Vol.

40(3) [May–June 2014]

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Mar. 14, 2014 [Link]

BRIEFING
203 High-Performance Thin-Layer Chromatography Procedure for Identification
of Articles of Botanical Origin. This new general chapter describes a standardized
high-performance thin-layer chromatography (HPTLC) procedure for use in identification
of articles of botanical origin. Careful control of the HPTLC variables is critical to
obtaining reproducible results. These results may be used in the Identification test of
official botanical articles in USP's compendia as a drug substance or drug product, as
an ingredient or a dietary supplement, and as an herbal medicine. The proposed
procedure is used by analysts to determine the identity of botanical ingredients and to
distinguish them from nonofficial but closely related materials. It may also be used to
detect possible adulterants. Necessary control of the procedure is achieved by
maintaining the relevant variables within specified, narrow ranges as provided in
accompanying equipment manuals. An informational general chapter Identification of
Articles of Botanical Origin Using High-Performance Thin-Layer Chromatography
Procedure 1064 proposed elsewhere in this issue of PF describes necessary control
approaches with illustrations. Monographs in USP–NF and the Herbal Medicines
Compendium should reference this general chapter when it becomes official if the
HPTLC technique is employed. Alternate procedures for the Identification test may also
be used subject to suitable review and acceptance.

(DS: N. Sarma.) Correspondence Number—C141899

Add the following:

▪
203 HIGH-PERFORMANCE THIN-
LAYER CHROMATOGRAPHY
PROCEDURE FOR IDENTIFICATION OF
ARTICLES OF BOTANICAL ORIGIN
INTRODUCTION
 This chapter describes a procedure for use in a USP Identification test that relies on
the technique of high-performance thin-layer chromatography (HPTLC). It is applicable
to the identification of articles of botanical origin in USP's compendia that serve as a
drug substance or drug product, or as an ingredient or a dietary supplement. Careful
control of the variables for the HPTLC technique is briefly described with references to
more detailed information provided in equipment manuals. Reproducibility of the results
allows comparison of closely related botanical materials that are not official ingredients.
The analytical technique uses high-performance plates, appropriate equipment to
control variables, and a system suitability test for purposes of performance qualification.
[NOTE—See Identification of Articles of Botanical Origin Using High-Performance Thin-
Layer Chromatography Procedure 1064 for additional information.]

REQUIRED EQUIPMENT
The equipment used for HPTLC technique consists of the following:

• Plates: Unless otherwise specified in the individual monograph, use plates

coated with a uniform 200-µm layer of porous (60-Å pore size) silica gel having
irregular particles of 2–10 µm and an average particle size of 5 µm, a polymeric
binder, and a fluorescence indicator (F254) of 20 × 10 cm. [Note—
Chromatographic methods using high-performance thin-layer chromatographic
glass plates are preferred over aluminum-backed sheets because of greater
mechanical stability.]
• A device suitable for the application of specified volumes of samples as bands
with specified length at the specified positions
• A suitable chromatographic chamber (for example, a twin trough chamber)
allowing for 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
• A device suitable for treatment of the plate with derivatization reagent, if required
• A device suitable for heating as part of the derivatization procedure, if required
• A system suitable for documentation of chromatograms under UV 254 nm, UV
366 nm, and white light

Each of these devices as well as the system as a whole should pass installation
qualification (IQ), operational qualification (OQ), and performance qualification (PQ) in
order to assure that the instruments are working according to their specifications to
control the variables within their intended ranges. [NOTE—See Analytical Instrument
Qualification 1058 for additional information.] Installation qualification and
operational qualification are typically performed by the instrument manufacturer. The
system suitability test is performed by the analyst as proof of performance qualification.

