Analysis of Fentanyl and Fentanyl Analogs Using Atmospheric Pressure Chemical Ionization Gas Chromatography–Mass Spectrometry (APCI-GC-MS)

Illicit fentanyl and its analogs pose a significant challenge in forensic and toxicological analysis due to their structural similarities. This study seeks to enhance the characterization of 74 fentanyl analogs by providing retention time data, precursor ions, and product ion spectra obtained through atmospheric pressure chemical ionization-gas chromatography–mass spectrometry (APCI-GC-MS) using a triple quadrupole mass analyzer.

By applying collision-induced dissociation (CID) at various energy levels, the study investigates fragmentation patterns, particularly how different structural modifications influence dissociation pathways. The findings highlight that APCI efficiently produces product ions from the piperidine and N-alkyl chain but provides limited information on the acyl group. Additionally, variations in N-alkyl and amide group substitutions impact fragmentation preferences, offering critical insights into fentanyl analog identification.

The original article

Analysis of Fentanyl and Fentanyl Analogs Using Atmospheric Pressure Chemical Ionization Gas Chromatography–Mass Spectrometry (APCI-GC-MS)

Karen A. Reyes Monroy, Richard McCrary, Isabelle Parry, Catherine Webber, Teresa D. Golden, and Guido F. Verbeck

J. Am. Soc. Mass Spectrom. February 2, 2025

https://doi.org/10.1021/jasms.4c00455

licensed under CC-BY 4.0

Soon after its discovery, fentanyl, which is a synthetic opioid originally developed by Dr. Paul Janssen in 1960, was approved for use as pain relief medication and as an anesthetic in medical settings. (1,2) Shortly after its integration into the medical field, fentanyl abuse as well as overdoses began to increase, and this caused stricter measures to be placed on the pharmaceutical use of fentanyl, which made it a scheduled II drug. (3) The demand for this highly addictive and potent opioid continued to increase well after stricter measures were placed on its medicinal use. (3,4) The Drug Enforcement Administration (DEA) began to see fentanyl arrive in the illicit drug market; this posed a great risk to the community, since, like morphine, fentanyl is a potent analgesic but is 50–100 times more powerful and serves as the foundation for a family of analogs. (5−9) To mitigate the production of illicit fentanyl, the DEA placed restrictions on commonly used precursors of the drug such as 4-anilino-N-phenethylpiperidine (A-NPP). (5) Data accumulated by the DEA show that fentanyl is rarely produced within the states and is commonly made in foreign laboratories. The majority of fentanyl-producing laboratories are in China; these laboratories can either directly export the fentanyl to the US or export it to Mexico for transit to the US. (10−12) To evade current laws against fentanyl, clandestine laboratories are implementing new reagents and precursors to obtain fentanyl analogs. The core structure of fentanyl is readily modified; therefore, structural modifications of the base structure of fentanyl are quite unchallenging. (13,14) Clandestine manufacturers implement chemical reactions using a wide variety of reagents to modify the core structure of fentanyl. Such practices produce analogs with different structures and unknown potencies, which is dangerous since fentanyl is increasingly used as a “cutting agent”. (15−17) Fentanyl as a cutting agent poses a significant threat because of its high potency, making the chance of a drug overdose higher. In 2022, fentanyl and xylazine drug mixtures were found in multiple intoxication deaths; both of these drugs have narrow therapeutic windows and thus increase the chance of overdoses. (18) While cutting agents are used primarily for economic reasons such as for maximizing drug profits, they pose a public health hazard because the users may not be aware of the substances they are ingesting or the true potency of the drugs they consume. (19) This increases the likelihood of overdoses, adverse reactions, and fatalities. (5,20,21)

