3^rd International Conference and Exhibition on Advances in Chromatography & HPLC Techniques July 13-14, 2017 Berlin, Germany

Jian-Yong Wu is an Associate Professor in the Department of Applied Biology and Chemical Society in the Hong Kong Polytechnic University. His research mainly focuses on biotechnology and bioprocesses with medicinal fungi and plant tissue cultures; food science and engineering on functional foods (natural and bioactive polysaccharides; dietary fiber, prebiotics and microbiota) and ultrasound-assisted extraction and processing of food and medicinal products.

Vitamin D, also known as “sunshine vitamin”, is a fat-soluble hormone which plays an important role in maintaining bone health. Vitamin D is the general name for a group of vitamins having similar biological activities with cholecalciferol (vitamin D3). Ergocalciferol (vitamin D2) is the synthetic form of vitamin D from ergosterol (provitamin D2). Mushrooms that are cultivated in dark environment, without exposure to UV light, usually contain a negligible amount of vitamin D2 but an abundant amount of ergosterol naturally. Ergosterol can be eventually converted to vitamin D2 under UV irradiation with a series of reaction steps. In this study, the UV-B irradiation effects on the conversion of ergosterol into vitamin D2 in Cordyceps sinensis Cs-HK1 mycelia was investigated under various irradiation time and temperature. Ergocalciferol (vitamin D2) and ergosterol contents in the extract solution was analyzed by high performance liquid chromatography system with a reversed phase at 282 nm, using acetonitrile/ methanol (75:25) as mobile phase, qualitatively and quantitatively. The results showed that an increase in UV-B irradiation time had a positive effect on the conversion of ergosterol to vitamin D2, ranging from 145.8μg vitamin D2 to 406.8 μg vitamin D2 per gram sample when compared to the original (14.7 μg vitamin D2 per gram sample). The optimum irradiation time was found to be 60 minutes. Moreover, an increase in irradiation temperature also had a positive effect on the above conversion. At the optimum UV-B irradiation time (60 minutes), the vitamin D2 content in the Cs HK1 mycelia at irradiation temperature 20ºC was analyzed to be 230.3 μg vitamin D2 per gram sample, which was only about 56.6% of that at irradiation temperature 35ºC (406.8μg vitamin D2).

Yui-Bing Lee has her expertise in analytical chemistry and product testing and certification. She obtained her PhD from the University of Hong Kong, majoring in Organometallic Chemistry. She has been working on green product development, analytical instrumentations and product testing. She is currently working in the Faculty of Science and Technology at the Technological and Higher Education Institute of Hong Kong as a Teaching Fellow.

Plasticizers are an important class of polymer additives, which can help to improve the flexibility and mechanical properties of polymers by lowering the glass transition temperature (Tg) of the polymer. However, in recent years, petroleum based polymers additives, such as phthalate plasticizers, are gradually fading out in the European Union and other major countries due to their high toxicity and environmental impact. One of the mostly concern plasticizers is the phthalate family. Although plasticizers can modify the properties of polymer, these organic molecules are not ightly bound to polymer itself. They were found to be readily migrated out from the polymers during the life time of the polymers and then released into the environment and human body. Therefore, the design of new non toxic plasticizers for polymer (in particular PVC) used in food packaging, medical use and children products is of particular high concern, as the potential for migration of plasticizers in these products is relatively high. Several studies have already proven that epoxidized or esterified triglyceride vegetable oils (including but not limited to soybean oil, linseed oil, castor oil, sunflower oil and palm oil) can give promising results as primary or secondary plasticizers in the production of flexible polyvinyl chloride (PVC). From recent studies, it was found that when hydrophobic tails of the triglyceride bear more polar functional groups (such as –OH and C=O) can help to enhance the compatibility of the plasticizers with polar polymers i.e., polyvinyl chloride (PVC). However, toxicity study has shown that epoxidized vegetable oil will impose health issues to liver, kidney, testis and uterus of rats. This project uses HPLC as the analytical instrumentation on the analysis of the structural change and molecular weight distribution of a new series of bio-based plasticizers from modified vegetable oils with high degree unsaturation enhance the biodegradability and stability of vinyl polymers.

Angela Wong has experience of working in the testing and certification industry, with the expertise in the laboratory testing on consumer products ensuring quality and safety (including physical, chemical and functional evaluation). She is also familiarized with global regulations on hazardous chemicals in consumer products, especially textiles and apparel, electrical and electronic products and food contact materials. Currently, she is working at the Technological and Higher Education Institute of Hong Kong (THEi) as a Teaching Fellow; her research is focused on the subject areas of analytical chemistry, food chemistry and food analysis.

