Day 3 :
- Track 1: Applications of Mass Spectrometry Track 4: Advances in Sample Preparation Techniques Track 11: Advances in Chromatography
Chair
Sam F.Y. Li
National University of Singapore, Singapore
Co-Chair
Kalluru Seshaiah
Sri Venkateswara University, India
Session Introduction
Kalluru Seshaiah
Sri Venkateswara University, India
Title: A Rugged Bio-analytical Method for Determination of Hydralazine in Human Plasma using Gas Chromatography coupled with Tandem Mass spectrometry
Biography:
K. Seshaiah has completed his PhD from Sri Venkateswara University, Tirupati, India and postdoctoral studies from Chaoyang University of Technology, Taiwan (ROC). He is the Chairman, Board of Studies in Chemistry, Sri Venkateswara University, India. He has guided 20 Ph.D., students and published more 80 papers in reputed journals. He has completed several research projects sponsored by DST, CSIR, UGC, Govt. of India and has international collaboration with University of Surrey, UK.
Abstract:
A simple, sensitive and rugged quantitative method for the determination of Hydralazine in human serum by gas chromatography-tandem mass spectrometry (GC-MS/MS) has been developed and validated. Hydralazine (1-hydrazinylphthalazine) is a vasodilator used to treat severe hypertension, congestive heart failure, myocardial infarction and preeclampsia. The HPLC methods reported for assays of Hydralazine in plasma are complicated because of rapid cooling and or separation of plasma immediately after withdrawing each blood samples, and Donnell et al.,1 and Woodworth et al.,2 have reported about the instability of Hydralazine at physiological pH. In the present study, a simple, specific and selective method has been developed for the determination of Hydralazine from plasma samples using GC-MS-MS. The sample preparation technique, described here involves rapid processing of samples and also rugged according to commonly acceptable FDA guidelines. Chromatographic separation has been achieved by GC with MS detector in positive ionization mode. Helium was used as the carrier gas and DB 5ms column, (30m X 0.25mm, 0.25µm (J& W; DB 5ms) film thickness Capillary column coated with cross Linked Polymer was used) gave a good peak shape for both analyte and internal standard. The retention time of Hydralazine and 4-methyl Hydralazine (IS) were 12.99 and 14.17 minutes respectively. The total run time of 20.00 min for each sample was freezed upon. A simple liquid-liquid extraction (LLE) technique was employed for the sample preparation that provided a good recovery of both analyte and IS. The LLOQ was 10.000ng/mL Hydralazine hydrochloride. The precision and accuracy of analyte at LLOQ concentration was found to be 6.423 % and 88.093 %, respectively. The CV % was 3.21% and 3.81% and % accuracy was 97.82% and 100.373% for LQC and HQC respectively.
Sam F.Y. Li
National University of Singapore, Singapore
Title: NMR and MS-based Metabolomics of Microcystis spp. Unravel the Involvement of Microcystin in Thylakoid Membrane Stabilization and Photoinhibition Resistance
Biography:
Professor Sam Li is a faculty member at the Department of Chemistry, National University of Singapore. He received his BSc, PhD and DSc degrees from Imperial College, UK. His research interests include environmental science and technology, biosensors, metabolomics and nanotechnology. He has authored/co-authored 325 publications in international peer review journals, more than 100 conference presentations and 10 US patents. He serves/served on editorial advisory boards of several international scientific journals, including Electrophoresis and Journal of Chromatographic Science.
Abstract:
Microcystin (MC), a potent hepatotoxin commonly produced by several freshwater cyanobacterial species, especially Microcystis, pose serious threats to water safety worldwide, making them unsuitable for recreational uses, as well as drinking water supplies. Here, the metabolomes of MC-producing and MC-free Microcystis species chronically exposed to strong light illumination were compared using complementary metabolomics tools; NMR and LC-MS. NMR and MS-based metabolomics were capable of revealing MC-influenced metabolic changes in Microcystis spp. The up-regulation of thylakoid membrane building blocks (phosphotidylglycerol), photosynthetic pigments (chlorophyll-a), and carbon reserve sources (sucrose, glycogen, 3-hydroxybutyreate) in toxic Microcystis strains, indicated the involvement of MC in photosynthesis apparatus stabilization under high light illumination. The lowered levels of intermediates involved in phospholipid degradation revealed that Microcystis strains were more resistant to light-induced oxidative stress in the presence of MC. Taken together, our comparative metabolomic data suggest a photoinhibition resistance role for MC in light-stressed Microcystis spp., and confirm its involvement in thylakoid membrane and chlorophyll-a stabilization..
