International Conference and Exhibition on Advances in HPLC & Chromatography Techniques March 14-15, 2016 London, UK

Julie Le Grandois has completed her PhD in Analytical Chemistry in 2009 from University of Strasbourg (France). She is now Research Scientist in charge of the development of analytical methods at Aérial. She has published 11 papers in peer-reviewed journals.

Vitamins are essential for health but most of them are not synthesized by humans. Thus, vitamins have to be introduced through diet. Vitamins are an added value for food products. Their labeling requires identification and quantification. For that purpose, HPLC with UV or fluorimetric detections has been intensively used in the last years. These methods usually require specific chromatographic conditions for each vitamin, which is time consuming and expensive. Despite the democratization of analytical approaches such as mass spectrometry, the simultaneous determination of B-group vitamins remains challenging due to several reasons: i) their different structures and chemical properties; ii) their low stability; iii) their presence at trace levels in foods; iv) the complexity of food matrices. These features make it difficult to develop a single method for the simultaneous extraction, separation and quantification of B vitamins in foods. This work aims at combining a single enzymatic extraction step, with a single chromatographic run using ESI-MS² (MRM mode) to evaluate B vitamin content in foodstuffs. The design of experiments was used to determine the optimal pH and temperature conditions for the simultaneous extraction of B1, B2, B3 (nicotinic acid, nicotinamide), B5 (pantothenic acid), B6 (pyridoxine, pyridoxal, pyridoxamine) and B8 (biotin, biocytin) from food matrices. The efficiency of the procedure was investigated on four matrices (liver, yeast, powder milk, peas) and results were compared to the individual protocols. Results were conclusive and prefigured a good chance to develop an efficient mutivitaminic protocol.

Julie Le Grandois has completed her PhD in Analytical Chemistry in 2009 from University of Strasbourg (France). She is now Research Scientist in charge of the development of analytical methods at Aérial. She has published 11 papers in peer-review journals.

Since several years, antioxidant compounds are of great interest due to their implication in protection against radicals. Several studies have tried to understand which kinds of interactions occur in complex mixtures or extracts, since a potent regeneration of an antioxidant by another one can increase or decrease the activity of a mixture of antioxidants. In this study, individual TEAC (Trolox Equivalent Antioxidant Capacity) values of pure lipophilic compounds were first determined using a microplate assay and an online LC-AOx method, with post-column reaction. Then, TEAC values of lipophilic extracts from Solanum lycopersicum L. were also determined using both methods. Differences were observed and significant interactions were suspected. To evaluate these interactions, mixtures of lipophilic compounds were prepared in variable proportions. TEAC value (n=3) of each mixture was determined using the microplate TEAC assay. Interaction effects were calculated and additive, antagonist or synergic effects were observed. Results showed HPLC-ABTS provides individual TEAC values without interactions but helps in the identification of compounds involved in antioxidant capacity, whereas microplate assay only provides TEAC values of mixtures including interactions.

Andrija Ciric has completed his PhD at Faculty of Science, University of Kragujevac, Serbia and Post-doctoral studies at Faculty of Chemistry, University of Warsaw, Poland. He is presently working as a Teaching Assistant at Faculty of Science in Kragujevac, Serbia.

A simple, sensitive, specific, precise and accurate isocratic reversed phase-high performance liquid chromatography (RP-HPLC) method was developed and validated for analysis of some flavonoids (quercetin, kaempferol, rutin, kaempferol-3-glucoside, luteolin-7-glucoside) in comercial juices from berries (black currant and cherry). The developed chromatographic method was optimized for the separations of flavonoids using Phenomenex Luna C8(2) column (150 × 2.0 mm, 3 μm) as stationary phase with mobile phase consisting of a mixture of formic acid, at pH 2.8 and acetonitrile (80 : 20 v/v). Flow rate of the mobile phase was 0.5 ml/min and UV detection at 370 nm was employed. The column was thermostated at 30°C. Chromatographic separations of analytical peaks were achieved within 30 min. Linearity of the method was established over the concentration ranges of 0.05 – 20 μg mL−1 for all compounds with correlation coefficients greater than 0.9985 with LOQ ca. 0.039 μg/ml and LOD ca. 0.013 μg/ml for all analytes. SPE method was employed to extract the bioflavonoids from samples of juices. The efficiency of extraction was better than 95%. The obtained results (mg/100 mL of juice) for different bioflavonids in black currant and cherry juice were for quercetin, 0.34 and 0.21, for kaempferol, 0.09 and 0.06 mg, for rutin, 0.54 and 0.41, for luteolin-7-O-glucosid, 9.32 and 15.6 and kaempferol-3-O-glucosid, 0.02 and 0.34, respectively. The proposed method was found to be sensitive, specific and was successfully applied for the estimation of flavonoids in commercial juices.

