EUROPEAN PHARMACOPOEIA 5.

0 Belladonna leaf tincture, standardised

and again heat the residue in an oven at 100 °C to 105 °C for Top of the plate
15 min. Dissolve the residue in a few millilitres of methylene _______ _______
chloride R, add 20.0 ml of 0.01 M sulphuric acid and remove
the methylene chloride by evaporation on a water-bath. Chlorogenic acid : a light blue A light blue fluorescent zone
Titrate the excess of acid with 0.02 M sodium hydroxide fluorescent zone (chlorogenic acid)
using methyl red mixed solution R as indicator. A yellow or yellowish-brown
fluorescent zone
Calculate the percentage content of total alkaloids, expressed _______ _______
as hyoscyamine, from the expression :
Rutin : a yellowish-brown A bluish-grey fluorescent zone
fluorescent zone
A yellow fluorescent zone
A yellowish-brown fluorescent
n = volume of 0.02 M sodium hydroxide used, in zone
millilitres, Reference solution Test solution
m = mass of the substance to be examined, in grams.
B. Examine the chromatograms obtained in the test for
STORAGE atropine, detection A.
Store in an airtight container, protected from light. Results A : see below the sequence of zones present in
the chromatograms obtained with the reference solution
and the test solution. Faint secondary zones may appear,
particularly in the middle of the chromatogram obtained
01/2005:1812 with 40 µl of the test solution or near the starting point
in the chromatogram obtained with 20 µl of the test
solution.
BELLADONNA LEAF TINCTURE,
STANDARDISED Top of the plate
Hyoscine : a brownish-orange A brownish-orange zone
zone (hyoscine)
Belladonnae folii tinctura normata _______ _______

DEFINITION Faint secondary zones

Tincture produced from Belladonna leaf (0221).
Content : 0.027 per cent to 0.033 per cent of total alkaloids,
calculated as hyoscyamine (C17H23NO3 ; Mr 289.4). The
alkaloids consist mainly of hyoscyamine together with small
quantities of hyoscine.
PRODUCTION
The tincture is produced from 1 part of the powdered
drug (355) and 10 parts of ethanol (70 per cent V/V) by a
suitable procedure.
IDENTIFICATION
A. Thin-layer chromatography (2.2.27).
Test solution. Evaporate to dryness 10.0 ml of the tincture
to be examined in a water-bath at 40 °C under reduced
pressure. Dissolve the residue in 1.0 ml of methanol R.
Reference solution. Dissolve 1.0 mg of chlorogenic
acid R and 2.5 mg of rutin R in 10 ml of methanol R.
Plate : TLC silica gel plate R.
Mobile phase : anhydrous formic acid R, water R, methyl
ethyl ketone R, ethyl acetate R (10:10:30:50 V/V/V/V).
Application : 40 µl, as bands.
Development : over a path of 15 cm.
Drying : at 100-105 °C.
Detection : spray the warm plate with a 10 g/l solution of
diphenylboric acid aminoethyl ester R in methanol R ;
subsequently spray the plate with a 50 g/l solution of
macrogol 400 R in methanol R ; allow the plate to dry in
air for 30 min and examine in ultraviolet light at 365 nm.
Results : see below the sequence of zones present in the
chromatograms obtained with the reference solution and
the test solution. Furthermore, other fluorescent zones
may be present in the chromatogram obtained with the
test solution.
_______ _______
Hyoscyamine : a A brownish-orange zone
brownish-orange zone (hyoscyamine)
Faint secondary zones
Reference solution Test solution
TESTS
Atropine. Thin-layer chromatography (2.2.27).
Test solution. To 15.0 ml of the tincture to be examined
add 15 ml of 0.05 M sulphuric acid. Filter. Add 1 ml of
concentrated ammonia R to the filtrate and shake with
2 quantities, each of 10 ml, of peroxide-free ether R. Separate
by centrifugation if necessary. Dry the combined ether layers
over anhydrous sodium sulphate R. Filter and evaporate to
dryness on a water-bath. Dissolve the residue in 0.5 ml of
methanol R.
Reference solution. Dissolve 50 mg of hyoscyamine
sulphate R in 9 ml of methanol R. Dissolve 15 mg of
hyoscine hydrobromide R in 10 ml of methanol R. Mix
1.8 ml of the hyoscine hydrobromide solution and 8 ml of
the hyoscyamine sulphate solution.
Plate : TLC silica gel plate R.
Mobile phase : concentrated ammonia R, water R, acetone R
(3:7:90 V/V/V).
Application : 20 µl and 40 µl of each solution, as bands.
Development : over a path of 10 cm.
Drying : at 100-105 °C for 15 min.
Detection A : spray with potassium iodobismuthate
solution R2.
Detection B : spray with sodium nitrite solution R until the
plate is transparent. Examine after 15 min.

