Programme
Separation Science 2012, now in its fourth year comes to Kuala Lumpur for a world-class regional conference and exhibition, covering fundamentals, best practice, method development strategies and applications in chromatographic and related analytical techniques. This industry-leading event, will also feature two unique troubleshooting panel sessions (one on LC, one on GC):
LC Troubleshooting Discussion Panel
Wednesday, 27 June: 4.30-6.00pm
This session will assemble a panel of experts for interaction with the audience regarding HPLC-related topics of current interest. Some of the topics that are fair game for discussion are:• Problems and solutions with new technology (such as UHPLC)
• Troubleshooting problem separations
• When compendial methods don’t work
• How to extend column lifetime
• Best practices for reliable HPLC operation
GC Troubleshooting Discussion Panel
Thursday, 28 June: 4.30-6.00pm
This session will assemble a panel of experts for interaction with the audience regarding GC-related topics of current interest. Some of the topics that are fair game for discussion are:
• How to speed up separations using existing hardware
• Understanding GC injection techniques (e.g., splitless, on-column and settings)
• Strategies for extending column life
• Troubleshooting using chromatograms and peak shape
• Maximizing sensitivity in trace analysis
• Column coupling (when and how to implement)
For more information on these panel discussion sessions, CLICK HERE>>
Oral Programme Highlights
Using Quality by Design Principles to Enhance HPLC Method Validation
John Dolan (LC Resources, USA)
The use of Quality by Design (QbD) principles for the development and validation of HPLC methods is based on the ICH Q8 initiative, which states that quality cannot be tested into a product, but that it must be designed into it. The product in this case is an HPLC method. Variables, such as the %B, pH, temperature, and column type, influence the ability to separate analytes in an HPLC separation, but they also can be important factors in how robust the separation is to unintentional changes in conditions. Better HPLC methods can be obtained by using QbD to evaluate the effect of these variables and then demonstrate during validation that a method can tolerate unavoidable changes.
This talk will focus on how to select the important variables during method development and how to evaluate their influence on the separation. Software tools are available that enable one to accurately predict chromatographic results based on a limited amount of experimental data. By using QbD during validation, one can construct a final method that includes information about how to adjust a method to keep it operational even when unexpected changes are encountered.
Application and Limitation of using Adsorbents as Stationary Phases in Gas Chromatography for the Separation of Volatile Compounds
Jaap de Zeeuw (Restek, Netherlands)
Adsorbents are widely used in many petrochemical applications for several reasons. They have high selectivity for dedicated classes of compounds which allows short run times, high retention which allows higher analysis temperatures and there extreme stability because the retention is based on adsorption, which make them very durable. When adsorbents are coated into a capillary, we speak of PLOT – type columns. (Porous Layer Open Tubular). Such adsorption layers are formed by particles which need to be concentric and stabilized. Here is also the challenge, as particles can be swept from the wall and cause blockage. Today’s PLOT columns are highly stabilized and perform well in most hardware configurations. They are chosen often for portable and process type applications where also metal capillaries have found a new application. Developments have been made on alumina, molecular sieve as well on porous polymers adsorbents.
Practical Applications and limitations of using adsorbents in GC will be presented and discussed.
Characterising (bio-)Macromolecules using Hyphenated and Comprehensively Coupled Chromatographic Techniques
Hans-Gerd Janssen (Unilever Research Vlaardingen, Netherlands)
Bio-macromolecules, such as proteins, lignins and polysaccharides, as well as man-made synthetic polymers are often very complex mixtures of individual molecules. This is especially true if the native molecules are modified using enzymatic- or chemical derivatisation methods. For the separation of the resulting highly complex mixtures single dimensional chromatographic separation methods can not deliver the required peak capacity. It is for this reason that multidimensional chromatographic techniques or hyphenated methods involving dedicated detection devices are widely used for characterising such mixtures. Interesting detection systems for hyphenation to LC or GC include of course mass spectrometric detectors, next to techniques like e.g. NMR or selective electrochemical detection. An alternative route towards the more detailed characterisation of complex mixtures of (bio) polymers and their conversion products are systems exploiting hyphenated- or comprehensively coupled chromatographic dimensions.
In the presentation we will discuss various multidimensional systems for the characterisation of complex mixtures of in-tact and (partially) degraded (bio-)macromolecules. The systems studied include LC-NMR, hyphenated- and comprehensive LC – LC and LC × LC, LC – pyrolysis GC, SEC × LC, LC × GC, LC × GC × GC etc., all either or not in combination with MS or other selective detection methods. In particular attention will also be devoted to the selection of the optimum multidimensional method. Finally, the theoretical and practical aspects of comprehensive two- and three-dimensional methods will be discussed.
Moving Supercritical Fluid Chromatography into the Ultra Performance Arena to Become The Interface of GC and LC separations
Mark Ritchie (Waters Pacific, Singapore)
Supercritical fluid chromatography (SFC) is a form of normal phase chromatography that utilises carbon dioxide under super-critical pressures as a mobile phase. With the intermediate properties between liquids and gases, the low viscosity and high diffusivity of supercritical CO2 as a mobile phase component results in higher optimal linear velocities and a lower pressure drop across a column for analyses. It has also been a very attractive technique in the analytical separation of isomers, when used with an appropriate mobile phase. In addition it has been touted as a “green” technique as the solvent use is greatly decreased, and the CO2 for the mobile phase is a by product of other industrial processes.
Recent developments in pumping and fluidic control combined with the application of smaller particle (sub 2 micron) stationary phases and decreased system dispersion have allowed this technique to enter the ultra performance arena. This new analytical ability has been explored in a variety of application areas from biochemical studies through to food safety analysis (e.g. pesticides) for both for chiral and achiral species. Initial investigations have shown good separation of both volatile and non volatile species at a variety of polarities, for example essential oils through to phospholipids and hydrocarbons through to sulphonamides. Many of these species are favoured by either GC or LC techniques, lending the term “convergence chromatography” to the interface between these techniques afforded by this extension of SFC.
