Aadil Bajoub

Comparing two metabolic profiling approaches (liquid chromatography and gas chromatography coupled to mass spectrometry) for extra-virgin olive oil phenolic compounds analysis: A botanical classification perspective.

Over the last decades, the phenolic compounds from virgin olive oil (VOO) have become the subject of intensive research because of their biological activities and their influence on some of the most relevant attributes of this interesting matrix. Developing metabolic profiling approaches to determine them in monovarietal virgin olive oils could help to gain a deeper insight into olive oil phenolic compounds composition as well as to promote their use for botanical origin tracing purposes. To this end, two approaches were comparatively investigated (LC-ESI-TOF MS and GC-APCI-TOF MS) to evaluate their capacity to properly classify 25 olive oil samples belonging to five different varieties (Arbequina, Cornicabra, Hojiblanca, Frantoio and Picual), using the entire chromatographic phenolic profiles combined to chemometrics (principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA)). The application of PCA to LC-MS and GC-MS data showed the natural clustering of the samples, seeing that 2 varieties were dominating the models (Arbequina and Frantoio), suppressing any possible discrimination among the other cultivars. Afterwards, PLS-DA was used to build four different efficient predictive models for varietal classification of the samples under study. The varietal markers pointed out by each platform were compared. In general, with the exception of one GC-MS model, all exhibited proper quality parameters. The models constructed by using the LC-MS data demonstrated superior classification ability.

Evaluating the potential of LC coupled to three alternative detection systems (ESI-IT, APCI-TOF and DAD) for the targeted determination of triterpenic acids and dialcohols in olive tissues.

Herewith the development of a rapid and powerful LC methodology (with three different detectors) is presented to determine triterpenic acids and dialcohols in extracts from Olea europaea tissues (olive skin, pulp and leaves). After the proper optimization of the LC, DAD and MS conditions and the comprehensive characterization of the behavior of each analyte in ESI and APCI (with accurate m/z signals and, in ESI, with MS/MS data too), the method was fully validated. DAD, ESI-IT MS and APCI-QTOF MS were used as detection systems to give different alternatives to carry out the accurate determination of these analytes, evaluate their analytical performance, advantages and drawbacks, and check whether the quantitative results achieved by the three platforms were in good agreement. ESI-IT MS gave the lowest detection limits (3-455 μg/L) followed by APCI-QTOF MS (22-408 μg/L); in contrast, DAD (83-600 μg/L) had the widest dynamic range. The RSD values for inter-day repeatability were found below 11.82% in all the cases. No statistically significant differences were found among the quantitative results from the three detectors. Olive leaves showed the highest concentration levels of ursolic acid (1.8 mg/g), erythrodiol (1.6 mg/g) and uvaol (1.2mg/g), whereas the olive skin was the richest matrix in terms of maslinic (80 mg/g), betulinic (0.20mg/g), and oleanolic (26 mg/g) acids. Concentration values of triterpenic acids were established by first time for skinless olive pulp, and were found around 65, 1.2, 55 and 4.4 μg/g for maslinic, betulinic, oleanolic and ursolic acids, respectively.

Olive Oil Authentication: A Comparative Analysis of Regulatory Frameworks with Especial Emphasis on Quality and Authenticity Indices, and Recent Analytical Techniques Developed for Their Assessment. A Review

ABSTRACT Over the last decades, olive oil quality and authenticity control has become an issue of great importance to consumers, suppliers, retailers, and regulators in both traditional and emerging olive oil producing countries, mainly due to the increasing worldwide popularity and the trade globalization of this product. Thus, in order to ensure olive oil authentication, various national and international laws and regulations have been adopted, although some of them are actually causing an enormous debate about the risk that they can represent for the harmonization of international olive oil trade standards. Within this context, this review was designed to provide a critical overview and comparative analysis of selected regulatory frameworks for olive oil authentication, with special emphasis on the quality and purity criteria considered by these regulation systems, their thresholds and the analytical methods employed for monitoring them. To complete the general overview, recent analytical advances to overcome drawbacks and limitations of the official methods to evaluate olive oil quality and to determine possible adulterations were reviewed. Furthermore, the latest trends on analytical approaches to assess the olive oil geographical and varietal origin traceability were also examined.

In-Depth Two-Year Study of Phenolic Profile Variability among Olive Oils from Autochthonous and Mediterranean Varieties in Morocco, as Revealed by a LC-MS Chemometric Profiling Approach.

