Analytical Methods for the Characterization of Vegetable Oils.

The determination of the authenticity of extra virgin olive oils (EVOOs) has become more interesting in recent years. Italy is the first country in Europe in terms of number of Protected Designation of Origin (PDO) oils, which connects consumers to a feeling of tradition and thus to higher quality standards. This work focused on the consideration of the inorganic content as a possible marker of EVOOs. Ten vegetable oils (VOs), eight Italian EVOOs and seven not Italian EVOOs were analyzed. After pretreatment by acid mineralization, Al, Ba, Ca, Cu, Fe, K, Li, Mg, Mn, Na, P, Sb, Se and Zn were determined by ICP-OES. The electrochemical properties of a selected group of EVOOs and other vegetal oils of different botanical origin were investigated by voltammetry. Carbon paste electrodes (CPEs) were prepared. The features observed in the voltammograms reflect the reactions of electroactive compounds, which are present in the oils. A chemometric treatment of the results was performed to assess the possibility to distinguish (i) the region of provenience considering the inorganic profile; and (ii) the plant species from which each oil was obtained on the basis of the current profile registered during voltammetric analysis. Inorganic composition seems to be a useful marker for the assessment of the geographical origin of an EVOO. The EVOO-CPEs voltammetry seems to have a good ability to distinguish the plant species of origin. This method could be useful to monitor the conservation status of the oils, as the redox profile is linked to the oxidative degradation state.

Exploring extra dimensions to capture saliva metabolite fingerprints from metabolically healthy and unhealthy obese patients by comprehensive two-dimensional gas chromatography featuring Tandem Ionization mass spectrometry.

This study examines the information potential of comprehensive two-dimensional gas chromatography combined with time-of-flight mass spectrometry (GC×GC-TOF MS) and variable ionization energy (i.e., Tandem Ionization™) to study changes in saliva metabolic signatures from a small group of obese individuals. The study presents a proof of concept for an effective exploitation of the complementary nature of tandem ionization data. Samples are taken from two sub-populations of severely obese (BMI > 40 kg/m2) patients, named metabolically healthy obese (MHO) and metabolically unhealthy obese (MUO). Untargeted fingerprinting, based on pattern recognition by template matching, is applied on single data streams and on fused data, obtained by combining raw signals from the two ionization energies (12 and 70 eV). Results indicate that at lower energy (i.e., 12 eV), the total signal intensity is one order of magnitude lower compared to the reference signal at 70 eV, but the ranges of variations for 2D peak responses is larger, extending the dynamic range. Fused data combine benefits from 70 eV and 12 eV resulting in more comprehensive coverage by sample fingerprints. Multivariate statistics, principal component analysis (PCA), and partial least squares discriminant analysis (PLS-DA) show quite good patient clustering, with total explained variance by the first two principal components (PCs) that increases from 54% at 70 eV to 59% at 12 eV and up to 71% for fused data. With PLS-DA, discriminant components are highlighted and putatively identified by comparing retention data and 70 eV spectral signatures. Within the most informative analytes, lactose is present in higher relative amount in saliva from MHO patients, whereas N-acetyl-D-glucosamine, urea, glucuronic acid γ-lactone, 2-deoxyribose, N-acetylneuraminic acid methyl ester, and 5-aminovaleric acid are more abundant in MUO patients. Visual feature fingerprinting is combined with pattern recognition algorithms to highlight metabolite variations between composite per-class images obtained by combining raw data from individuals belonging to different classes, i.e., MUO vs. MHO.Graphical abstract.

Comprehensive two-dimensional gas chromatography as a boosting technology in food-omic investigations.

This review focuses on the role that comprehensive two-dimensional gas chromatography can play within the investigation workflows of food-omics and related disciplines and subdisciplines, including food metabolomics, nutrimetabolomics, sensomics, and food safety. After a short introductory survey, discussing the intriguing context of system biology and integrationist approaches of investigation, the concepts of analytical dimensions and the key characteristics of comprehensive two-dimensional gas chromatography are introduced. Through a selection of relevant examples, the boosting role of comprehensive two-dimensional gas chromatography within food-omics is described, providing to the reader evidence of how comprehensive multidimensional separations based platforms have introduced new concepts and tools in the analytical measurement of complex biological samples.

Climate and Processing Effects on Tea (Camellia sinensis L. Kuntze) Metabolome: Accurate Profiling and Fingerprinting by Comprehensive Two-Dimensional Gas Chromatography/Time-of-Flight Mass Spectrometry.

