Fentanyl and fentanyl analogs are so potent that response teams now don full hazmat suits if the drug is suspected at a scene. Photo: DEA

Last year, the leading cause of death for people under the age of 50 wasn’t cancer or heart problems or even motor vehicle accidents. So what is the reason for the death of more than 59,000 in 2016? Drugs.

According to preliminary estimates, more than 59,000 people under the age of 50 died last year due to some involvement with drugs. It could be heroin, cocaine, opioids or a variety of new fentanyls that hit the street seemingly every day. The drug crisis is escalating—as it has been for the past few years—and scientists are having a hard time keeping up.

“It’s a moving target,” said Jennifer Colby, associate director of clinical chemistry at Vanderbilt University. “Novel psychoactive substances (NPS) change so rapidly. It’s hard to predict what will come and when it will come. What we see in one geographic area can be different than what we see in another. In addition to the fact that they’re constantly changing, there is no good way to predict which ones will be important in your population, and which ones won’t.”

NPSs are structurally unique from target drugs, allowing them to evade identification by the Drug Enforcement Agency (DEA), as well as detection in laboratories. For example, fentanyl is one of the most popular drugs available illegally on the street currently. But as its popularity grows, so too do the detection capabilities of clinical and forensic laboratories. But by tweaking just a couple compounds, fentanyl can turn into carfentanil, acrylfentanyl and other analogs that can cause an individual to overdose just by touch. Of course, these analogs are structurally different enough from target fentanyl to evade detection.

While working in San Francisco, Colby had success using broad-spectrum drug screening via high-resolution mass spectrometry to quickly identify the compounds intoxicated patients had ingested. The sample preparation is easy—for urine, it is a dilute and shoot. In the case of an emergency, a blood sample undergoes a protein dump and dry down, re-suspend and inject. Altogether, it is about 15 minutes to injection time—10 minutes for the actual liquid chromatography, and 5 minutes for re-equilibration.

Colby and her group always start with a known target list of about 200 drugs, but they also have a “suspect list.” One of the advantages of using a high-res instrument is chemists only need to match the predicted math with whatever is measured in the sample. Researchers can then use isotope pattern matching or a spectrum in a library or case report to make a highly educated guess.

Colby contends it is not the strength of the method that defines it (there are about 10 other labs in the country doing the same thing), it’s the collaboration behind the analytical technique that really drives results.

“The first people to find a new drug in an area are the ones who work for the municipality or county, the actual drug chemists—the ones analyzing seized materials,” Colby explained to Forensic Magazine. “If it’s a new drug, you don’t necessarily know what the health events will be, so poison control will be the next to hear about the reported health events. So now we have a new drug, and we know some information about it. What we need to do it communicate that information to the lab so they can work on developing a test for it. But the communication doesn’t always happen smoothly when people are not in the same area or when we’re thinking about it differently. The drug chemists don’t often communicate with the ER personnel. We have all the pieces, but there’s not a bridge built between them.”

On the hundreds of cases Colby worked, she collaborated closely with poison control—creating a team featuring an analytical chemist and a physician with emergency medicine experience that could correlate and disseminate the clinical information behind a drug or metabolite.

Although Colby has since moved on, the method and poison control collaboration continues in San Francisco for Ph.D.s training to be lab directors and clinical chemists, as well as medical toxicologists. Currently, the group is looking into how the platform could be used in routine practice, and if the data analysis and conceptual interpretation could be done by someone without a Ph.D. The ultimate goal is to get the instrumentation and method to a point where anyone in the ER can run a sample and receive a result in minutes.

“Overall, it’s really important for all of us, clinical [chemists], poison control [personnel], drug chemists and forensic toxicologists to work together to track the drugs so we know where they are and when, and what they are doing to people,” Colby said. “That’s the only way to prevent the spread of this. The collaborative nature is going to be really critical in addressing this.”

Chemist Nicholas Manicke has developed a low-cost cartridge that can help emergency room physicians identify and treat overdoses. Photo: IUPUI

An MS-based cartridge
While the San Francisco-based group is trying to work out an MS method for ER personnel, a chemist at Indiana University-Purdue University Indianapolis (IUPUI) has developed a prototype that may be well on its way to solving the problem.

Assistant Professor of Chemistry Nicholas Manicke has developed a rapid, sensitive, low-cost, MS-based screening method to help emergency room physicians treat overdoses.

