High Performance TLC

as part of PhEur monographs

Seoul February 2012

Eike Reich
CAMAG Laboratory
2

Disclaimer

The statements made in this presentation are

personal. They reflect only the opinion of the

author and are neither endorsed by nor made on

behalf of the European Pharmacopoeia

3

Overview
 Structure of monographs on herbal drugs in the PhEur

 The definition of HPTLC

 Examples of recently adopted monographs

 Current efforts in harmonization

 Practical aspects concerning development of monographs for TCM

 Open questions
4

Structure of monographs on herbal drugs

General monograph on herbal drugs 1433
DEFINITION

PRODUCTION

IDENTIFICATION
Herbal drugs are identified using their macroscopic and microscopic descriptions
and any further tests that may be required (for example, thin-layer chromatography)

TESTS
Foreign matter … . An appropriate specific test may apply to herbal drugs liable to
be adulterated. …

ASSAY
Unless otherwise prescribed or justified and authorised, herbal drugs are assayed
by an appropriate method.

STORAGE
5

Guide for the elaboration of monographs

NOMENCLATURE
ENGLISH TITLE
LATIN TITLE
DEFINITION
 The state of the drug: whole, fragmented, peeled, cut, fresh or dried.
 The complete scientific name of the plant (genus, species, subspecies, variety,
author) as obtained from the Kew Index and its supplements (International Plant
Names Index IPNI).
 Commonly used synonyms may be mentioned.
 The part or parts of the plant used (written in the singular).
 Where appropriate, the stage in the growth cycle when harvesting takes place,
or other necessary information.
 Wherever possible, the minimum content of quantified constituents
(constituents with known therapeutic activity or markers). Herbal drugs very
often contain a mixture of related substances. In such cases, the total content of
quantified constituents is determined and expressed as one of the
constituents, usually the major constituent. separate limits may be given for
different forms of the drug (whole/cut).
6

Guide for the elaboration of monographs

CHARACTERS
ORGANOLEPTIC CHARACTERS
 Only if highly characteristic
MACROSCOPIC AND MICROSCOPIC BOTANICAL CHARACTERS
 Only if highly variable and considered not compulsory for the identification17

IDENTIFICATION
MACROSCOPIC BOTANICAL CHARACTERS
 The important macroscopic botanical characters of the drug are specified to
permit
MICROSCOPIC BOTANICAL CHARACTERS
 The microscopic examination of the drug reduced to a powder describes the
dominant or the most specific characters, including, if necessary, examination of
the stomata and stomatal index. The colour of the powder, the sieve number
(No. 355) and the reagents used for the microscopic examination are specified.
 Illustrations of the main microscopic features of powders may be included. a
clear identification.
7

Identification cont.: THIN-LAYER CHROMATOGRAPHY

Two types of presentation are possible.
TLC PRESCRIBED ONLY FOR THE IDENTIFICATION OF THE DRUG
 TLC is used under Identification, even if other chromatographic methods, such
as gas chromatography (GC) and liquid chromatography (LC) are subsequently
used in the monograph. In this context the TLC is aimed at elucidating the
chromatogram of the drug with respect to selected reference compounds that
are described for inclusion as reagents.
 Availability of reference compounds as commercial reagents must be verified
during monograph elaboration. Where they are not readily available, a chemical
reference substance will have to be established.
 The commercial name of the TLC plate used during monograph development is
included as a footnote to the monograph and after adoption by the Commission
is transferred to the EDQM Knowledge database.
 A minimum of 2 reference compounds must be used to validate the separation
and spacing between the zones, otherwise a resolution test is necessary.
 All the information concerning the preparation of the reference solution and the
test solution and the chromatographic conditions is clearly stated. The
methodology used, where possible, must be such that the application volume of
the reference solution and the test solution is the same.
 8

THIN-LAYER CHROMATOGRAPHY cont.

 The general chapter on thin-layer chromatography covers both classical TLC
and HPTLC. Where these two methods give equivalent results with the
development solvent and visualisation method prescribed both may be
included in the working conditions [HPTLC in brackets], otherwise preference
is given to classical TLC unless HPTLC is essential for proper identification.
 The chromatograms are described in the form of a table, which shows the upper,
middle and lower third of the plate.
 Only the principal zone(s) in the chromatogram obtained with the test solution are
described in the table in relation to the position of the zones due to the reference
compounds in the chromatogram obtained with the reference solution.
 The names of the constituents detected in the chromatogram obtained with the
reference solution are always given. The names of the constituents detected in the
chromatogram obtained with the test solution are given only if these constituents are
present in the reference solution or if the nature of the substance is well established.
 Chromatograms are never described in terms of Rf values.
 It is usually necessary to indicate that zones other than those described (usually
more faint) are also present in the chromatogram of the test solution.
 A copy in colour of the suitable chromatogram has to be provided to the Secretariat.
 9

THIN-LAYER CHROMATOGRAPHY cont.

