Determination of astaxanthin and astaxanthin esters in three samples of shrimp waste (Parapenaeus longirostris) by high performance liquid chromatography coupled photo-diode array and mass spectrometry detection.

The present study aimed to identify the content of astaxanthin and its esterified forms using high-performance liquid chromatography coupled with diode array and atmospheric pressure chemical ionisation mass spectrometry detection in three samples of shrimp waste. The analyses revealed twenty-one astaxanthin derivatives, including astaxanthin in free form, across all three extracts with the highest number of derivatives observed in the head extract. The shell extract had a lower content of astaxanthin and its esterified forms, with monoesterified astaxanthins being the major components, with contents ranging from 0.5-1 mg g-1. On the other hand, in both global waste and head extracts, astaxanthin diesters were found to be the dominant bioactive compounds, with contents ranging from 0.7-5.2 mg g-1 and 10.2-18.2 mg g-1, respectively. Notably, the astaxanthin content extracted from head was significantly higher compared to other extracts, indicating its potential as a valuable source of bioactive compounds.

Development and validation of a multi-class analysis of pesticides in corn products by comprehensive two-dimensional liquid chromatography-tandem mass spectrometry.

Due to the growing trend of organic food, there is still concern over the use of chemicals and pesticides in agriculture. In recent years, several procedures have been validated for the control of pesticides in food. In the present research, a comprehensive two-dimensional liquid chromatography coupled with tandem mass spectrometry is proposed for the first time for a multi-class analysis of 112 pesticides in corn-based products. Notably, a "reduced" QuEChERS-based method as extraction and clean-up procedure prior to the analysis, was successfully employed. Limits of quantification values were lower than the ones fixed by the European legislation; intra-day and inter-day precision were lower than 12.9% and 15.1%, respectively (at the 500 µg/kg concentration levels). Over 70% of the analytes provided recoveries between 70% and 120% range (at 50, 500 and 1000 µg/kg concentration levels) with standard deviation values below 20%. In addition, matrix effect values were in the range between 13% to 161%. The method was applied to the analysis of real samples, and three pesticides were detected at trace levels in both samples. The findings of this work pave the way for the treatment of complex matrices such as corn products.

Metabolomic profiling and antianginal activity of the bark of Sterculia setigera from Mali.

The present work focuses on the phytochemical characterization and evaluation of antianginal activity of the bark of Sterculia setigera. It was collected and authenticated in the African region of Mali, where the local population largely employs this plant for the treatment of several diseases. In the context of traditional or folk medicine and recent progresses in alternative medicine practices, it is essential to expand the knowledge about the chemical composition of such medicinal plants. In this research, a direct-Mass Spectrometry (MS) technique, known as Rapid Evaporative Ionization Mass Spectrometry (REIMS) was used for the identification of the main constituents of the Sterculia setigera bark. The REIMS source is here coupled with an electroknife as sampling device, so that the dried and pulverized bark was directly cut through the electroknife to generate a vapor, which was online transferred to the source via a Venture tube. In this way, an ambient MS approach was realized, which avoids any sample preparation procedure or pretreatment; the sample was analyzed in its native state according to a time-saving analytical process. A quadrupole-time of flight MS/MS analyzer was exploited for the identification process, based on mass accuracy data and MS/MS experiments for structure elucidation purposes. Lipids, including triterpenes, fatty acids, γ-sitosterol and α-tocopherol, and phenolic compounds were identified, some of them reported for the first time in a plant of the Sterculia genus and further confirmed through a gas chromatography-mass spectrometry analysis. The obtained metabolomic profile was successfully correlated to the antianginal activity of this plant.

Determination of the polyphenolic content of berry juices using focusing-modulated comprehensive two-dimensional liquid chromatography coupled to mass spectrometry detection.