“Some form of mass spectrometry-based testing is going to be needed for detection of NPSs,” Manicke told Forensic Magazine. “The disadvantage to typical approaches is the fairly complicated workflows. What we’re trying to do is simplify that process. It’s still based upon mass spectrometry. It’s still taking advantage of the power of that instrument. But we’re simplifying the sample prep process.”

Manicke’s cartridge device can interface with any off-the-shelf mass spectrometer. The general idea is to have the sampling cartridge perform all the sample prep, extraction and ionization, before transferring the sample into the mass spectrometer for analysis. Manicke was able to remove the chromatography step, as well as the method that couples LC to MS—resulting in reduced sample prep and handling, less bench work and a short analysis time.

“For this project, we added biofluid on the cartridge, and built into it a pre-concentration step that, from a couple hundred microliters of plasma, can concentrate and pull out the drug we’re trying to detect, and then directly analyze that from the cartridge,” Manicke explained. “The cartridge is small, just a few centimeters in length, and contains everything that would typically be done at the bench.”

Manicke works with multiple toxicology labs to get a sense of the newest compounds hitting the street, and builds a target list off that information. Already, the list has about 40 fentanyl analogs and synthetic cannabinoids. But the device also boasts a non-targeted capability where researchers can examine data retrospectively to see if there are any signatures they didn’t know they should be looking for the first time.

It’s a judgement call between target and non-targeted analysis, Manicke said, but he feels this technology is in the middle—right where it’s supposed to be.

“We think we have the sensitivity that is good enough to detect relevant concentrations of these compounds, but still have enough of a non-targeted side of the method to go back and look for things we didn’t know to look for previously,” he said.

Without sample purification, analysis of a blood sample using the device is about 30 seconds. And the materials in the cartridge are low-cost—less than 10 cents altogether, in fact. As with most R&D discoveries, scale-up is a problem. The technology has to get to a point where enough cartridges can be made for the manufacturing process to churn them out inexpensively.

This summer, Manicke began collaborating with Ezkenazi Health and IU Health Methodist Hospital to test his MS-based cartridge device. As of August, the team had just started to analyze the samples taken at the hospital in Manicke’s lab at IUPUI. For the next phase of the project, Manicke would like to move the entire process into the ER, enabling rapid lab-scale analysis at the point of care.

While the immediate goal of the project is to broaden the reach of the cartridge to more state-wide monitoring programs and emergency rooms, the intended long-term goal could revolutionize ER care.

“Long term, we want to equip these ERs with the instrumentation they need to do the entire analysis start to finish,” Manicke said. “There’s no question MS will go into the clinical space—it’s more a question of when and what will be the killer application. It’s sometimes said mass spectrometers are too expensive, too big, or too complicated to be used in the clinic, but every hospital runs big, expensive, complicated equipment, including an MRI, which is much bigger, more expensive and more complicated than an MS. But hospitals use it because they have absolutely have to. That is going to become the point for MS as well. There are going to be more and more applications for which MS is demanded in the clinic.

“As applications continue to be developed in the academic community, I think that is going to start making the case for using MS in the clinic. These technical hurdles that have prevented the use of MS in the past will be resolved.”

Researchers have identified 16 genetic mutations in the brain reward pathways that could help identify patients at high risk for addiction.

Time-of-flight mass spectrometry
Mass spectrometry may be waiting for its turn in the clinical lab, but it’s well-established in the forensic laboratory—one of the places hit hardest by the growing opioid crisis.

In a forensic setting, for legal reasons, analysts need to be able to confirm a sample’s identity against a certified reference material (CRM) or traceable analytical standard. However, that can be difficult when new analogs and NPSs sprout up overnight. The period of time between when an analyst sees a new drug and when a CRM is available for verification is, essentially, dead time.

Sabra Botch-Jones, a forensic toxicologist at Boston University, turned to Direct Sample Analysis (DSA) Time-of-Flight MS (TOF/MS) to help solve this problem.

In as little as 15 seconds, DSA TOF/MS can identify NPS compounds on blotter paper. The instrumentation can analyze a sample of a drug once extracted, or it can do analysis on a sample with absolutely no preparation. The sample is loaded onto a mesh screen, and the mesh screen is introduced into the ion source of the MS. The sample is then ionized—and because it’s a TOF/MS and is highly sensitive—it can measure the molecules of a drug sample out to four decimal places.