Advantages of Using Atomic Emission Detection to Measure Free and Bound Glycerides in Biodiesel
Matthew S. Klee (JAS Inc., USA)
The determination of free and bound glycerides in biodiesel is important for both product quality and process control. Approved method ASTM D6584 specifies a GC-FID configuration for the analysis of free and total glycerin in biodiesel. The method involves the use of internal standards and derivatization using N-methyl-N-trimethylsily-trifluroacetamide (MSTFA). Since the method uses an FID, peak identification relies solely on accuracy of the retention times of the derivatized glycerin and bound glycerides and proper separation of the target peaks from matrix.
The purpose of this study was to demonstrate the advantages of using an AED for ASTM D6584, while meeting method requirements. Correlation coefficients for glycerol, mono, di- and tri-glycerides all exceeded 0.999. The determined detection limit is estimated to be pasting
£ 2 pg Si /sec for silanized diglycerides and £ 30 pg Si/sec for C in triglycerides. Parameters that affect the performance in the GC-AED will be discussed.
Advances in Chiral Separation Technology
Tan Soo Choon (University of Penang, Malaysia)
During the “Golden Age” of synthetic chemistry and drug discovery (1950s to the 1970s) stereochemistry was largely neglected, resulting in approximately 25% of pharmaceuticals being marketed as racemates by the 1980s. The increasing awareness that many of nature’s molecules and that the natural ligands of drug targets are chiral in nature resulted in strong interest in the stereoselectivity of drug action. This lead to the discovery that individual enantiomers possess rather different pharmacological and pharmacokinetic properties. Such advancement in knowledge was possible due to the parallel advancement in separation technologies, allowing the chiral chromatographic separation of drug enantiomers. Chiral separations are only possible in a chiral chromatographic environment. Such an environment is afforded by either chiral additives in the mobile phases or chiral mobile phases. Alternatively, enantiomers can be separated by after conversion to diastereomers with chiral derivatizing reagents. However results from such indirect separation methods need to be interpreted carefully due to the possibility of racemisation during derivatization. Traditionally used chiral stationary phases (CSPs) are the Pirkle or brush type, cyclodextrin, protein and carbohydrate type CSPs. With the advancement in LC-MS instrumentation, newer generation LC-MS friendly CSPs have been developed. These include the macrocyclic glycopeptides phases and newer reversed phase carbohydrate phases. Antibodies have also been shown to be extremely good chiral selectors, as is with their synthetic alternatives like molecularly imprinted polymers and these have the potential to be used for semi-preparative separations.
Basic Principles of the Quadrupole Mass Spectrometer
Mustafa Ali Mohd (University of Malaya, Kuala Lumpur)
The paper discusses the basic concept, mechanisms and functions of the quadrupole in a mass spectrometer. The lecture will enable the users to understand the processes involved during the data generation and the data acquisition and ways to enhance data acquisition with respect to improving the quadrupole functions. Understanding the quadrupole function and the mass filtering concept will help in the understanding of the tuning and calibration aspect of the mass spectrometer. This will further lead to users to understand the concept of sensitivity and the possibility of improving sensitivities in their instruments.
New Approaches in Sample Preparation for Bioanalysis
Ronald E. Majors (Agilent Technologies, USA)
For many years, techniques of classical liquid-liquid extraction and solid-phase extraction (SPE) have been used to isolate drugs and their metabolites in biological fluids (e.g. plasma, urine, CSF). For these approaches, large quantities of biofluids were often required and extensive manual labor and time were required to process samples, often with poor analyte recovery. There has been a renaissance in sample preparation techniques that are scaled down to work with smaller samples and are simpler to perform. For example, QuEChERS, which initially used for pesticides in fruits and vegetables, has been found to be more universal and can handle biofluids, such as blood, directly. Automation techniques such as protein precipitation in 96-well plates have revolutionized high-throughput sample prep such that many samples can be processed in a single day that used to require multiple days. In this lecture, attendees will learn about some of these newer techniques of sample preparation as well as improved sample collection/transport approaches such as dried matrix (blood) spotting where microliter size samples can be collected a long way from the laboratory and be analyzed later using newer measurement techniques such as LC-MS/MS. The result of these new approaches is faster sample processing, lower detection limits, better recovery, and less manual labor.
Pharmaceutical Analysis by Ion Chromatography with Universal Detection
Paul Haddad (University of Tasmania, Australia)
Pharmaceutical analysis is routinely performed by reversed-phase high performance liquid chromatography (RPLC). However, many pharmaceutically-related compounds are either ionic or ionogenic, so ion-exchange offers an alternative separation mode which can provide complementary separation selectivity to RPLC. Use of two such complementary separation tools enhances the capability of separating and identifying minor impurities during drug discovery and production. Two facets of pharmaceutical analysis by ion chromatography (IC) will be addressed. The first relates to rapid, computerised optimisation of separation conditions for organic pharmaceuticals. A complicating factor here is that the eluents used must normally contain considerable amounts of organic solvents to minimise secondary hydrophobic adsorption of the analytes onto the polymeric stationary phase. For this reason, both electrostatic and hydrophobic retention mechanisms need to be considered. Two facets of pharmaceutical analysis by ion chromatography (IC) will be addressed. The first relates to rapid, computerised optimisation of separation conditions for organic pharmaceuticals. A complicating factor here is that the eluents used must normally contain considerable amounts of organic solvents to minimise secondary hydrophobic adsorption of the analytes onto the polymeric stationary phase. For this reason, both electrostatic and hydrophobic retention mechanisms need to be considered. The second topic relates to new uses of suppressors, which have long been used in IC to enhance the detection signal when conductivity detectors are used. Suppressors operate by exchanging eluent ions for regenerant ions and when this results in the formation of water, the suppressor effectively acts as a de-salting device. Thus, suppressors can be used for de-salting of IC eluents prior to detection using universal detection modes such as mass spectrometry, evaporative light scattering detection (ELSD) and charged aerosol detection (CAD). Studies showing how the suppression reactions affect recoveries of organic analyte ions passing through the suppressor are described, and new purpose-built suppressors are also evaluated. The use of suppressors coupled to nebulisation detectors and mass spectrometric detection will be illustrated, showing that ion-exchange separations of non-chromophoric analytes of pharmaceutical interest can be achieved under gradient elution conditions.