General Notices (1) apply to all monographs and other texts 1061
Belladonna, prepared
Results B : the zones due to hyoscyamine in the
chromatograms obtained with the test solution and
the reference solution change from brownish-orange to
reddish-brown but not to greyish-blue (atropine) and any
secondary zones disappear.
Ethanol (2.9.10) : 64 per cent V/V to 69 per cent V/V.
ASSAY
Evaporate 50.0 g of the tincture to be examined to a volume
of about 10 ml. Transfer quantitatively to a separating
funnel, with the minimum volume of alcohol (70 per
cent V/V) R. Add 5 ml of ammonia R and 15 ml of water R.
Shake with not fewer than 3 quantities each of 40 ml of a
mixture of 1 volume of methylene chloride R and 3 volumes
of peroxide-free ether R, carefully to avoid emulsion, until
the alkaloids are completely extracted. Combine the organic
layers and concentrate the solution to a volume of about
50 ml by distilling on a water-bath. Transfer the resulting
solution quantitatively to a separating funnel, rinsing with
peroxide-free ether R. Add a quantity of peroxide-free
ether R equal to at least 2.1 times the volume of the solution
to produce a layer having a density well below that of water.
Shake the resulting solution with not fewer than 3 quantities
each of 20 ml of 0.25 M sulphuric acid until the alkaloids are
completely extracted. Separate the layers by centrifugation
if necessary and transfer the layers to a separating funnel.
Make the combined layers alkaline with ammonia R and
shake with not fewer than 3 quantities each of 30 ml of
methylene chloride R until the alkaloids are completely
extracted. Combine the organic layers, add 4 g of anhydrous
sodium sulphate R and allow to stand for 30 min with
occasional shaking. Decant the methylene chloride and filter.
Wash the sodium sulphate with 3 quantities each of 10 ml
of methylene chloride R. Combine the organic extracts,
evaporate to dryness on a water-bath. Heat the residue in an
oven at 100-105 °C for 15 min. Dissolve the residue in a few
millilitres of methylene chloride R, evaporate to dryness on
a water-bath and heat the residue in an oven at 100-105 °C
for 15 min again. Dissolve the residue in a few millilitres
of methylene chloride R. Add 20.0 ml of 0.01 M sulphuric
acid and remove the methylene chloride by evaporation on
a water-bath. Titrate the excess of acid with 0.02 M sodium
hydroxide using methyl red mixed solution R as indicator.
Calculate the percentage content of total alkaloids, expressed
as hyoscyamine, from the expression :
n shake for 15 min and filter. Wash the filter with 0.05 M
= volume of 0.02 M sodium hydroxide used, in
millilitres,
m = mass of drug used, in grams.
01/2005:0222 in 0.5 ml of methanol R.
BELLADONNA, PREPARED
Belladonnae pulvis normatus
DEFINITION
Prepared belladonna is belladonna leaf powder (180)
adjusted if necessary by adding powdered lactose or
belladonna leaf powder with a lower alkaloidal content to
contain 0.28 per cent to 0.32 per cent of total alkaloids,
calculated as hyoscyamine (Mr 289.4) with reference to the
dried drug.
1062 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 5.0
CHARACTERS
Slightly nauseous odour.
IDENTIFICATION
A. The powder is dark green. Examine under a microscope,
using chloral hydrate solution R. The powder shows the
following diagnostic characters : fragments of leaf lamina
showing sinuous-walled epidermal cells, a striated cuticle
and numerous stomata predominantly present on the
lower epidermis (anisocytic and also some anomocytic) ;
multicellular uniseriate covering trichomes with smooth
cuticle, glandular trichomes with unicellular heads and
multicellular, uniseriate stalks or with multicellular
heads and unicellular stalks ; parenchyma cells including
rounded cells containing microsphenoidal crystals of
calcium oxalate ; annular and spirally thickened vessels.
The powdered drug may also show the following :
fibres and reticulately thickened vessels from the
stems ; subspherical pollen grains, 40 µm to 50 µm in
diameter, with three germinal pores, three furrows and
an extensively pitted exine ; fragments of the corolla, with
a papillose epidermis or bearing numerous covering or
glandular trichomes of the types previously described ;
brownish-yellow seed fragments containing irregularly
sclerified and pitted cells of the testa. Examined in
glycerol (85 per cent) R, it may be seen to contain lactose
crystals.
B. Shake 1 g with 10 ml of 0.05 M sulphuric acid for
2 min. Filter and add to the filtrate 1 ml of concentrated
ammonia R and 5 ml of water R. Shake cautiously with
15 ml of ether R, avoiding formation of an emulsion.
Separate the ether layer and dry over anhydrous sodium
sulphate R. Filter and evaporate the ether in a porcelain
dish. Add 0.5 ml of fuming nitric acid R and evaporate
to dryness on a water-bath. Add 10 ml of acetone R and,
dropwise, a 30 g/l solution of potassium hydroxide R in
alcohol R. A deep violet colour develops.
C. Examine the chromatogram obtained in the
chromatography test. The principal zones in the
chromatogram obtained with the test solution are similar
in position, colour and size to the principal zones in the
chromatogram obtained with the same volume of the
reference solution.
TESTS
Chromatography. Examine by thin-layer chromatography
(2.2.27), using silica gel G R as the coating substance.
Test solution. To 0.6 g add 15 ml of 0.05 M sulphuric acid,
sulphuric acid until 20 ml of filtrate is obtained. To the
filtrate add 1 ml of concentrated ammonia R and shake
with two quantities, each of 10 ml, of peroxide-free ether R.
If necessary, separate by centrifugation. Dry the combined
ether layers over anhydrous sodium sulphate R, filter and
evaporate to dryness on a water-bath. Dissolve the residue
Reference solution. Dissolve 50 mg of hyoscyamine
sulphate R in 9 ml of methanol R. Dissolve 15 mg of
hyoscine hydrobromide R in 10 ml of methanol R. Mix
1.8 ml of the hyoscine hydrobromide solution and 8 ml of
the hyoscyamine sulphate solution.
Apply separately to the plate as bands 20 mm by 3 mm
10 µl and 20 µl of each solution, leaving 1 cm between
the bands. Develop over a path of 10 cm using a mixture
of 3 volumes of concentrated ammonia R, 7 volumes
of water R and 90 volumes of acetone R. Dry the plate
at 100 °C to 105 °C for 15 min, allow to cool and spray
with potassium iodobismuthate solution R2, using about