilute to 1.0 L with the
same solvent ; adjust to pH 2.5 with a 2.9 g/L solution of
phosphoric acid R (approximately 600 mL is required) ; to
740 mL of the buffer solution add 260 mL of acetonitrile R1 ;

Mr 354.5 mobile phase B : dissolve 3.9 g of sodium dihydrogen

phosphate R in water R and dilute to 1.0 L with the
same solvent ; adjust to pH 2.5 with a 2.9 g/L solution of
DEFINITION
phosphoric acid R (approximately 600 mL is required) ; to
200 mL of the buffer solution add 800 mL of acetonitrile R1 ;
7-[(1R,2R,3R)-3-Hydroxy-2-[(1E,3S)-3-hydroxyoct-1-enyl]-5oxocyclopentyl]heptanoic acid.
Time
Mobile phase A
Mobile phase B
C20H34O5
[745-65-3]

Content : 95.0 per cent to 102.5 per cent (anhydrous substance).

(min)
0 - 75

(per cent V/V)

100

(per cent V/V)

0

CHARACTERS

75 - 76

100 0

0 100

Appearance : white or slightly yellowish, crystalline powder.

76 - 86

100

86 - 87

0 100

100 0

87 - 102

100

Solubility : practically insoluble in water, freely soluble in

ethanol (96 per cent), soluble in acetone, slightly soluble in
ethyl acetate.
IDENTIFICATION
A. Specic optical rotation (2.2.7) : 70 to 60 (anhydrous
substance).
Immediately before use, dissolve 50 mg in ethanol (96 per
cent) R and dilute to 10.0 mL with the same solvent.
B. Infrared absorption spectrophotometry (2.2.24).
Comparison : alprostadil CRS.
C. Examine the chromatograms obtained in the assay.
Results : the principal peak in the chromatogram obtained
with the test solution is similar in retention time and size
to the principal peak in the chromatogram obtained with
the reference solution.
TESTS

Flow rate : 1 mL/min.

Detection : spectrophotometer at 200 nm.
Injection : 20 L.
Retention time : alprostadil = about 63 min.
System suitability :
resolution : minimum 1.5 between the peaks due to
impurity H and alprostadil in the chromatogram obtained
with reference solution (b).
System B
Use the same conditions as for system A with the following
mobile phase and elution programme :
mobile phase A : dissolve 3.9 g of sodium dihydrogen
phosphate R in water R and dilute to 1.0 L with the
same solvent ; adjust to pH 2.5 with a 2.9 g/L solution of
phosphoric acid R (approximately 600 mL is required) ; to
600 mL of the buffer solution add 400 mL of acetonitrile R1 ;

Related substances. Liquid chromatography (2.2.29). Prepare

mobile phase B : use mobile phase B as described under
the solutions protected from light.
system A ;
Test solution. Dissolve 10.0 mg of the substance to be
Time
Mobile phase A
Mobile phase B
examined in a mixture of equal volumes of acetonitrile R1
(min)
(per cent V/V)
(per cent V/V)
and water R and dilute to 10.0 mL with the same mixture of
solvents.
0 - 50
100
0
Reference solution (a). Dilute 100 L of the test solution to
20.0 mL with a mixture of equal volumes of acetonitrile R1
and water R.
Reference solution (b). Dissolve 1.0 mg of dinoprostone
impurity C CRS (alprostadil impurity H) and 1.0 mg of the
substance to be examined in a mixture of equal volumes of
acetonitrile R1 and water R and dilute to 20.0 mL with the
same mixture of solvents.
Reference solution (c). In order to prepare impurities A
and B in situ, dissolve 1 mg of the substance to be examined
in 100 L of 1 M sodium hydroxide (the solution becomes
brownish-red), wait for 3 min and add 100 L of a 112 g/L
solution of phosphoric acid R (yellowish-white opalescent
solution) ; dilute to 5.0 mL with a mixture of equal volumes of
acetonitrile R1 and water R.
General Notices (1) apply to all monographs and other texts

50 - 51

100 0

0 100

51 - 61

100

61 - 62

0 100

100 0

62 - 72

100

Relative retention with reference to alprostadil

(retention time = about 7 min) : impurity A = about 2.4 ;
impurity B = about 2.6.
System suitability :
resolution : minimum 1.5 between the peaks due to
impurity A and impurity B in the chromatogram obtained
with reference solution (c).
Carry out the test according to system A and B.