Olive oil phenolic fraction considerably contributes to the sensory quality and nutritional value of this foodstuff. Herein, the phenolic fraction of 203 olive oil samples extracted from fruits of four autochthonous Moroccan cultivars (“Picholine Marocaine”, “Dahbia”, “Haouzia” and “Menara”), and nine Mediterranean varieties recently introduced in Morocco (“Arbequina”, “Arbosana”, “Cornicabra”, “Frantoio”, “Hojiblanca”, “Koroneiki”, “Manzanilla”, “Picholine de Languedoc” and “Picual”), were explored over two consecutive crop seasons (2012/2013 and 2013/2014) by using liquid chromatography-mass spectrometry. A total of 32 phenolic compounds (and quinic acid), belonging to five chemical classes (secoiridoids, simple phenols, flavonoids, lignans and phenolic acids) were identified and quantified. Phenolic profiling revealed that the determined phenolic compounds showed variety-dependent levels, being, at the same time, significantly affected by the crop season. Moreover, based on the obtained phenolic composition and chemometric linear discriminant analysis, statistical models were obtained allowing a very satisfactory classification and prediction of the varietal origin of the studied oils.

A metabolic fingerprinting approach based on selected ion flow tube mass spectrometry (SIFT-MS) and chemometrics: A reliable tool for Mediterranean origin-labeled olive oils authentication

Selected Ion flow tube mass spectrometry (SIFT-MS) in combination with chemometrics was used to authenticate the geographical origin of Mediterranean virgin olive oils (VOOs) produced under geographical origin labels. In particular, 130 oil samples from six different Mediterranean regions (Kalamata (Greece); Toscana (Italy); Meknès and Tyout (Morocco); and Priego de Córdoba and Baena (Spain)) were considered. The headspace volatile fingerprints were measured by SIFT-MS in full scan with H3O+, NO+ and O2+ as precursor ions and the results were subjected to chemometric treatments. Principal Component Analysis (PCA) was used for preliminary multivariate data analysis and Partial Least Squares-Discriminant Analysis (PLS-DA) was applied to build different models (considering the three reagent ions) to classify samples according to the country of origin and regions (within the same country). The multi-class PLS-DA models showed very good performance in terms of fitting accuracy (98.90-100%) and prediction accuracy (96.70-100% accuracy for cross validation and 97.30-100% accuracy for external validation (test set)). Considering the two-class PLS-DA models, the one for the Spanish samples showed 100% sensitivity, specificity and accuracy in calibration, cross validation and external validation; the model for Moroccan oils also showed very satisfactory results (with perfect scores for almost every parameter in all the cases).

Development and validation of LC-MS-based alternative methodologies to GC–MS for the simultaneous determination of triterpenic acids and dialcohols in virgin olive oil

Pentacyclic triterpenes are minor, but very relevant compounds found in virgin olive oil (VOO). A rapid and reliable LC-MS method for determining the triterpenic acids and dialcohols (after ultrasound assisted extraction) from VOO has been developed, giving an alternative to the widely used GC (FID/MS) methodologies. The analytical parameters of the proposed method were exhaustively checked, establishing limits of detection (from 1 to 95µg/l) and quantification, precision (RSD values for inter-day repeatability were found between 4.2 and 7.3% considering area values), trueness (within the range 92.7 and 100.5%) and evaluating possible matrix effect (which was no significant). The method was applied to the analysis of six triterpenic compounds in 11 monovarietal VOOs and the results compared with the quantitative GC-MS data. Moreover, the direct injection (after a simple dilution) of the samples into the LC-MS system was also tested, in an attempt to proffer an even simpler sample treatment.

Deep insight into the minor fraction of virgin olive oil by using LC-MS and GC-MS multi-class methodologies

Several analytical methods are available to evaluate virgin olive oil (VOO) minor compounds; however, multi-class methodologies are yet rarely studied. Herewith, LC-MS and GC-MS platforms were used to develop two methods capable of simultaneously determine more than 40 compounds belonging to different VOO minor chemical classes within a single run. A non-selective and highly efficient liquid-liquid extraction protocol was optimized for VOO minor components isolation. The separation and detection conditions were adjusted for determining phenolic and triterpenic compounds, free fatty acids and tocopherols by LC-MS, plus sterols and hydrocarbons by GC-MS. Chromatographic analysis times were 31 and 50 min, respectively. A comparative assessment of both methods in terms of analytical performance, easiness, cost and adequacy to the analysis of each class was carried out. The emergence of this kind of multi-class analytical methodologies greatly increases throughput and reduces cost, while avoiding the complexity and redundancy of single-chemical class determinations.