Application of High Performance Liquid Chromatography and Gas Chromatography in Palm Oil Analyses
Halimah Muhamad (Malaysian Palm Oil Board, Malaysia)
Palm oil offers great potential for use in many areas, such as a source of energy, food products, pharmaceuticals, and also cosmetics. As Malaysia is one of the world’s largest palm oil producers and exporters, it has an important role in fulfilling the global need for quality control and safe palm oil for consumption. Therefore, methods for quality measurement of palm oil and its products have been developed by MPOB to ensure that only good quality palm oil is produced.
There are many analyses being carried out by MPOB which involved wet analysis such as Peroxide Value (PV), Free Fatty Acid (FFA) and Iodine Value (IV), used to determine the quality of palm oil. Other than that, research in MPOB also involves chemical analyses and development of methods for palm oil and its products and environmental samples that require instrumental analysis. For instance, instruments such as Liquid Chromatography and Gas Chromatography with Mass Spectrometer (MS) are used to separate and identify the compound of interest and at the same time, check the purity of the components. Instruments with other detectors such as ultraviolet (UV), Fluorescence (FL) and Refractive Index (RI) are used to detect the presence of a specific compound in samples. Therefore, this paper reviews research areas involving volatile compounds, minor components, fatty acid composition, organic compounds and pesticides residue in palm oil and its products using High Performance Liquid Chromatography and Gas Chromatography with different detectors.
Improving Method Speed and Quality with Modern Time of Flight (TOF) LC/MS Systems
Silverio Iactone (PerkinElmer, USA)
There is a continuous drive to obtain more information from samples, faster and easier. Having to complete solid phase extraction protocols for sample matrices adds a layer of complexity, cost and risk of reduced reproducibility.
Modern Time of Flight (TOF) LC/MS systems offer advantages over other LC/MS techniques due to their ease of use, their full scan sensitivity, combined with accurate mass capability and stability. The ability to quantify with speed, over the entire mass range enables the user to be confident of structural insight gained whilst also realising the benefits of the UHPLC separation. Employing different phase chemistries or stationary phase types (traditional porous silicas vs. superficially porous silicas) provides further scope to increase speed of analysis.
With the ambient sampling technique of Direct Sample Analysis (DSA) coupled with TOF MS, scientists have rapid qualitative and quantitative analysis at their fingertips. Little or No sample clean up required and automated sampling provides incredibly fast and efficient work flows that take seconds to generate exceptional full scan, mass accurate information, from solids or liquids.
Solvent-Based and Sorbent-Based Microscale Sample Preparation for Onsite Applications: Possibilities and Challenges
Hian Kee Lee (NUS, Singapore)
Although there have been many examples of in-house solvent-based and sorbent-phase microscale sample preparation that, on paper, appear to lend themselves to potential onsite application, the reality is that they are faced with many practical challenges and problems. This talk will discuss the microscale sample preparation procedures that our laboratory has developed, and demonstrate how they may be applied to onsite use. The presentation will also describe how some of these procedures may be integrated with chromatography-mass spectrometric analysis for automated onsite operations.
Recent Advances in In-silico Chromatographic Method Development for Pharmaceutical Analysis
Roman Szucs (Pfizer Global R&D, Sandwich, UK)
Chromatographic analysis represents a large proportion of the analytical testing used to support pharmaceutical drug development as well as quality control. Even though some recent trends in pharmaceutical analysis seem to favour Process Analytical Technology (PAT) and spectroscopic tools, chromatography remains the number one choice mainly for its ability to deal with complex matrices. Chromatographic method lifecycle typically consists of three steps, method development, method validation and method application. This process is continuously applied in parallel with product development as the changes in synthetic process and formulation usually trigger the need for method development or optimisation.
In this contribution we demonstrate how recent advances in separation sciences have led to significant simplification of the chromatographic method development. Using practical examples, we will show how increased resolution using state of the art techniques and rational selection of stationary phase selectivity can increase the probability of complete resolution of even the most complex samples without a corresponding need for method development or optimisation. Finally, we will also demonstrate how the latest computational tools can further improve the effectiveness of the chromatographic method development process, reducing waste, and speeding up the process as well reducing the negative impact on the environment
Low Level POP Analysis Using GCxGC-TOFMS
Jayne de Vos (National Metrology Insitute of South Africa)
There is an accelerating trend towards persistent organic pollutant (POP) analysis at lower and lower levels. Many countries, especially those in the developing world, do not have gas chromatography coupled with high resolution mass spectrometry (GC-HRMS) systems capable of doing this analysis. Comprehensive two-dimensional gas chromatography combined with Time-of-Flight mass spectrometry (GCxGC-TOFMS) has been investigated as a screening tool for POP analysis, and this presentation details the extension of this approach to encompass these lower levels. As a screening tool GCxGC-TOFMS has the advantage of being able to screen for various POPs in a single analytical run. With this in mind, the technique of Concurrent Solvent Recondensation Large Volume Splitless Injection (CSR-LVSI) was used for analyte introduction, using up to 10µL onto 0.53mm retention gap connected to the primary analytical column (30m x 0.25mm x 0.25µm). The larger amount of analyte introduced onto the primary column increases the ability to reach the lower detection limits needed for trace analysis. This set-up is simple, cost effective and easily implemented as is required in laboratories in the developing world.