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EUROPEAN PHARMACOPOEIA 8.4

Alprostadil

Limits :
correction factors : for the calculation of content, multiply
the peak areas of the impurities listed in Table 1488.-1 by
the corresponding correction factor ;

IMPURITIES

Table 1488.-1.
Impurity
impurity G

Relative retention Relative retention Correction factor

(system A)
(system B)
0.80
0.7
-

0.8

0.90

1.0

0.96

0.7

impurity E

1.10

0.7

impurity C

1.36

1.9

impurity K

1.85

0.06

impurity A

2.32

0.7

impurity B

2.45

1.5

impurity I

4.00

1.0

impurity J

5.89

1.0

impurity F

0.88

impurity D
impurity H

A. 7-[(1R,2S)-2-[(1E,3S)-3-hydroxyoct-1-enyl]-5oxocyclopent-3-enyl]heptanoic acid (prostaglandin A1),

B. 7-[2-[(1E,3S)-3-hydroxyoct-1-enyl]-5-oxocyclopent-1enyl]heptanoic acid (prostaglandin B1),

C. 7-[(1R,2R,3R)-3-hydroxy-2-[(1E)-3-oxooct-1-enyl]-5 impurity A : not more than 3 times the area of the principal
oxocyclopentyl]heptanoic acid (15-oxoprostaglandin E1),
peak in the chromatogram obtained with reference
solution (a) (1.5 per cent) ;
impurity B : not more than the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.5 per cent) ;
any other impurity : not more than 1.8 times the area
of the principal peak in the chromatogram obtained
D. 7-[(1R,2R,3R)-3-hydroxy-2-[(1E,3R)-3-hydroxyoctwith reference solution (a) (0.9 per cent), and not more
1-enyl]-5-oxocyclopentyl]heptanoic acid
than 1 such peak has an area greater than the area of
(15-epiprostaglandin E1),
the principal peak in the chromatogram obtained with
reference solution (a) (0.5 per cent). Evaluate impurities
appearing at relative retentions less than 1.2 by system A
and impurities appearing at relative retentions greater than
1.2 by system B ;
total : not more than 3 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
E. 7-[(1R,2R,3S)-3-hydroxy-2-[(1E,3S)-3-hydroxyoct-1-enyl](1.5 per cent) ;
5-oxocyclopentyl]heptanoic acid (11-epiprostaglandin E1),
disregard limit : 0.1 times the area of the principal peak in
the chromatogram obtained with reference solution (a)
(0.05 per cent).
Water (2.5.32) : maximum 0.5 per cent, determined on 50 mg.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances, system A. Prepare the solutions protected
from light.
Test solution. Dissolve 10.0 mg of the substance to be
examined in a mixture of equal volumes of acetonitrile R1
and water R and dilute to 25.0 mL with the same mixture of
solvents. Dilute 3.0 mL of the solution to 20.0 mL with a
mixture of equal volumes of acetonitrile R1 and water R.
Reference solution. Dissolve 5.0 mg of alprostadil CRS in
a mixture of equal volumes of acetonitrile R1 and water R
and dilute to 25.0 mL with the same mixture of solvents.
Dilute 6.0 mL of the solution to 20.0 mL with a mixture of
equal volumes of acetonitrile R1 and water R.
Injection : 20 L.
Calculate the percentage content of C20H34O5 taking into
account the assigned content of alprostadil CRS.
STORAGE
At a temperature of 2 C to 8 C.

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F. 7-[(1S,2R,3R)-3-hydroxy-2-[(1E,3S)-3-hydroxyoct-1-enyl]5-oxocyclopentyl]heptanoic acid (8-epiprostaglandin E1),

G. (5Z)-7-[(1R,2R,3R)-3-hydroxy-2-[(1E,3S)-3-hydroxyoct-1enyl]-5-oxocyclopentyl]hept-5-enoic acid (dinoprostone),

H. (5E)-7-[(1R,2R,3R)-3-hydroxy-2-[(1E,3S)-3-hydroxyoct-1-enyl]-5-oxocyclopentyl]hept-5-enoic acid
((5E)-prostaglandin E2),
See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.4

