Applying UHPLC–HRMS/MS to Differentiate Plant- and Drug-Derived Salicylic Acid Residues in Eggs

Using ultrahigh-performance liquid chromatography high resolution tandem mass spectrometry (UHPLC–HRMS/MS), researchers at Wageningen Food Safety Research, part of Wageningen University & Research (The Netherlands)identified methyl 2-(sulfo-oxy)benzoate as a novel biomarker that differentiates pharmaceutical from plant-derived salicylic acid in eggs. The findings address regulatory challenges, enable detection of illicit salicylate use in poultry, and strengthen food safety and consumer protection frameworks. LCGC International spoke to Serena Rizzo, corresponding author of the paper resulting from this work (1), about the group’s findings.

What advantages did high-resolution mass spectrometry (HRMS) offer over targeted liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods for identifying unexpected salicylic acid–related metabolites in eggs?

High-resolution mass spectrometry provided key advantages over unit-resolution LC–MS/MS for the identification of unexpected salicylic acid–related metabolites because of its intrinsic mass accuracy, resolving power, and full-scan acquisition capability. The high mass accuracy enabled confident assignment of an elemental composition to an unknown feature, which is essential when no prior structural information or reference standard is available. The high resolving power allowed effective discrimination of the analyte ion from near-isobaric background signals originating from the complex egg matrix, thereby reducing the risk of false-positive assignments. Furthermore, HRMS enabled the acquisition of high-quality, accurate-mass MS/MS spectra, which supported rational structural elucidation of the previously unreported metabolite, methyl 2-(sulfo-oxy)benzoate. Collectively, these characteristics make HRMS particularly well-suited for untargeted metabolite discovery compared with unit-resolution mass spectrometric approaches.

Can you describe how the untargeted ultrahigh-performance liquid chromatography–high resolution tandem mass spectrometry (UHPLC–HRMS/MS) workflow was designed to capture both known salicylates and previously unreported metabolites such as methyl 2-(sulfo-oxy)benzoate?

The untargeted UHPLC–HRMS/MS workflow was designed to be selective for salicylic acid and related compounds, while remaining sufficiently open to capture previously unreported metabolitesassociated with different exposure sources.

The study aimed to differentiate salicylic acid residues originating from plant-based dietary sources from those resulting from pharmaceutical treatments in laying hens. To this end, an animal study was conducted involving six groups of hens receiving different feeds or treatments, including an untreated control group, four groups supplemented with plant materials (lucerne, willow bark, and two plant-based feed supplements, FS1 and FS2), and one group treated with solacyl, a veterinary medicine containing sodium salicylate as its active ingredient.

First, the sample preparation protocol (routinely used in our laboratory for the analysis of salicylic acid and structurally related compounds in eggs) was selected because it provides efficient extraction and clean-up of both free salicylates and conjugated forms. This ensured selective extraction and effective clean-up of salicylate-related compounds from the complex egg matrix.

Second, the UHPLC separation was optimized using a set of 28 salicylates for which authentic reference standards were available. Chromatographic conditions were adjusted to achieve adequate retention and resolution of these compounds, which in turn increased the likelihood that additional, structurally related salicylate metabolites with similar physicochemical properties would also be retained, separated, and detected.

For mass spectrometric acquisition, full-scan HRMS was combined with data-dependent MS/MS. The deprotonated precursor ions of the 28 reference salicylates were included in an inclusion list to ensure their prioritized fragmentation and acquisition of high-quality HRMS/MS spectra. In parallel, the Top-N parameter was set to 4, allowing the most intense additional precursor ions detected after each full-scan event to also be selected for fragmentation. This approach ensured that both known salicylates and abundant, previously uncharacterized features were systematically subjected to MS/MS analysis.

Finally, the differential data-analysis workflow, including feature extraction and comparative evaluation across sample groups, was applied to identify ions uniquely associated with each treatment. This approach enabled clear discrimination between untreated, plant-supplemented, and pharmaceutical-treated samples, and was crucial in detecting methyl 2-(sulfo-oxy)benzoate as a treatment-specific, previously unreported metabolite.

What mass spectral features (accurate mass, isotopic pattern, fragmentation behavior) were critical in tentatively assigning methyl 2-(sulfo-oxy)benzoate prior to nuclear magnetic resonance (NMR) confirmation?

The tentative assignment of methyl 2-(sulfo-oxy)benzoate prior to NMR confirmation was based on several critical mass spectral features. First, the accurate mass of the precursor ion at m/z 230.9971 (C₈H₇O₆S) allowed calculation of the elemental composition consistent with a methylated, sulfonated derivative of salicylic acid. Second, the MS/MS fragmentation pattern closely resembled that of methyl salicylate, with characteristic product ions at m/z 151.0401 (C₈H₇O₃), 121.0295 (C₇H₅O₂), and 91.0189 (C₆H₃O), but included a unique ion at m/z 110.9758 (CH₃O₄S), indicating sulfonation at the C-2 position of the aromatic ring. The observation that the product ion at m/z 151.0401 could be rationalized as resulting from the neutral loss of 79.96 Da from the precursor ion further supported the presence of a sulfonic acid group. Together, these features (accurate precursor mass, the characteristic fragmentation pattern, and the presence of a sulfonation-specific product ion) provided strong evidence for assigning the compound as methyl 2-(sulfo-oxy)benzoate, distinguishing it from other salicylates before NMR verification.

