Poster Session - British Mass Spectrometry Society

AstraZeneca Conference FacilityAlderley Park33rd BMSS MEETING17th and 18th April 2012oeculesCelebrating the technological innovationsin mass spectrometry on the30th Anniversary ofFast Atom Bombardment (FAB)BOOK OF ABSTRACTS

Table of ContentsTable of Contents ............................................................................................................................3ChairPersons Letter .......................................................................................................................10BMSS Meeting Sponsors ...............................................................................................................12BMSS 2012 Exhibitor List .............................................................................................................13BMSS 2-Day meeting Alderley Park 2012 Programme ...................................................................15Programme Tuesday 17th April 2012 ...................................................................................16Programme Wednesday 18th April 2012 .............................................................................19ORAL PRESENTATIONS ....................................................................................................23LOWERING THE LIMITS .............................................................................................................24Lowering the limits - catching the sports drug cheat David A Cowan ...........................24Using LCMS and pyGCMS to identify pigments in microsamples from works of artJustin J Perry Joanna Russell, Elke Cwiertnia and Brian W Singer .........................................24Determination of a Urinary Drug Metabolite using LC-FAIMS-MS andLC-FAIMS-In-source CID-MS Robert W Smith Danielle E Toutoungi, James C Reynolds,Ashley Sage, Anthony Bristow, Andrew Ray, Daniel J Weston, Ian Wilson, Billy Boyle andColin S Creaser* ..................................................................................................................25Deamidation of collagen peptides in ancient bones using FT-ICR-MS.Pilar P. Hurtado Matthew Collins Peter B. O’Connor .............................................................25Clinical Testing of Rapid Evaporative Ionization Mass Spectrometry-basedIntraoperative Tissue Identification László Sasi-Szabó, Laura Muirhead, James Kinross,László Damjanovich, Balázs Dezs , Robert Goldin, Júlia Balog3 and Zoltán Takáts .................26Enhancing the detection limits of direct-real time MS in breath analysis Iain White ...26SPEEDING THINGS UP Dr Andy Pitt ............................................................................................27The use of an on-line two-dimensional (RP/RP) liquid chromatography mobilityenabledapproach for the characterisation of the cellular proteomesJames H. Scrivens Baharak Vafadar-Isfahani Nisha A. Patel Susan E. Slade Lee GethingsChris Hughes Jim Langridge ....................................................................................27LC-MS/MS Analysis of Eicosanoids and Isoprostanes: Understanding The Role ofNanoparticles in Generating Inflammation and Oxidative StressBen Maskrey, Janet Adamson, Wan-Seob Cho, Rodger Duffin, Ken Donaldson,Ian Megson, Phillip Whitfield ...............................................................................................28Monitoring the effects of physiological stress by metabolic profiling of saliva usingultra performance liquid chromatography-ion mobility-mass spectrometry.Aditya Malkar, Helen J Martin, Pareen Patel, Matthew A Turner, Phillip Watson,Ron J Maughan, Helen J Reid, C L Paul Thomas and Colin S Creaser ....................................28Surface analysis using micro-PADI Mass Spectrometry in ambient conditionsAndrew Bowfield and James W. Bradley. ..............................................................................29Developing automated dried blood spot direct analysis techniques for high samplethroughput quantitative bioanalysis Paul Abu-Rabie, ....................................................29TAKING MS TO THE EXTREME ...................................................................................................30Mass Spectrometers in Space! William B. Brinckerhoff .............................................30LCMS to support drug discovery and early phase clinical trials in theDivision of Cancer Therapeutics at The Institute of Cancer Research.Florence I Raynaud ..................................................................................................30

Alderley Park 2012The use of GC-MS and LC-MS to support manufacturing ofpharmaceutical products Alice Laures ...............................................................................31Exploring structural differences between two similar fibril morphologiesof beta-2 microglobulin Lucy A.Woods, C. J. Sarell, S.E. Radford, A.E. Ashcroft. ..............31Improved data acquisition and data handling methods for MALDI-MS andMALDI-MSI of small molecules in tissue Josephine Bunch, Rian Griffiths,Andrew Palmer, Alan Race, Rory Steven, Joscelyn Sarsby and Sarah Turker ...........................32Binding site identification of glyoxal in Substance P by mass spectrometryAndrea F. Lopez-Clavijo, Mark P. Barrow, Naila Rabbani, Paul J. Thornalley andPeter B. O’Connor ...............................................................................................................33PIONEERING MS GREAT AND SMALL ........................................................................................34Structural Biology by Mass Spectrometry: 3D Proteomics ofSupramolecular Assemblies Juri Rappsilber , , Salman Tahir, Lutz Fischer, Colin Combe,Jimi-Carlo Bukowski-Wills, Zhuo Angel Chen. ......................................................................34The advantages of absorption mode spectra.Peter B. O’Connor, Yulin Qi, Mark P. Barrow.........................................................................34Self reporting oligonucleotide probes – a new design for small mass tagsJo-Anne Riley, G. John Langley, Julie Herniman. ...................................................................35The metamorphic protein lymphotactin undressed by IM-MS and top down ECDSophie R. Harvey, Albert Konijnenberg, David J. Clarke, Robert C. Tyler,Brian F. Volkman and Perdita E. Barran .................................................................................35Application of nanoUPLC®-MS/MS to quantify human insulin-likegrowth factor I prohibited in sportFilipe Lopes, Mark Parkin and David Cowan .............................................................36Finding small needles in a hay stack: how large-scale proteomics helps toidentify key players in powdery mildew infection of barley.Laurence V Bindschedler, Dana M Gheorghe, Liam J McGuffin, Celia J Smith,Clara M Pliego Prieto, Pietro D Spanu and Rainer Cramer ........................................36POSTER SESSIONS .....................................................................................37Posters will be displayed in the Helix Room.There are no formal poster sessions – all posters will be up for both days with opportunities to viewat tea/coffee breaks, at lunch and during the Wine Reception held at the end of the day on Tuesday(17.00 – 18.00). Presenters should therefore be by their posters at some of these times.Poster Boards are portrait – 1 metre wide and 2 metres high (from top to bottom) with Velcro fixingPOSTER SESSION – LOWERING THE LIMITS .............................................38POSTER 1 – Pharmacokinetic analysis of montelukast in healthy Korean volunteersby high perfomance liquid chromatography-tandem mass spectrometryMin-Ho Jo ...........................................................................................................................38POSTER 2 – The Analysis of primary environmental pollutants by GasChromatography-Mass Spectrometry using generated hydrogen carrier gas.M Wilkinson, J Heseltine .....................................................................................................384 BMSS © 2012

Alderley Park 2012POSTER 19 – Simultaneous Quantitative and Qualitative measurements for PrimaryMetabolism Investigations using a Quadrupole-Time-of-Flight MassSpectrometer. George McLeod Mark Savage and Angus Nedderman ................................47POSTER 20 – Simultaneous quantitative and qualitative data generation from driedblood spots Angus N R Nedderman, Mark E Savage, Julian J Haynes, Michelle Gleave .......48POSTER 21 – Improving Assay Stability by Turbulent Flow ChromatographyTony Edge, Luisa Pereira and Francois Espourteille ................................................................48POSTER 22 – Core enhanced technology column performance L. Pereira, T. Edge,H. Ritchie, S. Luke ................................................................................................................49POSTER 23 – Investigation into use of IDCube as a potential open access systemin support of medicinal chemistry Andrew D Ray ...........................................................49POSTER 24 – Computational Analysis of the Effects of Variable Raster Speedsand Laser Pulse Repetition Rates in MALDI-MSI Rory T. Steven, Alan Race,Andrew Palmer, Ata Kaban, G. Ed Rainger and Josephine Bunch .........................................50POSTER 25 – The Effect of Amino-terminal basic groups upon b-ion Rearrangementduring Collision Induced Dissociation (CID) Ross Chawner , Simon J Gaskell ,Claire E Eyers ......................................................................................................................50POSTER 26 – Direct thermal desorption electrospray ionisation mass spectrometry for theanalysis of potentially genotoxic impurities in active pharmaceutical ingredients.James C Reynolds and Colin S Creaser .................................................................................51POSTER 27 – Detection of Triacetone Triperoxide (TATP) and its Complexes with Metal IonsUsing Nanoelectrospray-Ion Mobility-Mass Spectrometry. Alex R Hill, James C Reynolds,Paul F Kelly and Colin S Creaser ...........................................................................................51POSTER 28 – DFT guided Ion-Mobility Studies Offer a Powerful Strategy for UnderstandingPharmacodynamic Interactions within Antimalarial Haem Complexes Fyaz M. D. Ismail,Harry Morris, Nicola M. Dempster, Imran Y. Saleem Frank Kasaija Hieu V. TruongLaura E. Randle Said Alizadeh-Shekalgourabi Giles Edwards Jonathan P. WilliamsMichael. J. Dascombe & Michael G. B. Drew .......................................................................52POSTER 29 – Nanoelectrospray liquid extraction surface analysis of intact proteins frompolyvinylidene fluoride (PVDF) membranes Nicholas J. Martin, Josephine Bunchand Helen J. Cooper ............................................................................................................52POSTER 30 – Improving Efficiency of GC-MS Analysis within a PharmaceuticalDevelopment Environment by Implementation of a Single ChromatographyPlatform Martin Sims Karen Rome John Moncur Scott Campbell .......................................53POSTER 31 – Xenobiotic Metabolism: Chemical Characterisation, MicrosomalIncubations and HPLC-Electrospray Mass Spectrometry Analysis of VariousAntimalarials Including Artemisinin, Pyronaridine and Novel BisquinolinesEspecially Metaquine Fyaz M. D. Ismail, Harry Morris, Nicola M. Dempster,Imran Y. Saleem, Frank Kasaija, Hieu V. Truong, Elizabeth K. Y. Yeung, Dorothy Dafimu,Sarmad M. Jasim, Laura E. Randle, Said Alizadeh-Shekalgourabi Giles Edwards,Jonathan P. Williams Michael. J. Dascombe & Michael G. B. Drew. .......................................53POSTER 32 – Liquid Extraction Surface Analysis Imaging of in-situ Lipids and Proteins fromHuman Liver Analysed by High Resolution Mass Spectrometry Joscelyn Sarsby, AlanRace, Helen Cooper,Patricia Lalor, Hamid Dehghani and Josephine Bunch. ...........................546 BMSS © 2012

Alderley Park 2012POSTER 33 – Direct gradient extraction of urinary analytes from undevelopedreversed-phase thin-layer chromatography plates combined with on-lineelectrospray ionisation and ion mobility-mass spectrometry Neil A DevenportJames C Reynolds Ian D Wilson Daniel J Weston Colin S Creaser. ........................................54POSTER 34 – Structural determination of N-linked glycans by ion mobility and negativeion fragmentation David J. Harvey , , Christopher N. Scanlan, Max Crispin,Charlotte A. Scarff, Matthew Edgeworth, Frank Sobott, James H. Scrivens. ..........................55POSTER 35 – Determining structural changes resulting from deamidation of nativestate gas phase proteins using FTICR-MS and ion mobility methods.Andrew J Soulby , Peter B O’Connor James H Scrivens ...........................................................55POSTER 36 – Issues with Dried Blood Spots and Hematocrit - Possible SolutionsDr Philip Denniff*, Mr Adam Hughes, Ms Aida Merchan ......................................................56POSTER 37 – Transfer of Methods in LC and UHPLC, what calculations do I need?Ken Butchart; Mark Woodruff ............................................................................................56POSTER 38 – Automated, high-throughput workflowAutomated, high-throughputworkflow for the analysis of serum 25-hydroxyvitamin D using theThermoFisher Scientific Versette and multiplexed TurboFlow LC-MS/MSLewis Couchman, Caje Moniz Sarah Robinson .....................................................................57POSTER 39 – How to Optimise Your UHPLC Performance – Connect Properly!Ken Butchart; Mark Woodruff. ...........................................................................................57POSTER 40 – A Simple Method for Resolution of 22 Amino Acids in LCKen Butchart; Mark Woodruff ............................................................................................58POSTER SESSION – TAKING MS TO THE EXTREME .................................59POSTER 41 – Identification of penile tumour tissue in-situ using MALDI imagingand classification algorithms Brian Flatley , , Chris-Jude Quaye, Elizabeth Johnston,Fawaz H Musa, Peter R Malone and Rainer Cramer. .............................................................59POSTER 42 – Monitoring of an Imidazolidinone-Catalysed Reaction Using IonMobility-Mass Spectrometry Victoria E. Wright, James C. Reynolds, Andrew Poulton,Steven D. R. Christie and Colin S. Creaser . ..........................................................................59POSTER 43 – Classification and segmentation for molecular histology of phenotypicalregions of NASH human liver disease tissue from MALDI Mass SpectrometryImages Andrew D Palmer Alan Race, Patricia Lalor, Josephine Bunch..................................60POSTER 44 – Nitrate salts as useful additives in MALDI-MS and MS/MS analysis of lipidsR L Griffiths, J Bunch .........................................................................................................60POSTER 45 – An investigation into the pH-dependent selectivity of porous graphiticcarbon for phosphopeptides prior to their analysis by tandem mass spectrometry.John R Griffiths, Simon Perkins, Yvonne Connolly, Valeria Barattini, Luisa Pereira,Anthony Edge, Harald Ritchie and Duncan L Smith. ............................................................61POSTER 46 – Measurement of the Natriuretic Hormone Uroguanylin UsingUPLC-IMS/MSE to Characterise Individual Topoisomers Involved inCardiovascular Disease Charlotte Daly , , Leong L. Ng, Richard Willingale;Kevin Giles and Donald J. L. Jones .......................................................................................61POSTER 47 – High-resolution time-of-flight mass spectrometry using folded flightpath technology for profiling of steroids and steroid metabolites in urineJürgen Wendt, Kevin Siek, Jeffrey S. Patrick, Erica Guice ......................................................62BMSS © 2012 7

Alderley Park 2012POSTER 48 – An investigation into the concentration of coenzyme Q10 (ubiquinoland ubiquinone) in over the counter health supplements using high resolutionliquid chromatography mass spectrometry. Edward Goucher, Katerina Klagkou. ...........62POSTER 49 – The separation and quantification of diglycerides in biodiesel byHPLC followed by sequential quantification and identification by GC coupledwith Mass spectrometric and flame ionisation detection. D.Lorenzi and C.Hopley . ..63POSTER 50 – Multiplatform Metabolic Fingerprinting of Leishmania donovaniresponse to Miltefosine Gisele A. B. Canuto , , Emerson A. Castilho-Martins, Luís Rivas,Antonia García, Marina F. M. Tavares, Ángeles López-Gonzálvez and Coral Barbas. .............63POSTER SESSION – PIONEERING MS GREAT AND SMALL .......................64POSTER 51 – Absorption-Mode Fourier Transform Mass Spectrometry: Advantagesfor Protein and Petroleum Spectra Yulin Qi, Mark P. Barrow, Joseph E. Meier,Steve L. VanOrden, Christopher J. Thompson, Peter B. O’Connor .........................................64POSTER 52 – Structural Characterisation of Oligomeric Intermediates on Pathway toAmyloid Formation Aneika C Leney, Charlotte C Scarff, Sheena E Radford,Alison E Ashcroft ................................................................................................................64POSTER 53 – Investigating Solvent-Assisted Inlet Ionisation (SAII) Celia Smith,Laurence Bindschedler and Rainer Cramer .........................................................................65POSTER 54 – The Role of MS in the Physiochemical Characterisation of BiosimilarsAshleigh Wake, Betty Flitroft, Helen Brittan ..........................................................................65POSTER 55 – Enrichment of phosphopeptides/-proteins by lanthanum chlorideprecipitation Chhaya B Patole, Laurence V Bindschedler, Jim Dunwelland Rainer Cramer .............................................................................................................66POSTER 56 – Illustrate the breakdown of chlorophyll a by tandem mass spectrometryJuan Wei and Peter O’Connor ..............................................................................................66POSTER 57 – N-glycosylated Glycoproteins characterized by Mass Spectrometry –an Integrated Software Approach Rod Watson , Ulrike Schweiger-Hufnagel,Arndt Asperger, Anja Resemann, Detlev Suckau ...................................................................67POSTER 58 – Performance of a QMS operating in stability zones 1 and 3 underthe influence of magnetic field Sarfaraz U. A. H. Syed, S. Maher, J.R. Gibsonand S. Taylor. .......................................................................................................................67POSTER 59 – The effect of quadrupole non-linear resonances on ion trajectoriesvisualised in phase-space David P A Kilgour, Peter B O’Connor and John F J Todd ..........68POSTER 60 – Metabolomic lipid profiling by LC-MS; identifying changes in ethanolmetabolism and endogenous metabolites following the administration ofalcohol as a liquid diet to mice Alan Barnes, Simon Ashton, Neil J Loftus,Ian D Wilson, Filippos Michopoulos and Ji Cheng ................................................................68POSTER 61 – Single pass principal component analysis algorithm for use onlarge mass spectrometry imaging data sets Alan Race , , Andrew D Palmer,Rory T Steven, Joscelyn Sarsby, Rian L Griffiths, Iain Styles, Josephine Bunch. .......................69POSTER 62 – MALDI MS imaging of on-tissue digested proteins aided by highefficiency ion mobility separation. Ian Edwards, Emmanuelle Claudeand James Langridge. ..........................................................................................................698 BMSS © 2012

Alderley Park 2012POSTER 63 – Proximity Effects in the Fragmentation of IonisedDihalogenostyrylbenzoxazoles Stephen Ayrton, Jetakerina Panova,Richard D Bowen, Adam R Michalik and Richard T Gallagher ..............................................70POSTER 64 – Using Electron Induced Dissociation to Characterise Polyketides andPolyketide Synthase Intermediates Rebecca H Wills, Manuela Tosinand Peter B O’Connor ........................................................................................................70POSTER 65 – Towards Top-Down identification, determination of sequencetermini of intact proteins in mixtures of moderate complexity Laura Main,Anja Resemann, Eckhardt Belau and Detlev Suckau ............................................................71POSTER 66 – Mass Spectra of Tricyclic Indoles, Ketonindoles and IndoleninesAmie Saidykhan, William Martin, Richard D Bowen, Richard T Gallagher .............................71POSTER 67 – Identification of CRIP1 as a Novel Protein Biomarker for HER2-positiveBreast Cancer Sandra Rauser, Claudio Marquardt, Benjamin Balluff, , Detlev Suckau,Katja Specht, Matthias P. Ebert, Manfred Schmitt, Michaela Aubele, Heinz Höfler,Axel Walch and Julia Smith ..................................................................................................72POSTER 68 – Post-translational modifications to translation initiation factorsas a response to stress Jemma Keenan, Paul F. G. Sims, Mark P. Ashe ..............................72BMSS © 2012 9

