EUROPEAN PHARMACOPOEIA 9.

0 Rosemary leaf

Test solution. To 1 g of the powdered herbal drug (355) 01/2013:1560

(2.9.12) add 10 mL of ethanol (60 per cent V/V) R. Shake
for 15 min and filter.
Reference solution. Dissolve 2.5 mg of quinaldine red R and
2.5 mg of sulfan blue R in 10 mL of methanol R.
ROSEMARY LEAF
Plate : TLC silica gel plate R (5-40 µm) [or TLC silica gel
plate R (2-10 µm)]. Rosmarini folium
Mobile phase : anhydrous formic acid R, water R, butanol R
DEFINITION
(10:12:40 V/V/V).
Whole, dried leaf of Rosmarinus officinalis L.
Application : 5 µL [or 2 µL] as bands of 10 mm [or 8 mm]. Content :
Development : over a path of 10 cm [or 6 cm]. – minimum 12 mL/kg of essential oil (anhydrous drug) ;
Drying : in air. – minimum 3 per cent of total hydroxycinnamic derivatives,
expressed as rosmarinic acid (C18H16O8 ; Mr 360.3)
Detection : examine immediately in daylight. (anhydrous drug).
Results : see below the sequence of zones present in the CHARACTERS
chromatograms obtained with the reference solution and Strongly aromatic odour.
the test solution. Furthermore, other faint zones may
be present in the chromatogram obtained with the test IDENTIFICATION
solution. A. The leaves are sessile, tough, linear or linear-lanceolate,
1-4 cm long and 2-4 mm wide, with recurved edges. The
Top of the plate
upper surface is dark green, glabrous and grainy, the lower
_______ _______ surface is greyish-green and densely tomentose with a
prominent midrib.
Quinaldine red : an orange-red
zone
An intense violet zone

Sulfan blue : a blue zone

An intense violet-blue zone

_______ _______

Reference solution Test solution

TESTS
Foreign matter (2.8.2) : maximum 2 per cent of fragments of
fruits (red funicles and parts of the 5-caverned capsule with
yellowish-grey pericarp, whose thin walls consist of several
layers of differently directed fibres ; flattened, reniform seeds
with a dotted surface) and maximum 2 per cent of other
foreign matter.
Loss on drying (2.2.32) : maximum 11.0 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 10.0 per cent.
Colouring intensity. Reduce 100 g to a coarse powder (1400)
(2.9.12) and homogenise. Reduce about 10 g of this mixture
to a very fine powder (355) (2.9.12). To 1.0 g of this powder
in a 100 mL flask add 25 mL of boiling water R and heat for
15 min on a water-bath with frequent shaking. Filter the hot
mixture into a 50 mL graduated flask ; rinse successively the
100 mL flask and the filter with 3 quantities, each of 5 mL, of
warm water R. After cooling, dilute to 50 mL with water R.
Dilute 5 mL of this solution to 50 mL with water R. Measure
Figure 1560.-1. – Illustration for identification test B of
the absorbance (2.2.25) at 520 nm using water R as the
powdered herbal drug of rosemary leaf
compensation liquid. The absorbance is not less than 0.350
for the whole drug and not less than 0.250 for the cut drug. B. Microscopic examination (2.8.23). The powder is
greyish-green or yellowish-green. Examine under a
microscope using chloral hydrate solution R. The powder
ASSAY shows the following diagnostic characters (Figure 1560.-1) :
Shake 1.00 g of the powdered herbal drug (355) (2.9.12) with fragments of the lower epidermis (surface view [B, J])
100.0 mL of carbon dioxide-free water R for 15 min. Filter. with straight or sinuous-walled cells [Ba] and numerous
To 50.0 mL of the filtrate add 100 mL of carbon dioxide-free diacytic stomata (2.8.3) [Bb] and glandular trichomes [Ja]
water R. Titrate with 0.1 M sodium hydroxide to pH 7.0, or covering trichomes or their scars [Bc, Bd] ; numerous
determining the end-point potentiometrically (2.2.20). multicellular, mostly branched, covering trichomes of the
lower epidermis, usually fragmented [A, C, D] ; fragments
1 mL of 0.1 M sodium hydroxide is equivalent to 6.4 mg of the upper epidermis (surface view [F]) with cells
of citric acid. with straight, thickened and pitted walls [Fa], and an

