Detecting Fentanyl and Its Analogs

Opioid-related deaths have dramatically increased in recent years, prompting officials to declare an opioid crisis. In the first three months of 2018, approximately 1,000 opioid-related deaths occurred in Canada, with 94 percent considered accidental.

In 2017, there were more than 72,000 drug overdose deaths in the United States, a dramatic increase from previous years. Why the sudden, significant increase? Fentanyl. Of these accidental overdose deaths in Canada, 73 percent involved fentanyl or its analogs. Of the 72,000 in the United States, nearly 30,000 were attributable to fentanyl. Fentanyl is a powerful synthetic opioid that is 50- to 100-fold more potent than morphine. It has become popular among recreational drug users because it is relatively inexpensive and easily accessible. Unfortunately, many unsuspecting users consume fentanyl mixed into heroin or cocaine and suffer the lethal consequences. Opioids exert their effects through opioid μ-receptors, located primarily in the spinal cord and brain. Following receptor binding, fentanyl depresses the central nervous system (CNS) and respiration, potentially resulting in death. In cases of overdose, naloxone hydrochloride (Narcan) administration can alleviate CNS and respiratory depression through its antagonistic activity, allowing more time to seek medical assistance. Greater awareness of the dangers of fentanyl has led to increased availability of emergency naloxone kits, a small effort to address the growing problem.

The Problem with Potency

Bradley Urquhart, PhD, is an associate professor in the Department of Physiology and Pharmacology at Western University. As a trained pharmacologist, his research is focused on why patients respond to medications differently. His laboratory uses mass spectrometry to measure drug and metabolite concentrations and perform advanced metabolomics analysis to identify novel metabolites that predict patient response. According to Dr. Urquhart, fentanyl is “one of the drugs at the center of the [opioid] problem.” While fentanyl is very useful for the treatment of pain, it is “much more potent than some other opioids on the market.” Its potency makes it beneficial for pain management but can be very dangerous for unsuspecting street drug users. “When street drug users think they are buying a drug like heroin, it often also contains fentanyl,” Urquhart explains.

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Bhushan Kapur, DPhil, CChem, FRSC, FACB, FCACBj, is the president of Clini Tox Inc. and has worked as a scientist in the Department of Clinical Pathology, Sunnybrook Health Sciences Center, and is an associate professor in the Department of Laboratory Medicine and Pathobiology at the University of Toronto. He also has acted as a consultant on toxicology at The Hospital for SickKids. He stresses the same point as Dr. Urquhart makes: the potency of fentanyl is especially dangerous. When used in a clinical setting, fentanyl is administered in very small doses (usually in micrograms). When a street drug user purchases fentanyl, or another drug spiked with fentanyl, they aren’t aware of its potency and are at risk of overdose. He describes one of his first experiences with patients abusing fentanyl that occurred after he delivered a lecture and learned of patients extracting fentanyl from transdermal patches that were prescribed for chronic and severe pain. “Unfortunately, Fentanyl is just the start of the problem,” says Urquhart. “Analogs of fentanyl [molecules with very similar structure to fentanyl] have similar strength and can be even stronger than fentanyl.” These analogs also complicate testing, requiring forensic laboratories to update their analytical methods to detect known analogs. Given the dangers of fentanyl contamination of other drugs, there is an urgent need for accurate, sensitive testing strategies. Dr. Kapur explains that the first test for fentanyl was an immunoassay. It is highly sensitive and can easily detect the presence of a class of drugs such as opioids however, a lack of specificity means it is not suitable for separating the analogs in the sample. Chromatographic separation followed by mass spectrometry for detection enables greater specificity and can therefore be used to identify fentanyl analogs in a sample.