Alderley Park 2012ChairPersons LetterDear DelegateWelcome to the 33rd Annual BMSS Meeting. Due to the International MS Conference in Japanin September we have moved to an Easter date for this year so it comes close on the heels of the32nd Meeting. We don’t usually arrange an Annual Meeting in the year of the IMSC, but as thefrequency of these has increased to every two years we have no choice. The BMSS cannot continuein its current form if it only has income from an Annual Meeting every other year. So ratherthan scale down our funding of activities we will trial running a meeting every year – a truly annualmeeting.BMSS used to hold a smaller meeting at Easter in the past so the date is not completely new tous. However, as organisationally it is so close to the Cardiff event last September, it has givenus an opportunity to make some changes in the format. There are so many meetings these daysthat we want to identify what format will ensure future BMSS Annual Meetings continue to be assuccessful as ever. Having broken away from the University environs in 2010 we have chosen tostick with that decision and come north to the AstraZeneca Conference Centre. Firstly it becauseit balances out our two years in South Wales somewhat, but more importantly the venue suits theshorter format meeting.The 32nd Annual Meeting sees us return to a two day event and runs with just one oral sessionat a time. Our aim is to continue bringing together a wide range of MS practitioners so the foursessions are open, rather than focussed on specific topics, to enable us to celebrate the incrediblywide frontiers at which our technique operates.As we have the exciting prospect of hosting the Olympics in the UK this Summer we open witha presentation by Professor David Cowan (The Drug Control Centre, King’s College London)who has led the organisation of the complex drug-testing labs set up for the Games. This sessionwill then provide a platform for others to talk about the ever lower limits of detection that MS ispushed towards and the challenges that face them. After lunch Dr Andy Pitt (Aston University)will give the second Plenary Lecture describing how he now produces more data more quickly.This will be followed by a number of examples of how MS is used to generate complex data-setsthat were unthinkable just a few years ago, or where time is saved by analysing samples withalmost no clean-up beforehand.Our Conference Dinner will be at a local restaurant and provides a time for more informal networkingbefore the second day gets underway. We are pleased to welcome Will Brinckerhoff(NASA Goddard Space Flight Center) to start day two with a Plenary about the use of MS in theextremes of space research. This will be followed by a range of examples of MS operating at theextremes. Finally Professor Juri Rappsilber (University of Edinburgh) will tell us about the incrediblemacromolecules now amenable to MS, leading us into a session highlighting the greatand the small of applications of MS. You will notice that, unlike at recent meetings, we don’t havea dedicated Young Generation session but opportunities have been given to young researchersthroughout the main program.10 BMSS © 2012

Alderley Park 2012The Exhibition at this meeting is less elaborate than normal but is still the UK’s focal point foranyone who wants to approach the breadth of companies involved, in one way or another, withMS. In response to feedback at Cardiff we want to increase the opportunities for technical interactionsand discussions so we have mixed the posters and the Exhibitors to stimulate this. It meansthat at coffee breaks and lunchtimes all the delegates can stay together in the one place, and wewelcome your feedback on this format or any other aspect of the meeting.As with all our conferences we owe a great deal of thanks to those who have been working behindthe scenes, in this case on a very short timeline since September. We have a new team in placearranging our meetings so a very big thank you to Don Jones and Colin Creaser (outgoing andincoming Papers Secretaries), Tony Bristow (Vice-Chair and host for the venue), and of courseAnna Upton (Administrator) for keeping us all on track. Our new Meetings Secretary, Tony Sullivan,will be learning the ropes as we go and will be out and about asking you what you want fromfuture meetings. In addition I’d like to thank Rainer Cramer and all the other tutors for organisingand delivering the Introduction to MS course before the meeting. Since September they have managedto review the content of every lecture in order to ensure that they minimise duplication andkeep the content right up to date.Last, but not least, thanks to you the delegates. It’s always great to welcome old friends back, butin particular I’d like to welcome anyone attending for their first BMSS meeting. We know not allof you will be able to attend the IMSC under the current economic climate so we hope that youfind this a useful opportunity to keep up-to-date with the latest developments and applications ofmass spectrometry. And remember; please do take the time to give us your feedback so we canbuild ideas into our plans for 2013.Dr Susan CroslandBMSS ChairApril 2012BMSS © 2012 11

Alderley Park 2012Sponsors BMSS Meeting 201217th – 18th April 2012AstraZeneca Conference Facility, Alderley ParkThanks are extended to the following for their sponsorship of the ReceptionDrinks and the Wine at the Conference DinnerAgilent Technologies LtdThermo Fisher ScientificWaters Corporation12 BMSS © 2012

Alderley Park 2012Exhibitors BMSS Meeting 2012AB SciexAdvanced Chemistry Development IncAgilent Technologies LtdBruker UK LtdCrawford Scientific LtdExpedeon LtdFortis Technologies LtdHichrom LtdIn House Gas LtdJaytee Biosciences LtdKR Analytical LtdKRSS LtdLECO Instruments (UK) LtdMicrosaic SystemsPeak Scientific InstrumentsPerkin ElmerPresearch LtdProteaBio EuropeProvidionSAI LtdShimadzuSpectral Works LtdThermo Fisher ScientificVRSWaters CorporationBMSS © 2012 13

Alderley Park 201214 BMSS © 2012

Alderley Park 2012BMSS 2-Day Meeting17th &18th April 2012AstraZeneca Conference FacilityAlderley ParkBMSS © 2012 15

Alderley Park 2012Programme Tuesday 17th April 20128.00 Registration with coffee available from 9.00 to 9.459.45 Chair’s WelcomeSession LOWERING THE LIMITSTitle Chaired by Susan Crosland10.00 PLENARYProfessor David CowanKing’s College London‘Lowering the limits – catching the sports drug cheat’11.00 Justin PerryUsing LCMS and pyGCMS to identify pigments in microsamples from works of art11.20 Tea/Coffee11.40 Robert SmithDetermination of a Urinary Drug Metabolite using LC-FAIMS-MSand LC-FAIMS-in-source CID-MS16 BMSS © 2012

Alderley Park 201212.00 Pilar Perez HurtadoDeamidation of collagen peptides in ancient bones using FT-ICR-MS12.20 Zoltan TakatsClinical Testing of Rapid Evaporative Ionization Mass Spectrometry-basedIntraoperative Tissue Identification12.40 Iain WhiteEnhancing the detection limits of direct-real time MS in Breadth analysis13.00 LUNCH withto POSTER SESSION14.00 & EXHIBITIONSession SPEEDING THINGS UPTitle Chaired by Gavin O’Connor14.00 PLENARYDr Andrew PittAston University‘Bigger, faster …. better? The application of rapid scanning ToF MS’15.00 Jim ScrivensThe use of an on-line two-dimensional (RP/RP) liquid chromatography mobility-enabledapproach for the characterisation of the cellular proteomesBMSS © 2012 17

Alderley Park 201215.20 Ben MaskreyLC-MS/MS analysis of eicosanoids and isoprostanes:Understanding the role of nanoparticles in generating inflammation and oxidative stress15.40 Aditya MalkarMonitoring the effects of physiological stress by metabolic profiling of salivausing ultra performance liquid chromatography-ion mobility-mass spectrometry.16.00 Tea/Coffee16.20 Andrew BowfieldSurface analysis using micro-PADI mass spectrometry in ambient conditions16.40 Paul Abu-RabieDeveloping automated dried blood spot direct analysis techniques for high samplethroughput quantitative bioanalysis17.00 End of SessionFrom 17.00 WINE RECEPTION with Posters & Exhibition hosted by the BMSSto 18.00 Open to all delegates and exhibitors19.30 for RECEPTION AND CONFERENCE DINNER - Gusto Restaurant – Alderley Edge20.00 pre-booked delegates/exhibitors onlySponsored by Agilent Technologies, ThermoFisher Scientific and Waters Corporation18 BMSS © 2012

Alderley Park 2012Programme Wednesday 18th April 20128.00 Registration8.50 AnnouncementsSession TAKING MS TO THE EXTREMETitle Chaired by Tony Bristow9.00 PLENARYWill BrinckerhoffNASAMass Spectrometers in Space10.00 Florence ReynaudLCMS to support drug discovery and early phase clinical trials in theDivision of Cancer Therapeutics at The Institute of Cancer Research10.20 Tea/Coffee10.40 Alice LauresThe use of GC-MS and LC-MS to support manufacturing of pharmaceutical products11.00 Lucy WoodsExploring structural differences between two similar fibril morphologies of beta-2 microglobulinBMSS © 2012 19

Alderley Park 201211.20 Josephine BunchImproved data acquisition and data handling methods for MALDI-MS and MALDI-MSIof small molecules in tissue.11.40 Andrea Lopez-ClavijoBinding site identification of glyoxal in Substance P by mass spectrometry.12.00 LUNCH withPOSTER SESSION& EXHIBITIONSession PIONEERING MS GREAT AND SMALLTitle Chaired by Rainer Cramer13.00 PLENARYProfessor Juri RappsilberUniversity of EdinburghBiology by Mass Spectrometry : 3D Proteomics of Supermolecular Assemblies14.00 Peter O’ConnorThe advantages of absorption mode spectra.14.20 Jo-Anne RileySelf reporting oligonucleotide probes – a new design for small mass tags14.40 Tea/Coffee20 BMSS © 2012

Alderley Park 201215.00 Perdita BarranThe metamorphic protein lymphotactin undressed by IM-MS and top down ECD15.20 Filipe LopesApplication of nanoUPLC-MS/MS to quantify human insulin-like growth factor Iprohibited in sport15.40 Laurence BindschedlerFinding small needles in a hay stack: how large-scale proteomics helps to identifykey players in powdery mildew infection of barley.16.00 End of Session and Close of Meeting RemarksBMSS © 2012 21

Alderley Park 2012Notes22 BMSS © 2012

ORAL SESSIONSBMSS © 2012 23

Oral PresentationsLowering the limitsLOWERING THE LIMITS - CATCHING THE SPORTS DRUG CHEATDavid A CowanDrug Control Centre, Department of Forensic Science and Drug Monitoring, King’s College London, LondonSE1 9NH, UKPreparations are nearly complete to accredit the Anti-Doping Science Centre in Harlow to analyse morethan 5,000 urine and 1,000 blood samples from athletes competing in the 2012 Olympic and ParalympicGames.Several new chromatography coupled mass spectrometric methods have been developed to provide “superfast, super sensitive” analysis. The new methods range from using isotope ratio mass spectrometry (C-IRMS)for measuring 12CO2 and 13CO2 to evidence testosterone administration to the analysis of peptides for thepresence of synthetic insulin and quantifi cation of IGF-I to help prove growth hormone administration. TheC-IRMS method incorporates a solvent-free injection system, GC-GC with heart-cutting followed by samplesplitting to a scan MS and the IRMS. Our UPLC®-MS instruments include a bespoke bar-coding system(now a standard option) and, for the fi rst time at an Olympics, we will screen samples using UPLC® withhigh-resolution mass spectrometry for total data capture. This will enable us to target or to look at the dataretrospectively for any suspicion of designer drug use.Our aim is to deter drug misuse in sport and this presentation will illustrate how bioanalytical science withmass spectrometry can help.Keywords: Olympic Games, Bioanalysis, Anti-DopingUSING LCMS AND PYGCMS TO IDENTIFY PIGMENTS IN MICROSAMPLES FROM WORKS OF ARTJustin J Perry 1 Joanna Russell, 2 Elke Cwiertnia 1 and Brian W Singer 21Department of Chemical and Forensic Sciences, School of Life Sciences, Northumbria University,Newcastle upon Tyne NE1 8ST2Conservation of Fine Art, School of Arts and Social Sciences, Northumbria University,Newcastle upon Tyne NE1 8STArt conservators and historians often need to identify the pigments used by artists whether it be forrestoration or authentication. Usually this identifi cation can only be via micro-samples which ethicallycan only be obtained from damaged areas or unseen margins of priceless objects. Mass spectrometryhas hence proved to be an essential tool in identifi cation of trace components in these microsamples.”Lake” pigments based on plant dyes have been used since antiquity as sources of colour in works of art.We have used HPLC-electrospray MS to identify the fl avanoid dyes in the yellow portions of works of artsuch as Munch’s “The Scream”, and hence suggest plant origins for pigments to be used by conservatorstasked with repair.More modern artists materials have proved more challenging to identify due to the greater diversity of dyesand pigments used since the genesis of the modern chemical industry. In a project funded by the Estate ofFrancis Bacon (1909-1992), we have been charting this artist’s use of paint through his career by analysisof materials in his studio and of microsamples from his paintings. To do this we have developed pyrolysisGCMS methodology which has enabled us to track paint from can to work of art.Keywords: electrospray, pyrolysis GCMS, pigment, art24 BMSS © 2012

Oral PresentationsDETERMINATION OF A URINARY DRUG METABOLITE USING LC-FAIMS-MS AND LC-FAIMS-IN-SOURCE CID-MSRobert W Smith 1 Danielle E Toutoungi 2 , James C Reynolds 1 , Ashley Sage 3 , Anthony Bristow 4 , Andrew Ray 4 ,Daniel J Weston 5 , Ian Wilson 5 , Billy Boyle 2 and Colin S Creaser 1 *1Centre of Analytical Science, Department of Chemistry, Loughborough University, Leicestershire, LE11 3TU,2Owlstone Ltd, 127 Cambridge Science Park, Cambridge, CB4 0GD, 3 Agilent Technologies, Lakeside, CheadleRoyal Business Park, Stockport, SK8 3GR, 4 Pharmaceutical Development, AstraZeneca, Macclesfield, SK102NA, 5 DMPK Innovative Medicines, AstraZeneca, Alderley Park, Cheshire, SK10 4TGHigh-field asymmetric waveform ion mobility spectrometry (FAIMS) distinguishes ions at atmospheric pressurebased on their differential mobility under low and high fi eld conditions. FAIMS separation is orthogonalto liquid chromatography (LC) and mass spectrometry (MS) detection, making hyphenation with thesetechniques highly desirable. A chip-based FAIMS device has been inserted in the source region of a TOFMS to allow rapid FAIMS pre-selection of gas-phase ions prior to mass analysis (LC-FAIMS-MS). In-sourcecollision-induced dissociation of FAIMS pre-selected ions (LC-FISCID-MS) may also be carried out prior tomass analysis. The quantitative analysis of FAIMS-enhanced (R/S)-ibuprofen 1-â-O-acyl glucuronide (IAG),spiked into urine, has been evaluated using the LC-FAIMS-MS and LC-FISCID-MS techniques. A CF scanof directly infused ibuprofen and the ibuprofen acyl glucuronide metabolite shows selective transmission ofthe metabolite can be achieved. IAG spiked standards determined with FAIMS pre-selection gave a linearresponse in the range 0.13-52 ìg/ml with an R2 value of >0.99. In-source CID of the FAIMS-selected IAG[M-H]- ion (LC-FISCID-MS) was used to generate fragments of the precursor ion to further enhance theselectivity of the analysis compared to LC-MS.Keywords: FAIMS-MS, LC-FAIMS-MS, in-source CID, urine, drug metaboliteDEAMIDATION OF COLLAGEN PEPTIDES IN ANCIENT BONES USING FT-ICR-MS.Pilar P. Hurtado 1 Matthew Collins 2 Peter B. O’Connor 11Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK2Department of Archaeology, University of York, York YO10 5YW, UKCollagen is the major component of skin, tendons, ligaments, teeth and bones, it provides the framework thatholds most multi-cellular animals together, and type I collagen constitutes the major fibrillar collagen of bone.Due to the complexity of collagen’s structure the study of post-translational modifications such as deamidationfor this protein is challenging. Although there is no evidence of this protein being used for age assessment;it has been shown that deamidation of collagen is remarkably increased in old bones from mammals.Nonspectrometric methodologies have been used for the determination of the extent of deamidation bymeasuring the amount of amide nitrogen released in ammonia as well as the rate constants for deamidationof asparagine in collagen. In general these methodologies required more sample and separation processes.To understand if collagen plays a signifi cant role in the aging process of fossil materials, we aim to apply themethodology recently developed in our lab to determine the extent of deamidation in collagen, but this time incollagen extracted from ancient bones. The present work shows how to determine the extent of deamidationin collagen from ancient bones (1,000 to 5,000 years old) using Fourier-Transform Ion Cyclotron ResonanceMass Spectrometry (FT-ICR-MS) along with Collisionally Activated Dissociation (CAD) and Electron CaptureDissociation (ECD). The measured deamidation half-life for the tryptic peptides from collagen (I) was foundto range from 4000-5000 seconds under high temperature conditions (~62 °C) and pH 7.5.Keywords: Deamidation, Collagen, Ancient bones, FT-ICR-MSBMSS © 2012 25

Oral PresentationsCLINICAL TESTING OF RAPID EVAPORATIVE IONIZATION MASS SPECTROMETRY-BASEDINTRAOPERATIVE TISSUE IDENTIFICATIONLászló Sasi-Szabó 1 , Laura Muirhead 2 , James Kinross 2 , László Damjanovich 1 , Balázs Dezsõ 1 , Robert Goldin 2 ,Júlia Balog 3 and Zoltán Takáts 21Institute of General Surgery, University of Debrecen, Móricz Zsigmond krt. 22. Debrecen,Hungary H-40242Department of Surgery and Cancer, Imperial College, London, Sir Alexander Fleming Building SW7 2AZ3Medimass Ltd., Széher út. 77. Budapest, Hungary H-1021The recently introduced Rapid Evaporative Ionization Mass Spectrometry (REIMS) technique allows thein-situ, in-vivo chemical characterization of biological tissues. Chief application area of the technique iscancer surgery, where it can be used to establish surgical margins. Following exploratory studies, we haveperformed the fi rst large scale study aimed at the validation of the tissue identifi cation capability of themethod in surgical environment.REIMS-based tissue identifi cation was established by using standard electrosurgical handpieces. Massspectrometer (LTQ, ThermoScientifi c) was enclosed into special chassis. The study involved 250 patientsundergoing bowel resection or liver metastasis resection. REIMS data was collected throughout interventionswhen electrosurgery was used. Data was identifi ed using a database of authentic REIMS data and amultivariate statistical approach. Data analysis is performed in 0.5-0.9 s, giving real-time identifi cationcapability. Results of MS-based identifi cation were compared to corresponding histopathology results for3340 individual data points. Mass spectrometric identifi cation results were in agreement with histopathologyin 97.84% of the cases, identifi cation failed in 1.35% of the cases while in 0.81 % of the cases the twoidentifi cation results were different. Results indicate that REIMS is a solution for rapid intraoperative tissueidentifi cation yielding results superior to the current gold-standard intraoperative histology.ENHANCING THE DETECTION LIMITS OF DIRECT-REAL TIME MS IN BREATH ANALYSISPaul MonksDepartment of Chemistry, University of Leicester, Leicester.The talk will detail new technological advances in using RF funnel technology to enhance the sensitivityof Proton transfer time of fl ight mass spectrometry (PTR-TOF-MS). Details will be given on the how theapplication of a RF fi eld in the drift tube enhances the sensitivity by an order of magnitude without apparentlychanging the fragmentation pattern for a wide range of test compounds. The talk will further explore theapplication of PTR-TOF-MS to breath analysis as a medical diagnostic in a novel holistic non-invasivediagnostic suite been developed in the A&E department at the Leicester Royal Infi rmary. Results will beshown that indicate the power and challenges for the real-time analysis of breath.26 BMSS © 2012

