Determination of Pyrrolizidine Alkaloids in Food via LC-cIM-HRMS

Plant-produced pyrrolizidine alkaloids (PAs) pose health risks due to their toxicity and frequent occurrence in foods like teas, spices, and honey. A joint study by the University of Parma (Italy) and the Waters Corporation (UK) proposes the use of ion mobility technologies to improve the targeted and semi-targeted detection of 35 regulated PAs. Previously, a linear ion mobility approach, such as travelling wave ion mobility spectrometry (TWIMS), was evaluated and the first CCS database for PAs was reported. However, epimeric compounds could still not be distinguished. Therefore, cyclic ion mobility spectrometry (cIMS), which provides higher resolving power, was employed to address challenges related to co-elutedisomers.

In the resulting paper published in Analytica Chimica Acta,¹ the authors write that their work “highlights, for the first time, the integration of multiple targeted multipass cIMS separations within an untargeted liquid chromatography-high-resolution mass spectrometry (LC–HRMS) workflow, underscoring its potential to expand separation power and analytical confidence for structurally related small molecules. The proposed workflow is especially valuable for the analysis of complex mixtures where epimeric compounds co-occur, as commonly found in naturally contaminated foods.”

LCGC International spoke to Laura Carbonell-Rozas, lead author of that paper, about the study.

Can you explain how ion mobility spectrometry (IMS) separates ions and what physical properties determine their mobility through the gas-filled device?

IMS separates ions in the gas phase by pulling them through a gas filled device under the influence of an electric field. Ions experience repeated collisions with the gas molecules; the balance between the electric force and the collisional drag determines their drift velocity.

The mobility of the ions through such devices depends mainly on ion size and shape (conformation), charge state, and ion–gas interactions such as polarizability, dipole moment, and clustering, with smaller ions being more mobile than larger ions and displaying shorter arrival times. These properties are reflected in the ion’s effective collision cross section (CCS).

What is the significance of CCS values in compound identification, and how do they complement traditional mass spectrometry (MS) data?

The CCS is a quantitative descriptor of an ion’s gas-phase size and shape under defined experimental conditions, which is commonly used as a proxy for ion shape.CCS provides an orthogonal identification parameter that complements accurate mass and fragmentation information obtained from MS.

Two compounds may share the same mass-to-charge ratio (m/z), retention times, and produce similar tandem mass spectrometry (MS/MS) spectra (such as isomers or epimers), yet still differ slightly in their CCS values. Matching experimentally measured CCS values with those stored in reference libraries increases confidence in compound identification. Additionally, CCS tolerances can be applied as filtering criteria in suspect screening and non-target workflows.

How has IMS technology been applied to address food safety challenges, particularly in detecting plant toxins such as pyrrolizidine alkaloids?

IMS has been increasingly applied in food safety analysis to improve selectivity in complex matrices. By introducing an additional gas-phase separation step after traditional separation techniques such as liquid chromatography and before mass detection, IMS enables the separation of isomeric and isobaric compounds and reduces chemical background noise. This additional separation dimension provides complementary information for challenging plant toxins such as pyrrolizidine alkaloids (PAs), whose separation remains particularly difficult using conventional liquid chromatography-mass spectrometry (LC–MS) workflows.

Initially, traveling wave ion mobility spectrometry (TWIMS) was firstly evaluated at the University of Parma²; however, the separation of some compounds was not achieved. Therefore, cyclic IMS (cIMS), which provides higher resolving power, was investigated in our work at Waters Corporation.¹

In general, for plant toxins such as PAs, IMS facilitates:

-Suspect screening using CCS-enabled libraries.

-Improved discrimination of co-eluting or structurally similar compounds.

-Generation of cleaner extracted ion chromatograms, improving identification reliability when LC or MS/MS alone is insufficient.

What advantages does ion mobility spectrometry offer for separating structurally similar compounds such as isomers and epimers that are difficult to resolve with conventional LC–MS alone?

For structurally similar molecules that are co-eluted chromatographically and share identical exact masses to be separated based on differences in gas-phase conformation. such as isomers or epimers, IMS can exploit subtle conformational differences, increase peak capacity and reduce interferences. As a result, IMS improves the reliability of compound identification and quantification without requiring major modifications to chromatographic conditions.

How does cIMS differ from traditional linear IMS approaches, and what benefits does the multiple-pass capability provide for complex mixture analysis?

Traditional linear IMS instruments have a fixed path length, which limits resolving power. In contrast, cIMS uses a closed-loop separation device that allows ions to travel through the mobility cell multiple times.

Each additional pass increases the effective separation length, thereby enhancing resolving power. This multiple-pass capability allows resolution to be tuned according to analytical needs, making cIMS particularly effective for separating closely related compounds such as epimers in complex mixtures. In the case of pyrrolizidine alkaloids (PAs), the increased resolution power allowed us to resolve some PA-epimers pairs that presented similar arrival times and therefore CCS values when using traditional linear IMS such as TWIMS. Nevertheless, with multipass CCS, we observed differences that help us to distinguish between both compounds.

Compare traveling wave ion mobility spectrometry (TWIMS) with drift tube ion mobility spectrometry (DTIMS) in terms of CCS determination and practical applications.