Lilian Regina Barros Mariutti
University of Campinas, Brazil
Title: Carotenoids and carotenoid esters from fruits: strategies for identification by LC-MS
Biography:
Lilian Mariutti is a Researcher in the Department of Food Science of the Faculty of Food Engineering at University of Campinas (Brazil). She received her Doctorate degree in Food Science from Unicamp in 2009. She also holds a M.S. in Food Science and B Sc in Food Engineering. She was a Researcher Fellow in the Laboratory of Veterinary Drug Residues of the Brazilian Ministry of Agriculture, Livestock and Food Supply. Her research focuses on the identification and bioaccessibility of bioactive compounds (carotenoids and phenolic compounds), antioxidant activity of bioactive compounds in different systems and action of natural antioxidants in food.
Abstract:
Carotenoids are yellow to red liposoluble pigments found in plants, animals and microorganisms. The health benefits of the carotenoids have been attributed mainly to their antioxidant properties, which would minimize the in vivo oxidative damages induced by reactive oxygen (ROS) and nitrogen species (RNS). Their basic structure is a tetraterpene with a series of conjugated double bounds, and they can be divided into two main groups: carotenes (composed only of carbon and hydrogen atoms) and xanthophylls (contain at least one oxygen atom). Xanthophylls possessing hydroxyl groups can be found in nature acylated to several fatty acids, generating the carotenoid esters. The fact that a single carotenoid can be found acylated in several different combinations with distinct fatty acids further increases the variability of structures that can be found and the complexity of the analyzed matrix. Due to the high number of possible carotenoid esters in fruits and since all the esters of the same free carotenoid present exactly the same UV-visible spectrum, their identification must include tandem mass spectrometry (MSn) studies. Therefore, separation of carotenoids in reversed-phase liquid chromatography using C30 columns in combination with DAD and APCI-MS/MS detection allows high sensitivity and selectivity and is the technique of choice for carotenoid identification in complex matrices. Sample preparation and strategies for the identification of carotenoids and carotenoid esters based on UV-vis, MS and MS/MS spectra will be presented and discussed.
Biography:
Lokesh Gupta, has completed his PhD at the age of 26 years by researching in University of Delhi and Ch. CS University Meerut, India. He is an analytical research scientist and serving as Chief Manager of Analytical R&D team with TEVA API (a world leader in generic pharmaceuticals) in India, focusing on analytical research/methods development, spectroscopic characterization of organic pharmaceuticals, technology validation, impurity isolation and characterization etc. He is participating and discussing his commended research in several national/international seminars/conferences. He had published more than 42 papers in peer reviewed reputed journals of chemistry & spectroscopy and serving as an eminent referee for several journal of international repute.
Abstract:
From decades spectrometry has been accepted as a key analytical tool for understanding and characterization of molecules in chemistry, the level of world-wide research activity in this field promises that these capabilities will continue to improve, rapidly. Over the period of time sensitivity of spectroscopy tools have been improved to work at micro level and in more depth, ultimately become a faster research tool; such tools now being used as process analytical tools (PAT) giving online understanding of characteristics of a molecule during research/development and in production. Same time their utilization has widen up by coupling these tools with liquid chromatography, and thermal measurement tools e.g. (LC-MS-MS, LC-NMR, DSC-FT etc.)Mass spectroscopy has achieved horizons from single quadrupole to triple quadrupole, MS-TOF & the Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) these have become a powerful and widespread analytical tool in life science and health sector. The dynamic mass range (1-300 kDa), high accuracy and sensitivity make it a superior method for analysis of all kinds of biomolecules including proteins, nucleic acids, metabolites and carbohydrates. Particularly in drug discovery, where compound identification and purity from synthesis and early pharmacokinetics are determined, MS has proved indispensable outcomes. Today, the MS practitioner can choose among a range of ionization techniques which have become robust and trustworthy on a variety of instruments with demonstrated capabilities. In combination with 2D-elelectrophoresis, MALDI-TOF-MS is particularly suitable for the identification of protein spots via mass fingerprint or micro sequencing. Same time MS-TOF is widely used in pharmaceutical word, TOF has improved the sensitivity by increasing the path length in TOF tube, so ion remain in path of light for longer time increasing sensitivity, on other side measurement tolls has been improved to see the mass number in several digits can differentiate molecules having closure mass and differentiating isotopes. Software calculates elemental formula, for which confirmation performed by comparing theoretical fragments to the obtained TOF -MS/MS of molecule. In this review I had evaluated and focused on advancement and updates in MS field, with respect to technology update & applications.