Capillary Electrophoresis in the Pharmaceutical and Biotech Industries
Cari Sänger (Kantisto, Netherlands)
Myths, facts, use, issues and needs. In this presentation, an overview will be given about the current use of the capillary electrophoresis techniques (CE) in the pharmaceutical and biotech industry. Regularly returning questions will be addressed: Why would one use capillary electrophoresis and why not, what are the myths and the facts? Does industry use capillary electrophoresis? How is the regulatory acceptance? What are the prerequisites for successful application? Several examples will be presented, both of small molecules and protein therapeutics. Finally, current issues with the use of the capillary electrophoresis techniques in industry will be discussed.
After this lecture, you will understand the position of the capillary electrophoresis techniques within industry and the regulatory agencies, illustrated with many application examples. You will also be aware of what is needed for further successful application and implementation of CE.
The Core-shell Advantage: The Current and Future Standard of Ultra High Performance Liquid Chromatography
Phil Koerner (Phenomenex, USA)
This presentation examines the extent to which the various beneficial properties of coreshell particles manifest themselves over a wide range of particle and pore sizes, including those already commercially available (1.7, 2.6, 2.7 and 3.6 μm - in various pore sizes) as well as several experimental particles with unique particle geometries. The various contributors to the “core-shell advantage,” include 1) exceptional reduced plate-height maintained over a wide range of linear velocities including extremely high ones, 2) manageable frictional heating due to high thermal conductivity and 3) outstandingly low separation impedance. The results presented here demonstrate that the aforementioned benefits are maintained over the entire range of core-shell particle morphologies studied to date, and thus are intrinsic to the coreshell technology. This technology platform is well positioned to continue its leading position in providing the separation solutions required for both the fastest and the most demanding analyses of the future.
Development of Rapid Chromatographic Systems for the Screening of Improvised Explosives
Greg Dicinoski (University of Tasmania, Australia)
The identification of explosives is an indispensable tool during the investigations and prevention of terrorist attacks. A large percentage of bombings or attempted bombings are performed employing improvised explosive devices (IEDs) based on mixtures from legal and readily available inorganic compounds. The identification of residues of IEDs in real situations demands analytical methods with high requirements in terms of speed of analysis, sensitivity and portability of the instrumentation. Capillary electrophoresis offers an attractive combination of characteristics for the identification of inorganic IEDs residues including high separation efficiency and simplicity of instrumentation. This presentation will discuss our recent research in both capillary and microchip format electrophoresis to perform rapid sub-minute separations of the anions and cations critical to the identification of IEDs. Rapid chromatographic systems have also been developed to provide confirmation analysis and these will also be presented.
Prefractionation Strategies for the Analysis of the Proteome of Hevea brasiliensis: Impact on Protein Coverage using Nano LC-MS with Multiple Activation Methods
Aishah Latiff (Doping Control Centre, Universiti Sains Malaysia, Penang, Malaysia)
Hevea brasiliensis is a latex producing tree that is an important commodity in Malaysia. Recently, the Centre for Chemical Biology, Universiti Sains Malaysia, has sequenced the whole genome of this plant, and characterized a number of biosynthetic pathways relating to latex production. This study was undertaken to characterize the proteome of the organism through a comprehensive analysis of three fractions obtained from latex collected using a routine trunk tapping protocol, and from leaves of a juvenile plant. Protein identification results were correlated with proteins predicted from the genome sequence, and subsequently compared with the suspected biosynthetic pathways.
It was decided to opt for a prefractionation protocol, simplifying the protein mixture first by molecular weight separation into 12 liquid based fractions on a GELFREE 8100. The collected fractions were individually reduced and aklylated before being subjected to protease digestion with trypsin. Each of the digests was separated by nano-flow reverse phase chromatography coupled to an Orbitrap Velos. Data was acquired in the MS mode at 60,000 resolution and MS/MS data acquired using three activation methods – CID, HCD, and ETD.
This pre-fractionation protocol led to the successful identification of more than 9,758 proteins including allergen, latex elongation factor, protease inhibitors, enzymes, and latex cystatin.
Improving UPLC Performance for the Routine Analysis of Drug Final Formulations
Doug McCabe (Waters Corp., USA)
An application area where the sample throughput, analyte response and chromatographic resolution benefits of UPLC technology have translated into significant business productivity gains is the routine analysis of drug final formulations. In this very practical seminar we will describe how UPLC technology can best be utilized in laboratories that follow chromatographic test methods found in compendial monographs. We will provide examples of drug final formulations assays taken from the USP-NF that were successfully transferred to UPLC along with routine analysis studies where the long-term robustness of the UPLC methods was evaluated.
Multidimensional Analytical Screening Technologies for Comprehensive Metabolic and Natural Extract Profiling
Jeroen Kool (VU University Amsterdam, The Netherlands)
For profiling of complex bioactive mixtures, different integrated bioaffinity screening approaches are pursued. This presentation focuses on on-line screening technologies and high resolution nanofractionation approaches. On-line systems combine separation sciences, mass spectrometry and biochemical methodologies in single integrated platforms. In some cases, when on-line analysis is less suitable, nanofractionation strategies are excellent alternatives. MS based metabolite profiling and natural extract screening in combination with comprehensive bioaffinity, selectivity and functional activity assessment will be discussed.
Optimization of Solid Phase Extraction for the Analysis of Benzodiazapines from Plasma
Tony Edge (Thermo Fisher Scientific, UK)
The use of sample preparation techniques can significantly reduce the effects of ion suppression; however optimization of this process is not routinely performed. This presentation will look at the use of solid phase extraction and the optimization of the load, wash, and elution steps to determine the effect that this has on the degree of ion suppression. A series of benzodiazapines has been investigated to demonstrate the effect of optimization of the extraction procedure in the removal of endogenous material. Of particular interest is the effect of the elution conditions, which typically defaults to 100% organic, which is not always optimal for selective extraction which will be demonstrated in this talk.
Data demonstrates how the optimization of the wash and elution profiles maximizes recovery of the benzodiazepines and the selectivity of the assay by minimizing interferences from the matrix.
