Hicham Benabdelkamel

Assay of tyrosol and hydroxytyrosol in olive oil by tandem mass spectrometry and isotope dilution method

Hydroxytyrosol and tyrosol, the strong antioxidant present in large amount in virgin olive oil have been assayed by LC-MS/MS under MRM condition and isotope dilution method, using d(2)-labelled internal standards obtained by simple synthetic procedures. The assay has been performed under MRM condition monitoring two transitions for each analyte to improve the specificity. This paper deals with a modern approach for assaying the content of this polyphenols in virgin olive oil down to a limit of a few hundreds of parts per billion. Tyrosol and hydroxytyrosol ranged from 10 to 47ppm and from 5 to 25ppm in commercial olive oil, respectively. The accuracy (98-107%) and analytical parameters values confirm the reliability of the proposed approach. The method can be extended to any natural matrices, including mill wastes, after a simple step of sample preparation.

Authenticity of PGI “Clementine of Calabria” by Multielement Fingerprint

Clementine is a citrus fruit that has found a peculiar habitat in specific areas of Calabria, a region located in southern Italy. Due to its peculiar characteristics it was recently awarded with protected geographical indications (PGI) from the European Union. In this work, stepwise linear discriminant analysis (S-LDA), soft independent modeling of class analogy (SIMCA), and partial least-squares discriminant analysis (PLS-DA) were used to build chemometric models able to protect PGI Clementine from others of different origin. Accordingly, the concentration of 24-26 elements was determined in peel and juice samples, respectively, obtained from Calabrian PGI clementine and from fruits cultivated in Algeria, Tunisia, and Spain. A cross-validation procedure has shown very satisfactory values of prediction ability for both S-LDA (96.6% for juice samples and 100% for peel samples) and SIMCA (100% for both peel and juice samples). PLS-DA models also yielded satisfactory results.

Comprehensive assay of flavanones in citrus juices and beverages by UHPLC-ESI-MS/MS and derivatization chemistry.

Flavanones, a class of flavonoids present in large amounts in fruits and vegetables, have been assayed by LC-MS/MS and derivatization chemistry using d0/d3-labelled derivatized internal standards obtained by simple reaction procedures which involves d0/d3 methoxyamine. The assay method considers 13 flavanones including aglycones, neohesperidosides, rutinosides and 3-hydroxy-3-methyl glutaryl derivatives. The strengths of the method consist in a relative short analysis time (16 min) and good repeatability and reproducibility values which are in most cases under 10% (RSD%). The accuracy values range from 95.4% to 111.3% whilst the LOQ values ranges from 0.05 to 0.29 mg/L depending on the analyte.

Light and heavy dansyl reporter groups in food chemistry: amino acid assay in beverages

5-Dimethylamino-1-sulfonyl naphthalene (DNS, commonly referred as dansyl) is a functionality, bearing well-established properties in directing the fragmentation, by mass spectrometry (MS), of the corresponding ionized sulfonylated derivatives. This property is shared also by its labeled analogs. The use of d(0)/d(6) DNS derivatives is now exploited in the application of the well-established isotope dilution mass spectrometric approach in the assay of complex mixtures. A new method for the quantitation of amino acids (AAs) in beverages is therefore presented, which relies on liquid chromatographic separation of their N-dansylated derivatives followed by comparative electrospray tandem MS/MS of the d(0)/d(6) isobaric mixtures. Labeled and unlabeled DNS derivatives of the selected AAs are readily available by microwave-assisted synthetic protocols. The novelty of the method is represented by the use of heavy and light DNS-isotopologue providing suitable reporter groups. Multiple-reaction monitoring has been applied in the assay of AAs in wine, pineapple juice and bergamot juice with good-to-excellent results as proved by both relative standard deviation, lower than 15%, and by the accuracy values in the range 90-110%.