Hong Wang
Shanghai Institute for Food and Drug Control, Shanghai, China
Title: Comparative Assessment of Glycosylation of a Recombinant Human FSH and a Highly Purified FSH Extracted from Human Urine
Biography:
Hong Wang received her PhD degree in 2012 from the Institutes of Biomedical Sciences, Fudan University, China. Using MS and a novel lectin microarray, she revealed the glycosylation of biomarkers. She currently studies the quality of biotechnology drugs in Shanghai Institute of Food and Drug Control. Based on the innovation of MS, she contributes to the analysis of protein glycosylation, the structural characterization of mAbs, the consistency of biosimilars. She is the leader of Shanghai Sailing Program. She has taken part in several national and provincial R&D programs. She has published more than 10 scientific papers in high level journals.
Abstract:
Human follicle-stimulating hormone (hFSH) plays a key role in the development and function of the reproductive system. Two classes of hFSH-containing pharmaceutical preparations currently exist; those derived from the urine of postmenopausal women (uhFSH) and those manufactured using recombinant DNA technology (rhFSH). Comparative clinical studies have revealed the differences in oocyte quality and clinical outcome between rhFSH and uhFSH. The oligosaccharide moiety is critical in determining the pharmacological properties of therapeutic glycoproteins. hFSH is a glycoprotein and glycan significantly influences the biological properties of hFSH. Considering the importance of hFSH glycosylation to the biological activity of hFSH, evaluating the difference in glycosylation between rhFSH and uhFSH intended for clinical use is essential. Using a glycoproteomic strategy, this study compared the glycosylation of uhFSH with that of rhFSH. Intact and subunit masses, N-glycans, N-glycosylation sites, and intact N- and O-glycopeptides were analyzed and compared by mass spectrometry. Classic and complementary analytical methods, including SDS-PAGE, isoelectric focusing, and the Steelman−Pohley bioassay were also employed to compare their intact molecular weights, charge variants, and specific activities. Results showed that highly sialylated, branched, and macro-heterogeneity glycans are predominant in the uhFSH compared with those in rhFSH. A high degree of heterogeneity was observed in the N-glycopeptides of both hFSHs. The O-glycopeptides of both hFSHs, which have not been described previously, were characterized herein. The differences in glycosylation provide useful information in elucidating and in further investigation the critical glycan structures of hFSH.
Mahmoud A. Saleh
Texas Southern University, USA
Title: Mass Spectrometry Regiospecific Analysis of neutral triacylglycerols
Biography:
Mahmoud A Saleh has completed his PhD in physical organic chemistry at the age of 28 years from University of California, Davis and postdoctoral studies at University of California, Berkeley in environmental toxicology. He is a professor of chemistry at Texas Southern University. He has published more than 150 papers in reputed journals and has been serving on editorial board member of several journals.