In this work, a comprehensive two-dimensional liquid chromatography system, comprised of a ZIC-HILIC and C18 columns in the first and second dimensions, respectively, was tuned and employed for attaining high resolution profiles of the polyphenolic pattern in seven commercial berry juices. The developed HILIC × RP-LC method was validated in terms of linearity range, correlation coefficients, limit of detection, limit of quantification, precision (intra- and inter-day), and recovery. A total of 104 polyphenolic compounds belonging to different chemical classes (hydroxybenzoic and cinnamic acid derivatives, flavone glycosides, flavonols, flavonol glycosides, dihydroflavonols, and anthocyanin glycosides) have been characterized and quantified in the juices investigated. Despite the constituents being similar, a notable quantitative variation among the analyzed berry species was observed. Elderberry contained the highest amount of polyphenols (918 ± 1.10 mg 100 mL-1), followed by chokeberry (516 ± 0.08 mg 100 mL-1). On the other hand, raspberry contained the lowest amount (104 ± 1.21 mg 100 mL-1). Further, total phenolic, flavonoid, and anthocyanin contents were determined spectrophotometrically, yielding consistent results. The free-radical scavenging activity (DPPH test) and reducing power of the juices, expressed as IC50 (µL mL-1) and mg ASE mL-1, varied from 2.79 ± 0.03 (honeyberry) to 31.66 ± 0.02 (blueberry) and from 1.71 ± 0.01 (blueberry) to 8.89 ± 0.12 (chokeberry), respectively. Such a ZIC-HILIC × C18 platform based on focusing modulation, never employed so far for berry juices, showed a remarkable separation capability with high values of corrected peak capacity (up to 1372) and orthogonality (Ao up to 0.80), thus providing a great applicability to be advantageously employed for other complex food samples.

Phytochemical Characterization of Rhus coriaria L. Extracts by Headspace Solid-Phase Micro Extraction Gas Chromatography, Comprehensive Two-Dimensional Liquid Chromatography, and Antioxidant Activity Evaluation.

Rhus coriaria L. (Anacardiaceae), commonly known as sumac, has been used since ancient times for many different applications, and nowadays is used mostly as a spice obtained from its in the Mediterranean and the Middle ground fruits and employed for flavoring and garnishing food, predominantly Eastern regions. Traditionally, sumac has been also used in popular medicine for the treatment of many ailments including hemorrhoids, wound healing, diarrhea, ulcers, and eye inflammation. Sumac drupes are indeed rich in various classes of phytochemicals including organic acids, flavonoids, tannins, and others, which are responsible of their powerful antioxidant capacity, from which treatment of many common diseases such as cardiovascular disease, diabetes, and cancer could benefit. In this work we evaluated the influence of fruit ripeness, conservation, and processing. To this aim, a phytochemical characterization of six different samples of Rhus coriaria L. was carried out. Specifically, headspace solid-phase micro extraction gas chromatography coupled to mass spectrometry and comprehensive two-dimensional liquid chromatography coupled to photodiode array and mass spectrometry detection, were employed. A total of 263 volatile compounds, including terpene hydrocarbons, acids, and aldehydes, as well as 83 polyphenolic compounds, mainly gallic acid derivatives, were positively identified. All samples showed a significant antioxidant activity by means of oxygen radical absorbance capacity, in line with their polyphenolic content and composition. Such findings set a solid ground to support the utilization of this plant as an attractive target for novel nutraceutical approaches and for drug discovery.

Elucidation of Antioxidant Compounds in Moroccan Chamaerops humilis L. Fruits by GC-MS and HPLC-MS Techniques.

