Equisetum stem
Mobile phase : concentrated ammonia R, methanol R,
methylene chloride R (1:10:40 V/V/V).
Application : 10 μL [or 1 μL] as spots with a diameter of
5 mm [or 2 mm].
Development : over a path of 10 cm [or 6 cm].
Drying : in air.
Detection : spray with a 2 g/L solution of ninhydrin R in
ethanol (96 per cent) R ; heat at 110 °C for 10 min and
examine immediately in daylight.
Results : see below the sequence of spots present in the
chromatograms obtained with the reference solution and
the test solution. Furthermore, other faint spots may
be present in the chromatogram obtained with the test
solution.
Top of the plate
_______
2-Indanamine : a purple spot
A purple spot may be present
_______
Ephedrine : a purple spot at the A purple spot (ephedrine) at the
border between the middle and
lower thirds
border between the middle and
lower thirds
Reference solution Test solution
TESTS
Loss on drying (2.2.32) : maximum 10.0 per cent, determined
on 1.000 g of the powdered herbal drug (355) (2.9.12) by
drying in an oven at 105 °C for 2 h.
Total ash (2.4.16) : maximum 9.0 per cent.
ASSAY
Liquid chromatography (2.2.29).
Test solution. To 0.200 g of the powdered herbal drug (355)
(2.9.12) add 25.0 mL of methanol R, weigh and sonicate for
45 min. Allow to cool, weigh and adjust to the original mass
with methanol R, shake well and filter. Transfer 1.0 mL of the
filtrate to a small column (1 cm in diameter) packed with
1.50 g of neutral aluminium oxide R (60-210 μm). Elute with a
mixture of equal volumes of methanol R and water R. Collect
about 9 mL of the eluate, add 0.5 mL of phosphoric acid R
and dilute to 10.0 mL with a mixture of equal volumes of
methanol R and water R.
Reference solution (a). Dissolve 10.0 mg of ephedrine
hydrochloride CRS in methanol R and dilute to 100.0 mL with
the same solvent. Dilute 2.0 mL of the solution to 25.0 mL
with the mobile phase.
Reference solution (b). Dissolve 1 mg of ephedrine
hydrochloride CRS and 1 mg of terbutaline sulfate CRS in
methanol R and dilute to 10 mL with the same solvent. Dilute
2 mL of the solution to 25 mL with the mobile phase.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : acetonitrile R1, 0.1 per cent V/V solution of
phosphoric acid R (15:85 V/V).
Flow rate : 2.0 mL/min.
Detection : spectrophotometer at 207 nm.
Injection : 10 μL.
Run time : 3 times the retention time of ephedrine.
System suitability : reference solution (b) :
– resolution : minimum 3.5 between the peaks due to
terbutaline and ephedrine.
1426
EUROPEAN PHARMACOPOEIA 10.
Calculate the percentage content of ephedrine using the
following expression :
A1 ´ m 2 ´ p ´ 165.2
A 2 ´ m1 ´ 5 ´ 201.7
A1 = area of the peak due to ephedrine in the
chromatogram obtained with the test solution ;
A2 = area of the peak due to ephedrine in the
chromatogram obtained with reference
solution (a) ;
m1 = mass of the herbal drug to be examined used to
prepare the test solution, in grams ;
m2 = mass of ephedrine hydrochloride CRS used to
prepare reference solution (a), in grams ;
p = percentage content of ephedrine hydrochloride in
_______
ephedrine hydrochloride CRS.
04/2012:1825
_______
EQUISETUM STEM
Equiseti herba
DEFINITION
Whole or cut, dried sterile aerial parts of Equisetum arvense L.
Content : minimum 0.3 per cent of total flavonoids, expressed
as isoquercitroside (C21H20O12 ; Mr 464.4) (dried drug).
IDENTIFICATION
A. It consists of fragments of grooved main stems, branches
with longitudinal sharp ridges and leaves in whorls, united
at the base into a sheath, light green or greenish-grey. The
fragments are rough to the touch, brittle and crunchy when
crushed. The main stems are about 1-4.5 mm in diameter,
hollow, jointed at the nodes, which occur at intervals of
about 1.5-4.5 cm ; distinct vertical grooves are present on
the internodes, ranging in number from 4 to 14 or more.
The central hollow is less than 50 per cent but more than
25 per cent of the diameter of the main stem. Verticils of
widely spaced and erect branches, usually simple, each
about 1 mm thick with 3-5 longitudinal, sharp ridges,
occur at the nodes ; at the end of each ridge is a protruding,
distinct collenchymatic bundle under the epidermis. The
branches are not hollow. The leaves are small, linear,
verticillate at each node, concrescent at the base ; they form
a toothed sheath around the stem with the number of teeth
corresponding to the number of grooves on the stem. Each
tooth, often brown, is lanceolate-triangular. The lowest
internode of each branch is longer than the sheath of the
stem to which it belongs.
B. Microscopic examination (2.8.23). The powder is
greenish-grey. Examine under a microscope using chloral
hydrate solution R. The powder shows the following
diagnostic characters (Figure 1825.-1) : fragments of the
epidermis (surface view [B, C]) composed of rectangular
cells with wavy walls and paracytic stomata (2.8.3) in
2-4 rows, the 2 subsidiary cells are in the same plane
as the epidermis, cover the guard cells and show radial
ridges ; small silica pilulae are scattered on the surface