How did tandem MS fragmentation help differentiate methyl 2-(sulfo-oxy)benzoate from structurally similar salicylate conjugates, such as glucuronides or sulfates?

Tandem MS fragmentation was essential for distinguishing methyl 2-(sulfo-oxy)benzoate from structurally similar salicylate conjugates, by revealing both class-specific and structure-informative fragmentation behavior. Consistent with sulfate ester fragmentation, the MS/MS spectrum of methyl 2-(sulfo-oxy)benzoate displayed a neutral loss of approximately 80 Da (SO₃) from the precursor ion at m/z 230.9971 to the product ion at m/z 151.0401, confirming the presence of a sulfated moiety. Moreover, the MS/MS spectrum of methyl 2-(sulfo-oxy)benzoate closely resembled that of methyl salicylate, with characteristic product ions at m/z 151.0401, 121.0295, and 91.0189, but also included a unique ion at m/z 110.9758, indicative of sulfonation at the C-2 position of the aromatic ring. This fragment was most readily rationalized by cleavage of the aryl–O–SO₃ bond,a pathway commonly observed for aryl sulfates, and was not consistent with fragmentation patterns expected for glucuronide conjugates (which typically show a dominant neutral loss of approximately 176 Da. Combined with the accurate precursor mass and chromatographic behavior (the sulfo-oxy derivative eluted later than free salicylic acid), these features provided multiple orthogonal criteria to differentiate methyl 2-(sulfo-oxy)benzoate from other salicylate conjugates.

What challenges did matrix effects from egg components pose for HRMS detection, and how were these addressed during method development and validation?

Eggs contain complex components, including lipids and proteins, which can co-elute and cause matrix effects that suppress or enhance ionization in HRMS. To mitigate these challenges, we employed a selective extraction and cleanup compatible with both salicylic acid and related compounds, included an isotopically labeled internal standard (salicylic acid-¹³C₆) to correct for sample preparation and instrumental variability, and applied matrix-matched calibration and standard-addition approaches to account for extraction efficiency and residual suppression. Highly concentrated samples were diluted within the linear calibration range to avoid detector saturation and ensure accurate quantification. The method’s performance was assessed in terms of extraction recovery, linearity, sensitivity (limit of detection (LOD) and limit of quantitation (LOQ)), intra-day repeatability, specificity, and matrix effects, ensuring reliable detection and accurate quantification of methyl 2-(sulfo-oxy)benzoate in the complex egg matrix.

Why was a combination of untargeted HRMS discovery and targeted quantitative analysis essential for establishing methyl 2-(sulfo-oxy)benzoate as a reliable biomarker?

Untargeted HRMS was essential for discovering methyl 2-(sulfo-oxy)benzoate, as it allowed unbiased detection of a previously unreported, treatment-specific feature without requiring predefined transitions or standards. Once the structure was confirmed (including NMR) and a reference standard synthesized, targeted analysis using multiple reaction monitoring (MRM) on a triple quadrupole enabled sensitive quantification at the ppb level. The MRM approach achieves higher sensitivity and selectivity than full-scan HRMS because it monitors specific precursor-to-product ion transitions with optimized dwell times, which is difficult to achieve withorbital ion trap data-dependent MS/MS due to limited duty cycle and lower signal intensity at very low concentrations. Together, this workflow allowed both discovery and accurate quantification, establishing methyl 2-(sulfo-oxy)benzoate as a potential biomarker suitable for regulatory consideration.

How did the metabolite-to-parent (methyl 2-(sulfo-oxy)benzoate–salicylic acid) ratio strengthen the spectrometric evidence for distinguishing pharmaceutical from plant-derived exposure?

The metabolite-to-parent ratio strengthened the spectrometric evidence by providing a quantitative and source-dependent indicator of exposure, rather than relying on simple presence or absence of the metabolite. Although methyl 2-(sulfo-oxy)benzoate was detected at trace levels in the willow bark feed administered to one plant-supplemented group and in the feed supplement suspected of adulteration, it was not detectable in eggs from any plant-supplemented groups, including willow bark. In contrast, eggs from pharmaceutical treatment and the suspected adulterated supplement contained measurable levels of the metabolite, allowing calculation of a metabolite-to-salicylic-acid ratio that reflected the extent of salicylic acid biotransformation in vivo.

The higher relative conversion observed in hens fed the suspected adulterated supplement indicates that salicylic acid in this formulation was metabolized differently from naturally occurring plant-derived salicylates. This finding supports the suspicion of pharmaceutical-level exposure, while also highlighting that the ratio should be interpreted quantitatively and in context rather than as evidence of exclusivity. Several factors may contribute to the enhanced conversion, including the presence of additional compounds in the supplement matrix that modulate absorption, distribution, or hepatic metabolism, or the presence of other salicylates that converge on the same metabolic pathway but are not directly detected. What is unambiguous is that methyl 2-(sulfo-oxy)benzoate originates from salicylic acid metabolism, as its formation was also observed following administration of Solacyl, which contains only pharmaceutical salicylic acid and no other salicylates.