The aim of this study was to characterize the phytochemical content as well as the antioxidant ability of the Moroccan species Chamaerops humilis L. Besides crude ethanolic extract, two extracts obtained by sonication using two solvents with increased polarity, namely ethyl acetate (EtOAc) and methanol-water (MeOH-H2O) 80:20 (v/v), were investigated by both spectroscopy and chromatography methods. Between the two extracts, the MeOH-H2O one showed the highest total polyphenolic content equal to 32.7 ± 0.1 mg GAE/g DM with respect to the EtOAc extract (3.6 ± 0.5 mg GAE/g DM). Concerning the antioxidant activity of the two extracts, the EtOAc one yielded the highest value (1.9 ± 0.1 mg/mL) with respect to MeOH-H2O (0.4 ± 0.1 mg/mL). The C. humilisn-hexane fraction, analyzed by GC-MS, exhibited 69 compounds belonging to different chemical classes, with n-Hexadecanoic acid as a major compound (21.75%), whereas the polyphenolic profile, elucidated by HPLC-PDA/MS, led to the identification of a total of sixteen and thirteen different compounds in both EtOAc (major component: ferulic acid: 104.7 ± 2.52 µg/g) and MeOH-H2O extracts (major component: chlorogenic acid: 45.4 ± 1.59 µg/g), respectively. The attained results clearly highlight the potential of C. humilis as an important source of bioactive components, making it a valuable candidate to be advantageously added to the daily diet. Furthermore, this study provides the scientific basis for the exploitation of the Doum in the food, pharmaceutical and nutraceutical industries.

Phytochemical Profile, Antioxidant Capacity, α-Amylase and α-Glucosidase Inhibitory Potential of Wild Moroccan Inula viscosa (L.) Aiton Leaves.

Medicinal plants offer imperative sources of innovative chemical substances with important potential therapeutic effects. Among them, the members of the genus Inula have been widely used in traditional medicine for the treatment of several diseases. The present study investigated the antioxidant (DPPH, ABTS and FRAP assays) and the in vitro anti-hyperglycemic potential of aerial parts of Inula viscosa (L.) Aiton (I. viscosa) extracts through the inhibition of digestive enzymes (α-amylase and α-glucosidase), responsible of the digestion of poly and oligosaccharides. The polyphenolic profile of the Inula viscosa (L.) Aiton EtOAc extract was also investigated using HPLC-DAD/ESI-MS analysis, whereas the volatile composition was elucidated by GC-MS. The chemical analysis resulted in the detection of twenty-one polyphenolic compounds, whereas the volatile profile highlighted the occurrence of forty-eight different compounds. Inula viscosa (L.) Aiton presented values as high as 87.2 ± 0.50 mg GAE/g and 78.6 ± 0.55mg CE/g, for gallic acid and catechin, respectively. The EtOAc extract exhibited the higher antioxidant activity compared to methanol and chloroform extracts in different tests with (IC50 = 0.6 ± 0.03 µg/mL; IC50 = 8.6 ± 0.08 µg/mL; 634.8 mg ± 1.45 AAE/g extract) in DPPH, ABTS and FRAP tests. Moreover, Inula viscosa (L.) Aiton leaves did show an important inhibitory effect against α-amylase and α-glucosidase. On the basis of the results achieved, such a species represents a promising traditional medicine, thanks to its remarkable content of functional bioactive compounds, thus opening new prospects for research and innovative phytopharmaceuticals developments.

Phytochemical Profile and Antioxidant Activity of the Aerial Part Extracts from Matthiola incana subsp. rupestris and subsp. pulchella (Brassicaceae) Endemic to Sicily.

As part of a project aimed at investigating the Matthiola taxa endemic to Sicily (Italy), this study focused on Matthiola incana, an edible species used in the traditional medicine of various countries. Herein, the characterization of phenolic and volatile compounds, the antioxidant capacity in vitro (1,1-diphenyl-2-picrylhydrazil (DPPH), reducing power and Fe2+ chelating activity assays) and the toxicity test (Artemia salina lethality bioassay) of the hydroalcoholic extracts from the aerial parts of M. incana subsp. rupestris from Mt. Pellegrino (Palermo) and Mt. Erice (Trapani), and of M. incana subsp. pulchella are reported. The results are compared with those previously shown for M. incana subsp. incana, to achieve a comprehensive overview of the three subspecies. The HPLC-PDA/ESI-MS and SPME-GC/MS analyses led to the identification of 13 phenolics and 54 volatile compounds. Differences in the qualitative-quantitative profile of these phytochemicals have been highlighted between the M. incana subspecies. The antioxidant tests showed different activity for the extracts, which were found to possess better chelating properties. At last, none of the tested extracts displayed toxicity against brine shrimp larvae. These findings enrich the knowledge on the Matthiola taxa growing wild in Sicily, both from the strictly systematic point of view and for the possible applications as sources bioactive compounds that can be used in the nutraceutical field.

