Chromatographic and Mass Spectrometric Screening of Novel Psychoactive Substances in Seized Drug Analysis

The rapid emergence of novel psychoactive substances (NPS) has created major challenges for forensic laboratories, as many of these drugs are specifically designed to avoid existing drug laws and are difficult to identify using traditional screening methods. Conventional presumptive techniques such as color tests, thin-layer chromatography, and gas chromatography with flame ionization detection often lack the speed, sensitivity, or structural detail needed to accurately detect new synthetic drugs. As a result, advanced analytical approaches combining chromatography and mass spectrometry have become increasingly important for rapid drug screening and identification. Direct analysis in real time–mass spectrometry (DART-MS) is a fast and convenient technique that requires little sample preparation and can quickly provide molecular information about seized drug samples. However, because DART-MS does not include chromatographic separation, interpretation of the resulting spectra can be challenging, especially when unexpected oxygen-containing ions are formed during analysis.

A recent study focused on understanding the formation of these oxygenated species in nitazene analogs and other seized drug-related compounds. Improving the interpretation of DART-MS spectra can support faster and more reliable screening of emerging drugs, helping forensic laboratories reduce case backlogs and respond more effectively to the ongoing opioid crisis. LCGC International spoke to Emma Hardwick, lead author of the paper that resulted from this work.¹

How does direct analysis in real time-mass spectrometry (DART-MS) differ from traditional chromatographic techniques like gas chromatography (GC) in terms of sample preparation, speed, and data interpretation?

DART-MS is an ambient ionization technique that provides chemical information about a sample without the use of chromatography. The ability to ionize an analyte under ambient, open-air conditions enables analysis without extensive sample preparation. Typically, samples are dissolved in an appropriate solvent and either deposited onto a glass rod or metal mesh, or these materials are dipped directly into the sample before analysis. DART-MS has also been used to analyze a variety of samples, including currency, intact tablets, plastics, and even drug testing strips.^2,3 Analysis times are extremely rapid due to the lack of chromatographic separation, which means a full chemical profile can be achieved in seconds. However, this can result in complex data, particularly if more than one substance is present in a sample. Without chromatographic separation, the mass spectra will contain information about all analytes present, which can require deconvolution for accurate interpretation.

Why is the lack of chromatographic separation in DART-MS both an advantage and a limitation for screening novel psychoactive substances?

One of the primary advantages of DART-MS is the ability to rapidly analyze unknown samples without the necessity of chromatographic separation before detection. This makes DART-MS an ideal screening technique because it can provide chemical information about a sample in seconds, which is much faster than typical chromatographic-based screening methods that may take 10-15 minutes or more. This is particularly important for novel psychoactive substances (NPS) that may be difficult to identify using traditional screening techniques like color tests. However, without chromatographic separation, components in a mixture will all appear in the same mass spectrum, increasing the difficulty of mass spectral interpretation. One challenge with NPS is the prevalence of isomers, or compounds with the same molecular formula. Without the added information from a retention time, isomers may be indistinguishable using DART-MS alone. This was a limitation we noticed in our previous work looking at the DART-MS analysis of nitazene analogs, a recent class of novel synthetic opioids (NSOs) with many sets of positional isomers.⁴ Therefore, DART-MS is better suited as a screening technique coupled with a confirmatory technique that utilizes chromatography.

In mass spectrometry, what are the differences between detecting a molecular ion (M^+•) and a protonated molecule ([M+H]⁺), and why is this distinction critical for compound identification?

A molecular ion (M^+•) and a protonated molecule [(M+H]⁺) both inform us about the molecular weight of a compound but may be formed under different conditions. With gas chromatography-electron ionization-mass spectrometry (GC-EI-MS), molecular ions are commonly formed through the removal of an electron from the analyte. In contrast, liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) typically forms protonated molecules in solution through the addition of a proton to the analyte of interest. DART-MS is interesting because it is an ambient ionization technique, and ionization can therefore occur through a variety of mechanisms. In a review of DART-MS published in 2014, Dr. Jürgen Gross explains how both protonated molecules and molecular ions can be formed with DART ionization.⁵ Although protonated molecules are more likely to form and are observed more frequently than molecular ions, both outcomes can still occur. However, the focus of our study was exploring the formation of oxygenated species, an alternative ionization outcome. If these species are confused with the protonated molecule, they could mislead an analyst about the weight of the analyte of interest.

How does the choice of ionization gas (helium vs. nitrogen) influence ion formation mechanisms in DART ionization?

