Ipecacuanha tincture, standardised EUROPEAN PHARMACOPOEIA 11.

Ash insoluble in hydrochloric acid (2.8.1): maximum 3.0 per Top of the plate
cent. _______ _______

ASSAY Emetine : a blue fluorescent zone A blue fluorescent zone (emetine)

Place 7.5 g of the powdered herbal drug (180) (2.9.12) in a dry Cephaeline : a blue fluorescent zone A blue or faint blue fluorescent
flask, add 100 mL of ether R and shake for 5 min. Add 5 mL zone (cephaeline)
of dilute ammonia R1, shake for 1 h, add 5 mL of water R and _______ _______
shake vigorously. Decant the ether layer into a flask through a
plug of cotton. Wash the residue in the flask with 2 quantities, Reference solution Test solution
each of 25 mL, of ether R, decanting each portion through
the same plug of cotton. Combine the ether solutions and Detection B : treat with a 5 g/L solution of iodine R in ethanol
eliminate the ether by distillation. Dissolve the residue in (96 per cent) R, heat at 60 °C for 10 min and examine in
2 mL of ethanol (90 per cent V/V) R, evaporate to dryness ultraviolet light at 365 nm.
and heat at 100 °C for 5 min. Dissolve the residue in 5 mL of System suitability : reference solution :
previously neutralised ethanol (90 per cent V/V) R, warming
on a water-bath. Add 15.0 mL of 0.1 M hydrochloric acid and – the blue fluorescent zone due to cephaeline and the yellow
titrate the excess acid with 0.1 M sodium hydroxide using fluorescent zone due to emetine are clearly separated.
0.5 mL of methyl red mixed solution R as indicator. Results B : see below the sequence of zones present in the
1 mL of 0.1 M hydrochloric acid is equivalent to 24.03 mg chromatograms obtained with the reference solution and
of total alkaloids, expressed as emetine. the test solution. Furthermore, other faint fluorescent zones
may be present in the chromatogram obtained with the test
STORAGE solution.
In an airtight container. Top of the plate
_______ _______

04/2016:1530 Emetine : a yellow fluorescent zone A yellow fluorescent zone (emetine)

Cephaeline : a blue fluorescent zone A blue fluorescent zone (cephaeline)

_______ _______

Reference solution Test solution

IPECACUANHA TINCTURE,
STANDARDISED TESTS
Ethanol (2.9.10): 95 per cent to 105 per cent of the quantity
Ipecacuanhae tinctura normata stated on the label.
DEFINITION ASSAY
Tincture produced from Ipecacuanha root (0094). Transfer 10.00 g of the tincture to be examined to a
