Thyme EUROPEAN PHARMACOPOEIA 9.

Mobile phase : 07/2014:0865

– mobile phase A : glacial acetic acid R, water R (0.1:99.9 V/V) ;
– mobile phase B : acetonitrile R ;

Time Mobile phase A Mobile phase B

THYME
(min) (per cent V/V) (per cent V/V)
0 - 16.5 90 → 71 10 → 29 Thymi herba
DEFINITION
Flow rate : 3.0 mL/min. Whole leaves and flowers separated from the previously dried
stems of Thymus vulgaris L. or Thymus zygis L. or a mixture
Detection : spectrophotometer at 260 nm. of both species.
Injection : 10 μL. Content :
– essential oil : minimum 12 mL/kg (anhydrous drug) ;
Identification of peaks : use the chromatogram supplied with – sum of the contents of thymol and carvacrol (both C10H14O ;
kudzuvine root dry extract HRS and the chromatogram Mr 150.2) : minimum 40 per cent in the essential oil.
obtained with the reference solution to identify the peaks
due to the isoflavonoids (puerarin, 3-methoxypuerarin, CHARACTERS
6-O-D-xylosylpuerarin and daidzin). Strong odour reminiscent of thymol.
Relative retention with reference to puerarin (retention IDENTIFICATION
time = about 3.4 min) : 6-O-D-xylosylpuerarin = about 1.15 ; A. The leaf of Thymus vulgaris is usually 4-12 mm long and
daidzin = about 1.4. up to 3 mm wide, sessile or with a very short petiole. The
lamina is tough, entire, lanceolate or ovate, covered on
System suitability : reference solution : both surfaces by a grey or greenish-grey indumentum ; the
– peak-to-valley ratio : minimum 10, where Hp = height above edges are markedly rolled up towards the abaxial surface.
the baseline of the peak due to 3-methoxypuerarin and The midrib is depressed on the adaxial surface and is very
Hv = height above the baseline of the lowest point of the prominent on the abaxial surface. The calyx is green, often
curve separating this peak from the peak due to puerarin. with violet spots and is tubular ; at the end are 2 lips of
which the upper one is bent back and at the end has 3 lobes,
Calculate the percentage content of puerarin using the the lower is longer and has 2 hairy teeth. After flowering,
following expression : the calyx tube is closed by a crown of long, stiff hairs. The
corolla, about twice as long as the calyx, is usually brownish
in the dry state and is slightly bilabiate.
The leaf of Thymus zygis is usually 1.7-6.5 mm long and
0.4-1.2 mm wide ; it is acicular or linear-lanceolate and
A1 = area of the peak due to puerarin in the the edges are markedly rolled towards the abaxial surface.
chromatogram obtained with the test solution ; Both surfaces of the lamina are green or greenish-grey and
the midrib is sometimes violet ; the edges, in particular at
A2 = area of the peak due to puerarin in the
the base, have long, white hairs. The dried flowers are very
chromatogram obtained with the reference
similar to those of T. vulgaris.
solution ;
m1 = mass of the herbal drug to be examined used to B. Microscopic examination (2.8.23). The powder of both
species is greyish-green or greenish-brown. Examine under
prepare the test solution, in grams ; a microscope using chloral hydrate solution R. The powder
m2 = mass of kudzuvine root dry extract HRS used to shows the following diagnostic characters (Figure 0865.-1
prepare the reference solution, in grams ; and Figure 0865.-2) : fragments of the outer epidermis
p = percentage content of puerarin in kudzuvine root of the corolla (surface view [A, C, F]), consisting of cells
dry extract HRS. with wavy and slightly thickened [Fc] or unthickened
[Ac] walls, numerous uniseriate, multicellular, covering
Calculate the percentage content of total isoflavonoids trichomes, often with 1 cell collapsed [Aa], glandular
(puerarin, 6-O-D-xylosylpuerarin and daidzin) using the trichomes with a unicellular head and a unicellular [Ca,
following expression : Fb] or multicellular [Ab] stalk, diacytic stomata (2.8.3) [Fa]
and glandular trichomes generally with 12 cells [D] ; cells
of the epidermis from the base of the corolla, isodiametric
with slightly thickened walls [C] ; pollen grains, relatively
rare, spherical and smooth, with 6 germinal slit-like pores,
measuring about 35 μm in diameter [B] ; the powder of
A1 = sum of the areas of the peaks due to the
T. zygis also contains numerous thick bundles of fibres
isoflavonoids (puerarin, 6-O-D-xylosylpuerarin from the main veins and from fragments of stems ; the
and daidzin) in the chromatogram obtained with epidermises of the leaves (surface view [G, K]) have cells
the test solution ; with anticlinal walls that are sinuous and beaded [Ga, Ka],
A2 = area of the peak due to puerarin in the and diacytic stomata (2.8.3) [Gb] ; numerous glandular
chromatogram obtained with the reference trichomes made up of 12 secretory cells, the cuticle of
solution ; which is generally raised by the secretion to form a globular
m1 = mass of the herbal drug to be examined used to or ovoid, bladder-like covering [Kb] ; glandular trichomes
prepare the test solution, in grams ; with a unicellular stalk and a globular or ovoid head [Kc] ;
m2 = mass of kudzuvine root dry extract HRS used to in both species, the adaxial epidermis bears covering
prepare the reference solution, in grams ; trichomes with warty walls that are shaped as pointed
teeth [Gc], and is usually associated with underlying
p = percentage content of puerarin in kudzuvine root palisade parenchyma [Gd, Kd] ; the abaxial epidermis
dry extract HRS. (transverse section [H, L]) bears covering trichomes of