Reversed phase versus hydrophilic interaction liquid chromatography as first dimension of comprehensive two-dimensional liquid chromatography systems for the elucidation of the polyphenolic content of food and natural products.

Comprehensive two-dimensional liquid chromatography is a well-established method for the unraveling of very complex real-world samples. With regard to food and natural products such a technique turned out to be a very promising approach due to its high resolving power and improved identification capability, especially in combination with mass spectrometry. In this context, polyphenols comprise a particular complex class of bioactive compounds, due to their nature and content in commonly consumed foodstuffs, making their analysis challenging. The present contribution shows an overview of the two commonly employed approaches used for polyphenol analysis, viz. RP-LC × RP-LC and HILIC × RP-LC. Furthermore, the latest implementations as well as limitations and future perspectives are critically reported.

Multidimensional liquid chromatography approaches for analysis of food contaminants.

In the past years, multidimensional liquid chromatography became very widespread for the complete separation of non-volatile analytes in complex matrices. The main advantage of these techniques, especially when coupled with mass spectrometry, is the enhancement of separation power or peak capacity, due to an increase in selectivity and sensitivity of the two systems. With respect to conventional one-dimensional liquid chromatography, multidimensional liquid chromatography allows us to resolve potential co-elutions and also minimize the matrix effect thus providing a more accurate quantitative analysis. This review provides an overview on the presence of contaminants in food, the main sources of contamination, and finally, the techniques used to reveal their presence. All different modes, investigated so far on this topic for heart-cutting and comprehensive techniques were described. Advantages and disadvantages of each method are reported in addition to the main food applications.

Comprehensive two-dimensional liquid chromatography-based quali-quantitative screening of aqueous phases from pyrolysis bio-oils.

Pyrolysis processes are an alternative to minimize the environmental problem associated to agrifood industrial wastes. The main product resulting from these processes is a high-value liquid product, called bio-oil. Recently, the use of comprehensive two-dimensional liquid chromatography (LC × LC) has been demonstrated as a useful tool to improve the characterization of the water-soluble phases of bio-oils, considering their complexity and high water content. However, the precise composition of bio-oils from different agrifood byproducts is still unknown. In the present study, the qualitative and quantitative screening of eight aqueous phases from different biomasses, not yet reported in the literature, using LC × LC is presented. The two-dimensional approach was based on the use of two reverse phase separations. An amide column in the first dimension together with a C18 column in the second dimension were employed. Thanks to the use of diode array and mass spectrometry detection, 28 compounds were identified and quantified in the aqueous phase samples with good figures of merit. Samples showed a distinct quali-quantitative composition and a great predominance of compounds belonging to aldehydes, ketones and phenols, most of them with high polarity.

Chemical Characterization of Three Accessions of Brassica juncea L. Extracts from Different Plant Tissues.

