International Conference and Expo on Separation Techniques August 10-12, 2015 San Francisco, USA

Lactulose is a synthetic disaccharide which is produced by the isomerization reaction of lactose in an alkaline environment. Nowadays, the beneficial properties of lactulose in pharmaceutical and nutrition industry have attracted the attentions to explore various perspectives of this disaccharide. Therapeutic effects of lactulose on Portal Systemic Encephalopathy (PSE) and chronic constipation diseases show the importance of lactulose in the pharmaceutical industry. The main challenge associated with laculose synthesis is a considerable amount of unreacted lactose in the reaction media. So far, various methods have been suggested to separation of lactose as an impurity from the lactulose syrup. In this study effect of ethanol and operating temperature on in the separation process of lactose from lactulose syrup was statistically investigated. Taguchi model was utilized to address the importance and effectiveness of the involved factors as well as statistical optimum condition. For this purpose, proportion of ethanol: syrup and operating temperature was were selected as the effective factors. Ethanol: syrup Syrup ratio contains six levels which are 0:2, 2:2, 3:2, 4:2 ,5:2 and 6:2 and operating temperature levels are consist of three levels of 25⁰C (room temperature), 31⁰C, and 37⁰C. Based on the Taguchi model response, Ethanol concentration has the higher the effect than operating temperature on separation of lactose from syrup. Also, Taguchi model revealed that the optimum separation point is at the highest operating temperature and highest ethanol, concentration, however, engineering considerations suggest optimum operating area between 31-37⁰C for operating temperature and 4:2 to 6:2 for ethanol: syrup ratio for.

Irina Khachian has extensive experience in analytical chemistry. She is an Associate Scientist in the Structure Guided Chemistry Department at Dart NeuroScience. Her main focus is on the purification and analysis of small molecules using SFC-MS and HPLC-MS techniques. She is using the latest generation of instruments such as UPC2 and QDa and designs novel components to improve the performance of analytical chemistry systems. She received her MS in Analytical Chemistry from the University of Louisville, Kentucky.

Supercritical Fluid chromatography (SFC) is recognized as a powerful technology for the separation and purification of small chiral and achiral compounds. The flow splitter is a critical instrument component in Preparative SFC-MS instruments that enable efficient and reliable fraction collection by mass. In this study, the evaluation and optimization of tunable and passive flow splitters for preparative SFC-MS are discussed. It has been shown that the passive splitter designed for the Prep100 SFC-MS system can successfully replace the tunable splitter. After several modifications of the passive splitter we were able to minimize the solvent usage for the make-up pump and sustain high resolution, high reproducibility, good peak shape and good recovery of standards. These studies helped us to simplify the instrument hardware, decreased instruments downtime due to splitter clogging and improved the overall reliability and robustness of the Prep100 SFC-MS system.

Warfarin is the most commonly prescribed oral anticoagulant but its use is complicated by its narrow therapeutic index. The biotransformation of warfarin involves both oxidative and reductive processes. In contrast to oxidative pathways, the reductive metabolism of warfarin has not been yet investigated thoroughly. Reduction of warfarin generates warfarin alcohols of two diastereomers. Because warfarin alcohols retain pharmacological activity, the reductive metabolism of warfarin may have impact on treatment outcomes. Two HPLC and SFC methods have been developed and applied for analysis of warfarin alcohols formed after incubation with human liver subcellular fractions and purified recombinant enzymes. One of the methods was the UPLC method for detection of diastereomers of warfarin alcohol, i.e. RS/SR-warfarin alcohol (alcohol 1) and RR/SS-warfarin alcohol (alcohol 2). The second chiral HPLC method allowed analysing the formation of individual enantiomers of warfarin alcohols. Notably, this is the first chiral HPLC method developed so far for the detection of enantiomers of warfarin alcohols. The formation of warfarin alcohols in cytosol exceeded that in microsomes by 10-fold. From the tested enzymes, significant activity toward warfarin reduction has been exhibited by cytosolic enzyme AKR1C3. Another well-known cytosolic enzyme implicated in xenobiotic reductive metabolism, CBR1, formed only low amounts of warfarin alcohols. Even lower activity has been detected for AKR1C1 and AKR1C2. The predominant metabolite formed after incubation with AKR1C3 was RS-warfarin alcohol. This is in agreement with in vivo results from human patients. In conclusion, AKR1C3 was suggested to be the main enzyme responsible for warfarin reduction.