This study applied an untargeted-targeted (UT) fingerprinting approach, based on comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOF MS), to assess the effects of rainfall and temperature (both seasonal and elevational) on the tea metabolome. By this strategy, the same compound found in multiple samples need only to be identified once, since chromatograms and mass spectral features are aligned in the data analysis process. Primary and specialized metabolites of leaves from two Chinese provinces, Yunnan (pu'erh) and Fujian (oolong), and a farm in South Carolina (USA, black tea) were studied. UT fingerprinting provided insight into plant metabolism activation/inhibition, taste and trigeminal sensations, and antioxidant properties, not easily attained by other analytical approaches. For example, pu'erh and oolong contained higher relative amounts of amino acids, organic acids, and sugars. Conversely, black tea contained less of all targeted compounds except fructose and glucose, which were more similar to oolong tea. Findings revealed compounds statistically different between spring (pre-monsoon) and summer (monsoon) in pu'erh and oolong teas as well as compounds that exhibited the greatest variability due to seasonal and elevational differences. The UT fingerprinting approach offered unique insights into how differences in growing conditions and commercial processing affect the nutritional benefits and sensory characteristics of tea beverages.

Corylus avellana L. Natural Signature: Chiral Recognition of Selected Informative Components in the Volatilome of High-Quality Hazelnuts.

The volatile fraction of plant-based foods provides useful functional information concerning sample-related variables such as plant genotype and phenotype expression, pedoclimatic and harvest conditions, transformation/processing technologies, and can be informative about the sensory quality. In this respect, the enantiomeric recognition of the chiral compounds increases the level of information in profiling studies, being the biosynthesis of native compounds often stereo-guided. Chiral native volatiles mostly show an enantiomeric excess that enables origin authentication or support correlation studies between chemical patterns and sensory profiles. This study focuses, for the first time, on the enantiomeric composition of a large set of chiral compounds within the complex volatilome of Corylus avellana L. belonging to different cultivars (Tonda Gentile Romana, Tonda Gentile Trilobata, Anakliuri) and harvested in different geographical areas (Italian and Georgian). Besides native components profiled in raw kernels, volatiles formed after technological treatment (i.e., roasting) are also considered. Headspace solid-phase microextraction combined with enantioselective gas chromatography-mass spectrometry enables the accurate tracking and annotation of about 150 compounds across many samples. The results show that chiral compounds have diagnostic distribution patterns within hazelnut volatilome with cultivar and harvest region playing the major role. Moreover, being some of these chiral molecules also key-aromas, their distribution has a decisive impact on the sensory properties of the product. In particular, the enantiomeric composition of (E)-5-methyl-2-hepten-4-one (filbertone) resulted to be discriminant for origin authentication. The enantiomeric distribution showed, according to literature, an excess of the (S)-enantiomer in both raw and roasted samples volatilome with larger differences in raw samples. The amount of both (R) and (S)-filbertone increases during roasting; the most marked increase for (R)-enantiomer is observed in Italian samples, thus supporting evidence of better hedonic properties and more pleasant odor and aroma.

Integrated Strategy for Informative Profiling and Accurate Quantification of Key-Volatiles in Dried Fruits and Nuts: An Industrial Quality Control Perspective.

Edible nuts and dried fruits, usually traded together in the global market, are one of the cornerstones of the Mediterranean diet representing a source of essential nutrients and bioactives. The food industry has an interest in the selection of high-quality materials for new product development while also matching consumers' expectations in terms of sensory quality. In this study, walnuts (Juglans regia), almonds (Prunus dulcis), and dried pineapples (Ananas comosus) are selected as food models to develop an integrated analytical strategy for the informative volatile organic compounds (VOCs) quali- and quantitative profiling. The study deals with VOCs monitoring over time (12 months) and in the function of storage conditions (temperature and atmosphere).VOCs are targeted within those: (i) with a role in the product's aroma blueprint (i.e., key-aromas and potent odorants); (ii) responsible for sensory degradation (i.e., rancidity); and/or (iii) formed by lipid autoxidation process. By accurate quantitative determination of volatile lipid oxidation markers (i.e., hexanal, heptanal, octanal, nonanal, decanal, (E)-2-heptenal, (E)-2-octenal, (E)-2-nonenal) product quality benchmarking is achieved. The combination of detailed VOCs profiling by headspace solid phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS) and accurate quantification of rancidity markers by multiple headspace-SPME (MHS-SPME) answers many different questions about shelf-life (i.e., aroma, storage stability, impact of temperature and storage atmosphere, rancidity level), while providing reliable and robust data for long-range studies and quality controls. The quantification associated with HS-SPME profiling is demonstrated and critically commented on to help the industrial research in a better understanding of the most suitable analytical strategies for supporting primary materials selection and new product development.