Content : 0.18 per cent m/m to 0.22 per cent m/m of total chromatography column about 0.2 m long and about 15 mm
alkaloids, expressed as emetine (C29H40N2O4 ; Mr 480.6). in internal diameter, containing 8 g of basic aluminium
oxide R. After infiltration into the aluminium oxide layer, rinse
PRODUCTION the internal wall of the column with 3 quantities, each of 2 mL,
The tincture is produced from the herbal drug by a suitable of ethanol (70 per cent V/V) R. Elute in portions, with 40 mL
procedure using ethanol (70 per cent V/V). of ethanol (70 per cent V/V) R. Avoid disturbance or drying of
the surface of the aluminium oxide layer. Collect the whole
CHARACTERS of the eluate. Evaporate the eluate on a water-bath to about
Appearance : yellowish-brown liquid. 10 mL. Allow to cool. Add 10.0 mL of 0.02 M hydrochloric
acid and 20 mL of carbon dioxide-free water R. Titrate the
IDENTIFICATION excess acid with 0.02 M sodium hydroxide using 0.15 mL of
Thin-layer chromatography (2.2.27). methyl red mixed solution R as indicator.
Test solution. To 2.0 mL of the tincture to be examined add Perform a blank assay replacing the tincture to be examined
2 mL of water R and 0.1 mL of concentrated ammonia R. Add with 10.0 mL of ethanol of the strength stated on the label.
10 mL of ether R and shake. Separate the ether layer, dry it 1 mL of 0.02 M hydrochloric acid is equivalent to 4.807 mg of
over about 2 g of anhydrous sodium sulfate R and filter. total alkaloids, expressed as emetine.
Reference solution. Dissolve 2.5 mg of emetine
hydrochloride CRS and 3 mg of cephaeline hydrochloride CRS
in methanol R and dilute to 10 mL with the same solvent. 01/2019:2566
Plate : TLC silica gel F254 plate R (5-40 μm) [or TLC silica gel
F254 plate R (2-10 μm)].
Mobile phase : concentrated ammonia R, methanol R, ethyl
acetate R, toluene R (2:15:18:65 V/V/V/V).
Application : 10 μL [or 5 μL] as bands of 10 mm [or 8 mm]. ISATIS ROOT
Development : over a path of 10 cm [or 6 cm].
Drying : in air. Isatidis radix
Detection A : examine in ultraviolet light at 365 nm.
DEFINITION
Results A : see below the sequence of zones present in the
chromatograms obtained with the reference solution and Whole or fragmented, dried root of Isatis tinctoria L.
the test solution. Furthermore, other faint fluorescent zones (I. indigotica Fortune ex Lindl.) collected in autumn.
may be present in the chromatogram obtained with the test Content : minimum 1.0 per cent of arginine (C6H14N4O2 ;
solution. Mr 174.2) (dried drug).