Indian mustard or Brassica juncea (B. juncea) is an oilseed plant used in many types of food (as mustard or IV range salad). It also has non-food uses (e.g., as green manure), and is a good model for phytoremediation of metals and pesticides. In recent years, it gained special attention due to its biological compounds and potential beneficial effects on human health. In this study, different tissues, namely leaves, stems, roots, and flowers of three accessions of B. juncea: ISCI 99 (Sample A), ISCI Top (Sample B), and "Broad-leaf" (Sample C) were analyzed by HPLC-PDA/ESI-MS/MS. Most polyphenols identified were bound to sugars and phenolic acids. Among the three cultivars, Sample A flowers turned were the richest ones, and the most abundant bioactive identified was represented by Isorhamnetin 3,7-diglucoside (683.62 µg/100 mg dry weight (DW) in Sample A, 433.65 µg/100 mg DW in Sample B, and 644.43 µg/100 mg DW in Sample C). In addition, the most complex samples, viz. leaves were analyzed by GC-FID/MS. The major volatile constituents of B. juncea L. leaves extract in the three cultivars were benzenepropanenitrile (34.94% in Sample B, 8.16% in Sample A, 6.24% in Sample C), followed by benzofuranone (8.54% in Sample A, 6.32% in Sample C, 3.64% in Sample B), and phytone (3.77% in Sample B, 2.85% in Sample A, 1.01% in Sample C). The overall evaluation of different tissues from three B. juncea accessions, through chemical analysis of the volatile and non-volatile compounds, can be advantageously taken into consideration for future use as dietary supplements and nutraceuticals in food matrices.

Polyphenolic compounds with biological activity in guabiroba fruits (Campomanesia xanthocarpa Berg.) by comprehensive two-dimensional liquid chromatography.

This study aimed to evaluate the polyphenolic composition along with the biological activity of guabiroba (Campomanesia xanthocarpa Berg.) fruits using comprehensive two-dimensional liquid chromatography (LC × LC). A simplex centroid design comprising three solvents (methanol, 2% acetic acid, and acetonitrile) was used to optimize the extraction mixture for polyphenols from ripe and unripe guabiroba fruits. A quantitative LC × LC platform was proposed to characterize the guabiroba extracts using a RP-Amide column and a C18 column in the first and second dimensions, respectively. Antidiabetic properties, using in vitro enzyme assay models and in vivo antioxidant activity with the eukaryote model Saccharomyces cerevisiae, was measured. Total phenolics compounds were more efficiently extracted with 2% acetic acid solution and acetonitrile (50:50, v/v). A total of 37 different compounds were identified and quantified using the proposed LC × LC method (linearity ranging from 0.9990 to 0.9994, intra- and interday precision from 0.40 to 10.57% and, accuracy from 81.89 to 108.98%). Significant differences were observed between ripe and unripe guabiroba fruits, especially for the compounds geraldone and methyl galangin isomer. Guabiroba fruits showed significant antidiabetic and antioxidant properties and may be potentially adopted as part of dietary strategies in the management of early stages of type 2 diabetes and associated complications.

8-Hydroxyquinoline-2-Carboxylic Acid as Possible Molybdophore: A Multi-Technique Approach to Define Its Chemical Speciation, Coordination and Sequestering Ability in Aqueous Solution.

8-hydroxyquinoline-2-carboxylic acid (8-HQA) has been found in high concentrations (0.5-5.0 mmol·dm-3) in the gut of Noctuid larvae (and in a few other lepidopterans), in which it is proposed to act as a siderophore. Since it is known that many natural siderophores are also involved in the uptake and metabolism of other essential elements than iron, this study reports some results on the investigation of 8-HQA interactions with molybdate (MoO42-, i.e., the main molybdenum form in aqueous environments), in order to understand the possible role of this ligand as molybdophore. A multi-technique approach has been adopted, in order to derive a comprehensive set of information necessary to assess the chemical speciation of the 8-HQA/MoO42- system, as well as the coordination behavior and the sequestering ability of 8-HQA towards molybdate. Chemical speciation studies have been performed in KCl(aq) at I = 0.2 mol·dm-3 and T = 298.15 K by ISE-H+ (glass electrode) potentiometric and UV/Vis spectrophotometric titrations. CV (Cyclic Voltammetry), DP-ASV (Differential Pulse-Anodic Stripping Voltammetry), ESI-MS experiments and quantum mechanical calculations have been also performed to derive information about the nature and possible structure of species formed. These results are also compared with those reported for the 8-HQA/Fe3+ system in terms of chemical speciation and sequestering ability of 8-HQA.

Evaluation of matrix effect in one-dimensional and comprehensive two-dimensional liquid chromatography for the determination of the phenolic fraction in extra virgin olive oils.

