An Introduction to Drugs-in-Sport Performance Enhancing Drug (PED) Detection
Jim Healy (Limerick Institute of Technology, Irealnd)
Sport plays an important role in the modern world, for both active participants and spectators. In fact, sport can now be regarded as a multi-million dollar industry. The desire to come first has led some participants to choose to use performance enhancing drugs (PEDs). The financial rewards available have increased the pressure on athletes. Drug abuse in sport is often called doping. This presentation will concentrate on presenting, in a very accessible manner, an introduction to the area of drugs in sport, with particular reference to the methodology of detection used in the modern doping control laboratory. However, this field of activity does not solely involve samples from human sports events. Analysis of samples taken from animal athletes (horses and greyhounds) is, arguably, of equal importance.
Fast, Flexible and Fit for Purpose Methods for Industrial Problem Solving
Sam Whitmarsh (BP Technology Centre, Reading, UK)
In industry, analytical problem solving requires a flexible approach where instrumentation can be readily applied to a diverse range of potential problems. Time scales are often very limited and achieving fit for purpose results is the name of the game. Therefore, analysts do not always have the time to be able to develop fully optimised methods. Often these analysis types entail the use of sample preparation (either online or offline) to LC or GC coupled to multiple detector suites which typically include a single quadrupole mass spectrometer. This paper will describe real world examples of how coupling multiple separation techniques with quadrupole mass spectrometers can provide unique and rapid solutions to real world problems. Techniques will include GC and HPLC-MS, GC-ICP-MS, TD/Pyrolysis-GC-MS/NPD and Headspace-GC-MS. Examples will include matrices such as petrochemicals, fuel, lubricants, engine deposits, polymers and from Motorsport.
Analysis of Polycyclic Compounds in Environmental Sample Extracts using GC×GC-TOFMS - Challenges and Solutions
Anthony Gravell (National Laboratory Service, UK)
The current practice of taking grab samples followed by laboratory analysis may not always provide a useful indication of the chemical quality of the water course. The reliability of this method is questionable as there is a chance that pollutants can be missed because a sample was taken at the wrong time; the 'spot check' approach is only able to capture any pollutants present in the column of water, the moment the sample is taken. It would therefore be advantageous to detect potential impacts that may not be found through conventional chemical monitoring. Passive sampling is such a technique where pollutants are concentrated by the sampler over time thereby indirectly lowering detection limits. They also allow for the detection of trace, yet toxicologically relevant, contaminant mixtures over extended periods of time. Samplers can also be deployed over extended periods, typically 2 to 4 weeks in a river system, thereby increasing substantially the likelihood of capturing pollution events, whether the source be point or diffuse. Recent advances in the range of analytical instrumentation and especially the coupling of Gas Chromatography (GC) with fast mass spectrometric techniques, such as Time of Flight (TOF) detectors, have had a significant effect on the successful identification of pollutants. GC is a powerful technique for the separation and determination of semi (volatile) compounds and even with the use of high resolution capillary columns, GC is unable to separate the multitude of compounds that can be present in complex (e.g. biota, sediments, passive sampler) environmental samples. Comprehensive two-dimensional gas chromatography (GC x GC) is particularly suited to the separation of compounds from such difficult matrices and compound identification is more reliable with GC x GC, as each substance has two specific retention times, one in each dimension. Using two columns with different retention characteristics peaks are usually grouped by carbon number along the first (x-axis) dimension and by chemical class (e.g. alkanes, mono-aromatics, di-aromatics) along the second (y-axis) dimension. This type of separation effect greatly increases confidence in group-type analysis and the provisional assignment of unknown substances. GCXGC was successfully used to resolve the stereo-isomers of polycyclic musks in passive sampling extracts.
HPLC Troubleshooting: Listen to the Chromatogram
John W. Dolan (LC Resources, USA)
In our hurry to fix HPLC problems and get back to productive work, we tend to look for the quick-fix. Often the chromatogram is screaming at us to pay attention. If we would just compare chromatographic performance using standard chromatographic measurements, we might be surprised to find that our quick-fix diagnosis merely is putting the problem off for another day. In this presentation, delegates will see through case studies: how first impressions can be wrong; how estimates of “normal” chromatography can be made; how to determine which problem to address first; how to select conditions that are less likely to fail.
Using Temperature in HPLC – Turning on the Heat
Anthony Edge (Thermo Fisher Scientific, UK)
Temperature is often a forgotten parameter in HPLC and yet it can provide the solutions to many of today’s challenges. In this presentation the use of temperature to alter selectivity without the need to change a column or the mobile phase is discussed. The use of isothermal systems and also thermal gradients will also be reviewed and how these can be best applied to the separation process. Another advantage of raising the temperature in HPLC is that more environmentally friendly solvents can be employed which has the added benefit of allowing for coupling of detection systems that would be typically incompatible with a standard mobile phase.