Corylus avellana L. Aroma Blueprint: Potent Odorants Signatures in the Volatilome of High Quality Hazelnuts.

The volatilome of hazelnuts (Corylus avellana L.) encrypts information about phenotype expression as a function of cultivar/origin, post-harvest practices, and their impact on primary metabolome, storage conditions and shelf-life, spoilage, and quality deterioration. Moreover, within the bulk of detectable volatiles, just a few of them play a key role in defining distinctive aroma (i.e., aroma blueprint) and conferring characteristic hedonic profile. In particular, in raw hazelnuts, key-odorants as defined by sensomics are: 2,3-diethyl-5-methylpyrazine (musty and nutty); 2-acetyl-1,4,5,6-tetrahydropyridine (caramel); 2-acetyl-1-pyrroline (popcorn-like); 2-acetyl-3,4,5,6-tetrahydropyridine (roasted, caramel); 3-(methylthio)-propanal (cooked potato); 3-(methylthio)propionaldehyde (musty, earthy); 3,7-dimethylocta-1,6-dien-3-ol/linalool (citrus, floral); 3-methyl-4-heptanone (fruity, nutty); and 5-methyl-(E)-2-hepten-4-one (nutty, fruity). Dry-roasting on hazelnut kernels triggers the formation of additional potent odorants, likely contributing to the pleasant aroma of roasted nuts. Whiting the newly formed aromas, 2,3-pentanedione (buttery); 2-propionyl-1-pyrroline (popcorn-like); 3-methylbutanal; (malty); 4-hydroxy-2,5-dimethyl-3(2H)-furanone (caramel); dimethyl trisulfide (sulfurous, cabbage) are worthy to be mentioned. The review focuses on high-quality hazelnuts adopted as premium primary material by the confectionery industry. Information on primary and secondary/specialized metabolites distribution introduces more specialized sections focused on volatilome chemical dimensions and their correlation to cultivar/origin, post-harvest practices and storage, and spoilage phenomena. Sensory-driven studies, based on sensomic principles, provide insights on the aroma blueprint of raw and roasted hazelnuts while robust correlations between non-volatile precursors and key-aroma compounds pose solid foundations to the conceptualization of aroma potential.

Shelf-Life Evolution of the Fatty Acid Fingerprint in High-Quality Hazelnuts (Corylus avellana L.) Harvested in Different Geographical Regions.

Hazelnuts are characterized by a relatively high abundance of oleic acid and poly-unsaturated fatty acids, which give this fruit a high nutritional value. As a counterbalance, such a lipid profile is more susceptible to autoxidation and/or degradation reactions under enzymatic catalysis. Lipid oxidation occurs on fatty acids (FAs), both esterified on triacylglycerols and in free form (after lipolysis), but with favorable kinetics on the latter. In this study, the quali-quantitative changes in FA profiles (both free and esterified) were monitored during the shelf life (time 0, 6, and 12 months) as a function of different drying and storage conditions and different cultivars and geographical areas. A derivatization/extraction procedure was performed to quantify the profile of free and esterified fatty acids accurately. The overall profile of the free and esterified fatty acids concurred to create a biological signature characteristic of the cultivar and of the harvest region. The free and esterified forms' characterization enabled the efficient monitoring of the effects of both the hydrolytic activity (increment in overall free fatty acids) and the oxidative process (decrease in unsaturated free fatty acids versus esterified fatty acids) over the 12 months of storage.

Exploring the Extra-Virgin Olive Oil Volatilome by Adding Extra Dimensions to Comprehensive Two-Dimensional Gas Chromatography and Time-of-Flight Mass Spectrometry Featuring Tandem Ionization: Validation of Ripening Markers in Headspace Linearity Conditions.

