Methods Based on Thin Layer
Chromatography (TLC) Usage to Identity Counterfeit
Drugs
Dr. Gary Witman
Dr. Mark Moskovitz
Identification of counterfeit
drugs has become a serious international issue. By
definition counterfeit drugs are mislabeled
medicines with substandard safety, quality and
effectiveness. Counterfeit products include drugs
with the correct or wrong ingredients, without
active ingredients, with insufficient active
ingredients or fake packaging.
The counterfeiting of
pharmaceuticals has only been detected for the past
20 years. The poor quality of drugs has been linked
to counterfeiting of medicines, chemical instability
(especially in tropical climates) and poor quality
control during manufacture. Pharmaceutical compounds
have specific biological properties and mechanisms
of action and individuals are dependent upon
consistent and reproducible drug performance. In
most countries the quality of pharmaceutical
products is assured through inspection systems by
governmental regulatory agencies and by the
utilization of current good manufacturing (cGMP)
practices. Counterfeit drugs may be placebo in
quality, or worse, containing materials that do
harm.
The World Health Organization
estimates that 25% of medicines in developing
countries are counterfeit, with a figure as high as
50% in some places. During 2006 the European
Commission reported that customs agents intercepted
2.7 million counterfeit drugs at EU borders – an
increase of 384% from a year earlier. In just two
months in 2008 European Union officials seized more
than 34 million fake prescription pills. The problem
with fake and suboptimal medications has been much
worse with the advent of the World Wide Web, as a
significant proportion of medications sold through
Internet pharmacies are counterfeit. The most common
counterfeit drug agents are those that are
prescribed for the treatment of malaria,
tuberculosis, HIV, and other opportunistic
infections. Furthermore, it has been stated that the
greatest health risk in the world today is the lack
of appropriate medicines for treating both common
and life threatening illnesses. It is known that
some counterfeit drugs have caused kidney failure in
patients. The growing challenge is halting the
distribution and marketing of such medicines at
every level of the distribution chain.
To understand the magnitude of the problem, the US
Center for Medicine in the Public Interest has
predicted that by this year, the global sales of
counterfeit drugs will reach $75 billion USD, which
is a 95% increase in just the past 5 years. At least
20% of the Indian drug supply is fake. Concurrently
the World Health Organization believes that India is
responsible for at least 35% of the world’s
counterfeit medications. A recent study in the
British medical journal, The Lancet, reported that
around 40% of the drug artesunate, used for the
treatment of malaria, was a completely inert placebo
in pills being sold in Thailand and Nigeria. In
Southeast Asia and Africa the preferred counterfeit
antimicrobials appear to be older antibiotics – such
as penicillin, tetracycline,
trimethoprim-sulfamethoxazole, quinolones,
chloramphenicol and anti-malarials such as
chloroquine, mefloquine quinine and artesunate, that
are all globally no longer patent protected.
Almost everything connected with
drug manufacturing is being counterfeited – the
active ingredients, dosage forms, package inserts,
packaging, manufacturer’s names, batch numbers,
expiration dates and documentation relating to
quality control. Fake pharmaceuticals often have the
same appearance as the brand name and generic drugs
they mimic. They are often indistinguishable in
packaging, pill color shape and markings. Indeed,
they have electronic bar codes, that pharmacists
scan to verify drug authenticity.
Main Types of Counterfeit Drugs
| Categories of
Drugs |
% of Total
Counterfeits |
Antibiotics
Hormones and Steroids
Anti-asthma and anti-allergy
Anti-malarial
Analgesics and anti-pyretics
Others (14 therapeutic classes)
|
28
18
8
7
6
33 |
The Chinese are scared of the
safety of their domestic drug supply, and with good
reason. As many as 5 million Chinese work in the
drug counterfeit market, that includes fake drugs,
patent infringement and trademark infringement. In
January, 2009 two people died and nine were
hospitalized using counterfeit diabetes drugs in
Xinjiang province. Fake products are often
deliberately engineered to pass basic quality tests,
using a substitute ingredient that mimics the
chemical activity of the authentic ingredient
closely. In 2008 a chemical added to heparin as a
blood thinning agent was responsible for at least 95
deaths in China, Europe and the United States. In
March, 2009 the United Kingdom seized nearly
$750,000 of counterfeit pharmaceuticals which
originated in China.
To counter this enormous
commitment to illegal drugs, the Chinese government
has spent nearly $ 1 billion through their State
Food and Drug Administration to monitor illegal drug
production and has shut down more than 1,000 illegal
drug facilities. Despite these efforts, China’s
intellectual property laws are not yet consistent or
compliant with the World Trade Organization’s
international Intellectual Property agreement. China
still allows drugs that infringe trademarks and may
be substandard to be resold (after repackaging)
rather than being destroyed. China implemented a
Fast Chemical Identification System in 2005, which
is usually targeted at the molecular structure and
functional groups in the drug molecule. This is
comprised of two color reactions based on functional
groups in the molecules and two TLC methods for
screening of fake macrolide antibiotics.