The use of Bisphenol A (BPA) in food containers and food packaging is highly concerned in the recent years, due to its potential endocrine disrupting effect on humans, especially infants and children. In the European Union (EU), it is classified as a chemical that possesses toxic effects on our ability to reproduce, according to the European Chemicals Agency (ECHA). It is commonly used in the industry as the monomer of polycarbonate plastics and epoxy-resins. The use of BPA-based polymer in food containers is extensive. Polycarbonate is commonly used in the fabrication of infant feeding bottles, while epoxy resins are used to coat the inner walls of metal cans that are in contact with foods and beverages. Due to its highly lipophilic nature, BPA can leach from the container or the plastic lining of cans to the food or beverage inside, resulting in contamination, human exposure and bioaccumulation, especially when the food is comparatively high in fat such as cow’s milk and dairy products. BPA has been banned from infant feeding bottles in the EU since 2011, and there is a specific migration limit from food contact plastics. In France, it has also been banned in all food packaging, containers and utensils. In Hong Kong, however, there is no legislation regulating the BPA levels migrated from food containers or food packaging. In this study, a high-performance liquid chromatography method with UV detector was used to determine the BPA levels in milk and dairy products (including cream, evaporated milk and condensed milk) sold in Hong Kong market.

Shun-Wan Chan is an Associate Professor in the Department of Food and Health Sciences, Faculty of Science and Technology, Technological and Higher Education Institute of Hong Kong. His research is focused on studying the molecular mechanisms of functional foods on preventing chronic diseases, such as cardiovascular diseases and diabetes mellitus. His research interests in food science also include food safety, food microbiology and toxicology.

The dried roots of Polygonum cuspidatum Sieb. et Zucc. (HZ, Hu Zhang in Chinese) have been used for the treatment of hepatitis, scald and hyperlipidemia in oriental counties. However, the effects of water extract of HZ on lipid-lowering activity still remains in a black box due to lack of appropriate methods. The present investigations described the major components of HZ water extract using high-performance liquid chromatography with diode-array detection (HPLC-DAD) and ultra performance liquid chromatography with orbitrap mass spectrometry (UPLC-Orbitrap-MS). Additionally, the effects of HZ water extract on serum in rats fed with high fat diet (HFD) was analyzed using metabolomics approach by ultra performance liquid chromatography quadrupole time of flightmass spectrometry (UPLC-QTOF-MS). The major components of HZ water extract have been identified as polydatin, emodin, resveratrol, emodin 8-O-β-D-glucopyranoside, physcion O-β-D-glucopyranoside and piceatannol (Fig. 1). Data from serum analysis showed that the supplementation of HZ extract significantly lowered the levels of total cholesterols. Multivariate statistics revealed that treatment of HZ significantly elevated serum levels of chenodeoxycholate, deoxycholate, their glycine conjugated bile acids and glycocholate level when compared with HFD model, indicating that the underlying cholesterol-lowering mechanism of HZ might be associated with bile acid biosynthesis. In order to verify the mechanism, bile acid biosynthesis related proteins, including cytochrome P450 7A1(CYP7A1) and mitogen-activated protein kinase (MAPK) [P38, extracellular regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK)] pathway were investigated using Western blot assay. The expressions of these proteins were found to be upregulated by HZ treatment, supporting that the cholesterol lowering effect of HZ is associated with bile acid metabolisms.

Maddalena Cagnone is a PhD student in Biomedical Science in the Department of Molecular Medicine at the University of Pavia, Italy. She graduated in Experimental and Applied Biology at the University of Pavia. After graduation, she obtained a scholarship to study the expression of CD44 on mesenchymal cells from patients with different degree of chronic lung rejection. In the course of PhD studies, she has been studying the role of AAT in BALf of transplant patients.