BACKGROUND: Comprehensive two-dimensional gas chromatography (GC×GC) combined with time-of-flight (TOF) MS is the most informative analytical approach for chemical characterization of the complex food volatilome. Key analytical features include separation power and resolution enhancement, improved sensitivity, and structured separation patterns from chemically correlated analytes. OBJECTIVE: In this study, we explore the complex extra-virgin olive oil volatilome by combining headspace (HS) solid-phase microextraction (SPME), applied under HS linearity conditions to GC×GC-TOF MS and featuring hard and soft ionization in tandem. METHOD: Multiple analytical dimensions are combined in a single run and evaluated in terms of chemical dimensionality, method absolute and relative sensitivity, identification reliability provided by spectral signatures acquired at 70 and 12 eV, and dynamic and linear range of response provided by soft ionization. RESULTS: Method effectiveness is validated on a sample set of oils from Picual olives at different ripening stages. Ripening markers [3,4-diethyl-1,5-hexadiene (RS/SR), 3,4-diethyl-1,5-hexadiene (meso), (5Z)-3-ethyl-1,5-octadiene, (5E)-3-ethyl-1,5-octadiene, (E, Z)-3,7-decadiene and (E, E)-3,7-decadiene, (Z)-2-hexenal, (Z)-3-hexenal and (Z)-3-hexenal, (E)-2-pentenal, (Z)-2-pentenal, 1-pentanol, 1-penten-3-ol, 3-pentanone, and 1-penten-3-one] and quality indexes [(Z)-3-hexenal/nonanal, (Z)-3-hexenal/octane, (E)-2-pentenal/nonanal, and (E)-2-pentenal/octane] are confirmed for their validity in HS linearity conditions. CONCLUSIONS: For the complex olive oil volatilome, the proposed approach offers concrete advantages for the validation of the informative role of existing analytes while suggesting new potential markers to be studied in larger sample sets. HIGHLIGHTS: The accurate fingerprinting of volatiles by HS-SPME operating in HS linearity conditions followed by GC×GC-TOF MS featuring tandem ionization gives the opportunity to improve the quality of analytical data and reliability of results.

Chemical fingerprinting strategies based on comprehensive two-dimensional gas chromatography combined with gas chromatography-olfactometry to capture the unique signature of Piemonte peppermint essential oil (Mentha x piperita var Italo-Mitcham).

Accurate, reliable, and informative mapping of untargeted and targeted components across many samples is here performed by combining off-line GC-Olfactometry (GC-O) and comprehensive two-dimensional gas chromatography (GC×GC) coupled to time-of-flight mass spectrometry with variable ionization energy (TOF MS featuring Tandem Ionization™). In particular, untargeted and targeted (UT) features patterns are processed by chromatographic fingerprinting, giving differential priority to potent odorants' retention-times regions. Distinguishing peppermint essential oil (EO) from Piedmont (Italy - Mentha × piperita L. var. Italo-Mitcham - Menta di Pancalieri EO), with its unique sensory fingerprint (i.e., freshness and long-lasting sweetness), from high-quality peppermint EOs produced in other areas poses a great challenge. Chromatographic UT fingerprinting provided a great chemical dimensionality by mapping more than 350 peak-regions at 70 eV and 135 at 12 eV. From them, 95 components were identified and responses compared to available literature. Then, potent odorants, detected by GC-O using the aroma extraction dilution analysis (AEDA), were tracked over the chromatographic space and tentatively identified. With the highest flavor dilution (FD), 1,8-cineole (eucalyptus, fresh, camphoraceous); menthone (minty, herbaceous); and menthofuran (minty, musty, petroleum-like) were highlighted. Responsible for creamy and coumarinic notes were the diasteroisomers of (3,6)-dimethyl-4,5,6,7-tetrahydrobenzo[b]-furan-2(3H)-one (i.e., menthofurolactones), detected in higher relative abundance in Pancalieri EOs. By prioritizing the investigation of volatiles on higher LogFD retention regions, including 131 untargeted/targeted features, Pancalieri EOs were separately clustered from United States samples. Besides pre-targeted analytes, additional untargeted features were post-processed for identification within marker chemicals. Myrtenyl methyl ether, ethyl 3-methyl butanoate, propyl-2-methylbutanoate, and (E)-2-hexenal were putatively identified. Of the "unknown - knowns" with diagnostic roles, all metadata were collected including low energy spectra at 12 eV, which were found to be highly complementary to 70 eV spectra.

Chromatographic Fingerprinting Enables Effective Discrimination and Identitation of High-Quality Italian Extra-Virgin Olive Oils.