Olea europaea, meaning "European olive," is a small tree belonging to the family Oleaceae, occurring in the Mediterranean Basin. Olive oil is an essential component of a balanced diet because of its nutritional value. Among micronutrients, phenolic compounds did show important beneficial effects for human health. The majority of the research studies on the phenol content are carried out by liquid chromatography combined to photodiode array and/or mass spectrometry detection; however, because of matrix complexity, one-dimensional liquid chromatography cannot be sometimes sufficient to obtain rewarding separations, requiring more advanced analytical techniques. In this work, comprehensive two-dimensional liquid chromatography, incorporating RP-Amide and C18 stationary phases, in the first and second dimension, respectively, both under reversed phase conditions, was investigated for the determination of the phenolic fraction in extra virgin olive oil samples. As far as detection is concerned, triple quadrupole mass spectrometry was employed under multi reaction monitoring mode offering superior selectivity and sensitivity. The reduction of matrix effects, when using comprehensive two-dimensional liquid chromatography with respect to conventional one-dimensional liquid chromatography, was assessed by comparing the slopes of calibration curves built from standard solutions and spiked olive oil samples.

Determination of the Metabolite Content of Brassica juncea Cultivars Using Comprehensive Two-Dimensional Liquid Chromatography Coupled with a Photodiode Array and Mass Spectrometry Detection.

Plant-based foods are characterized by significant amounts of bioactive molecules with desirable health benefits beyond basic nutrition. The Brassicaceae (Cruciferae) family consists of 350 genera; among them, Brassica is the most important one, which includes some crops and species of great worldwide economic importance. In this work, the metabolite content of three different cultivars of Brassica juncea, namely ISCI Top, "Broad-leaf," and ISCI 99, was determined using comprehensive two-dimensional liquid chromatography coupled with a photodiode array and mass spectrometry detection. The analyses were carried out under reversed-phase conditions in both dimensions, using a combination of a 250-mm microbore cyano column and a 50-mm RP-Amide column in the first and second dimension (2D), respectively. A multi (three-step) segmented-in-fraction gradient for the 2D separation was advantageously investigated here for the first time, leading to the identification of 37 metabolites. In terms of resolving power, orthogonality values ranged from 62% to 69%, whereas the corrected peak capacity values were the highest for B. juncea ISCI Top (639), followed by B. juncea "Broad-leaf" (502). Regarding quantification, B. juncea cv. "Broad-leaf" presented the highest flavonoid content (1962.61 mg/kg) followed by B. juncea cv. ISCI Top (1002.03 mg/kg) and B. juncea cv. ISCI 99 (211.37 mg/kg).

Exploration of Rapid Evaporative-Ionization Mass Spectrometry as a Shotgun Approach for the Comprehensive Characterization of Kigelia Africana (Lam) Benth. Fruit.

Rapid evaporative-ionization mass spectrometry (REIMS) coupled with an electroknife as a sampling device was recently employed in many application fields to obtain a rapid characterization of different samples without any need for extraction or cleanup procedures. In the present research, REIMS was used to obtain a metabolic profiling of the Kigelia africana fruit, thus extending the applicability of such a technique to the investigation of phytochemical constituents. In particular, the advantages of REIMS linked to a typical electrosurgical handpiece were applied for a comprehensive screening of this botanical species, by exploiting the mass accuracy and tandem MS capabilities of a quadrupole-time of flight analyzer. Then, 78 biomolecules were positively identified, including phenols, fatty acids and phospholipids. In the last decade, Kigelia africana (Lam.) Benth. fruit has attracted special interest for its drug-like properties, e.g., its use for infertility treatments and as anti-tumor agent, as well as against fungal and bacterial infections, diabetes, and inflammatory processes. Many of these properties are currently correlated to the presence of phenolic compounds, also detected in the present study, while the native lipid composition is here reported for the first time and could open new directions in the evaluation of therapeutic activity.