1538 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 9.0 Thyme

different types : unicellular, straight or slightly curved [Ha,

La] ; bicellular or tricellular, articulated and most often
elbow-shaped [Hb, J] (T. vulgaris) ; bicellular or tricellular,
more or less straight [N], or very large, multicellular [M],
at the base of the lamina (T. zygis) ; fragments of calyx
covered by numerous, uniseriate trichomes with 5-6 cells
and a weakly striated cuticle (surface view [E]).

C. Thin-layer chromatography (2.2.27).

Test solution. To 0.5 g of the powdered herbal drug (355)

(2.9.12) add 5 mL of methanol R. Sonicate for 10 min.
Centrifuge or filter ; use the supernatant or the filtrate.

Reference solution. Dissolve 1 mg of rutoside trihydrate R

and 1 mg of rosmarinic acid R in 5 mL of methanol R.

Plate : TLC silica gel F254 plate R (5-40 μm) [or TLC silica
gel F254 plate R (2-10 μm)].

Mobile phase : anhydrous formic acid R, water R, ethyl

acetate R (1:1:15 V/V/V).

Application : 20 μL [or 5 μL] as bands of 20 mm [or 8 mm].

Development : over a path of 15 cm [or 6 cm].

Drying : in air.

Detection : heat at 100 °C for 3 min, treat the still-hot plate

with a 5 g/L solution of diphenylboric acid aminoethyl
ester R in ethyl acetate R, then treat with a 50 g/L solution Figure 0865.-1. – Illustration for identification test B of
of macrogol 400 R in methylene chloride R ; examine in powdered herbal drug of thyme
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 faint fluorescent
zones may be present in the chromatogram obtained with
the test solution.

Top of the plate

2 red fluorescent zones

Rosmarinic acid : a blue A blue fluorescent zone

fluorescent zone (rosmarinic acid)
_______ _______

1 or 2 blue fluorescent zones

_______ _______

2 yellow or orange fluorescent

zones
A green fluorescent zone may be
present

Rutoside : an orange-yellow
fluorescent zone
Reference solution Test solution

D. Examine the chromatograms obtained in the assay for

thymol and carvacrol.

Results : the characteristic peaks in the chromatogram

obtained with the test solution are similar in retention time
to those in the chromatogram obtained with reference Figure 0865.-2. – Illustration for identification test B of
solution (a). powdered herbal drug of thyme

General Notices (1) apply to all monographs and other texts 1539
Thyme oil, thymol type EUROPEAN PHARMACOPOEIA 9.0

