Chromatographic Analysis of Antimicrobial Residues in Legume-Based Foods Using LC–MS/MS

The increasing global demand for sustainable food sources, driven by rapid population growth and environmental challenges, has raised concerns regarding antimicrobial contamination in agricultural systems and its contribution to antimicrobial resistance (AMR). Antimicrobial drugs used in human healthcare, livestock production, and crop cultivation can enter soil, water, and food crops through wastewater, agricultural runoff, and manure, posing risks to both environmental and human health. Legumes, widely promoted as sustainable plant-based protein sources, are particularly important to monitor because they may accumulate antimicrobial residues during cultivation. Due to the complexity of legume matrices, sensitive and selective analytical techniques are required for reliable detection. Chromatographic methods, especially liquid chromatography–tandem mass spectrometry (LC–MS/MS), have become the preferred approach for multi-residue analysis because of their high sensitivity, selectivity, and capability for simultaneous determination of multiple antimicrobial classes.

Researchers recently developed and validated a rapid and efficient LC–MS/MS method combined with a dilute-and-shoot sample preparation technique for the simultaneous detection of antibacterial, antifungal, and antiparasitic residues in legume-based alternative protein products, providing a valuable tool for food safety monitoring and environmental contamination assessment. LCGC International spoke to Awanwee Petchkongkaew and Chanita Boonkanon, corresponding and lead authors, respectively, of the paper¹ outlining this work.

Why is liquid chromatography–tandem mass spectrometry (LC-MS/MS) considered the gold standard for multi-class antimicrobial residue analysis compared to single-stage LC or gas chromatography (GC) methods?

LC–MS/MS is a state-of-the-art analytical instrument for analyzing non-volatile compounds using a mobile phase. It performs two stages of mass-to-charge (m/z) analysis, enabling selective detection of precursor and product ions with high precision. This provides greater accuracy and selectivity than conventional LC systems, which measure only a single m/z value.

What chromatographic challenges arise when separating chemically diverse antimicrobial classes (such as antibiotics, antifungals, and antiparasitics) within a single LC method, and how can mobile phase composition and gradient design address them?

A key challenge is that all three antimicrobial classes, along with other compounds, can dissolve in the same solvent, leading to co-elution and peak overlap at the same retention time despite distinct m/z values, which increases the risk of human error. Gradient elution resolves this by separating compounds over time, while appropriate mobile phase selection further optimizes elution for faster analysis or improved signal intensity.

How do matrix components in legume-based alternative proteins influence chromatographic performance and ionization efficiency in LC-MS/MS analyses?

The complex matrices in legume-based alternative proteins can induce significant matrix effects in LC–MS/MS, particularly ion suppression caused by co-extracted lipids that interfere with analyte ionization. These lipids may also contaminate the ion source, leading to electrospray ionization (ESI) emitter clogging, reduced spray stability, decreased signal intensity, and peak shifts, and in severe cases, column contamination and deterioration.

What are the advantages and limitations of the Dilute-and-Shoot (DnS) sample preparation approach from a chromatographic perspective, particularly regarding peak shape, column lifetime, and system robustness?

The main advantage is efficient matrix removal, improving MS stability, extending column lifetime, and enhancing signal intensity. Although this approach requires an additional sample preparation step, this limitation is minimal, as DnS is a simple and highly convenient method.

How can chromatographic separation help mitigate matrix effects when extensive sample cleanup steps such as solid-phase extraction (SPE) are intentionally avoided?

DnS involves only two main steps, as implied by its name: dilution with a suitable solvent followed by centrifugation used to isolate the supernatant for direct injection. In contrast, SPE requires multiple steps and specialized cartridges, increasing both complexity and cost. As a result, DnS provides a simpler, faster, and more cost-effective approach with higher throughput and a reduced risk of analyte loss.

What criteria should be used to select an appropriate LC column chemistry for the simultaneous separation of polar antibiotics and more hydrophobic fungicides?

For the analysis of nonpolar to moderately polar compounds, a C18 column is commonly employed. The compatibility between the stationary phase and the mobile phase must be carefully evaluated, while column length is selected based on the complexity and number of target analytes.

How do retention time stability and chromatographic resolution contribute to meeting regulatory requirements for confirmatory analysis of antimicrobial residues at maximum residue levels (MRLs)?

Retention time is essential for confirming compound identity, while good resolution ensures that the detected peak corresponds to the target analyte rather than interference or noise, thereby improving accuracy at trace levels. Well-resolved peaks can then be reliably used for quantitative analysis with regulatory guidelines.

In method validation, how are chromatographic parameters such as resolution, selectivity, and peak symmetry evaluated alongside MS/MS performance metrics?

Analytical confirmation is based on (1) verification of m/z values; (2) consistent retention time; and (3) sufficient signal intensity, with a signal-to-noise ratio ≥ 3 for qualitative analysis and ≥ 10 for quantitative analysis.

What role does chromatographic optimization play in improving method sensitivity for trace-level antimicrobial detection in high-protein, high-carbohydrate legume matrices?

Improved chromatographic separation improves sensitivity for multiple classes of antimicrobial drugs, which is critical for routine high-throughput analysis. Well-resolved, sharp peaks and a stable analytical process enable accurate and reliable detection while minimizing instrument maintenance, prolonging column lifetime, and ensuring cost-effective operation.

How could advances in chromatographic techniques, such as ultra-high performance liquid chromatography (UHPLC), alternative stationary phases, or two-dimensional LC, further improve high-throughput monitoring of antimicrobial residues in plant-based foods?

Multiple advanced chromatographic techniques play a key role in assessing potential risks in food, supporting a safer food chain. Sustained methodological development further expands analytical capabilities by improving speed, resolution, selectivity, and matrix separation, while enabling efficient, high-throughput analysis without excessive operational burden.

Reference

1. Boonkanon, C.; Uawisetwathana, U.; Waesoh, N. et al. A Novel LC-MS/MS Multi-Group Method for Simultaneous Determination of Antimicrobial Residues in Legume-Based Alternative Proteins. NPJ Sci. Food 2026. DOI: 10.1038/s41538-025-00678-3