of the subsidiary cells and appear more frequent at the
margin forming a distinct ring surrounding the subsidiary
cells [C] ; 2-celled papillae on the ridges, less distinct on the
main stem [A] but large and rectangular on the branches,
oriented longitudinally [F] ; in surface view, the epidermis
of the main stems consists of elongated cells [G], the
epidermis of the secondary branches shows the 2-celled
papillae which resemble pairs of small cells separated by a
See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 10.0
larger cell [D] ; fragments of large-celled parenchyma [H]
and groups of long unlignified fibres with narrow lumens ;
small vessels with spiral or annular thickening [E].
Figure 1825.-1. – Illustration for identification test B of
powdered herbal drug of equisetum stem
C. Examine the chromatograms obtained in the test for
Equisetum palustre.
Results : see below the sequence of zones present in the
chromatograms obtained with reference solution (b) and
the test solution. Furthermore, other weak fluorescent
zones may be present in the chromatogram obtained with
the test solution.
Top of the plate
2 red fluorescent zones
Caffeic acid : a greenish-blue
fluorescent zone
2 greenish-blue fluorescent zones
_______
An orange fluorescent zone
Hyperoside : an orange fluorescent
zone
2 greenish-blue fluorescent zones
_______
Rutoside : an orange fluorescent
zone
Reference solution (b) Test solution
TESTS
Foreign matter (2.8.2) : maximum 5 per cent.
Equisetum palustre. Thin-layer chromatography (2.2.27).
Test solution. To 1.0 g of the powdered herbal drug (355)
(2.9.12) add 10 mL of methanol R. Heat in a water-bath at
60 °C for 10 min with occasional shaking. Allow to cool. Filter. acid R in methanol R.
General Notices (1) apply to all monographs and other texts
Equisetum stem
Reference solution (a). To 100.0 mg of Equisetum palustre HRS
add 10 mL of methanol R. Heat in a water-bath at 60 °C for
10 min with occasional shaking. Allow to cool. Filter.
Reference solution (b). Dissolve 1.0 mg of caffeic acid R, 2.5 mg
of hyperoside R and 2.5 mg of rutoside trihydrate R in 20 mL
of methanol R.
Plate : TLC silica gel plate R (2-10 μm).
Mobile phase : anhydrous formic acid R, glacial acetic acid R,
water R, ethyl acetate R (7.5:7.5:18:67 V/V/V/V).
Application : 5 μL as bands of 8 mm.
Development : over a path of 6 cm.
Drying : in a current of cold air for 5 min.
Detection : heat at 100 °C for 3 min and treat the still-warm
plate with a 10 g/L solution of diphenylboric acid aminoethyl
ester R in methanol R, then treat with a 50 g/L solution of
macrogol 400 R in methanol R ; allow to dry in a current of cold
air and examine after 10 min in ultraviolet light at 365 nm.
System suitability : the chromatogram obtained with reference
solution (a) shows 2 greenish fluorescent zones just above the
line of application.
Results : in the chromatogram obtained with the test
solution, any greenish fluorescent zones just above the line
of application are not more intense than the corresponding
zones (characteristic of E. palustre L.) in the chromatogram
obtained with reference solution (a).
Loss on drying (2.2.32) : maximum 10.0 per cent, determined
on 1.000 g of the powdered herbal drug (355) (2.9.12) by
drying in an oven at 105 °C for 2 h.
Ash insoluble in hydrochloric acid (2.8.1) : minimum 3.0 per
cent and maximum 15.0 per cent.
Total ash (2.4.16): minimum 12.0 per cent and maximum
27.0 per cent.
ASSAY
Stock solution. In a 100 mL round-bottomed flask, introduce
0.800 g of the powdered herbal drug (355) (2.9.12) and
add 1 mL of a 5 g/L solution of hexamethylenetetramine R,
20 mL of acetone R and 2 mL of hydrochloric acid R1. Boil
the mixture under a reflux condenser for 30 min. Filter the
liquid through a plug of absorbent cotton into a flask. Add the
absorbent cotton to the residue in the round-bottomed flask
and extract with 2 quantities, each of 20 mL, of acetone R,
_______ each time boiling under a reflux condenser for 10 min. Allow
to cool and filter each extract through a plug of absorbent
cotton into the flask. After cooling, filter the combined
acetone extracts through a filter paper into a volumetric flask
and dilute to 100.0 mL with acetone R by rinsing the flask
and the filter paper. Introduce 20.0 mL of the solution into a
_______ separating funnel, add 20 mL of water R and shake the mixture
with 1 quantity of 15 mL and then 3 quantities, each of 10 mL,
of ethyl acetate R. Combine the ethyl acetate extracts in a
separating funnel, wash with 2 quantities, each of 50 mL, of
water R, and filter the extracts over 10 g of anhydrous sodium
sulfate R into a volumetric flask. Dilute to 50.0 mL with ethyl
acetate R.
Test solution. To 10.0 mL of the stock solution add 1 mL of
aluminium chloride reagent R and dilute to 25.0 mL with a
5 per cent V/V solution of glacial acetic acid R in methanol R.
Compensation solution. Dilute 10.0 mL of the stock solution
to 25.0 mL with a 5 per cent V/V solution of glacial acetic
1427
Eucalyptus leaf EUROPEAN PHARMACOPOEIA 10.0