Analysis of Trace Level Phthalate Esters in Environmental Water Samples by Online-SPE-LC-MS
Ichiro Hirano (Shimadzu Corporation, Kyoto, Japan)
Phthalate esters are nearly ubiquitous in today’s society as these compounds are produced in large quantities throughout the world and are widely used as not only primary plasticizers in plastic industries but also the ingredients of toiletry products. However, these compounds draw global concern due to their suspected endocrine disrupting potential. Furthermore, when phthalate di-esters are introduced into the human body, they are metabolized into mono-esters and excreted in urine. Subsequently these compounds may pass from wastewater to river water primarily due to insufficient wastewater treatment. Since both phthalate di- and mono-esters are likely to exist as a mixture in the environment, it is important to develop a simultaneous quantification method for both forms. Here, we report the development of a simultaneous analysis technique for trace amounts of phthalate di- and mono-esters by using an online-SPE-LC system coupled to a triple quadruple mass spectrometer. In order to achieve a single high throughput method, rapid polarity switching was critical as phthalate di-esters are preferentially ionized in ESI positive but mono-esters are more sensitive in ESI negative.
Chromatographic Optimization for High-Throughput Quantitative Proteomics
Chris Loran (Technology Consultant, USA)
Nano flow LC-MS/MS has become a standard tool for the identification, characterization and quantitation of low abundance proteins in complex proteome samples. Although this technique provides sensitivity and resolution, the low flow rates used with this technique often result in long sample loading times, long gradient delays and long system re-equilibration times. Since the MS only generates useful MSMS spectra during the LC gradient time, these long delays can result in MS utilization only 20-40% of the total run time (inject to inject).
This presentation will investigate the tools and techniques commercially available to optimize the LCMS system, reducing the chromatographic delay and re-equilibration times, while increasing efficient usage of the mass spectrometer. The techniques presented will enable the user to tune their LCMS resulting in higher throughput and enhanced protein identification. The data presented will include 33% reduction in run time with 5-fold increase in peak capacity for simple protein digests. These techniques will then be applied to complex proteome samples to achieve 2X protein identifications through chromatographic optimization prior to MSMS analysis.
Preparation and Release Analysis of Encapsulated Herbals for Slow Release Dosage Forms Containing Multiple Bioactive Components
Paul Heng (National University of Singapore)
There had been much interest to prepare slow release bioactive products as it allows more sustained and prolonged availability of the bioactives in the systemic circulatory system. The slow release system may allow for better bioactive absorption as well as more sustained blood concentration for the bioactive to exert its pharmacological or therapeutic functions. However, the in vitro release assessment of bioactives from herbal products such as traditional Chinese medicines in slow release formulations is difficult to determine with a high level of confidence. Part of this difficulty comes with the lack of the clear identity of the main therapeutic agent. It is commonly conceived that the biological action from herbal product is more akin to singing by a chorus rather than a soprano. Thus, the identity of the main bioactive compound is not clearly defined, making release testing rather complicated. It may be necessary to consider the net release of all the multiple components. Application of multivariate analysis to the analysis of bioactive release from herbal products may be an option. This presentation will explore the formation and release testing of coated multi-particulate herbal products for the design of slow release bioactive dosage form.
Application of the Hyphenated System, LC-ICP-MS to Perform Arsenic Speciation Analysis in Rice
Joanne Chan (HSA, Singapore)
Arsenic has organic and inorganic species, of which the inorganic form has been evaluated by International Agency for Research on Cancer (IARC) to be the more toxic form. Arsenic is naturally present in the environment, particularly in the soils and water. Hence, amongst all crop plants, rice is known to generally have higher levels of arsenic. As rice is the primary dietary source to the Asian population, it is important to develop a sensitive analytical method to accurately determine the inorganic arsenic levels in rice. This study also establishes more comprehensive occurrence data on inorganic arsenic in Asian rice.
Comprehensive Multidimensional Gas Chromatography with Resistively Heated Capillary Columns
Robert Shellie (University of Tasmania, Australia)
Comprehensive multidimensional gas chromatography (GCxGC) is an enabling technology for environmental assessment of fuel-contaminated sites. GCxGC was first described in the open literature in 1991. GCxGC relies on modulating the transfer of solute from a first-dimension separation column to a second-dimension separation column where a series of contiguous discrete analyses are performed. Various approaches exist for performing the necessary modulation, these include: i) thermal modulation – where cooling and / or heating is used to manipulate solute retention, ii) flow switching using mechanical valves and iii) pneumatic modulation using live switching to modulate the flow of chromatographic effluent from the first- to second-column. We have embraced the latter of these approaches in search of a field-based approach for environmental assessment of fuel-contaminated sites. Initial evaluations were performed using conventional instrumentation with highly satisfactory results. Recently we have transferred our methodology to a more portable format. Namely a lightweight, low-energy gas chromatograph that employs resistively heated capillary columns in place of conventional oven. This paper will discuss our progress towards the development of a field-deployable comprehensive multidimensional gas chromatograph.
Modern Extraction Methods Combined with CE for the Analysis of Phenolic Compounds
Bahruddin Saad (Universiti Sains Malaysia)
The beneficial effects of phenolic compounds as natural antioxidants has been long established. Epidemiological studies have shown that consumption of foods and beverages rich in phenolics is correlated with reduced incidence of heart diseases. Fruits, vegetables and spices are the primary sources of naturally occurring antioxidants for humans. Very few studies investigate on the type and levels of phenolic compounds present in vegetable oils. The determination of these phenols in vegetable oils normally involves liquid-liquid extraction (LLE), followed by reversed phase HPLC separation. Although the LLE is a proven technique, it is time-consuming, labour intensive and is further complicated by the frequent formation of emulsions. Furthermore, the use of some of the extracting solvents can also pose adverse effects on the operator and the environment. In this presentation, we outline an new method for the determination of 13 phenolic compounds (caffeic, gallic, cinnamic, ferulic, chlorogenic, syringic, vanillic, benzoic, p-hydroxybenzoic, 2,4-dihydroxybenzoic, o-coumaric, m-coumaric and p-coumaric acids) in vegetable oil samples. The analytes are selective extracted using a solventless in-vial liquid-liquid microextraction technique before being separated using capillary electropohoresis (CE). The combination of the microextraction and the high resolving power CE procedure resulted in a new method that not only consumes markedly reduced amounts of solvents but is also rapid and simple. The validated method was applied to the determination of phenolic acids in several vegetable oil samples (palm, olive, walnut and grapeseed).