Fentanyl and its analogs act upon μ-opioid receptors and manifest significant psychoactive effects including profound sedation, loss of consciousness, and, in severe cases, death. (22,23) The potency of these fentanyl analogs varies significantly; for example, carfentanil exhibits potency levels 5,000–10,000 times higher than those of morphine, while sufentanil is only 500 times more potent. (24) According to the National Center for Health Statistics, in 2022, 22.7 deaths per 100,000 standard population from synthetic opioids other than methadone were reported in the US. (25) Structural characterization of fentanyl analogs is crucial for determining its analgesic and anesthetic effects. Such information can be used to help treat fentanyl analogue overdoses with naloxone, an opioid antagonist that binds to opioid receptors, which blocks and reverses the effects of other opioids but has no effect on people who have no opioids in their system. Structural identification can be used to enact the “Anti-Drug Abuse Act of 1986″ which allows for the prosecution of structurally similar chemical compounds as schedule 1 or 2 controlled substances if it is intended for human consumption. (26) Yet, as more new psychoactive substances (NPS) emerge, the ability to characterize them is becoming more strenuous; this in turn complicates the process of identifying the appropriate treatment and potential risks involved. There are many ways to identify fentanyl and its analogs. Characterization of these analogs often requires analysis of product ions produced, and for this reason, gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) is currently the gold standard for illicit drug characterization. (27−30) In GC, gas-phase molecules are introduced into the mass spectrometer under a high vacuum. These neutral molecules are then bombarded with electrons from a heated filament, causing them to ionize and fragment. (31−33) EI is considered a hard ionization technique because it ionizes the molecule of interest directly, and this causes a lack of or low abundance of molecular ions produced. (34−40) Thus, during GC-EI-MS analysis of fentanyl structures, the GC retention time and the product ions observed are used to determine the molecular ion, which is often absent in an EI spectrum. A major reason this technique is widely used is that resulting EI mass spectra can be standardized since each molecule has a unique “fingerprint”. Thus, drug identification using this technique can readily be accomplished if reference data is available.

Yet, while electron ionization generates fingerprint data that is vital during analogue identification, as previously stated, it produces little to no molecular ion data, which can simplify the characterization process. This presents a challenge when several fentanyl analogs are observed to undergo similar dissociation pathways. The lack of molecular ion mass-to-charge (m/z) peaks makes the identity assignment difficult. Furthermore, identification becomes more cumbersome when new and emerging drugs that do not have reference data are analyzed. Thus, while GC-EI-MS is a great tool for identifying known fentanyl substances, due to the lack of reference data for emerging substances, the same cannot be said for new and emerging drugs. Unlike EI, in gas chromatography chemical ionization (CI), a high abundance of intact molecular ion species is generated. (41−44) Yet, this “soft” ionization technique can more easily ionize low polarity analytes that are not as readily ionized in ESI. (45) Furthermore, its sensitivity is much lower than that of EI in GC-MS analysis due to reagent gas interference. The appeal for a soft ion source capable of producing an abundance of molecular ions as well as fragment data with high sensitivity and selectivity brought about the development of atmospheric pressure chemical ionization (APCI).

APCI is a powerful instrumental analysis technique known for its high sensitivity, high selectivity, high resolution, and soft ionization capabilities. (46−48) It is typically used in conjunction with liquid chromatography–mass spectrometry (LC-MS), but it has also been coupled to GC-MS. Furthermore, APCI can generate both protonated and deprotonated molecular ions, as well as full dissociation data which is very beneficial for drug profiling and screening applications. (45,49−51) The identity of analytes can thus be determined based on the molecular ion’s mass and/or the protonated molecule and produced fragments. (52) This is extremely valuable for the analysis and characterization of new and emerging designer drugs that have never been cataloged.