Oral PresentationsSpeeding things upDr Andy PittAston University, BirminghamBigger, faster …. better? The application of rapid scanning ToF MSQqToF mass spectrometry instruments continue to develop rapidly in both sensitivity and speed, to theextent that ToF-based MRM scanning is becoming feasible. This has applications across proteomics andmetabolomics, but requires that the data quality is suffi cient and the software keeps pace. I will presentsome of our data generated using this new instrumentation and discuss whether this offers improvementsover traditional methods.THE USE OF AN ON-LINE TWO-DIMENSIONAL (RP/RP) LIQUID CHROMATOGRAPHY MOBILITY-ENABLED APPROACH FOR THE CHARACTERISATION OF THE CELLULAR PROTEOMESJames H. Scrivens 1 Baharak Vafadar-Isfahani 1 Nisha A. Patel 1 Susan E. Slade 1 Lee Gethings 1 Chris Hughes 2Jim Langridge 21School of Life Sciences, University of Warwick, Coventry, CV4 7AL, United Kingdom2Waters Corporation, Atlas Park, Simonsway, Manchester, M22 5PP, United KingdomIt has previously been shown that the use of an online, two-dimensional RP-RP, high/low pH separation stepincreases both the number of peptides and proteins identified using the MSE, non-labelled, data-independentapproach. This is due to an increase in sample size combined with the reduction in peptide complexity anddynamic range associated with the various steps. Recently it has also been shown that the use of a mobilityenabledseparation stage in the proteomics experiment improves the quality of the product ion spectraobtained and again increases the number of confi dent peptide (and therefore protein) identifi cations. The2D LC experiment comes with a cost in experimental time proportional to the number of separation stepsand the mobility experiment, whilst not adding to the experimental time, slightly reduces the experimentallyeffective dynamic range. Here the combination of the two approaches, coupled with the use of analyticalcolumns that reduce the overall experimental time produces an effective approach that, whilst increasingthe experiment time provides data of signifi cantly higher quality. Experimental data has been collected fora number of cellular protein extracts and compared in terms of number of confi dent peptide and proteinidentifi cations and effective dynamic range of protein concentrations.Keywords: proteomics, 2D LC, ion mobility, cellularBMSS © 2012 27

Oral PresentationsLC-MS/MS ANALYSIS OF EICOSANOIDS AND ISOPROSTANES: UNDERSTANDING THE ROLE OFNANOPARTICLES IN GENERATING INFLAMMATION AND OXIDATIVE STRESSBen Maskrey 1 , Janet Adamson 2 , Wan-Seob Cho 3 , Rodger Duffi n 3 , Ken Donaldson 3 , Ian Megson 2 , PhillipWhitfi eld 11Lipidomics Research Facility,2Free Radical Research Facility, Department of Diabetes and Cardiovascular Science,University of the Highlands and Islands, United Kingdom,3ELEGI/Colt Laboratory, Centre for Infl ammation Research, University of EdinburghCellular metabolism leads to the generation of reactive oxygen species, oxidative damage and inflammation.Eicosanoids participate in the regulation of the infl ammatory process and are formed from arachidonic acid(20:4; n-6) through the cyclooxygenase, lipoxygenase and cytochrome P450 pathways. The free radicalmediatedperoxidation of arachidonic acid also leads to the production of isoprostanes, which are recognisedto be effective markers of oxidative stress. Due to the close relationship between oxidative stress andinflammation there is increasing interest in the measurement of these lipids, however their analysis representsa considerable challenge. We have developed a rapid, sensitive and specifi c liquid chromatography-tandemmass spectrometry (LC-MS/MS) method for the characterisation and quantifi cation of eicosanoids andisoprostanes in cells, tissues and body fl uids. The application of this assay to cultured lung epithelial cellsexposed to a variety of nanoparticles (e.g. carbon-based, silicon-based, metal-derived) revealed an increasein the concentration of a range of prostaglandin, HETE (hydroxyeicosatetraenoic acid) and isoprostaneisomers upon exposure to certain nanoparticles. Further understanding of the characteristics and propertiesof nanoparticles that elicit infl ammatory and oxidative stress responses may allow the development andmanufacture of a range of nanoparticles with reduced cytotoxic effects.Keywords: Isoprostanes, eicosanoids, LC-MS/MS, nanoparticles, oxidative stressMONITORING THE EFFECTS OF PHYSIOLOGICAL STRESS BY METABOLIC PROFILING OFSALIVA USING ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY-ION MOBILITY-MASSSPECTROMETRY.Aditya Malkar 1 , Helen J Martin 1 , Pareen Patel 1 , Matthew A Turner 1 , Phillip Watson 2 , Ron J Maughan 2 , HelenJ Reid 1 , C L Paul Thomas 1 and Colin S Creaser 11Centre for Analytical Science, Department of Chemistry, Loughborough University, Loughborough,Leicestershire, LE11 3TU, UK2School of Sport, Exercise and Health Sciences (SSEHS), Loughborough University, Loughborough,Leicestershire, LE11 3TU, UKWhole saliva offers the advantage of non-invasive sample collection and thus constitutes an excellent biofluidfor metabonomics. Liquid chromatography-mass spectrometry has been used extensively for the non-targeted,global metabonomic analysis of various biofl uids such as urine, blood and plasma for biomarker discovery.However, the use of this technique in combination with ion mobility-mass spectrometry for the metabolicprofi ling of saliva remains under explored. A method has been developed for the salivary metabolite profi lingbased on protein precipitation and ultra performance liquid chromatography-ion mobility-mass spectrometry(UPLC-IM-MS). The developed method has been applied in conjunction with multivariate analysis to explorethe potential biomarkers of physiological stress.Physiological stress was induced in healthy participants by exercise on a cyclometer set at a resistance of 2W.kg-1 of body mass. Saliva was collected by passive drool pre and post-exercise. Samples were cleanedup by acetonitrile protein precipitation and were concentrated by evaporation prior to analysis using UPLC-IM-MS. Analysis was carried out on an Acquity UPLC coupled with a Synapt HDMS mass spectrometer(Waters, Manchester, UK). The use of UPLC-IM-MS provides increased analytical space and allows rapidglobal metabonomic profi ling for biomarker discovery with a cycle time of 15 minutes with good reproducibly,which is essential for profi ling of biofl uids.Keywords: Metabonomics, Saliva, UPLC, Ion-mobility, Mass Spectrometry28 BMSS © 2012

Oral PresentationsSURFACE ANALYSIS USING MICRO-PADI MASS SPECTROMETRY IN AMBIENT CONDITIONSAndrew Bowfi eld and James W. Bradley.Department of Electrical Engineering and Electronics, University of Liverpool, L69 3GJ, UKOne of the most exciting developments in new ambient ionisation is called plasma assisted desorption/ionisation (PADI). Conventional PADI is based on the application of RF (13.56 MHz) to a sharpenedTungsten needle connected coaxially to an independent gas feed stock (He etc.). This set-up produces anon-thermal non-equilibrium plasma (300 K) preventing surface damage of the exposed analyte. It is simplein construction and instrumentation, runs at low voltages and produces a relatively localised plasma plumeat the needle tip allowing it to remotely activate the surface through direct contact of the plasma. PADI haspotential advantages over electro-spraying techniques (DESI), since it does not require extensive samplepreparation and there are less precise requirements for plasma-sample angles.This study reports that a modifi ed PADI can also be applied to ambient MS. This modifi ed PADI can beoperated without the requirement of noble gas flow as a discharge gas. Alternatively, the device breaks-downambient air by creating a substantial electric fi eld at the tip of a 200 μm diameter Tungsten wire. Ambient MSspectra of polytetrafl uoroethene (PTFE) and Ibuprofen and Paracetamol tablets produced by the modifi edPADI device are then compared to those produced by a traditional PADI needle.Keywords: Ambient MS, PADI,DEVELOPING AUTOMATED DRIED BLOOD SPOT DIRECT ANALYSIS TECHNIQUES FOR HIGHSAMPLE THROUGHPUT QUANTITATIVE BIOANALYSISPaul Abu-Rabie 1,21Platform Technology and Science, Drug Metabolism and Pharmacokinetics, GlaxoSmithKlineResearch and Development, Park Road, Ware, Hertfordshire, SG12 0DP, UK.2School of Science, University of Greenwich at Medway, Central Avenue, Chatham Maritime,Kent, ME4 4TB, UKDirect analysis of dried matrix spots (DMS) will aid bioanalytical effi ciency and acceptance of this samplingtechnique in multiple fi elds, such as healthcare, environmental, forensic and pharmaceutical studies. Proofof concept has been demonstrated for a number of on-line, direct elution, direct desorption/ambient ionizationand microfl uidic extraction DMS techniques coupled to mass spectrometers. The relative performance,advantages and disadvantages, robustness, and detector compatibility of these techniques will be discussed,as will the possibility of eliminating liquid chromatographic separation.Development is now also focused on automating and adding additional functionality to make these techniquescompatible with high throughput requirements. Latest developments and future plans in this area will bereported, such as internal standard addition, visual recognition systems, how to accommodate sampledilution, and controlling carry over.Overcoming the hematocrit issue could be vital if DBS is to maximise its potential to be used in regulatedpharmaceutical development environments. Potential methods of doing so will be reported, as will alternativemicrosampling techniques that could bypass the current issues.Keywords: Dried Blood Spots, Direct Analysis, Ambient Ionization, Automation, HPLC-MS/MSBMSS © 2012 29

Oral PresentationsTaking MS to the ExtremeMASS SPECTROMETERS IN SPACE!William B. BrinckerhoffNASA Goddard Space Flight CenterExploration of our solar system over several decades has benefi tted greatly from the sensitive chemicalanalyses offered by spacefl ight mass spectrometers. When dealing with an unknown environment, thebroadband detection capabilities of mass analyzers have proven extremely valuable in determining thecomposition and thereby the basic nature of space environments, including the outer reaches of Earth’satmosphere, interplanetary space, the Moon, and the planets and their satellites. Numerous mass analyzertypes, including quadrupole, monopole, sector, ion trap, and time-of-fl ight have been incorporated in fl ightinstruments and delivered robotically to a variety of planetary environments. All such instruments wentthrough a rigorous process of application-specifi c development, often including signifi cant miniaturization,testing, and qualifi cation for the space environment. Upcoming missions to Mars and opportunities formissions to Venus, Europa, Saturn, Titan, asteroids, and comets provide new challenges for fl ight massspectrometers that push to state of the art in fundamental analytical technique. The Sample Analysis at Mars(SAM) investigation on the recently-launch Mars Science Laboratory (MSL) rover mission incorporates aquadrupole analyzer to support direct evolved gas as well as gas chromatograph-based analysis of martianrocks and atmosphere, seeking signs of a past or present habitable environment. A next-generation linearion trap mass spectrometer, using both electron impact and laser ionization, is being incorporated into theMars Organic Molecule Analyzer (MOMA) instrument, which will be fl own to Mars in 2018. These and othermass spectrometers and mission concepts at various stages of development will be described.LCMS TO SUPPORT DRUG DISCOVERY AND EARLY PHASE CLINICAL TRIALS IN THE DIVISIONOF CANCER THERAPEUTICS AT THE INSTITUTE OF CANCER RESEARCH.Florence I RaynaudCancer Research UK Cancer Therapeutics Unit, Division of Cancer Therapeutics, The Institute of CancerResearch, Sutton, Surrey, UK.Liquid chromatography mass spectrometry is an essential technology in drug development from targetvalidation through to clinical trials. My team focuses on evaluation of pharmacokinetic properties inorder to guide the selection of those compounds that are likely to become drugs, and supports the firstin man pharmacokinetic evaluations at the Royal Marsden Hospital. In the discovery process, a numberof in vitro assays are used that provide rapid evaluation of compounds ahead of in vivo testing. Furtherinvestigations characterise pharmacokinetic properties in rodents and establish the optimal dosing scheduleto obtain biomarker modulation and in vivo activity. In addition, analyses of samples from clinical trialsallow determination of pharmacokinetic parameters in fi rst in man studies. Robotics, automated methoddevelopment and autoMSMS function allow adequate throughput for characterisation of early ADMEproperties such as permeability and metabolism in a non-regulated environment. The improvement ininstrumentation (mass accuracy, sensitivity) facilitates collection of qualitative and quantitative informationin support of early metabolism studies. The improvement in sensitivity of triple quadrupole instrumentsallows full pharmacokinetics in individual mice. The same triple quadrupole instruments can be switchedfrom non-regulated to regulated environment to support clinical studies. The acquisition of full scan accuratemass data from samples of various matrices allows identifi cation of metabolites that vary under differentconditions in metabolomic studies. This requires the ability to compare large datasets by multivariate analysisand identify features that are different between groups. The identity of these metabolites can be ascertainedusing databases and MSMS information. These various activities will be illustrated with examples fromdifferent drug discovery programs.30 BMSS © 2012

Oral PresentationsTHE USE OF GC-MS AND LC-MS TO SUPPORT MANUFACTURING OF PHARMACEUTICALPRODUCTSAlice LauresStructural and Chemical Analysis, GlaxoSmithKline Research Center, Stevenage, UKGas chromatography mass spectrometry and liquid chromatography mass spectrometry can beapplied as an investigation tool to support manufacturing of various pharmaceutical products.The use of accurate mass LC-MS instruments enables the acquisition of high quality MS and MSMSdata for the structure elucidation of unknown compounds. ESI, APCI and APPI are commonlyused with LC-MS to extend the range of compounds that can be analysed by this technique.GC-MS offers the advantage of library matching of electron ionization mass spectra, which is particularlyuseful when dealing with possible contaminations and formulation issues. The sample introduction systemfor GC-MS presents versatility and enables the analysis of gas, liquids and can be automated for sampleextraction.The presentation will describe some real examples from support for the manufacture of drug products whichdemonstrate the utility of LC-MS and GC-MS for tackling a wide range of problem types from this fi eld. Theexamples will show the importance of selecting the right technique for the right problem and the versatilityof these techniques in a problem-solving environment.Keywords: GCMS, LCMS, manufacturingEXPLORING STRUCTURAL DIFFERENCES BETWEEN TWO SIMILAR FIBRIL MORPHOLOGIES OFBETA-2 MICROGLOBULINLucy A.Woods, C. J. Sarell, S.E. Radford, A.E. Ashcroft.The Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds. LS2 9JT, UKThe ability of amyloidogenic proteins to assemble into fi brillar structures is associated with over twenty-fi vedifferent disease states and is both well known and characterised. Such fibrils are able to assemble in vitro undera variety of conditions, for example, by denaturation, upon the addition of metal ions or through the introductionof mutations and deletions within the protein sequence. Under each of these conditions, fi brils formed are ofa similar morphology, however, it has long been debated as to whether differences in the fibril structure exist.Here, ion mobility spectrometry-mass spectrometry and limited proteolysis have been used to characterisethe assembly of two morphologically similar fi bril types formed by beta-2 microglobulin under differentconditions. Analysis of both the intermediates formed prior to fi bril formation and of the fi nal fi bril structurereveals differences in the assembly pathways.Keywords: amyloid, non-covalent, ion-mobility, limited-proteolysisBMSS © 2012 31

Oral PresentationsIMPROVED DATA ACQUISITION AND DATA HANDLING METHODS FOR MALDI-MS AND MALDI-MSIOF SMALL MOLECULES IN TISSUEJosephine Bunch, Rian Griffi ths, Andrew Palmer, Alan Race, Rory Steven, Joscelyn Sarsby and SarahTurkerMALDI MS is a powerful method for imaging small and large molecules directly in tissue with applicationsin a diverse range of disciplines. In this presentation we address sample preparation, laser operation anddata handling for MALDI MS imaging of lipids in human and animal tissue.The addition of salts to matrix solutions has been shown to enhance MALDI MS data. We present nitratesas new matrix additives and show their usefulness in lipid analysis across wider concentration ranges thanpreviously reported additives.Raster mode imaging permits signifi cantly shorter analysis times in MALDI imaging. Faster stage speedsrequire access to high repetition rates in order to generate suffi cient ions for detection from each pixelarea. Through systematic survey of laser characteristics we reveal new insights into the effects of variablerepetition rates in raster mode imaging.Optimised methods are applied to the study of human liver disease. Images of stained sections labeledby a histologist are projected onto a MALDI image and used to label all pixels in the dataset. We presenteffective classifi cation of disease type and tissue region from MS data afforded by memory effi cient PCAroutines and higher order clustering of PCA results.32 BMSS © 2012

Oral PresentationsBINDING SITE IDENTIFICATION OF GLYOXAL IN SUBSTANCE P BY MASS SPECTROMETRY.Andrea F. Lopez-Clavijo 1 , Mark P. Barrow 1 , Naila Rabbani 2 , Paul J. Thornalley 2 and Peter B. O’Connor 11Warwick Centre for Analytical Science, Chemistry Department, 2 WMS - Clinical Sciences ResearchInstitute, University of Warwick, Coventry, CV4 7AL, United Kingdom.During hyperglycemia the high concentration of glucose leads to protein function and structure damageextracellularly and intracellularly as well. Glucose during its non-enzymatic reaction with proteins and peptidesgenerates fructosamines (an early glycation adduct), which decomposes to form, among others, glyoxal(G). Glyoxal is also produced by glucose auto-oxidation and lipid peroxidation in the human body. Thus,during diabetes, glucose, glyoxal, fructosamine, and other a-dicarbonyl compounds generate the glycationproducts commonly named Advanced Glycation End-product (AGEs). These AGEs also react with proteinsin biological systems1 forming the irreversible toxic AGE adducts;2,3 which are strongly associated withdiabetic complications4,1 such as diabetic neuropathy,5 nephropathy, retinopathy, cataract,6 and hearthdisease.7 Hence, glycation is a post-translational modifi cation (PTM) that involves a variety of reactionsbeing the products formed or AGEs a heterogeneous group of compounds. Thus, glycation was studiedhere using Substance P as a model for binding to glyoxal. The binding site (under the conditions of theexperiment), was determined at the arginine residue using Fourier Transform Ion Cyclotron ResonanceMass Spectrometry (FT-ICR-MS). Binding site identifi cation has been achieved due to the ability to performElectron Capture Dissociation (ECD),8 double resonance ECD (DR-ECD),9,10 and Collisionally ActivatedDissociation (CAD),11 with a confi dent assignment of the modifi ed amino acid with a mass error < 1 ppm.During hyperglycemia the high concentration of glucose leads to protein function and structure damageextracellularly and intracellularly as well. Glucose during its non-enzymatic reaction with proteins andpeptides generates fructosamines (an early glycation adduct), which decomposes to form, among others,glyoxal (G). Glyoxal is also produced by glucose auto-oxidation and lipid peroxidation in the human body.Thus, during diabetes, glucose, glyoxal, fructosamine, and other a-dicarbonyl compounds generate theglycation products commonly named Advanced Glycation End-product (AGEs). These AGEs also react withproteins in biological systems1 forming the irreversible toxic AGE adducts; which are strongly associatedwith diabetic complications1 such as diabetic neuropathy, nephropathy, retinopathy, cataract, and hearthdisease. Hence, glycation is a post-translational modification (PTM) that involves a variety of reactions beingthe products formed or AGEs a heterogeneous group of compounds. Thus, glycation was studied here usingSubstance P as a model for binding to glyoxal. The binding site (under the conditions of the experiment), wasdetermined at the arginine residue using Fourier Transform Ion Cyclotron Resonance Mass Spectrometry(FT-ICR-MS). Binding site identifi cation has been achieved due to the ability to perform Electron CaptureDissociation (ECD), double resonance-ECD (DR-ECD), and Collisionally Activated Dissociation (CAD), witha confi dent assignment of the modifi ed amino acid with a mass error < 1 ppm.(1) Rabbani, N.; Sebekova, K.; Heidland, A.; Thornalley, P. J. Kidney Int. 2007, 72, 1113-21.Glycation, AGEs, ECD, CAD, Mass SpectrometryBMSS © 2012 33

