José Julián Ríos

Dynamic headspace gas chromatographic method for determining volatiles in virgin olive oil

Abstract Dynamic headspace sampling methods prior to capillary gas chromatography are especially suitable in the determination of volatile compounds at a wide range of concentrations, and numerous methods have been developed and applied to very different kinds of samples. In this work, a simple and rapid dynamic headspace technique was developed to determine volatiles present in virgin olive oil samples. Headspace components were swept from 0.5 g of sample at low temperature (40°C) and concentrated on Tenax TA, thermally desorbed and subsequently trapped in a fused-silica cold trap previously cooled to −110°C. They then passed to the capillary column. This system was connected to a mass spectrometer to identify the most important compounds and a comparative study of the main volatiles identified in virgin olive oil samples using other methods was carried out. Sniffing of the components eluted from the chromatographic column was also performed. Different virgin olive oil samples showing different chromatographic profiles were analysed. The differences were mainly quantitative because most compounds were present in all oils analysed, and only the proportions in which these compounds are present varied. Discriminant analysis of these compounds allowed the origin of each sample to be determined with a probability of greater than 90%.

Analysis of secreted flavonoids of Cistus ladanifer L. by high-performance liquid chromatography–particle beam mass spectrometry

Abstract The analysis of secreted flavonoids of the leaf Cistus ladanifer was carried out by high-performance liquid chromatography–mass spectrometry coupled through a particle beam interface. The mass spectra were recorded using electron impact. Apigenin, 4′-methyl-apigenin, 7-methyl-apigenin, 7,4′-dimethyl-apigenin, 3-methyl-kaempferol, 3,4-dimethyl-kaempferol, 3,7-dimethyl-kaempferol and 3,7,4′-trimethyl-kaempferol were identified in the leaf resin.

A new high-performance liquid chromatographic method with evaporative light scattering detector for the analysis of phospholipids. Application to Iberian pig subcutaneous fat

A new method for the analysis of phospholipids by normal-phase HPLC is described using a silica column. Addition of ammonia and triethylamine to a gradient based on chloroform/methanol/water promoted a good and rapid separation of phospholipid classes (20 min run). The use of an evaporative light scattering detector permitted an accurate analysis of a mixture of phospholipids. Calibration curves were linear within different range for each phospholipid class. The LOD and LOQ obtained were below 0.03 and 0.05 mg kg⁻¹ for all cases, respectively. Besides, a new method for the separation of phospholipids from total lipids before HPLC analysis by a solid-phase extraction (SPE) with Si cartridges has been developed. This methodology gave a good recovery ranging from 97 to 117%. The method was validated with a standard mixture of phospholipids. This method has been applied to characterize the phospholipid fraction of subcutaneous fat from Iberian pig. Cardiolipin, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, and sphingomyelin have been described for first time in these samples. The fatty acid composition of the different phospholipid classes and their HPLC electrospray ionization mass spectrometry have been used for characterizing the molecular species present in each one.

Nonfluorescent chlorophyll catabolites in loquat fruits (Eriobotrya japonica Lindl.).

Nonfluorescent chlorophyll catabolites (NCCs) and nonfluorescent dioxobilane chlorophyll catabolites (NDCCs) are the terminal compounds of the chlorophyll degradation pathway that may display beneficial properties to human health related to their antioxidant properties, which were recently shown. A profile of NCCs/NDCC of the loquat fruit Eriobotrya japonica Lindl. is described. From the 13 known different NCC structures described to date, three have been identified in loquats. Two new structures not defined so far were characterized in loquat fruits: Ej-NCC2, which corresponds to the methyl ester at C13(2) of Bn-NCC1 and in very low amount Ej-NDCC1, the only NDCC found in loquats. Keto-enol tautomerism at the C13(1) position in NCCs is described for the first time as a regular process in chlorophyll catabolism, probably through a nonspecific mechanism since almost all the chlorophyll catabolites structures detected in fruits of loquat present keto and enol tautomers. The results obtained have been possible through a high-performance liquid chromatography coupled with electrospray ionization ion trap and quadropole time-of-flight mass spectrometry fitted with a powerful postprocessing software.

Isolation and characterization of sucrose polyesters

Various chromatographic techniques for isolation and separation of highly esterified sucrose polyesters (SPE) of olive oil are described. High-performance size-exclusion chromatography was used to check the purity of the samples, particularly to show that SPE were free from unreacted fatty acid methyl esters. Thin-layer chromatography (TLC), Iatroscan TLC flame-ionization detection (FID) and highperformance liquid chromatography (HPLC) on reversed-phase were applied for separation of octa-, hepta- and hexaesters of sucrose. Pure fractions from the total mixture, obtained by column chromatography, were identified by infrared and nuclear magnetic resonance spectroscopy. When necessary, specific reactions were applied; particularly, silylation and lead acetate-dichlorofluorescein (in toluene) spray were used to ascertain the degee of esterification of sucrose. Finally, octa-, hepta- and hexaesters of sucrose were quantitated by silica column chromatography, TLC/FID and HPLC on reversed-phase.