A microextraction method for the isolation of phenolic compounds is described. Complex mixtures can be resolved in a relatively short time using capillary electrophoresis.
GC-QTOF – A New Tool for Challenging Applications
Tan Lay Peng (Agilent Technologies, Singapore)
The new Agilent 7200 Q-TOF combines the proven, rugged performance of the Agilent 7890A GC with the high spectral resolution of a Q-TOF analyzer that also features time-tested designs for high-confidence results.
Discover how the 7200 GC-QTOF delivers a new level of chromatographic and spectral resolution to a wide range of biological and chemical analyses to help researchers coax more qualitative and quantitative information out of increasingly complex samples. Applications include environmental analysis, food safety, sports-doping detection, energy research, natural products research, and energy research.
This presentation will provide an overview of this new technology and why it can be used for challenging applications with examples of real applications.
Food Flavours and Composition – Advanced Methods of Analysis Based on Gas Chromatography
Philip Marriott (Monash University, Australia)
Many compositional parameters in foods may be studied by use of GC methods. We have been at the forefront of development of novel methods based on multidimensional GC and comprehensive 2D GC for a wide range of analytes, from pesticides, to petrochemicals and oils, drugs, and perfumes/essential oils. Amongst these, we have focussed some effort on foods and beverage components. This includes GC-olfactometry (O) and MDGC-O detection for aroma-active compounds in a variety of samples such as herbs and spices, coffee, and wine. We have also applied our methods to fatty acid (FA) analysis, since this can provide a superior approach to profiling the composition of FA in foods. In this presentation, we shall describe how we set up a multidimensional GC experiment, and why MDGC with sensory evaluation (olfactometry) is a better – more reliable – way to conduct this experiment. Since identification also requires mass spectrometry, we incorporate MS with our ‘O’ studies, and sometimes need to develop ‘multi-platform’ technologies to provide identification. Finally, we will briefly describe the role of our methods for improved characterisation of FA in a variety of sample types. After this presentation, we hope that we will provide an improved appreciation of why advanced methods of separation – based on newly emerging multidimensional approaches – can lead to the best possible characterisation of complex chemical samples.
Application of Mass Spectrometry for Rapid Identification of Microbes
Matthew Lau (Nanyang Polytechnic, Singapore)
Detection and confirmation of microbial specimens in food safety has always been a time consuming process, taking between 2 days to more than a week for a good identification of the organism. It also requires the lab to be suitably manned by experts as well as appropriately provisioned for the various types of media and identification kits for the suspected organism.
This presentation will provide an example of how Mass Spectrometry could be applied in the food safety testing lab. Application of MALDI ToF for the identification and of not only single organisms but also mixtures of organisms of diverse origins would be covered with examples. The overall benefit of this protocol and the flexibility it affords to the food safety labs could potentially transform food safety testing in the future.
A Generic Method Development Strategy For Single and Coupled Column Systems
Gert Desmet (Free University, Belgium)
One of the most difficult tasks in analytical liquid chromatography is method development (MD), i.e., the search for the combination of chromatographic parameter settings that leads to the complete resolution of a sample in all its individual constituents. Nowadays, the MD process still involves a lot of trial and error because of the high probability for peak overlap in the obtained chromatograms. Many separation problems can only be cracked after a few weeks' or even a months’ work, despite the fact that the analyst has a vast arsenal of chromatographic parameter settings at his disposal to try to reposition the compound peaks in the chromatogram: type of organic modifier, type of stationary phase, temperature, gradient profile, pH, ionic strength, etc. In the present contribution, we report on the possibilities of an MD algorithm that explores the chromatographic parameter space in a blind search-mode, but uses the information on the retention properties of some of the peaks collected during this search to shift and stretch the elution window over different parts of the time-axis using a model-based prediction of the position of these peaks.
Improved Analysis of Fatty Acids by Capillary GC columns based on Ionic Liquids
Frank Michel (Supelco/Sigma-Aldrich R&D, Germany)
Analyses of fatty acids (FAs) after transesterification to the corresponding FA methyl esters (FAMEs) are continuing to gain importance as more research is focusing on their biomedical impacts. This includes the analysis of saturated and polyunsaturated FAs along with the positional geometric (cis and trans) FA isomers. Unsaturated trans FAs (which are created e.g. during hydrogenation of vegetable oil) and saturated FAs are considered to have a negative impact on human health. Although bicyanopropyl polysilicone columns are the current standard for the separation of geometric FAME isomers there are still some limitations especially in the separation of both mono- and polyunsaturated FAMEs and conjugated linoleic acids (CLA).
Ionic liquids are a class of non-molecular ionic solvents with low melting points. These compounds exhibit ideal properties for a stationary phase in gas chromatography such as very low vapor pressure and high thermal stability [1]. These liquids are unique combinations of cations and anions and can provide a variety of different selectivities when used as capillary GC phases. The application of new dicationic liquids as stationary phases improved the efficiency as well as the temperature stability [2].
This work introduces the technology of ionic liquids as stationary phases for GC and focuses on the challenging separation of selected geometric and positional isomers of the FAMEs 18:1, 18:2 and 18:3. Separations of several artificial and original multiunsaturated fatty acid (MUFA) mixtures from partially hydrogenated vegetable oil on this new polar GC column based on ionic liquids will be shown and be compared with those obtained from traditional bicyanopropyl polysiloxane columns. The ionic liquid GC column provided resolution of some geometrical isomers that require complimentary techniques for their analysis using bicyanopropyl polysiloxane columns.