Oral PresentationsPioneering MS great and smallSTRUCTURAL BIOLOGY BY MASS SPECTROMETRY: 3D PROTEOMICS OF SUPRAMOLECULARASSEMBLIESJuri Rappsilber 1,2 , Salman Tahir 1 , Lutz Fischer 1 , Colin Combe 1 , Jimi-Carlo Bukowski-Wills 1 , Zhuo AngelChen 1 .1Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK;2Technische Universität Berlin, Berlin, GermanyCurrent structural biology methods leave an information gap in the mid-resolution range at which proteininteractions or conformation changes are defi ned at domain or sub-domain level. Mass spectrometry inconjunction with cross-linking is providing exactly this information. We have applied our tools to complexesup to 670 kDa in size, endogenous, tagged complexes and even whole cell lysates. We have furthermoreanalysed conformation changes in solution using stable isotope labelling for quantitative analyses.We have transformed cross-linking/mass spectrometry from an expert approach to routine application byestablishing an integrated workfl ow through having: (1) developed an enrichment strategy for cross-linkedpeptides based on charge; (2) characterised in detail the fragmentation behaviour of cross-linked peptidesin a high resolution mass spectrometer; (3) derived lessons from this for a search algorithm that does notrequire isotope-labelled cross-linkers and overcomes the n2 problem of database searching for cross-links;and (4) written user friendly web-based search software that includes a revolutionary spectrum viewer formatch evaluation with implications reaching beyond this fi eld and a cross-link map viewer for fast hypothesisgeneration that expands the current visualisation concepts of protein network viewers such as used inSTRING.We believe that cross-linking/mass spectrometry is now ready for deployment into structural and molecularbiology laboratories for routine application.THE ADVANTAGES OF ABSORPTION MODE SPECTRA.Peter B. O’Connor, Yulin Qi, Mark P. BarrowDepartment of Chemistry, University of Warwick, Coventry, CV4 7AL, UKFourier transform mass spectra can be plotted in magnitude mode, as normal, or in absorption mode asis done in NMR, provided the phase function can be defi ned. We’ve recently found a new method tocalculate the phase function and can now plot all FT mass spectra in the absorption mode. This algorithmwill be presented.Absorption mode offers substantial resolution, mass accuracy, signal/noise, and dynamic range advantagesover magnitude mode. Most importantly, these advantages come at no additional cost in instrumentationand minimal computational cost - roughly equivalent to calibrating the instrument. These advantages willbe presented for protein, peptide, polymer, and petroleomic data.Keywords: absorption mode fourier transform34 BMSS © 2012

Oral PresentationsSELF REPORTING OLIGONUCLEOTIDE PROBES – A NEW DESIGN FOR SMALL MASS TAGSJo-Anne Riley, G. John Langley, Julie Herniman.University of Southampton, Southampton, United KingdomRecent discovery of microRNAs and siRNAs has revealed short oligonucleotides to be a potential new genetherapy method, therefore novel techniques for their analysis are required. Mass spectrometry providesadvantages compared to standard bioanalysis such as increased multiplexing potential. However, directanalysis of oligonucleotides can be problematic. ESI produces multiply charged species and alkali metaladducts which reduces sensitivity and hinders quantification. An alternative is indirect analysis using cleavablesmall molecule mass tags, conjugated to biomolecules via enzyme cleavable or photocleavable linkers.This project aims to eliminate the need to design and synthesise a compatible cleavable linker. RNasedigestion of custom designed oligonucleotides was undertaken to generate mono-, di- and trinucleotideproducts which could themselves serve as cleavable small molecule mass tags for mass spectral analysis.Labelling of one or more bases comprising these small molecule mass tags with atoms such as bromine,with its distinctive isotope pattern, makes signals from the mass tags easily distinguishable from backgroundsignals. Initial experiments using RNase A indicate that the specifi city of the enzyme is inadequate andcleavage at undesired sites was observed, which would limit the multiplexing potential of the approach.Redesign of the oligonucleotide sequence is underway to circumvent this problem.Keywords: Oligonucleotides, RNA, Mass-TagsTHE METAMORPHIC PROTEIN LYMPHOTACTIN UNDRESSED BY IM-MS AND TOP DOWN ECDSophie R. Harvey 1 , Albert Konijnenberg 1 , David J. Clarke 1 , Robert C. Tyler 2 , Brian F. Volkman 2 and PerditaE. Barran 11EastChem, University of Edinburgh,Edinburgh UK EH9 3JJ2Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226 USALymphotactin is a unique chemokine, a small signalling protein, and is the only member of the C sub classof this family of secreted proteins. Lymphotactin contains only one disulfi de bond as opposed to the twonormally found in the other classes of chemokines. It contains a unique extended C terminus sequence,which forms an intrinsically disordered tail. In addition, it assumes two distinct conformations in equilibrium,with interconversion between these two folds involving a complete restructuring of the core residues. Witheach structure performing separate roles; the monomeric, conserved chemokine fold, Ltn 10 activates theG protein coupled receptor XCR1. Whilst, the unique dimeric conformation, Ltn 40, is required for bindingof the extracellular glycosaminoglycans.Wild type (WT) lymphotactin has been investigated using nano electrospray ionisation mass spectrometryunder a variety of ammonium acetate buffer strengths and conditions. In addition two mutants have beenstudied, CC3 and W55D. The CC3 mutant contains an additional disulfi de bond, preferentially stabilising thechemokine fold, while the single amino acid substitution in W55D results in a preference for the dimeric fold.Conformations adopted by these proteins in the gas phase have been studied using a home built linear drifttube ion mobility mass spectrometer. In addition both the monomeric and dimeric conformations adoptedby the WT lymphotactin have been further probed using electron capture dissociation on a 12T Bruker ApexQe Fourier transform ion cyclotron resonance mass spectrometer.Keywords: Ion Mobility Mass Spectrometry, Protein Folding, Instrinsically Disordered Proteins,BMSS © 2012 35

Oral PresentationsAPPLICATION OF NANOUPLC®-MS/MS TO QUANTIFY HUMAN INSULIN-LIKE GROWTH FACTOR IPROHIBITED IN SPORTFilipe Lopes, Mark Parkin and David CowanDrug Control Centre, King’s College London, 150 Stamford Street, London SE1 9NH, UKMass spectrometry (MS) is preferred over immunoassays to evidence drug misuse in sport. Insulin-likegrowth factor (IGF-I) is a 7.6 kDa protein prohibited by the World Anti-Doping Agency and is also a reliablebiomarker for growth hormone administration, which has anabolic and lipolytic properties. Quantifi cation ofintact IGF-I by MS in very small sample volumes (

POSTER SESSIONSBMSS © 2012 37

PostersPoster session – Lowering the limitsPOSTER 1PHARMACOKINETIC ANALYSIS OF MONTELUKAST IN HEALTHY KOREAN VOLUNTEERS BYHIGH PERFOMANCE LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRYMin-Ho JoDepartment of Pharmaceutical Biochemistry, College of Pharmacy, Kyung Hee University, South KoreaA specifi c and effective high performance liquid chromatography-tandem mass (LC/MS/MS) method for theanalysis of montelukast in human plasma has been developed and validated. After cold acetonitrile-inducedprecipitation of proteins from the plasma samples, montelukast and glipizide (internal standard, IS) wereeluted on a reverse-phase C18 column by isocratic mobile phase consisted of 10 mM ammonium formatebuffer (adjusted to pH 3.5 with formic acid) and acetonitrile (3:97, v/v). Acquisition was performed with multiplereaction monitoring (MRM) mode by monitoring the transitions: m/z 587.2 ! 423.2 for montelukast and m/z446.0 ! 321.2 for IS. Ranges of concentration for calibration curves (10-1000 ng/mL), showed correlationcoeffi cients (r2) better than 0.9948. Precision of intra- and inter-day ranged from 3.70 to 11.68% and from3.04 to 12.95%, The accuracy of intra-day and inter-day ranged from 93.34 to 102.75% and from 100.79 to107.63%, respectively. Method has been applied to the analysis in 16 healthy Korean volunteers. AUC0-t andCmax for montelukast sodium 10 mg were 4910.537 ± 916.391 ng•hr/mL, 670.3 ± 149.8 ng/mL, respectively.Tmax was observed 2.7 ± 1.0 hr. The developed method provides information for the pharmacokinetics andbioequivalence study of montelukast.Keywords: Montelukast, Pharmacokinetics, LC/MS/MSPOSTER 2THE ANALYSIS OF PRIMARY ENVIRONMENTAL POLLUTANTS BY GAS CHROMATOGRAPHY-MASS SPECTROMETRY USING GENERATED HYDROGEN CARRIER GAS.M Wilkinson, J HeseltineParker Hannifi n Ltd, Gateshead, UKGas Chromatographers are using hydrogen carrier gas more frequently as an inexpensive and readilyavailable substitute to helium, as well as to gain some performance improvements. The purpose of thisstudy is to investigate the infl uence that choosing generated hydrogen carrier gas has, as an alternative tohelium, on analytical performance using gas chromatography-mass spectrometry (GC-MS). The EnvironmentProtection Agency (EPA) has designated 16 Polycyclic Aromatic Hydrocarbons (PAH) as primary pollutants.The detection and quantification of these compounds, especially in water and soils is of paramount importancefor human health and the environment, due to the toxic and carcinogenic nature. This study uses GC-MS toeliminate non-required peaks using single ion monitoring to ensure robust data interpretation. The anticipatedoutcome of this laboratory study is the rapid and robust identifi cation and quantifi cation of the 16 primaryPAH pollutants using generated hydrogen carrier gas to yield shorter run times, increased sensitivity andresolution when compared to helium. The fi ndings may be useful in maximising the productivity of the busyenvironmental laboratory.Keywords: hydrogen carrier gas38 BMSS © 2012

PostersPOSTER 3SIMULTANEOUS DETERMINATION OF METABOLIC STABILITY, METABOLITE IDENTIFICATIONAND PROFILING USING THE AGILENT 6550 IFUNNEL Q-TOF LC/MS SYSTEMAshley B Sage, Yuqin Dai, Michael Flanagan, Keith WaddellAgilent Technologies, Stockport, Cheshire, UKTimely and rapid assessment of metabolic stability, metabolite identifi cation and profi ling is critical foraccelerating lead optimization and enhancing the success rate of drug candidates entering into drugdevelopment. Triple quadrupole LC/MS instruments using multiple-reaction-monitoring (MRM) have beenthe workhorse for quantitative analysis such as metabolic stability and profi ling. However, this platform isoptimized for high sensitivity target quantitation and not well suited for non-targeted qualitative analysis. Forthese reasons, metabolite identifi cation (qualitative) is often performed in a separate analysis on differenttypes of mass spectrometers. Furthermore, due to the limitation of sensitivity on traditional tandem massspectrometers, a relatively high substrate concentration (i.e.10-20 μM) is often required in order to identifymetabolites with a broad coverage. The ability to obtain quantitation and identifi cation in a single analysismakes metabolic stability, metabolite identifi cation and profi ling studies much more effi cient. This alsohas the potential to increase assay productivity and decrease costs in drug discovery and development.Herein, we present an integrated Qual/Quan workfl ow. The utility of the Agilent 6550 iFunnel Q-TOF LC/MSsystem for determination of metabolic stability, metabolite identifi cation and profi ling in a single experimentis demonstrated in the in vitro buspirone (1μM) metabolism study in rat liver microsomes.Keywords: metabolite ID & profi ling, quantitation, sensitivityPOSTER 4INCREASING PROTEIN IDENTIFICATIONS ON A LC-QTOF MASS SPECTROMETERA. B. Sage, P. D. Perkins, H. Yin, C. Miller, J. Meza, N. Kitagawa, W. TangAgilent Technologies, Stockport, Cheshire UKEfficient protein identification by LC-MS/MS depends on both the chromatographic separation of the peptidesand optimized MS/MS analysis. Improved separation decreases the number of peptides presented to the massspectrometer at any given instant and also increases the instantaneous signal for the same amount injected,yielding better detection of lower abundance species. Improved data dependent acquisition efficiently selectsand fragments precursors to produce ‘information rich’ spectra. This work describes the combined benefi t ofimproved LC and data-dependent MS/MS algorithms. Experiments were performed on a nanoflow LC QTOFmass spectrometer with a multilayer polyimide microfluidic chip. Proteins from E. coli lysate were used for analysisby one-dimensional nanoflow LC-MS/MS. A 1% global peptide FDR was used for database searching of results.A new peptidic isotope cluster algorithm was implemented which improves precursor identifi cation of themonoisotopic m/z and centering of the quad isolation window for optimal transmission. Precursor candidatesare ranked based on their abundance and precursor “purity”. Precursors that would co-isolate with otherpeptides, yielding chimeric peptide spectra, were fi ltered based on low purity score. MS/MS accumulationtime was modulated to achieve a user-specifi ed target for MS/MS TIC. Results showed a 44% increase inpeptides identifi ed and 42% increase in proteins identifi ed (at a

PostersPOSTER 5CHALLENGES OF TRANSFERRING AN ASSAY FROM A SCIEX QTRAP 5500 TO A XEVO TQSOlamide Ottun, Zoe Cobb, Kirsten Wilcox.Quotient Bioresearch Ltd, Newmarket Road, Fordham, Cambridgeshire CB7 5WW, UKThe ability to achieve as low as possible lower limit of quantifi cation to use for the quantifi cation ofpharmaceuticals in low dose samples is a major challenge in bioanalysis and very high sensitivity massspectrometers are being designed to help overcome this challenge. The AB Sciex QTRAP 5500 and WatersXevo TQS LC-MS/MS systems are new technologies developed to be highly selective and sensitive.On transferring an assay from the 5500 to the TQS improvements in sensitivity were seen that led to areduced sample volume, however it was necessary to modify the chromatography extensively in order toremove signifi cant interferences that were not observed on the 5500.The aim of this poster is to discuss the challenges of assay development for a compound and its metaboliteencountered on both instruments and the advantages and disadvantages of each method.Keywords: Sensitivity, Xevo, 5500POSTER 6DETERMINATION OF 25-HYDROXY VITAMIN D2 AND 25-HYDROXY VITAMIN D3 FROM HUMANPLASMA USING SPE-LC/MS/MSJoanne Jones, Steve WestwoodThermo Fisher Scientifi c, Manor Park, Runcorn, UK, WA7 1TVitamin D occurs in two forms: vitamin D2 (ergocalciferol) found naturally in plants and commonly used asa dietary vitamin D supplement; vitamin D3 (cholecalciferol) occurs naturally in mammals and is formedin the skin by exposure to sunlight. Vitamin D2 and D3 are hydroxylated in the liver to form 25-hydroxyvitamin D2 (25-OHD2) and 25-hydroxy vitamin D3 (25-OHD3) respectively. Measurement of 25-OHD2and 25-OHD3 is the preferred method of assessing a subject’s total vitamin D status due to the circulatingconcentrations (nanogram per mL levels) and serum half-life. An optimum concentration of 25-OH-D withinthe body is within the range of 32-80ng/mL, concentrations below are considered defi cient and above thisrange regarded as toxic.A quick, simple and sensitive method for the extraction and quantifi cation of 25-OHD2 and 25-OHD3 inhuman plasma using Thermo Scientific SOLA solid phase extraction (SPE) has been developed. SOLATM isa novel product which provides greater extraction recoveries, better reproducibility and increased sensitivitycompared with conventional SPE systems. The method is coupled with liquid chromatography-tandemmass spectrometry (LC-MS/MS) and the accuracy, precision, sensitivity and extraction recoveries will bedemonstrated.Keywords: Solid phase extraction, extraction recovery, sensitivity40 BMSS © 2012

PostersPOSTER 7ACHIEVING REPRODUCIBILITY IN THE BIO-ANALYTICAL PROCESSWilliam Faulkner, Ken Meadows, Tim Liddicoat, Mike Oliver, Steve WestwoodThermo Fisher Scientifi c, Runcorn, Cheshire, UK, WA7 1TASolid Phase Extraction (SPE) plays a vital role in sample preparation for many fi elds of analytical chemistryincluding, bioanalytical, clinical, forensic, environmental and food safety. SPE usually involves cartridgesor well plates packed with a loose powder of silica or polymeric material positioned between two frits.These packed beds are potentially prone to settling and voiding in production or transport leading to phasechannelling as well as irreproducibility in packing, all resulting in poor recovery and reproducibility in analyticalresults.The work presented in this paper describes and demonstrates the advantages of a novel SPE cartridgeand well plate device (Thermo Scientifi c SOLA) that eliminates the issues associated with classical SPEbeds. These SPE beds have signifi cant advantages for the analyst when processing compounds in complexmatrices particularly in high throughput bioanalytical and clinical LC/MS assays where reduced failure rate,higher analysis speed and lower sample/solvent requirements are critical in gaining greater confi dence inanalytical results and reducing cost.Data presented in this poster exemplifi es this for the SPE-LC/MS assay for Rosuvastatin in human plasmaand includes data on assay accuracy, precision and analyte recovery.Keywords: Solid Phase Extraction, bioanalytical, clinical, analyte recovery, complex matricesPOSTER 8REDEFINING SPE PERFORMANCE IN BIOANALYTICAL AND CLINICAL WORKFLOWSKen Meadows, Tim Liddicoat, Mike Oliver, Steve WestwoodThermo Fisher Scientifi c, Runcorn, Cheshire, UK, WA7 1TASolid Phase Extraction (SPE) plays a vital role in sample preparation for many fi elds of analytical chemistryincluding, bioanalytical, clinical, forensic, environmental and food safety. SPE usually involves cartridgesor well plates packed with a loose powder of silica or polymeric material positioned between two frits.These packed beds are potentially prone to settling and voiding in production or transport, leading to phasechannelling as well as irreproducibility in packing, all resulting in poor recovery and reproducibility in analyticalresults.The work presented in this poster describes and demonstrates the advantages of a novel SPE cartridgeand well plate device (Thermo Scientifi c SOLA) that eliminates the issues associated with classical SPEbeds. These SPE beds have signifi cant advantages for the analyst when processing compounds in complexmatrices particularly in high throughput bioanalytical and clinical LC/MS assays in which reduced failure rate,higher analysis speed and lower sample/solvent requirements are critical in gaining greater confi dence inanalytical results and reducing cost.Data presented also compares this new material to classical loose packed material and exemplifi es:• Improved reproducibility• Improved recovery even at low elution volumes• Lower solvent consumption• Higher extract cleanliness from high quality media and reduced extractablesKeywords: complex matrices, solid phase extraction, improved recovery, extract cleanlinessBMSS © 2012 41