C. Thin-layer chromatography (2.2.27).
Test solution. To 0.3 g of the powdered drug (355) add 3 ml of methanol R. Heat on a water-bath at 60 °C
for 1 min. Filter.
Reference solution. Dissolve 50 mg of coumarin CRS and 20 mg of o-coumaric acid R in 50 ml of methanol
R.
Plate: TLC silica gel plate R or TLC silica gel plate R (2-10 μm).
Mobile phase: upper phase of the following mixture: dilute acetic acid R, ether R, toluene R (10:50:50 V/V/V).
Application: 25 μl [5 μl] as bands of 15 mm [8 mm].
Development: over a path of 12 cm [6 cm].
Drying: in air.
Detection: treat with 2 M alcoholic potassium hydroxide R. Examine in 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 zones of various colours may be present in the chromatogram
obtained with the test solution.
10

Example of table description

11

TLC PRESCRIBED UNDER TESTS AND IDENTIFICATION

 If a TLC test is used both for the control of adulterations and for identification,
the method is described entirely under Tests with a cross-reference under
Identification.
Example:
Angelica root
 C. Examine the chromatograms obtained in the test for lovage root.
 Results: see below the sequence of the zones present in the chromatograms
obtained with the reference solution and the test solution.
12

Guide for the elaboration of monographs

CHEMICAL REACTIONS FOR IDENTIFICATION
 Chemical reactions are included only TLC/HPTLC does not give sufficient identification and
if the reaction is particularly characteristic of a constituent or a group of constituents. The
must allow rapid identification without 1 the use of complex equipment and not be so
sensitive as to give a false positive result.
TESTS
TYPICAL TESTS
TOTAL ASH (2.4.16)
ASH INSOLUBLE IN HYDROCHLORIC ACID (2.8.1)

THIN-LAYER CHROMATOGRAPHY (2.2.27)

 TLC can be used under Tests to detect plant species that are not part of the definition. The
TLC method is described entirely under Tests and wherever feasible is also used to identify
the herbal drug. The name of the unwanted plant species or their constituent(s) (e.g. :
thujone in Three-lobed sage-leaf) is used as the title of the test. In the chromatogram
obtained with the test solution only the position and colour of the zone(s) of the
constituent(s) which must be absent are described by comparison with the chromatogram
obtained with the reference solution. The zones present in the chromatogram obtained with
the test solution are not described under Tests but under Identification.
13

Guide for the elaboration of monographs

FOREIGN MATTER (2.8.2)
HEAVY METALS
 A general method Heavy metals in herbal drugs and fatty oils (2.4.27) is included
in the PhEur. The test is prescribed where there is potential contamination with
heavy metals. A test for a specific heavy metal may be needed where a
particular herbal drug is known to accumulate that metal.
LOSS ON DRYING (2.2.32)
WATER (2.2.13)
 For herbal drugs containing more than 10 ml/kg (1 per cent) of essential oil, the
determination of water by distillation (2.2.13) is carried out instead of the test for
loss on drying. The fineness of the powder using a sieve number (2.1.4) is
indicated if required.
SWELLING INDEX (2.8.4)
BITTERNESS VALUE (2.8.15)
 14

Guide for the elaboration of monographs

EXTRACTABLE MATTER
 Applicable to herbal drugs: Eleutherococcus, Restharrow root, Hop strobile, Gentian
root, Couch grass rhizome. It is considered useful to determine extractable matter only
where no constituent suitable for an assay is known or when the material is used to
produce a preparation with a dry residue.
OTHER TESTS

ASSAY
 Wherever possible, an assay is included. Substances used for quantification are
established as Chemical Reference Substances; availability of a sufficient quantity of a
batch of suitable quality must be verified during monograph elaboration.
 Wherever possible, liquid or gas chromatography are the methods of choice to
determine the content of specific constituents rather than a global determination by
spectrophotometry
15

Assay cont.
ULTRAVIOLET AND VISIBLE ABSORPTION SPECTROPHOTOMETRY

DETERMINATION OF TANNINS IN HERBAL DRUGS (2.8.14)

VOLUMETRIC TITRATION

DETERMINATION OF ESSENTIAL OILS IN HERBAL DRUGS (2.8.12)

LIQUID CHROMATOGRAPHY (2.2.29) AND GAS CHROMATOGRAPHY

(2.2.28).