Separation Is Key for Identification

Scientists may use gas or liquid chromatography (GC and LC, respectively) to separate compounds in a sample. Gas chromatography often requires an inert gas as a mobile phase and a liquid or polymer layer in a column as a stationary phase. Similarly, liquid chromatography requires a mobile phase that often consists of a mixture of solvents such as water, acetonitrile, and methanol, and a stationary phase such as silica in the column. Liquid chromatography has recently become more common because it requires less extensive sample processing and extraction; however, “both GC and LC are used in toxicology laboratories,” says Kapur. “When coupled with a mass spectrometry [MS] detector, these methods are both sensitive and specific and can help in differentiation of the opiates.” Once compounds are separated by the GC or LC column, mass spectrometry is used to identify and quantify the presence of different compounds. A mass analyzer consists of an ion source, a mass analyzer, and an ion detector. When a sample enters the mass spectrometer from either gas or liquid chromatography, it is ionized by the ion source. The mass-to-charge ratio of ions within a sample is used to identify the chemical structure of the compounds. According to Dr. Kapur, tandem mass spectrometry (MS/MS) greatly increases the specificity of MS fragments because mass analysis occurs twice. “Once a precursor ion is isolated, it can then be fragmented again to produce product ions and neutral fragments.” Using LC-MS/MS, scientists are able to quantify drugs and compounds present at much lower concentrations, explains Dr. Urquhart. “The fact that a fragmentation pattern is used [by following transitions] allows the analyst to be reasonably certain that the molecule they see in their analysis is indeed the drug of interest. This is because they have several levels of evidence, including the chromatography retention time and how the molecule fragments when energy is applied.”

Tandem mass spectrometry also aids in the resolution of compounds with “overlapping peaks that can or may not be separated by either of the separating methods,” according to Kapur. What’s more, Kapur affirms, when the overlapping peaks have different molecular weights or structures, tandem mass spectrometry “helps in the resolution of these compounds.” Another detection method, LC-HRMS (liquid chromatography and high-resolution mass spectrometry), is “essentially taking it further and coming up with the molecular weight for use in identifying the compound,” says Kapur. This strategy is useful for identification of unknown drugs; however, “the sacrifice that HRMS instruments makes is that they have decreased sensitivity,” says Dr. Urquhart. “This means they are not able to measure concentrations as low as LC-MS/MS systems are.”

Challenges and Considerations

Chromatography coupled to mass spectrometry is certainly one of the most powerful tools for identifying fentanyl and its analogs in various samples. As with all analytical methods, there are some considerations and difficulties with the approach. “One of the biggest difficulties in identifying a compound is not having a library standard,” says Dr. Kapur. Analytical standards are pure compounds of known concentration. The presence of a specific compound in a sample can be confirmed by comparing the sample compound chromatogram to the analytical standard chromatogram. There are several online libraries with information and chromatograms for thousands of individual compounds; however, it is entirely possible that a compound of interest has not yet been added to a library and that an analytical standard is not available. In these cases, explains Dr. Kapur, a scientist can purchase a sample for analysis from a drug company and enter the resulting chromatogram into a reference library. Fentanyl and its analogs are uniquely challenging because “there often is very little or no information about the chemical formula and structure. In addition, analytical standards may not be available for purchase” says Urquhart. In these instances, the process of updating the reference library described by Dr. Kapur is often completed by forensic laboratories. According to Dr. Urquhart, these forensic laboratories are “often the ones identifying new analogs when they run tests on drugs seized by police. They are able to do this mostly by employing liquid or gas chromatography coupled to mass spectrometry.” Opioid abuse is a complex and growing problem. These drugs are potent pain relievers and are effective when prescribed following surgery or for other severe pain.

However, they are often abused by patients who become addicted to their rapid, powerful effects. To further complicate the matter, street drugs such as heroin often contain unknown concentrations of fentanyl and its analogs with varying potency. As such, many recreational drug users have unknowingly consumed narcotics containing fentanyl and suffered an overdose. The severity and size of this problem have increased the need for accurate and reliable analytical methods for detecting fentanyl and various fentanyl analogs. Gas or liquid chromatography coupled to mass spectrometry enables scientists to tackle this challenge.