6^th International Conference and Exhibition on Advances in Chromatography & HPLC Techniques August 02-03, 2018 Barcelona, Spain

Sayaka Hashimoto received her BSc in Applied Chemistry at Tokyo University of Science, Japan, in 2018. Her main research interest and work includes “Establishment of high resolution-separation technique of reversed-phase high-performance liquid chromatography (RP-HPLC)”. She has recently succeeded in separating gold-silver alloy cluster by improving the resolution of RP-HPLC.

By doping heteroatoms to thiolate-protected gold clusters, we can add more functionality to the cluster depending on the number of doped heteroatoms and the difference in doping positions. However, in the synthesis of alloy clusters, the mixture of clusters with a different number of heteroatoms is produced. Therefore, a precise separation of each cluster is required to understand their properties. In this study, we attempted to separate gold-silver alloy clusters precisely, furthermore investigated isomer change by reversed-phase high-performance liquid chromatography (RP-HPLC). Au38-nAgn(SC4H9)24 was used as a sample. This cluster was synthesized by two methods, (1) by adding [Ag(SC4H9)] complex to Au38(SC4H9)24 (metal exchange) and gold and silver ions were reduced in the presence of butanethiol in solution (co-reduction). The mixture of alloy clusters was separated by RP-HPLC using gradient program for controlling mobile phase, and core-shell type column. The peaks obtained from the chromatogram were evaluated by electrospray ionization (ESI) mass spectrometry connected to RP-HPLC directly. Figure.1 (a) shows the chromatogram of Au38-nAgn(SC4H9)24 obtained by the metal exchange. Some clear peaks were observed in chromatogram. Each peak was attributed to the cluster having a precise number of silver atoms (Figure. 1(b)). These results indicate that the mixture of Au38-nAgn(SC4H9)24 was precisely separated according to the number of silver atom. Furthermore, the shape of chromatogram of Au38-nAgn(SC4H9)24 prepared by the metal exchange changed by leaving this cluster in toluene for 6 days (Figure. 1(c)). Interestingly, the shape was similar to that of Au38-nAgn(SC4H9)24 prepared by the co-reduction (Figure. 1 (d)). These results suggest that Au38-nAgn(SC4H9)24 prepared by the metal exchange contains metastable clusters, these are transformed to the stable clusters by leaving in toluene. In conclusion, we have succeeded in the high-resolution separation of alloy clusters according to each chemical composition, and observation of isomer transformation.

Kana Yoshida has completed her BSc in 2017 in Applied Chemistry at Tokyo University of Science, Japan. Her main research interests are high resolution separation of noble metal clusters protected by hydrophilic thiolate ligands. She has presented her work many times at conferences. Furthermore, her research was published to Nanoscale (IF=7.367) which is one of the high impact factor journal in nanoscience field.

Small thiolate-protected gold clusters show size-specific physical and chemical properties, which are not observed in bulk gold. Especially, hydrophilic thiolate-protected gold clusters exhibit a good affinity for biomaterials. Therefore, much research has been conducted in this field. However, it is difficult to selectively synthesize the clusters with specific chemical composition. Therefore, to evaluate properties of hydrophilic thiolate-protected gold clusters accurately, we need to separate single cluster from the mixture of these clusters with high resolution. In this work, we report on precise separation of various hydrophilic thiolate-protected gold clusters (Aun(SR)m, SR=SG (glutathionate), NALC (N-acetyl-L-cysteine), p-MBA (p-mercaptobenzoic acid)) by hydrophilic interaction liquid chromatography (HILIC). Furthermore, we attempted to evaluate the chemical composition of each cluster by introducing LC/MS which was directly connected the chromatograph with the mass spectrometer. The mixture of clusters used in this work was synthesized by reducing gold ions in the presence of thiols. Figure 1(a) shows the UV chromatogram of Aun(SG)m.¬ Multiple peaks were observed in the chromatogram. Figure 1(b) shows the ESI-mass spectrum of each peak observed in the chromatogram. It was found that almost only one cluster was contained in each peak. These results indicate that mixture was separated with high resolution according to the chemical composition of clusters. Similar separation has also been achieved for Aun(NALC)m and Aun(p-MBA)m. Therefore, it was revealed that the use of HILIC columns is powerful tool for separating of gold clusters protected by many kinds of hydrophilic thiolates. Furthermore, Although chemical compositions observed for Aun(SG)m and Aun(NALC)m were similar each other, that for Aun(p-MBA)m was less compared with these clusters. These results indicate that chemical compositions of the Aun(SR)m vary depending on the ligand structure. In this manner, we have succeeded in revealing how difference of ligand affects synthesis of gold clusters protected by hydrophilic thiolate ligands.