General Notices (1) apply to all monographs and other texts 1499
Rosemary oil EUROPEAN PHARMACOPOEIA 9.0

underlying hypodermis composed of large, irregular cells TESTS

with thickened and beaded anticlinal walls [Fb] ; fragments Foreign matter (2.8.2): maximum 5 per cent of stems and
of the lamina (transverse section [G]), showing the maximum 2 per cent of other foreign matter.
epidermis covered by a very thick cuticle [Ga], hypodermal
cells extending across the mesophyll [Gb] at intervals, Water (2.2.13) : maximum 100 mL/kg, determined on 20.0 g
separating 1 or 2 layers of palisade parenchyma into large, of the powdered herbal drug (355) (2.9.12).
crescent-shaped areas [Gc] ; glandular trichomes of 2 types, Total ash (2.4.16) : maximum 9.0 per cent.
the majority with a short, unicellular stalk and a radiate
head composed of 8 cells (surface view [E], side view [H]), ASSAY
others, less abundant, with a uni- or bicellular stalk and a Total hydroxycinnamic derivatives
spherical, unicellular head [Ja, K]. Stock solution. To 0.200 g of the powdered herbal drug (355)
C. Thin-layer chromatography (2.2.27). (2.9.12) add 80 mL of ethanol (50 per cent V/V) R. Boil in
a water-bath under a reflux condenser for 30 min. Allow
Test solution. Dissolve 20 µL of the oil obtained in the assay
to cool and filter. Rinse the filter with 10 mL of ethanol
in 1 mL of hexane R.
(50 per cent V/V) R. Combine the filtrate and the rinsings in a
Reference solution. Dissolve 5 mg of borneol R, 5 mg of volumetric flask and dilute to 100.0 mL with ethanol (50 per
bornyl acetate R and 10 µL of cineole R in 1 mL of hexane R. cent V/V) R.
Plate : TLC silica gel plate R. Test solution. To 1.0 mL of the stock solution add 2 mL
Mobile phase : ethyl acetate R, toluene R (5:95 V/V). of 0.5 M hydrochloric acid, 2 mL of a solution prepared
by dissolving 10 g of sodium nitrite R and 10 g of sodium
Application : 10 µL as bands. molybdate R in 100 mL of water R, and then add 2 mL of
Development : over a path of 15 cm. dilute sodium hydroxide solution R and dilute to 10.0 mL with
Drying : in air. water R ; mix.
Compensation solution. Dilute 1.0 mL of the stock solution to
Detection : treat with anisaldehyde solution R, heat at 10.0 mL with water R.
100-105 °C for 10 min and examine in daylight.
Measure immediately the absorbance (2.2.25) of the test
Results : see below the sequence of zones present in the solution at 505 nm.
chromatograms obtained with the reference solution and Calculate the percentage content of total hydroxycinnamic
the test solution. derivatives, expressed as rosmarinic acid, using the following
Top of the plate expression :
A red zone

Bornyl acetate : a yellowish-brown A yellowish-brown zone of low

zone intensity i.e. taking the specific absorbance of rosmarinic acid to be 400.
A coloured zone of low intensity A = absorbance of the test solution at 505 nm ;
Cineole : a violet zone A violet zone m = mass of the substance to be examined, in grams.
Coloured zones of low intensity
Essential oil (2.8.12). Use 25.0 g of the crushed herbal drug, a
Borneol : a violet-brown zone A violet-brown zone 1000 mL flask and 300 mL of water R as the distillation liquid.
A coloured zone of low intensity
Distil at a rate of 2-3 mL/min for 3 h.

Reference solution Test solution 01/2008:1846

D. Thin-layer chromatography (2.2.27).
Test solution. Grind 1.0 g of the herbal drug in 10 mL of
methanol R and filter.
Reference solution. Dissolve 1.0 mg of caffeic acid R and ROSEMARY OIL
5.0 mg of rosmarinic acid R in 10 mL of methanol R.
Plate : TLC silica gel plate R. Rosmarini aetheroleum
Mobile phase : anhydrous formic acid R, acetone R, DEFINITION
methylene chloride R (8.5:25:85 V/V/V). Essential oil obtained by steam distillation from the flowering
Application : 10 µL of the test solution and 20 µL of the aerial parts of Rosmarinus officinalis L.
reference solution, as bands.
CHARACTERS
Development : over a path of 8 cm.
Appearance : clear, mobile, colourless or pale yellow liquid.
Drying : in air.
Characteristic odour.
Detection : examine in ultraviolet light at 365 nm.
IDENTIFICATION
Results : see below the sequence of zones present in the
chromatograms obtained with the reference solution and First identification : B.
the test solution. Second identification : A.
Top of the plate A. Thin-layer chromatography (2.2.27).
A pink fluorescent zone Test solution. Dissolve 0.5 mL of the substance to be
examined in toluene R and dilute to 10 mL with the same
Caffeic acid : a light blue A blue fluorescent zone of low solvent.
fluorescent zone intensity
Reference solution. Dissolve 50 mg of borneol R, 50 mg of
Rosmarinic acid : a light blue An intense light blue fluorescent
fluorescent zone zone
bornyl acetate R and 100 µL of cineole R in toluene R and
dilute to 10 mL with the same solvent.
Reference solution Test solution
Plate : TLC silica gel plate R.

