Alprazolam
TESTS
Specific absorbance (2.2.25) : maximum 0.2, determined
at the absorption maximum at 270 nm. The ratio of the
absorbance measured at 232 nm to that measured at 270 nm
is greater than 7.
To 0.100 g add cyclohexane R and dilute to 10.0 mL with the
same solvent. Adapt the concentration of the solution so that
the absorbance lies between 0.5 and 1.5, measured in a 1 cm
cell.
Acid value (2.5.1) : maximum 2.0, determined on 5.0 g.
Peroxide value (2.5.5, Method A) : maximum 15.0.
Unsaponifiable matter (2.5.7): maximum 0.9 per cent,
determined on 5.0 g.
Composition of fatty acids. (2.4.22, Method A). Use the
mixture of calibrating substances in Table 2.4.22.-3.
Composition of the fatty-acid fraction of the oil :
– saturated fatty acids of chain length less than C16 : maximum
0.1 per cent,
– palmitic acid : 4.0 per cent to 9.0 per cent,
– palmitoleic acid : maximum 0.8 per cent,
– margaric acid : maximum 0.2 per cent,
– stearic acid : maximum 3.0 per cent,
– oleic acid : 62.0 per cent to 86.0 per cent,
– linoleic acid : 20.0 per cent to 30.0 per cent,
– linolenic acid : maximum 0.4 per cent,
– arachidic acid : maximum 0.2 per cent,
– eicosenoic acid : maximum 0.3 per cent,
– behenic acid : maximum 0.2 per cent,
– erucic acid : maximum 0.1 per cent.
Sterols (2.4.23).
Composition of sterol fraction of the oil :
– cholesterol : maximum 0.7 per cent,
– campesterol : maximum 4.0 per cent,
– stigmasterol : maximum 3.0 per cent,
– β-sitosterol : 73.0 per cent to 87.0 per cent,
– Δ5-avenasterol : minimum 10.0 per cent,
– Δ7-stigmastenol : maximum 3.0 per cent,
– Δ7-avenasterol : maximum 3.0 per cent,
– brassicasterol : maximum 0.3 per cent.
Water (2.5.32) : maximum 0.1 per cent, determined on 1.00 g.
STORAGE
In a well-filled container, protected from light.
01/2008:1065
corrected 10.0
ALPRAZOLAM
Alprazolamum
C17H13ClN4 Mr 308.8 100.0 mL with dimethylformamide R. Dilute 0.5 mL of this
[28981-97-7]
1788
EUROPEAN PHARMACOPOEIA 10.
DEFINITION
8-Chloro-1-methyl-6-phenyl-4H-[1,2,4]triazolo[4,3-a][1,4]-
benzodiazepine.
Content : 99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : practically insoluble in water, freely soluble in
methylene chloride, sparingly soluble in acetone and in
ethanol (96 per cent).
It shows polymorphism (5.9).
IDENTIFICATION
First identification : B.
Second identification : A, C.
A. Dissolve the substance to be examined in the smallest
necessary quantity of ethyl acetate R and evaporate to
dryness on a water-bath. Thoroughly mix 5.0 mg of the
substance to be examined with 5.0 mg of alprazolam CRS.
The melting point (2.2.14) of the mixture does not differ
by more than 2 °C from the melting point of the substance
to be examined.
B. Infrared absorption spectrophotometry (2.2.24).
Preparation : discs.
Comparison : alprazolam CRS.
If the spectra obtained in the solid state show differences,
dissolve the substance to be examined and the reference
substance separately in the minimum volume of ethyl
acetate R, evaporate to dryness on a water-bath and record
new spectra using the residues.
C. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 10 mg of the substance to be
examined in methanol R and dilute to 10 mL with the same
solvent.
Reference solution (a). Dissolve 10 mg of alprazolam CRS
in methanol R and dilute to 10 mL with the same solvent.
Reference solution (b). Dissolve 10 mg of alprazolam CRS
and 10 mg of midazolam CRS in methanol R and dilute to
10 mL with the same solvent.
Plate : TLC silica gel GF254 plate R.
Mobile phase : glacial acetic acid R, water R, methanol R,
ethyl acetate R (2:15:20:80 V/V/V/V).
Application : 5 μL.
Development : over a path of 12 cm.
Drying : in air.
Detection : examine in ultraviolet light at 254 nm.
System suitability : reference solution (b) :
– the chromatogram shows 2 clearly separately spots.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position and size to
the principal spot in the chromatogram obtained with
reference solution (a).
TESTS
Related substances. Liquid chromatography (2.2.29).
Buffer solution. Dissolve 7.7 g of ammonium acetate R in
1000 mL of water for chromatography R and adjust to pH 4.2
with glacial acetic acid R.
Test solution. Dissolve 0.100 g of the substance to be examined
in dimethylformamide R and dilute to 10.0 mL with the same
solvent.
Reference solution (a). Dissolve 2 mg of alprazolam CRS and
2 mg of triazolam CRS in dimethylformamide R and dilute to
100 mL with the same solvent.
Reference solution (b). Dilute 5.0 mL of the test solution to
solution to 10.0 mL with dimethylformamide R.
See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 10.0 Alprazolam

Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : end-capped extra-dense bonded phenylsilyl
silica gel for chromatography R (5 μm).
Mobile phase :
– mobile phase A : buffer solution, methanol R (44:56 V/V);
– mobile phase B : buffer solution, methanol R (5:95 V/V) ;
– temperature : 40 °C ;
C. [5-chloro-2-[3-methyl-4H-1,2,4-triazol-4-
Time Mobile phase A Mobile phase B yl]phenyl]phenylmethanone,
(min) (per cent V/V) (per cent V/V)
0 - 15 98 2

15 - 35 98 → 1 2 → 99

35 - 40 1 99

Flow rate : 2 mL/min.

Detection : spectrophotometer at 254 nm.
Injection : 10 μL ; inject dimethylformamide R as a blank.
Retention time : triazolam = about 9 min ; alprazo-
lam = about 10 min.
D. 8-chloro-1-ethenyl-6-phenyl-4H-[1,2,4]triazolo[4,3-
System suitability : reference solution (a) : a][1,4]benzodiazepine,
– resolution : minimum 1.5 between the peaks due to
triazolam and alprazolam.
Limits :
– total : not more than the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.25 per cent) ;
– disregard limit : 0.2 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.05 per cent).
E. (2-amino-5-chlorophenyl)phenylmethanone,
Loss on drying (2.2.32): maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.
ASSAY
Dissolve 0.140 g in 50 mL of a mixture of 2 volumes of acetic
anhydride R and 3 volumes of anhydrous acetic acid R.
Titrate with 0.1 M perchloric acid, determining the end-point
potentiometrically (2.2.20). Titrate to the 2nd point of
inflexion.
1 mL of 0.1 M perchloric acid is equivalent to 15.44 mg F. [5-chloro-2-[3-(chloromethyl)-5-methyl-4H-1,2,4-triazol-
of C17H13CIN4. 4-yl]phenyl]phenylmethanone,
STORAGE
Protected from light.
IMPURITIES

A. (4RS)-3-amino-6-chloro-2-methyl-4-phenyl-3,4- G. 7-chloro-1-methyl-5-phenyl[1,2,4]triazolo[4,3-a]quinolin-
dihydroquinazolin-4-ol, 4-amine,

B. [5-chloro-2-[3-(hydroxymethyl)-5-methyl-4H-1,2,4- H. bis[[4-(2-benzoyl-4-chlorophenyl)-5-methyl-4H-1,2,4-
triazol-4-yl]phenyl]phenylmethanone, triazol-3-yl]methyl]amine,

General Notices (1) apply to all monographs and other texts 1789
Alprenolol hydrochloride EUROPEAN PHARMACOPOEIA 10.0