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EUROPEAN PHARMACOPOEIA 11.0 Isatis root

IDENTIFICATION Reference solution. Dissolve 4 mg of arginine R and 4 mg

A. Whole drug. It is cylindrical, slightly tortuous, 8-22 cm of cysteine hydrochloride R in 1 mL of ethanol (70 per
long, 1.5 cm in diameter, externally greyish-yellow or cent V/V) R.
brownish-yellow, wrinkled longitudinally and lenticellate Plate : TLC silica gel F254 plate R (5-40 μm) [or TLC silica
transversally, with rootlets or rootlet scars. The root crown gel F254 plate R (2-10 μm)].
is slightly expanded, exhibiting dark green or dark brown Mobile phase : anhydrous formic acid R, water R,
petiole bases arranged in whorls, and dense tubercles. The acetonitrile R (2:8:30 V/V/V).
texture is compact and easily broken. The transversely cut
surface shows a yellowish-white, brown or dark brown Application : 4 μL as bands of 10 mm [or 8 mm].
bark, darkest near the cambium, sometimes appearing as a Development : over a path of 8.5 cm [or 6 cm].
thin dark line, and a yellow or brown wood. Drying : in air.
Fragmented drug. It occurs as transverse or oblique slices, Detection : expose to concentrated ammonia R vapour for
rounded or elliptical, or as short, thin, cylindrical pieces, 5 min, treat with ninhydrin solution R4, then heat at 120 °C
0.3-1.3 cm in diameter. It is externally greyish-yellow or for 3 min.
brownish-yellow, wrinkled longitudinally, and transverse Results : see below the sequence of zones present in the
lenticels, rootlets and rootlet scars are sometimes visible. chromatograms obtained with the reference solution and
The transversely cut surface shows a yellowish-white, the test solution. Furthermore, other faint coloured zones
brown or dark brown bark, darkest near the cambium, may be present in the chromatogram obtained with the
sometimes appearing as a thin dark line, and a yellow or test solution.
brown wood.
B. Microscopic examination (2.8.23). The powder is Top of the plate
whitish-yellow, yellow or light brown. Examine under a _______ _______
microscope using chloral hydrate solution R. The powder
_______ _______
shows the following diagnostic characters (Figure 2566.-1) :
fragments of cork consisting of thin-walled cells (surface A prominent brown zone
view [A], transverse section [B]) ; fragments of xylem [D]
consisting of reticulate, pitted or, more rarely, spiral Cysteine : a brown zone
vessels [Da] included in thin-walled parenchyma A brown zone
cells [Db] ; groups of lignified xylem fibres, occasionally
accompanying vessels, with sparse irregularly-positioned
pits may be present ; thin-walled parenchyma cells Arginine : a brown zone A brown zone (arginine)
(longitudinal section [E], transverse section [F]) ; groups
of greenish-yellow sclereids with thick, pitted walls A faint brown zone
embedded in parenchyma tissue may be present. Examine Reference solution Test solution
under a microscope using a 50 per cent V/V solution
of glycerol R. The powder shows abundant, single or
compound (mostly 2, or less frequently, 3 or 4) starch TESTS
granules, 1.5-18 μm in diameter, free [C] or included Loss on drying (2.2.32) : maximum 9.0 per cent, determined
in parenchyma [G], with a punctiform, slit-shaped or on 1.000 g of the powdered herbal drug (355) (2.9.12) by
V-shaped hilum. drying in an oven at 105 °C for 2 h.
Total ash (2.4.16) : maximum 5.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1) : maximum 1.0 per
cent.
ASSAY
Liquid chromatography (2.2.29).
Test solution. To 0.100 g of the powdered herbal drug (355)
(2.9.12) add 20 mL of ethanol (70 per cent V/V) R, sonicate for
20 min, filter, and evaporate the filtrate to dryness. Dissolve
the residue in ethanol (70 per cent V/V) R and dilute to
10.0 mL with the same solvent.
Reference solution (a). Dissolve 25.0 mg of arginine CRS in
ethanol (70 per cent V/V) R and dilute to 50.0 mL with the
same solvent.
Reference solution (b). Dissolve 3.0 mg of cysteine
hydrochloride R in 6.0 mL of reference solution (a) and dilute
to 10.0 mL with ethanol (70 per cent V/V) R.
Reference solutions (c), (d), (e), (f), (g), (h). Dilute reference
solution (a) to obtain 6 reference solutions of arginine, the
concentrations of which span the expected value in the test
solution.
Column :
– size : l = 0.15 m, Ø = 4.6 mm ;
Figure 2566.-1. – Illustration for identification test B of – stationary phase : base-deactivated end-capped octadecylsilyl
powdered herbal drug of isatis root silica gel for chromatography R (3 μm) ;
C. Thin-layer chromatography (2.2.27). – temperature : 30 °C.
Test solution. To 0.5 g of the powdered herbal drug (355) Mobile phase : trifluoroacetic acid R, water for
(2.9.12) add 5 mL of ethanol (70 per cent V/V) R and chromatography R (0.2:99.8 V/V).
sonicate for 10 min. Centrifuge and use the supernatant. Flow rate : 0.2 mL/min.

General Notices (1) apply to all monographs and other texts 1569
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Ispaghula husk EUROPEAN PHARMACOPOEIA 11.0