India has taken a step forward
by agreeing to partner with the Food and Drug
Administration (US) to allow more foreign
inspectors. And the FDA has now placed eight full
time permanent positions in China.
Thin layer chromatography is the
main screening method being used today in order to
determine if a drug product meets label
specifications and is legal. TLC can be employed for
the identification of drug substances, to estimate
drug substance content and to detect related
substances that may be regarded as impurities. This
technique allows the active ingredient to be
recognized by comparison with a known drug standard.
The approach using TLC chromatography is cheap,
specific and sensitive. The TLC procedures are
therefore preferred, as they are capable of giving
semi-quantitative information on the active
ingredient and also on any related substances in the
dosage forms.
The Center for Disease Control (CDC, Atlanta,
GA) recommends testing for suspicious drugs,
especially for travelers.
According to the CDC, “Drug
quality can be evaluated in the field by two simple,
effective and low cost techniques: thin layer
chromatography and colorimetry…the TLC technique
consists of placing a spot of drug sample on a thin
layer of silica attached to a plate of glass,
aluminum or plastic. The plate is then inserted into
a vessel containing a solvent mixture. By capillary
action, the solvent creeps up the adsorbent
material, silica, alumina, or cellulose, and
dissolves the sample. The drug sample consists of a
mixture of drug and inactive ingredients. These
compounds will have various affinities to the
adsorbent matrix and will migrate with the solvent
at various speeds. This characteristic effectively
separates out a mixture of compounds. After
migration of the solvent is complete, individual
components can be visualized by chemical treatment
or ultraviolet (UV) adsorbance. The distance that
the components migrate is characteristic for each
compound; therefore the active ingredient can be
recognized by comparison with a known drug standard.
The solvent can be modified to increase resolution
between various components. This method is
relatively inexpensive, specific and sensitive. It
is commonly used to assess drug quality.”
Over 300 German Pharma Health
Fund e.V. Mini-labs (GPHF-Minilab) are being used in
70 countries to help identify counterfeit and
substandard drugs. These minilabs, which contain
simple materials (as described below) and that
utilize standard TLC technology are able to analyze
some 40 commonly adulterated counterfeit drug
compounds. One thing unique about the GPHF minilab
is that iodine-KI solution is replaced by iodine
vapor sublimed from crystals. The Minilab supplies a
collection of authentic secondary standard tablets
and capsules in sealed plastic tubes. The Minilab
analysis identifies the active ingredient by
comparison of distance of travel (RF value) between
the sample spot and an authentic standard spotted on
the same plate, and semi-quantitative proof of
content is made visually comparing the color, size,
and intensity between the sample spot and reference
spots for each method of detection.
In response to effective
anti-counterfeit measures, counterfeiters have often
introduced small quantities of the genuine
pharmaceutically active substances into the dosage
forms. TLC procedures are able to provide
semi-quantitative information on the active
ingredient and also on the related substances,
providing valuable information on the drug being
tested. When the counterfeit compound is extremely
sophisticated in design, it may require more
advanced analytical tools such as elemental
analyzer-isotope mass spectrometry (EA-IRMA), LC/MS,
Raman spectrometry, X-ray powder diffraction method,
near infrared spectrophotometry (NIR) and nuclear
magnetic resonance to help more carefully define the
drug contents. NIR has become portable, but unlike
TLC, this technique is not quantitative. However,
the use of these more sophisticated technologies is
beyond the scope of this paper.
Separations to determine the
activity of drug agents can be performed on for
example silica gel layers containing fluorescent
indicator, and separated spots are detected under
ultraviolet lamps or with iodine detection reagent.
This technology can be performed out in the field by
inspectors with a limited scientific expertise.
Development and iodine detection are carried out
either in glass jars or in polyethylene bags.
Tablets are ground into a fine powder and the proper
volume of solvent is added.
In most instances TLC
identification is carried out on multi channeled
soft layer silica gel GF254 plates or hard layer
silica gel F254 plates (20 cm x 20 cm). Mobile
phases may consist of a multitude of solvent systems
depending upon the class of drugs such as hexane,
ethyl acetate, toluene, alcohols, ammonia, and
others. TLC plates are allowed to air dry. If a more
elaborate TLC separation is required the plate can
be run in a two dimension solvent system.
The presence of a fluorescent
indicator is necessary for the detection of drugs
that quench fluorescence under 254 nm UV light. Blue
fluorescent quenched spots may be viewed on a bright
green background under 254 nm ultraviolet (UV) light
and of brown spots in white light after dipping
plates in iodine-KI solution or exposing the plates
to an iodine vapor mist. The staining of a Thin
Layer Plate with iodine vapor is among the oldest
methods for the visualization of organic compounds.
It is based upon the observation that a iodine vapor
has a high affinity for both unsaturated and
aromatic compounds.
Dynamic Adsorbents provides the
widest range of TLC plates in the industry and can
assist you with your use of this technology.
Back to Technical Info
