Elena Hurtado-Fernández

Application and potential of capillary electroseparation methods to determine antioxidant phenolic compounds from plant food material.

Antioxidants are one of the most common active ingredients of nutritionally functional foods which can play an important role in the prevention of oxidation and cellular damage inhibiting or delaying the oxidative processes. In recent years there has been an increased interest in the application of antioxidants to medical treatment as information is constantly gathered linking the development of human diseases to oxidative stress. Within antioxidants, phenolic molecules are an important category of compounds, commonly present in a wide variety of plant food materials. Their correct determination is pivotal nowadays and involves their extraction from the sample, analytical separation, identification, quantification and interpretation of the data. The aim of this review is to provide an overview about all the necessary steps of any analytical procedure to achieve the determination of phenolic compounds from plant matrices, paying particular attention to the application and potential of capillary electroseparation methods. Since it is quite complicated to establish a classification of plant food material, and to structure the current review, we will group the different matrices as follows: fruits, vegetables, herbs, spices and medicinal plants, beverages, vegetable oils, cereals, legumes and nuts and other matrices (including cocoa beans and bee products). At the end of the overview, we include two sections to explain the usefulness of the data about phenols provided by capillary electrophoresis and the newest trends.

Profiling LC-DAD-ESI-TOF MS method for the determination of phenolic metabolites from avocado (Persea americana).

A powerful HPLC-DAD-ESI-TOF MS method was established for the efficient identification of the chemical constituents in the methanolic extracts of avocado (Persea americana). Separation and detection conditions were optimized by using a standard mix containing 39 compounds belonging to phenolic acids and different categories of flavonoids, analytes that could be potentially present in the avocado extracts. Optimum LC separation was achieved on a Zorbax Eclipse Plus C18 analytical column (4.6×150 mm, 1.8 μm particle size) by gradient elution with water+acetic acid (0.5%) and acetonitrile as mobile phases, at a flow rate of 1.6 mL/min. The detection was carried out by ultraviolet-visible absorption and ESI-TOF MS. The developed method was applied to the study of 3 different varieties of avocado, and 17 compounds were unequivocally identified with standards. Moreover, around 25 analytes were tentatively identified by taking into account the accuracy and isotopic information provided by TOF MS.

Ultra high performance liquid chromatography-time of flight mass spectrometry for analysis of avocado fruit metabolites: Method evaluation and applicability to the analysis of ripening degrees

We have developed an analytical method using UHPLC-UV/ESI-TOF MS for the comprehensive profiling of the metabolites found in the methanolic extracts of 13 different varieties of avocado at two different ripening degrees. Both chromatographic and detection parameters were optimized in order to maximize the number of compounds detected and the sensitivity. After achieving the optimum conditions, we performed a complete analytical validation of the method with respect to its linearity, sensitivity, precision, accuracy and possible matrix effects. The LODs ranged from 1.64 to 730.54 ppb (in negative polarity) for benzoic acid and chrysin, respectively, whilst they were found within the range from 0.51 to 310.23 ppb in positive polarity. The RSDs for repeatability test did not exceed 7.01% and the accuracy ranged from 97.2% to 102.0%. Our method was then applied to the analysis of real avocado samples and advanced data processing and multivariate statistical analysis (PCA, PLS-DA) were carried out to discriminate/classify the examined avocado varieties. About 200 compounds belonging to various structural classes were tentatively identified; we are certain about the identity of around 60 compounds, 20 of which have been quantified in terms of their own commercially available standard.

Determination of changes in the metabolic profile of avocado fruits (Persea americana) by two CE-MS approaches (targeted and non-targeted).