DTIMS measures ion mobility in a uniform electric field. CCS values can be calculated directly from first principles because the relationship between drift time and ion mobility is well defined. DTIMS is therefore often considered a reference method for CCS measurements.

TWIMS separates ions using traveling voltage waves that propel ions through the mobility cell. In this case, CCS values are typically obtained through calibration using reference standards with known CCS values.

While DTIMS provides highly accurate CCS measurements, TWIMS is widely implemented in commercial instruments and is commonly used in routine analytical workflows and high-throughput applications. The CCS values should present low bias in inter-platform and inter-laboratory studies, particularly when the drift gas used is the same.

What are the key considerations when developing a liquid chromatography–cyclic Ion mobility–high resolution mass spectrometry (LC–cIM–HRMS) workflow for small molecule analysis, particularly for natural toxins?

To develop a LC-Cim-HRMS method firstly we should investigate de cyclic conditions (cyclic sequences) to separate the co-eluting natural toxins. Once we have obtained the corresponding separations, we would integrate them into the LC-HRMS workflow using the corresponding software. Thus, different retention time windows can be analyzed using high definition mass spectrometry (HDMS) or high definition tandem mass spectrometry (HDMS/MS) modes, with the specific cyclic sequence set by selecting the mass of the epimer pair in the quadrupole. The key considerations can be summarized as:

• Acquisition design: determining when to apply single-pass or multipass mobility separations according to retention time windows or targeted m/z values, together with the selection of appropriate acquisition modes (HRMS or HRMS/MS
• Avoiding wrap-around effects, and signal loss during cyclic sequences optimization.
• Adduct selection: determining whether protonated ions ([M+H]+) or alternative adducts ([M+Na]+) provide better mobility separation.
• Calibration and quality assurance: ensuring accurate single and multi-pass CCS calibration, mass accuracy, and retention time alignment.
• Data processing: constructing robust libraries containing m/z, retention time (RT), CCS values, and fragment ions.

How can increasing the number of passes through a cyclic IMS device improve the separation of closely related compounds?

Increasing the number of passes effectively increases the separation path length, which improves the resolving power of the instrument. As ions travel longer distances through the mobility cell, small differences in mobility translate into larger differences in arrival times, allowing more effective separation of closely related species. In the case of PAs, we noticed that peak-to-peak resolution was achieved with 10 passes, while almost baseline separation is observed at 25 passes.

However, practical limitations such as wrap-around effects, duty cycle constraints, and potential signal loss must be considered. In this regard, the number of passes was not increased above 25 to avoid wrap-around that happens when slower ions are overtaken by speedier ion populations.

Why are epimeric pairs of PAs particularly challenging to separate, and how might advance IMS techniques address these limitations?

Epimeric PAs often have identical exact masses, very similar polarity, and nearly identical fragmentation patterns. These compounds frequently co-elute chromatographically (typically differing by only ± 0.1 min) and exhibit very similar CCS values, particularly under single-pass IMS conditions. Their gas-phase conformations may also be highly similar since they only differ in the spatial configuration of a functional group. In this case, single-pass CCS can be used in CCS libraries for suspect and unknown analysis for confirmation purposes.

Advanced IMS approaches, such as multipass cyclic IMS can enhance mobility differences by providing high resolution CCS values, increasing the CCS differences between epimers. In this work, for instance, we observed that PA-epimers with a CCS difference of around 0.5% allowed us to differentiate among them. In addition, the exploration of different ion adducts can enable improved separation in some cases as was reported using both instruments (linear TWIMS and cIMS).

What approaches would you use to validate a new LC–cIM–HRMS method for regulatory compliance in food safety testing involving more than 35 regulated compounds?

Although CCS values are not yet included in regulatory criteria for confirmatory analysis, they can provide valuable complementary information for compound identification/separation. Method validation for an LC–cIM–HRMS workflow should therefore consider the same parameters used when validated LC-MS methods such as linearity, calibration range, accuracy and precision, matrix effects and recovery. In our work, we applied the method to green tea extract demonstrates the applicability as a proof of concept for food safety; however, a full validation is needed for quantification purposes.

When including more PAs, I would proceed as indicated in question 7, considering all the potential new co-eluting PAs. The method would then be validated for the PAs for which reference standards are available. For PAs lacking analytical standards, semi-quantification would be performed using the calibration curve of a structurally similar PA available as a surrogate.

References

1. Carbonell-Rozas, L.; Dreolin, N.; Dall’Asta, C. et al. Cyclic Ion Mobility-Mass Spectrometry to Enhance the Separation of Pyrrolizidine Alkaloid Epimers. Anal Chim Acta 2026, 1395, 345207. DOI: 10.1016/j.aca.2026.345207
2. Carbonell-Rozas, L.; Dreolin, N.; Foddy, H. et al. Enhancing Pyrrolizidine Alkaloid Separation and Detection: LC-MS/MS Method Development and Integration of Ion mobility Spectrometry into the LC-HRMS Workflow, J Chromatogr A, 2025, 1748, 465863. DOI: 10.1016/j.chroma.2025.465863