6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September 01-03, 2015 Valencia, Spain

Bronopol (2-bromo-2-nitropropane-1,3-diol) is an effective preservative employed in cosmetic formulations. However, under specific conditions, bronopol can decompose releasing low levels of formaldehyde and nitrites . These last compounds can react with any secondary amines or amides to produce significant levels of carcinogenic nitrosamines. The production of nitrosamines in cosmetic products has to be avoided. For this reason, although the use of bronopol is authorized in the current European Regulation, the maximum allowed concentration of bronopol in cosmetic products is 0.1%. Nevertheless, there is still no official method of analysis for the determination of this compound. The aim of this work is to propose a fast and simple analytical method to determine bronopol in cosmetic products. The proposed method is based on a vortex-assisted emulsification extraction, as one-step solution extraction process to prepare cosmetic samples, followed by liquid chromatography with spectrophotometric detection at 250 nm. The best results were achieved by using a C18 column at the following optimized conditions: Temperature, 40ºC; Flow rate, 0.5 mL min-1; Mobile phase, ethanol and a 1% acetic acid aqueous solution were used to perform the separation by elution gradient. Satisfactory results were obtained for the analysis of 19 cosmetic samples including creams, shampoos and bath gels, with good recoveries and repeatability. Limits of detection and quantification were at the low Bg mL−1 levels. These good analytical features, as well as its environmentally-friendly characteristics, make the presented method suitable for the determination of bronopol in cosmetic products.

Asmaa El-Zaher has completed her PhD at Cairo University in 1999 and teaches pharmaceutical and medicinal chemistry in Cairo, Ain Shams, Misr International Universities in Egypt and Umm Al-Qura University in Saudi Arabia. She is specialized in drug analysis. She supervised more than 12 master and PhD awarded thesis and about the same number in progress. She has been awarded for her international publication for more than 4 times by Cairo University. She has published more than 25 papers in reputed journals and more than 10 conference abstracts. She has been serving as reviewer for many reputable journals.

A rapid, precise and selective RPLC method was developed for the simultaneous determination of the widely used oral anti-diabetic; metformin hydrochloride (MTF), along with some of the most commonly prescribed oral anti-diabetic medications representing different pharmacological classes, namely; sitagliptin phosphate (SIT), pioglitazone hydrochloride (PGZ), gliclazide (GLZ), glibenclamide (GLB) and repaglinide (RPG), in bulk, laboratory prepared mixtures and their pharmaceutical formulations. The developed method is recommended for application in the quality control of the herbal anti-diabetic products to detect possible counterfeits. The chromatographic separation carried out using gradient elution mode with acetonitrile: 0.05 M potassium di hydrogen phosphate (MKP) and 0.01 M sodium octane sulphonate (SOS) (pH 3.55) at a flow rate of 0.85 ml/min on a Kromasil 100- C18, (30 x 0.4 cm, 10 µm) at a temperature of 40°C. UV detection carried out at 220 nm. The method validated according to ICH guidelines. Linearity, accuracy and precision were satisfactory over the concentration ranges of 0.05-205 µg/ml for MTF, at 0.05-100 µg/ml for PGZ, GLB and SIT, at 0.1-100 µg/ml for RPG and at 1-100 µg/ml for GLZ. The correlation coefficients were > 0.99 for all analytes. Limits of quantification (LOQs) found to be 0.002, 0.003, 0.009, 0.012, 0.007 and 0.024 µg/mL for MTF, SIT, PGZ, GLZ, GLB and RPG respectively. The method was successfully applied for the determination of each of the studied drugs in their synthetic mixtures and pharmaceutical dosage forms. The developed method is specific and accurate for the quality control and routine analysis of the cited drugs in their pharmaceutical preparations

Dennis Bernieh is a PhD student in the Leicester School of Pharmacy at De Montfort University. His research interests lie in drug treatment optimization and dried blood spot analysis. He is researching into the quantification of therapeutic drugs from dried blood spots based on LC-MS and LC-MS/MS studies for medicine optimization. Prior to starting his PhD studies, he was a KTP Research Associate working for the Department of Chemistry of the University of Hull-UK, and Cobalt Light Systems Ltd in Oxfordshire UK.

