how chemists found the mystery drug is explained

Pill testing services are well equipped to quickly identify most of the things that come through their doors. So what happens when you encounter a truly mysterious substance?

“On the site, we target 10 of the most anticipated drugs – and most of the time, people come in saying, ‘I’ve got one of these drugs,'” says Professor Mal McLeod, a chemist at the Australian National University. works with CanTEST, Australia’s first permanent drug testing service.

“So the story is often very simple – ‘you do’ or ‘no you don’t’ and ‘here’s the purity.’

But when a customer 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 dive into the black box of analytical chemistry.

Three terms to watch

Chemists can use dozens of different techniques to identify substances, but the three terms to watch are spectroscopy, spectrometry, and chromatography. These include many, but not all, ways of recognizing molecules.

Spectroscopy can be thought of as shining light on molecules and seeing what kind of shadows they cast. Different types of light will give you different shadows.

Spectrometry can tell you the mass of atoms – and therefore, what elements you’re working with. It can also tell you a little about how these atoms bond in a molecule.

Chromatography separates mixtures – among other things, it can tell you a lot about the purity of a substance.

The CanTEST clinic. Credit: Tracey Nearmy/ANU

At the pill testing clinic

Pill testing sites have machines that work quickly and can give a lot of information about known substances: such as their purity and the presence of contaminants – and whether the drug is actually what the customer expects it to be.

Read more: How does pill testing work at a concert?

For example, CanTEST, which publishes monthly summaries of the substances it tests for, came up with four samples of suspected methamphetamine in its first month. Three were methamphetamine, but the fourth was sugar.

In CanTEST, there are three techniques: two types of spectroscopy and one type of chromatography.

Man wearing rubber gloves holds small metal tube over a box-like device sitting on a bench - an infrared spectroscope
ANU PhD student Cassidy Whitefield loads a sample for IR spectroscopy into CanTEST. Credit: Tracey Nearmy/ANU

First, there is Fourier transform infrared spectroscopy, or FTIR. This device is about the size and shape of a traditional tabletop microscope and provides information very quickly.

A small amount of the substance is placed on a plate. When infrared light penetrates the substance, different bonds in the molecule will absorb slightly different hues.

Close-up of a metal plate with a very small amount of white powder funneled into the center
The infrared plate. Testers only need a few milligrams of the substance for each test. Credit: Tracey Nearmy/ANU

Ultimately, this creates a unique “fingerprint” for each different molecule – a graph showing where infrared light is absorbed and how much.

This fingerprint is compared to a database of 30,000 known substances.

Infrared spectrum of ethanol: a graph with a line that peaks and falls across the graph
Ethanol ‘fingerprint’: this is the graph you get if you put alcohol through infrared spectroscopy. Credit: Mfomich – Own work, CC0,

“We use a range of criteria to give us a level of confidence about it – so if the score is high enough, we know what it is, basically, with very little doubt,” says McLeod.

The testers then tested ultra-high performance liquid chromatography with a photodiode array (UPLC-PDA).

It is two techniques in one: chromatography and spectroscopy.

Chromatography uses a liquid to pump the sample through a very thin tube filled with resin. Different samples will stick more to the resin or liquid, depending on their properties, and will come out of the tube at different times.

This separates the components of the mixture, telling you how pure it is. The time it takes for a substance to pass through the tube can also give you some clues as to what it is.

Two computers on a bench with two devices on either side: the IR spec on the left is roughly the size of a laptop, the gas chromatography/uv spec on the right is the size of a large printer
Infrared spectroscopy on the left, liquid chromatography/UV spectroscopy on the right. Credit: Tracey Nearmy/ANU

Then there’s more spectroscopy: this time, with UV instead of infrared light, which produces different information – usually not as rich as infrared, but still handy.

UPLC-PDA takes approximately five minutes to generate results.

In CanKet’s case, none of these techniques could give testers a definitive answer. The next step was to move it to a better equipped laboratory at the Australian National University.

In the university lab

McLeod and colleagues then used a gas chromatography-mass spectrometry (GCMS) machine.

“It’s about the size of a kitchen dishwasher, sitting on the counter, with a little nozzle on top,” says McLeod.

Like the other techniques, it only takes a small amount of whatever you’re testing, dissolved in a liquid.

“We’re going to inject a microliter of substance—so a millionth of a liter, 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 in the pill testing clinic, but uses a gas to pump the mystery chemical through rather than a liquid.

“It vaporizes the sample and passes it through the column, which separates the different components so we can isolate our drug,” says McLeod.

The sample then hits a mass spectrometer, whose main 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 crushes the molecule, so you get a kind of fingerprint for the compound,” says McLeod.

Read more: A quick way to weigh molecules

This assay was compared to a library for sample matching.

The mystery substance turned out to be very similar to a substance called fluororexetamine, but not identical. Actually, the two compounds are isomers: substances that have the same number and type of atoms, but the atoms bond together in different ways.

Large beige tub with a base around it and a ladder that slopes up to the top of the tub
An NMR device at the University of Bergen in Norway. Credit: Adville – Own work, CC BY-SA 4.0,

“We’re mindful that isomers might be involved, so we went to NMR,” says McLeod.

Nuclear Magnetic Resonance Spectroscopy – NMR – is the best way to definitively identify a substance.

So why not start with it in CanTEST? It produces very complex data, takes several hours to get results – and a single NMR machine is on the order of $1 million or so. Most universities will have at least one, but they are too bulky and expensive to install in smaller outfits like pill testing labs.

“The NMR is a very large machine. It has a huge, super-cooled magnet,” says McLeod.

The machines are wide drums, slightly taller than humans, suspended by legs and filled with liquid helium and liquid nitrogen to keep the magnet cool.

“We’re exploring the molecule in a tiny little tube in the middle of this magnet with radio frequency radiation,” says McLeod.

This radiation causes specific atoms in the molecule to send signals. From these signals, chemists can predict how the atoms in the molecule relate to each other.

NMR data is a bit like a logic puzzle. It can tell you, for example, how the hydrogen atoms in a molecule are placed close to each other, or how the hydrogen atoms relate to the carbon atoms in the molecule – and from that, you can eventually work out the shape of the molecule itself.

“Using this approach, we were able to generate 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.

Zippered bag: powder against a 10 cm ruler
The mystery drug. Credit: Mal McLeod / ANU

So we’ve reached CanKet – now what?

The substance, currently referred to as CanKet, had also been identified by Chinese and Taiwanese law enforcement.

“As far as we know we are the first drug enforcement agency to identify this substance and I believe we are the first in Australia to see this,” says McLeod.

But now they know what it is, will it be easier for other drug controllers to spot it? Absolutely, according to McLeod.

“We’ve seen this compound five or six times now in the CanTEST pilot, and in each case, we can use the instruments on the ground to identify it without a doubt,” he says.

Because the researchers now have all the fingerprints from their previous spectroscopy and chromatography ventures, new presentations of CanKet can be identified immediately.

What they don’t know yet is what the drug can 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 for clinical data to know for sure.

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