Nutrient and Bioactive Fraction Content of Olea europaea L. Leaves: Assessing the Impact of Drying Methods in a Comprehensive Study of Prominent Cultivars in Morocco.

This study explores the potential of olive leaves, long integral to Mediterranean traditional medicine, as a rich source of valuable compounds. The challenge lies in their considerable water content, hindering these compounds' full valorization. Four drying methods (air-drying, oven-drying, freeze-drying and solar-drying) were investigated for their impact on nutrient and bioactive compound content in the leaves of four olive varieties ("Arbequina", "Koroneiki", "Menara" and "Picholine Marocaine") cultivated in Morocco. In their fresh state, "Picholine Marocaine" exhibited the highest protein levels (6.11%), "Arbequina" had the highest phenolic content (20.18 mg gallic acid equivalents/g fresh weight (FW)), and "Koroneiki" and "Menara" were highest in flavonoids (3.28 mg quercetin equivalents/g FW). Specific drying methods proved optimal for different varieties. Oven-drying at 60 °C and 70 °C effectively preserved protein, while phenolic content varied with drying conditions. Air-drying and freeze-drying demonstrated effectiveness for flavonoids. In addition, an analytical approach using high-performance liquid chromatography and diode array detection (HPLC-DAD) was applied to investigate the effects of the different drying methods on the bioactive fraction of the analyzed samples. The results showed qualitative and quantitative differences depending on both the variety and the drying method used. A total of 11 phenolic compounds were tentatively identified, with oleuropein being the most abundant in all the samples analyzed. The freeze-dried samples showed the highest content of oleuropein in the varieties "Arbequina" and "Picholine Marocaine" compared to the other methods analyzed. In contrast, "Koroneiki" and "Menara" had higher oleuropein content when air dried. Overall, the obtained results highlight the importance of tailored drying techniques for the preservation of nutrients and bioactive compounds in olive leaves.

Study of the minor fraction of virgin olive oil by a multi-class GC-MS approach: Comprehensive quantitative characterization and varietal discrimination potential.

For the first time, a multi-class GC-MS method was applied to perform the quantitative-profiling of the minor fraction of VOOs (considering >40 compounds) in a single run. This comprehensive methodology has demonstrated a remarkable profiling ability on five groups of compounds (phenolic and triterpenic compounds, tocopherols, sterols and free fatty acids) with wide range of polarities/volatilities and chemical entities. After the complete analytical validation of the method, 32 VOO samples from eight different cultivars (some of them very scarcely studied before) were analyzed and the quantitative results were subjected to both non-supervised and supervised multivariate statistics for testing the capability of the determined VOO minor compounds to discriminate the varietal origin of the samples. Typical compositional profiles were defined for each cultivar and promising potential varietal markers were pointed out. The models built to discriminate Cayon and Maurino samples from the rest exhibited the best quality parameters. The relative levels of tocopherols together with characteristic concentration of luteolin, ß-sitosterol and tyrosol were, for instance, the most specific features of Cayon VOOs.

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.

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.

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.

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.

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.

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.

Assessing the varietal origin of extra-virgin olive oil using liquid chromatography fingerprints of phenolic compound, data fusion and chemometrics.

High Performance Liquid Chromatography (HPLC) with diode array (DAD) and fluorescence (FLD) detection was used to acquire the fingerprints of the phenolic fraction of monovarietal extra-virgin olive oils (extra-VOOs) collected over three consecutive crop seasons (2011/2012-2013/2014). The chromatographic fingerprints of 140 extra-VOO samples processed from olive fruits of seven olive varieties, were recorded and statistically treated for varietal authentication purposes. First, DAD and FLD chromatographic-fingerprint datasets were separately processed and, subsequently, were joined using "Low-level" and "Mid-Level" data fusion methods. After the preliminary examination by principal component analysis (PCA), three supervised pattern recognition techniques, Partial Least Squares Discriminant Analysis (PLS-DA), Soft Independent Modeling of Class Analogies (SIMCA) and K-Nearest Neighbors (k-NN) were applied to the four chromatographic-fingerprinting matrices. The classification models built were very sensitive and selective, showing considerably good recognition and prediction abilities. The combination "chromatographic dataset+chemometric technique" allowing the most accurate classification for each monovarietal extra-VOO was highlighted.

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.

Potential of LC Coupled to Fluorescence Detection in Food Metabolomics: Determination of Phenolic Compounds in Virgin Olive Oil.