Introduction: Over 355 million prescriptions were dispensed for cardiovascular diseases in the UK in 2013. Half of these, costing the NHS £2.3 billion, were wasted because patients do not take their medicines as prescribed. A method using dried blood spot (DBS) sample collection followed by liquid chromatography-high resolution mass spectrometry (LC-HRMS) was developed and validated for quantification of eleven commonly UK prescribed cardiovascular drugs: amlodipine, atenolol, atorvastatin, bisoprolol, diltiazem, doxazosin, lisinopril, losartan, ramipril, simvastatin and valsartan. Thus medication efficiency, adherence or drug/drug interactions can be assessed from reference pharmacokinetic data. Methods: For the preparation of DBS calibration samples whole blood was spiked with eleven target analytes to produce 30 µl blood spots on specimen cards. 8 mm disc was punched out and extracted with methanol: water (70:30 v/v) containing the internal standard, atenolol D7. Chromatography analysis was performed using gradient elution with a run time of 2.5 min. MS detection was carried out in electrospray positive ion mode for all target analytes and internal standard. Results: The LC-HRMS method showed good linearity and the accuracy (relative error) and precision (coefficient of variation) values were within the pre-defined limits of ≤15% at all tested concentrations for eight of the target drugs. Drug recoveries from spiked blood spots were ≥ 82% for atenolol, bisoprolol, diltiazem, doxazosin, losartan, ramipril and valsartan. Results from volunteers were within expected levels except where elevated levels indicated a recognised drug/drug interaction. Conclusions: Quantitative DBS analyses can be used as a means to optimize medication for heart disease patients.

Eliska Cechova is a 3rd year PhD student of environmental chemistry in Masaryk University, Czech Republic with the main focus on development of new analytical methods for determination of organic pollutants and their metabolites in human matrices such as human milk. She is working on EU project Denamic, which is looking for relationship between the presence of mixture of common environmental pollutants and possible effects to child health.

Recently, there is a growing number of children with developmental disorders (autism spectra disorders ASD, attention deficit hyperactivity disorder ADHD, learning problems). The EU project DENAMIC investigates harmful neurotoxic effects of selected compounds in order to reveal possible links between exposure to the compound mixtures and the cause of developmental problems of children. One part of the research includes development of a sensitive method for determination of possible neurotoxic compounds, i.e. persistent and non-persistent pesticides (organochlorine pesticides OCPs and pyrethroids), polybrominated diphenyl ethers (PBDEs), novel flame retardants (NFRs), marker polychlorinated biphenyls (PCBs) and PBDE and pyrethroid metabolites in human matrices, especially in breast milk and human urine. The chemical properties of many of these compounds (part of OCPs and NFRs, all pyrethroids and their metabolites) require non-destructive clean-up methods during sample preparation. After pressurized liquid extraction of a milk sample, the dialysis with LDPE membrane was chosen as a simple and efficient method for fat removal. Additional clean-up with solid phase column with two different sorbents was added for complete lipid removal. Different instrumental methods were tested in order to lower limits of quantification and reduce the effect of matrix. GC-HRMS and APGC-MS/MS for pyrethroid analysis with softer ionization allowed us to determine ultra-trace levels of developmental neurotoxicants in breast milk from European cohorts.

John Ogwu completed his undergraduate degree in Biochemistry from the University of Jos, Plateau State, Nigeria and an MSc with distinction in Advanced Biomedical Science (Chemical Pathology) from De Montfort University Leicester, United Kingdom. He is currently a doctoral research student in the School of Pharmacy, Faculty of Health and Life Sciences, De Montfort University Leicester, UK. His research under the supervision of Dr Sangeeta Tanna and Dr Graham Lawson is titled: ‘Rapid Instrumental Identification of Counterfeit Medicines’.

From therapeutic to lifestyle medicines, the counterfeiting of medicines has been on the rise in recent times. Estimates indicate that about 10% of medicines worldwide are counterfeits with much higher figures in developing countries. Currently, the rapid screening of medicines is a challenge leaving many patients at risk. This study considered the potential use of Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR) for rapid quantitative analysis of tablet formulations. ATR-FTIR requires minimal sample preparation as it only requires crushing the tablet before analysis unlike the conventional methods where time-consuming solvent extraction of the Active Pharmaceutical Ingredient (API) is necessary. Reference spectra for pure API and excipients were recorded as part of a reference library for identification purposes. Preliminary studies were carried out with tablets having a single API (Paracetamol). API could be identified down to 5% w/w of the tablet. Tablet samples with multiple APIs were also identified. For quantitative analysis, IR spectra of standard mixtures of Paracetamol in excipient were recorded and used in calibration. Paracetamol tablets from Europe, Africa and Southeast Asia were then quantified based on calibration data. Quantification data for selected characteristic peak areas for each API/excipient mixture was linear. Results were in the expected range with conventional UV analysis confirming data obtained. Therefore, ATR-FTIR can be applied in the rapid identification and quantification of tablet formulations. The faster sampling time, simplicity and portability of ATR-FTIR makes it valuable in the authentication of medicines especially in developing countries where facilities are not readily available.