Metabolic profiling approach to determine phenolic compounds of virgin olive oil by direct injection and liquid chromatography coupled to mass spectrometry

A LC-MS method involving direct injection of extra-virgin olive oil (EVOO) - after a simple dilution - for determining its phenolic compounds has been developed. Optimization of the most appropriate solvent for sample dilution, selection of the optimum oil/solvent ratio, and establishment of column cleaning strategy and maximum number of injections were some of the most relevant steps. Then, the analytical parameters of the method were evaluated, establishing LOD (from 3.3 to 31.6µg/L) and LOQ, precision (RSD values for inter-day repeatability were found between 3.49 and 6.12%), and trueness (within the range 89.9-102.3% for 1.0mg/L) and checking possible matrix effect (which was no significant). Three kinds of calibration were used: external standard, standard addition and calibration in a phenols-free matrix, which was subsequently applied to quantify the phenolic compounds in 16 EVOOs (from 6 cultivars). A total of 21 compounds were determined without the need of using any extraction protocol.

Targeted LC-MS Approach to Study the Evolution over the Harvesting Season of Six Important Metabolites in Fruits from Different Avocado Cultivars

Herewith, a longitudinal study where the metabolic evolution of six relevant metabolites (p-coumaric, ferulic, chlorogenic, pantothenic and abscisic acids, and epicatechin) in four commercially important avocado varieties collected at different times over their harvest season is presented with the aim of evaluating the metabolic changes that take place during fruit development and maturation. The selection of these metabolites was based on the fact that they have been previously identified as potential varietal and/or ripening markers of avocado fruits and are produced by the phenylpropanoid pathway. Avocado metabolic profiles can be highly variable within a system, showing variations even from day to day. Thus, a longitudinal study design was selected to overcome possible negative effects of this variability on the interpretation of data, considering multiple sampling over time. A powerful HPLC-ESI-IT MS method was optimized and fully validated, and subsequently, a very unique sample selection (composed by 172 avocado extracts) was analyzed and the selected metabolites quantified. The content of pantothenic, p-coumaric, and ferulic acids was quite similar in the four avocado varieties studied. However, epicatechin, chlorogenic, and abscisic acid concentrations showed significant differences; ‘Bacon’ was the cultivar with the highest content of chlorogenic acid, ‘Hass’ and ‘Bacon’ of epicatechin, and ‘Fuerte’ and ‘Reed’ were the richest in terms of abscisic acid.

Establishing the Phenolic Composition of Olea europaea L. Leaves from Cultivars Grown in Morocco as a Crucial Step Towards Their Subsequent Exploitation

In Morocco, the recovery of olive agro-industrial by-products as potential sources of high-added value substances has been underestimated so far. A comprehensive quantitative characterization of olive leaves’ bioactive compounds is crucial for any attempt to change this situation and to implement the valorization concept in emerging countries. Thus, the phenolic fraction of olive leaves of 11 varieties (‘Arbequina’, ‘Hojiblanca’, ‘Frantoio’, ‘Koroneiki’, ‘Lechín’, ‘Lucque’, ‘Manzanilla’, ‘Picholine de Languedoc’, ‘Picholine Marocaine’, ‘Picual’ and ‘Verdal’), cultivated in the Moroccan Meknès region, was investigated. Thirty eight phenolic or related compounds (including 16 secoiridoids, nine flavonoids in their aglycone form, seven flavonoids in glycosylated form, four simple phenols, one phenolic acid and one lignan) were determined in a total of 55 samples by using ultrasonic-assisted extraction and liquid chromatography coupled to electrospray ionization-ion trap mass spectrometry (LC-ESI-IT MS). Very remarkable quantitative differences were observed among the profiles of the studied cultivars. ‘Picholine Marocaine’ variety exhibited the highest total phenolic content (around 44 g/kg dry weight (DW)), and logically showed the highest concentration in terms of various individual compounds. In addition, chemometrics (principal components analysis (PCA) and stepwise-linear discriminant analysis (s-LDA)) were applied to the quantitative phenolic compound data, allowing good discrimination of the selected samples according to their varietal origin.