A CZE method with two different MS detection conditions (MRM and Full Scan) was developed to determine qualitative and quantitative changes in the metabolic profile of avocado fruits (Persea americana). LODs in MRM approach were found between 20.1 and 203.0 ppb for abscisic acid and perseitol, respectively, whilst in Full Scan, varied within the range 0.22–1.90 ppm for the same metabolites. The RSDs for reproducibility test did not exceed 11.45%. The two MS approaches were used to quantify 10 metabolites (phenolic acids, flavonoids, a carbohydrate, an organic acid, a vitamin and a phytohormone) in 18 samples of avocado at different ripening states, and the achieved results were compared. Perseitol, quinic, chlorogenic, trans‐cinnamic, pantothenic and abscisic acids, as well as epicatechin and catechin decreased during the ripening process, whereas ferulic and p‐coumaric acids showed the opposite trend. Moreover, some other unknown compounds whose concentration changed largely during ripening were also studied by MS/MS and QTOF MS to get a tentative identification.

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.

Online spectral library for GC-atmospheric pressure chemical ionization–ToF MS

BACKGROUND Invented more than three decades ago by Horning, GC-MS under atmospheric pressure chemical ionization (GC-APCI-MS) has only recently emerged from years of obscurity. However, the general acceptance of GC-APCI-MS is certainly constrained by the lack of spectral libraries, which make the traditional GC-MS approaches so powerful. RESULTS Here we present a concept of a GC-APCI-QqToF spectral library. The library is web-based, fully searchable and at moment includes spectra of 150 compounds from the most common chemical families. The fragmentation pattern of some chemical families is explained and a protocol for de novo identification has been provided in order to facilitate the identification of unknown compounds. CONCLUSION A library for GC-APCI-QqToF is now publicly available online.

Metabolomic analysis of avocado fruits by GC-APCI-TOF MS: effects of ripening degrees and fruit varieties

In order to investigate avocado fruit ripening, nontargeted GC-APCI-TOF MS metabolic profiling analyses were carried out. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were used to explore the metabolic profiles from fruit samples of 13 varieties at two different ripening degrees. Mannoheptulose; pentadecylfuran; aspartic, malic, stearic, citric and pantothenic acids; mannitol; and β-sitosterol were some of the metabolites found as more influential for the PLS-DA model. The similarities among genetically related samples (putative mutants of “Hass”) and their metabolic differences from the rest of the varieties under study have also been evaluated. The achieved results reveal new insights into avocado fruit composition and metabolite changes, demonstrating therefore the value of metabolomics as a functional genomics tool in characterizing the mechanism of fruit ripening development, a key developmental stage in most economically important fruit crops.

Merging a sensitive capillary electrophoresis-ultraviolet detection method with chemometric exploratory data analysis for the determination of phenolic acids and subsequent characterization of avocado fruit.

Herein we present the development of a powerful CE-UV method able to detect and quantify an important number of phenolic acids in 13 varieties of avocado fruits at 2 ripening stages. All the variables involved in CE separation were exhaustively optimized and the best results were obtained with a capillary of 50 μm i.d. × 50 cm effective length, sodium tetraborate 40 mM at a pH of 9.4, 30 kV, 25 °C, 10s of hydrodynamic injection (0.5 psi) and UV detection at 254 nm. This optimal methodology was fully validated and then applied to different avocado samples. The number of phenolic acids determined varied from 8 to 14 compounds; in general, they were in concentrations ranging from 0.13 ppm to 3.82 ppm, except p-coumaric, benzoic and protocatechuic acids, which were found at higher concentrations. Principal component analysis (PCA) was applied to highlight the differences between varieties and ripening degrees, looking for the most influential analytes.

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.

Avocado fruit—Persea americana

Abstract Avocado (Persea americana) is a tropical or subtropical fruit native from South America, which has been referred to as the most nutritious of all fruits. It is highly valued not only for its unique texture, exquisite taste and aroma, and nutritional profile, but also for the numerous healthy benefits that it possesses. For all of this, avocado has gained worldwide recognition and its consumption has considerably increased in the last years. This chapter gives an overview about different aspects of the avocado, including, the origin of this tropical fruit, its physiology, biochemistry, chemical composition, sensory characteristics, harvest season and conservation, as well as information about its production and industrial applications.