Jan Rejšek graduated from Charles University in Prague in analytical chemistry. Now, he is doing his PhD on ambient mass spectrometry at the Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic. He worked for three years in the State Veterinary Institute Prague in the laboratory of atomic spectrometry. He is specialized in the development and routine use of spectrometric analytical techniques.

Desorption atmospheric pressure photoionization (DAPPI) is the ionization technique examined in this study. This method employs solvent spray for desorption and ionization of analytes from the solid surfaces. The aim of this study was to develop a rapid analytical technique enabling to localize separated compounds on the TLC plate and secretory gland openings on the insect body surface and characterize compounds of the secretion using a setup combining DAPPI-MS with a software-controlled moveable motorized sample holder. Six lipid standards and then vernix caseosa extract were separated on TLC plates. Developed TLC plates were mounted on software-controlled moveable motorized sample holder, and spatial distribution of separated compounds was tracked. Soldiers of the termite Prorhinotermes simplex and adult stink bugs Graphosoma lineatum were killed by freezing and fixed on the glass slide using the correction fluid. Body surface was scanned by means of DAPPI to map the spatial distribution of defense compounds. DAPPI in negative ion mode was used to map the spatial distribution of (E)-1-nitropentadec-1-ene on the body surface of P. simplex soldier. Opening of the frontal gland was localized. DAPPI in positive ion mode was used to track the spatial distribution of selected unsaturated aldehydes on the body surface of G. lineatum. Opening of the metathoracic scent glands was localized in the posterior part of the thorax. Effect of surface roughness was evaluated and limit of detection was calculated In brief, development of non-destructive imaging technique suitable for the examination of TLC plates and biological samples was done.

Maha Said Ahmed Abd El-Tawab has graduated from the Faculty of Pharmacy, Cairo University in 2008 with a grade of excellent with honour degree and completed her Master degree in Pharmaceutical Sciences (Pharmaceutical Chemistry) from the same faculty. She is currently an assistant lecturer at the Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University. She has contributed to a conference at Cairo University in 2013 and published a paper in the same year concerning the analysis of some cardiovascular drugs. Also, she is serving as a reviewer at a reputed journal in analytical chemistry.

Aliskiren hemifumarate (ALS) is the first member of a new class of non-peptide renin inhibitors, used as anti-hypertensive agent. Degradation of ALS under alkaline conditions was studied in order to investigate the degradation kinetics of the drug. The analysis of the degraded samples was performed by an isocratic stability indicating liquid chromatographic method. Good separation of ALS from its alkaline degradation product was obtained using Kromasil C18 column (150 x 4.6 mm, 5 µm) at room temperature. The mobile phase was composed of acetonitrile: phosphate buffer (50 mM adjusted to pH 3.5 with o-phosphoric acid) (40: 60, v/v) at a flow rate of 1 ml/min. UV detection was carried out at 229 nm. Linearity was obtained over the concentration range 1.5 – 300 µg/ml. ICH guidelines were adopted for method validation. The method showed high sensitivity, good accuracy and precision. The proposed method was applied to the drug determination in pharmaceutical formulation. The kinetics parameters, such as order of reaction, rate constants and half-life (t1/2) were determined. The degradation of ALS followed pseudo-first order reaction and the degradation rate constant was found to increase by increasing Na OH concentration (up to 1.5 N) and temperature (up to 90°C). The obtained results confirm the reliability of the chromatographic method for the kinetic investigation of the alkaline degradation of ALS and for determining the intact drug in presence of its alkaline degradation product.

The proposed eMALDI approach combines the application of electro-wetting during sample preparation (drying) and regular MALDI MS analytical procedures. The introduction of electro-wetting step allows suppressing the coffee stain effect, decreasing the area of dried sample residue distribution and thus leads to enhancement of MS signal and decrease of signal to noise ratio in the same time not affecting the chemical integrity of sample. Therefore, the introduction of eMALDI as the sample preparation step has a potential to significantly improve the performance of MALDI-MS instruments. The more uniform distribution of examined dry sample residue over smaller area allows decreasing the time for analysis and opens opportunities for further automatization in high throughput MALDI MS techniques. As the e MALDI procedure is used on the sample preparation step, its introduction into MALDI MS protocols would not require further modification of the existing MALDI-MS equipment and would help to provide fast, reliable and fault free MALDI MS analysis.