1500 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 9.0 Rosemary oil

Mobile phase : ethyl acetate R, toluene R (5:95 V/V). Temperature :

Application : 10 µL, as bands. Time Temperature
(min) (°C)
Development : over a path of 15 cm. 0 - 10 50
Column
Drying : in air. 10 - 85 50 → 200
Detection : spray the plate with vanillin reagent R and heat 85 - 110 200
the plate at 100-105 °C for 10 min. Examine immediately
in daylight. Injection port 200

Results : see below the sequence of the zones present in Detector 250
the chromatograms obtained with the reference solution
and the test solution. Furthermore, several violet-blue to Detection : flame ionisation.
violet-grey zones of medium intensity (terpene alcohols) Injection : 1 µL.
are present in the lower third of the chromatogram Elution order : order indicated in the composition of the
obtained with the test solution. reference solution. Record the retention times of these
Top of the plate
substances.
System suitability : reference solution :
An intense violet zone
– resolution : minimum 1.5 between the peaks due to
A violet-grey zone limonene and cineole and minimum 1.5 between the peaks
_______ _______ due to α-terpineol and borneol.
Bornyl acetate : a bluish-grey zone A bluish-grey zone of low Using the retention times determined from the chromatogram
of low intensity intensity (bornyl acetate) obtained with the reference solution, locate the components
A violet-pink zone of the reference solution in the chromatogram obtained with
the test solution.
_______ _______
Determine the percentage content of these components.
Cineole : an intense blue zone An intense blue zone (cineole)
For rosemary oil, Spanish type, the percentages are within the
Borneol : a violet-blue zone of A violet -blue zone of medium following ranges :
medium intensity intensity (borneol)
– α-pinene : 18 per cent to 26 per cent,
Reference solution Test solution
– camphene : 8.0 per cent to 12.0 per cent,
B. Examine the chromatograms obtained in the test for – β-pinene : 2.0 per cent to 6.0 per cent,
chromatographic profile. – β-myrcene : 1.5 per cent to 5.0 per cent,
Results : the characteristic peaks in the chromatogram – limonene : 2.5 per cent to 5.0 per cent,
obtained with the test solution are similar in retention time – cineole : 16.0 per cent to 25.0 per cent,
to those in the chromatogram obtained with the reference
solution. – p-cymene : 1.0 per cent to 2.2 per cent,
– camphor : 13.0 per cent to 21.0 per cent,
TESTS – bornyl acetate : 0.5 per cent to 2.5 per cent,
Relative density (2.2.5) : 0.895 to 0.920. – α-terpineol : 1.0 per cent to 3.5 per cent,
Refractive index (2.2.6) : 1.464 to 1.473. – borneol : 2.0 per cent to 4.5 per cent,
Optical rotation (2.2.7) : − 5° to + 8°. – verbenone : 0.7 per cent to 2.5 per cent.
Acid value (2.5.1) : maximum 1.0. For rosemary oil, Moroccan and Tunisian type, the percentages
are within the following ranges :
Chromatographic profile. Gas chromatography (2.2.28) : use
the normalisation procedure. – α-pinene : 9.0 per cent to 14.0 per cent,
– camphene : 2.5 per cent to 6.0 per cent,
Test solution. Dissolve 0.20 mL of the substance to be
examined in hexane R and dilute to 10.0 mL with the same – β-pinene : 4.0 per cent to 9.0 per cent,
solvent. – β-myrcene : 1.0 per cent to 2.0 per cent,
Reference solution. Dissolve 20 µL of α-pinene R, 10 mg of – limonene : 1.5 per cent to 4.0 per cent,
camphene R, 20 µL of β-pinene R, 10 µL of β-myrcene R, – cineole : 38.0 per cent to 55.0 per cent,
20 µL of limonene R, 50 µL of cineole R, 10 µL of p-cymene R,
50 mg of camphor R, 30 mg of bornyl acetate R, 10 mg of – p-cymene : 0.8 per cent to 2.5 per cent,
α-terpineol R, 10 mg of borneol R and 10 µL of verbenone R in – camphor : 5.0 per cent to 15.0 per cent,
hexane R and dilute to 10.0 mL with the same solvent. – bornyl acetate : 0.1 per cent to 1.5 per cent,
Column : – α-terpineol : 1.0 per cent to 2.6 per cent,
– material : fused silica, – borneol : 1.5 per cent to 5.0 per cent,
– size : l = 30 m (a film thickness of 1 µm may be used) to 60 m – verbenone : maximum 0.4 per cent.
(a film thickness of 0.2 µm may be used), Ø = 0.25-0.53 mm,
STORAGE
– stationary phase : macrogol 20 000 R. At a temperature not exceeding 25 °C.
Carrier gas : helium for chromatography R.
LABELLING
Flow rate : 1 mL/min.
The label states that the content is Spanish type or Moroccan
Split ratio : 1:50. and Tunisian type.

General Notices (1) apply to all monographs and other texts 1501