The DART ionization process involves the creation of metastable excited-state species of the source gas. These metastable species then interact with components of the ambient environment, such as nitrogen, oxygen, or water, forming charged species that then ionize the analyte. Helium is the most common source gas utilized because it has a high enough internal energy (19.8 eV) to ionize most molecules found in ambient environments. One of the most accepted theories about DART ionization using helium is that the metastable helium atoms from the DART source interact with the most common species found in air, nitrogen, through what is known as Penning ionization, resulting in a charged nitrogen species.⁵ This ion will then interact with other nitrogen species, forming an alternative species that can ionize water through a charge transfer reaction. Ionized water will then undergo a cascade of reactions that lead to protonated water clusters, which can in turn ionize the analyte, resulting in an [M+H]⁺ protonated molecule.

On the other hand, nitrogen has a lower internal energy and is unable to efficiently ionize many compounds in the air. Song and associates studied the formation of ions using nitrogen as the DART-MS source gas and established that [M+H]⁺ ions of polar compounds may be formed through Penning ionization followed by self-protonation.⁶ Alternatively, non-polar compounds may gain their charge through proton transfer. Due to the lower ionization efficiency of nitrogen, the signal of the analyte is typically much lower with nitrogen than with helium; however, nitrogen is a more affordable, renewable resource, and therefore provides a useful alternative to helium.

What challenges arise when interpreting full-scan spectra without prior precursor ion selection, compared to using tandem MS (MS/MS)?

Full scan mass spectra and MS/MS product ion spectra provide different types of information. Full scan mass spectra provide information about the molecular weight of an unknown species, whereas product ion spectra provide information about the structure of the compound. To gain this structural information, precursor ion selection is required, so that the origin of the product ions can be determined. Precursor ion selection requires a tandem mass spectrometer, which can be more expensive and complicated to operate. Typically, DART ionization is coupled with single-stage high-resolution mass spectrometers, such as a time-of-flight (TOF) mass spectrometer. These types of instruments provide very accurate measurements of the protonated molecules (for example, precursor ions), but they cannot provide precursor ion selection. Instead, fragmentation can be induced through a process known as in-source-collision induced dissociation (IS-CID), which involves increasing the voltages used to help ions transition from atmospheric pressure to the high vacuum of the mass analyzer. Without IS-CID, most full scan mass spectra only contain the protonated molecule and any background ions or adducts that may form. For example, our work found that oxygenated adducts were formed using DART-MS, and these species were present in the full scan mass spectra.

Explain the formation of adduct ions such as [M+NH4]⁺ in ambient ionization techniques. Under what conditions are these adducts more likely to form?

Adducts are relatively common with DART-MS, because anything present in the DART source transient microenvironment can play a role in ionization. [M+NH₄]⁺ ions were first observed by Cody and associates when a bottle of dilute ammonium hydroxide was opened near the DART source.³ Song and colleagues observed [M+NH₄]⁺ more frequently when using nitrogen as the source gas and proposed that the metastable nitrogen will ionize ammonia, which will then interact with water to form an ammonium cation that can attach to an analyte through hydrogen bonding.⁶ However, our work only explored oxygenated adducts, such as [M+H+O]⁺, rather than ammoniated adducts.

Why are traditional screening methods like thin layer chromatography or flame ionization detection insufficient for distinguishing structurally similar novel psychoactive substances (NPS) compounds?

Traditional screening methods often focus on chemical or class characteristics as a relatively quick and affordable method of detection. Color tests, thin layer chromatography (TLC), and GC coupled with a flame ionization detector (GC-FID) are common examples that are utilized in forensic laboratories. However, these techniques are not well-suited for NPS for a variety of reasons. Color tests were created for traditional drugs, and many NPS have very different chemical structures compared to traditional drugs. Therefore, many classes of NPS do not have appropriate color tests. GC-FID and TLC are dependent on the chromatographic separation of NPS and rely on a comparison of the retention behavior with a known standard. The nature of NPS is such that reference standards may not yet exist for these compounds. Therefore, screening techniques for NPS need to provide structural information that can aid with identification.

What mechanisms could explain the formation of oxygenated ions (e.g., [M+H+O]⁺) in DART-MS, even when using helium as the source gas?

The ionization mechanism for both helium and nitrogen include the formation of many different radical or cationic species. For example, the ionization process with helium as the source gas involves the formation of water clusters. Through this process, ^•OH radicals are formed, which are highly reactive and could be a source of oxygen. Another species that could be responsible for oxygenation is NO⁺, which has been observed under a variety of different conditions and instrumental configurations.⁷ Although our work did not outline specific mechanistic pathways for the oxygen adducts, the [M+H]⁺ ions are likely formed first through the normal ionization process, and then the NO⁺ or ^•OH species can react with the protonated molecule, resulting in the formation of either an [M+H+O-2H]⁺ or an [M+H+O]⁺ ion. Through the data we collected, we propose that the [M+H+O-2H]⁺ ion corresponds to an amide, and the [M+H+O]⁺ ion corresponds to a secondary alcohol.

How does in-source fragmentation (for example, in-source collision-induced dissociation [IS-CID] or all ions fragmentation [AIF]) impact spectral complexity and compound identification in rapid screening workflows?