A powerful chromatographic method coupled to a fluorescence detector was developed to determine the phenolic compounds present in virgin olive oil (VOO), with the aim to propose an appropriate alternative to liquid chromatography-mass spectrometry. An excitation wavelength of 285 nm was selected and four different emission wavelengths (316, 328, 350 and 450 nm) were simultaneously recorded, working therefore on "multi-emission" detection mode. With the use of commercially available standards and other standards obtained by semipreparative high performance liquid chromatography, it was possible to identify simple phenols, lignans, several complex phenols, and other phenolic compounds present in the matrix under study. A total of 26 phenolic compounds belonging to different chemical families were identified (23 of them were susceptible of being quantified). The proposed methodology provided detection and quantification limits within the ranges of 0.004-7.143 µg·mL(-1) and 0.013-23.810 µg·mL(-1), respectively. As far as the repeatability is concerned, the relative standard deviation values were below 0.43% for retention time, and 9.05% for peak area. The developed methodology was applied for the determination of phenolic compounds in ten VOOs, both monovarietals and blends. Secoiridoids were the most abundant fraction in all the samples, followed by simple phenolic alcohols, lignans, flavonoids, and phenolic acids (being the abundance order of the latter chemical classes logically depending on the variety and origin of the VOOs).

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.

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.

Comprehensive 3-year study of the phenolic profile of Moroccan monovarietal virgin olive oils from the Meknès region.

The phenolic fraction of monovarietal virgin olive oils (VOOs) from the main Moroccan cultivar Picholine marocaine (142 samples from three different subareas of the Meknès region) was studied over three consecutive crop seasons (2011, 2012, and 2013) using a powerful LC-MS methodology. First, LC-ESI-TOF MS was used to get a comprehensive characterization of the phenolic fraction; afterward, LC-ESI-IT MS was utilized for further identification (MS/MS experiments) and quantitation purposes. A total of 28 phenolic compounds (and quinic acid) were determined, revealing the complex profile of Meknès VOO, composed, in order of abundance, by secoiridoids, phenolic alcohols, lignans, flavonoids, and phenolic acids. Tukey's test was applied to ascertain possible significant intraregional and/or interannual variations of the phenolic content of the Meknès VOOs under study. Results showed that the content of phenolic compounds was mainly related to the crop season.

Quality and chemical profiles of monovarietal north Moroccan olive oils from "Picholine Marocaine" cultivar: registration database development and geographical discrimination.

Current knowledge of the quality and composition of Moroccan olive oil is still incomplete and no consistent database compiling its properties is available. This study was carried out to achieve a comprehensive characterisation of north Moroccan olive oils. Thus, 279 olive samples of "Picholine Marocaine" cultivar grown in 7 Moroccan regions were collected, and oils extracted over two consecutive crop seasons (2011 and 2012) and analysed (considering physicochemical quality parameters and purity criteria). Results indicated that all the studied samples showed values fulfilling the established limits set by the International Olive Council (IOC) standards, with the exception of 32 samples that had a linolenic acid content higher than 1%, which is the maximum value fixed by the IOC regulation. Furthermore, the usefulness of the evaluated parameters for tracing the geographical origin of the studied samples was tested by using canonical discriminant analysis. A good rate of correct classification and prediction was achieved.

First comprehensive characterization of volatile profile of north Moroccan olive oils: A geographic discriminant approach.

Herewith we have evaluated the variability of the composition in terms of volatile compounds of monovarietal "Picholine marocaine" olive oils and checked the possible influence of their geographical origin. For this purpose, 92 olive samples were collected during the harvesting period 2012/2013 from 7 north Moroccan regions, and the analysis of the volatile profiles of the obtained oils was performed by using headspace solid-phase microextraction coupled to gas chromatography with flame ionization and mass spectrometry detectors (HS-SPME/GC-FID-MS). A total of 40 volatile compounds belonging to different chemical classes were identified and quantified. Significant differences in the concentration levels of volatile constituents from oils of different geographical origins were found. Furthermore, for testing the ability of the identified volatile compounds for the geographical origin discrimination of the investigated oils, a stepwise linear discriminant analysis (s-LDA) was applied. Results revealed a very satisfactory classification of the studied oils according their geographic origin.

Potential of LC-MS phenolic profiling combined with multivariate analysis as an approach for the determination of the geographical origin of north Moroccan virgin olive oils.

The applicability of two different platforms (LC-ESI-TOF MS and LC-ESI-IT MS) as powerful tools for the characterisation and subsequent quantification of the phenolic compounds present in north Moroccan virgin olive oils was assessed in this study. 156 olives samples of "Picholine Marocaine" cultivar grown in 7 Moroccan regions were collected and olive oils extracted. The phenolic profiles of these olive oils were studied using a resolutive chromatographic method coupled to ESI-TOF MS (for initial characterisation purposes) and coupled to ESI-IT MS (for further identification and quantification). 25 phenolic compounds belonging to different chemical families were identified and quantified. Secoiridoids were the most abundant phenols in all the samples, followed by phenolic alcohols, lignans and flavonoids, respectively. For testing the ability of phenolic profiles for tracing the geographical origin of the investigated oils, multivariate analysis tools were used, getting a good rate of correct classification and prediction by using a cross validation procedure.