Liquid-Liquid Chromatography: Why a Liquid Stationary Phase is So Exciting!
Ian Garrard (Brunel University, UK)
There are a number of distinct advantages to having a liquid stationary phase for chromatographic separations including: a very high sample loading, the ability to inject crude particulates, 100% recovery of all components, no drifting of peak retention times, easy recycling of the solvent used, and no expensive columns to replace. Variously known as dynamic extraction, centrifugal partition chromatography or counter current chromatography according to the instrument used, all these techniques use two immiscible liquids as the mobile and the stationary phase of a separation. After briefly describing the separation mechanism in each instrument, recent advances in both machine design and understanding will be detailed, concentrating in particular on instruments designed for biological separations. Current applications of the technology in the field of chromatography will be presented with a number of case-study examples including:
• The one step purification of a natural product from crude extract to pure material.
• The use of aqueous two phase systems for protein extraction and purification.
• The latest research on the purification of active biological molecules such as antibodies and enzymes.
Improving Electrospray LODs by Use of Narrow Bore HPLC Columns
Frederick E. Klink (Mass-spec-training.com, San Ramon, California USA)
Electrospray (ES) has unique characteristics among LC/MS interfaces. It is a concentration-sensitive detector, i.e. higher concentrations of analyte in the interface result in higher response. ES is also a flow-sensitive detector where lower flow rates yield improved response. As narrow-bore HPLC columns (1–2 mm I.D.), operate at much lower flow-rates than standard 4.6 mm columns and exhibit much higher “in-peak” analyte concentrations, they are an ideal match to electrospray interfaces where improved limits-of-detection (LODs), are required. Narrow bore columns are also a desirable solution for most laboratories because, unlike capillary columns, they do not require significant investment in new HPLC hardware. In this paper we will present a theoretical discussion of the improvement in ‘in-peak’ concentrations showing the magnitude of improvement expected for various columns. We will also discuss how to select the optimum flow-rate when scaling down an analysis from a 4.6 mm column to a narrow-bore column. In addition, the characteristics of electrospray interfaces which result in the concentration and flow sensitivity described above will be presented. This includes how solvent saturation levels in the interface can affect ion evaporation efficiency.
Novel Derivatization Strategies for Targeted LC-MS
Jeroen Kool (VU University Amsterdam, The Netherlands)
The most important issues in LC-MS-based approaches are matrix effects, sensitivity and selectivity. In general, analyte derivatization can be performed for various reasons: increase the stability of the analyte, improve the separation from matrix components in sample pretreatment and/or chromatography, enhance the ionization efficiency, and/or alter the fragmentation characteristic in MS. Using a number of, recently developed, the advantages and limitations of LC-MS-based derivatization will be discussed. This topic will be beneficial for researches in the fields of screening and/or targeted analysis of organic compounds in complex materials in which LC-MS-based procedures are the method of choice, and where sensitivity and/or selectivity are important issues.
Rapid Analysis by Ambient MS and On-Chip Sample Clean-up
Teris A. van Beek (Wageningen University, The Netherlands)
Desorption Electrospray Ionisation and Direct Analysis in Real Time MS are ambient MS techniques capable of probing plants without any sample clean-up. Pros and cons and examples in phytochemical analysis will be presented. Miniaturized three-phase microreactors offer prospects for the rapid sample clean-up of alkaloid-containing plants. Developments will be discussed. Delegate benefits from attending will include introduction into ambient MS and learning about its scope and limitations, understanding the complementary nature of DESI-MS and DART-MS, knowing when to apply these MS techniques in phytochemical analysis, understanding the theoretical background of 3-phase microreactors, and learning about lab-on-a-chip applications in alkaloid analysis.
Computational Approaches to Chromatographic Method Development
Roman Szucs (Pfizer, UK)
Chromatographic method development often relies on intensive laboratory experimentation which can sometimes be based on simple trial and error optimisation. Although such approach can be ultimately successful, it is very often time and resource consuming. In addition, such non-systematic approach cannot guarantee desired robustness of the final product (chromatographic method). Systematic approach based on screening of multiple experimental conditions is typically costly and environmentally unfriendly. In this presentation we will discuss an alternative approach which minimises laboratory experimentation through the use of computational methods and tools. We will compare various in-silico approaches and critically evaluate their usefulness for selection of optimal chromatographic methods.