Abstract:
Different mass spectrometry ionization techniques were compared for their use in regiospecific analysis of neutral triacyglycerol (TAG) in different samples of seeds oil. Atmospheric pressure chemical ionization, atmospheric pressure photo ionization, electro spray ionization, ion trap and QTOF accurate mass were used in the LCMS experiments. Electron ionization, chemical ionization and QTOF accurate mass were used in GCMS experiments. MSMS QTOF and MSMS1-n ion trap experiments were used for structural conformation. Seed oil of Silybum marianum and 9 species of the Apiaceae family (dill, caraway, cumin, coriander, anise, carrot, celery, fennel and Khella) were used in this investigation. Results for regiospecific analysis showed a non-random fatty acids distribution in both S. marianum and Apiaceae seeds. Petroselinic acid was predominantly located at the sn-1 and sn-3 positions in carrot, celery and parsley seed oils, while it was mainly at the sn-2 position in caraway seed oil. The distribution of linoleic acid was opposite to that of petroselinic acid. Oleic acid was mostly located at the sn-2 position, except for caraway, where it was evenly distributed between the sn-1(3) and sn-2 positions. Both the saturated FA, palmitic and stearic acid, were located mainly at the sn-1(3) position. Based on our finding, it can be concluded that fragmentation pattern for TAGs follow the following scheme: The most abundant ions observed in the mass spectra of TAG are positive ions containing two complete fatty acid chains and those containing only one complete chain corresponding to the loss of one or two fatty acids respectively. Two ions were found corresponding to the ion remaining after the loss of one acyloxy group from the molecular ion [M-RCOO]+, and the other corresponding [M-RCOOH]+. If the three fatty acids are different, three different [M-RCOO]+ and [M-RCOOH]+ ions corresponding to the loss of each different acyloxy group. However, due to steric and electronic property, the loss of the fatty acid located in the sn2 is energetically less favorable than one at the end positions (sn1 and sn3). Therefore, ions corresponding from the loss of fatty acids from the sn1 and sn3 positions are much more abundance the ion corresponding to the loss of fatty acid at the sn2 position.
Oana Teodora Marisca
Babes Bolyai University, Romania
Title: An alternative diabetic retinopathy therapy: a laser triggered microsystem for controlled release of Resveratrol
Biography:
Oana Teodora Marisca is a 25 years old PhD student at Babes Bolyai University, Romania. Her research focuses on gold nanoparticles-cell interaction and targeted therapies using gold and silver nanoparticles. She is currently member of a Romanian government funded research project entitled “A controlled-release target delivery system for the alternative therapy of retina diseases-a novel perspective”. She published 4 articles in reputed journal
Abstract:
A severe ocular complication that causes visual impairment and even worse, blinding, due to a damage of the retina and the optic nerves is diabetic retinopathy (DR). During recent years, there was shown a significantly increase in using natural Resveratrol (RV) (3,5,4’-trihydroxystilbene) for the prevention or as complementary therapies for eye diseases. Due to existing challenges in resveratrol delivery, our current approach is to load RV into polyelectrolyte multilayer microcapsules (PEM), whose release is controlled by a laser triggered procedure into retina pigmented epithelial cells. Mimicking the physiologic concentrations of resveratrol and hyperglycemia, which occurs in DR patients by using D407 cells, the unique in vitro experimental models for eye disease, we assess the therapeutic potential of RV-loaded PEM. The synthesis process of the complex is based on a simple Layer by layer assembly approach. During this procedure the capsules walls are decorated with collagen-coated gold nanoparticles for laser controlled release facilitation (figure on the left). The so obtained RV-loaded PEM were characterized using spectroscopic and microscopic methods. Next, the controlled release process of RV was optimized. The Raman signal from RV molecules is detected once the microcapsule is open by exposure to 532 or 633 nm laser light and the entrapped RV is released. Quantification of the RV released from microcapsule is assessed by HPLC-PDA. Based on the information obtained and on the specific formulas the RV loading content, RV entrapment efficiency and yield of production are determined for quantification of the therapeutic agent who reaches the target site.
Min Li
Huahai Pharmaceutical Co. Ltd, China
Title: Application of LC-MSn molecular fingerprinting in the rapid elucidation of drug impurities
Biography:
Min Li received his PhD in Organic Chemistry from Johns Hopkins University in 1991. Starting from 1994, he had worked for several multi-national pharmaceutical companies including Roche, Merck, and Schering-Plough/Merck with increasing responsibilities. Since 2014, he is Vice President for Analytical Operation at Huahai Pharmaceutical, a leading Chinese pharmaceutical company with operations in China and US. Dr. Li is a leading expert in drug degradation chemistry and he published a single-authored book, “Organic Chemistry of Drug Degradation” (by RSC) in 2012. He is the first/communicating author of 40 publications in organic, medicinal, bioconjugate, and analytical chemistry (including mass spectrometry).