Detection : evaporative light-scattering detector ; the following Test solution. To 10 mg of the powdered herbal drug
settings have been found to be suitable ; if the detector has (355) (2.9.12) in a thick-walled centrifuge tube, add 2 mL
different setting parameters, adjust the detector settings so of a 230 g/L solution of trifluoroacetic acid R and shake
as to comply with the system suitability criterion for the vigorously. Stopper the test tube and heat at 120 °C for 1 h.
signal-to-noise ratio : Centrifuge the hydrolysate, transfer the clear supernatant
– carrier gas : nitrogen R ; into a 50 mL flask, add 10 mL of water R and evaporate to
– pressure : 330 kPa ; dryness under reduced pressure. Take up the residue in
10 mL of water R and evaporate again to dryness under
– evaporator temperature : 80 °C. reduced pressure. Take up the residue with 2 mL of
Injection : 10 μL. methanol R.
Run time : 25 min. Reference solution (a). Dissolve 10 mg of arabinose R
System suitability : in a small quantity of water R and dilute to 10 mL with
– resolution : minimum 1.5 between the peaks due to cysteine methanol R.
and arginine in the chromatogram obtained with reference Reference solution (b). Dissolve 10 mg of xylose R in a small
solution (b); quantity of water R and dilute to 10 mL with methanol R.
– signal-to-noise ratio : minimum 50 for the peak due to Reference solution (c). Dissolve 10 mg of galactose R in
arginine in the chromatogram obtained with reference a small quantity of water R and dilute to 10 mL with
solution (a). methanol R.
Establish a calibration curve with the logarithm of the Plate : TLC silica gel plate R.
concentration (in milligrams per 10 mL) of reference Mobile phase : water R, acetonitrile R (15:85 V/V).
solutions (c), (d), (e), (f), (g) and (h) (corrected by the Application : 10 μL, as bands.
assigned percentage content of arginine CRS) as the abscissa
and the logarithm of the corresponding peak areas as the Development : over a path of 15 cm.
ordinate. Detection : spray with aminohippuric acid reagent R and
Calculate the percentage content of arginine using the heat at 120 °C for 5 min ; examine in daylight.
following expression : Results : the chromatogram obtained with the test solution
shows 2 orange-pink zones (arabinose and xylose) and
10 A a yellow zone (galactose) similar in position and colour
m ´ 10 to the zones in the chromatograms obtained with the
reference solutions.
A = logarithm of the concentration of arginine in the
test solution, determined from the calibration TESTS
curve ; Foreign matter (2.8.2). Carry out the determination using
m = mass of the herbal drug to be examined used to 5.0 g.
prepare the test solution, in grams. Loss on drying (2.2.32) : maximum 12.0 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 4.0 per cent.
Swelling index (2.8.4) : minimum 40, determined on 0.1 g of
the powdered herbal drug (355) (2.9.12).
01/2008:1334
corrected 6.0 01/2008:1333
corrected 6.0

ISPAGHULA HUSK
ISPAGHULA SEED
Plantaginis ovatae seminis tegumentum
DEFINITION Plantaginis ovatae semen
Episperm and collapsed adjacent layers removed from the DEFINITION
seeds of Plantago ovata Forssk. (P. ispaghula Roxb.). Dried ripe seeds of Plantago ovata Forssk. (P. ispaghula
IDENTIFICATION Roxb.).
A. The husk consists of pinkish-beige fragments or flakes up IDENTIFICATION
to about 2 mm long and 1 mm wide, some showing a light A. Ispaghula seed is pinkish-beige, smooth, boat-shaped and
brown spot corresponding to the location of the embryo curved. It is 1.5 mm to 3.5 mm long, 1.5 mm to 2 mm
before it was removed from the seed. wide and 1 mm to 1.5 mm thick. The concave surface
B. Microscopic examination (2.8.23). The powder is pale shows in the centre a light coloured spot corresponding to
yellow. Examine under a microscope using lactic reagent R. the hilum. The convex surface shows a light brown spot
The powder shows the following diagnostic characters : corresponding to the location of the embryo and takes up
mainly fragments of the episperm with polygonal cells about one quarter of the length of the seed.
filled with mucilage ; fragments of the inner layers of the B. Microscopic examination (2.8.23). The powder is pale
testa with brownish thin-walled cells often associated brown. Examine under a microscope using lactic reagent R.
with the outer layers of the endosperm. Examine under a The powder shows mainly fragments of the episperm
microscope using a 50 per cent V/V solution of glycerol R. with polygonal cells filled with mucilage ; fragments of the
The powder shows occasional starch granules, single or in inner layers of the testa with brownish thin-walled cells
groups of 2-4, measuring 3-25 μm in diameter. often associated with the outer layers of the endosperm ;
C. Thin-layer chromatography (2.2.27). fragments of the endosperm with cells with thick cellulose

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