High-Throughput Assay of Oleopentanedialdheydes in Extra Virgin Olive Oil by the UHPLC-ESI-MS/MS and Isotope Dilution Methods

The quality of extra virgin olive oil is associated with the presence of microcomponents whose healing effects have been proved in some special cases. The enzymatic hydrolysis of oleuropein and ligstroside, and of their demethylated analogues, affords four different pentanedialdehydes, and for one of which, 2-(4-hydroxyphenyl)ethyl (3S,4E)-4-formyl-3-(2-oxoethyl)hex-4-enoate, also known as oleocanthal, an anti-inflammatory effect was quite recently carefully assessed. Extra virgin olive oil is now worldwide considered as a functional food whose daily intake, as for the Mediterranean diet, helps consumers in keeping a constant level of nonsteroidal anti-inflammatory drug (NSAID) in the blood. The presence of these active principles provides, therefore, olive oil with an important added value. In the framework of the actions of the recently funded Agrifood Regional Center, which should coordinate the scientific research and production worlds, an absolute analytical method was developed for the mass spectrometric detection of the two most abundant NSAIDs, Tyr-OLPD and HTyr-OLPD (oleopentanedialdehydes (OLPDs) conjugated to p-hydroxyphenylethanol and 3,4-dihydroxyphenylethanol, respectively), by UHPLC-ESI-MS/MS.

Effect of H/D isotopomerization in the assay of resveratrol by tandem mass spectrometry and isotope dilution method.

Resveratrol is a phytoalexin found in several plant tissues and present in wines, which is supplied as a nutritional supplement. Different studies have revealed its beneficial effects as anticancer, antiviral, neuroprotective, antiaging, and anti-inflammatory natural active principle. The assaying of resveratrol by mass spectrometry and isotope dilution method, using a stable [(2)H(4)] analogue, has required a full elucidation of its gas-phase H/D isotopomerization. Either selected ion monitoring (SIM) or multiple reaction monitoring (MRM) methods have been used for the evaluation of the amount of resveratrol present in wine and plasma samples in the negative ionization mode. In all instances the acquired accuracy, limit of quantitation (LOQ), and limit of detection (LOD) are fit for the intended purpose of the assay.

Proteomic analysis of mature adipo cytes from obese patients in relation to aging

Obesity and aging are interrelated conditions that both cause changes in adipocyte metabolism and affect the distribution of fat in both subcutaneous and visceral depots. In addition, both weight gain and aging can lead to similar clinical outcomes such as insulin resistance, cardiovascular disease, type 2 diabetes mellitus, atherosclerosis and stroke. Our objective was to examine the changes in protein expression within the subcutaneous adipose tissue of obese patients, matched for BMI, in relation to age. Mature adipocytes were isolated from liposuction samples of abdominal subcutaneous adipose tissue collected from both young (26.2±4.3 (mean age±SD); n=7) and old (52.2±4.7 (mean age±SD); n=7) obese individuals. Total protein extracts were then compared by two-dimensional difference in gel electrophoresis (2D DIGE). Thirty differentially expressed protein spots (ANOVA test, p≤0.05; fold-change ≥1.8) were detected, of which, 15 were identified by MALDI-TOF mass spectrometry. These were comprised of a total of thirteen unique protein sequences. Nine proteins were more abundant in the adipocytes isolated from old vs. young individuals. These proteins included prohibitin 1, protein disulphide isomerase A3, beta actin, profilin, aldo-ketoreductase 1 C2, alpha crystallin B and the annexins A1, A5 and A6. Four other proteins were less abundant in the adipocytes from old, obese subjects and these included keratin type 2 cytoskeletal 1, keratin type 2 cytoskeletal 10 and hemoglobins A and B. The differentially abundant proteins were investigated by Ingenuity Pathway Analysis (IPA) to reveal their associations with known biological functions. This analysis identified signal transducer and activator of transcription 3 as the central molecule in the connectivity map and the apoptotic pathway as the pathway with the highest score. Differences in the abundances of several proteins were confirmed by immunoblotting: i.e., prohibitin 1, protein disulphide isomerase A3, beta actin, profilin and signal transducer and activator of transcription 3 proteins. In conclusion, proteomic analysis of subcutaneous adipose tissue reveals differences in the abundance of proteins in adipocytes isolated from young vs. old individuals. These differentially abundant proteins are involved in the regulation of apoptosis, cellular senescence and inflammatory response. All these are common pathologic events in both obesity and aging.