Lukas Stiburek has completed his PhD at the age of 29 years from Charles University in Prague. He is an Assistant Professor of molecular and cellular biology at First Faculty of Medicine, Charles University in Prague.

Blue-Native Polyacrylamide Gel Electrophoresis (BN-PAGE) was originally described by Schägger and von Jagow as a technique used for fractionation of enzymatically active membrane protein complexes. Prior to electrophoretic separation, which is carried in the presence of the anionic dye Coomassie Blue G-250, protein complexes are extracted from the membrane using non-ionic detergent (digitonin, dodecyl maltoside, Triton X-100). Subsequently, Coomassie Blue G-250 is added to the solubilized protein complexes. Equilibrium between protein-associated detergent-lipid micelle and protein-bound G-250 dye is established and the samples are ready to load onto the gels. From this moment on, the electrophoretic run closely resembles that of conventional denaturing PAGE. We have used this technique coupled to second dimension denaturing (SDS) PAGE and subsequent western blotting to study assembly state of mitochondrial oligomeric protein complexes I and IV. We have identified several assembly intermediates of the complexes ranging from approx. 10 to 950 kDa. Very recently, we used BN-PAGE together with second dimension denaturing run successfully for assessment of native molecular weight/oligomeric state of mitochondrial protease YME1L as well as mitochondrial ATPase LACE1. To summarize, when employed in conjunction with preceding protein mixture prefractionation and subsequent protein identification using either western blotting or mass spectrometry, this relatively simple and straightforward technique still proves very useful when studying mitochondrial membrane protein complexes.

The present study consists on the characterization and evaluation of antioxidant activity of phenolic compounds of fruits of Eucalyptus globulus (E. globulus). A method based on high-performance liquid chromatography coupled with diode array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESI-MS) following fractionation by chromatography on a Sephadex LH-20 column has been developed. The presence of 18 gallotannins, 26 ellagitannins, and 2 flavonols was established. Tentative identification is provided for these compounds on the basis of UV-visible spectra and mass spectrometry data. Most compounds described in this study have not previously detected in fruit of E. globulus. Moreover, this is the first report of methyl digalloyl diglucose, 3, 3-0 -dimethylellagic acid 4- -β-glucopyranoside, ellagic acid hexose, methyl ellagic acid pentose, methyltetragalloylglucose, and valoneic acid isomers (sanguisorbic, flavogallic acid dilactone) in the genus. Quantitatively, ellagic acid and its derivatives, including ellagitannins, are largely predominant.

The initiator moieties were successfully attached on the inner surface of silica capillary (50 µm ID and 50 cm length) by binding 4-chloromethyl phenyl isocynate to the silanol groups via isocyanate-hydroxyl reaction with an aid of dibutyl tin dichloride as a catalyst, followed by reaction with sodium diethyl dithiocarbamate. Then N-phenylacrylamide-styrene co-polymer layer was attached via reversed addition-fragmentation chain transfer polymerization. The resultant open tubular silica capillary column showed excellent separation performance for saccharide isomers. Five structural isomers were separated for maltotriose, and α and β anomers were separated for D-glucose with very high separation efficiency (N-values over one million) in CEC in 90/10 (v/v) ACN/30mM sodium acetate of pH 6.6.

Kinetics of bioethanol production from glucose using Saccharomyces cerevisiae (S. cerevisiae) was experimentally studied in a batch membrane bioreactor (MBR) using pervaporation process as well as conventional bioreactor (CBR). For pervaporation, dense hydrophobic polydimethylsiloxane (PDMS) membrane was used. The MBR resulted in increase of cell density, improved productivity and yield. A generic model was developed which can give a unique description for production of bioethanol within both MBR and CBR. Coupled describing equations of the model were solved by means of genetic algorithm approach. The logistic model considered for expression of growth kinetic and kinetic parameters calculated through genetic algorithm. The results demonstrated that this generic model is capable to describe reasonably the behavior of both CBR and MBR.

Reza Ahmadkhaniha has completed his PhD from Tehran University of Medical Sciences (TUMS) and Postdoctoral studies from the same University. He is a member of both “TUMS Scientific Board” and “advisory board of Pharmaceutical Quality Assurance Research Center” of TUMS. He has published more than 50 papers in reputed journals and has been serving as an external reviewer of some reputed journals such as Journal of Chromatography A and B. Currently he is also serving as director of central laboratory of faculty of public health.