STORAGE
REAGENTS
16

The definition of TLC: 2.2.27

THIN-LAYER CHROMATOGRAPHY
Thin-layer chromatography is a separation technique in which a stationary
phase consisting of an appropriate material is spread in a uniform thin layer
on a support (plate) of glass, metal or plastic. Solutions of analytes are
deposited on the plate prior to development. The separation is based on
adsorption, partition, ion-exchange or on combinations of these mechanisms
and is carried out by migration (development) of solutes (solutions of
analytes) in a solvent or a suitable mixture of solvents (mobile phase)
through the thin-layer (stationary phase).
17

TLC in chapter 2.2.27 of PhEur

 Chromatographic tank with a flat bottom or twin trough, of inert, transparent
material, of a size suitable for the plates used and provided with a tightly fitting
lid. For horizontal development the tank is provided with a trough for the mobile
phase and it additionally contains a device for directing the mobile phase to the
stationary phase.
 Micropipettes, microsyringes, calibrated disposable capillaries or other
application devices suitable for the proper application of the solutions.
 Fluorescence detection device to measure direct fluorescence or the inhibition
of fluorescence.
 Visualization devices and reagents. Suitable devices are used for
derivatization to transfer to the plate reagents by spraying, immersion or
exposure to vapor and, where applicable, to facilitate heating for visualization of
separated components.
 Documentation. A device may be used to provide documentation of the
visualized chromatogram, for example a photograph or a computer file.
18

Sample application
 Sample application. Apply the prescribed volume of the solutions at a suitable
distance from the lower edge and from the sides of the plate and on a line
parallel to the lower edge; allow an interval of at least 10 mm (5 mm on high-
performance plates) between the centers of circular spots and 5 mm (2 mm on
high-performance plates) between the edges of bands. Apply the solutions in
sufficiently small portions to obtain circular spots 2-5 mm in diameter (1-2 mm on
high-performance plates) or bands 10-20 mm (5-10 mm on high-performance
plates) by 1-2 mm.

 In a monograph, where both normal and high-performance plates may be used,

the working conditions for high-performance plates are given in the brackets [ ]
after those for normal plates.
19

TLC or HPTLC
 Pharmacopoeias see difference primarily in the plate
yet assume similar results

HPTLC plate

Average plate height [μm]

e
at
pl
C
TL
HP
TLC plate

0 10 20 m
20

Development
 Vertical development. Line the walls of the chromatographic tank with filter
paper. Pour into the chromatographic tank a sufficient quantity of the mobile
phase for the size of the tank to give after impregnation of the filter paper a layer
of appropriate depth related to the dimension of the plate to be used. For
saturation of the chromatographic tank, replace the lid and allow to stand at 20-
25 °C for 1 h. Unless otherwise indicated in the monograph, the
chromatographic separation is performed in a saturated tank. Apply the
prescribed volume of solutions as described above. When the solvent has
evaporated from the applied solutions, place the plate in the chromatographic
tank, ensuring that the plate is as vertical as possible and that the spots or
bands are above the surface of the mobile phase. Close the chromatographic
tank, maintain it at 20-25 °C and protect from sunlight. Remove the plate when
the mobile phase has moved over the prescribed distance, measured between
the points of application and the solvent front. Dry the plate and visualize the
chromatograms as prescribed.
21

Quality and reproducibility of results?

The central problem:
 When two (or more) labs do the „same“ or think that they are doing the
same, the results are not necessarily equal.
 Many parameters that can be chosen freely affect the final result.
 There is little guidance available: the general method description in the
Pharmacopoeias (EP 2.2.27, USP <201>, <621>, PhPRC ap. VI, JPXV
2.03 ) define „suitable” equipment and give ranges instead of values.
 A table (EP) or a result description (USP, JP) can only define the most
Most text books are even worse …
important aspects of a TLC chromatogram. That leaves room for
interpretation and uncertainty about what to expect. ATLAS?
 Example: how can a color be described correctly?
22

What is blue?