Figure 1 highlights the mechanism for molecules to ionize through a gas-phase ion–molecule reaction as in APCI. When nitrogen gas is used, nitrogen plasma ionization is initiated by electrons, produced from a corona discharge needle. Ionized nitrogen plasma results in N₂^+• or N₄^+• and via charge transfer, N₄^+• will ionize analytes with a low ionization potential. And since this process occurs at atmospheric pressure, water at the ion source is highly likely. H₂O⁺ formed at the source reacts with another water molecule and produces H₃O^+•and this ion further reacts to produce water clusters (H₃O⁺(H₂O)_n). These water clusters are also known to ionize the analyte molecule via proton transfer, thus promoting the protonated precursors [M + H]⁺. When comparing APCI and EI ion sources in GC-MS, they both perform similarly in reproducibility, dynamic range, limit of detection, and quantification. The main difference between the two is APCI’s ability to readily produce molecular ions, which are not commonly observed during EI. While obtaining a mass spectrum with molecular ion data is valuable, scientists have continuously opted out of using it due to the lack of availability of commercial mass spectral libraries. Thus, the main advantage of using GC-EI-MS is the availability of such reference data which can be used to identify components in question. While the implementation of EI for the identification of known drugs has been very successful, the rise of designer drugs that have not been cataloged requires a new approach, one that provides not only fingerprint data but also the mass of the protonated molecular ion. Thus, we believe that APCI libraries containing precursor and fingerprint data will be required for the rapid and efficient analysis of new and emerging designer drugs as they continue to be a problem. (53,54) Therefore, this paper aims to introduce an APCI-GC-MS method capable of analyzing a wide range of fentanyl analogs of varying masses and chemical composition with the intention to be used alongside screening tools, such as DART-MS or Raman spectroscopy. Here we provide chromatographic and mass spectral data obtained using a triple quadrupole mass analyzer by varying collision energies in the second quadrupole for 74 fentanyl analogs. Also analysis of fragmentation tendencies of these moieties was conducted with the goal of information to aid in characterizing unknown fentanyl analogs.

J. Am. Soc. Mass Spectrom. 2025, XXXX, XXX, XXX-XXX - Figure 1. Atmospheric pressure chemical ionization mechanism for APCI..jpg

Experimental Section

Analysis and Data Processing

Qualitative Masslynx software 4.1 was used for APCI-GC-MS control, mass spectral peak integration, and mass spectra evaluation, as well as to extract GC retention times for all 74 analyzed compounds. The relative retention time (rrt) for each analyte was calculated by dividing each individual analyte’s retention time (rt) by an internal standard (caffeine) retention time (rt). Retention and relative retention time values are listed in Table S2 in the Supporting Information. MSMS data obtained from implementing different collision energies (10, 20, 30, 40, 50 V) was plotted using Origin 2023b with m/z values on the x-axis and relative intensity on the y-axis. Peak detection with a 30% threshold was implemented, and all data that met such threshold was tabulated.

Conclusion

APCI-GC-MS instrumentation was used to analyze 74 fentanyl analogs at varying CID voltages of 10, 20, 30, 40, and 50 V. MSMS data for each analogue was successfully obtained and used for trend analysis. Our analysis indicates that the fentanyl analogs examined through APCI-GC-MS predominantly follow pathways A, B, and C, with pathways D and E (Figure 7) occurring less frequently. Analogs with one or more substituents at R1, R2, or R3 tend to fragment preferentially at site A. This fragmentation results in a high abundance of product A at voltages between 20 and 30 V, followed by the formation of product B, which becomes the base peak at 30–40 V. Fentanyl analogs featuring only a single substituent on the N-alkyl chain exhibit the same fragmentation pattern. In contrast, highly substituted fentanyl derivatives, particularly at the N-terminus, generally fragment along the N-alkyl chain. This leads to the predominant formation of structures B and C. Product ion A is typically observed as the base peak at low voltages and decreases in intensity at higher voltages for most fentanyls regardless of weight. In our analysis, we identified only seven analogs that produced product ion E1 and only one produced product ion E2. The acyl groups associated with the analogs that produced E1 were notably stable, including highly stabilized structures such as benzodioxole, phenyl rings, and furanyl rings. Therefore, if a stable enough acyl R1 group is present, fragmentation shifts from site A to site E; thus, it can be stated that pathway E is a competing pathway of A in these instances. Out of the 74 fentanyl analogs analyzed, only four yielded product ion D, and these were compounds that either lacked the R1 or R4 group.