Micro SPE – The Emerging Technique of Microextraction by Packed Sorbent and its Application in Bioanalysis
Andrew Gooley (SGE, Australia)
While the value of sample preparation is well understood it is not always executed efficiently or economically. Many laboratories run with a minimum of labour and consequently the development or improvement of sample preparation methodologies is rarely a priority. MEPS is a miniaturised form of SPE and is a promising approach since the coupling of the micro-SPE format to both GC-MS and LC-MS can be fully automated. The short extraction times, small sample & solvent volumes and the seamless integration with automation make the MEPS technique attractive for rapid sample preparation. This presentation will focus on the features and benefits of adopting micro SPE in sample preparation as well as published examples associated with bioanalysis including therapeutic drug monitoring and drug metabolism analysis in human extracellular fluids.
Application of Full Evaporation Dynamic Headspace (FEDHS) and GC-MS to the Analysis of Odor Compounds in Green Tea
Nobuo Ochiai (GERSTEL K.K., Japan)Since odor compounds in green tea present at trace level (from pg mL-1 to ng mL-1), analytical methods should include powerful extraction and enrichment steps before GC analysis. However, traditional approach such as steam distillation followed by solid phase extraction (SPE) requires large sample amount (typically more than 1 L) and tedious sample preparation process. Miniaturized methods such as solid phase microextraction (SPME) and stir bar sorptive extraction (SBSE), which allow extraction and concentration in a single step, provide enhanced sensitivity even with small sample amount (typically 10- 20 mL) because the whole extracted fraction can be introduced into GC by thermal desorption. However, these techniques in both immersion and headspace mode are generally biased toward recovering more hydrophobic compounds in aqueous samples. Recently, we developed a novel headspace technique called “Full evaporation dynamic headspace (FEDHS)” [1] based on full evaporation technique (FET) [2]. FEDHS allows complete vaporization of 100 μL of an aqueous sample, and drying it in an adsorbent packed tube, while recovering a wide range of odor compounds including more hydrophilic compounds, and leaving most of the low volatile matrix behind. The FEDHS-GC-MS method showed high sensitivity (sub-ng mL-1 to ng mL-1) even with the scan mode in the conventional MS. In this presentation, delegate will be shown application of FEDHS-GC-MS for analysis of odor compounds at sub-ng mL-1 to ng mL-1 in green tea. For a Japanese green tea sample, 48 compounds including 19 potent odorants were positively identified from only 100 μL of sample. Heat-induced artifact formation for potent odorants was also examined and the proposed method does not affect the additional formation of thermally generated compounds.
Enantioseparation of (±)-threo-Methylphenidate in Human Plasma by Cyclodextrin-modified Sample Stacking Capillary Electrophoresis
Shou-Mei Wu (Kaohsiung Medical University, Taiwan)
The (±)-threo-methylphenidate ((±)-threo-MP) is widely used for treatment of attention-deficit hyperactivity disorder (ADHD). According to clinical evidence, (+)-threo-MP possesses higher potency than (-)-threo-MP. Due to ppb level in plasma, till now, none of the capillary electrophoresis (CE) methods have been able to provide adequate sensitivity for therapeutic (±)-threo-MP monitoring. In this study, a cyclodextrin-modified field-amplified sample stacking CE method (CD-FASS-CE) for enantioseparation of (±)-threo-MP in human plasma was established for clinical applications. Phosphate buffer was filled into uncoated fused silica capillary, followed by a water plug. Electrokinetic injection was used to load samples and to enhance sensitivity. Stacking and separation were performed at 20 kV and 200 nm using phosphate buffer containing HP-CD and triethanolamine. Analytes were separated simultaneously by using CD-FASS-CE and had a lower detection limit of equal to the sub-ppb level. Linear calibration curves were obtained from 1 to 80 ng/mL (r=0.998). The limit of detection for both isomers was 600 pg/mL. RSD and RE of precision and accuracy in intra- and inter-day assays were below 7.89%. This method was further applied to analyze (±)-threo-MP in four healthy Asian volunteers and that provided some relevant information for clinical treatments.
A Compound-based Approach to Simplify GC-MS/MS Method Development and Data Processing for Analysis of Pesticides
Kefei Wang (Bruker Daltonics, USA)
Tandem mass spectrometry coupled to chromatography, such as GC-MS/MS, operated in MRM mode, is rapidly becoming the method of choice for targeted screening of multi-residues analysis in complex food matrix samples. This is due to its high specificity and capability of simultaneous monitoring of hundreds of MRMs from a large number of compounds in complex sample matrices such as food and environmental samples. However, MRM method development, including setting up the MRM acquisition table and data processing, is complicated and time consuming. In this study, a new compound-based screening (CBS) approach is introduced and demonstrated for the analysis of 49 pesticides in an extract of pumpkin on a Bruker SCION TQ GC-MS/MS system. The CBS based algorithm was designed to significantly simplify MRM method development and data processing by automatically linking data acquisition and processing together, eliminating the need for separate method set-up. In addition, CBS is able to optimize the dwell time for each MRM for each compound without the use of chromatographic segments.
Quantitative Determination of Volatile Compounds in Citrus Drink using SBSE-Thermal Desorption
Bin Yu (Firmenich, Singapore)
Since stir bar sorptive extraction (SBSE) was first developed in the 1990s, it has been widely applied to enrichment and sensitive analyses of environmental such as determination of odorous compounds in drinking water, endocrine-disrupting compounds in wastewater and etc. In couple with SBSE, a thermal desorption unit (TDU) and a cooled injection system (CIS) are always the crucial parts in desorbing and trapping, thus reducing the analyte discrimination during injection step. Particularly, better recoveries of thermally labile and volatile chemicals, and less pronounced adverse effects of non-volatiles present in the sample during the injection process could be achieved. To our knowledge, there are no systematic studies, prior to the present one, concerning the multivariate optimization of thermal desorption process of SBSE for the GC-MS analysis of citrus drink. In order to improve the sensitivity and reproducibility, it is necessary to optimize those variables that affect the efficiency, e.g., vent flow, thermal desorption time and cryotrapping temperature. By means of an experimental design strategy, a central composite design was employed to obtain optimum values of the significant factors for the analysis and also understand the interactions between factors.
