Lucía Olmo-García

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.

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.

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.

Impact of industrial hammer mill rotor speed on extraction efficiency and quality of extra virgin olive oil

Crushing is a key step during olive oil extraction. Among commercial crushers, the hammer mill is the most widely used due to its robustness and high throughput. In the present work, the impact of hammer mill rotor speed on extraction yield and overall quality of super-high-density Arbosana olive oils were assessed in an industrial facility. Our results show that increasing the rotor speed from 2400rpm to 3600rpm led to a rise in oil yield of 1.2%, while conserving quality parameters. Sensory analysis showed more pungency with increased rotation speed, while others attributes were unaffected. Volatile compounds showed little variation with the differences in crusher speed; however, total phenols content, two relevant secoiridoids, and triterpenoids levels increased with rotor speed. Hammer mill rotor speed is a processing variable that can be tuned to increase the extraction efficiency and modulate the chemical composition of extra virgin olive oil.

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.

Unravelling the Distribution of Secondary Metabolites in Olea europaea L.: Exhaustive Characterization of Eight Olive-Tree Derived Matrices by Complementary Platforms (LC-ESI/APCI-MS and GC-APCI-MS)

In order to understand the distribution of the main secondary metabolites found in Olea europaea L., eight different samples (olive leaf, stem, seed, fruit skin and pulp, as well as virgin olive oil, olive oil obtained from stoned and dehydrated fruits and olive seed oil) coming from a Picudo cv. olive tree were analyzed. All the experimental conditions were selected so as to assure the maximum coverage of the metabolome of the samples under study within a single run. The use of LC and GC with high resolution MS (through different ionization sources, ESI and APCI) and the annotation strategies within MetaboScape 3.0 software allowed the identification of around 150 compounds in the profiles, showing great complementarity between the evaluated methodologies. The identified metabolites belonged to different chemical classes: triterpenic acids and dialcohols, tocopherols, sterols, free fatty acids, and several sub-types of phenolic compounds. The suitability of each platform and polarity (negative and positive) to determine each family of metabolites was evaluated in-depth, finding, for instance, that LC-ESI-MS (+) was the most efficient choice to ionize phenolic acids, secoiridoids, flavonoids and lignans and LC-APCI-MS was very appropriate for pentacyclic triterpenic acids (MS (−)) and sterols and tocopherols (MS (+)). Afterwards, a semi-quantitative comparison of the selected matrices was carried out, establishing their typical features (e.g., fruit skin was pointed out as the matrix with the highest relative amounts of phenolic acids, triterpenic compounds and hydroxylated fatty acids, and seed oil was distinctive for its high relative levels of acetoxypinoresinol and tocopherols).

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.

Geographical Indication Labels in Moroccan Olive Oil Sector: Territorial Dimension and Characterization of Typicality: A Case Study of Meknès Region

Geographical indications (GIs) implementation is, nowadays, one of the most promi‐ nent differentiation strategies used in olive oil market. The proliferation of these labels, however, causes debate and controversy, in particular regarding their usefulness, effectiveness, and suitability of some protected areas to acquire them. This chapter discusses the use of GI labels in olive oil market, and proposes a four‐stage methodo‐ logical approach to examine the potential of Meknès region—a Moroccan olive growing area—to acquire a GI label. Based on this approach, Meknès region territorial dimen‐ sions were defined, the typicality of its olive oil was characterized, a general scheme for the GI recognition was proposed, and the adopted strategy to enhance the meaning of this label on domestic, national, and international markets was highlighted. The main findings of this study justify the suitability of Meknès region to protect its olive oil with a GI label.