“That is considered accurate mass spectrometry,” Botch-Jones told Forensic Magazine. “What it allows is for analysts to get a more precise measurement of the molecules and the molecules’ weight. That can be really important when you’re looking at drugs that have similar structures and looking to differentiate those drugs from one another.”

Unlike other MS techniques, TOF/MS runs off non-targeted analysis—which is different than using traditional commercial mass spec libraries. In this case, researchers populate the library themselves based on analyzed samples and analytical standards. What’s unique
about that is researchers can retrospectively pull data to confirm a match.

For example, a researcher may come across an as-yet-unidentified fentanyl sample one day. A couple months later, however, a new analog is making headlines and there is now a CRM to confirm identification. Using this DSA TOF/MS method, a researcher can reevaluate the original data using the new information to see if it can be matched—all without actually reanalyzing the sample itself.

Botch-Jones said in one specific case she applied the technique to actual forensic evidence, and was able to detect one of the new fentanyl analogs in a case sample that, when run previously on a GCMS, could not detect the compound.

“I think that shows how powerful the tool is,” she said.

Because of its rapid analysis time Botch-Jones said in the near future, she would like to see how the method can be applied to biological fluids for drug detection.

Genetic testing
A team of researchers led by Sherman Chang, vice president of R&D at AutoGenomics, Inc., is taking a different approach to the growing opioid crisis—stop prescription drug abusers before they can start.

After an extensive three years of literature review and two clinical trials, Chang believes he has identified 16 genetic mutations in the brain reward pathways that could help identify patients at a high risk for addiction.

“Many of the opioid drugs and pain management drugs are targeted to these brain reward pathways,” Fareed Kureshy, president and CEO of AutoGenomics, told Forensic Magazine. “So, if there is any alteration in these pathways, then the performance of these drugs are either subdued or enhanced, based on how the pathways are genetically altered in a specific person. We completed several clinical trials to determine what these genetic pathways are going to be—both mathematically and statistically—to see if they could be used to predict tendency of addiction.”

The AutoGenomics team used the Addiction Risk Assessment Panel to test 70 patients diagnosed with prescription opioid and/or heroin addiction and 68 non-affected individuals as the control.

After the genotyping results came in, the researchers designed a risk model that computes a score from one to 100, with any score over 52 representing an elevated risk of addiction. Of the 70 people with addiction that were tested, 53 had an addiction risk score greater than 52, while 49 of the 68 healthy controls scored under 52. Both the positive and negative predictive values of the model were determined to be 74 percent.

A second independent clinical study was conducted by a team of researchers at Prescient Medicine, led by CEO Keri Donaldson, M.D.
The company compared the frequency of the identified mutations in 37 patients with prescription opioid or heroin addiction versus 30 age- and gender-matched individuals with no history of addiction. The researchers also tested 138 additional patient samples with the genetic panel and algorithm. From this, they found that the test—called the NeuR Assay—was highly accurate, with a sensitivity of 97 percent and a specificity of 87 percent.

“The genetic part of the test is really personalized medicine,” Chang said. “There is no such thing as one drug fits all. Some drugs work well for certain people, but not others. It’s a genetic thing.”

The actual laboratory-developed test is the genetic component of the panel, but there’s even more. AutoGenomics produces a report that details how a person metabolizes a specific drug, as well as if a patient has taken the right dosage and even the correct drug.

For example, the popular pain-killer Vicodin needs to be metabolized by the gene 2D6. But if there is a defect on 2D6, a patient will not be able to metabolize the drug, rendering it completely ineffective. AutoGenomics’ test alerts physicians to those kinds of genetic limitations.

Physicians can use that information, along with a patient’s addiction risk level (low, medium or high), to make more informed decisions, such as prescribing a 4- or 5-day drug regimen, as opposed to a 30-, 60- or 90- day one. There are essentially no alternatives to opioids for pain management, but there are 56 different medications associated with hydrocodone, morphine and opioids. Knowing how a patient genetically metabolizes certain drugs helps physicians select the “right” drug.

“We need to keep educating the user group and the public so that tools like these become effective in pain management,” Kureshy said.
“AutoGenomics is doing many more clinical trials to keep improving the algorithm and keep improving the utility of this product. We are reaching out to addiction centers, pain management centers and chronic pain clinics.”