And how about the

color blind...
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Proposal (for USP): General description of HPTLC

High Performance Thin-Layer Chromatography (HPTLC)
HPTLC allows highly reproducible results and traceable records
through a standardized methodology and the use of suitable
instruments (typically controlled by software) for all steps of the
analysis. A system suitability test is used to qualify results.
Stationary Phase: HPTLC glass plates or aluminum sheets are
coated with a uniform thin layer (typically 200 micron) of porous
particles (2 - 10 μm) with an average particle size of 5 μm. The layer
typically consists of silica gel with a pore size of 60 Angstroms, a
polymeric binder and a so called fluorescence indicator (F254). The
standard format of the plate is 20x10 cm. Other stationary phases
and plate formats may be specified in a monograph.
24

Apparatus
 A device suitable for application of samples as bands providing
control of dimension and position of the application as well as
applied sample volume
 A suitable chromatographic chamber (typically a twin trough
chamber) providing control of saturation and developing
distance
 A device suitable for controlling the activity of the stationary
phase via relative humidity
 A device suitable for reproducible drying of the developed plate
 Suitable devices for reagent transfer and heating as part of the
derivatization procedure
 A device suitable for electronic documentation of
chromatograms under UV 254 nm, UV 366 nm, and white light
 For quantitative determinations a densitometer or image
evaluation software
25

Procedure:
Sample application: Solutions are applied in small volumes as narrow bands of 8
mm width, 8 mm above the lower edge of the plate. The left and right margins of the
plate are at least 15 mm, the minimum space between bands is 2 mm.
Other application patterns may be specified in a monograph.

Chromatogram development
1. Saturate the chamber for 20 min using a filter paper saturated with developing
solvent and positioned against the rear wall of the chamber. The solvent level in the
chamber must be 5 mm.
2. Condition the plate at a relative humidity between 30 and 40%
3. Place the plate in the front trough of the chamber in a vertical position so that the
stationary phase faces the filter paper.
4. Develop the plate to a distance of 70 mm from the lower edge, then remove it from
the chamber and dry it.
[Note: other chamber configurations or developments may be described by a
monograph]
5. Visualize, document and evaluate the chromatograms as prescribed
26

SOP for HPTLC

 Should be the basis for all work

 Applies to all methods

 All deviation need to be recorded

 Our SOP is in full compliance with PhEur, USP, ChP

27

Successful standardization
Echinacea June 30, 2005 – CSI Laboratory

Original image
published 2001
28

Variability of Actaea racemosa

UV 366nm Validated method:
Toluene, ethyl formate,
formic acid = 5 : 3 : 2
Deriv.: sulfuric acid reagent

WRT

1: Actein, 2-9: different

Actaea racemosa, rhizome

1 2 3 4 5 6 7 8 9 10
 29

Adulterants of Actaea racemosa

UV 366 nm UV 254nm, before deriv.

white light 5 6 7 8 9 10 11

1 Isoferulic acid
2 Norcimifugin
3 Actein
4 23-epi-26-Deoxyactein
5 Cimifugin
6 Actaea racemosa, rhizone 1
7 Actaea racemosa, rhizome 2
8 A. foetida, rhizome
9 A. heracleifolia, rhizome
10 A. dahurica, rhizome
1 2 3 4 5 6 7 8 9 10 11 11 A. americana, Yellow cohosh

Toluene, ethyl formate, formic acid = 5:3:2

 30
Collaborative trial (I)
Actaea racemosa with 5 % adulteration
* R1 R2 R3 R4 Appl. volume: 20 μl
Actein

Actein
Derivatization with
boric acid/oxalic acid,
120 °C during 5 min

System suitability
* G1 G2 G3 G4 test: Actein Rf ≈
0.37
Actein

Actein

Fluorescent zone with

Rf = 0.24 (Rf = 0.06)
 5 % A. foetida in C.
racemosa
31

Collaborative trial with 5 % mixtures (II)

Derivatization with
* R1 R2 R3 R4
antimony (III) chloride
Actein

Actein
120 °C for 10 min

* G1 G2 G3 G4
Actein

Actein

Fluorescent zone with

Rf = 0.39  5 % A.
dahurica or A. heraclei-
folia in A. racemosa
32

Collaborative trial with 5 % mixtures (III)

* R1 R2 R3 R4
Evaluation: UV254 nm

* G1 G2 G3 G4

Fluorescence
quenching zone at
Rf=0.30  5 % of
A. americana in A.
racemosa
33
34

PhEur 7.4 Black cohosh

knowledge database of
PhEur (www.edqm.eu)
35

Screening test for Aristolochic acid PhEur 7.3

Solvent mixture: anhydrous formic acid R, water R, methanol R (1:9:40 V/V/V).

