Rapid DART-HRMS Fingerprinting for 1%–Level Detection of Extra-Virgin Olive Oil Adulteration

A recent paper published in Food Chemistry¹ presented a high-throughput, green analytical chemistry approach for authenticating extra-virgin olive oil (EVOO) using direct analysis in real time–high resolution mass spectrometry (DART-HRMS). The method enabled direct analysis of extra-virgin olive oil without any sample preparation, delivering results in ~3 s per sample. Using ambient ionization coupled to high-resolution MS, the study captured full spectral fingerprints from 110 authentic extra-virgin olive oils (EVOOs) and nine adulterant classes, including vegetable oils and lower-grade olive oils. A two-stage experimental design, combining Plackett–Burman design with central composite optimization, was applied to optimize ionization conditions and improve sensitivity and reproducibility. Multivariate statistical analysis discriminated authentic from adulterated samples, achieving detection of 1% substitution. This solvent-free workflow reduced cost, chemical waste, and analysis time compared to chromatography, providing the industry with a rapid, robust and eco-friendly tool for routine EVOO authenticity screening and fraud prevention.

LCGC International spoke to Dimitrios Papelis, lead author of the aforementioned paper, about the method.

How does direct analysis in real time (DART) differ from traditional electrospray or atmospheric pressure chemical ionization (APCI) ionization, and why is it particularly well suited for rapid authenticity screening of edible oils?

DART differs from traditional electrospray ionization (ESI) and APCI in several ways. DART is an ambient ionization technique that operates in open air, whereas ESI and APCI require a closed interface and controlled sample introduction. In addition, DART does not require chromatographic separation. Ions are generated directly from the sample surface by being exposed to a stream of metastable gas species under atmospheric conditions. This direct approach does not require extensive sample preparation and reduces analysis time, enabling measurements to be performed within seconds.

These characteristics make DART particularly well suited for rapid authenticity screening of edible oils. Olive oil is a viscous matrix that can be analyzed directly without dilution or extraction, thereby preserving the chemical profile of both major lipid components and minor constituents, such as phenols. The absence of chromatography enables high sample throughput. Consequently, DART can be employed as a rapid screening tool for edible oils, where it allows efficient authenticity assessment and detection of adulterated samples.

What is meant by a “full spectral fingerprint” in DART-high resolution mass spectrometry (HRMS) analysis, and how does this untargeted approach improve the detection of olive oil adulteration compared to targeted marker analysis?

In DART-HRMS analysis, a “full spectral fingerprint” refers to the complete high-resolution mass spectrum acquired from a sample under specific conditions, rather than focusing on a limited set of predefined compounds. This fingerprint includes all detected signals from different chemical classes such as lipids, phenols, sterols, and other constituents in the case of extra-virgin olive oil (EVOO). Together, these features form a characteristic spectral pattern that reflects the overall chemical composition of the olive oil, rather than the presence or absence of individual markers.

Adulteration often leads to subtle changes in the relative abundances of many spectral features and not just in a small number of known compounds. Furthermore, full spectral fingerprinting allows multivariate models to detect differences between the authentic EVOO profile and the adulterated one even when the exact adulterant or marker compounds are unknown.

Why is high mass accuracy and resolving power (e.g., quality time of flight [QTOF]-MS) critical when using DART for EVOO authentication, especially in complex lipid matrices?

High mass accuracy and resolving power are critical when using DART for EVOO authentication. Since no chromatography is performed, the key factor of retention time, which is normally used for compound identification, is lost. Complex lipid matrices like EVOO, contain many compounds with similar masses. By using low-resolution measurements, these compounds can be incorrectly assigned. On the contrary, high-resolution instruments such as QTOF-MS provide accurate mass measurements and MS/MS fragmentation offers structural information, both of which are essential to confidently identify compound. In a lipid-rich matrix like olive oil, these capabilities are crucial for reliable identification.

Compared to chromatographic MS methods, what are the main limitations of DART-HRMS in terms of sensitivity, selectivity, and isomer separation, and how are these mitigated in the proposed workflow?

Compared to chromatographic MS methods, DART-HRMS has limitations in sensitivity and selectivity because there is no prior separation of compounds before ionization. As a result, simultaneous ionization of multiple species can occur, leading to overlapping signals and reduced discrimination between structurally similar compounds. In addition, DART-HRMS has limited capability to separate isomers, since compounds with identical masses and similar structures are introduced simultaneously into the mass spectrometer. In the proposed workflow, these limitations are mitigated by the use of high-resolution mass spectrometry and careful optimization of ionization conditions, Optimization not only improved spectral reproducibility but also enhanced overall signal intensity, thereby increasing sensitivity and allowing reliable classification despite the lack of chromatography.

