Ellen Phiddian
Cosmos science journalist
Pill-testing services are well-equipped to quickly identify most of the things that come through their doors. So what happens when you come across a truly mystery substance?
“At the site, we target 10 of the most expected drugs – and most of the time, people are coming in saying ‘I’ve got one of these drugs’,” says Professor Mal McLeod, a chemist at the Australian National University who’s been working with CanTEST, Australia’s first fixed-site drug checking service.
“So the story is often actually very simple – ‘you do’, or ‘no you don’t’, and ‘here’s the purity’.”
But when a client brought in a brand new substance, later referred to as CanKet, it took researchers several weeks to figure out what the unknown compound was.
How did they do it? It’s time to take a dive into the black box of analytical chemistry.
Three terms to keep an eye on
Chemists can use dozens of different techniques to identify substances, but the three terms to keep an eye on are spectroscopy, spectrometry and chromatography. These encompass many, but not all, ways to identify molecules.
Spectroscopy can be thought of as shining a light on molecules, and seeing what sorts of shadows they cast. Different types of light will give you different shadows.
Spectrometry can tell you about the mass of atoms – and therefore, which elements you’re working with. It can also tell you a little about how those atoms are connected in a molecule.
Chromatography separates mixtures – among other things, it can tell you a lot about the purity of a substance.
At the pill testing clinic
Pill testing sites have machinery that’s quick to run and can yield plenty of information about known substances: like their purity and the presence of contaminants – and whether the drug is in fact what the client expects it to be.
Read more: How does pill testing work at a concert?
For instance, CanTEST, which has been publishing monthly summaries on the substances it’s tested, was presented with four samples of suspected methamphetamine in its first month. Three were methamphetamine, but the fourth was sugar.
At CanTEST, there are three techniques at play: two types of spectroscopy, and one type of chromatography.
First up, there’s Fourier Transform Infrared Spectroscopy, or FTIR. This device is about the size and shape of a traditional desk bound microscope, and yields information very quickly.
A small amount of the substance is put on a plate. When infrared light is shone through the substance, different bonds in the molecule will absorb slightly different shades.
Eventually, this churns out a unique “fingerprint” for each different molecule – a graph showing where the infrared light is absorbed, and how much.
That fingerprint is compared to a database of 30,000 known substances.
“We use a series of criteria to give us a level of confidence for that – so if the score is high enough, we know what it is, basically, with very little doubt,” says McLeod.
Next, the testers tried ultra-high performance liquid chromatography with photodiode array (UPLC-PDA).
This is two techniques in one: chromatography and spectroscopy.
Chromatography uses a liquid to pump the sample through a very thin tube filled with a resin. Different samples will stick more to the resin, or to the liquid, depending on their properties, and exit the tube at different times.
This separates out the components of the mixture, telling you how pure it is. The amount of time a substance takes to get through the tube can also give you some hints on what it is.
Then there’s more spectroscopy: this time, with UV light instead of infrared light, yielding different information – usually not as rich as infrared, but still handy.
UPLC-PDA takes around five minutes to generate results.
In CanKet’s case, none of these techniques could get the testers a certain answer. The next step was to take it to a better-equipped laboratory at the Australian National University.
At the university lab
McLeod and colleagues next used gas chromatography-mass spectrometry (GCMS) machine.
“It’s about the size of the kitchen dishwasher, it sits up on the bench, with a little injector on the top,” says McLeod.
Like the other techniques, it only needs a tiny amount of whatever you’re testing, dissolved in a liquid.
“We will inject a microlitre of substance – so one millionth of a litre, and that’s enough to get that measurement,” says McLeod.
GCMS is also two techniques in one. The first – chromatography – uses the same theory as the chromatograph at the pill testing clinic, but it uses a gas to pump the mystery chemical through rather than a liquid.
“It vaporises the sample, and passes it down column, which separates the different components so we could isolate our drug,” says McLeod.
Then, the sample hits a mass spectrometer, whose chief job is to find the mass of atoms in the substance – so it can tell you the amount of carbon, oxygen, and other elements in the sample.
“It also smashes up the molecule, so you get a sort of fingerprint for the compound,” says McLeod.
Read more: A quick way to weigh molecules
This analysis was compared to a library for matching samples.
The mystery substance turned out to be very similar to a substance called fluorexetamine, but not identical. In fact, the two compounds are isomers: substances that have the same number and type of atoms, but the atoms are bonded together in different ways.
“We were mindful that there could be isomers involved, so that’s why we went to NMR,” says McLeod.
Nuclear magnetic resonance spectroscopy – NMR – is the best way to conclusively identify a substance.
So why not start with it at CanTEST? It generates very complicated data, takes several hours to get results – and a single NMR machine is on the order of around $1 million. Most universities will have at least one, but they’re too bulky and expensive to install at smaller outfits like pill testers.
“NMR is quite a big machine. It’s got a huge, super-cooled magnet,” says McLeod.
The machines are wide drums, a little taller than people, suspended on legs and filled with liquid helium and liquid nitrogen to keep the magnet cool.
“We probe the molecule in a tiny little tube in the middle of that magnet with radiofrequency radiation,” says McLeod.
This radiation causes specific atoms in the molecule to send out signals. From those signals, chemists can divine how the atoms in the molecule relate to each other.
NMR data is a little like a logic puzzle. It can tell you, for instance, how hydrogen atoms in a molecule are placed near one another, or how hydrogen atoms correlate to carbon atoms in the molecule – and from this, you can eventually work out the shape of the molecule itself.
“Using that approach, we were able to build up the structure for the molecule,” says McLeod.
The result? A substance called 2’-fluoro-2-oxo-phenylcyclohexylethylamine: something like ketamine, but with a few key differences.
So we’ve arrived at CanKet – what now?
The substance, which is currently being referred to as CanKet, had also been spotted by Chinese and Taiwanese law enforcement.
“As far as we’re aware that we’re the first drug checking service to identify this substance, and I believe we’re the first in Australia to see this,” says McLeod.
But now they know what it is, will it be easier for other drug checkers to spot it? Absolutely, according to McLeod.
“We’ve seen this compound five or six times now at the CanTEST pilot, and in each case, we can use the instrumentation on site to identify this without doubt,” he says.
Because the researchers now have all the fingerprints from their previous spectroscopy and chromatography ventures, new presentations of CanKet can be spotted straightaway.
What they don’t know yet is what the drug might do to a person who takes it.
Knowing the chemical structure means they can draw some possible conclusions about its effects, but we’ll have to wait on clinical data before we know for sure.