Statement of the Problem: Early and long-term graft and patient survival after lung transplantation are challenged by chronic lung allograft dysfunction (CLAD), whose main phenotype is represented by bronchiolitis obliterans syndrome (BOS), a condition characterized by an irreversible obstructive graft dysfunction due to fibro obliterative lesion of small airways. Patients with BOS have a poor survival (5-year) after its onset. Based on the anti inflammatory and anti-protease properties of alpha-1 antitrypsin (AAT), specifically versus neutrophil elastase (HNE), a possible role of this protease inhibitor in short and long term outcome after lung transplantation has been recently underlined by an increasing number of studies. Methodology & Theoretical Orientation: 1-D and 2-D polyacrylamide gel electrophoresis combined with Western blotting and LC-MS/MS were applied to the analysis of bronchoalveolar lavage fluid (BALf) from 80 subjects among which were selected stable lung transplant recipients and individuals affected by BOS at different grade of severity (BOS 0p to BOS 3). This proteomic study was carried out to understand whether long term transplant outcome could be correlated with AAT and HNE levels in patients’ BALf. Findings: The complex between HNE and AAT, that was expected to be present in all subjects, was observed only in a limited number of stable as well as of BOS patients. We speculated that the inability of AAT to inhibit completely HNE in BALf of these individuals was due to circulating inactive forms of this inhibitor. The identification of several degraded forms (ranging from 45 to 27 kDa) of AAT, strengthened our hypothesis. Conclusion & Significance: Comparison of protein profiles in BALf of lung transplant patients at different levels of severity have shown differentially expressed proteins among them. In particular, AAT seems to be the involved in disease progression and pathogenesis.

Natalia Y Marchenko has completed her PhD from Kharkiv National University (Ukraine) and Institute of Protein Research, RAS (Russia). Her research is focused on protein protein interactions, specifically on studying chaperone-protein interaction (especially, chaperonin GroEL from Escherichia coli) in various conditions using different physical and chemical methods, in particular, affinity chromatography. The method of affinity chromatography on a base of denatured proteins was developed and validated with her direct participation, and the application of this method for research and medical purposes is her main goal. She has published 10 papers in peer-reviewed journals.

Statement of the Problem: Molecular chaperones are a large group of proteins (presented in both prokaryotes and eukaryotes) that assist other proteins in their folding. The chaperones play important roles under normal conditions as well as in stress response and in disease development. Thus, detection of changes in chaperone content in cells under different conditions is important. Methodology & Theoretical Orientation: Tight interaction with denatured proteins is a general property of molecular chaperones. That is why affinity chromatography on a base of denatured proteins is a perspective method for chaperone analysis. Affinity sorbents were made on the base of Sepharose and a small target protein (e.g. lysozyme) covalently attached to it. The experiments were carried out in the conditions which are native for chaperones and denaturing for the target protein (pH 7.5 and presence of dithiothreitol). Findings: Cell lysates of bacteria (Escherichia coli, Thermus thermophilus, and Yersinia pseudotuberculosis), archaea (Halorubrum lacusprofundi) as well as lysate of rat liver mitochondria (in normal conditions and after oxidative stress) and human blood serum were analyzed. It is shown that Hsp60 chaperonins were the main proteins that bind to the affinity sorbent (except for the case of blood serum where albumin was the main bound protein). It is demonstrated that, apart from Hsp60, other proteins with a molecular weight of about 100, 50, 40, and 20 kDa are able to interact with denatured lysozyme. Conclusion & Significance: We are able to evaluate the quantity of bound proteins and to detect stress changes in protein content. So, this approach can be useful for studying various diseases connected with changes in chaperone content.

Mi Hee Woo is a Professor at the College of Pharmacy, Catholic University of Daegu, Republic of Korea. She obtained her MS degree in Pharmacy from Catholic University of Daegu, Republic of Korea, and PhD degree in Pharmacy from Yeungnam University, Republic of Korea. She is perennially engaged in research and teaching for spectroscopy, analytical chemistry, and natural pharmaceutical chemistry in the College of Pharmacy. Her research activities deal with development of new methods for quality control from traditional herbal medicine by chromatographic techniques. Furthermore, her scientific interest is focused on the separation, purification, and identification of new bioactive natural products from herbal medicine.

The genus of Artemisiae was used either health food or medicinal plant for the treatment of stomachache, fever, pain, asthma, enteritis, and bronchitis. In this study, we developed an HPLC/PDA method by using seven marker compounds [chlorogenic acid (1), hyperoside (2), 3,4-di O-affeoylquinic acid (3), 3,5-di O-caffeoylquinic acid (4), 1,5-di O-caffeoylquinic acid (5), 4,5-di Ocaffeoylquinic acid (6) and eupatilin (7)]. Seven marker compounds were well separated with a Kinetex C18 (250x4.6 mm, 5 μm) by gradient elution using 0.3% formic acid in water and acetonitrile as mobile phase. The flow rate was 1.0 mL/min and the UV detector wavelength was set at 327 nm. The method was successfully used to distinguish between Artemisiae argyi Folium and Artemisiae iwayomogi Herba by qualification of these marker compounds 1-7. Furthermore, this method also evaluated the linearity, recovery, precision, accuracy, stability and robustness. In conclusion, the results revealed the successful identification of the two herbal species according to qualification of seven marker compounds (1-7).