A Comparison of Recommended Strategies for Reversed Phase HPLC Method Development
Oona McPolin (Mourne Training Services, UK)
The benefits of using a strategic approach for developing HPLC methods are easily apparent. The numerous possible chromatographic parameters in a typical HPLC method make choosing the most suitable ones for a particular separation very daunting. In particular, how to select one column from the hundreds available? There are a number of different strategies which can be applied, these include: trial and error, changing one variable at a time; finding a method in the literature or finding a method in the literature for a similar compound; and sophisticated column screening experiments combined with computer modelling, peak tracking methods, experimental design and column comparison tools. In this presentation, current recommended method development strategies are reviewed and compared to give delegates an appreciation of the types of strategies which may be applied, so that they can identify the one which is most applicable for their method development needs.
Flow Optimized Separations using Coreshell Particles
James Rudge (Phenomenex, UK)
When compared to 1.7 µm fully porous materials, the ultra-high efficiency and low back pressures provided by Kinetex core-shell 2.6 µm columns, provides users opportunities to go beyond what is traditionally accepted for UHPLC runs. To match back pressures exerted by 1.7µm fully porous columns, large increases in core-shell column length and/or system flow rate is required. When gradient conditions are maintained, increases in flow rate and core-shell column length result in a significant increase in peak capacity. When MS conditions are optimized to compensate for new flow rates, sensitivity increases are also observed.
QbD for HPLC - Method Transfer Challenges
Kate Monks (Molnár-Institute, Berlin, Germany)
Transfering methods across continents, a number of problems are typically encountrered. In the global economy where products and their HPLC methods are distributed world wide, there are often discrepencies between the orignial method and its local variant. Due to these and other problems in method transfer the regulatory authorities (FDA, ICH, etc.) are requiring nowadays a more systematic approach to HPLC method development. This presentation shows how to apply Quality by Design (QbD) principles to the development of HPLC methods focusing on method understanding and good science. It will be shown how modeling creates a platform for Control Strategy and Design Space, helping also to visualize robustness in a unique way. This provides a deep chromatographic understanding and the necessary QbD framework for HPLC, achieving optimal robust methods in a transparent and demostrable way.
Diamonds are Not Forever: Use of GCxGC ToF MS to Identify Complex Mixtures of Polar Pollutants
Steven Rowland (University of Plymouth, UK)
Attendees at the lecture will learn how recent improvements in ToF MS design now allow library-searchable, fully interpretable, electron ionisation mass spectra of chemicals such as esters of diamondoid carboxylic acids to be obtained, even from components of the most complex of mixtures. In contrast to virtually all published GCxGC ToF mass spectra, the examples will show evidence of clear molecular and fragment ions, even in spectra of relatively high molecular weight esters. In addition, our optimisation of GCxGC conditions, in what has sometimes been referred to as 'reverse phase' GCxGC (polar separation in the first, apolar in the second, dimension), allows optimisation of the separation of, for example, phenols, from esters of aromatic acids, from esters of alicyclic acids. The improvements have made accessible, improved understandings of the toxicological significance of the components of numerous important pollutant mixtures, including naphthenic acids of the oil sands of Canada.
‘Hot and Spicy’, TD-GC-MS Characterisation of Flavours and Fragrances in Foods and Beverages
Gareth Roberts (Markes International, UK)
The technique of thermal desorption (TD) is widely used within a range of industries, these include food, flavour, and fragrance (FFF), environmental, material emissions, and typically combined with GCMS analysis. TD provides sufficient preconcentration enhancement to facilitate detailed chemical analysis of VOC/SVOC compounds from a variety of samples.
This presentation focuses on the FFF industry and in particular the flavour and aroma profiling of some food, beverage and tobacco products. An overview will be provided describing novel TD sampling techniques supplied by Markes International which are used to accommodate the variety of sample matrices typically encountered. Specialist TD accessories will also be reviewed as these can extend both the range of samples to be analysed and enhance levels of detection. Examples and applications of these using GCMS will be discussed and include:
Direct desorption. This technique applies to food products which are solid (powder) liquids or gels. Small (volume) samples are placed directly into a TD tube for desorption and GCMS analysis, however for larger bulk samples the implementation of a micro chamber thermal extraction system (µ-CTE) is described.
Sorptive extraction. Using small coated Titanium based cartridges the characteristic flavour/aroma components of beverages can be identified.
Headspace – TD. This technique combines the performance characteristics of both headspace and TD. The net result is at least a sensitivity enhancement by one to two orders of magnitude.
GCMS data analysis employed a new deconvolution/chemomteric based software, allowing specific target compounds to be identified within the complex TIC profiles.
