International Summit on Current Trends in Mass Spectrometry July 13-15, 2015 New Orleans, USA

The renin- angiotensin system (RAS) has long been recognized as an important regulator of systemic blood pressure and electrocyte homeostasis. Our view of RAS has experienced remarkable change over the past decades- new enzymes and products as well as their local formation and action have been described. Tissue RAS plays an important role in development of various diseases. All components of classical RAS have been found in various type of cancer tissue. It has been demonstrated that some angiotensins peptides can contribute to development and progression of tumour. Ang II stimulates cell proliferation and angiogenesis (also Ang III and Ang IV). On the other hand, Ang- (1-7) plays opposite role to Ang II, showing antiproliferative effects and reducing fibrosis, angiogenesis, and tumour volume and weight. However, there is still the lack of studies on role and action of newer angiotensins like Ang-(1-12), which is an alternative substrat for Ang II formation. The aim of this study was to estimate the main pathways and characterize the main products of metabolism of angiotensinogen fragments (Ang-(1-14), Ang-(1-12 and Ang I) in cancer cells. Using an LC/MS/MS method, we assessed the ability of breast cancer cells (MCF7, MDA-MB-231 and T47D) to produce main active angiotensins peptides. Better understanding of RAS role in cancer may represent a new approach for cancer prevention and treatment.

Diagnosis of disease biomarkers like cancer, diabetes, etc. have been conducted by performing mass spectrometry techniques on human tear fluid. Apart from this, tear proteomics and lipidomics have been studied extensively by researchers worldwide for identification of disease biomarkers pertaining to the ocular surface disorders. One prime example of such disease is dry eye, where by applying mass spectrometry techniques researchers have shown absence or presence of certain proteins and lipids characterizing the disease. But in this study, we have shown that apart from the inter-individual change between diseased state and control, there are differences intra-individually both at the proteomics and lipidomics level. The results obtained indicate that there are changes in left and right eye proteomics and lipidomics profile with an individual. This study in future could help in diagnosis of ocular disorders pertaining to a particular eye as opposed to the general notions adapted by ophthalmologists that if one is in a diseased state the other one is also prone to be leading to that state. The reason for this difference may be of neurobiological origin which needs more detailed future study.

Gotgam Chamoe, a native oriental melon in Korea, is known to possess the aroma of a dried persimmon, an agronomic relevance for melon breeding program. The quantitative distribution of components were investigated in cultivar (Ohbokggul) and native (Gotgam) Chamoes for development and application of solid-phase micro extraction method (SPME). The volatile components were analyzed by gas chromatography-mass spectrometry and identified by comparison with the mass spectra in the literature. A total of 69 volatile components were identified and quantified, and 28 volatile compounds were specific to either the cultivar Chamoe or the native Chamoe. The amounts of volatile alcohol, unsaturated hydrocarbon, and ketone compounds were 2.2, 1.3, and 1.2 times higher in Gotgam Chamoe, respectively, and that of saturated hydrocarbon volatiles were 2.4 times higher in Ohbokggul Chamoe, whereas the total amounts of esters were similar. Among them, the functional groups of major components were ester and alcohol, including 2-methyl-1-butyl acetate (131/115/44 or 131/61/44 m/z), n-butyl acetate (117/101/44 or 117/61/44 m/z), and 3-methyl-1-butanol (89/71/43 m/z). As a result, volatile compounds were formed differently in the two Chamoes within different parts of the fruits, which contributes to aromatic differences between cultivar and native melon fruits.

The Fatty Acid Methyl Esters (FAMEs) profiles of eight isolates of unialgal samples, collected from various locations along the Lebanese coast, were determined using GC-MS analysis. The eight isolates were cultured in 20 L flat photo-bioreactors containing seawater fertilized with Guillards F/2 medium. After studying the growth dynamics and the dry matter yield of each isolate, the FAMEs profiles were conducted using two main steps: Extraction of the lipid fraction followed by derivatization to the desired FAMEs and the determination of fatty acid profiles after simultaneous washes with water. The profiles were carried out using a Thermo Gas Chromatograph equipped with an automatic injector (Triplus AS) and a Mass Spectroscopy detector (MS). The palmitic acid, linoleic acid, linolenic acid and eicosapentanoic acid are common to all eight isolates, and other fatty acids are also present in minor percentages. The percentages of saturated, monounsaturated and polyunsaturated fatty acids varied among isolates. The best microalgal isolate tested gave an Omega-3 FA percentage of around 18%, which seems to be useful for providing significant health benefits, as dietary supplements, and for medical purposes. To improve the percentage of PUFA on isolates, other studies are in progress for the optimization of culture conditions, and the search of new monoalgal samples having high percentages of PUFA.