Tamara Alvarez-Segura completed her Master degree in Experimental Techniques in Chemistry in 2014, at the University of Valencia. She is now performing diverse research activities in the Department of Analytical Chemistry to get her PhD degree. She has received two Pre-doctoral fellowships. During her Master studies, she began her collaboration with María Celia García-Alvarez-Coque and José Ramón Torres-Lapasió in the field of the modulation of the selectivity in HPLC, using serially coupled columns and other strategies to analyse complex samples. She has written 11 research articles and presented several communications in international meetings. The PhD period included a three-month stay (September to November 2017) in the Analytical Chemistry Department at the University of Barcelona (Spain), under the supervision of Prof Martí Rosés, working in the field of Hydrophilic Interaction Liquid Chromatography (HILIC).

Model-based chromatographic optimizations provide the best separation conditions efficiently. Unfortunately, with the routine usage, columns inevitably suffer some deterioration and, as a result, actual chromatograms may differ from the forecasted expectancies; new optimal separation conditions are then needed, but unfortunately, models are not valid anymore. The chromatographer could then decide repeating the whole modelling process, but this solution is undesirable and too time consuming, particularly when several coupled columns are involved. We propose a shortcut to correct time and peak profile predictions. The original models are corrected by introducing parameters accounting the deterioration, obtained using a small subset of compounds, selected from those studied in the training set before the column had suffered performance decline. The ageing parameters are not solute dependent. These are fitted in such a way that they minimize the discrepancies between the data predicted with the original retention models for the brand-new column and the experimental data measured for the aged column. The approach was developed and tested to predict the chromatographic behaviour of 15 sulphonamides, analysed with individual and tandem columns, using isocratic and gradient elution. From the gathered information, predictions more in line with the deteriorated performance were forecasted for the whole family of compounds analysed with that column (or set of coupled columns). The results suggest that, whenever the columns keep sufficient performance, we can take advantage of the extensive experimental work carried out when the system was initially modelled with the brand-new columns. With this information and a minimal extra experimental effort, accurate enough predictions in the degraded situation are possible. The full modelling of the chromatographic behaviour is thus only made with the brand-new columns. The agreement between predicted and experimental chromatograms in the aged columns was excellent.

Tamara Alvarez-Segura has completed her Degree in Chemistry in 2013 and Master degree in Experimental Techniques in Chemistry in 2014, both at the University of Valencia. She is now performing diverse research activities in the Department of Analytical Chemistry to get her PhD degree. She has received two pre-doctoral fellowships. During her Master studies, she began her collaboration with María Celia García-Alvarez-Coque and José Ramón Torres-Lapasió in the field of the modulation of the selectivity in HPLC, using serially coupled columns and other strategies to analyse complex samples. She has written 11 research articles and presented several communications in international meetings. The PhD period included a three-month stay (September to November 2017) in the Analytical Chemistry Department of the University of Barcelona (Spain), under the supervision of Prof Martí Rosés, working in the field of Hydrophilic Interaction Liquid Chromatography (HILIC).