Effects of conventional heating on the stability of major olive oil phenolic compounds by tandem mass spectrometry and isotope dilution assay.

The quality of olive oils is sensorially tested by accurate and well established methods. It enables the classification of the pressed oils into the classes of extra virgin oil, virgin oil and lampant oil. Nonetheless, it would be convenient to have analytical methods for screening oils or supporting sensorial analysis using a reliable independent approach based on exploitation of mass spectrometric methodologies. A number of methods have been proposed to evaluate deficiencies of extra virgin olive oils resulting from inappropriate technological treatments, such as high or low temperature deodoration, and home cooking processes. The quality and nutraceutical value of extra virgin olive oil (EVOO) can be related to the antioxidant property of its phenolic compounds. Olive oil is a source of at least 30 phenolic compounds, such as oleuropein, oleocanthal, hydroxytyrosol, and tyrosol, all acting as strong antioxidants, radical scavengers and NSAI-like drugs. We now report the efficacy of MRM tandem mass spectrometry, assisted by the isotope dilution assay, in the evaluation of the thermal stability of selected active principles of extra virgin olive oil.

The assay of pterostilbene in spiked matrices by liquid chromatography tandem mass spectrometry and isotope dilution method

Pterostilbene (trans-3,5-dimethoxy-4-hydroxystilbene) is an active component found in several plant species, exhibiting important pharmacological properties. A new and reliable method of assaying this phyto compound in various matrices is presented; the assay is based on (1) the selectivity of liquid chromatography (LC) hyphenated with electrospray ionisation (ESI), (2) the specificity of a two-step mass spectrometric analysis (MS/MS) and (3) the accuracy of the isotope dilution method. The labelled analogue may be conveniently synthesised in a few steps. The sensitivity of the method is confirmed by the very low limit of detection (LOD) and limit of quantitation (LOQ) values achieved in the assay of pterostilbene in two distinct fortified matrices, and is further supported by the observed accuracy values.

Hyaluronic Acid Coated Chitosan Nanoparticles Reduced the Immunogenicity of the Formed Protein Corona

Studying the interactions of nanoparticles (NPs) with serum proteins is necessary for the rational development of nanocarriers. Optimum surface chemistry is a key consideration to modulate the formation of the serum protein corona (PC) and the resultant immune response. We investigated the constituent of the PC formed by hyaluronic acid-coated chitosan NPs (HA-CS NPs). Non-decorated chitosan NPs (CS NPs) and alginate-coated chitosan NPs (Alg-CS NPs) were utilized as controls. Results show that HA surface modifications significantly reduced protein adsorption relative to controls. Gene Ontology analysis demonstrates that HA-CS NPs were the least immunogenic nanocarriers. Indeed, less inflammatory proteins were adsorbed onto HA-CS NPs as opposed to CS and Alg-CS NPs. Interestingly, HA-CS NPs differentially adsorbed two unique anti-inflammatory proteins (ITIH4 and AGP), which were absent from the PC of both controls. On the other hand, CS and Alg-CS NPs selectively adsorbed a proinflammatory protein (Clusterin) that was not found on the surfaces of HA-CS NPs. While further studies are needed to investigate abilities of the PCs of only ITIH4 and AGP to modulate the interaction of NPs with the host immune system, our results suggest that this proof-of-concept could potentially be utilized to reduce the immunogenicity of a wide range of nanomaterials.