PostersPOSTER 9SIMULTANEOUS QUANTIFICATION OF 17-Â-OESTRADIOL AND OESTRONE IN HUMAN PLASMABY LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRYW. Faulkner 1 , L. Pereira 1 , Tony Edge 1 , Xiang He 2 , Marta Kozak 21Thermo Fisher Scientifi c, Runcorn, Cheshire, UK, WA7 1TA2Thermo Fisher Scientifi c, San Jose, CA, USAA simple, rapid and sensitive procedure for the simultaneous quantification of 17-â-oestradiol and oestrone inserum and human plasma using a combination of liquid-liquid extraction and liquid chromatography-tandemmass spectrometry (LLE-LC/MS/MS) has been developed and validated. Employing standards and qualitycontrol samples prepared in a matrix of human plasma and charcoal stripped serum, the analytes wereisolated and sub-sequently chromatographically separated on a 150 x 2.1 mm column under reversed-phaseconditions. Operating under Atmospheric Pressure Chemical Ionization (APCI) conditions and in negativepolarity mode, multiple reaction monitoring (MRM) was utilised to observe transitions of E1 (m/z 269 ! 145and E2 ( m/z 271 ! 183).The LLE-LC/APCI/MS/MS procedure presented in this poster possesses good accuracy, specifi city,sensitivity and precision, and may be adopted as a convincing alternative to immunological approaches forthe measurement of oestrogens in routine, clinical investigations.Keywords: oestrogens, clinical, quantifi cationPOSTER 10SPE-LC-MS/MS ANALYSIS OF BIOLOGICAL EXTRACTSWilliam Faulkner, Anthony Edge, Ken Meadows, Mike Oliver, Tim LiddicoatThermo Fisher Scientifi c, Tudor Road, Manor Park, Runcorn, Cheshire WA7 1TA, UKThe use of solid phase extraction is now commonplace for the sample pre-treatment of many biologicalsamples. The combination of this technique with LC-MS/MS provides the analytical scientist with a verypowerful approach for the determination of compounds in a biological matrix. This technique has beenapplied to single molecule analysis. This presentation will demonstrate how effectively SPE-LC-MS/MScan be when applied to the analysis of these types of samples. In particular, data will be presented onendogenous material, both from the matrix and also of more concerned from the SPE cartridge itself, andhow to reduce this by optimisation of the extraction process. In particular the use of mix mode phasesand then latterly the use of a novel fritless technology which have been specifi cally treated to improve thecleanliness of the SPE tube itself. Data will be presented which demonstrates the advantages of using cleancartridges . Data obtained from monitoring for phospholipids and also for general background ions will bepresented which demonstrates the importance of using the appropriate sample preparation technique forsingle molecule analysis.Keywords: matrix effects, extraction optimisation,fritless technology,42 BMSS © 2012

PostersPOSTER 11THE APPLICATION OF GC-HRT TO THE IDENTIFICATION OF TOBACCO SMOKE CONSTITUENTSJ van Heemst 1 , J Frosina 1 , L Bishop 1 , A Kettle 2 , C Wright 11British American Tobacco Group Research & Development, Southampton SO15 8TL UK2Leco Separation Sciences UK, Hazel Grove, Cheshire, UKTobacco smoke is a complex aerosol containing many chemical constituents, with recent research indicatingthe presence of over 5600 identified components. The total number of tobacco smoke constituents may exceed100 000. In conjunction with exploring GC x GC - ToF approaches for the separation of complex mixturesand identifi cation of components, we are evaluating the application of gas chromatography coupled to highresolutiontime-of-fl ight mass spectrometry for the deconvolution of chromatographic data and identifi cationof tobacco smoke contituents. Results will be presented to demonstrate the identifi cation of substances infortifi ed smoke samples and the comparison of results for control samples with published data.Keywords: GC, high resolution, TOF, smokePOSTER 12THE ANALYSIS OF ETHYL GLUCURONIDE AND ETHYL SULPHATE BY ULTRA HIGH RESOLUTION,ULTRA HIGH SENSITIVITY TIME OF FLIGHT MASS SPECTROMETRYW Weinmann 1 & J Hillis 21Abteilungsleiter Forensische Toxikologie und Chemie (FTC) Universität Bern2Bruker UK Ltd, Banner Lane, Coventry, United KingdomEthyl glucuronide (EtG) and ethyl sulphate (EtS) are well-established biomarkers for alcohol ingestion. Asthey are detectable in human hair and urine respectively, they offer an opportunity to monitor alcohol intakewithout invasive sampling. Matrix effects are encountered when analyzing human hair or urine and currenttechniques are seriously challenged by the sensitivity required.The latest generation of UHR-TOF Mass Spectrometer, the maXis impact, combined with an innovativenew ion source, gives a performance of e”40k resolution, d” 1ppm mass accuracy, while maintaining highisotopic fi delity without sensitivity compromise. While acquisition rates of up to 50 Hz enable the maXisimpact to handle the fastest of UHPLC separations with ease. Spectacular sensitivity improvements ofup to 50-fold over the previous generation of instruments mean that for the fi rst time, accurate massMass Spectrometry offers all the benefi ts of a high-performance Time of Filght platform coupled withsensitivity levels previously obtainable only with the most sensitive triple-quadrupole instruments.EtG (LOD of 1 pg on column) and EtS (LOD 400 fg on column) were quantifi ed over the ranges were 2-2500pg on column and 2-1000 pg on column respectively. This equates to an LLOQ for ethyl glucuronide of 2pg/mg of hair sample.Keywords: ethyl glucuronide, ethyl sulphate, High Resolution, High Sensitivity,BMSS © 2012 43

PostersPOSTER 13IMPLICATIONS OF USING BIOLOGICAL MATRIXES AND SURROGATES IN BIOANALYSIS.Henry E PrattQuotient Bioresearch Ltd, Bioanalysis (Fordham)An over view of matrix types handled in pharmaceutical research and their inherent properties and implicationsof use.Topics covered will include how samples are collected then stored, what problems protein bindingcan cause, the potential effects of multiple freeze thaw cycles, enzymatic and bacterial degradationand the effects of matrix to suppression or enhance ionisation within mass spectrometers.Methodologies applied to overcome matrix issues will be discussed such as sample cleanup, the use of stabilising agents, using surrogates and dosing labelled analytes.The poster fi nishes with the new matrix validation procedures that have been implement by QuotientBioresearch as a result of the July 2011 EMEA guidelines (‘Guideline on Bioanalytical Method Validation’bought into effect on 01Feb2012).Keywords: Matrix, storage, sample preparation44 BMSS © 2012

PostersPoster Session – Speeding things upPOSTER 14ELECTROSPRAY IONISATION AND DESORPTION ELECTROSPRAY IONIZATION ION MOBILITY-MASS SPECTROMETRY STUDIES OF ANTIOXIDANTS USED IN COMMERCIAL LUBRICANTS.Caitlyn Da Costa 1 , James C. Reynolds 1 , Duncan R. Leetch 2 , Samuel Whitmarsh 2 , Tom Lynch 2 , Colin S.Creaser 1 .1Centre for Analytical Science, Department of Chemistry, Loughborough University,Loughborough, LE11 1BY, UK.2Castrol, Technology Center, Whitchurch Hill, Pangbourne, Reading, RG8 7QR, UK.Commercial lubricants contain a variety of additives that assist in enhancing both the performancecharacteristics and the lifetime of the product. One such group of additives are antioxidants, which arecommonly aromatic amines and sterically hindered phenols that help stabilize the lubricant by reducing theformation of sludge caused by oxidation reactions within the engine. The analysis of commercially availableantioxidants by electrospray ionization (ESI) and desorption electrospray ionization (DESI) is described.Antioxidant compounds were analysed by ion mobility-mass spectrometry (Waters Synapt HDMS) using ESI.Detection of the antioxidants was achieved both with and without ion mobility (IM) separation of the targetanalysis. In addition, fragmentation patterns have been examined using tandem mass spectrometry (MS/MS)for structural elucidation to aid identifi cation. Modifi cation of the Waters Synapt HDMS Z-spray ion sourcewas carried out in order to allow the analysis of antioxidants on surfaces using DESI. DESI enables the directanalysis of compounds on a variety of surfaces with little or no sample preparation. Optimisation of the DESIparameters has been investigated for the rapid and direct analysis of the antioxidants of interest.Keywords: Electrospray ionisation, Desorption electrospray ionisation, Ion mobility-MS, Antioxidants,AmbientPOSTER 15COUPLING LIQUID AND GAS-PHASE SEPARATIONS FOR COMPREHENSIVE AND QUANTITATIVEANALYSIS OF VISCERAL FAT PROTEINS IN RATS OF DIFFERING AEROBIC CAPACITYJoanne B. Connolly 1 , Lauren G. Koch 3 , Steven L. Britton 3 , Jatin G. Burniston 21Waters Corporation, Manchester, UK;2Research institute for sport and exercise science, Liverpool John Moore’s University, Liverpool, UK;3Department of Anesthesiology, University of Michigan, Ann Arbor, MI, USALow aerobic fi tness is strongly associated with metabolic syndrome and development of chronic disease.Selection of low-capacity and high-capacity running rats (LCR and HCR) has provided insight into aerobiccapacity and disease susceptibility. LCR rats exhibit features of human metabolic syndrome whereas HCRare relatively protected from disease. Visceral adipose tissue changes are intimately associated with theprogression of metabolic syndrome but have not previously been investigated in this animal model. Herewe used ion mobility mass spectrometry and data-independent label-free quantitative analysis to discovernovel differences between LCR and HCR adipose tissue proteomes. Preliminary data using ion trap massspectrometry had yielded 430 protein identifi cations in 3hr analyses. On moving to the IMS coupled ToFbased analytical strategy in this study protein identifi cation numbers were increased to 639 and 1023,with the SYNAPT G2 and G2S (the latter at a quarter of the protein loading) respectively using 90 minutegradients. Principal component analysis of the data shows a clear differentiation between the HCR andLCR phenotypes. Relative quantifi cation analysis has identifi ed 85 regulated proteins (p

PostersPOSTER 16DEVELOPMENT AND VALIDATION OF A HPLC-MS/MS ASSAY TO QUANTIFY THE NON-NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITOR (NNRTI) ANTI-RETROVIRAL DRUGEFAVIRENZ IN DRIED BLOOD SPOTS (DBS).A. B. Amara 1 , J. Tjia 1 L. J. Else 1 S. Khoo 2 D. J. Back 21Liverpool Bioanalytical Facility, Royal Liverpool University Hospital, Prescot Street, Liverpool, L69 3GA2Department of Pharmacology and Therapeutics, University of Liverpool, Liverpool, United KingdomIntroduction: Efavirenz (EFV) is one of the preferred components of fi rst line antiretroviral treatment. EFVis characterised by a long plasma half-life (40-55h) and exhibits large inter-patient variability which makesit a candidate for individualisation of therapy. Analyses of plasma concentration of EFV require specialisedfacilities (cold storage/transport) which, in resource-limited environments, can be problematic. Under theselimitations, DBS-EFV measurements thus provide a cheap, easy alternative. Our objective was to developand validate a LC-MS/MS method to quantify EFV in DBS to be used in clinical trials in developing countries.Methods: Spots for standards, QC and patient samples, were excised in duplicate and extracted with ethylacetate/n-hexane (50/50) after addition of 20mL each of working internal standard and 1M K2CO3. Theorganic phase was evaporated to dryness, the residue reconstituted in mobile phase and the resultingsolution analysed directly by LC-MS/MS on a Thermo Access Triple Quadrupole mass spectrometer.Hexobarbital was used as internal standard. Gradient elution was on a reverse-phase C18 column using1mM ammonium acetate in water and acetonitrile. Quantifi cation was by SRM in negative ionisation mode.Results: The ISTD and EFV were eluted at 2.68 and 3.54 minutes respectively within a 5 minute run time.Matrix effects were minimal (-5.39%). Calibration curves were validated over a concentration range of 25-5000 ng/mL. Intra and inter assay variation ranged between 6.74-8.69% for precision and 100.27-104.20%for accuracy. Recovery was within 78 - 112%.Conclusions: The validated assay is currently applied to clinical samples to measure DBS-EFV for PKanalysis. The methodology is robust, accurate and sensitive.Keywords: Efavirenz, Anti-retroviral, Pharmacokinetics, Dried Blood Spots, HPLC-MS/MS,POSTER 17SEQUENCING OF OLIGORIBONUCLEIC ACIDS BY COLLISION INDUCED DISSOCIATION TANDEMMASS SPECTROMETRYHenry Fisher 1 , Marco Smith 2 , Ute Gerhard 2 and Alison E. Ashcroft 11Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS16 9DG, UK2GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire, UKOligoribonucleotides are important molecules that play many vital roles in cellular systems from the regulationof gene expression to cell defence from viral RNA. RNAs have not been studied by mass spectrometry tothe extent of proteins due to their inherent instability and the high level of salt adduction. As the interestin RNA therapeutics increases it is becoming necessary for analytical techniques of greater sensitivity andthroughput to be developed. NanoESI-MS is an ideal tool for the analysis of large biomolecules as it is fast,sensitive and versatile. The adduction of salt ions was fi rst addressed because of the decreased sensitivityand complex spectra caused by these adducts. Solutions of 40 mM RNA with 50 mM ammonium acetate or25 mM imidazole/piperidine buffers were compared. It was found that both greatly decreased salt adductionbut that the imidazole/piperidine buffer resulted in greater sensitivity. In order to increase detection limitsfurther, the broad charge state distribution was reduced by judicious use of solvents. Collision induceddissociation tandem mass spectrometry sequencing was also optimised with these buffer/solvent conditions.Full sequence coverage was achieved for fi fteen RNA molecules ranging from 5-mers to 21-mers.Oligoribonucleotides, Mass Spectrometry, Sequencing, RNA46 BMSS © 2012

PostersPOSTER 18IDENTIFICATION OF A GROUP OF PUTATIVE DAPHNIA KAIROMONES AFFECTED BY ZNONANOPARTICLES BY FT-ICR MS BASED METABOLOMICSJulia Fabrega 1 , Ulf Sommer 2 , Lisa M Bishop 2 , Tamara S Galloway 1 , Mark R Viant 2 , Charles R Tyler 1 .1School of Biosciences, Hatherly Laboratories, University of Exeter, Prince of Wales Road,Exeter EX4 4PS, UK2NERC Biomolecular Analysis Facility – Metabolomics Node (NBAF-B), School of Biosciences,University of Birmingham, Edgbaston, Birmingham B15 2TT, UKManufactured nanoparticles are found in many consumer products and eventually can be released into thenatural environment. Currently their potential toxicity is poorly understood. Here we investigated the effect ofzinc oxide nanoparticles on the metabolism of Daphnia magna, a model freshwater organism in toxicity testing.Daphnia were treated with different concentrations of nanoparticles, while using treatments with soluble ZnCl2,bulk ZnO and no treatment as controls. Ten metabolite extracts per treatment group were then analyzed byFT-ICR MS in three replicates in both ion modes. Multivariate analysis of the spectral datasets discovered,in both ion modes, a group of compounds that was signifi cantly decreased by the nanoparticles. As thesecompounds could not be identified with database searches, they were characterised by fragmentation studiesusing CID and IRMPD. The aliphatic sulphates that were identifi ed show structural similarity to invertebrategut anionic surfactants, and some have been reported to act as kairomones in Daphnia spp., i.e. signallingmolecules that are excreted by the Daphnia that change the morphology of algae upon which they graze.We therefore propose a novel mode of toxicity for the ZnO nanoparticles which was discovered by virtue ofusing a high-throughput non-targeted mass spectrometry based approach.Keywords: FT-ICR MS, high-throughput metabolomics, nanoparticle toxicityPOSTER 19SIMULTANEOUS QUANTITATIVE AND QUALITATIVE MEASUREMENTS FOR PRIMARYMETABOLISM INVESTIGATIONS USING A QUADRUPOLE-TIME-OF-FLIGHT MASSSPECTROMETER.George McLeod 1 Mark Savage 2 and Angus Nedderman 21Bruker UK Ltd, Banner Lane, Coventry, CV4 9GH.2Unilabs Bioanalytical Solutions, Discovery Park, Ramsgate Road, Sandwich, Kent, CT13 9NJ.Recently there has been considerable interest in simultaneously performing both quantitative and qualitativeDMPK analyses in support of small molecule drug discovery. This work describes an investigation into thepossibility of using a Quadrupole-Time-of-Flight mass spectrometer to obtain clearance data, metaboliteidentifi cation, structure elucidation and metabolite profi les from P450 microsomal incubations at a drugconcentration of 1 mM from a single sample set.P450 microsomal incubations of commercially available drug substances including Pindolol, Verapamiland Haloperidol were prepared at 1 mM concentration. The incubations were sampled and quenched, atintervals to provide a time course over a period of 60 minutes. The analysis of the samples was carried outby LC-MS using a Bruker Maxis Impact Quadrupole-Time-of-Flight mass spectrometer to obtain data suitablefor measuring clearance and plotting metabolic profi les. Data dependent MSMS spectra were collected inorder to identify and elucidate the structures of the observed metabolites. The clearance profi les and valuesare compared to those obtained using a triple quadrupole mass spectrometer to analyse the same sampleset.Keywords: Quantitative, Qualitative, Metabolite, DMPK, Time-of-FlightBMSS © 2012 47

PostersPOSTER 20SIMULTANEOUS QUANTITATIVE AND QUALITATIVE DATA GENERATION FROM DRIED BLOODSPOTSAngus N R Nedderman, Mark E Savage, Julian J Haynes, Michelle GleaveMetabolism and Discovery Services Group, Unilabs Bioanalytical Solutions, Discovery Park, RamsgateRoad, Sandwich, Kent , UK, CT13 9NJAmongst the recent initiatives within DMPK is the use of dried blood spots (DBS) for quantitative bioanalysis,offering improved data quality and reduced animal usage via serial sampling of small blood volumes fromindividual animals. In addition, the simultaneous generation of qualitative and quantitative metabolism data(often referred to as ‘qual/quan’ data), intended to provide rapid information on metabolic clearance duringdrug discovery, has grown in popularity. This work describes an investigation of qual/quan data generationfor the literature compounds verapamil and diclofenac from small volume DBS samples. Whole blood wasspiked at varying concentrations with samples generated from microsomal incubations and analysed by LC-MS following simple extraction. Quantitative and qualitative data were generated using both nominal masstriple quadrupole and high-resolution mass spectrometry and compared to data generated directly from invitro incubations with a particular focus on sensitivity, recovery and matrix effects. The results showed thatreliable quantitatve data can be acquired from DBS samples using either high-resolution or triple quandrupolemass spectrometry, whilst high-resolution approaches also enabled the generation of detailed metabolisminformation. Overall, the work demonstrated the feasibility of using qual/quan approaches on DBS samplesto provide simultaneous information on metabolic clearance and compound exposure.Keywords: Qual/quan, DBS, metabolism.POSTER 21IMPROVING ASSAY STABILITY BY TURBULENT FLOW CHROMATOGRAPHYTony Edge 1 , Luisa Pereira 1 and Francois Espourteille 21Thermo Fisher Scientifi c, Runcorn, UK,2Thermo Fisher Scientifi c, Franklin, TN, USATurbulent fl ow chromatography (TFC) is a sample preparation procedure that has been used extensivelyin the fi eld of bioanalysis. The technique relies on the removal of proteins and other high molecular masscomponents from a biological matrix, by differences in the rates of diffusion of these categories of molecules,which are particularly emphasised when the fl ow in the column is dominated by inertial forces. This processis coupled with the selectivity of solid phase extraction, as that range of sorbent materials can be used toimprove the extraction selectivity towards certain classes of analytes.This presentation investigates the advantages that turbulent flow chromatography has in terms of removal ofendogenous materials when compared to simpler extraction techniques such as protein precipitation. Theremoval of phospholipids will also be discussed in relation to both approaches. Approaches to improve thelifetime of the analytical columns that are routinely used within these analytical systems, in particular, the useof solid core materials will be investigated. Data will demonstrate that it is possible to have a chromatographicsystem that is stable for many hundreds of injections of large volumes (up to 50mL) of serum without affectingthe chromatographic integrity of the analytical data.Keywords: Bioanalysis, on-line sample preparation, column lifetime, sample matrix, phospholipids48 BMSS © 2012