Both IS-CID and AIF are non-targeted acquisition techniques, making them more applicable for screening techniques than traditional targeted CID. However, the non-targeted nature means that everything in the sample or transient microenvironment of the source region will be ionized, and if collision energy is applied, fragmented. Therefore, fragment ions cannot be easily attributed to their parent ions as they could be with CID. This could be a particular issue for mixtures or compounds that fragment similarly. We noticed this in our previous work, which compared these three different activation techniques for nitazene analogs.⁴ The CID product ion spectra were much cleaner and had fewer product ions than the AIF or IS-CID spectra, which contained fragments from a variety of different precursors, including background species. Nitazene analogs fragment very similarly, and many nitazene isomers are indistinguishable based on their product ion spectra alone. This is further complicated when using IS-CID or AIF, and these techniques are therefore recommended for screening purposes only. It should be noted that the limitation of isomer identification is applicable for other techniques as well, like electron ionization-mass spectrometry (EI-MS) and electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Our previous works have structurally characterized nitazene analogs using both and found that positional isomers could not be distinguished using the mass spectra alone.^8,9

When analyzing emerging compounds like nitazene analogs, what strategies would you use to improve confidence in presumptive identification using DART-MS data?

Nitazene analogs are a sub-class of NSOs and therefore require a screening technique to provide structural information because of the limitations with other traditional screening techniques. DART-MS is a great option for screening nitazene analogs because of its rapid nature. I would recommend using a non-targeted technique, such as AIF or IS-CID that doesn’t require the prior knowledge of a precursor ion to isolate. With AIF, all the ions generated in the source region will be fragmented, and the analyst can look at the full scan and fragment ion spectra separately to gain both molecular weight and structural information. Something to look for in the full scan mass spectra is oxygenated species at a relative abundance of ~3-5% or higher. Our work explored the formation of these adducts, and some compounds, such as 5-aminoisotonitazene, consistently produced very high abundances of the oxygenated species. This information can all be used in tandem to make a presumptive identification of a nitazene analog. However, it is important to note that there is instrument-to-instrument variability due to the nature of DART ionization and various instrumental configurations, so the lack of the observed oxygenated species does not necessarily preclude presumptive nitazene analog identification. Full scan mass spectra and potential oxygenated species need to be thoroughly assessed based on individual instrumental configurations.

References

1. Hardwick, E. K.; Couch, A. N.; Davidson, J. T. Addressing the Oxygen in the Room: Exploring the Formation of Oxygenated Species for Nitazene Analogs Using DART-MS. J Am Soc Mass Spectrom. 2026. DOI: 10.1021/jasms.6c00059
2. Appley, M. G.; Pyfrom, E. M.; Elkasabany, R. A. et al. R. Development of an Optimized Extraction Method to Recover Drug Material From Used Test Strips for Comprehensive Drug Checking. Drug Test Anal 2025, 17 (10), 2054-2065. DOI: 10.1002/dta.3911
3. Cody, R. B.; Laramee, J. A.; Durst, H. D. Versatile New Ion Source for the Analysis of Materials in Open Air Under Ambient Conditions. Anal Chem 2005, 77, 2297-2302. DOI: 10.1021/ac050162j
4. Hardwick, E. K.; Couch,A. N.; Davidson, J. T. Comparison of Activation Techniques for the Identification of Nitazene Analogs Using the NIST/NIJ Data Interpretation Tool. Rapid Commun Mass Spectrom 2026, 40 (5), e70004. DOI: 10.1002/rcm.70004
5. Gross, J. H. Direct Analysis in Real Time--A Critical Review on DART-MS. Anal Bioanal Chem 2014, 406 (1), 63-80. DOI: 10.1007/s00216-013-7316-0
6. Song, L.; Chuah, W. C.; Lu, X. E. et al. Ionization Mechanism of Positive-Ion Nitrogen Direct Analysis in Real Time. J Am Soc Mass Spectrom 2018, 29 (4), 640-650. DOI: 10.1007/s13361-017-1885-7
7. Cody, R. B. Aperture Size Influences Oxidation in Positive-Ion Nitrogen Direct Analysis in Real Time Mass Spectrometry. J Am Soc Mass Spectrom 2022, 33 (7), 1329-1334. DOI: 10.1021/jasms.2c00115
8. Hardwick, E. K.; Davidson, J. T. Structural Characterization of Nitazene Analogs Using Electron Ionization-Mass Spectrometry (EI-MS). Forensic Chem 2024, 40. DOI: 10.1016/j.forc.2024.100605
9. Hardwick, E. K.; Davidson, J. T. Structural Characterization of Nitazene Analogs Using Electrospray Ionization-Tandem Mass Spectrometry (ESI-MS/MS). Drug Test Anal 2025, 17 (11), 2127-2140. DOI: 10.1002/dta.3921