Systematic HPLC/ESI-High Resolution-qTOF-MS Methodology for Metabolomic Studies in Nonfluorescent Chlorophyll Catabolites Pathway

Characterization of nonfluorescent chlorophyll catabolites (NCCs) and dioxobilane-type nonfluorescent chlorophyll catabolite (DNCC) in peel extracts of ripened lemon fruits (Citrus limon L.) was performed by HPLC/ESI-high resolution-qTOF-MS method. Compounds were identified in samples on the basis of measured accurate mass, isotopic pattern, and characteristic fragmentation profile with an implemented software postprocessing routine. Three NCC structures already identified in other vegetal tissues were present in the lemon fruit peels (Cl-NCC1; Cl-NCC2; Cl-NCC4) while a new structure not defined so far was characterized (Cl-NCC3). This catabolite exhibits an exceptional arrangement of the peripheral substituents, allowing concluding that the preferences for the NCC modifications could be a species-related matter.

Development of an accurate and high-throughput methodology for structural comprehension of chlorophylls derivatives. (II) Dephytylated derivatives

Dephytylated chlorophylls (chlorophyllides and pheophorbides) are the starting point of the chlorophyll catabolism in green tissues, components of the chlorophyll pattern in storage/processed food vegetables, as well as the favoured structural arrangement for chlorophyll absorption. In addition, dephytylated native chlorophylls are prone to several modifications of their structure yielding pyro-, 13(2)-hydroxy- and 15(1)-hydroxy-lactone derivatives. Despite of these outstanding remarks only few of them have been analysed by MS(n). Besides new protocols for obtaining standards, we have developed a new high throughput methodology able to determine the fragmentation pathway of 16 dephytylated chlorophyll derivatives, elucidating the structures of the new product ions and new mechanisms of fragmentation. The new methodology combines, by first time, high resolution time-of-flight mass spectrometry and powerful post-processing software. Native chlorophyllides and pheophorbides mainly exhibit product ions that involve the fragmentation of D ring, as well as additional exclusive product ions. The introduction of an oxygenated function at E ring enhances the progress of fragmentation reactions through the β-keto ester group, developing also exclusive product ions for 13(2)-hydroxy derivatives and for 15(1)-hydroxy-lactone ones. Consequently, while MS(2)-based reactions of phytylated chlorophyll derivatives point to fragmentations at the phytyl and propionic chains, dephytylated chlorophyll derivatives behave different as the absence of phytyl makes β-keto ester group and E ring more prone to fragmentation. Proposals of the key reaction mechanisms underlying the origin of new product ions have been made.

Determination of ent-kaurene in subcutaneous fat of Iberian pigs by gas chromatography multi-stage mass spectrometry with the aim to differentiate between intensive and extensive fattening systems

This work presents a gas chromatography multi-stage mass spectrometry (GC-MS(3)) method for the determination of ent-kaurene in subcutaneous fat of Iberian pig, present in adipose tissue of animals due to pasture ingestion (extensive fattening system). The method comprises a saponification and a liquid-liquid extraction of the unsaponifiable fraction, followed by an isolation of the hydrocarbon fraction by high performance liquid chromatography (HPLC) and analysis by GC-MS(3) (ion trap) with electronic ionization. The GC-MS(3) analysis allows the isolation and characterization of specific fragments from the original (MS(1)) molecular structure, and particularly, those fragments originated from the precursor ion (m/z=229) characteristic of ent-kaurene. The MS/MS product fragment m/z=213 is used as a further precursor fragment giving rise to a MS(3) spectrum specific for ent-kaurene. The limit of detection of the MS(3) technique is lower than 0.2 microg kg(-1) and a linear regression has been found between 0.2 and 112 microg kg(-1). This method is applicable for the determination of the fattening system of the Iberian pig.

Characterization of sucrose polyesters-triacylglycerols mixtures

High-performance size-exclusion chromatography (HPSEC) and thin-layer chromatography/flame-ionization detection (TLC/FID) have been used for the characterization of mixtures of either monoacid sucrose octaesters with triacylglycerols (TAG) or sucrose polyesters (SPE) prepared from oils with natural oils. In mixtures of monoacid sucrose octaesters/TAG, no significant differences were found between the values obtained by either HPSEC or TLC/FID and the actual component proportions. Additionally, components could be separated by TLC, which was confirmed by fatty acid composition data of each fraction. Analysis of SPE/oil mixtures was attainable by HPSEC, but alternative quantitation by TLC/FID required previous silylation. Likewise, fatty acid composition could be determined only in the total mixture and in the sucrose octaester fraction. A formula derived for calculation of oil fatty acid composition, based on analytical data, showed the validity of the approach used in this study to determine component proportions in functional SPE/oil mixtures.

Physico-chemical properties of chickpea lipoxygenases

Abstract Purified lipoxygenase isoenzymes from chickpea ( Cicer arietinum ) cv Pedroxillano are single polypeptide chains with M r s of 96 000–97 000, determined by hydrodynamic and electrophoretic methods. Both isoenzymes difrered in pI , 4.92 for CL-1 and 4.74 for CL-2. Each isoenzyme contained one atom of iron in a divalent oxidation state. CL-1 contained 760 amino acid residues and CL-2 753 residues, with a high proportion of hydrophobic amino acids, particularly proline and leucine. Eight and seven cysteines were also present, two and three of which were as free sulphydryl residues, in CL- 1 and CL-2, respectively. Thiols, sulphur bridges and carboxyl and amine groups seemed to be essential in the maintenance of a catalytically active tertiary structure in both isoenzymes. CL-2 formed mainly 13-hydroperoxy-(9 Z , 11 E )-octadecadienoic acid from linoleic acid, while CL- 1 yielded almost equal proportions of the 9-and 13-hydroperoxides, and the 9- and 13-ketodienes.