As more and more Wisconsinites die of opioid overdoses, public and private toxicology labs are ground zero for understanding an ever-evolving mix of illicit drugs. Most of the county medical examiners and coroners around the state don't have their own labs, so they send blood, urine or vitreous fluid samples from suspected overdose victims to the Wisconsin State Laboratory of Hygiene, the Wisconsin State Crime Lab and any number of private labs around the state and nation.

Toxicologists at these labs are worried about tracking the impact of emerging drugs as more chemically novel opioids, often analogs of fentanyl, are manufactured and enter the market. "Staying ahead would be ideal, but I think we're usually on the back end of it," said Jennifer Greene, toxicology technical unit leader for the state crime lab.

In the standard approach to toxicology testing, pathologists know what they're looking for, chemically speaking, and if a drug has a history of legitimate medical use (like fentanyl and various prescription painkillers), they also know what concentrations of it are toxic. But compounds including furanyl fentanyl, acetyl fentanyl and U-47700 tend to lack one or both of those reference points. Some of these drugs are illicitly manufactured variations on the chemical structure of fentanyl or other more familiar opioids. Even if they've been legally formulated in the past, like U-47700, they've never been tested on or prescribed to people, so the medical community doesn't fully understand how their effects might differ from opioids that have been around longer and are used much more widely, like heroin or oxycodone.

To further complicate things, these compounds are often present in small amounts in a victim's system, alongside other and more easily detected substances — overdose deaths often involve more than one drug. But many of these novel chemicals are lethal in very small amounts.

One approach that would help scientists track down chemicals within a certain category, even when they don't know exactly what they're looking for, is to use more sensitive lab equipment that conduct more advanced chemical analysis. One kind of technology that can identify rare chemicals in drugs is based on time-of-flight mass spectrometry. The specific machine is called a quadrupole time-of-flight mass spectrometer, or Q-ToF.

Amy Miles, forensic toxicology director at the state hygiene lab, said this machine costs about half a million dollars. She's been trying to get funding for a Q-ToF for the past three years, and says purchasing one would be cheaper in the long run than outsourcing more overdose toxicology testing to private labs, especially if overdose deaths continue to increase. The instrumentation isn't necessarily hard to find in Wisconsin, but the state hygiene lab can't really get much use out of the Q-ToF technology that exists in University of Wisconsin System labs or elsewhere in the state.

"The instrumentation would be used almost daily, so it would not be efficient to share the instrument with the [UW-Madison] campus — that is why we would need to pursue a Q-ToF on our own," Miles said. "Private labs do have this capability, along with some publicly funded forensic laboratories, across the country. This instrumentation is being purchased by most laboratories as it is the way our field is moving. However, we have not been successful in garnering funding at our lab."

It might not be possible to anticipate every single novel compound an illegal opioid manufacturer synthesizes. But if toxicologists know to keep an eye out for things with a "fentanyl backbone" as Miles calls it, Q-ToF instrumentation could help them cast a wider net and catch up a bit faster with new variations.

"The advantage of a Q-ToF is if you break apart a particular molecule, it might have a building block that's similar throughout a class of compounds," explained Greg Sabat, a proteomics specialist at the UW-Madison Biotechnology Center's Mass Spectrometry and Proteomics Facility. "You can maybe extrapolate or propose that they have arisen from the same starting molecule."

The facility's director, Greg Barrett-Wilt, elaborated that Q-ToF's ability to accurately measure molecular weight helps it catch chemicals more conventional toxicology testing instrumentation may not.

"When you have novel compounds that may have additional chemical properties that change slightly the structure or the molecular weight or some other aspect of the compound, then a lot of the pre-made or pre-established techniques will fail because of those exact things," he said. "It changes something about the chemistry of this molecule that renders it invisible."

Searching for traces of unknown drugs

The state crime lab — which has several locations around Wisconsin — is already working on tackling the problem of unknown opioids from another angle, and with a different kind of instrumentation. At its Milwaukee facility, the lab recently acquired liquid chromatography-mass spectrometry instrumentation, and is currently developing methods for its use, said toxicology technical unit director Jennifer Greene said.

"That instrumentation is going to give us more power to be able to detect some of the newer drugs and at lower concentrations," she said.

The State Crime Lab's facility in Milwaukee is preparing to use new liquid chromatography-mass spectrometry instrumentation to detect novel opioid compounds.

Wisconsin Department of Justice

So far the state crime lab has relied on gas chromatography-mass spectrometry, a technology very commonly used in toxicology testing. A gas chromatograph heats up liquid-based samples to help separate out molecules for analysis. The disadvantage with this approach is that some compounds aren't very stable when heated and can break down easily, which is especially frustrating if a substance is present only in small amounts and tough to identify in the first place. Liquid chromatography-mass spectrometry instruments, on the other hand, use a variety of approaches to separate out molecules in a sample.

Wisconsin Department of Justice

The Wisconsin State Crime Lab has used gas chromatography-mass spectrometry instrumentation in its toxicology testing, but this tool isn't as useful in identifying novel opioids.

Greene said this approach is less likely to obscure unstable traces of a novel compound.

"Especially with some of the newer drugs that we don't know much about pharmacologically, it may not take much for them to be toxic or lethal," she explained. "If someone has what appears to be a relatively normal amount of morphine, but there's a fentanyl analog in a very small amount and it's a very potent drug, that would be helpful to know."

Greene also believes that Q-ToF technology would be a helpful addition to the state's toxicology resources. She points out that liquid chromatography-mass spectrometry instrumentation is good for confirming the presence and quantity of a novel compound, while Q-ToF allows a lab to look for many things at the same time. That means having one doesn't eliminate the need for the other.

"Those two methods will still complement each other," Greene said.