The challenging process of high-quality food authentication takes advantage of highly informative chromatographic fingerprinting and its identitation potential. In this study, the unique chemical traits of the complex volatile fraction of extra-virgin olive oils from Italian production are captured by comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry and explored by pattern recognition algorithms. The consistent realignment of untargeted and targeted features of over 73 samples, including oils obtained by different olive cultivars (n = 24), harvest years (n = 3), and processing technologies, provides a solid foundation for sample identification and discrimination based on production region (n = 6). Through a dedicated multivariate statistics workflow, identitation is achieved by two-level partial least-square (PLS) regression, which highlights region diagnostic patterns accounting between 58 and 82 of untargeted and targeted compounds, while sample classification is performed by sequential application of soft independent modeling for class analogy (SIMCA) models, one for each production region. Samples are correctly classified in five of the six single-class models, and quality parameters [i.e., sensitivity, specificity, precision, efficiency, and area under the receiver operating characteristic curve (AUC)] are equal to 1.00.

An effective chromatographic fingerprinting workflow based on comprehensive two-dimensional gas chromatography - Mass spectrometry to establish volatiles patterns discriminative of spoiled hazelnuts (Corylus avellana L.).

The volatile fraction of hazelnuts encrypts information about: cultivar/geographical origin, post-harvest treatments, oxidative stability and sensory quality. However, sensory features could be buried under other dominant chemical signatures posing challenges to an effective classification based on pleasant/unpleasant notes. Here a novel workflow that combines Untargeted and Targeted (UT) fingerprinting on comprehensive two-dimensional gas-chromatographic patterns is developed to discriminate spoiled hazelnuts from those of acceptable quality. By flash-profiling, six hazelnut classes are defined: Mould, Mould-rancid-solvent, Rancid, Rancid-stale, Rancid-solvent, and Uncoded KO. Chromatographic fingerprinting on composite 2D chromatograms from samples belonging to the same class (i.e., composite class-images) enabled effective selection of chemical markers: (a) octanoic acid that guides the sensory classification being positively correlated to mould; (b) Æ´-nonalactone, Æ´-hexalactone, acetone, and 1-nonanol that are decisive to classify OK and rancid samples; (c) heptanoic and hexanoic acids and Æ´-octalactone present in high relative abundance in rancid-solvent and rancid-stale samples.

Delineating the extra-virgin olive oil aroma blueprint by multiple headspace solid phase microextraction and differential-flow modulated comprehensive two-dimensional gas chromatography.

Comprehensive two-dimensional gas chromatography with parallel mass spectrometry and flame ionization detection (GC × GC-MS/FID) enables effective chromatographic fingerprinting of complex samples by comprehensively mapping untargeted and targeted components. Moreover, the complementary characteristics of MS and FID open the possibility of performing multi-target quantitative profiling with great accuracy. If this synergy is applied to the complex volatile fraction of food, sample preparation is crucial and requires appropriate methodologies capable of providing true quantitative results. In this study, untargeted/targeted (UT) fingerprinting of extra-virgin olive oil volatile fractions is combined with accurate quantitative profiling by multiple headspace solid phase microextraction (MHS-SPME). External calibration on fifteen pre-selected analytes and FID predicted relative response factors (RRFs) enable the accurate quantification of forty-two analytes in total, including key-aroma compounds, potent odorants, and olive oil geographical markers. Results confirm good performances of comprehensive UT fingerprinting in developing classification models for geographical origin discrimination, while quantification by MHS-SPME provides accurate results and guarantees data referability and results transferability over years. Moreover, by this approach the extent of internal standardization procedure inaccuracy, largely adopted in food volatiles profiling, is measured. Internal standardization yielded an average relative error of 208 % for the fifteen calibrated compounds, with an overestimation of + 538% for (E)-2-hexenal, the most abundant yet informative volatile of olive oil, and a -89% and -80% for (E)-2-octenal and (E)-2-nonenal respectively, analytes with a lower HS distribution constant. Compared to existing methods based on 1D-GC, the current procedure offers better separation power and chromatographic resolution that greatly improve method specificity and selectivity and results in lower LODs and LOQs, high calibration performances (i.e., R2 and residual distribution), and wider linear range of responses. As an artificial intelligence smelling machine, the MHS-SPME-GC × GC-MS/FID method is here adopted to delineate extra-virgin olive oil aroma blueprints; an objective tool with great flexibility and reliability that can improve the quality and information power of each analytical run.