Roman Polacek graduated at Slovak University of Technology in June 2012 with a Master of Science degree in Analytical chemistry. He began post-gradual study at Institute of Analytical Chemistry with topic Chiral analysis by spectroscopic methods with multivariate calibration. He has great experience in the development and application of various spectroscopy techniques and multivariate calibration methods to determine enantiomeric composition of drug in pharmaceutical and biological samples. He has published five research papers in international journals.

Fluoxetine is a selective serotonin reuptake inhibitor in presynaptic neurons. It proves that stereospecific antidepressant drugs could have very high therapeutic and commercial potential. It is used to treat mental depression, obsessive compulsive disorder, premenstrual dysphoric disorder and nervous bulimia. The enantiomeric composition identification of biologically active chiral compound poses special challenges to scientist in the areas of environmental and pharmaceutical research. The new strategies of the enantiomers resolution include chiral analysis by non chiro-optical methods, such as molecular spectroscopy with chemometric evaluation of data. The rapidity and simplicity of these methods is appealing and reduces the analytical time and the cost of chiral analysis because there is no need of expensive chiral columns and large amount of solvents. The three modes of fluorescence spectrometry, emission, excitation and synchronous, were performed to obtain the data for determination of the enantiomeric composition of the fluoxetine in tablets. Multivariate calibration methods, namely principal component regression (PCR) and partial least square (PLS), were used to evaluation of the data and comparison of the fluorimetric modes. Chiral recognition of fluoxetine enantiomers in the presence of β-CD was based on diastreomeric complexes. The smallest residuals between reference results and predicted values were achieved by PLS model constructed from synchronous fluorescence data. This conclusion is supported by calculated values of figure of merit.

The Kalenjins living in Kericho and the Maasai in the Narok region of Kenya, process and use fermented milk as food after addition of food preservatives. In the present study lead (Pb), cadmium (Cd), mercury (Hg) and chromium (Cr) in fermented cow milk was determined. Samples were collected from selected areas of Kericho and Narok counties of south Rift in Kenya. An atomic absorption spectrometric (AAS) was used for analysis. The amounts of trace metals, copper (Cu) and zinc (Zn) were also similarly quantified. Fermented milk from Narok had higher amounts of copper (0.013±0.03ppm) compared to the other metals Cd (0.012±0.03 ppm), Pb (0.206±0.03 ppm), Zn (0.222±0.03 ppm) and Hg (0.002±0.03 ppm). On the other hand, milk samples from Kericho also had a higher copper content (0.127±0.03ppm) than the other metals, Cd (0.004±0.03 ppm), Pb (0.100±0.03 ppm), and Zn (0.085±0.03 ppm). Chromium was not detected in milk samples for the two regions while mercury was undetected only in milk samples from Narok. Lead levels were higher (0.206±0.03 ppm) in milk samples from Narok than in those from with Kericho County with 0.100±0.03 ppm. In addition, mercury residues were present in the Narok samples averaging 0.002 ppm. Nevertheless, no significant statistical differences were found between the quantities of heavy and trace elements between Kericho and Narok cow milk in reference to the permissible limits by Codex/ WHO. The presence of these heavy and trace metals in fermented milk samples calls for further monitoring in other areas where milk is thus processed and consumed. In addition, some caution in the use of such milk is necessary.

Zeynep AydoÄŸmuÅŸ has completed her PhD from Istanbul University and Postdoctoral studies from University of Illinois at Chicago, College of Pharmacy and Free University of Brussels. She is a full-time Professor at Istanbul University, Faculty of Pharmacy since 2012. She has published more than 35 papers in reputed journals and has been serving as an Editorial Board Member of repute.