Liquid chromatography with single columns often does not succeed in the analysis of complex samples, in terms of resolution and analysis time. A relatively simple solution to enhance chromatographic resolution is the modulation of the stationary phase through the serial coupling of columns. This can be implemented with any type of column using compatible elution conditions and conventional instruments. This review describes the key features of column coupling and published procedures, where two or more columns were coupled in series to solve separation problems in chiral liquid chromatography. In most reports, two chiral columns are coupled. However, coupling chiral and achiral stationary phases is also an active field, to analyze samples containing mixtures of chiral and achiral compounds. In all reports, the authors could not resolve their samples with single columns, whereas significant enhancement in chromatographic performance was obtained when the columns were combined. Particularly interesting is the reduction in the analysis time in the isocratic mode, which alleviates the general elution problem of liquid chromatography, and may represent a stimulus for the proposal of new procedures, especially in combination with mass spectrometric, electrochemical and refractometric detection. Developments proposed to make the serial coupling of columns useful in routine and research laboratories are outlined, including optimisation strategies that facilitate the selection of the appropriate column combination and elution conditions (solvent content, flow rate or temperature) in both isocratic and gradient modes. The availability of zero dead volume couplers, able to connect standard columns, and the commercialisation of short columns with multiple lengths, has expanded the possibilities of success.

Susana Jesus is a PhD student in the Chemistry Engineering at Instituto Superior Técnico. She is a Researcher in the MISAGE project-mitigation strategies of acrylamide and advanced glycation end-products in bread. The aim of this project is to mitigate acrylamide in bread through the application of extracts of spices based on antioxidant profile. She has experience in acrylamide determination by UPLC-MS/MS and in simultaneous quantification of sugars with UPLC-MS/MS in food products.

Statement of the Problem: Acrylamide is a carcinogenic contaminant produced during food processing at high temperatures. This contaminant is a Maillard Reaction product that results from a reaction between asparagine and reducing sugars. Therefore, the determination of sugar profile is an important form to understand the formation of acrylamide in foods, such as bread, potatoes or coffee. In the particular case of bread, it is important to understand the effect of fermentation on the formation of acrylamide. Until now, several techniques have been developed to determine saccharides in food, GC-MS, HPLC-DAD, HPLC-FLD, HPLC-ELSD. However, these methods have disadvantages, such as low selectivity and some methods require derivatization. Therefore, the aim of the present work was to develop a method to analyze the mono and disaccharides by UPLC-MS/MS in flour and bread dough. Methodology & Theoretical Orientation: Six flours (oat, rye, wheat flours) and four bread dough were selected. For the detection and quantification of sugars ultra-performance liquid chromatographic coupled to a mass detector (UPLC-MS/MS) was used. To identify sugars and optimize the parameters of the detector, direct injections of the sugars under study (sucrose, glucose, fructose, maltose, and mannose) was carried out. The following chromatographic conditions were then tested: mobile phase A with acetonitrile/water (10:90) with 0.1% ammonium hydroxide and mobile phase B with acetonitrile/water with 0.1% ammonium hydroxide. The method has a flow of 0.2 ml/min with a 32 minutes gradient: 0-30 min, 100-70% B; 30-30.1 min, 70-60% B; 30.1-31 min, 60-80% B; 31-32 min, 80-100% B. Result: With this method, it was observed that there are no matrix interferences in the chromatogram (Figure 1), concluding that this method is suitable for sugar analysis in flour and bread dough. Conclusion & Significance: We developed a simple and rapid method for simultaneous determination of sugars in flour and bread dough to understand the formation of acrylamide in bread.

Lucia Váhovská is a Researcher in the Department of Chemistry, Biochemistry and Biophysics at the University of Veterinary Medicine and Pharmacy in Košice. She has completed her PhD in Inorganic Chemistry at University of Pavol Jozef Šafárik in Košice in 2014. Her academic training and work experience include the preparation of some transition metal complexes with subsequent physico-chemical characterization using infrared spectroscopy, UV-VIS spectroscopy, elemental analysis, RTG structure analysis, magnetic measurement. At present, she participates in a project with the aim to study selected drug releasing (naproxen, 5-fluorouracile) from mesoporous (modified) silica SBA15 using HPLC method.

