CASCARA Rhamni purshianae cortex

Cascara EUROPEAN PHARMACOPOEIA 5.0
— stationary phase: octylsilyl silica gel for
chromatography R (5 µm),
— temperature: 55°C.
Mobile phase: dissolve 1.77 g of potassium dihydrogen
phosphate R in water R anddiluteto650mlwiththesame
solvent; adjust to pH 2.0 with phosphoric acid R and add
350 ml of acetonitrile R.
Flow rate: 1.0ml/min.
Detection: spectrophotometer at 240 nm.
Injection: 20µl.
Run time: 8 times the retention time of carvedilol.
Relative retention with reference to carvedilol
(retention time = about 4 min): impurity A = about 0.6;
impurity C = about 3.5; impurity B = about 6.7.
System suitability: reference solution (b):
— resolution: minimum 17 between the peaks due to
carvedilol and to impurity C.
Limits:
— correction factor: for the calculation of content, multiply
the peak area of impurity A by 2,
— impurity A: not more than twice the area of the principal
peak in the chromatogram obtained with reference
solution (a) (0.2 per cent),
— impurity C: not more than twice the area of the
corresponding peak in the chromatogram obtained with
reference solution (c) (0.02 per cent),
— any other impurity: not more than the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.1 per cent),
— total: not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.5 per cent),
— disregard limit: the area of the principal peak in the
chromatogram obtained with reference solution (c)
(0.01 per cent).
Heavy metals (2.4.8): maximum 10 ppm.
2.0gcomplieswithlimittestC.Preparethestandardusing
2.0 ml of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32): maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 100-105 °C.
Sulphated ash (2.4.14): maximum 0.1 per cent, determined
on 1.0 g.
ASSAY
Dissolve 0.350 g in 60 ml of anhydrous acetic acid R.Titrate
with 0.1 M perchloric acid, determining the end-point
potentiometrically (2.2.20).
1mlof0.1 M perchloric acid is equivalent to 40.65 mg of
C24H26N2O4. IMPURITIES
A. 1-[[9-[2-hydroxy-3-[[2-(2-methoxyphenoxy)ethyl]amino]propyl]-9H-carbazol-4-yl]oxy]-3-[[2-(2-methoxyphenoxy)ethyl]amino]propan-2-ol,
B. 1,1′-[[2-(2-methoxyphenoxy)ethyl]nitrilo]bis[3-(9Hcarbazol-4-yloxy)propan-2-ol],
C. (2RS)-1-[benzyl[2-(2-methoxyphenoxy)ethyl]amino]-3-(9Hcarbazol-4-yloxy)propan-2-ol.
CASCARA
Rhamni purshianae cortex
01/2005:0105
DEFINITION
Cascara consists of the dried, whole or fragmented bark
of Rhamnus purshianus D.C. (Frangula purshiana (D.C.)
A. Gray ex J. C. Cooper). It contains not less than 8.0 per
cent of hydroxyanthracene glycosides of which not less
than 60 per cent consists of cascarosides, both expressed
as cascaroside A (C27H32O14; Mr 580.5) and calculated with
reference to the dried drug.
CHARACTERS
It has the macroscopic and microscopic characters described
under identification tests A and B.
IDENTIFICATION
A. The bark occurs in slightly channelled or nearly flat
pieces, usually 1 mm to 5 mm in thickness, usually
varying greatly in length and width. The outer surface is
grey or dark greyish-brown and shows occasional lenticels
that are orientated transversally. It is usually more or less
completely covered by a whitish coat of lichens, epiphytic
moss and foliaceous liverwort. The inner surface is yellow
to reddish-brown or almost black with fine longitudinal
striations; it turns red when treated with alkali. The
yellow fracture is short and granular in the outer part and
somewhat fibrous at the inner part.
