Cinnamomum cassia – twig

1. Scope
This method identifies dried twigs of Cinnamomum cassia (L.) J. Presl (syn. Cinnamomum
aromaticum Ness) by HPTLC fingerprint and discriminates the bark of Cinnamomum verum J.
Presl.
2. Authors and adoptions
HPTLC Association
Adopted in USP41-N36 S1, Dietary Supplements Compendium 2019 (change to SST
requirements)
3. Procedure
Test solution Mix 2 g of finely powdered sample with 10 mL of methanol,
sonicate for 10 min, centrifuge, and transfer the extract to a round-
bottom flask. Evaporate to dryness under reduced pressure.
Dissolve the residue in 2 mL of toluene, sonicate for 2 min,
centrifuge, and use the supernatant as test solution (stable for at
least 8 h).
Reference solutions Standard solution A: 0.5 mg/mL each of USP Cinnamaldehyde RS
and coumarin in methanol.
Standard solution B: Mix 2 g of USP Cinnamomum cassia Twig
Powder RS with 10 mL of methanol (1 in 5). Sonicate for 10 min,
centrifuge, and evaporate the solvent. Suspend the residue in
2 mL of toluene, sonicate for 2 min, centrifuge, and use the
supernatant.
Stationary phase HPTLC Si 60 F 254 (Merck)
Application volume 15 tracks, band length 8 mm, track distance 11.4 mm, distance
from left edge 20 mm, distance from lower edge 8 mm, application
volume 5 µL* of reference and test solutions
*Deviation from monograph with lower application volume
Developing solvent Toluene, ethyl acetate 19:1 (v/v)
Developing distance 70 mm from lower edge
Saturation time 20 min, with a saturation pad
Relative humidity 33%, saturated MgCl 2
Temperature 22 ± 5°C
Derivatization reagent Anisaldehyde reagent
Preparation: Slowly mix 85 mL of ice-cold methanol with 10 mL of
acetic acid and 5 mL of sulfuric acid. Allow to cool to room
temperature, add 0.5 mL of anisaldehyde (p-methoxy
benzaldehyde).
Use: Derivatize (Immersion device: time 0, speed 5; Derivatizer:
nozzle blue, spraying level 3), heat at 100°C for 4 min
Detection A) Underivatized, UV 254 nm
B) *Underivatized, UV 366 nm
C) *Underivatized, white light
D) Derivatized, UV 366 nm
E) *Derivatized, white light
*Deviation from monograph with additional detection modes

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A non-profit organization dedicated to the promotion of HPTLC in plant analysis and other analytical fields • www.hptlc-association.org
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4. Results
Note: These chromatographic fingerprints are representative of the samples used in this
analysis. Fingerprints obtained may vary from sample to sample. Analysts must validate the
most appropriate fingerprint for their identity standard.

Figure 1: HPTLC profiles under UV 254 nm (A), UV 366 nm (B) and white light (C) prior to derivatization, and under
UV 366 nm after derivatization (D), and white light after derivatization (E).

Track Sample Origin

Coumarin (0.5 mg/mL), USP Cinnamaldehyde RS (0.5 mg/mL) (with
1 --
increasing R F )
2 USP Powdered Cinnamomum cassia Twig RS (200 mg/mL) --
3-4 Cinnamomum cassia powdered bark (200 mg/mL) Unknown
Cinnamomum verum powdered bark, not compliant, similar to
5 Unknown
Cinnamomum cassia bark (200 mg/mL)
Cinnamomum cassia powdered bark, not compliant, similar to
6 Unknown
Cinnamomum verum (200 mg/mL)
Cinnamomum powdered bark, similar to Cinnamomum verum
7-8 Unknown
(200 mg/mL)
9 Cinnamomum loureiroi powdered bark (200 mg/mL) Unknown
10 Cinnamomum loureiroi powdered twig (200 mg/mL) Unknown
11 Cinnamomum aromaticum powdered bark (200 mg/mL) Unknown

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System suitability test (under UV 254 nm prior to derivatization)
• Cinnamaldehyde: a quenching zone at R F ~ 0.39
• Coumarin: a quenching zone at R F ~ 0.26

Identification
Compare the results with reference images. The fingerprint of the test solution prepared from
the sample is similar to those obtained from the corresponding botanical reference materials.
Additional weak zones may be present.
Under UV 254 nm, the chromatogram of standard solution B shows an intense quenching
zone, at the higher R F, and a weak quenching zone corresponding to cinnamaldehyde and
coumarin, respectively, in standard solution A. There is a quenching zone close to the starting
position due to co-elution of cinnamic acid and 2-methoxycinnamic acid, and a quenching zone
between coumarin and cinnamic acid due to cinnamyl alcohol. The chromatogram of the test
solution shows the most intense quenching zone in the middle-third section, corresponding in
R F and color to the cinnamaldehyde band in standard solution A. There are additional zones
corresponding to similar zones in standard solution B.
Under UV 366 nm after derivatization, the chromatogram of standard solution B exhibits a zone
corresponding in R F and color to the cinnamaldehyde zone in standard solution A, and a yellow
zone immediately below the cinnamaldehyde zone. The chromatogram of the test solution
exhibits a zone corresponding in R F and color to the cinnamaldehyde zone in standard
solutions A and B, and a yellow zone immediately below the cinnamaldehyde zone. There is
no red zone immediately above the cinnamaldehyde zone (a distinction from Cinnamomum
verum, yellow arrow).

Version Revision history Released by

1 Created by: TD/14 Jul 2015 VW/01 Dec 2015
2 Harmonized with USP40-NF35 monograph, images updated, MHS/28 Jan. 2019
VW/03 Oct 2018

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A non-profit organization dedicated to the promotion of HPTLC in plant analysis and other analytical fields • www.hptlc-association.org
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