Over the last decades, the development of controlled drug delivery systems has increased rapidly. However, recently, there has been growing interest in the use of mesoporous materials as controlled drug delivery matrixes because they have several attractive features, such as stable uniform mesoporous structures, high surface areas, tunable pore sizes with narrow distributions, and well-defined surface properties. The delivery of different drugs using the mesoporous silica materials was investigated, up to now. Our attention has been focused on the study of naproxen release from drug delivery system, based on ordered hexagonal mesoporous silica SBA15. Naproxen, a non-steroidal anti-inflammatory drug (NSAID) derived from propionic acid, is widely used to moderate pain relief in the treatment of many diseases. NSAIDs, including naproxen, are commonly employed to reduce ongoing inflammation, pain and fever, because they are able to block the cyclooxygenase (Cox) enzymes that both produce prostaglandins; these classes of compounds have several important functions, such as the promotion of inflammation, pain and fever. In this work, the released amount of naproxen from SBA15 was monitored in selected time intervals by reversed-phase high-performance liquid chromatography (RP-HPLC). A Dionex Ultimate 3000RS system equipped with a diode array detector (DAD) and programmable Chromeleon Chromatography Data System, Version 7.2, was used for analysis. The mixture of acetonitrile and water (55:45, v/v) adjusted with ortho-phosphoric acid to pH 3 was selected as the best mobile phase. The naproxen was monitored by UV detection at 229 nm.

James A O’Hanlon is pursuing his PhD at University of Manchester, School of Chemistry, in the group of Prof Melissa A Denecke. He has an Industrial Cooperative Awards in Science and Technology, studentship specially funded by the EPSRC (Engineering and Physical Sciences Research Council) to promote mutually beneficial research collaboration between academic and partner organizations.

Ethylene diamine tetra acetic acid (EDTA) is found in decontamination agents used throughout the nuclear industry, therefore, is often found in repository consigned wastes. The Low-Level Waste Repository (LLWR) is the UK’s Centre for the disposal of low-level radioactive waste. LLWR maintain strict limits of acceptance on EDTA because, when present in the waste, the ligand potentially solubilizes otherwise surface-bound radionuclides, making them more susceptible to groundwater transportation into the wider geo/biosphere. A significant quantity of EDTA and radioactively-contaminated ion-exchange resin sourced from decontamination operations during nuclear submarine maintenance is in radioactive decay-storage in UK Naval dockyards. Before the material can be accepted for disposal at LLWR, it must be thermally conditioned to remove EDTA; and then analyzed to confirm EDTA destruction. A simple method has been developed for the extraction and quantification of EDTA from an incinerated ion-exchange resin matrix using reversed-phase ion-pair high-performance liquid chromatography (HPLC) with ultraviolet detection. EDTA is extracted directly into an aqueous Fe (III) solution to undergo complexation, separated on a monolithic silica column (Chromolith® HighResolution, Merck) and detected at 258 nm. The linearity of the response is high (R2; 0.9999) and the limit of detection/quantification for the method has been determined to be 0.23/0.62 mg/kg, respectively. From the standard addition method on four samples of incinerated resin containing 6% EDTA prior to treatment, high recoveries were obtained (mean value; 78.3±3.34 %), with reasonably high intra- and inter-day repeatability (RSD; 1.42–12.4%). Absorption peaks at similar retention times were observed and, as they do not occur for the resin incinerated without EDTA, are attributed to ferric complexes of EDTA thermal degradation products. Interfering peaks were resolved by applying a least squares fit to the data.

Aysel Berkkan has been an Associated Professor at Gazi University, Faculty of Pharmacy, Division of Analytical Chemistry in Turkey since 2014. She received her Ph.D. degree in 2004 at Gazi University, Faculty of Pharmacy, Division of Analytical Chemistry about Hydride Generation Atomic Absorption Spectrometry with Professor Nusret Ertas. She has been an Assistant Director in the Institude of Medical Sciences at Gazi University in 2016. Dr Berkkan’s reseachs involve Hydride Generation Atomic Absorption Spectroscopy, Inductively Couple Plasma- Mass Spectrometry, and Head Space Gas Chromatography.