PostersPOSTER 22CORE ENHANCED TECHNOLOGY COLUMN PERFORMANCEL. Pereira, T. Edge, H. Ritchie, S. LukeThermo Fisher Scientifi c, Manor Park, Runcorn, UK, WA7 1TAThe use of sub-2μm particle packed columns in fast LC and LC/MS assays is now well established in theanalytical laboratory. However, fully porous sub-2μm particle packed columns do not always produce theeffi ciency enhancements predicted by the theory based on particle size, and the backpressure generatedgenerally prevents the use of conventional HPLC systems.Solid-core materials present an alternative as they provide fast, high effi ciency separations at pressuresavailable in conventional HLPC systems. The enhanced mass transfer properties of solid-core particlesresult in high efficiency separations at high mobile phase linear velocities, which facilitates highthroughput analysis. As these particles are in the range of 2.6 – 2.7 mm in diameter, the backpressuresgenerated are significantly lower than those obtained with sub-2μm particle packed columns.The work presented in this poster compares the performance of 5, 3, and sub-2μm fully porous particles withthat of a newly developed 2.6 mm solid core material, using the kinetic plot method. Parameters investigatedare analysis time, resolution, selectivity, peak capacity and sensitivity of the assay.Keywords: High throughput, solid-core, effi cient separations, low backpressurePOSTER 23INVESTIGATION INTO USE OF IDCUBE AS A POTENTIAL OPEN ACCESS SYSTEM IN SUPPORTOF MEDICINAL CHEMISTRYAndrew D RayPharmaceutical Development, AstraZeneca R&D, Silk Road Business Park, Charter Way, Macclesfi eld,Cheshire, SK10 2NALC/MS open access systems are now commonly used in support of medicinal chemistry. These ionise awide range of compounds and provide useful information quickly to the chemist. However not all samplesare amenable to this technique, insoluble samples for example, and queues of samples may build up onthe system dependent on the length of the chromatographic run. There is also a desire from the chemiststo have open access accurate mass measurementModern ambient ionisation techniques (such as DART, ASAP, DESI etc) have been shown to be widelyapplicable to a range of compounds and are of particular use for the analysis of solid material. The IDCubeuses a small spot of liquid or solid onto a metal gauze held on a card which is inserted into the IDCubeand the sample is then ionised directly into the mass spectrometer with the spectra being observed withinseconds.The potential of the IDCube to support chemistry was investigated by analysis of a variety of compounds,in solution and as powders.Keywords: DART ChemistryBMSS © 2012 49

PostersPOSTER 24COMPUTATIONAL ANALYSIS OF THE EFFECTS OF VARIABLE RASTER SPEEDS AND LASERPULSE REPETITION RATES IN MALDI-MSIRory T. Steven, Alan Race, Andrew Palmer, Ata Kaban, G. Ed Rainger and Josephine BunchThe University of Birmingham, Birmingham, UK.MALDI-MS is an extremely powerful technique for detection and imaging of small molecules. Thecharacteristics of the laser used (wavelength, pulse width, repetition rate and energy per pulse) are veryimportant in ablation and desorption processes and therefore strongly affect sensitivity. Altering these variableshas a signifi cant effect on ion counts detected, the rate at which suffi cient ion counts can be obtained andsuitability of a particular matrix. In this work a frequency tripled high repetition rate Nd:YVO4 laser (1-25 kHz)is employed at varying raster speeds for the analysis and imaging of lipids. The on-going aim of this workis the improved understanding of next generation instrumentation for the improvement of high throughputhigh sensitivity imaging of lipids.Initial studies were carried out on thin layer depositions of lipid standard PC 34:1 with á-CHCA pre mixedand applied via airspray deposition.Preliminary results show that repetition rate had a direct impact on mean ion count under raster modeimaging conditions. At the higher fl uences studied (28 and 34 Jm-2) the largest ion counts were obtained ata repetition rate of 1 kHz and raster speed of 0.2 mms-1.Keywords: MALDI-MSIPOSTER 25THE EFFECT OF AMINO-TERMINAL BASIC GROUPS UPON B-ION REARRANGEMENT DURINGCOLLISION INDUCED DISSOCIATION (CID)Ross Chawner 1 , Simon J Gaskell 2 , Claire E Eyers 11Michael Barber Centre for Mass Spectrometry, Manchester Interdisciplinary Biocentre,131 Princess Street, Manchester, M1 7DN, UK.2Queen Mary University of London, Mile End Road, London, E1 4NS, UK.Proteomic studies typically employ tandem mass spectrometry for identifi cation and quantifi cation of largenumbers of peptides and proteins. However there are limitations in the assignment of fragmentation productsdue to incomplete understanding of peptide fragmentation pathways. Consequently, potentially informativeinformation is often ignored. Recent studies (1-2) show that rearrangement of b-ion products can occur duringCID resulting in scrambling of the native peptide sequence. Such rearranged species may cause incorrectfragment ion assignment and false identification of the peptide in question. Furthermore such rearrangementcan decrease scores from automated search algorithm spectral matching due to unaccounted product ions.These possibilities become increasingly likely with increasing extent of rearrangement to give a more intensemass spectrometric signal.The rearrangement process involves head to tail cyclisation of the b-ion giving a macrocycle structure thatcan ring open at various amide bonds yielding a scrambled peptide sequence, for which we have defi neda novel nomenclature (3). Here we present data comparing the observed scrambling during analyses ofpeptides derived from Lys-C and Lys-N proteolysis of standard proteins. Modification of the lysine side-chainto homoarginine is shown to impact upon the observed scrambling with the potential benefi t of reducingspectral complexity in proteomic studies.(1) Riba-Garcia I. et al., J Am Soc Mass Spectrom 2008, 19, 1781–1787(2) Harrison A.G., J Am Soc Mass Spectrom 2008, 19, 1776–1780(3) Chawner R et al., Rapid Comm Mass Spectrom 2012 26, 205-6Keywords: CID, Fragmentation, Ion Mobility, Rearrangement50 BMSS © 2012

PostersPOSTER 26DIRECT THERMAL DESORPTION ELECTROSPRAY IONISATION MASS SPECTROMETRY FORTHE ANALYSIS OF POTENTIALLY GENOTOXIC IMPURITIES IN ACTIVE PHARMACEUTICALINGREDIENTS.James C Reynolds and Colin S CreaserCentre for Analytical Science, Loughborough University, Ashby Road, Loughborough LE11 3TU, UKSulfonic acids are commonly used reagents in pharmaceutical synthesis where they are used as salt counterions and acid catalysts. However, sulfonic acids may react with alcohols present in the reaction mixtureforming sulfonic acid esters, these species are potential alkylating agents and therefore may have genotoxicproperties. Current guidelines for the accepted daily dose of a potentially genotoxic impurity (PGI) state thatthe daily dose should be limited to less than 1.0-1.5 μg/day which gives a rough upper limit of 1.0 ppm inthe drug assuming a dose of 1g per day.Here we describe a rapid thermal desorption method for the direct analysis of solid APIs spiked with variousPGIs using electrospray ionisation mass spectrometry. Preliminary data obtained using benzene and toluenesulfonate esters show strong and reproducible mass spectrometric responses with limits of detection below the1.0 ppm control limit. However dialkyl sulfonate esters are labile and decompose in the ion source resultingin reduced sensitivity. Doping the electrospray solvent with an alkali metal salt is shown to be capable offorming stable [M+alkali metal]+ adduct ions in the ion source, enhancing the sensitivity of the approachand enabling accurate determination of dialkyl sulfonate esters.Keywords: Thermal desorption, Electrospray, Pharmaceutical analysis, Direct analysisPOSTER 27DETECTION OF TRIACETONE TRIPEROXIDE (TATP) AND ITS COMPLEXES WITH METAL IONSUSING NANOELECTROSPRAY-ION MOBILITY-MASS SPECTROMETRY.Alex R Hill, James C Reynolds, Paul F Kelly and Colin S CreaserCentre for Analytical Science, Department of Chemistry, Loughborough University,Leicestershire, LE11 3TU, UKThe peroxide based explosive triacetone triperoxide (TATP) has gained notoriety through its use by terroristorganisations. Its straightforward synthesis and an explosive power comparable to TNT, make it the explosiveof choice for illicit use. Many explosive detection systems rely upon the presence of a nitro group or metallicelement within the explosive, the lack of these in the cyclic TATP makes detection diffi cult. Reaction of theperoxide with metal ions can improve the detection ability of the explosive. The analysis of these compoundshas been carried out by nanoelectrospray mass spectrometry (nano-ESI-MS) and tandem mass spectrometry(MS/MS), both coupled with ion mobility (IM) spectrometry. The use of a nanospray source allows the analysisof the weakly bound metal complexes with good sensitivity and IM provides structural information on thecomplexes and their fragments based on collision cross section.Keywords: Triacetone triperoxide, Nanoelectrospray, Ion mobility-MSBMSS © 2012 51

PostersPOSTER 28DFT GUIDED ION-MOBILITY STUDIES OFFER A POWERFUL STRATEGY FOR UNDERSTANDINGPHARMACODYNAMIC INTERACTIONS WITHIN ANTIMALARIAL HAEM COMPLEXESFyaz M. D. Ismail,1 Harry Morris,1 Nicola M. Dempster, 1 Imran Y. Saleem 1 Frank Kasaija 1 Hieu V. Truong 1Laura E. Randle 1 Said Alizadeh-Shekalgourabi 2 Giles Edwards 3 Jonathan P. Williams 4 Michael. J. Dascombe 5& Michael G. B. Drew 6 .1Institute for Health Research, School of Pharmacy and Biomolecular Sciences,Liverpool John Moores University, Liverpool, L3 3AF, U.K.;2Department of Chemistry, University of Hertfordshire, Hatfi eld AL10 9AB, U.K.,3Daris Centre for Scientifi c Research and Technology Development, The University of Nizwa,P.O.Box 33, PC 616, Birkat Al Mouz Nizwa, Sultanate of Oman;4Waters Corporation, Manchester, UK;5Faculty of Life Sciences, The University of Manchester, Manchester M13 9PT, U.K.6School of Chemistry, University of Reading, Reading RG6 6AD, U.KThe mechanism of anti-malarial action of 4-aminoquinolines and 9-aminoacridines remains imperfectlyunderstood despite considerable effort. Understanding the bonding interaction and geometry betweenan anti-malarial drug (e.g. chloroquine; CQ) and its receptor (haem; Hm) lays the foundations for rationaldrug design and consequently drug action. It is commonly and, incorrectly, assumed that the interactionsbetween haem and various drugs are only pi...pi or dispersive in nature. Using a combination of ion mobilityelectrospray mass spectrometry, CID studies and density functional theory, it is possible for the fi rst time todefine the geometry of chloroquine bound to its receptor in the gas phase as “edge on” and involving hydrogenbonding between acceptors-nitrogen atoms and propionic acids donors within haem. It is also possible tonow reject the haem mu-oxo dimer as a receptor. Our data refute assumed pi...pi or orthogonal interactionssimilar to that between haem and histidine as being responsible for aminoquinoline and acridine containingdrugs binding to haem. Evidence that this novel bonding arrangements persists in the solution and solidphase will also be presented. The new geometry reveals useful avenues for rational drug design and also,retrospectively, explains existing conundrums involving antimalarial drug action at the haem receptor.Keywords: ion-mobility, DFT, antimalarials, haem, pharmacodynamicsPOSTER 29NANOELECTROSPRAY LIQUID EXTRACTION SURFACE ANALYSIS OF INTACT PROTEINS FROMPOLYVINYLIDENE FLUORIDE (PVDF) MEMBRANESNicholas J. Martin 1 , Josephine Bunch 2 and Helen J. Cooper 11School of Biosciences, 2School of Chemistry, University of Birmingham, Birmingham United KingdomTop down proteomics is an emerging fi eld in mass spectrometry, largely down to improvements ininstrument resolution and fragmentation techniques. One factor limiting its development is the diffi cultyof fractionating and purifying proteins prior to analysis. PVDF is an absorbent polymeric matrix used in avariety of laboratory applications, most notably for protein blotting following separation by gel electrophoresis.We present a method for sampling proteins dried onto PVDF membranes by liquid extraction surface analysis(LESA) by use of an Advion Triversa Nanomate (TVNM) coupled with a Thermo Fisher LTQ Orbitrap Velosmass spectrometer. Previously, we have demonstrated the application of LESA of dried blood spots inthe top-down analysis of haemoglobin variants. The LESA technique has also been applied to thin layerchromatography plates, dried blood spots and thin tissue sections for the analysis of small molecules. Massspectra were obtained from proteins extracted from PVDF membranes with as little as 5 picomoles totalprotein. In further work, we show that the TVNM advanced user interface can be used to incorporate proteinpurifi cation by reversed phase C4¬ Ziptips into the LESA sampling routine.Keywords: Top Down Proteomics, LESA, PVDF52 BMSS © 2012

PostersPOSTER 30IMPROVING EFFICIENCY OF GC-MS ANALYSIS WITHIN A PHARMACEUTICAL DEVELOPMENTENVIRONMENT BY IMPLEMENTATION OF A SINGLE CHROMATOGRAPHY PLATFORMMartin Sims 1 Karen Rome 1 John Moncur 2 Scott Campbell 21Analytical Science, Pharmaceutical Development, Astrazeneca, Macclesfi eld, SK10 2NA, UK2SpectralWorks Ltd, Runcorn, WA7 4QX, UKWhile open access LC-MS is widely used within Pharmaceutical Development there has long been a desireto introduce a similar system for GC-MS analysis. Such a system is currently being implemented for bothsynthetic organic chemists and specialised analytical chemists that will increase productivity by presentingan affordable, simple, user-friendly, platform independent and easily accessible GC-MS solution. The typicaluser will use the same simple log-in regardless of the make and model of mass spectrometer being used andwill be able to view the results simply and easily from their desk. The system has the potential to be furtherexpanded to include LC-MS and other instruments. An overview of the selection and implementation, and alonger term view of future developments and improvements will be presented to demonstrate that a powerfuland fl exible open-access GC-MS system is feasible in the todays large industrial laboratory.Keywords: gc-ms open accessPOSTER 31XENOBIOTIC METABOLISM: CHEMICAL CHARACTERISATION, MICROSOMAL INCUBATIONSAND HPLC-ELECTROSPRAY MASS SPECTROMETRY ANALYSIS OF VARIOUS ANTIMALARIALSINCLUDING ARTEMISININ, PYRONARIDINE AND NOVEL BISQUINOLINES ESPECIALLYMETAQUINEFyaz M. D. Ismail 1 , Harry Morris 1 , Nicola M. Dempster 1 , Imran Y. Saleem 1 , Frank Kasaija 1 , Hieu V. Truong 1 ,Elizabeth K. Y. Yeung 1 , Dorothy Dafimu 1 , Sarmad M. Jasim 1 , Laura E. Randle 1 , Said Alizadeh-Shekalgourabi 2Giles Edwards 3 , Jonathan P. Williams 4 Michael. J. Dascombe 5 & Michael G. B. Drew 6 .1Institute for Health Research, School of Pharmacy and Biomolecular Sciences,Liverpool John Moores University, Liverpool, L3 3AF, U.K.;2Department of Chemistry, University of Hertfordshire, Hatfi eld AL10 9AB, U.K.3Daris Centre for Scientifi c Research and Technology Development, The University of Nizwa,P.O.Box 33, PC 616, Birkat Al Mouz Nizwa, Sultanate of Oman;4Faculty of Life Sciences, The University of Manchester, Manchester M13 9PT, U.K.5School of Chemistry, University of Reading, Reading RG6 6AD, U.K.Drug-induced liver injury is a major cause of drug withdrawal and constitutes a signifi cant challenge duringpharmaceutical drug development. Candidate compounds are usually screened in the early stages ofdrug development, for their ability to form chemically reactive metabolites during bioactivation using livermicrosomes. These reactive metabolites have the potential to bind and irreversibly and modify cellularmacromolecules. Small molecules bound onto cellular macromolecules can prime the immune systemsand subsequently lead to pathological conditions. It is, therefore, imperative to understand these processesand eliminate potentially harmful drug prototype at the earliest possible stage of the drug discovery anddevelopment process. In order to eliminate resistant Plasmodia, combinations of various drugs with artemisininhave been developed to eliminate persistent parasites during antimalarial chemotherapy. Using a HPLC-MSwe have investigated the potential for various known and experimental antimalarials to undergo Phase I andPhase II metabolism modulating the PK profile allowing further development. Compounds included mepacrine,pyronaridine, Ro 47-7737 (various isomers), ortho-, meta and para-quine as well as the sesquiterpeneendoperoxide antimalarial artemisinin and dihdroartemisinin. Several unusual metabolites, including thoseillustrating demethoxy-thiolation were discovered, which may explain the superiority of some drugs such aspyronaridine over original prototypes such as mepacrine.Keywords: metabolism, electrospray pharmacokinetics, demethoxy-thiolationBMSS © 2012 53

PostersPOSTER 32LIQUID EXTRACTION SURFACE ANALYSIS IMAGING OF IN-SITU LIPIDS AND PROTEINS FROMHUMAN LIVER ANALYSED BY HIGH RESOLUTION MASS SPECTROMETRYJoscelyn Sarsby 1 , Alan Race 1 , Helen Cooper 2 ,Patricia Lalor 3 , Hamid Dehghani 4 and Josephine Bunch 1 .1Physical Science of Imaging in the Biomedical Sciences and School of Chemistry,2School of Biosciences,3Centre for Liver Research Institute of Biomedical Research School of Immunity andInfection College of Medical and Dental Sciences,4School of Computer Science, University of Birmingham, Edgbaston, Birmingham B15 2TTLiquid extraction surface analysis (LESA) using the Advion Triversa Nanomate is presented as a new methodfor detecting and imaging small and large molecules in tissue. In LESA, a liquid micro-junction between asample surface and conductive pipette tip allows extraction of molecules from the sample. The droplet isretracted into the pipette tip and delivered to the back of the ESI Chip for infusion nano-electrospray highresolutionmass spectrometry and tandem mass spectrometry (MS/MS). Here we present the use of LESAfor analysis of lipids and proteins in human liver samples. Small volumes (~5uL) were aspirated from aconductive pipette tip and a liquid micro-junction between the pipette tip and tissue section was maintainedfor 5 seconds. The re-aspirated solution was injected using nano-electrospray into a Thermo Fisher Orbitrapmass analyser with a tip voltage of 1.75kV, gas pressure 0.5psi and capillary temperature of 250?C. Theprocess was repeated at defi ned intervals (1mm) across the tissue. Data for each location was collectedfor 2 min in the form of a series of 5 co-added microscans and resulting ion images were constructed usingin-house software.Keywords: LESA, Proteins, Lipids, NanomatePOSTER 33DIRECT GRADIENT EXTRACTION OF URINARY ANALYTES FROM UNDEVELOPED REVERSED-PHASE THIN-LAYER CHROMATOGRAPHY PLATES COMBINED WITH ON-LINE ELECTROSPRAYIONISATION AND ION MOBILITY-MASS SPECTROMETRYNeil A Devenport 1 James C Reynolds 1 Ian D Wilson 2 Daniel J Weston 2 Colin S Creaser 1 .1Centre for Analytical Science, Department of Chemistry, Loughborough University, Leics., LE11 3TU, UK2DMPK Innovative Medicines, AstraZeneca R&D, Alderley Park, Cheshire, SK10 4TG, UKAnalysis of human biofl uids provides a wealth of information, including the ability to track biomarkers,metabolites and monitor disease state. The non-invasive nature of urine collection enables large samplevolumes to be obtained, but the sample preparation required to remove the high concentrations of saltlimits its practicality for direct mass spectrometric analysis. The retention of urinary analytes depositedon undeveloped reverse phase-thin layer chromatography (RP-TLC) plates allows salts to be removedbefore on-line extraction. A rapid solvent gradient was used during the extraction process and was shownto reduce ion suppression effects, compared to direct infusion, providing higher quality mass spectral data.The selective retention of urinary components on the RP-TLC plate provides a degree of separation basedupon the interaction of individual analytes with the C18 stationary phase. The incorporation of an ion mobility(IM) separation is shown to further enhance the gradient profiling technique by distinguishing between targetanalytes and interfering (isobaric) ions. IM also provides an improvement in target identifi cation throughselection of a specifi c drift time window. The use of a solvent gradient in the extraction of urinary analytesby the TLC-ESI-IM-MS technique is a potentially powerful tool for the rapid profi ling of raw biofl uids.Keywords: Urinary profi ling, direct analysis, TLC, gradient extraction, ion mobility-mass spectrometry54 BMSS © 2012