Modern GCMS Analysis – Faster, Higher, Broader
Susanne Kräher (Shimadzu Europa GmbH, Germany)
In today’s laboratory productivity has become increasingly important due to the high sample throughput. Giving productivity the highest priority requires a focus on reducing the cycle time per analysis – including sample preparation, sample analysis, data processing and reporting. Modern equipment offers the flexibility for automated sample preparation and intuitive data handling software. Transferring methods from conventional GCMS to fast GCMS allows us to decrease sample running time, however, maintaining separation efficiency or even improving it is the true goal to show a real benefit in the method transfer. Modern equipment also allows routine use of fast GCMS in a high sample throughput environment. In order to convert a conventional GCMS method into a fast or ultra fast method, the hardware used must fulfil dedicated requirements including a compatible injection system with high, stable head pressures right through to a fast responding detection system. A high scanning speed single quadrupole allows all of these things, as well as “Simultaneous” SIM/Scan acquisition modes for comprehensive data acquisition in one run. Furthermore a fast responding single quadrupole enables the possibility for quantitative GCxGC applications, providing the highest separation capacity.
“Just Enough” Sample Preparation: A New Trend in Sample Handling
Ron Majors (Agilent Technologies, USA)
In the past, sample preparation has been a tedious, error-prone, multi-step process and frequently the rate determining step in the analytical cycle. More recently, with the advent of tandem MS with its high selectivity and sensitivity, clean-up procedures have been simplified to a “Just Enough” process. Procedures like protein precipitation (rather than SPE) for drugs in biological fluids, QueChERS for pesticide residues, and Dried Blood Spotting are examples of “Just Enough” sample preparation. This presentation will highlight these and other examples that are simplifyingchemical analysis. Attendees will learn about new techniques for sample preparation and how they are simplified by the use of LC-MS/MS and GC-MS/MS. Overall advantages include higher speed analysis, better sensitivity, better analyte recovery, and lower cost sample prep. Examples include the sample preparation for drugs and drug metabolites in biological fluids, the extraction of pesticides from fruits and vegetables, and new procedures for environmental samples will be featured.
Separations in Microfluidics
Gillian Greenway (University of Hull, UK)
Microfluidic systems have become increasingly popular for a wide range of applications. Their applications can be extend by the inclusion of solid phase materials within the systems, however these systems are usually designed to be low power and low pressure and high back pressure can occur. To overcome this separations are often achieved by electrokinetic approaches. Alternatively monolithic columns can be incorporated within the systems. These can be silica bases in which the monolith is first prepared and then incorporated into the systems. Otherwise they can be polymer based and prepared insitu by photoinitiation. Examples of integrated microfluidic systems will be discussed for DNA, clinical and water analysis.
The Ever-Shrinking HPLC
Han Gardeniers (MESA+ Institute for Nanotechnology, University of Twente, The Netherlands)
A large driving force in the development of new HPLC instrumentation is the ever-shrinking sample size. Following this trend, it would make sense to shrink not only the stationary phase particle size, but shrink all of the relevant components of the system, to achieve a better match between sample and instrument. This presentation will discuss the state-of-the-art in developments of microfluidic chips for the purpose of HPLC. Delegates will see how the introduction of cutting edge micro and nanofabrication technology may contribute to the next generation of analytical separation instruments.
It's All About Selectivity
Diane Turner (Anthias, UK)
Analysing a sample is very rarely straightforward, it often involves looking for very low concentration analytes, which may or may not be known in a very messy matrix, a bit like looking for a needle in a haystack! Selectivity, selectivity, selectivity. Selectively extracting analytes from the sample matrix,; selectively transferring the analytes onto the column; selectivity of the column phase for separation and where this fails, selectively transferring a part of the chromatogram onto a different column phase or separating the whole sample on two column phases with different selectivities; selectivity of the detector to see the analytes; selectively choosing the ions for data analysis. In gas chromatography, how do we put it all together to develop more sensitive and discriminating methods that can quantify analytes with less interference from other components? In this presentation we look at the whole process rather than just selectivity of the stationary phase.
Ionic Liquid Phases – a new era for Gas Chromatography?
Lisa Fitzpatrick (Sigma-Aldrich, UK)
GC is a well-established technology; however, there have been few new additions to traditional phase chemistries. Recently, innovations in phase chemistries have been developed using novel ionic liquids (ILs) as separation phases. Ionic liquids are a class of ionic solvents built of unique combinations of cations and anions with low melting points that remain a liquid at room temperature instead of forming crystals. IL chemistries (including multifunctional ILs) have shown usefulness in various application areas, including:
• Novel solvents in organic synthesis and liquid-liquid extraction,
• MALDI-MS matrices,
• Sensor electrolytes,
• For the ESI-MS of anions (positive ion mode),
• Mobile phase additives in HPLC
• Buffer additives in CE.