B. Reduce to a powder (355). The powder is yellowish-brown.
Examineunderamicroscopeusingchloral hydrate
solution R. Thepowdershows:bundlesofpartly
lignified phloem fibres accompanied by crystal sheaths
containing prisms of calcium oxalate; groups of sclereids
accompanied by crystal sheaths; cluster crystals of
calcium oxalate; some parenchymatous cells contain a
yellow substance which becomes deep red when treated
with alkali; cork cells and, frequently, epiphytes; the
latter may be liverworts, entire or in fragments, having a
1194 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 5.0 Cascara
lamina one cell thick without a midrib and composed of
isodiametric cells, or leaves of mosses, having a lamina
one cell thick composed of elongated cells and possessing
a midrib several cells thick.
C. Examine the chromatograms obtained in the test for
“Other species of Rhamnus; anthrones” after spraying
with a 50 g/l solution of potassium hydroxide R
in alcohol (50 per cent V/V) R and heating. The
chromatogram obtained with the test solution shows
several reddish-brown zones with different intensities:
there are four faint zones, three being situated at about
the mid-point of the chromatogram and one in the lower
third and there is a strong zone in the upper third of the
chromatogram. Examine in ultraviolet light at 365 nm.
The chromatogram obtained with the test solution shows
several zones with the same fluorescence, situated above
and particularly below (cascarosides) that corresponding
tobarbaloininthechromatogramobtainedwiththe
reference solution.
D. Heat 0.2 g of the powdered drug (180) with 50 ml of
water R on a water-bath for 15 min. Allow to cool and
filter. To 10 ml of the filtrate add 20 ml of hydrochloric
acid R1 and heat on a water-bath for 15 min. Allow to
cool, transfer to a separating funnel and shake with three
quantities, each of 20 ml, of ether R. Reserve the aqueous
layer (solution A).
(a) Combine the three ether extracts and shake with
10 ml of dilute ammonia R2. Theaqueouslayerbecomes
reddish-violet.
(b) Transfer solution A to a small flask, add 5 g of ferric
chloride R and heat on a water-bath for 30 min. Allow
to cool, transfer to a separating funnel and shake with
15 ml of ether R. Wash the ether layer with 10 ml of
water R, discard the water and shake the ether layer with
5mlofdilute ammonia R2. A red colour develops in the
aqueous layer.
solution of nitrotetrazolium blue R in methanol R. Examine
the chromatogram immediately. No violet or greyish-blue
zones appear.
Foreign matter (2.8.2). Not more than 1 per cent.
Loss on drying (2.2.32). Not more than 10.0 per cent,
determined on 1.000 g of the powdered drug (180) by drying
in an oven at 100 °C to 105 °C for 2 h.
Total ash (2.4.16). Not more than 7.0 per cent.
ASSAY
Carry out the assay protected from bright light in one day.
Stir 1.00 g of the powdered drug (180) into 100 ml of
boiling water R and continue boiling and stirring for 5 min.
Allow to cool, dilute to 100.0 ml with water R, shake,filter
and discard the first 20 ml of filtrate. Transfer 10.0 ml
of the filtrate to a separating funnel, add 0.1 ml of 1M
hydrochloric acid and shake with two quantities, each of
20 ml, of a mixture of 1 volume of ether R and 3 volumes of
hexane R. Wash the combined organic extracts with 5 ml of
water R, discard the organic layer and return the rinsings
to the aqueous layer. Shake the combined aqueous layers
with four quantities, each of 30 ml, of ethyl acetate R freshly
saturated with water R, (to150mlofethyl acetate R add
15 ml of water R, shake for 3 min and allow to stand) on
each occasion allowing separation to take place until the
organic layer is clear. Combine the ethyl acetate extracts.
Use the aqueous layer for the assay for cascarosides and the
organic layer for the assay for hydroxyanthracene glycosides
other than cascarosides.
Hydroxyanthracene glycosides other than cascarosides.
Transfer the organic layer to a suitable flask and remove
the solvent by distillation, evaporating almost to dryness.
Dissolve the residue in 0.3 ml to 0.5 ml of methanol R and
transfer to a volumetric flask, rinsing the first flask with
warm water R and adding the rinsings to the methanolic
solution. Allow to cool and dilute to 50.0 ml with water R.
