Belladonna leaf dry extract, standardised EUROPEAN PHARMACOPOEIA 5.

01/2005:1294 Reference solution. Dissolve 50 mg of hyoscyamine

sulphate R in 9 ml of methanol R. Dissolve 15 mg of
BELLADONNA LEAF DRY EXTRACT, hyoscine hydrobromide R in 10 ml of methanol R. Mix
1.8 ml of the hyoscine hydrobromide solution and 8 ml of
STANDARDISED the hyoscyamine sulphate solution.
Apply to the plate, as bands, 20 µl of each solution. Develop
Belladonnae folii extractum siccum over a path of 10 cm using a mixture of 3 volumes of
normatum concentrated ammonia R, 7 volumes of water R and
90 volumes of acetone R. Dry the plate at 100 °C to
DEFINITION 105 °C for 15 min, allow to cool and spray with potassium
iodobismuthate solution R2, until the orange or brown
Standardised belladonna leaf dry extract is produced from
zones become visible against a yellow background. The
Belladonna leaf (0221). It contains not less than 0.95 per
zones in the chromatogram obtained with the test solution
cent and not more than 1.05 per cent of total alkaloids,
are similar to those in the chromatogram obtained with
calculated as hyoscyamine (C17H23NO3, Mr 289.4), with
the reference solution with respect to their position
reference to the dried extract.
(hyoscyamine in the lower third, hyoscine in the upper third
PRODUCTION of the chromatogram) and their colour. Other faint zones
may be present in the chromatogram obtained with the test
The extract is produced from the drug and ethanol (70 per
solution. Spray the plate with sodium nitrite solution R
cent V/V) using an appropriate procedure.
until the coating is transparent. Examine after 15 min. The
CHARACTERS zones corresponding to hyoscyamine in the chromatograms
obtained with the test solution and the reference solution
A hygroscopic brown or greenish powder. change from orange or brown to reddish-brown but not to
IDENTIFICATION greyish-blue (atropine).
A. Examine by thin-layer chromatography (2.2.27), using a Loss on drying (2.8.17) : maximum 5.0 per cent.
suitable silica gel as the coating substance. Microbial contamination. Total viable aerobic count (2.6.12)
Test solution. To 1 g of the extract to be examined add not more than 104 micro-organisms per gram of which not
5.0 ml of methanol R. Shake for 2 min and filter. more than 102 fungi per gram, determined by plate count.
Reference solution. Dissolve 1.0 mg of chlorogenic It complies with the tests for Escherichia coli and for
acid R and 2.5 mg of rutin R in 10 ml of methanol R. Salmonella (2.6.13).
Apply to the plate, as bands, 20 µl of each solution. ASSAY
Develop over a path of 15 cm using a mixture of At each extraction stage it is necessary to check that the
10 volumes of anhydrous formic acid R, 10 volumes alkaloids have been completely extracted. If the extraction is
of water R, 30 volumes of methyl ethyl ketone R and into the organic phase this is done by evaporating to dryness
50 volumes of ethyl acetate R. Dry the plate at 100 °C to a few millilitres of the last organic layer, dissolving the
105 °C and spray the warm plate with a 10 g/l solution residue in 0.25 M sulphuric acid and verifying the absence
of diphenylboric acid aminoethyl ester R in methanol R. of alkaloids using potassium tetraiodomercurate solution R.
Subsequently spray the plate with a 50 g/l solution of If the extraction is into the acid aqueous phase, this is done
macrogol 400 R in methanol R. Allow the plate to dry in by taking a few millilitres of the last acid aqueous phase
air for 30 min and examine in ultraviolet light at 365 nm. and verifying the absence of alkaloids using potassium
The chromatograms obtained with the reference solution tetraiodomercurate solution R.
and the test solution show in the central part a light blue
fluorescent zone (chlorogenic acid) and in the lower part Disperse 3.00 g in a mixture of 5 ml of ammonia R and 15 ml
a yellowish-brown fluorescent zone (rutin). Furthermore, of water R. Shake with no fewer than three quantities, each
the chromatogram obtained with the test solution shows of 40 ml of a mixture of 1 volume of methylene chloride R
a little above the start a yellowish-brown fluorescent zoneand 3 volumes of peroxide-free ether R until the alkaloids
and directly above a yellow fluorescent zone, a yellow or are completely extracted. Concentrate the combined organic
yellowish-brown fluorescent zone between the zone due layers to about 50 ml by distilling on a water-bath and
to rutin and the zone due to chlorogenic acid. Further transfer the resulting liquid to a separating funnel, rinsing
zones may be present. with peroxide-free ether R. Add a quantity of peroxide-free
ether R equal to at least 2.1 times the volume of the liquid
B. Examine the chromatogram obtained in the test for to produce a layer having a density well below that of water.
atropine. The principal zones in the chromatogram Shake the resulting solution with no fewer than three
obtained with the test solution are similar in position quantities, each of 20 ml, of 0.25 M sulphuric acid until the
and colour to the principal zones in the chromatogram alkaloids are completely extracted. Separate the layers by
obtained with the reference solution. centrifugation, if necessary and transfer the acid layers to a
TESTS second separating funnel. Make the combined acid layers
alkaline with ammonia R and shake with no fewer than
Atropine. Examine by thin-layer chromatography (2.2.27), three quantities, each of 30 ml, of methylene chloride R
using a suitable silica gel as the coating substance. until the alkaloids are completely extracted. Combine the
Test solution. To 0.20 g of the extract to be examined add organic layers, add 4 g of anhydrous sodium sulphate R and
10.0 ml of 0.05 M sulphuric acid, shake for 2 min and filter. allow to stand for 30 min with occasional shaking. Decant
Add 1.0 ml of concentrated ammonia R and shake with the methylene chloride and wash the sodium sulphate with
two quantities, each of 10 ml, of peroxide-free ether R. If three quantities, each of 10 ml, of methylene chloride R.
necessary, separate by centrifugation. Dry the combined Combine the organic extracts, evaporate to dryness on
ether layers over about 2 g of anhydrous sodium sulphate R, a water-bath. Heat the residue in an oven at 100 °C to
filter and evaporate to dryness on a water-bath. Dissolve the 105 °C for 15 min. Dissolve the residue in a few millilitres of
residue in 0.5 ml of methanol R. methylene chloride R, evaporate to dryness on a water-bath

1060 See the information section on general monographs (cover pages)

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