Characterization of the polyphenolic fraction of pomegranate samples by comprehensive two-dimensional liquid chromatography coupled to mass spectrometry detection.

Punica granatum L., commonly known as pomegranate, is an ancient fruit widely consumed all over the world as fresh fruit or juice. In addition, it is extensively used in therapeutic formulas, cosmetics and food seasonings. The fruit is native to Afghanistan, Iran, China and the Indian sub-continent. The pomegranate market has steadily grown, presumably due to the increasing demand of health-conscious consumers for products with potential beneficial effects on human health, due to the synergistic presence of a unique and complex phytochemical composition that enclose anthocyanins, phenolic acids and hydrolysable tannins. Conventionally, for their analysis liquid chromatography is employed even though it can present some drawbacks in terms of resolving power. In this contribution, as a valuable alternative, comprehensive two-dimensional liquid chromatography with "shifted gradients" in the second dimension, was applied for the characterization of three pomegranate samples, leading to the identification of 37 different polyphenolic compounds.

Application of compressed fluid-based extraction and purification procedures to obtain astaxanthin-enriched extracts from Haematococcus pluvialis and characterization by comprehensive two-dimensional liquid chromatography coupled to mass spectrometry.

The green microalga Haematococcus pluvialis has been widely studied due to its capacity to accumulate great amounts of astaxanthin, a high-value carotenoid with biological activities. In the present work, two green compressed fluid-based processes, pressurized liquid extraction (PLE) and supercritical antisolvent fractionation (SAF), are integrated to obtain an astaxanthin-enriched extract from this microalga. PLE was carried out using pressurized ethanol as solvent, for 20 min, at 10 MPa, and 50 °C as extraction temperature. Subsequently, the obtained extract was processed by SAF to further purify the carotenoid fraction. The SAF process was optimized using a 3-level factorial experimental design and considering three experimental variables: (i) CO2 pressure (10-30 MPa), (ii) percentage of water in the PLE extract (20-50%), and (iii) PLE extract/supercritical-CO2 flow rate ratio (0.0125-0.05). Total carotenoid content was evaluated in both extracts and raffinates. Best results were obtained at 30 MPa, 0.05 feed/SC-CO2 mass flow rate, and 20% (v/v) of water in the feed solution, achieving values of 120.3 mg g-1 carotenoids in extract (in the SAF extract fraction), which were significantly higher than those obtained in the original PLE extract. In parallel, a new fast two-dimensional comprehensive liquid chromatography (LC×LC) method was optimized to get the full carotenoid profile of these extracts in less than 25 min. This is the first time that the use of a C30 column is reported in an on-line LC×LC system. Graphical abstract.

Quantitative analysis of aqueous phases of bio-oils resulting from pyrolysis of different biomasses by two-dimensional comprehensive liquid chromatography.

Agrifood by-products are perfect candidates to be further processed under the concept of circular economy, in order to produce their valorization. Although significant amounts of food-related wastes that are discarded are produced worldwide, these might still be rich in valuable compounds. A strategy to further valorize agrifood-related by-products is based on pyrolysis processes. The result of this process is a liquid product termed bio-oil which is composed of an organic phase and an aqueous phase. This bio-oil is rich on a variety of components and its analysis implies several challenges. In this work, quantitative on-line comprehensive two-dimensional liquid chromatography (LC × LC) is proposed for the first time to characterize several aqueous phases of different bio-oils. Rice husk, peanut shell, spent coffee grounds, peach core and Eucalyptus sawdust biomasses were analyzed. The developed quantitative LC × LC method presented very good linearity, precision, reproducibility, recovery and LODs and LOQs as low as 0.05 µg mL-1 and 0.16 µg mL-1, respectively. As much as 28 components were simultaneously separated and quantified in those samples. Our results found that the composition of these bio-oils was different but strongly related to the agrifood by-product submitted to pyrolysis. The developed methodology is foreseen as a valuable tool for the quantitative study of other bio-oils, considering the great complexity and high dimensionality of these samples.