Natalia Yu Marchenko has completed her PhD in the Kharkiv National University, Ukraine and Institute of Protein Research, RAS, Russia. Currently, she is a Researcher at the Institute of Protein Research, RAS. Her work is focused on protein-protein interactions, specifically on studying the chaperone-protein interaction using different physical and chemical methods, in particular, affinity chromatography. She has published 10 papers in peer reviewed journals.

Molecular chaperones are mainly members of the family of heat shock proteins (HSPs), which participates in folding, transmembrane transport and degradation of numerous cellular proteins. The common property of chaperones is an interaction with polypeptides, which have no rigid spatial structure. In the present work, we demonstrate the use of affinity chromatography on the base of denatured proteins for one-step purification and the analysis of chaperones in cell lysates of different origin. Affinity sorbents on the base of cheap commercial protein products such as lysozyme, cytochrome, α-lactalbumin and pepsin, covalently attached to BrCN-Sepharose, were prepared and tested using GroEL chaperonin. The conditions for the interaction and dissociation of GroEL and the substrate proteins in different conformational states both in solution and in the affinity column were studied. The dissociation constants of some complexes and capacity of some sorbents were evaluated using titration experiments. It was shown that the affinity sorbent on the base of denatured pepsin allows simple purification of native GroEL from fluorescent impurities, tightly bound to it after standard isolation from cells. Using affinity sorbent on the base of denatured lysozyme, cell lysates of different origin (bacteria, archaea, mitochondria) were analyzed. It was shown that mainly GroEL-like chaperonins tightly interact with denatured lysozyme, although other proteins (depending on the lysate origin) were also found upon elution from this affinity sorbent.

Sridhar Siddiraju has completed his PhD from Berhampur University, India. He is working as Associate Professor in Department of Pharmaceutical Chemistry, Malla Reddy College of Pharmacy, Hyderabad, India. He has published more than 30 papers in reputed journals and is serving as Editor-in-Chief of International Journal of Pharmacy.

Darunavir is a protease inhibitor medication used to treat HIV infection. Cobicistat is a pharmacokinetic enhancer used along with anti HIV agents. The US Food and Drug Administration has approved a fixed-dose combination of Darunavir (800 mg) and Cobicistat (150 mg) for HIV treatment in 2015. In the present work, we planned to develop a simple, fast, accurate and precise RP-HPLC method for the simultaneous estimation of Darunavir and Cobicistat in pharmaceutical dosage form. The chromatographic method was developed by using Kromosil C18 column (250mm x 4.6mm, 5µm) and the mobile phase was pumped with acetonitrile and water (pH was adjusted to 3.2 by using ortho phosphoric acid) in the ratio of 70:30 v/v. The mobile phase was pumped at 1ml/min flow rate and the temperature was maintained at 30oC. The retention times of Darunavir and Cobicistat were found to be 3.5 min and 2.7 min, respectively. The method developed was validated in accordance with ICH guidelines with respect to the stability indicating capacity of the method including system suitability, accuracy, precision, linearity, range, limit of detection, limit of quantification, solution stability and robustness. Darunavir and Cobicistat were found to be linear in the range of 40-240 µg/ml and 7.5-45 µg/ml, respectively. The assay results of Darunavir and Cobicistat in PREZCOBIX were found to be 100.4% and 100.9%, respectively. The sample solution was tested for degradation studies in acidic, basic, neutral, peroxide, heat, photolytic conditions and the percentage degradation was found to be within the limits. The developed methods can be used for the routine analysis of the drugs in pharmaceutical dosage forms.