Test solution. To 1.0 g of the powdered herbal drug add 10.0 mL of the solvent mixture, sonicate for 10 min and
centrifuge.
Reference solution (a). Disperse an amount of aristolochia HRS corresponding to 0.10 mg of aristolochic acid I in 20.0 mL
of the solvent mixture, sonicate for 10 min and centrifuge.
Reference solution (b). Dilute 1.0 mL of reference solution (a) to 25.0 mL with methanol R.
Plate: TLC silica gel F254 plate R (2-10 µm) = HPTLC
Mobile phase: anhydrous formic acid R, water R, ethyl acetate R, toluene R (3:3:30:60 V/V/V/V); use the upper layer.
Application: 20 µL as bands of 8 mm.
Development: over a path of 6 cm.
Drying: in a current of cold air for 5 min.
Detection: spray with a 100 g/L solution of stannous chloride R in dilute hydrochloric acid R until the plate is slightly wet,
heat at 100 °C for 1 min and examine in ultraviolet light at 365 nm.
System suitability:
— the chromatogram obtained with reference solution (a) shows 2 greenish-blue zones due to aristolochic acids I and II
between RF = 0.35 and RF = 0.55, which may not be completely separated;
— the chromatogram obtained with reference solution (b) shows at least 1 of these zones (corresponding to 2 ppm of
aristolochic acid I).
Results: in the chromatogram obtained with the test solution no zone is similar in position and fluorescence to any of the
zones due to aristolochic acids in the chromatogram obtained with reference solution (a).
If the chromatogram obtained with the test solution shows any zones similar in position and fluorescence to any of the
zones due to aristolochic acids I and II in the chromatogram obtained with reference solution (a), apply method B.
36

Limit test for Aristolochic acids

1: Ref (a)
2: Ref (b) (2 ppm AA1)
3: Stephania
4: Aucklandia
5: Vladimiria

6: Aristolochia debilis

1 2 3 4 5 6
37

Current efforts in harmonization

 Related drugs should use the same method
 Angelica, Levisticum, Ligusticum,
Notopterygium
 Lamiaceae
 Zingiberaceae
 Standardized methods for substance classes
 Essential oils
 Fatty oils
 Flavonoids
38

Identity of Ginger rhizome (Zingiber officinalis)

white light Toluene, ethyl acetate
(WRT) = 9:1
Deriv.: anisaldehyde
reagent

1 6-Gingerol
2 8-Gingerol
3 10-Gingerol
UV 366nm 4 6-Shogaol
5 Ginger rhizome 1
6 Ginger rhizome 2
7 Ginger rhizome 3
8 Ginger rhizome 4
9 Ginger rhizome 5
10 Ginger rhizome 6
11 Ginger rhizome 7
12 Alpinia officinarum, rhizome
13 Kaempferia galangal, rhizome
14 Alpinia oxyphylla, fruit
15 Alpinia katsumadai, seed
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
 acid
39

Rutin
Rosmarinic
Caffeic acid

Hyperoside
Melissa
Peppermint
Thyme
Marjoram
Sweet Basil
Holy Basil
Holy Basil
Holy Basil
Flavonoids of Laminaceae

Holy Basil
Holy Basil
Holy Basil
Holy Basil
Holy Basil
Quercetin
40

Thyme leaf

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Track Volume Sample Track Volume Sample

1 7 µL Thyme leaf #1 9 10 µL Oregano leaf #1

2 10 µL Thyme leaf #1 10 15 µL Oregano leaf #1

3 15 µL Thyme leaf #1 11 10 µL Oregano leaf #2

4 10 µL Thyme leaf #2 12 10 µL Oregano leaf #3

5 10 µL Wild thyme leaf #1 13 10 µL Oregano leaf #4

6 10 µL Wild thyme leaf #2 14 10 µL Oregano leaf #5

7 2 µL Rutin, Rosmarinic acid 15 Blank

8 7 µL Oregano leaf #1
41

Thyme leaf plates 1 - 3

42

Thyme leaf
43

Practical questions related to TCM

 Obtaining multiple and representative samples

 Setting acceptance criteria

 Defining referenc substances

 Harmonization
44

Example: Zanthoxylum
Two species: Z. schinefolii, Z. bungeanum

Samples on track 11 – 16
represent Z. bungeanum

Samples on track 3 – 5 represent

immature fruits of Z. bungeanum

Sample on track 17 is Z. shinifolii

(see test below)

The nature of the other samples is

still under investigation but they
should be regarded as adulterants.
45

Test for bergapten: Z. shinifolii

According to Chinese literature bergapten is a marker for Z. shinifolii. The
method of the Chinese TLC Atlas addresses this issue for identification, but
it can not discriminate the species as the method proposed above does
46

Ziziphus
47

The use of a reference extract

48

International harmonization?

 Chinese Pharmacopoeia 2015 edition

 Chinese TLC Atlas 2015 edition

 PhEur monogrphs on TCM

 USP will adopt monographs from ChP

49

Thank you!

Your questions please

eike.reich@camag.com

www.camag-laboratory.com