How do principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and support vector machines (SVM) differ in their roles for spectroscopic data interpretation, and why is SVM particularly effective for detecting low-level EVOO adulteration?

PCA, PLS-DA, and SVM differ in their roles for interpreting spectroscopic data based on whether class information is used and how separation is achieved. PCA is an unsupervised method that reduces data dimensionality and reveals natural clustering or trends without prior knowledge of sample classes. PLS-DA is a supervised technique that uses class labels to maximize separation between predefined groups. SVM is also a supervised model, but it focuses on defining an optimal decision boundary between classes and performs well in high-dimensional datasets. SVM is particularly effective for detecting low-level EVOO adulteration because it can identify subtle spectral differences near class boundaries, even when overall changes in the spectra are small.

How do Plackett–Burman and central composite designs contribute to optimizing DART-HRMS parameters, and why is systematic optimization especially important in ambient ionization spectroscopy?

Plackett–Burman and central composite designs contribute to optimizing DART-HRMS parameters by allowing systematic evaluation of multiple experimental variables and their effects on analytical performance. Plackett–Burman designs are used to screen many factors and identify those that significantly influence ionization efficiency and signal stability. Central composite designs are then applied to optimize these critical parameters and model their interactions. Systematic optimization is especially important in ambient ionization spectroscopy because small changes in experimental conditions can strongly affect ion formation, spectral patterns, and reproducibility, which can directly impact chemometric classification.

In what ways does DART-HRMS align with green analytical chemistry principles, and how does it compare environmentally to liquid chromatography (LC)-based spectroscopic and spectrometric workflows?

DART-HRMS aligns well with green analytical chemistry principles since it requires minimal or no sample preparation and generates minimal chemical waste. The technique also offers rapid analysis, reducing energy consumption per sample. Compared to LC-based spectroscopic and spectrometric workflows, DART-HRMS has a much smaller environmental burden, as it eliminates mobile phases and consumables associated with chromatographic separation. This makes it an environmentally favorable option for high-throughput screening applications, such as food authenticity testing.

What types of matrix effects can arise when analyzing raw olive oil by DART-MS, and how might these effects influence spectral reproducibility and classification performance?

Analyzing raw olive oil by DART-MS presents several matrix-related challenges due to its high lipid content. Dominant triacylglycerols can cause ion suppression, and different compound classes may compete during ionization, while variations in thermal desorption efficiency can affect signal intensity and spectral reproducibility. To address these challenges, different sample spotting techniques and volumes were tested. By combining optimized source conditions, standardized sampling procedures, and data normalization within chemometric models, spectral reproducibility was significantly enhanced, and more reliable analytical results were ensured.

How could the integration of trapped ion mobility spectrometry (TIMS) with DART-HRMS improve compound identification confidence in EVOO authentication studies?

The integration of trapped ion mobility spectrometry (TIMS) with DART-HRMS enhances the confidence of compound identification by introducing collisional cross-section (CCS) measurements as an additional analytical criterion. CCS values reflect the gas-phase size and shape of ions, providing structural information that complements conventional mass spectrometric data. This feature addresses the limitation of mass spectrometry, which often fails to resolve isomeric or isobaric species. By enabling their separation and incorporating CCS into the identification process, TIMS could strengthen the reliability of compound identification, thereby improving the robustness of EVOO authentication models.

How can authenticity markers discovered using DART-QTOF MS be transferred to more routine spectrometric platforms, such as triple quadrupole MS, without losing analytical reliability?

Authenticity markers discovered using DART-QTOF MS can be transferred to more routine spectrometric platforms such as triple quadrupole MS (e.g., EVOQ DART-TQ⁺) by first confirming their identities using high-resolution measurements and reference standards. Once validated, these compounds can be monitored using targeted multiple reaction monitoring (MRM) transitions. By focusing on a small panel of markers and validating their performance across authentic and adulterated samples, analytical reliability can be maintained while enabling routine, cost-effective analysis on more widely available instrumentation.

Reference

1. Papelis, D. A.; Drakopoulou, S. K.; Nastou, E. S. et al. Direct Analysis in Real Time Coupled to QTOF-MS for the Rapid Authentication and Fraud Detection of Extra Virgin Olive Oil. Food Chem. 2025, 502, 147618. DOI: 10.1016/j.foodchem.2025.147618