PROCEDURE
Preparation of Test Solution
 Unless otherwise stated in an individual monograph, 100 mg of a powdered botanical
ingredient, 10 mg of a dry extract or fraction, or the amount of a dosage form containing
the equivalent of the aforementioned quantities of the botanical ingredient is sonicated
for 15 min with 1 mL of methanol. After centrifugation the filtrate or supernatant is used
as the Sample solution. Unless otherwise stated in an individual monograph, 50 µL of
an essential oil is dissolved in 1 mL of toluene and used as the Sample solution.
Preparation of the Standard Solutions
Unless otherwise stated in an individual monograph, USP Reference Standards of
individual marker compounds are dissolved at a concentration of 1 mg/mL in methanol.
The USP Reference Standard extracts are shaken and sonicated in methanol at a
concentration of 10 mg/mL or, for essential oils, the USP Reference Materials are
dissolved in toluene at a concentration of 50 µL/mL.
Sample Application and Plate Layout
Samples are applied as narrow bands of 8.0 ± 0.5 mm length at a distance of 8.0 ± 0.5
mm from the lower edge of the plate. The system suitability standards are applied on
the lane nearest to the edge at NLT 20 mm from the edge of the plate. The distance
between tracks (center to center) is NLT 11.0 ± 0.5 mm. The developing distance is
marked with a pencil close to one of the edges of the plate before the development,
although an electronic solvent front detection device may be substituted.
Preconditioning of the Plate
Following sample application and unless otherwise stated in an individual monograph,
the plate is conditioned at a relative humidity of 33% for a minimum of 10 min (for
example, by standing in a closed chamber containing a saturated solution of
magnesium chloride).
Preparation of the Developing Chamber and Development of the Plate
Where a twin trough chamber is used, the rear trough is fitted with filter paper. The
chamber is charged with a sufficient volume of developing solvent to wet the filter paper
completely and achieve a level of developing solvent of exactly 5 mm in both troughs.
With the lid closed, the chamber is left 20 min for saturation. The plate is introduced in a
vertical position into the front trough of the chamber so that the coating layer faces the
filter paper. When the mobile phase has reached a distance corresponding to a
development path of 6 cm, the plate is removed from the chamber and dried in a vertical
position in a stream of cold air. Other chamber configurations and developing distances
may be specified in an individual monograph. [NOTE—Other development chambers may
be employed if the results obtained fulfill all of the system suitability criteria.]
Derivatization Procedure
Where derivatization reagents are used, defined volumes of reagents in solution
(typically 1–2 mL) are homogeneously sprayed onto the plate or the plate is immersed
into the reagent solution at a defined speed and for a defined dwell time. [NOTE—
Immersion speed of 50 mm/s and dwell time of 1 s works for most nonaqueous
reagents.]
Visualization
 Chromatograms on the plate are visualized as stated in an individual monograph.
Observation and evaluation may be performed under UV 254 nm, UV 366 nm, or white
light prior to and after derivatization.
System Suitability
To check the suitability of the system for resolution, position, and color of the bands,
unless otherwise stated in an individual monograph, two or more reference substances
are selected that have similar but just separable RF values under the chromatographic
conditions to be used; for example, chlorogenic acid (blue) and hyperoside (yellow-
orange) in chromatographic systems used for flavonoids. USP Reference Standard
mixtures for System suitability may be provided, or the substances designated to check
the system suitability for resolution, position, and colors of the bands may be included in
the USP Reference Standard extracts. Description of the resolution, position, and colors
for the key bands of the reference material fingerprint should match the description in
the monograph within a specified tolerance range. The System suitability requirements
in an individual monograph are satisfied when the results obtained comply with those
specified in the monograph.
Evaluation and Acceptance Criteria
Chromatograms of the Sample solution and Standard solution are compared against
the description in the Acceptance criteria section of the monograph with respect to zone
position, zone separation, color, and relative intensity.
Documentation
Documentation is necessary to record the results in an auditable manner to comply
with current good manufacturing practices. Proper documentation tools should be
employed; for example, a camera suitable for taking digital pictures under UV and white
light and an imaging software suitable for archiving, retrieving, and analyzing the results
makes it easy to maintain electronic records.

▪1S (USP38)