A reliable and sensitive quantification method based on stir bar sorptive extraction (SBSE) coupled with thermal desorption-gas chromatography-mass spectrometry (GC-MS) was described for the simultaneous determination of different organophosphorous pesticides in water samples. The analytes were extracted by hydroxyl modified multi-walled carbon nanotubes (MWCNTs-OH) as coating material. The adsorbed compounds were thermally desorbed using a thermal desorption unit connected to a programmed-temperature vaporization (PTV) injector. The desorbed analytes were determined by GC-MS analysis in 32.5 min run time. The method was optimized and validated using quality control samples at different concentrations levels. The optimum condition were found to be 5.5-7.5 for the pH, 30 % (g/v) for the amount of salt, 35 °C and 60 min for adsorption temperature and time respectively, 1500 rpm for stirring rate, programmed desorption at 20°C for 0.5 min, then to 300°C (at 60°C min-1, held for 5 min). The developed method showed good performance. The acquired calibration curves were linear (r2 ≥ 0.96) over the concentration range from ≤5 to 1000 ng L−1. For all of the analytes, the limit of detection was below 2.4 ng L−1. Interday precision values for the analytes were always ≤12% and the reproducibility between coated stir bars for extraction of PAHs was found acceptable (RSD <11 %). The recoveries were in the range of 80-88 % with low relative standard deviation (RSD) values in the range of 8.2-9.5%. Good solvent and thermal-resistance are the other advantages of these coating materials which make it suitable for extraction of target compounds exist in water samples. The developed method was successfully applied to determine target organophosphorous pesticides in real samples. Based on the results diazinon and malathion were found in more than 80% of the samples in the range of 10-150 ng mL-1.

Simple, rapid and sensitive high performance liquid chromatography (HPLC) method was developed and validated for identification and determination of flavonoids in Convolvulus pilosellifolius. The chromatographic separation was achieved in less than 6 min using C18 column (150 × 4.6 mm, 3 μm) with isocratic mixture of methanol and water containing 0.1% v/v formic acid in the ration of 80:20 at 258 nm with a flow rate of 0.4 mL/min. The method was validated in the linear calibration curve ranged between 1–300 μg/mL with detection limits of 0.39 and 0.26 μg/mL and quantification limits of 1.20 and 0.79 μg/mL for quercetin and kaempferol, respectively. Good repeatability of the method were achieved at percent relative standard deviation (RSD <2.18%) with respect to inter- and intra-days. Recovery values were found to be in the range of 98.2–100.2% indicating high accuracy of the method. The maximum flavonoid contents were 1.07 and 1.54 % for quercetin and kaempferol, respectively.

Noushin Rastkari has completed her PhD from Tehran University of Medical Sciences (TUMS) and Postdoctoral studies from the same University. She is a member of both “TUMS Scientific Board” and “advisory board of Pharmaceutical Quality Assurance Research Center” of TUMS. She has published more than 55 papers in reputed journals and has been serving as an external reviewer of some reputed journals such as Analytical Method and Journal of Environmental Health Science and Engineering currently.

A new technique of stir bar sorptive extraction (SBSE) using stir bar 10 mm long coated (50 µm to 175 µm) with hydroxyl modified single-walled carbon nanotubes (SWCNTs-OH) was used to determine polycyclic aromatic hydrocarbons (PAHs) in water samples. Quantitative HPLC analysis was carried out using a 35 min gradient elution and fluorescence detection with excitation and emission wavelengths optimized for each compound. Optimization of experimental conditions including different extraction and desorption factors was performed. The optimum condition were found to be 50°C and 1 h for adsorption temperature andtime respectively, 25°C and 15 min for desorption temperature and time respectively, acetonitrile for extraction solvent. Based on the validation study results, the developed method showed good performance. The acquired calibration curves were linear (r2≥0.97) over the concentration range from ≤1 to 100 ng L−1. For all of the analytes, the limit of detection was below 0.5 ng L−1. Interday precision values for the analytes were always <12% and the reproducibility between coated stir bars for extraction of PAHs was found acceptable (RSD <12 %). The total recoveries were 88.4 to 96.3%. In addition, the coated material showed good solvent and thermal-resistance that is especially advantageous to extraction of target compounds exist in aqueous media. The developed method was applied to the analysis of PAHs in different water samples. One of the samples was found to be polluted with naphthalene. Results of the present study demonstrate the feasibility of the SWCNTs as SBSE adsorbent for the determination of PAHs in water samples.

