1. HPTLC Method Validation and Identification of Chamomile Flower
Monika Arrhenius, Yanjun Zhang, Quanyin Gao, Peter Chang and Gary Swanson
Quality Control & Quality Assurance
Herbalife International, 950 W 190th Street, Torrance, CA 90502

2. Abstract
This validation study describes a rapid high performance thin layer chromatography (HPTLC) method for identification of chamomile (Matricaria recutita) flower powder.
Comparison to the chromatograms of Asteraceae family of Roman Chamomile (Chamaemelum nobile) flowers and Feverfew (Tanacetum parthenium) flowers and aerial parts is outlined.
The identification procedure consists of extraction of botanical material, flower powders, with methanol followed by the comparison of this sample solution to a botanical reference using a CAMAG HPTLC instrument.
A discussion of “finger print bands” exhibiting characteristic colors and Rf values is provided in the study.  

3. Introduction
The dried flowers of chamomile (Matricaria recutita, German chamomile) from the family Asteraceae are among the most popular, well-documented and universally recognized herbal materials.
This material possesses many beneficial effects [1,2] recognized as being related to the content of Apigenin and Apigenin-7-O-glucoside (A7G) [2, 3].
When obtaining chamomile (M. recutita) from diverse sources, other Asteraceae members resembling chamomile in appearance, i.e. Chrysanthemum, feverfew etc., may be present.

4. Pictures of Chamomiles Flowers and Feverfew Flowers
Fig. 1 Matricaria recutita (German Chamomile )
Fig. 2 Chamaemelum nobile (Roman Chamomile)
Fig. 3 Tanacetum parthenium (Feverfew)
Photos copy from Wikipedia, the free encyclopedia

5. Materials and Methods Materials
Botanical Reference Materials (BRMs) were purchased from American Herbal Pharmacopoeia as verified (AHP-Verified) botanical material.
Apigenin-7-O-Glucoside was purchased from ChromaDex.
All other reagents and solvents used for this study were HPLC or Analytical Grade, obtained from standard commercial vendors.
Chemical Reference Solution (CRS)
Weigh ~2 mg of Apigenin-7-Glucoside into a 15 mL screw cap test tube. Add 10 mL of methanol to the test tube, sonicate for 5 minutes. Transfer a portion of the solution to an HPLC vial for HPTLC analysis.
BRM & Test Solution:
Weigh ~500 mg of each of BRM and the test sample into individual 15 mL screw cap test tubes. Add 5 mL of methanol to each test tube; Vortex and then sonicate at 60˚C for 10 minutes. Allow to cool, then filter a portion of the BRM and test solution through µ syringe filters into individual HPLC vials.

6. Instrument and Reagents
CAMAG Automatic TLC Sampler 4
CAMAG ADC2 Automatic Developing Chamber
CAMAG TLC Visualizer or
CAMAG Chromatogram Immersion Device III
CAMAG TLC Plate Heater III
HPTLC silica gel 60 F254, 20 X 10 glass plates were used for the thin layer chromatography (TLC) and CAMAG Filter paper was employed for saturation of the ADC 2 development chamber.
Mobile Phase: A mixture of Ethyl Acetate: Acetic Acid; Formic Acid: Water.
Application Volume: 2.0 µL for chemical reference; 3.0 µL for botanical reference and 5.0 µL for test samples.
Derivatization Reagent: NP reagent.

7. Fig. 4 BRM of M. recutita, C. nobile and T. parthenium

8. Rf Value & Correspond Color of Fig. 4
Track # 1 2 3 4 5 6 7
RF Value
A7G
CGA
M. recutita F
M. recutita Aerial
C. nobile F
T. parthenium F
T. parthenium Aerial
~0.60
Green
N/A
Faint Green
Not Present
~0.40
Blue, bright
Blue, faint w/ shift to upward Bright Blue
Blue, bright & broad
~0.88
Blue
Yellow, tint with red
Yellow, bright & tint w/green
Yellow, tint w/ green
Yellow, tint w/ red
~0.80
Blue, bright & thin
~0.74
Blue, thin
~0.70
Blue, faint
~0.66
~0.63
~0.55
Yellow
~0.53
Greenish, tint w/Yellow on top
~0.45
Orange
~0.43
Yellow, faint

9. Fig. 5 M. Recutita Flower vs its Aerial Parts

10. Rf Value & Correspond Color of Fig. 5
Track # 1 2 3 4 5 6 7
RF Value
A7G
CGA
M. recutita F
M. recutita (F/A 10:1)
M. recutita Aerial
~0.60
Green
N/A
Faint Green
~0.40
Blue, bright
~0.88
Blue
Yellow, tint with red
~0.80
~0.74
Not Present
~0.70
Blue, thin
Blue, faint
~0.66
~0.63
~0.55
Yellow
~0.53
~0.45
Orange
~0.43

11. Fig. 6 M. recutita Flower & Mixture of Like-Materials

12. Fig. 7 Chromatogram of Flower Test Samples

13. Rf Value & Correspond Color of Fig. 7
Track # 1 2 3 4 5 6 7 8
RF Value
A7G
CGA
M. recutita F
Sample 1
Sample 2
C. nobile F
~0.60
Green
N/A
~0.40
Blue, bright
~0.88
Blue
Yellow, bright
~0.80
~0.74
~0.70
Blue, thin
~0.66
Not Present
~0.63
~0.55
Yellow
~0.53
~0.45
Orange
~0.43

14. Results & Discussion
A7G, used as a system reference in the test method, produces a green band at Rf ~0.6 (UV 366 nm).
The Botanical Reference of Matricaria recutita and Test sample should contain A7G at its corresponding Rf value and with correct color.
A key distinction of Matricaria recutita Chamomile from similar materials such as Roman Chamomile or Feverfew can be found in two major bands at Rf ~0.88 (blue) and Rf~0.45 (orange)at UV 366 nm
This result can be further confirmed by the combinations of different ratio of Matricaria recutita flower with its aerial part and other analogous materials including Roman and Feverfew.

15. Conclusions
The method described in this study is practical, validated and selective.
It is suitable for a fast paced quality control environment.
The method provides a means for differentiation of chamomile (M. recutita) flower powder from other closely related species (C. nobile) or physically similar flowers such as feverfew (T. parthenium).
The collective results obtained from this test method lend themselves well to providing control over the quality of this herbal material the quality of derived finished products.

16. References Journal of Chromatography A, 1385 (2015) 103-110
Journal of Pharmaceutical and Biomedical Analysis 107 (2015)
Journal of Separation Science 37( 2014)
Plant Drug Analysis, Hilbert Wagner. Sabine Bladt, 2nd Edition

17. Acknowledgement
Thanks to AOAC for the opportunity to present our work