Taken together, the metabolite-to-parent ratio provided a robust quantitative framework for distinguishing pharmaceutical exposure from plant-derived background by reflecting differences in the degree of metabolic conversion rather than relying on detection alone. At the same time, the observed variability underscores the influence of formulation and matrix effects on metabolism and points to the need for further investigation, which makes the identification of this metabolite particularly relevant for future residue and biomarker studies.

What role did accurate-mass confirmation and retention-time consistency play in excluding false positives, particularly given the natural occurrence of salicylates in plant-based feeds?

High mass accuracy narrowed elemental composition choices, reducing the likelihood of false positives. Consistent chromatographic retention times between sample extracts and the synthetic reference standard ensured exclusion of co-eluting matrix interferences. Matching MS/MS fragmentation patterns provided additional structural evidence. Together, these orthogonal criteria (accurate mass, retention time, and fragmentation behavior) minimized the risk of misassigning naturally occurring salicylates from plant-based feeds with the pharmaceutical biomarker.

In your view, how transferable is this HRMS-based biomarker approach to routine regulatory laboratories, considering instrument availability, data complexity, and expertise requirements?

In my view, implementing this HRMS-based biomarker approach in routine regulatory laboratories presents several challenges. High-resolution instruments and the expertise required to process untargeted metabolomics data sets are still relatively uncommon in many laboratories, and untargeted workflows generate complex data that require specialized bioinformatics. A practical pathway for adoption is to perform discovery and structural confirmation in specialized reference laboratories and then translate the identified biomarker into a routine-targeted monitoring method (such as MRM on a triple quadrupole) using certified reference materials and standardized sample preparation. This two-tier approach, with reference-laboratory discovery followed by routine- laboratory targeted monitoring, makes implementation feasible while maintaining regulatory throughput, reproducibility, and cost-effectiveness.

How could future advances in high-resolution spectrometry—such as ion mobility separation or data-independent acquisition (DIA)—further improve the discrimination of natural versus pharmaceutical salicylate residues in food?

Future advances in high-resolution mass spectrometry can substantially improve the discrimination between natural and pharmaceutical salicylate residues, but their effectiveness ultimately depends on the quality of the raw analytical data. Regardless of how sophisticated post-acquisition workflows become, newly formed or low-abundance metabolites cannot be detected with confidence if signal quality, chromatographic selectivity, or adequate ion counts for reliable fragmentation are insufficient at the acquisition stage. Therefore, robust sample preparation and chromatography remain essential, particularly when addressing unknown compounds in complex food matrices, where the challenge is to be selective for the relevant chemical space while minimizing co-extracted interferences.

Ion mobility spectrometry offers a valuable additional separation dimension by providing collision cross-section information, which can help distinguish isomeric or closely related conjugates and reduce co-elution effects. This orthogonal information strengthens structural confidence when combined with accurate mass, retention time, and MS/MS data, but its benefit is maximized only when precursor signals are well resolved and of adequate intensity.

With respect to acquisition strategies, no single approach is universally optimal. Both data-independent acquisition (DIA) and data-dependent acquisition (DDA) can be applied for suspect and non-target screening, increasing the likelihood of obtaining fragment information for unexpected metabolites. In DIA, MS/MS spectra are acquired for all ions within relatively broad m/z isolation windows, enabling the capture of low-abundance and unexpected precursors. While this ensures comprehensive coverage, the resulting spectra may contain fragments from multiple co-isolated species. In contrast, DDA isolates a narrow m/z range around a single precursor, producing higher-quality, cleaner MS/MS spectra for confident structural assignment. As such, the choice between DIA and DDA should be guided by the specific analytical objective (such as comprehensive screening versus confident structural elucidation) rather than treating the two approaches as competing or interchangeable.

Advanced data-processing tools, including molecular networking, further enhance untargeted analysis by organizing related MS/MS spectra into chemically meaningful clusters, allowing structural information to be propagated across metabolite families. The effectiveness of these approaches, however, strongly depends on the availability and quality of spectral libraries, which remain critical for both annotation confidence and discrimination between closely related natural and pharmaceutical-derived compounds.

Overall, improved discrimination will not arise from a single instrumental development alone but rather from a holistic analytical strategy that combines selective sample preparation, appropriate separation techniques, high-quality, high-resolution mass spectrometric acquisition, and robust computational data analysis. These elements must be selected and adapted to the research question at hand, reinforcing that analytical selectivity and data quality are prerequisites for confident interpretation in both untargeted discovery and suspect-screening applications.

References

1. Rizzo, S.; Vonsovic, S.; Thiyagarajan, S. et al. HRMS-Based Differential Analysis of Hen Eggs Reveals Biomarker Distinguishing Plant-Based and Pharmaceutical Salicylic Acid Administration. Food Chem. 2025, 501, 147612. DOI:10.1016/j.foodchem.2025.147612