Identification of aldose reductase inhibitory compounds from Zea mays L. by LC-ESI-MS: As part of our ongoing search of natural sources for therapeutic and preventive agents for diabetic complications, the aldose reductase inhibitory effect of purple corn (Zea mays L.) was investigated. In an attempt to identify bioactive components, seven polyphenols and five anthocyanins were identified by LC-ESI-MS and isolated by column chromatography, including protocatechuic acid (154 m/z), vanillic acid (168 m/z), 2,4,6-trihydroxy benzoic acid (170 m/z), p-hydroxycinnamic acid (164 m/z), ferulic acid (194 m/z), hirsutrin (464 m/z), 3’-methoxyhirsutrin (478 m/z), cyanidin-3-glucoside (449/287 m/z), pelargonidin-3-glucoside (433/271 m/z), peonidin-3-glucoside (463/301 m/z), cyanidin-3-(6”-malonyl-glucoside) (535/449/287 m/z), and peonidin-3-(6”-malonyl-glucoside) (549/301 m/z). These compounds were investigated via inhibitory assays of rat lens aldose reductase, kinetic study of recombinant human aldose reductase, and galactitol accumulation in rat lenses and erythrocyte. Among them, Zea mays L. derived anthocyanins hirsutrin showed most potent inhibitory effect of rat lens aldose reductase with IC50 values of 4.78 μM. In the kinetic analyses using Lineweaver-Burk plot of 1/velocity and 1/concentration of substrate, hirsutrin showed non-competitive inhibition against recombinant human aldose reductase. Furthermore, it inhibited galactitol formation in a rat lens incubated with a high concentration of galactose. Thus, hirsutrin could be offered as a leading compound for further study as a new natural products drug for diabetic complications.

Josilene Chaves Ruela Corrêa is graduated in Pharmacy-Industrial pharmacy (2006) at University of Minas Gerais State, MSc (2011) and PhD in Pharmaceutical Sciences (2014) at “Universidade Estadual Paulista - UNESP” in São Paulo, Brazil. She has experience in managing people and projects, coordinating activities in the pharmaceutical company in product development and quality control Currently she conducts is deepening her research with the drug darunavir for the development of her Postdoctoral research at the Faculty of Pharmaceutical Sciences of UNESP.

Darunavir, a protease inhibitor used in the treatment of HIV infection, is a pillar of therapy cocktail for patients with the virus. This work proposed an LC-MS method indicative of stability for the determination of darunavir in the complex darunavir:β-cyclodextrin, a recent advance for the use of antiretroviral. The method was completely validated according to the International Conference on Harmonization guidelines, showing accuracy, precision, selectivity, robustness and linearity. The separation was achieved on a 250 mm × 4.6 mm, 5.0 μm particle size CN Luna column with water + 0.1% glacial acetic acid : acetonitrile + 0.1% glacial acetic acid, 60:40 (v/v) at phase at a flow rate of 1.0 mL min-1. UV detection was performed at 268 nm at ambient room temperature (25°C). Mass spectral analyses were performed with ESI ion source and ion trap mass analyser. The method was linear over the concentration range of 10-60 μg mL-1 with correlation coefficient 1.000 and limits of detection and quantification of 1.13 and 3.43 μg mL-1, respectively. The drug was subjected to acid, basic, oxidative, neutral degradation and photolysis. Degradation products were identified by LC-MS and LC-MS/MS and they showed no interfere in the method of darunavir, therefore the method can be regarded as indicative of stability. The validated method is very useful to the routine quality control for quantification of darunavir in inclusion complex darunavir:β-cyclodextrin.