Abstract:
ICH guidelines require that impurities of drug substances and products be identified and/or qualified once they exceed certain thresholds. The identification of these impurities, including process impurities and degradation products, can be quite challenging and time-consuming. In this presentation, the use of LC-MSn molecular fingerprinting in conjunction with knowledge of process and/or drug degradation chemistry will be shown to be a very effective way in the rapid elucidation of unknown drug impurities. MSn fragmentation patterns (n is typically 2 to 3) generated with suitable collision energy are highly reproducible on same type of MS instrument and collectively, they provide a unique molecular fingerprint for a particular impurity. With the relevant process chemistry (for process impurities) or drug degradation chemistry (for degradation products) in mind, comparison of the MSn fingerprint of an unknown impurity with those of known compounds, especially the isomers of the unknown impurity, can quickly yield critical structural information, which will greatly expedite the process of structure elucidation. This approach will be demonstrated through several case studies involving structurally diverse drug
Sreenivasa RJ
National Institute of Nutrition, India
Title: Determination of Heavy Metal Contents in Indian foods Using ICP-MS after Closed Vessel Microwave Digestion
Biography:
Sreenivasa RJ is an Asst. Director at National Institute of Nutrition, (ICMR), Hyderabad, India. He has completed his post graduation in Biochemistry and doctoral studies from Osmania University, Hyderabad. He has published several research papers in reputed peer reviewed journals and presented his research contributions in several National and International conferences and workshops. He holds life membership in professional scientific organization. He has been an invited distinguished speaker for many universities for discourse on heavy and trace metal toxicity, food composition and nutritional health. Presently he is working on, plant secondary metabolites and heavy metals in Indian foods using ICPMS. His core area of research also includes evaluation of carotenoid metabolism. He is a board member of NIN Scientist Association and Security and maintenance committee.
Abstract:
Heavy metals are natural components of the earth's crust and are widely used in agricultural, manufacturing and food/material processing industries. Among a variety of substances entering the soil, inland waters and the ocean as waste products, heavy metals like Nickel, Arsenic, Cadmium, Mercury and Lead are prominent. Heavy metal toxicity is a result of long term, low level exposure to pollutants through air, water and numerous consumer food products. In the field of food and nutrition the increasing levels of heavy metals in the environment, their entry into the food chain, and the overall health effects on people who consume green leafy vegetables (GLV) and fish are of major concern to researchers. In the present study the heavy metal content was analyzed from GLV and fish samples. Closed vessel microwave digestion system (CEM-MARS-USA).of food samples assisted by HNO3+ H2O2 were used to determine the heavy metals using ICPMS. CRM samples procured from NIST were used for method standardization and validation for linearity, repeatability, limits of detection (LOD) and limits of quantification (LOQ). Heavy metal measurements were performed using ICP-MS (PerkinElmer Élan 9000-USA). For better operating conditions the ICP-MS was adjusted to Nebulizer gas flow 0.91 L/min, Radio frequency (RF) 1200 W, Lens voltage 1.6 V, Cool gas 13.0 L/min, and Auxiliary gas 0.70 L/min. Microwave digestion followed by analysis by ICP-MS has been shown to be a simple, reliable method for the multi-element determination of heavy and trace metals in GLV and Fish samples. Results were in accordance with recommended human daily intake values except for arsenic, which were in agreement with WHO/FAO recommended values. Several reliable analytical methods like Colorimetric and Atomic Absorption Spectrophotometer are available for monitoring heavy metals in food samples, but ICP-MS is the most sophisticated and reliable technique, fast quantitative, high sensitivity, good precision and accuracy. Isotope ratio measurements were also possible using ICP-MS and interferences relatively few in number compared to other analytical techniques.