Transfer 20.0 ml of the solution to a 100 ml round-bottomed
flask with a ground-glass neck and containing 2 g of ferric
chloride R and 12 ml of hydrochloric acid R.Attachareflux
condenser and place the flask in a water-bath so that the
level of the water is above that of the liquid in the flask
and heat for 4 h. Allow to cool, transfer the solution to
a separating funnel and rinse the flask successively with
3mlto4mlof1M sodium hydroxide and 3 ml to 4 ml of
water R, adding the rinsings to the separating funnel. Shake
the contents of the separating funnel with three quantities,
each of 30 ml, of a mixture of 1 volume of ether R and
3 volumes of hexane R. Washthecombinedorganiclayers
with two quantities, each of 10 ml, of water R and discard
the rinsings. Dilute the organic phase to 100.0 ml with
the mixture of ether and hexane. Take 20.0 ml, evaporate
carefully to dryness on a water-bath and dissolve the residue
in 10.0 ml of a 5 g/l solution of magnesium acetate R in
methanol R. Measuretheabsorbance(2.2.25) at515nm
using methanol R as the compensation liquid.
Calculate the percentage content of hydroxyanthracene
glycosides, expressed as cascaroside A, from the expression:
TESTS
Other species of Rhamnus; anthrones.Examineby
thin-layer chromatography (2.2.27), using a suitable silica
gel as the coating substance.
Test solution. To 0.5 g of the powdered drug (180) add 5 ml
of alcohol (70 per cent V/V) R and heat to boiling. Cool and
centrifuge. Decant the supernatant solution immediately
and use within 30 min.
Reference solution. Dissolve 20 mg of barbaloin R in
alcohol (70 per cent V/V) R and dilute to 10 ml with the
same solvent.
Apply separately to the plate as bands 10 µl of each solution.
Develop over a path of 10 cm using a mixture of 13 volumes
of water R, 17volumesofmethanol R and 100 volumes of
ethyl acetate R. Allow the plate to dry for 5 min, spray with
about10mlofa50g/lsolutionofpotassium hydroxide R
in alcohol (50 per cent V/V) R and heat at 100 °C to 105 °C
for 15 min. Examine the chromatograms immediately after
heating. The chromatogram obtained with the reference
solution shows, in the central part, a reddish-brown zone
corresponding to barbaloin. Examine in ultraviolet light
at 365 nm. The zone corresponding to barbaloin shows
intense yellowish-brown fluorescence. In the chromatogram i.e.takingthespecificabsorbancetobe180.
obtained with the test solution, no zone with orange-brown
fluorescence is seen between the zone of barbaloin and the
zones of cascarosides.
A
m
=
=
absorbance at 515 nm,
massofthesampleingrams.
Apply to another plate as a band 10 µl of the test solution Measure the absorbance of the test solution at 440 nm. If
and develop as described above. Allow the plate to dry for the ratio of the absorbance at 515 nm to that at 440 nm is
not longer than 5 min and spray immediately with a 5 g/l less than 2.4, the assay is not valid and must be repeated.
GeneralNotices(1)applytoallmonographsandothertexts 1195

Cassia oil EUROPEAN PHARMACOPOEIA 5.0
Cascarosides. Dilute the aqueous layer reserved for this
assay to 50.0 ml with water R. Treat 20.0 ml of the solution
as described above in the assay of hydroxyanthracene
glycosides other than cascarosides.
Measure the absorbance of the test solution at 440 nm. If
theratiooftheabsorbanceat515nmtothatat440nmis
less than 2.4, the assay is not valid and must be repeated.
Calculate the percentage content of cascarosides, expressed
as cascaroside A from the expression:
i.e.takingthespecificabsorbancetobe180.
A = absorbance at 515 nm,
m = massofthesampleingrams.
Measure the absorbance of the test solution at 440 nm. If
theratiooftheabsorbanceat515nmtothatat440nmis
less than 2.7, the assay is not valid and must be repeated.
STORAGE
Store protected from light.
CASSIA OIL
01/2005:1496
Cinnamomi cassiae aetheroleum
DEFINITION
Cassia oil is obtained by steam distillation of the leaves
and young branches of Cinnamomum cassia Blume
(C. aromaticum Nees).