PostersPOSTER 34STRUCTURAL DETERMINATION OF N-LINKED GLYCANS BY ION MOBILITY AND NEGATIVE IONFRAGMENTATIONDavid J. Harvey 1,2 , Christopher N. Scanlan 1 , Max Crispin 1 , Charlotte A. Scarff 2 , Matthew Edgeworth 2 , FrankSobott 3 , James H. Scrivens 2 .1Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford,South Parks Road, Oxford, OX1 3QU, UK;2Warwick/ Waters Center for Biomedical Mass Spectrometry and Proteomics,School of Life Sciences, Gibbet Hill Campus, University of Warwick, Coventry, CV4 7AL, UK;3Department of Chemistry, University of Antwerp, Groenenborgerlaan 171 2020, Antwerpen, Belgium.Although many studies have been published on structural determination of N-glycans (those attached toproteins in an Asn-Xxx-Ser(Thr) sequence where Xxx is any amino acid except proline), many methodsfail to elucidate all structural features such as, for example, branching patterns and the location of fucoseresidues. We have found that negative ion fragmentation is much superior to the more popular positive ionfragmentation in that it produces very specific cross-ring fragment ions defining many structural features thatare diffi cult to determine by traditional techniques. Furthermore, no derivatization is necessary, considerablyspeeding up analyses. Ion mobility in negative ion mode has added another dimension to the analysis ofthese carbohydrates. In particular, the technique has proved to be an excellent method for extracting theions of carbohydrates with good signal:noise ratios from samples that give electrospray or MALDI spectrathat initially show no signs of the presence of these compounds. Glycan structures can, consequently, bedetermined from very small amounts of purified or gel-separated glycoproteins. Furthermore, ion mobility drifttime differences provide information on the occurrence of isomers whose structures can then be elucidatedby negative ion fragmentation. Examples include glycans released from glycoproteins such as gp120 andIgG.Keywords: Ion mobility, N-glycans, MALDI, electrospray, isomersPOSTER 35DETERMINING STRUCTURAL CHANGES RESULTING FROM DEAMIDATION OF NATIVE STATEGAS PHASE PROTEINS USING FTICR-MS AND ION MOBILITY METHODS.Andrew J Soulby 1,2 Peter B O’Connor 1 James H Scrivens 21Department of Chemistry, 2 Department of Biological Sciences, University of Warwick, Coventry, UK.The post translational modifi cation of proteins occurs continuously in vivo and can result in changes ofprotein structure that alter functionality with potential health implications. Here, ion mobility, topdown HDX-ECD and infra red absorption dependant unfolding-ECD are used to assess the structural changes of nativestate gas phase Calmodulin and Beta-2-microglobulin following post translational modifi cation. Briefl y,proteins were deamidated (conversion of Asparagine to Aspartic Acid or Iso-Asp via amine group removaland water addition) in NaOH at a pH of 9 overnight before being desalted, purifi ed and resuspended in10mM Ammonium Acetate. The previously mentioned mass spectrometry methods were then carried outusing nanospray alongside non deamidated controls. Deamidation was selected as a modifi cation primarilybecause it occurs frequently with protein aging in vivo and also because it involves the conversion of anuncharged residue (Asn) to a negatively charged one (Asp) making it more likely for the modifi cation to havean impact on local and global structure. Ion mobility is a robust method for this type of study and results fromthe topdown FTICRMS methods will be compared with this data to assess the extra structural informationthey can provide over solely utilising ion mobility.Keywords: Deamidation, FTICR MS, IonMobility, HDX, PTMBMSS © 2012 55

PostersPOSTER 36ISSUES WITH DRIED BLOOD SPOTS AND HEMATOCRIT - POSSIBLE SOLUTIONSDr Philip Denniff*, Mr Adam Hughes, Ms Aida MerchanGlaxoSmithKline Ware UKThe need for micro sampling methods to perform bioanalysis is obvious, as witnessed by the speed withwhich the dried blood spot (DBS) approach has been taken up. Apart from the advantages that go with anymicrosampling method, the DBS technique also has the advantages of sampling simplicity, cost savingscombined with established protocols. However for quantitative use it has the disadvantage of an assay bias,if the samples and standards have a different hematocrit (HCT) content.The assay bias is composed of three components: Assay bias = Amount bias + Recovery bias + Suppressionbias. The amount bias is due to the variable amount of blood present in a punch taken from a DBS withchanging HCT. The recovery bias is due to the variation in analyte recovery that can occur as the HCTchanges. The suppression bias may arise from matrix differences between blood samples. Amount biassolutions: Whole spot elution; Novel dried matrix paper disc device; Correction factor; Sample modifi cation;Substrate development. Recovery bias solutions: Improve analyte recovery; Solvent selection; Substratemodifi cation; Mechanical aids. Suppression bias solution: Additional sample clean-up; Chromatographymodifi cationKeywords: DBS, method validation, haemocritPOSTER 37TRANSFER OF METHODS IN LC AND UHPLC, WHAT CALCULATIONS DO I NEED?Ken Butchart; Mark WoodruffFortis Technologies Ltd, 45 Coalbrookdale Road, Clayhill Industrial Park, Neston, Cheshire, CH64 3UG.UK.Much is made of the ability of UHPLC to speed up analysis of samples, improve high throughput screening,or to develop new methods quickly which can be scaled to/or from traditional HPLC.In order to performmethod transfer there are several ‘method development’ calculators available to help in making appropriatechanges to column dimension, fl ow rate and gradient conditions. If done properly method time will reducebut resolution and selectivity of solutes will not reduce but should remain constant or indeed improve.Inthis poster we discuss the equations involved so that the analyst has a fundamental understanding of thecalculations and can appreciate them in context of the method development taking place. Variables in methoddevelopment should be understood so that an effective method can be produced quickly and effi ciently.We believe that understanding of the fundamental chromatographic equations is necessary in an analyststraining so that chromatographic method integrity is maintained.We show how the changes suggested with the calculations lead to savings in both time and solvent for themethod. We discuss variables that are necessary such as HPLC columns that are the same as their UHPLCcolumn counterpart. We also discuss the use of selectivity of stationary phase’s in UHPLC as opposed tojust reliance on the increased effi ciency of small particles.Keywords: HPLC principles, transferring methods56 BMSS © 2012

PostersPOSTER 38AUTOMATED, HIGH-THROUGHPUT WORKFLOWAUTOMATED, HIGH-THROUGHPUT WORKFLOWFOR THE ANALYSIS OF SERUM 25-HYDROXYVITAMIN D USING THE THERMOFISHER SCIENTIFICVERSETTE AND MULTIPLEXED TURBOFLOW LC-MS/MSLewis Couchman 1 , Caje Moniz 1 Sarah Robinson 21Department of Clinical Biochemistry, King’s College Hospital NHS Foundation trust, London, UK2Thermo Fisher Scientifi c, Hemel Hempstead, UKHypovitaminosis D is now implicated in impaired bone health and a spectrum of other disorders.Increasingly the analysis of serum total 25-hydroxyvitamin D, considered the most reliable and robustmarker of vitamin D status, is performed using LC-MS/MS. Manual sample preparation techniques priorto LC-MS/MS analysis are time-consuming and therefore costly, and assay precision may not always bereproducible between operators. Such techniques are therefore not suited to high-throughput analyses.Samples arriving in the laboratory were portioned into a 1.3 mL deep 96-well plate, along with calibrationstandards and quality control samples (Chromsystems, Munich) regularly interspersed throughout theplate. Aliquots (100 μL) from each well were automatically transferred to a separate deep 96-well plate(ThermoFisher Scientifi c Versette), and the primary plate returned to storage. From a reagent reservoir,internal standard/precipitation reagent (200 μL) was automatically added to each of the sample wells.Plates were sealed with mats, vortex-mixed (10 min) and centrifuged (2,000 g, 10 min). Prepared plateswere returned to the automated system, and 150 μL supernatant from each well transferred to a cleanmicrotitre plate. The plate was sealed and transferred directly to the autosampler tray for analysis.Prepared samples were analysed by TurboFlow LC-MS/MS (ThermoFisher Scientifi c). Under turbulentflow (2.0 mL/min), samples (100 μL) were loaded onto a ThermoFisher Scientific C18 XL HTLCTurboFlow column (50 x 0.5 mm i.d.). Retained analytes were subsequently eluted from the TurboFlowcolumn and focussed onto a ThermoFisher Scientific Hypersil GOLD C18 analytical column (50 x2.1 mm i.d., 1.9 μm average particle size). Tandem MS (MS/MS) analysis was carried out followingpositive mode atmospheric pressure chemical ionisation (APCI). Two SRM transitions were monitoredfor each analyte. For increased throughput, Aria Transcend multiplexing technology was used.Manual sample preparation time was reduced from approximately 2 hrs per batch (typically 100 tubes) tojust 30 mins. Assay precision was improved by (i) better precision when pipetting from the primary plate and(ii) less well-to-well variation compared with mixing individual tubes.Keywords: automation, Multiplexing, 25-OH vitamin DPOSTER 39HOW TO OPTIMISE YOUR UHPLC PERFORMANCE – CONNECT PROPERLY!Ken Butchart; Mark Woodruff.Fortis Technologies Ltd, 45 Coalbrookdale Road, Clayhill Industrial Park, Neston,Cheshire, CH64 3UG. UK.Ultra high-pressure liquid chromatography (UHPLC) is now an established technique allowing faster methoddevelopment and analysis to be achieved in a shorter period of time. As a result an increasing number ofUHPLC instrumentation has entered the marketplace alongside new UHPLC columns and chemistries froma number of companies, not all systems and column hardware are compatible! It is therefore importantwhen evaluating the most suitable column for the analyst’s method/method development, that a number ofconsiderations regarding the system hardware and plumbing are taken in to account.In this poster we discuss the variability between system and column plumbing and the contributing factorsto poor chromatography. We show that by the correct selection of connections between the system andcolumn that the true capability of UHPLC columns can be achieved. We highlight approaches to improvingcolumn lifetime and the implications for the resulting chromatography.Automated, high-throughput workfl owfor the analysis of serum 25-hydroxyvitamin D using the ThermoFisher Scientifi c Versette and multiplexedTurboFlow LC-MS/MSKeywords: UHPLC, Vitamin DBMSS © 2012 57

PostersPOSTER 40A SIMPLE METHOD FOR RESOLUTION OF 22 AMINO ACIDS IN LCKen Butchart; Mark WoodruffFortis Technologies Ltd, 45 Coalbrookdale Road, Clayhill Industrial Park, Neston, Cheshire, CH64 3UG.UK.Amino acids are always of interest due to their availability in so many samples in biochemistry. Theyhave many functions in metabolism critical to life and are seen as the building blocks of proteins. Linearchains can be formed in many sequences to produce a variety of proteins. Key in nutrition and nutritionalsupplements, fertilizers, food technology and industry such as biodegradable plastics, drugs and chiralcatalysts. Many industries therefore rely on accurate results when trying to measure amino acids in theproducts or by-products. Amino acids are relatively simple molecules containing an amine group a carboxylicacid group and a side chain that alters. Due to the variability of the side chain and the potential for one ormore of the groups to be charged amino acids can prove troublesome to retain and resolve from each otherin LC or LC-MS. They can incorporate varying degrees of hydrophobic and hydrophilic nature, whose widerange can make it diffi cult to analyse them all in one run. In this poster we show a simple reversed phaseseparation of all of the amino acids commonly referenced, using a polar-endcapped stationary phase anda buffer:organic gradient. Good resolution of the compounds provides better qualitative results along withhigher sensitivity and quantifi cation levels. We discuss the methods that have been tried before and whythis particular method is simple and yet comprehensive.Keywords: Amino acids, separation, method development58 BMSS © 2012

PostersPoster Session – Taking MS to the extremePOSTER 41IDENTIFICATION OF PENILE TUMOUR TISSUE IN-SITU USING MALDI IMAGING ANDCLASSIFICATION ALGORITHMSBrian Flatley 1, 2 , Chris-Jude Quaye 3 , Elizabeth Johnston 3 , Fawaz H Musa 3 , Peter R Malone 2 and RainerCramer 1 .1Department of Chemistry, University of Reading, Reading, UK2Harold Hopkins Department of Urology, Royal Berkshire NHS Foundation Trust Hospital, Reading, UK3Department of Pathology, Royal Berkshire NHS Foundation Trust Hospital, Reading, UKPenile cancer is one of the rarest cancers with

PostersPOSTER 43CLASSIFICATION AND SEGMENTATION FOR MOLECULAR HISTOLOGY OF PHENOTYPICALREGIONS OF NASH HUMAN LIVER DISEASE TISSUE FROM MALDI MASS SPECTROMETRYIMAGESAndrew D Palmer 1 Alan Race 1 , Patricia Lalor 2 , Josephine Bunch 31PSIBS Doctoral Training Centre, University of Birmingham, Birmingham, UK, B15 2TT2School of Immunity and Infection, University of Birmingham, B15 2TT3School of Chemistry, University of Birmingham, Birmingham, UK, B15 2TTNon-alcoholic steatopeatitis (NASH) is a deadly liver disease. Currently, the only method for diagnosing NASHis histological examination of biopsy samples which show characteristic infl ammation and cirrhosis damage.MALDI Imaging of tissue sections can reveal molecular distributions mirroring distributions of diseased tissueand reveal the spatial extent and characteristic molecules associated with the disease tissue.Specifi c MALDI ion images of NASH disease tissue sections exhibit spatial features corresponding to thedistribution of tissue features, as identifi ed by a histologist. Manually identifying these features is a timeconsuming process so automated methods have been developed to identify and display tissue regions thatexhibit spectral similarity so patterns can be extracted and detect spectral signals that exhibit a distributioncorresponding to that of the disease phenotype.Automatic clustering of pixels provides a rapid and visually striking summary of the data and were suffi cientto discriminate between inflammation and liver tissue. Due to the large size of the datasets PCA was requiredto reduce the data size so clustering could be performed. Scores for principal components that displayedspatial distribution corresponding to disease morphology were observed and the loading for these scoresexamined. It was found that loading which featured strongly could be used to identify particular m/z valueswhich showed disease-linked distribution.Keywords: liver, disease, imaging, MALDI, multivariatePOSTER 44NITRATE SALTS AS USEFUL ADDITIVES IN MALDI-MS AND MS/MS ANALYSIS OF LIPIDSR L Griffi ths, J BunchSchool of Chemistry, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UKLipids are readily detected as [M+H]+, [M+Na]+ and [M+K]+ in positive ion MALDI-MS experiments. This candecrease sensitivity and/or lead to overlapping m/z values of various adducts of different lipids. Additivescan be used to promote formation of a particular adduct, improving sensitivity, reducing spectral complexityand enhancing structural characterization in CID (MS/MS) experiments. We report a systematic evaluationof new and existing additives in MALDI-MS and MS/MS of lipids. Salts of Li+, Na+, K+, Cs+ and NH4+ wereconsidered as acetates, chlorides and nitrates at concentrations between 5-80mM. We demonstrate theimportance of additive cation and anion choice and concentration for tailoring spectral results and presentnitrates as particularly useful additives for lipid analysis.The inclusion of sodium or potassium nitrate to the matrix increased average ion counts of the respectiveadduct to the greatest extent. Comparison of salts in MS/MS experiments indicates the use of nitrate saltsincreases ion counts of detected product ions. This is particularly useful for lipid characterisation. Nitratesalts are shown to exhibit little concentration dependence in MS or MS/MS experiments, which was not thecase with other salt types. Hence nitrate salts may be a viable alternative for a range of applications.Keywords: MALDI, lipid, MS/MS, additives, nitrates60 BMSS © 2012

PostersPOSTER 45AN INVESTIGATION INTO THE PH-DEPENDENT SELECTIVITY OF POROUS GRAPHITIC CARBONFOR PHOSPHOPEPTIDES PRIOR TO THEIR ANALYSIS BY TANDEM MASS SPECTROMETRY.John R Griffiths 1 , Simon Perkins 1 , Yvonne Connolly 1 , Valeria Barattini 2 , Luisa Pereira 2 , Anthony Edge 2 ,Harald Ritchie 2 and Duncan L Smith 1 .1Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road,Manchester. M20 4BX.2Thermo Fisher Scientifi c, Tudor Road, Manor Park, Runcorn, Cheshire, UK.The phosphorylation of proteins by kinases plays a key role in cell signalling and aberrations in these eventsare often implicated in diseases such as the cancers. Mass spectrometry combined with nanofl ow liquidchromatography has become the technique of choice for studying phosphorylation in biological systems.Porous graphitic carbon (PGC), Hypercarb, is a robust stationary phase capable of withstandingextremes of pH, which would cause catastrophic damage to C18 silica-based columns. We have recentlydemonstrated the successful application of PGC in 2D-LC-MS/MS in proteomics, and now extend thatwork to investigate how the unique properties of PGC may be exploited in studying the phosphoproteome.Using a Hypercarb column we investigate the effect of pH on the selectivity of this media for phosphorylatedpeptides. Initial data suggest that loading at high pH (0.1 % ammonia water) on PGC results in signifi cantlyless breakthrough of a population of these peptides compared to loading at low pH (0.1 % TFA) on both PGCand standard C18 columns. In addition, using stable isotope labelled phosphopeptides, we demonstrate thateluant pH has a dramatic effect on phosphopeptide retention on Aspire PGC tips. Finally, we incorporatePGC into a global phosphoproteomic workfl ow applied to cancer studies.Keywords: Porous graphitic carbon; phosphorylation; pH-selectivity, Hypercarb, proteomics.POSTER 46MEASUREMENT OF THE NATRIURETIC HORMONE UROGUANYLIN USING UPLC-IMS/MSE TOCHARACTERISE INDIVIDUAL TOPOISOMERS INVOLVED IN CARDIOVASCULAR DISEASECharlotte Daly 1,2 , Leong L. Ng 2 , Richard Willingale 3 ; Kevin Giles 4 and Donald J. L. Jones 11Department of Cancer Studies and Molecular Medicine,2Department of Cardiovascular Sciences and3Department of Physics and Astronomy, University of Leicester; 4 Waters Corporation,Atlas Park, ManchesterThe peptide uroguanylin is secreted by the gut, in response to salt or food intake, and acts in theintestine and kidney to regulate sodium balance as part of the wider family of natriuretic hormones. Inaddition, it may regulate appetite. Dysregulation of this system has been linked to heart failure, colorectalcancer and obesity. Uroguanylin is of particular interest due to its existence as two interconvertingtopological isomers which may have distinct pharmacology, e.g. on guanylate cyclase stimulation.This work shows the resolution and relative quantitation of the two topological isomers of uroguanylin, withUltra Performance Liquid Chromatography - Travelling Wave Ion Mobility – Data Independent Tandem MassSpectrometry, (UPLC-TWIMS-MSE). The isomers were identifi ed with collision cross section calculationsfrom experimental data and comparison to corresponding values from molecular dynamics simulations. Alsopresented is a validated quantitative method for the detection of the peptide on a Waters Synapt G2 HDMSinstrument as well as preliminary data showing the peptide in biological samples. Additionally, this analysiscan be used to help elucidate the signalling pathway of the hormone which is not fully understood, and tounderstand the pathophysiology of this system in different disease states.Keywords: Ion mobility, TWIMS, protein, biomarkers, cardiovascular, clinicalBMSS © 2012 61