Statement of the Problem: There are three major aspects in analytical measurements; first, to develop microanalytical methods. Second to perform rapid measurements on small sample volumes. Third, green analytical methodologies. Single Drop Microextraction (SDME) have special value in sample cleanup, rapid extraction and large enrichment factor. So, head space-single drop microextraction (HS-SDME) gas chromatography coupled to flame ionization detector (HS-SDME-GC-FID) was used to determine the fatty acids. Methodology & Theoretical Orientation: Fatty acids in oil samples were derivatized to improve volatility with esterification reaction. Methylation was carried out by trans-esterification with methanolic potassium hydroxide in order to convert the polar carboxylated groups to less polar methyl ester derivatives. Then, microextraction step was applied in 10 mL head space vial by using the GC microsyringe with a bevel tip that holds the drop. Extraction solvent (sodium dodecyl sulphate: 1-butanol, 1:3), concentration of ionic strength adjustment solution (NaCl), temperature (45°C), and extraction time (35 min) that affect the yield were optimized. Agilent Technologies, Inc. HP-5 column (30mx 0.320mm ID, 0.25 µm film thickness) was used in GC. Inlet and detector temperature were 270°C. Oven temperature program was as follow; 80C (2.0 min), 4 C/min ramp to 210C (5 min), 15C/min ramp to 300 C (5.0 min), helium was used as a carrier gas (1 mL/min flow rate). Injection sample volume was 1 µL with 100:1 split ratio. Findings: Chromatogram of fatty acids’ methyl esters in olive oil was shown in Figure. Conclusion & Significance: The identification of linoleic acid, and linolenic acid’s metyl esters in olive oil were performed by comparing their retention times with the standarts by HS-SDME-GC-FID. The amount of acids was expressed as a percentage (w/w) of all fatty acids’ esters detected, linoleic acid (3%), and linolenic acid’s (16%).

Prof.dr.sc. Nada Bezić has more than 45 papers published in international scientific journals in the field of virology, anatomy and physiology of xerophytes and their implementation in phytotherapy. Plant species that are the subject of research are spread along Mediterranean part of Croatia. Laboratory researches, apart from exploring plant structures, are focused on the isolation of plant compounds and their chemical characterization, as well as the determination of their biological activity. In the recent years our research was focusing on investigation of the antiviral activity of essential oils. As a professor at the University of Split, prof.dr.sc. Nada Bezić holds lectures and is lead mentor to students at the undergraduate, graduate and postgraduate level.

Genus Veronica L. (Plantaginaceae) is divided into 13 subgenuses and 270 species, of whom 40 are widespread in Croatia [1]. Samples of the investigated Veronica spicata L. were collected in two localities, mountains Dinara and Velebit. They are cosmopolitan and ecologically diverse species spread on a variety of habitats from aquatic, marshy and forest to rock, rock cracks, fields and ruderal habitats [2]. Some of the species from the genus Veronica have been used in traditional medicine. Literature reviews showed that the most investigated secondary metabolites for Veronica genus include iridoid glucosides, phenylethanoids and flavonoid glycosides [3], so this is the first study of the chemical composition of the essential waters - hydrolates from this species. Hydrolates are aqueous products of hydrodistillation from aerial parts of plant. Aerial parts of the two samples of V. spicata were air dried and subjected to hydrodistillation in Clevenger type apparatus. 2mL of pentane and 40 mL of water was added to the inner part of the graduated tube at each distillation. At the end of the process of distillation the organic and water layer were separated and refrigerated until the analyses [4]. The phenolic compounds from hydrolates have been analyzed by Perkin Elmer HPLC system (Waltham, Massachusetts, USA) using the C18 column (Ultra-Aqueous C-18, 250 x 4.6 mm, 5 Ã…) (Restek, USA). Components 3,4-dihydroxybenzoic acid and vanillin were identified, with the following values. Component in the Sample 1 contained 3.47 mg/L of 3,4-dihydroxybenzoic acid and the Sample 2 contained 6.12 mg/L. Component vanillin in the Sample 1 is represented with 0.12 mg/L and in the Sample 2 with 0.76 mg/L. Our previous studies have included research of volatile components in the composition of the essential oil of V. spicata analyzed by GC/MS [4]. Du and Jin found vanillic acid in the species Veronica peregrine [5]. The present study gives additional knowledge about volatile compounds in the hydrolates of the genus Veronica.

