Georgia Tech researchers have found a way to identify fake antimalarial medications using Desorption ElectroSpray Ionisation. John Toon* reports on how the technique is helping to close down the illegal operations of counterfeiters
Researchers from the Georgia Institute of Technology were recently part of a three-continent, multi-organisational effort known as "Operation Jupiter" that successfully identified and shut down manufacturers who were flooding Southeast Asia with counterfeit - and ineffective - antimalarial drugs.
With 11 different organisations, including the Centers for Disease Control and Prevention (CDC), the World Health Organisation (WHO), the Wellcome Trust - and ultimately the international law enforcement agency INTERPOL - the global effort provided Chinese officials with enough information to shut down the drug makers.
Beyond the human health cost of failing to treat hundreds of thousands of malaria cases effectively, the fake drugs could be fuelling development of malarial strains that may become resistant to the most sophisticated drug now available to treat the disease: artesunate. Counterfeiters sometimes include small quantities of the real drug in their fakes, possibly in an effort to fool simple quality tests. By not killing the malaria parasites, this small amount of active ingredient could facilitate development of drug resistance.
As their part of the investigation, Georgia Tech researchers used sensitive mass spectrometry techniques to analyse nearly 400 drug samples provided by public health authorities. They also developed methods to speed up analysis, including a desorption electrospray ionisation (DESI) mass spectrometry process that reduced the time required to test a drug sample from half an hour to just a few seconds.
Georgia Tech's efforts to develop faster analytical techniques were sponsored by the US National Science Foundation, while the sample analysis was supported by a small grant from WPRO/WHO.
Malaria kills more than a million people each year worldwide, and is a risk for about 40% of the world's population. Most victims would survive - if they had access to the proper drugs.
"About 50% of the samples obtained from the field in Southeast Asia were fakes," said Facundo Fernandez, an analytical chemist and assistant professor at Georgia Tech's School of Chemistry and Biochemistry. "They look very real, even down to the hologram in the packaging. It's very difficult to tell which ones are the fakes and which ones are real."
When Fernandez began analysis of the drug samples, he assumed that they would not include any real active ingredients. But his graduate students, Christina Hampton and Leonard Nyadong, soon discovered that the counterfeiters were making their fake antimalarials with a broad range of mostly expired pharmaceuticals.
"We found old and ineffective antimalarials like chloroquine," he said. "We found antibiotics like erythromycin. We found all sorts of drugs that basically have no effect on resistant malaria parasites. Acetaminophen was one of the most common chemicals we found." Fernandez speculates that the makers chose certain compounds, like acetaminophen, because they could temporarily make patients feel better by lowering the fever associated with malaria.
Mass spectrometry provides a very effective means of identifying samples by determining their accurate mole-cular weight. But conventional analysis can be time-consuming - especially in the preparation of samples.
Fernandez and his Georgia Tech group developed a faster method that allows them to analyse hundreds of samples in a single day. Their goal was to make mass spectrometry analyses responsive within the time constraints that surveys in developing countries and law enforcement agencies involved in anticounterfeiting tasks require.
"These are methods that let you analyse a solid sample without any significant preparation," he explained. "You can take a tablet, put it in front of the instrument with an ionisation source, and you get a quick snapshot of what's in the sample. It provides a very high throughput pipeline to identify samples quickly."
Ultimately, Fernandez hopes to help develop high-accuracy instrumental tests that could be used in the field to save the time and expense of shipping suspected fakes to labs (see box below).
Beyond the mass spectrometry, the effort also relied on analysis of pollen found in the drugs - a discipline known as forensic palynology - which was carried out by scientists in New Zealand who were part of Operation Jupiter. A study of calcium carbonate isotopes in the compounds, together with the pollen and active ingredients in the samples, pointed to two main groups of samples originating in different geographic regions of Asia.
"This is absolutely CSI - the techniques they use on the television programme really do work in real life," said Fernandez.
The team provided enough information for the Chinese authorities to be able to shut down the manufacturers, whose sophisticated operations were able to mimic accurately the packaging and holographic seals of legitimate pharma companies.
Fernandez and his students remain involved in anticounterfeiting activities and hope to obtain additional funding to continue supporting the efforts. They are now investigating fake antimalarials sold in Africa, analysing assortments of drugs sold in markets there, and studying other faked drugs, such as Tamiflu.
Fernandez got involved in the project in 2003 because of a chance encounter with Michael Green, a parasitic disease specialist at the CDC. He soon began working with Green and with Paul Newton, a physician from Oxford University, UK, who is based in Laos.
Large pharmaceutical companies can afford to pursue counterfeiting themselves, Fernandez noted, but in many cases, drugs sold for use in developing nations come from small companies that cannot afford private investigators and law firms to go after the counterfeiters.
"The problem is not over," he warned. "There are more fakes and more fake producers. But at least this is a beginning. Having an opportunity to do some good in this area is very satisfying."
How DESI techniques increase sample throughput
The quality of pharma formulations is usually assessed using high performance liquid chromatography (HPLC), Nuclear magnetic resonance (NMR) spectroscopy, and other techniques. The sample preparation steps usually include crushing the sample, dissolving it in an appropriate solvent, extraction, filtration, and centrifugation. These steps make these methods quite time consuming.
Recently, several high throughput methods for the qualitative screening of pharma preparations based on desorption electrospray ionisation (DESI) mass
spectrometry have been reported in the literature. The benefit of these methods is that they do not require sample preparation.
DESI makes use of a high-speed charged liquid spray directed at a sample held or deposited on a surface at atmospheric pressure. The DESI spray can be doped with selective reagents, to effect ion/molecule reactions at the interface between the charged microdroplets, and the solid surface bearing the condensed phase analyte with an enhancement in sensitivity.
Desorption electrospray ionisation mass spectrometry (DESI MS) is rapidly becoming accepted as a powerful surface characterisation tool for a wide variety of samples in the open air. Besides its well-established high throughput capabilities, a feature of DESI is that chemical reactions between the charged spray microdroplets and surface molecules can be exploited to enhance ionisation.
Researchers at Georgia Tech's Bioanalytical Mass Spectrometry department have developed a rapid screening assay for artesunate antimalarials based on reactive DESI.
The approach is based on the formation of stable non-covalent complexes between linear alkylamines dissolved in the DESI spray solution and artesunate molecules exposed on the tablet surface. The researchers found that, depending on amine type and concentration, a sensitivity gain can be obtained, in comparison with reagent-less DESI.
Once a reactive DESI assay was fully characterised, it was applied to a set of recently collected suspicious artesunate tablets purchased in shops and pharmacies in SE Asia. Not only did they find that these samples were counterfeits, but also detected the presence of several wrong active ingredients. Of particular concern was the positive
detection of artesunate traces in the surface of one of the samples, which was quantified with standard chromatographic techniques.