Chromatographic Fingerprinting by Template Matching for Data Collected by Comprehensive Two-Dimensional Gas Chromatography.

Data processing and evaluation are critical steps of comprehensive two-dimensional gas chromatography (GCxGC), particularly when coupled to mass spectrometry. The rich information encrypted in the data may be highly valuable but difficult to access efficiently. Data density and complexity can lead to long elaboration times and require laborious, analyst-dependent procedures. Effective yet accessible data processing tools, therefore, are key to enabling the spread and acceptance of this advanced multidimensional technique in laboratories for daily use. The data analysis protocol presented in this work uses chromatographic fingerprinting and template matching to achieve the goal of highly automated deconstruction of complex two-dimensional chromatograms into individual chemical features for advanced recognition of informative patterns within individual chromatograms and across sets of chromatograms. The protocol delivers high consistency and reliability with little intervention. At the same time, analyst supervision is possible in a variety of settings and constraint functions that can be customized to provide flexibility and capacity to adapt to different needs and goals. Template matching is shown here to be a powerful approach to explore extra-virgin olive oil volatilome. Cross-alignment of peaks is performed not only for known targets, but also for untargeted compounds, which significantly increases the characterization power for a wide range of applications. Examples are presented to evidence the performance for the classification and comparison of chromatographic patterns from sample sets analyzed under similar conditions.

A step forward in the equivalence between thermal and differential-flow modulated comprehensive two-dimensional gas chromatography methods.

Comprehensive two-dimensional gas chromatography (GC×GC) based on flow-modulation (FM) is gaining increasing attention as an alternative to thermal modulation (TM), the recognized GC×GC benchmark, thanks to its lower operational cost and rugged performance. An accessible, rational procedure to perform method translation between the two platforms would be highly valuable to facilitate compatibility and consequently extend the flexibility and applicability of GC×GC. To enable an effective transfer, the methodology needs to ensure preservation of the elution pattern, separation power, and sensitivity. Here, a loop-type thermal modulation system with dual detection (TM-GC×GC-MS/FID) used for the targeted analysis of allergens in fragrances is selected as reference method. Initially, six different columns configurations are systematically evaluated for the flow-modulated counterpart. The set-up providing the most consistent chromatographic separation (20 m x 0.18 mm dc x 0.18 µm df + 1.8 m x 0.18 mm dc x 0.18 µm df) is further evaluated to assess its overall performance in terms of sensitivity, linearity, accuracy, and pattern reliability. The experimental results convincingly show that the method translation procedure is effective and allows successful transfer of the target template metadata. Additionally, the FM-GC×GC-MS/FID system is suitable for challenging applications such as the quantitative profiling of complex fragrance materials.

Untargeted and Targeted Fingerprinting of Extra Virgin Olive Oil Volatiles by Comprehensive Two-Dimensional Gas Chromatography with Mass Spectrometry: Challenges in Long-Term Studies.

Comprehensive two-dimensional gas chromatography coupled with mass spectrometric detection (GC × GC-MS) offers an information-rich basis for effective chemical fingerprinting of food. However, GC × GC-MS yields 2D-peak patterns (i.e., sample 2D fingerprints) whose consistency may be affected by variables related to either the analytical platform or to the experimental parameters adopted for the analysis. This study focuses on the complex volatile fraction of extra-virgin olive oil and addresses 2D-peak patterns variations, including MS signal fluctuations, as they may occur in long-term studies where pedo-climatic, harvest year, or shelf life changes are studied. The 2D-pattern misalignments are forced by changing chromatographic settings and MS acquisition. All procedural steps, preceding pattern recognition by template matching, are analyzed and a rational workflow defined to accurately realign patterns and analytes metadata. Signal-to-noise ratio (SNR) detection threshold, reference spectra extraction, and similarity match factor threshold are critical to avoid false-negative matches. Distance thresholds and polynomial transform parameters are key for effective template matching. In targeted analysis (supervised workflow) with optimized parameters, method accuracy reaches 92.5% (i.e., % of true-positive matches) while for combined untargeted and targeted ( UT) fingerprinting (unsupervised workflow), accuracy reaches 97.9%. Response normalization also is examined, evidencing good performance of multiple internal standard normalization that effectively compensates for discriminations occurring during injection of highly volatile compounds. The resulting workflow is simple, effective, and time efficient.