Madhuri Singhal has completed her PhD at Dr Hari Singh Gaur University, Sagar and Postdoctoral work at Allahabad University, Allahabad. Her research area is the role of natural products from medicinal plants in drug discovery and development. She has presented research papers in international conferences in Australia in 2005 and was invited as visiting academic in 2006 at Australian University. She has presented papers in USA in 2009 and 2015. In 2010, she has presented research paper at Ubon Ratchathani University, Thailand. In 2011, she has presented paper in Hong Kong. She is an Editorial Board Member of an international research journal. More than 30 research papers have been published. At present, she is a Professor of Chemistry in Government Motilal Vigyan Mahavidyalaya, Bhopal.

Terminalia bellerica, known as bahera or beleric or bastard myrobalan, common on plains and lower hills in Southeast Asia, where it is also grown as an avenue tree. The leaves are about 15 cm long and crowded toward the ends of the branches. It is considered a good fodder for cattle. Terminalia bellirica seeds have an oil content of 40%, whose fatty-acid methyl ester meets all of the major biodiesel requirements in the USA (ASTM D 6751-02, ASTM PS 121-99). In traditional Indian Ayurvedic medicine, Beleric is known as "Bibhitaki" (Marathi: "Behada or Bhenda") (Terminalia bellirica). Its fruit is used in the popular Indian herbal rasayana treatment triphala. In Sanskrit it is called vibhīdaka. The fruit contains anthraquinones and tannins, It is anthelmintic, astringent (especially when ripe), digestive, tonic and laxative (especially when unripe) The fruit is used internally principally in the treatment of digestive and respiratory problems. In Indian herbal medicine the ripe fruit is used in cases of diarrhoea and indigestion, whilst the unripe fruit is used as a laxative in cases of chronic constipation. This paper contains quantitative analysis of ß-sitosterol in various part of Terminalia bellarica by using HPTLC method.

Areeg Z Alkarali has completed her Bachelor’s degree in Pharmaceutical Sciences at Misr International University in 2012 and Master’s degree in Pharmaceutical Analytical Chemistry at Suez Canal University in 2018.

Since animal products are a major source of human exposure to steroids, the extensive use of these hormones resulted in a critical request for accurate, sensitive, simple and applicable methods for the determination of these steroid hormones in pure form and in different matrices as hen eggs, chicken liver and tilapia farming pond water. The aim of this work is to develop a reliable liquid chromatography coupled with mass spectrometric method for the determination of selected steroid hormones in complicated matrices as hen egg, chicken liver and tilapia farming pond water. Using solid phase and liquid liquid extraction methods for sample preparation, in the present study LCMS/MS method was demonstrated for the simultaneous separation and quantification of four steroid hormones ethinylestradiol, 17 alpha methyl testosterone, testosterone and progesterone. Using mobile phase of methanol and 0.1% formic in ratio (70:30) at different m/z ratios, the method validation was carried out on each hormone showing: linearity for ethinylestradiol 0.5 μg/ml to 30 μg/ml r2=0.9997, 17α-methyl testosterone 0.5 μg/ ml-20 μg/ml r2=0.9999, Testosterone 0.5 μg/ml-20 μg/ml r2=0.9999 and progesterone 0.5 μg/ml-20 μg/ml r2=0.9999. LOD and LOQ values of ethinylestradiol, 17 α methyl testosterone and progesterone respectively (0.7 and 2.12), (0.23 and 0.69), (0.35 and 1.08) and (0.36 and 1.11). The method was validated using the ICH guidelines and successfully applied on egg, chicken liver and tilapia pond fresh water samples from Egypt.