Ammonium bromide

Test solution (b). Dilute 25.0 mL of test solution (a) to 50.0 mL

with water for chromatography R.
Reference solution (a). To 25.0 mL of test solution (a) add
1.0 mL of sulfate standard solution (10 ppm SO4) R and
12.0 mL of chloride standard solution (50 ppm Cl) R and dilute
to 50.0 mL with water for chromatography R.
I. ethyl 7-[(1R,2R,3R)-3-hydroxy-2-[(1E,3S)-3-hydroxyoctReference solution (b). Dilute 10.0 mL of test solution (a) to
1-enyl]-5-oxocyclopentyl]heptanoate (prostaglandin E1,
100.0 mL with water for chromatography R. To 2.0 mL of this
solution add 8.0 mL of chloride standard solution (50 ppm Cl) R
ethyl ester),
and dilute to 20.0 mL with water for chromatography R.
Blank solution : water for chromatography R.
Column :
size : l = 0.25 m, = 2 mm ;
stationary phase : strongly basic anion-exchange resin for
chromatography R (13 m).
Mobile phase : dissolve 0.600 g of potassium hydroxide R in
J. 1-methylethyl 7-[(1R,2R,3R)-3-hydroxy-2-[(1E,3S)water for chromatography R and dilute to 1000.0 mL with the
3-hydroxyoct-1-enyl]-5-oxocyclopentyl]heptanoate
same solvent.
(prostaglandin E1, isopropyl ester),
Flow rate : 0.4 mL/min.
Detection : conductivity detector equipped with a suitable ion
suppressor.
Injection : 50 L of test solution (b), reference solutions (a)
and (b) and the blank solution.
Run time : 2.5 times the retention time of bromide.
K. triphenylphosphine oxide.
Retention time : chloride = about 5 min ; bromide = about
8 min ; sulfate = about 16 min.
04/2015:1389 System suitability : reference solution (b) :
resolution : minimum 8.0 between the peaks due to chloride
and bromide.
AMMONIUM BROMIDE
Calculation of percentage contents :
for chlorides, use the concentration of chloride in reference
Ammonii bromidum
solution (a) ; correct the area of the peak due to chloride
in the chromatogram obtained with reference solution (a)
NH4Br
Mr 97.9
by subtracting the area of the peak due to chloride in the
[12124-97-9]
chromatogram obtained with test solution (b) ;
DEFINITION
for sulfates, use the concentration of sulfate in reference
solution (a) ; correct the area of the peak due to sulfate in
Content : 98.5 per cent to 101.0 per cent (dried substance).
the chromatogram obtained with reference solution (a)
CHARACTERS
by subtracting the area of the peak due to sulfate in the
chromatogram obtained with test solution (b).
Appearance : white or almost white, crystalline powder or
colourless crystals, hygroscopic.
Limits :
Solubility : freely soluble in water, sparingly soluble in ethanol chlorides : maximum 0.6 per cent ;
(96 per cent).
sulfates : maximum 0.01 per cent.
It becomes yellow when exposed to light or air.
Iodides. To 5 mL of solution S add 0.15 mL of ferric chloride
solution R1 and 2 mL of methylene chloride R. Shake and allow
IDENTIFICATION
to separate. The lower layer is colourless (2.2.2, Method I).
A. It gives reaction (a) of bromides (2.3.1).
Iron (2.4.9) : maximum 20 ppm.
B. 10 mL of solution S (see Tests) gives the reaction of
Dilute 5 mL of solution S to 10 mL with water R.
ammonium salts (2.3.1).
Magnesium and alkaline-earth metals (2.4.7): maximum
TESTS
200 ppm, calculated as Ca.
Solution S. Dissolve 10.0 g in carbon dioxide-free water R and 10.0 g complies with the test for magnesium and alkaline-earth
dilute to 100 mL with the same solvent.
metals. The volume of 0.01 M sodium edetate used does not
exceed 5.0 mL.
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
Heavy metals (2.4.8): maximum 10 ppm.
Acidity or alkalinity. To 10 mL of solution S add 0.05 mL
12 mL of solution S complies with test A. Prepare the reference
of methyl red solution R. Not more than 0.5 mL of 0.01 M
solution using lead standard solution (1 ppm Pb) R.
hydrochloric acid or 0.01 M sodium hydroxide is required to
Loss on drying (2.2.32) : maximum 1.0 per cent, determined
change the colour of the indicator.
on 1.000 g by drying in an oven at 105 C.
Bromates. To 10 mL of solution S add 1 mL of starch
Sulfated ash (2.4.14): maximum 0.1 per cent, determined on
solution R, 0.1 mL of a 100 g/L solution of potassium iodide R 1.0 g.
and 0.25 mL of 0.5 M sulfuric acid and allow to stand protected
from light for 5 min. No blue or violet colour develops.
ASSAY
Chlorides and sulfates. Liquid chromatography (2.2.29).
Dissolve 80.0 mg in water R, add 5 mL of dilute nitric acid R
and dilute to 50 mL with water R. Titrate with 0.1 M silver
Test solution (a). Dissolve 0.400 g of the substance to be
examined in 50 mL of water for chromatography R and dilute nitrate, determining the end-point potentiometrically (2.2.20).
to 100.0 mL with the same solvent.
1 mL of 0.1 M silver nitrate is equivalent to 9.794 mg of NH4Br.
General Notices (1) apply to all monographs and other texts

4711