Ashraf S Rasheed has done his MSc in Analytical Chemistry, College of Science, University of Baghdad, Iraq. She has done her PhD in Analytical Chemistry from the Faculty of Chemistry, Universität Marburg, Germany in 2014. His current interest of research is to study the retention mechanism in ZIC-HILIC. He has 21 papers published in reputed journals.

Even though the use of HILIC has been extensively growing during the last decade, its mechanism is still today partly unresolved. Hydrophilic interaction chromatography (HILIC) is an increasingly popular alternative to conventional HPLC for drug analysis. It offers increased selectivity and sensitivity, and improved efficiency when quantifying drugs and related compounds in complex matrices such as biological and environmental samples, pharmaceutical formulations, food, and animal feed. A set of four new covalently bond sulfobetaine exchangers with inner quaternary amines and outer sulfonic acids have been prepared. The preparation was achieved by the attachment of a series of zwitterionic precursors to a highly porous divinylbenzene polymer using a grafting reaction. The grafting reaction allows a flexible adjustment of the degree of functionalization. Due to the identical reactions, using a series of zwitterionic precursors, exchangers with the same core material, and identical spacers to the polymeric backbone as well as comparable capacities can be prepared. The resulting materials differ only in chain length between the charged functional groups. The interchange distance of the different precursors varies from one to four methylene groups. The study included four new applications for pharmaceuticals compounds: Dexamethasone sodium phosphate (DMSP), Tetracaine HCl (TCH), Pilocarpine HCl (PCH) and Chloramphenicol (CAP). Chromatographic properties of these exchangers are examined by separation of pharmaceuticals using sodium acetate/acetonitrile eluent with UV detection. The separation mechanism is explored by varying eluent ionic strength and eluent pH and acetonitrile content. The results of these variations show that pharmaceuticals separation can be achieved due to a hydrophobic interaction (RP-HPLC) and hydrophilic interaction (ZIC HILIC), cation and anion exchange mechanisms. This is a behavior of hydrophobic interaction and ion exchange was never observed before using zwitterionic exchangers. It contradicts the fact that sulfobetaine type materials used in zwitterionic ion chromatography (ZIC) usually are well suited for anion separation and only poorly suited for cation retention and non-polar compound separation.

Ramiro Quintanilla-Licea has done his BSc degree in Industrial Chemistry (1977) and a Master's degree in Organic Chemistry (1979), from the Universidad Autónoma de Nuevo León (Mexico). He did his PhD in Organic Chemistry from the University of Frankfurt am Main, German Federal Republic (1988). He is currently involved in the investigation of Mexican plants with anticancer, antiprotozoal and anti-diabetic activity, in collaboration with scientists from the UANL and different universities in Germany and the Japan.

Objectives: The aim of this work is to isolate bioactive substances from the medicinal plant G. glutinosum for further studies as antitumor agents. This plant is distributed from Central America to the South of the United States of America and in the scientific literature are different ethnobotanical reports about its biological activity. Materials & Methods: Dry and grind material from the aerial parts of the plant collected in the South of the city of Monterrey (Mexico) was subjected to a Soxhlet extraction with hexane. After removal of the solvent in an evaporator, the obtained extract showed excellent cytotoxic activity against the murine lymphoma L5178Y-R. Results: Fractionation of the hexane extract of G. glutinosum by adsorption column chromatography (silica gel, normal and reverse phase) and size exclusion chromatography (Sephadex) showed ent-labdane diterpenes 1, 2 and 3 as mainly responsible for the observed cytotoxic activity of this plant against the lymphoma murine L5178Y-R. The purity of compounds was monitored using silica gel thinlayer chromatography. The identification of the compounds was carried out by IR, MS, 1H-NMR, 13C NMR and 2D-NMR. Conclusions: The very similar structure of the diterpenes present in G. glutinosum (Fig. 1) hinders the separation of themselves with a single chromatographic technique, so the combined use of three different techniques of chromatography column is fundamental to achieve separation and purification. Also, the monitoring of the purity by thin layer chromatography requires several different techniques (UV light, iodine vapors, chemical reagents).