Measure the absorbance (2.2.25) of the test solution after

30 min, by comparison with the compensation solution at
425 nm. Calculate the percentage content of flavonoids,
expressed as isoquercitroside, using the following expression :
A ´ 1.25
m
i.e. taking the specific absorbance of isoquercitroside to be
500.
A = absorbance at 425 nm ;
m = mass of the substance to be examined, in grams.

07/2014:1320

EUCALYPTUS LEAF

Eucalypti folium
DEFINITION
Whole or cut, dried leaves of older branches of Eucalyptus
globulus Labill.
Essential oil content :
Figure 1320.-1. – Illustration for identification test B of
– for the whole drug, minimum 20 mL/kg (anhydrous drug) ; powdered herbal drug of eucalyptus leaf
– for the cut drug, minimum 15 mL/kg (anhydrous drug). C. Thin-layer chromatography (2.2.27).

CHARACTERS Test solution. Shake 0.5 g of the freshly powdered herbal

drug (355) (2.9.12) with 5 mL of toluene R for 2-3 min and
Aromatic odour of cineole. filter over about 2 g of anhydrous sodium sulfate R.

IDENTIFICATION Reference solution. Dissolve 50 μL of cineole R in toluene R

A. The leaves, which are mainly greyish-green and relatively
thick, are elongated, elliptical and slightly sickle-shaped
and usually up to 25 cm in length and up to 5 cm in width.
The petiole is twisted, strongly wrinkled and is 2-3 cm,
rarely 5 cm, in length. The coriaceous, stiff leaves are entire
and glabrous and have a yellowish-green midrib. Lateral
veins anastomose near the margin to a continuous line. The
margin is even and somewhat thickened. On both surfaces
there are minute, irregularly distributed, warty, dark brown
spots. Small oil glands may be seen in transmitted light.
B. Microscopic examination (2.8.23). The powder is
greyish-green. Examine under a microscope using chloral
hydrate solution R. The powder shows the following
diagnostic characters (Figure 1320.-1): fragments
of glabrous lamina (surface view [A, L], transverse
section [F, H]), with small, thick-walled epidermal cells
bearing a thick cuticle [Fa, Ha], numerous anomocytic
stomata (2.8.3) greater than 80 μm in diameter [Aa, La]
with occasional groups of brown cork cells, 300 μm
in diameter and brownish-black in their centre, and
underlying palisade parenchyma [Ab, Fb] ; fragments of
bilateral mesophyll (side view [G]), with 2-3 layers of
palisade parenchyma [Ga] on each side and in the centre
several layers of spongy mesophyll [Gb] with elongated
cells having the same orientation as the palisade cells
and containing prisms [B, Gd] and cluster crystals of
calcium oxalate [Gc, K] ; large schizogenous oil glands,
whole [E] or usually broken, accompanied by palisade
parenchyma [Ea] ; fragments of vessels [J] and thick-walled
and slightly channelled fibres [C] accompanied by crystal
sheaths [Ca, Ja] ; crystal sheaths containing prisms of
calcium oxalate [D].
and dilute to 5 mL with the same solvent.
Plate : TLC silica gel plate R.
Mobile phase : ethyl acetate R, toluene R (10:90 V/V).
Application : 10 μL as bands.
Development : over a path of 15 cm.
Drying : in air.
Detection : treat with anisaldehyde solution R and heat at
100-105 °C for 10-15 min ; examine in daylight.
Results : the chromatogram obtained with the reference
solution shows in the middle a zone due to cineole. The
chromatogram obtained with the test solution shows a
principal zone similar in position and colour to the zone
due to cineole in the chromatogram obtained with the
reference solution, it also shows an intense violet zone
(hydrocarbons) near the solvent front and there may also
be other fainter zones.
TESTS
Foreign matter (2.8.2) : maximum 3 per cent of dark and
brown leaves, maximum 5 per cent of stems and maximum
2 per cent of other foreign matter. Cordate or ovate sessile
leaves of young branches, with numerous glands on both
sides, visible as points in transmitted light, are not present.
Carry out the determination using 30 g of the herbal drug to
be examined.
Water (2.2.13) : maximum 100 mL/kg, determined on 20.0 g
of the powdered herbal drug (355) (2.9.12).
Total ash (2.4.16) : maximum 6.0 per cent.

1428 See the information section on general monographs (cover pages)