Innovations in UHPLC and MS/MS Technology for Identification and Quantitation of Food Contaminants, Steroids in Serum and Drugs of Abuse
Frank Rooney (AB SCIEX Australia and South East Asia)
Highly sensitive and reliable LC-MS/MS quantitation with QTRAP 4500 scanning functionality enabling compound identification with highest confidence based on MS/MS library searching, offers a comprehensive solution for food safety analysis steroids in human serum and drugs of abuse, in combination with the new Eksigent ekspert ultraLC. The new system with the option of QTRAP technology, which increases full scan sensitivity by 100x over basic triple quadrupoles by incorporating the sensitive Linear Accelerator Trap, providing high levels of confidence in results for screening applications. To simplify the adoption of this technology, the company has unveiled AB SCIEX Accelerated Lab Integration Packages. These packages consist of not only the mass spectrometer, but also the standards, software, training, validation services and a liquid chromatography (LC) system, including the Eksigent ekspert ultra 100 and 100-XL systems. With the 4500 series, LC systems and Accelerated Lab Integration packages, scientists and laboratory analysts are able to improve results for a variety of applications, including food and environmental contamination analysis, clinical research, forensic toxicology, protein identification, peptide quantitation and bioanalysis.
Food Safety Through Provenancing of the Food that We Eat
Angela Li (HSA, Singapore)
With the rise in food fraud and food contamination incidents around the world, there is a growing need to trace food items to their sources of origin. The prevalence of these food safety issues typically stems from the market drive for staple food items or premium food products. This talk will discuss about the stable isotope ratios and multi-element analysis techniques used in determining the geographical origins of rice that our laboratory has developed. The interrogation of the stable isotope ratios and multi-element data in rice by canonical discriminant analysis (CDA) is a useful tool in differentiating the rice by their provenance. The presentation will also highlight some of the challenges involved in establishing such food databases for geographical traceability.
A Practical Guide to High Resolution MS Analysis on Natural Products
Jason Neo (AB Sciex, Singapore)Recent developments in mass spectrometry technology allow high resolution data to be collected on both MS and MS/MS analysis at fast speed at the same time. This enables the use of high resolution high mass accuracy MS data for more confident screening of secondary plant metabolites and the same high resolution MS/MS data for structure elucidation, confirmation and identification of the compounds in a single analysis. This methodology complements the traditional approach in natural product research. Here we are able to show practical examples of different workflows on how natural product screening can be performed using Cistancea from China and Tongkat Ali from Malaysia.We had employed PCA statistical analysis for the differentiation of potential targets based on similarities and differences of different samples, and a non-targeted screening approach where a list of possible plant metabolites can also be used to screen and confirm molecules based on their mass accuracy, isotope distribution and MS/MS spectra.
HPLC Analysis of Biopharmaceuticals - New Application to Heterogeneity of mAb and PEG Protein
Hiroshi Tomizawa (Tosoh Corp., Japan)Biopharmaceuticals market, including monoclonal antibodies (mAbs), therapeutic proteins and vaccines has been expanding recently and expect to continue to grow. In comparison to synthetic drugs, which most of them have low molecular weight, biopharmaceuticals have high molecular weight and complex structure in general. More over, heterogeneity could be generated by post-translational modification in upstream and artificial modification in downstream processing. Characterization of biomolecules including heterogeneity is quite important in R&D and QC in biopharmaceuticals and methods by HPLC is typically used.In this presentation, I will present new applications to heterogeneity analysis of mAb and PEG protein by HPLC including analysis of vaccine. Also I will introduce brand-new size-exclusion chromatography columns for mAb separation and reversed-phase chromatography column for protein separation.
Analysis of Pesticides by GC-Triple Quad Made Easy with a New Modular Approach to GC Instrument Design, Novel Software and New MS Ion Source Design
Hans-Joachim Huebschmann (Thermo Fisher Scientific, Germany)
Pesticide analysis today is a true multi-class and multi-residue challenge for the screening of hundreds of compounds in one analytical run. The diverse chemical nature of pesticides, and also the requirements for productivity call for a short and fast sample preparation in food safety analysis. The extracts typically carry high loads of sample matrix limiting the practical use of classical ECD, NPD or FPD detection. Also GC-MS detection using single quadrupole instruments is affected by strong matrix interference on the selected ion mass traces (SIM). Residue pesticide concentrations require the high matrix selectivity for pesticide target compounds that is provided by GC-MS/MS analysis by using the selected reaction monitoring (SRM) mode for highly sensitive detection. For the analysis of pesticides this presentation offers powerful solutions for extended GC-MS/MS system productivity by reducing the system downtime with less manual maintenance operations. Critical aspects of applying large series of real life matrix samples in GC-MS/MS are discussed. Presented is a powerful new tool for automated SRM development of new compounds, as well as helpful new tools that facilitate easy method maintenance of complex, high throughput pesticide analyses.
Selected Applications of Comprehensive HILIC RP-LC for the Analysis of Phenolic Compounds
Andre de Villiers (University of Stellenbosch, South Africa)
The talk will provide an overview of the following aspects:
- The benefits of LC-LC, and HILIC RP-LC in particular, for improved phenolic analysis
- Practical aspects of coupling HILIC and RP-LC separations in on-line, off-line and stop-flow configurations
- A detailed fundamental evaluation of the performance of the various configurations.
- The potential of stop-flow LC-LC as an automated alternative to off-line LC-LC
- Practical aspects of on-line LC-LC-MS and data handling
- Wide applicability of HILIC RP-LC for various classes of phenolic compounds in diverse natural products on the hand of several real-life examples.
The programme will be will be updated regularly - please check back for more information.