TESTS 01/2012:1374
Foreign matter (2.8.2) : maximum 10 per cent of stems and
maximum 2 per cent of other foreign matter. Stems must not
be more than 1 mm in diameter and 15 mm in length.
Thymus serpyllum L. Adulteration with T. serpyllum L. is
indicated by the presence of leaves with long trichomes at
THYME OIL, THYMOL TYPE
their base and with weakly pubescent other parts.
Water (2.2.13) : maximum 100 mL/kg, determined on 20.0 g
Thymi typo thymolo aetheroleum
of the powdered herbal drug (355) (2.9.12). DEFINITION
Total ash (2.4.16) : maximum 15.0 per cent. Essential oil obtained by steam distillation from the fresh
Ash insoluble in hydrochloric acid (2.8.1): maximum 3.0 per flowering aerial parts of Thymus vulgaris L., T. zygis L. or a
cent. mixture of both species.
CHARACTERS
ASSAY
Appearance : clear, yellow or very dark reddish-brown, mobile
Essential oil (2.8.12). Use 30.0 g of the herbal drug, a 1000 mL liquid.
round-bottomed flask and 400 mL of water R as the distillation Odour reminiscent of thymol.
liquid. Distil at a rate of 2-3 mL/min for 2 h without xylene R
in the graduated tube. Solubility : miscible with anhydrous ethanol and with light
petroleum.
Thymol and carvacrol. Gas chromatography (2.2.28) : use
the normalisation procedure. IDENTIFICATION
Test solution. Filter the essential oil obtained in the First identification : B.
determination of essential oil over a small amount of Second identification : A.
anhydrous sodium sulfate R and dilute to 5.0 mL with A. Thin-layer chromatography (2.2.27).
heptane R by rinsing the apparatus and the anhydrous sodium Test solution. Dissolve 0.2 mL of the substance to be
sulfate. Dilute a volume of the filtered solution corresponding examined in methylene chloride R and dilute to 10 mL with
to 100 μL of the essential oil to 5.0 mL with heptane R. the same solvent.
Reference solution (a). Dissolve 0.20 g of thymol R and 50 mg Reference solution. Dissolve 5 mg of thymol R and 10 μL of
of carvacrol R in heptane R and dilute to 5.0 mL with the same carvacrol R in methylene chloride R and dilute to 10 mL
solvent. with the same solvent.
Reference solution (b). Dilute 10 μL of carvacrol R to 10.0 mL Plate : TLC silica gel plate R (5-40 μm) [or TLC silica gel
with heptane R. Dilute 100 μL of the solution to 10.0 mL with plate R (2-10 μm)].
heptane R. Mobile phase : methylene chloride R.
Column : Application : 10 μL [or 4 μL] as bands of 10 mm [or 8 mm].
– material : fused silica ; Development : over a path of 12 cm [or 6 cm].
– size : l = 30-60 m, Ø = 0.25 mm ; Drying : in air.
– stationary phase : macrogol 20 000 R (film thickness Detection : treat with anisaldehyde solution R and heat at
0.25 μm). 100-105 °C for 5-10 min ; examine in daylight.
Carrier gas : nitrogen for chromatography R or helium for Results : see below the sequence of zones present in the
chromatography R. chromatograms obtained with the reference solution and
the test solution. Furthermore, other faint zones may
Flow rate : 1-2 mL/min. be present in the chromatogram obtained with the test
Split ratio : 1:100. solution.
Temperature : Top of the plate
Time Temperature A pink zone
(min) (°C) _______ _______
Column 0 - 45 40 → 220
Thymol : an orange-brown zone An intense orange-brown zone
Injection port 190
(thymol)
Detector 210 Carvacrol : an orange-grey zone A faint orange-grey zone
(carvacrol) may be present
Detection : flame ionisation. _______ _______

Injection : 0.2 μL. A pink zone

Elution order : order indicated in the composition of reference A violet zone
solution (a) ; record the retention times of these substances.
A brownish-grey zone
System suitability : reference solution (a) :
Reference solution Test solution
– resolution : minimum 1.5 between the peaks due to thymol
and carvacrol. B. Examine the chromatograms obtained in the test for
Using the retention times determined from the chromatogram chromatographic profile.
obtained with reference solution (a), locate the components Results : the characteristic peaks in the chromatogram
of the reference solution in the chromatogram obtained with obtained with the test solution are similar in retention time
the test solution. to those in the chromatogram obtained with reference
Determine the percentage content of thymol and carvacrol. solution (a).
Disregard any peak due to the solvent or with an area less than
the area of the principal peak in the chromatogram obtained TESTS
with reference solution (b) (0.05 per cent). Relative density (2.2.5) : 0.915 to 0.935.

1540 See the information section on general monographs (cover pages)