For the first time, ILs can be used for GC, as high polarity phases that can operate at higher temperatures, compared with similar polar traditional phases. This widens the application range to 2D GC applications, as they are also in the position to utilize higher temperatures in the second (polar) dimension. There is potential for using ionic liquid GC phases with air as a carrier gas, making environmental field work a far simpler task. This presentation will outline the current R&D in IL phase chemistries and their applications as GC stationary phases in environmental and food analyses.
Separation Science Techniques for Air Quality Assessment
Alastair Lewis (National Centre for Atmospheric Science, UK)
Organic compounds play a major role in the chemistry of Earth’s atmosphere; they contribute toward the generation of air pollutants in cities and influence the formation and properties of fine particles and clouds. Separation science is central to unravelling the composition of the atmosphere and atmospheric scientists draw heavily on techniques such as thermal desorption, GC-MS, GCxGC and LC-MS. This presentation will provide an overview of latest advances in the atmospheric measurement of organic compounds in urban and background environments and from moving platforms, including high resolution separation techniques to determine semi-volatile organic compounds found in atmospheric aerosols. Trends towards field portable and low energy separation monitoring methods will also be discussed and examples of state of the art microfabricated GCxGC for air quality sensors presented.
A Novel Chromatographic Approach for mAb Aggregate Analytics
Judith Vajda (Tosoh Bioscience GmbH, Stuttgart, Germany)
The therapeutic use of antibodies has created a demand for reliable mAb aggregate analysis. Drug safety plays an important role for further success in treatment of severe illnesses. We found HIC to be a valuable analytic tool, providing the possibility of quantitative analysis due to the high efficiency of nonporous particles. We investigated the influence of different salts and salt mixtures on the chromatographic performance. Compared to classical SEC, our new method delivers for certain mixed electrolytes improved resolution of higher aggregates.
A comparison between SEC and the new HIC method is drawn, highlighting advantages and disadvantages of each chromatographic method. Finally, applying 2D chromatography we combine benefits to achieve the most accurate and sensitive result. This topic addresses to anyone who’s interested in Ab analysis or protein aggregate detection.
About the Development of 2nd Generation Monolithic HPLC Columns
Karin Cabrera (Merck KGaA, Darmstadt, Germany)
Monolithic HPLC columns are consisting of one piece of porous silica which is characterised by a well defined bimodal pore structure. Macropores of about 1-2µm in diameter offer high permeabilities and low column back pressures thus allowing the operation of these columns with conventional low pressure HPLC systems. Mesopores of about 11-15nm in diameter are located on the silica skeleton providing high surface areas which are needed for a sufficient chromatographic separation process.
We will present data on the development of a second generation of silica monoliths with improved separation efficiencies as well as improved peak shapes, especially for basic drugs, such as triptylines. For this purpose the domain size has been reduced with a corresponding macropore size of 1.2µm. The resulting columns show N/m of about 150.000–160.000 which is the double compared to the first generation. Furthermore, we have increased the mesopore size up to ca. 15nm with a corresponding surface area of about 250m2/g. The resulting monolithic columns show much better peak symmetries, especially for basic compounds. Since the chemistry of the surface modification to RP-18 endcapped hasn’t changed, we believe, that the increase of the mesopore size offer better mass transfer characteristics resulting in better peak shapes. Although the macropore size has been decreased, the resulting column backpressure of these new columns is only within the range of 55-65bar (800–950psi) at a flow rate of 2mL/min (ACN/ water, 60/40 (v/v), which permits an operation of these columns with conventional low pressure HPLC systems.
Production and Characterization of Polymer Monoliths: Trials and Tribulations
Damian Connolly (Dublin City University, Ireland)
The production of polymer monoliths, and their subsequant surface modification and physical characterization, is a technically challenging and continually evolving area of separation and material science. This presentation will provide some of the latest developments in this field, including the routes to obtaining nano-particle functionalized porous polymer monoliths within various analytical moulds, including capillary columns, pipette tips and within micro-fluidic chips. The ability to physical characterize these materials in-situ will be discussed, together with some new bioanalytical applications. Delegates attending this talk will be provided with an overview of monolith production, and approaches to their surface modification, including both surface grafting and nano-particle attachment. Delegates will also gain and appreciation of monolithic phase characterization techniques, including the application of scanning contactless conductivity dectection (sC4D) to evaluate monolith structure in-situ. Delegates will also be shown new bioanalytical applications, including selective glycoprotein extraction approaches