Nilotinib (NIL) is used to treat a type of blood cancer called Philadelphia chromosome positive chronic myeloid leukemia. Carbamazepine (CBZ) is used to treat seizures, nerve pain and bipolar disorder. The important drug interactions may occur when used in combination of carbamazepine with Nilotinib. Nilotinib is metabolized by CYP3A4. CBZ shows a strong CYP3A4 inducer feature. A simple, rapid and accurate high performance liquid chromatography method for the simultaneous determination of NIL and CBZ in their tablets were developed and validated. The developed method was applied to in vitro drug interaction studies in different pH environments, simulating empty and full stomach juice (pH 1 and 4), intestinal juice (pH 9), and blood pH (7.4) at 37oC. The method employs an isocratic elution using water and acetonitrile (30:70 v/v) and an RP-C18 HPLC column. Flow-rate was maintained at 1 mL/min, and the eluent monitored using a UV photo diode array detector at 281 nm. The calibration curves were linear over the concentration ranges of 0.0125-5 μg/mL for both drugs. For the interaction studies, in the presence of NIL, availability of CBZ was increased in all interaction medium. The increases of availability of CBZ in different pH medium from most to least were pH 7.4 > pH 4 > pH 9 > pH 1.2 > pH 6.8. For the application of NIL with CBZ, the availability of NIL increased mostly in pH 4 and moderate in pH 1.2 and pH 6.8. The availability of NIL showed decrease in pH 7.4 and 9.

Borzova V.A. has completed her graduation from Moscow State University, Department of Biochemistry. She is 28 years old now and is working currently on her PhD thesis at A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Laboratory of Structural Biochemistry of Proteins, as a Research Scholar. She has published 4 papers in international journals with impact factor > 3.

Thermal aggregation of bovine serum albumin (BSA) was studied at fixed temperatures (60 °C, 65 °C, 70 °C and 80 °C) using dynamic light scattering (DLS), asymmetric-flow field flow fractionation and analytical ultracentrifugation (AUC). Thermal denaturation of the protein was characterized by differential scanning calorimetry. Analysis of the experimental data allowed us to propose the mechanism of thermal aggregation of BSA. Protein unfolding results in the formation of three forms of the non-native protein with different propensity to aggregation. Highly reactive form (Uhr) is characterized by a high rate of aggregation. Aggregation of Uhr leads to the formation of the primary aggregates. Lowly reactive form (Ulr) possesses a low ability for self-aggregation. The Ulr form is able to participate in the aggregation process by attachment to the primary aggregates produced by the Uhr form. Non-reactive form (Unr) remains in the non-aggregated state during prolonged heating. The Unr form was purified and characterized by fluorescent spectroscopy, AUC and DLS. Thermal aggregation of BSA was proposed as a test-system for quantification of the anti-aggregation activity of arginine and its derivatives. The dual effect of arginine derivatives on the initial rate of aggregation was observed. The determination of the order of aggregation with respect to the protein at 70 C shows that the rate-limiting state of the general process of BSA aggregation is the stage of aggregation of the denatured protein molecules. Thus, the observed effects of arginine and its derivatives demonstrate their direct action on the stage of aggregation.

María José Ruiz-Ángel obtained her PhD from the University of Valencia (Spain) in 2003. In 2004‒2006, she was granted with a Post-doctorate fellowship in the Laboratoire des Sciences Analytiques at the University Claude Bernard in Lyon (France). In 2007, she was awarded with a Ramón y Cajal research position in the Department of Analytical Chemistry at the University of Valencia, where she is Professor since March 2012. She has written over 50 research articles, most focusing on secondary equilibria using surfactants and ionic liquids, fundamental studies in HPLC and development of analytical methods for pharmaceutical and clinical samples.

The addition of a surfactant to an aqueous mobile phase at micelle forming concentrations in reversed-phase liquid chromatography (RPLC) is the basis of the chromatographic mode named micellar liquid chromatography (MLC). The anionic surfactant sodium dodecyl sulphate (SDS) is selected in most reports. SDS enhances remarkably the efficiency and peak symmetry of basic compounds by covering the residual silanols in the stationary phase, which remains negatively charged. However, this increases significantly the retention of cationic basic compounds, forcing the addition of a certain amount of organic solvent. On the other hand, the use of a non-ionic surfactant such as Brij-35 increases the polarity of the stationary phase and allows the elution of compounds of low or intermediate polarity without the need of organic solvent. However, polar compounds are not retained. The use of micellar mobile phases containing mixtures of both surfactants (SDS and Brij-35) can modulate the favourable features of each micellar system, yielding successful resolution and practical analysis times for moderately polar basic compounds, such as many β-blockers, also without the addition of organic solvent. In this work, a procedure which employs mixed mobile phases of SDS and Brij 35 is applied to the analysis of a group of β-blockers in urine samples. The procedure is validated following the Food and Drug Administration guidelines in terms of limit of detection, sensitivity, linearity, precision, accuracy, recovery and robustness. The use of the mixed micellar mobile phase has the advantage of the direct injection of the urine sample, which expedites the routine analysis.