D. It gives reaction (a) of chlorides (2.3.1).

I. [5-chloro-2-[3-[[(6RS)-8-chloro-6-hydroxy-1-methyl-
6-phenyl-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-
5(6H)-yl]methyl]-5-methyl-4H-1,2,4-triazol-4-
yl]phenyl]phenylmethanone,
J. 2,17-dichloro-6,13-dimethyl-18b,19a-diphenyl-8b,19a-
dihydro-10H,18bH-[1,2,4]triazolo[4′′′,3′′′:1″,2″]-
quinolo[3″,4″:4′,5′]oxazolo[3′,2′-d]-1,2,4-triazolo[4,3-a]-
[1,4]benzodiazepine.
01/2017:0876
corrected 10.0
ALPRENOLOL HYDROCHLORIDE
Alprenololi hydrochloridum
C15H24ClNO2 Mr 285.8
[13707-88-5]
DEFINITION
(2RS)-1-[(1-Methylethyl)amino]-3-[2-(prop-2-enyl)phenoxy]-
propan-2-ol hydrochloride.
Content : 99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder or
colourless crystals.
Solubility : very soluble in water, freely soluble in ethanol
(96 per cent) and in methylene chloride.
IDENTIFICATION
First identification : B, D.
Second identification : A, C, D.
A. Melting point (2.2.14) : 108 °C to 112 °C.
B. Infrared absorption spectrophotometry (2.2.24).
Comparison : alprenolol hydrochloride CRS.
C. Examine the chromatograms obtained in the test for
impurity D.
Detection : examine in daylight, after exposure to iodine
vapour for 30 min.
Results : the principal spot in the chromatogram obtained
with test solution (b) is similar in position, colour and size
to the principal spot in the chromatogram obtained with
reference solution (a).
TESTS
Solution S. Dissolve 1.0 g in carbon dioxide-free water R and
dilute to 50 mL with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution B9 (2.2.2,
Method II).
Acidity or alkalinity. To 10 mL of solution S add 0.2 mL of
methyl red solution R and 0.2 mL of 0.01 M hydrochloric acid ;
the solution is red. Add 0.4 mL of 0.01 M sodium hydroxide ;
the solution is yellow.
Impurity C : maximum 0.1 per cent.
Dissolve 0.25 g in ethanol (96 per cent) R and dilute to 25 mL
with the same solvent. The absorbance (2.2.25) measured at
297 nm is not greater than 0.20.
Impurity D. Thin-layer chromatography (2.2.27).
Test solution (a). Dissolve 0.50 g of the substance to be
examined in methanol R and dilute to 10 mL with the same
solvent.
Test solution (b). Dilute 1 mL of test solution (a) to 50 mL
with methanol R.
Reference solution (a). Dissolve 10 mg of alprenolol
hydrochloride CRS in methanol R and dilute to 10 mL with
the same solvent.
Reference solution (b). Dissolve 10 mg of alprenolol
hydrochloride CRS and 10 mg of oxprenolol hydrochloride CRS
in methanol R and dilute to 10 mL with the same solvent.
Reference solution (c). Dilute 5 mL of test solution (b) to
50 mL with methanol R.
Plate : TLC silica gel G plate R.
Mobile phase : place 2 beakers each containing 30 mL of
ammonia R at the bottom of the tank containing a mixture of
5 volumes of methanol R and 95 volumes of ethyl acetate R.
Application : 5 μL.
Development : over a path of 15 cm in a tank saturated for at
least 1 h.
Drying : at 100 °C for 15 min.
Detection : expose to iodine vapour for up to 6 h.
System suitability : reference solution (b) :
– the chromatogram shows 2 clearly separated spots.
Limits : test solution (a):
– impurity D : any spot with an RF value greater than that of
the principal spot is not more intense than the principal
spot in the chromatogram obtained with reference
solution (c) (0.2 per cent).
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 20.0 mg of the substance to be
examined in the mobile phase and dilute to 10.0 mL with the
mobile phase.
Reference solution (a). Dissolve 4.0 mg of alprenolol
hydrochloride CRS and 0.8 mg of 4-isopropylphenol R in the
mobile phase and dilute to 100.0 mL with the mobile phase.
Reference solution (b). Dilute 4.0 mL of the test solution to
100.0 mL with the mobile phase. Dilute 1.0 mL of this solution
to 10.0 mL with the mobile phase.
Column :
– size : l = 0.15 m, Ø = 4 mm ;
– stationary phase : octylsilyl silica gel for chromatography R
(5 μm).
Mobile phase : mix 0.656 g of sodium octanesulfonate R with
150 mL of acetonitrile R and dilute to 500 mL with phosphate
buffer pH 2.8 prepared as follows : mix 1.78 g of phosphoric
acid R and 15.6 g of sodium dihydrogen phosphate R and dilute
to 2000 mL with water R.
Flow rate : 1 mL/min.

1790 See the information section on general monographs (cover pages)