CHARACTERS
A clear, mobile, yellow to reddish-brown liquid, with a
characteristic odour reminiscent of cinnamic aldehyde.
IDENTIFICATION
First identification: B.
Second identification: A.
A. Examine by thin-layer chromatography (2.2.27), using a
TLC silica gel plate R.
Test solution. Dissolve0.5mlofthesubstancetobe
examined in acetone R and dilute to 10 ml with the same
solvent.
Reference solution. Dissolve50µloftrans-cinnamic
aldehyde R,10µlofeugenol R and 50 mg of coumarin R
in acetone R and dilute to 10 ml with the same solvent.
Apply to the plate, as bands, 10 µl of each solution.
Develop over a path of 15 cm using a mixture of
10 volumes of methanol R and 90 volumes of toluene R.
Allow the plate to dry in air and examine in ultraviolet
light at 365 nm. The zone of blue fluorescence in the
chromatogram obtained with the test solution is similar
in position and colour to the zone in the chromatogram
obtained with the reference solution (coumarin). Spray
the plate with anisaldehyde solution R. Examinein
daylight while heating at 100-105 °C for 5-10 min. The
chromatogram obtained with the reference solution
shows in its upper part a violet zone (eugenol) and above
this zone a greenish-blue zone (trans-cinnamic aldehyde).
The chromatogram obtained with the test solution shows
a zone similar in position and colour to the zone due to
trans-cinnamic aldehyde in the chromatogram obtained
with the reference solution and may show a very faint
zone corresponding to eugenol. Other faint zones are
present.
B. Examine the chromatograms obtained in the test for
chromatographic profile. The retention times of the
principal peaks in the chromatogram obtained with the
test solution are similar to those in the chromatogram
obtained with the reference solution. Eugenol may be
absent from the chromatogram obtained with the test
solution.
TESTS
Relative density (2.2.5): 1.052 to 1.070.
Refractive index (2.2.6): 1.600 to 1.614.
Optical rotation (2.2.7): −1° to + 1°.
Chromatographic profile. Examinebygaschromatography
(2.2.28).
Test solution. Thesubstancetobeexamined.
Reference solution. Dissolve100µloftrans-cinnamic
aldehyde R, 10µlofcinnamyl acetate R, 10µl
of eugenol R, 20mgofcoumarin R and 10 µl of
trans-2-methoxycinnamaldehyde R in 1 ml of acetone R.
The chromatographic procedure may be carried out using:
— a fused-silica column 60 m long and about 0.25 mm in
internal diameter coated with macrogol 20 000 R as the
bonded phase,
— helium for chromatography R as the carrier gas at a flow
rate of 1.5 ml/min,
— a flame-ionisation detector,
— a split ratio of 1:100,
with the following temperature programme:
Time
(min)
Temperature
(°C)
Rate
(°C/min)
Comment
Column 0-10 60 – isothermal
10 - 75 60 → 190 2 linear gradient
75 - 160 190 isothermal
Injection port 200
Detector 240
Inject 0.2 µl of the reference solution. When the
chromatogram is recorded in the prescribed conditions, the
components elute in the order indicated in the composition
of the reference solution. Depending on the operating
conditions and the state of the column, coumarin may elute
before or after trans-2-methoxycinnamaldehyde. Record the
retention times of these substances.
Thetestisnotvalidunlesstheresolutionbetween
the peaks corresponding to coumarin and
trans-2-methoxycinnamaldehyde is at least 1.5.
Inject 0.2 µl of the test solution. Using the retention times
determined from the chromatogram obtained with the
reference solution, locate the components of the reference
solution in the chromatogram obtained with the test
solution. Determine the percentage content of each of these
components by the normalisation procedure.
The percentages are within the following ranges:
— trans-cinnamic aldehyde: 70percentto90percent,
— cinnamyl acetate: 1.0 per cent to 6.0 per cent,
— eugenol: lessthan0.5percent,
— coumarin: 1.5 per cent to 4.0 per cent,
— trans-2-methoxycinnamaldehyde: 3.0 per cent to 15 per
cent.
1196 See the information section on general monographs (cover pages)