Hirak V Joshi has completed his PhD at the age of 30 from Jodhpur National University, Jodhpur. He is Assistant Professor at Nootan Pharmacy College, Visnagar, Gujarat, India. He has published 08 research papers in various reputed journals and presented 6 papers at various national and international conferences. His area of interest is pharmaceutical analysis and analytical method development. His total experience is 8 year in the field of pharmaceutical science.

The present investigation describes about a simple, economic, selective, accurate, precise reverse phase high performance liquid chromatographic method for the simultaneous estimation of Trifluperazine HCl, and Isopropramide Iodide in pure and in pharmaceutical tablet dosage forms. Trifluperazine HCl, and Isopropramide Iodide were well separated using a Thermohypersil BDS C18column of dimension 100×4.6, 5µm and mobile phase was methanol (75%) and phosphate buffer (25%), where pH was previously adjusted to pH 3.5 consisting of at the flow rate 1 ml/min and the detection was carried out at 222 nm with UV detector. The retention time for trifluperazine HCl and isopropramide iodide were found to be 1.741 and 3.643 respectively. The developed method was validated for recovery, specificity, precision, accuracy, linearity according to ICH guidelines. The method was successfully applied to Trifluperazine HCl and Isopropramide Iodide in their combined tablet pharmaceutical dosage forms.

Jyothi Penta has completed her MPharm from Kakatiya University. She is now working as an Assistant Professor in a pharmacy college. She has 7 years of teaching and research experience. She has guided many Master’s students towards the achievement of their degree. She published 8 papers in reputed journals.

The intention of present investigation is to develop the glyburide liquisolid compacts containing varying amounts of carrier and coating materials to enhance the bioavailability of glyburide when compared to conventional tablets. MCC or Avicel pH 101/102 as carrier and Aerosil as the coating material are selected for the preparation of liquisolid compacts. Liquisolid technique is a new and promising method that can change the dissolution rate of drugs. This technique of liquisolid preparation is used to formulate drug solution in solid dosage form. From the in vitro drug release studies the optimized formulation F6 showed 92.57±0.54% drug release in 10 min where as the conventional tablets showed 23.87±1.13% in 10 min. The F6 formulation showed 9.25% /min initial dissolution rate, 2.14 relative dissolution rate and 67.52 dissolution efficiency. When compared to conventional tablets, dissolution efficiency increased by 4.5 times in optimized formulation F6. From the above results, it can be inferred that the liquisolid technique increases the wetting properties and surface area of glyburide drug particles by changing their properties achieved by dispersing the drug particles in a non-volatile liquid vehicle. It showed that there are no interactions between the drug and excipients. Differential Scanning Calorimetry (DSC) and Fourier Transform Infrared Spectroscopy (FTIR) studies revealed that there was no interaction between drug and polymer. In conclusion, liquisolid technology was successful in improving bioavailability of glyburide.

Ujashkumar A Shah did his PhD from Bharati Vidhyapeeth, Pune, India. He is currently working as Head, Department of Quality Assurance, Nootan Pharmacy College, Visnagar, Gujarat, India. He has published 06 international research papers with high impact factor international journals. His area of interest is advances in separation techniques and mass spectrometry.

Azelastine HCl and Fluticasone propionate in a nasal spray formulation is newly approved fixed dose combinations and widely used for the treatment of asthma and allergic rhinitis. The present investigation describes about a simple, economic, selective, accurate, precise reverse phase high performance liquid chromatographic method for the simultaneous estimation of azelastine HCl, and fluticasone in pure and in pharmaceutical tablet dosage forms. Estimation of AZL and FLU was carried out by RP-HPLC method, chromatographic separation which was achieved on ACE C18 (150 mm× 4.6 mm, 5 μm particle sizes) having mobile phase: Acetonitrile: Phosphate buffer (67.5: 32.5 %v/v) pH 3. The RP-HPLC method was linear over concentration range 14 to 70 μg/ml and 5 to 25 μg/ml for assay of azelastine HCl & fluticasone respectively. The retention time of azelastine HCl & fluticasone were found to be 1.99 and 5.9 min respectively. Validation of the proposed method was carried out. All above methods were found precise, accurate, rapid, specific and reproducible as all the results found within the acceptable range. All the method was found to be simple, accurate, economical, robust and reproducible. There was no interference of any excipient in the determination of drug in nasal spray formulation so all the methods can be successfully applied for simultaneous estimation of azelastine HCl and fluticasone propionate in nasal dosage form.