PostersPOSTER 47HIGH-RESOLUTION TIME-OF-FLIGHT MASS SPECTROMETRY USING FOLDED FLIGHT PATHTECHNOLOGY FOR PROFILING OF STEROIDS AND STEROID METABOLITES IN URINEJürgen Wendt 1 , Kevin Siek 2 , Jeffrey S. Patrick 2 , Erica Guice 31LECO Instrumente GmbH, Germany; 2 LECO Corporation, St.Joseph, Michigan (USA); 3 Western SlopeLaboratory.Analysis of endogenous steroid levels has many important applications. These assays are usually performedon plasma samples in a targeted manner. High-resolution time-of-fl ight mass spectrometry (TOFMS) allowssensitive measurement of targeted steroid metabolites while simultaneously capturing a global profi le ofother analytes present in the matrix. This technology allows qualitatively and quantitatively useful data tobe collected not just on the same instrument, but at the same time. The different qualitative and quantitativefunctions are accomplished post hoc with data analysis software, not by performing serial experiments orby compromising duty cycle in a single run.Endogenous steroid metabolites in urine samples were analyzed by liquid-chromatography / high-resolutiontime-of-fl ight mass spectrometry (LC-HRT). Collision-induced dissociation (CID) spectra of known analytesgenerated by LC-HRT were consistent with spectra previously reported from triple quadrupole and otherMS-MS instruments and provided favorable database search results. The accurate mass measurement andaccurate isotope ratio measurements included in the precursor and product ion spectra from the LC-HRTinstrument provided a level of confidence in analyte assignment superior to triple quadrupole instrument data.All this information was obtained from a single chromatographic analysis, demonstrating several advantagesof high resolution time-of-fl ight mass spectrometry.Keywords: steroid, metabolites, urine, Endogenous, time-of-fl ightPOSTER 48AN INVESTIGATION INTO THE CONCENTRATION OF COENZYME Q10 (UBIQUINOL ANDUBIQUINONE) IN OVER THE COUNTER HEALTH SUPPLEMENTS USING HIGH RESOLUTIONLIQUID CHROMATOGRAPHY MASS SPECTROMETRY.Edward Goucher, Katerina Klagkou.Unity Lab Services (Part of Thermo Fisher Scientifi c), Stafford House, Boundary Way, Hemel Hempstead,HP2 7GECoenzyme Q10 (CoQ10) is an endogenous enzyme cofactor that enables aerobic cellular respiratory functionsin the electron transport chain. It has a number of antioxidant properties and it is thought to benefi t a varietyof disease states. Although the compound is often recommended by doctors in specifi ed amounts, CoQ10is classed as a herbal remedy and there is no control over the content of the various types of formulationsavailable. Neither the presence of the active component nor its concentration are being established at anypoint. In addition, the wide variation in the cost of purchase can create doubts as to whether the amount ofactive ingredient, as indicated on the packaging, is truly present in any formulation. The aim of this researchwas therefore to investigate the content of CoQ10 in two popular over the counter health supplements andto develop a method for quick detection and quantitation using LC-MS.For the detection and quantifi cationof CoQ10, a high resolution accurate mass method was successfully developed on an Exactive TM massspectrometer. Chromatographic separation and retention was achieved using a C18 column. Samplesconsisting of tablets and gel capsules were accurately weighed and recorded and the CoQ10 was extractedusing a mixture of acetonitrile, tetrahydrofuran and water. For quantifi cation purposes, external calibrantsof CoQ10 were prepared and the [M+NH 4] + adduct was targeted. Results confi rm that CoQ10 was presentin both types of supplements and at concentrations relative to the amounts indicated on the packaging.A number of impurities were also detected, although accurate mass profi ling alone was not suffi cient forpurposes of identifi cation. Further investigations incorporating MS/MS, as featured on the Q Exactive TM ,would be required.62 BMSS © 2012

PostersPOSTER 49THE SEPARATION AND QUANTIFICATION OF DIGLYCERIDES IN BIODIESEL BY HPLC FOLLOWEDBY SEQUENTIAL QUANTIFICATION AND IDENTIFICATION BY GC COUPLED WITH MASSSPECTROMETRIC AND FLAME IONISATION DETECTION.D.Lorenzi and C.Hopley .Chemical Measurement and Calibration, LGC, Teddington, United KingdomThe continuing quest for renewable energy sources has seen a marked increase in the demand and productionof biodiesel. The search for alternative feedstock and the demand for the overall improvement of the qualityof the biofuel product have provided a number of analytical challenges.Biodiesels are a complex mixture of long chain fatty acid methyl esters (FAMES). However, the presenceof unsaturated FAMES and residues of the starting glycerides are known to be detrimental to their use asfuels. This has resulted in the development of industrial accepted methods, ASTM D6751 and EN 14214,which are mostly based around the use of GC-FID, for the analysis of these materials. However, referencematerials with known quantities of glycerides are still required for the validation of such methods in the fi eld.The purpose of this study was to develop methods which are suitable for the production of referencematerials. Therefore, the focus has been on the separation, identifi cation and quantifi cation of individualdiglycerides. The approach adopted consisted of fraction collecting the diglyceride portion from a rapeseedbasedbiofuel sample, after separation using high performance liquid chromatography (HPLC), followed bysimultaneous GC-FID-MS analysis. A number of HPLC methods were investigated. Reversed phase andnormal phase chromatography were used as the HPLC phase, and satisfactory isolation was obtained usingthe latter. The GC method involved the use of a wide bore column, capable of reaching high temperatures,and typically used for the analysis of the various components contained in biodiesel. In order to allow massspectra identification, the flow was split between the FID and the MS, using a splitter based on micro channeltechnology. In order to obtain a balance between GC and MS output, careful considerations were made forthe column dimensions and the fl ow. Therefore, satisfactory chromatographic resolution was obtained toenable quantifi cation by FID and identifi cation by MS.POSTER 50MULTIPLATFORM METABOLIC FINGERPRINTING OF LEISHMANIA DONOVANI RESPONSE TOMILTEFOSINEGisele A. B. Canuto 1,2 , Emerson A. Castilho-Martins 1 , Luís Rivas 3 , Antonia García 1 , Marina F. M. Tavares 2 ,Ángeles López-Gonzálvez 1 and Coral Barbas 1 . 1 CEMBIO, PharmacyFaculty, Campus Monteprincipe,Universidad CEU-San Pablo, 28668 Boadilla del Monte. Madrid, Spain. 2 Institute of Chemistry, Universityof São Paulo, São Paulo, SP, Brazil. 3 Centro de Investigaciones Biológicas (CSIC); Madrid, Spain.Metabolomics can be used to detect changes in a biological system, being separation techniques (GC,LC and CE) coupled to mass-spectrometry the most sensitive tools for these studies. Leishmaniasis, aparasitic disease affecting 12 million people, requires new drugs. Miltefosine is a candidate; however themechanism of action and potential resistance should be studied. Metabolomic approaches are being appliedin Leishmania’s studies with this aim. Different analytical platforms have been used: CE-MS, LC-MS/MSand GC-MS, to study miltefosine’s action on Leishmania donovani wild-type (controls), treated and resistantto the drug. The analytical methods used showed to be complementary with compounds with differentphysico-chemical properties but belonging to the same metabolic pathway identifi ed with different analyticaltechniques, i.e. different amino acids are better identified by CE-MS or CG-MS, while; long chain organic acidsare better identifi ed by reversed phase LC-MS. The effect of the treatment and resistance on parasites wasevaluated through proper multivariate data analysis, allowing to the identifi cation in Leishmania’s databasesof 17 analytes by GC-MS, 48 analytes by CE-MS and 55 analytes by LC-MS. The results of this analysiscontributed to the identifi cation of parasite’s metabolic pathways involved in the mechanism of drug actionand parasite resistance.Keywords: mass-spectrometry, Leishmania donovani, miltefosine, separations techniques, metabolomics.BMSS © 2012 63

PostersPoster Session – Pioneering MS great and smallPOSTER 51ABSORPTION-MODE FOURIER TRANSFORM MASS SPECTROMETRY: ADVANTAGES FORPROTEIN AND PETROLEUM SPECTRAYulin Qi 1 , Mark P. Barrow 1 , Joseph E. Meier 2 , Steve L. VanOrden 2 , Christopher J. Thompson 2 , Peter B.O’Connor 11Department of Chemistry, University of Warwick, Coventry, CV4 7AL, UK2Bruker Daltonics, 40 Manning Road, Billerica Massachusetts 01821, USThe Fourier transform ion cyclotron resonance (FT-ICR) mass spectrum is conventionally presented in themagnitude-mode. However, it is well known that the absorption-mode display gives a much narrower peakshape which greatly improves the spectrum. Our recent papers show that the successful solution of thephase equation allowed broadband phase correction which makes it possible to apply the absorption-moderoutinely in FT-ICR. The new spectra yield a 50-100% improvement in resolution, a 20-100% improvement inmass accuracy, and a 40% improvement in signal/noise ratio at No Cost in instrumentation! Here, we applythe method to both top-down protein and crude oil spectra, to demonstrate the advantages of the absorptionmodein the ultra-complex spectra. The result is substantially more advantageous than we had previouslyrealized. With all improvements above obtained simultaneously, the effect is similar in improvement ratio toincreasing the magnetic fi eld strength of our instrument from 12 Tesla to 17-24 Tesla.Keywords: FTICR, absorption-mode, applicationPOSTER 52STRUCTURAL CHARACTERISATION OF OLIGOMERIC INTERMEDIATES ON PATHWAY TOAMYLOID FORMATIONAneika C Leney, Charlotte C Scarff, Sheena E Radford, Alison E AshcroftAstbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, UKThe formation of insoluble amyloid fi brils in vivo is associated with over thirty different pathologicalconditions. Despite extensive studies, little is known about the mechanism by which these proteinsassemble into highly ordered, cross-beta structures. Here, along with solution studies, we use IMS-MS and CID-MS to characterise the on-pathway oligomers formed during amyloid fibril assembly.Beta-2 microglobulin is a 99-residue protein, whose aggregation is associated with the disease dialysisrelatedamyloidosis. Incubation of beta-2 microglobulin under physiological conditions in vitro does notlead to fi bril formation on a measurable timescale. Fibril formation can be initiated however, under acidicconditions yielding amyloid fi brils of similar morphology to those detected in vivo. Here we used theamyloid-binding fl uorescent dye, Thiofl avin T, to monitor the fi brillation kinetics of specifi c point mutationsin beta-2 microglobulin. ESI-IMS-MS was used to measure the mass, collision cross-sections, gasphase stability and structural dynamics of the oligomeric intermediates on-pathway to fi bril formation.Detailed characterisation reveals subtle differences in the subunit exchange dynamics of the oligomericspecies involved in fi bril formation, even though their gas phase stabilities, cross sections, and eventual fi brilmorphology remain comparable. Altogether, the comparison of the monomeric and oligomeric states of thesemutants along with their fi brillation kinetics reveal specifi c residues important in amyloidosis, highlighting thecomplexity of this fi bril-forming process.Keywords: ESI-IMS-MS, Amyloid, non-covalent protein complexes64 BMSS © 2012

PostersPOSTER 53INVESTIGATING SOLVENT-ASSISTED INLET IONISATION (SAII)Celia Smith, Laurence Bindschedler and Rainer CramerDepartment of Chemistry, University of Reading, Reading RG6 6AS, UKSeveral recent papers from Pagnotti et al. have described the use of solvent-assisted inlet ionisation(SAII) as an alternative to nanoelectrospray ionisation (nESI). This new technique, in which the sampleis introduced directly into the inlet tube without the application of spray voltage or gas fl ow, is reported toyield high sensitivity, and has the added advantage of not needing the ultra-low sample fl ow rate which isa prerequisite for nESI.The work presented here covers our independent examination of this new technique, the optimisation ofresponse and direct comparison of SAII and nESI using the same nanoLC and mass spectrometer. Thesuitability of this technique is investigated using individual peptides and complex digests of both proteinstandards and real, plant-derived samples, and the sensitivity achieved is reported.Keywords: solvent-assisted inlet ionisation (SAII), nano-electrospray ionisation (nESI), peptide, proteindigest, plant-derived samplePOSTER 54THE ROLE OF MS IN THE PHYSIOCHEMICAL CHARACTERISATION OF BIOSIMILARSAshleigh Wake, Betty Flitroft, Helen BrittanIntertek ASG, Hexagon Tower, Blackley, Manchester, M9 8ZSThe explosion of biopharmaceutical products under development/registration combined with ever increasingregulatory demands for characterisation thereof has presented huge analytical challenges over recent years.Mass Spectrometry plays a pivotal role in this characterisation.The aim of this poster is to discuss the application of mass spectrometry to the different analytical challengespresented by molecules of biological origin from the simplest determination of molecular weight to analyticsdesigned to assess post translational modifi cations such as glycosylation. Consideration will be given todiscussion of some of the expected challenges and the consequence of lack of clear guidance from theregulators.Keywords: Biologics Characterisation and Mass Spectrometry,.BMSS © 2012 65

PostersPOSTER 55ENRICHMENT OF PHOSPHOPEPTIDES/-PROTEINS BY LANTHANUM CHLORIDE PRECIPITATIONChhaya B Patole 1 , Laurence V Bindschedler 1 , Jim Dunwell 2 and Rainer Cramer 11Department of Chemistry, 2 School of Biological Sciences, University of Reading, Reading, RG6 6AS, UKProtein phosphorylation is a ubiquitous post-translational modifi cation that regulates almost all cellularprocesses. The low abundance/stoichiometry together with low MS-detectability of the modifi ed proteinsmake MS-based phosphoproteome research a challenging task. Various analytical methods have beenproposed to enrich phosphoproteins from complex samples e. g with calcium, barium salts, and morerecently with lanthanum salts (Pink M, et al., 2011). Lanthanum belongs to a group of metals that occur intrivalent oxidation state and are known to be strong phosphate binders. Here, we tested the application andreproducibility of lanthanum chloride in precipitating the phosphopeptides/-proteins from standard proteinmixture and complex biological sample, then compared this with other enrichment techniques. Proteins wererun on the SDS-PAGE gel to confirm the enrichment of phosphoproteins and subsequent protein identificationusing an LTQ orbitrap XL. This poster will present our results about the reproducibility of lanthanum chlorideprecipitation in comparison to other techniques in hand.Keywords: Protein/peptides, Phosphorylation, Lanthanum chloride, EnrichmentPOSTER 56ILLUSTRATE THE BREAKDOWN OF CHLOROPHYLL A BY TANDEM MASS SPECTROMETRYJuan Wei and Peter O’ConnorDepartment of Chemistry, University of Warwick, Coventry, UKThe breakdown of chlorophyll a is an enigma for a long time. As its irreplaceable role mainly depends on theasymmetric and conjugated structure, electron-based MS/MS can probably fragment it in a well-regulatedway, which may help us to understand the degradation mechanism of chlorophylls. Singly charged chlorophylla is fragmented by electron induced dissociation (EID) using fourier transform ion cyclotron resonance(FTICR) mass spectrometer. Fragments induced by EID are compared with those leaded by collisionallyactivated dissociation (CAD) and infrared multiphoton dissociation (IRMPD). Accordingly, the breakdown ruleof chlorophyll by MS/MS is illustrated, which can also provide some clues of how chlorophyll a degradationworks in vivo.Keywords: Electron induced dissociation, chlorophyll a, FTICR, tandem mass spectrometry66 BMSS © 2012

PostersPOSTER 57N-GLYCOSYLATED GLYCOPROTEINS CHARACTERIZED BY MASS SPECTROMETRY – ANINTEGRATED SOFTWARE APPROACHRod Watson 1 , Ulrike Schweiger-Hufnagel 2 , Arndt Asperger 2 , Anja Resemann 2 , Detlev Suckau 21Bruker UK Ltd, Banner Lane, Coventry, CV4 9GH2Bruker Daltonik GmbH, Fahrenheitstraße 4, 28359 Bremen, GermanyIn biochemistry, glycosylation is of increasing interest. Protein glycosylation, which is a key post-translationalmodification, is important e.g. for protein folding and for cell to cell adhesion. To study the biochemical function,a detailed structural characterization is required, and mass spectrometry is a highly suitable technique for thispurpose. However, the analysis of the N-glycosylation pattern present on glycoproteins is challenging dueto the heterogeneous glycan structures and ion suppression effects. For the interpretation of glycopeptideMS/MS spectra via a database search, the knowledge of the peptide and glycan mass is required. This canbe automatically handled in the presented software approach.To characterize the N-glycosylation patterns, a bottom-up approach was chosen. Tryptically digestedglycoprotein samples were separated by nano LC and analyzed by ESI and MALDI mass spectrometry. AllMS/MS spectra, which have been recognized as originating from glycopeptides, were applied to protein andglycan database searches in SwissProt and GlycomeDB, respectively.Keywords: Glycosylation, MALDI, ESI, glycopeptide, MSMSPOSTER 58PERFORMANCE OF A QMS OPERATING IN STABILITY ZONES 1 AND 3 UNDER THE INFLUENCEOF MAGNETIC FIELDSarfaraz U. A. H. Syed, S. Maher, J.R. Gibson and S. Taylor.Department of Electrical Engineering and Electronics, Brownlow Hill, University of Liverpool,Liverpool. L69 3GJThe work is mainly concerned with the performance of a quadrupole mass spectrometer (QMS) under theinfl uence of a static magnetic fi eld for operation in stability zones 1 and 3. Signifi cant improvement in QMSperformance was obtained under certain magnetic fi eld conditions, and these have been explained in termsof our theoretical model developed in the University of Liverpool. The theoretical approach assumed in themodel is that the QMS contains hyperbolic rods as electrodes and that the magnetic fi eld acts over the fulllength of the mass fi lter assembly. This model is capable of accurate simulation of spectra allowing the userto specify different values of mass spectrometer dimensions and applied input signals. Our latest analysisalso predicts for what values of operating parameters an enhancement of the QMS resolution is achievedwhen a magnetic fi eld is applied. The model predicts ultra high resolutions for identifi cation of low massisotopes; a resolution R > 3500 is obtained for a HT and D2 mixture with a 200 mm long mass fi lter operatingin stability zone 3 via application of a transverse magnetic fi eld.Keywords: Quadrupole Mass Filter (QMF); magnetic fi eld; Lorentz force; resolution.BMSS © 2012 67

PostersPOSTER 59THE EFFECT OF QUADRUPOLE NON-LINEAR RESONANCES ON ION TRAJECTORIESVISUALISED IN PHASE-SPACEDavid P A Kilgour 1 , Peter B O’Connor 1 and John F J Todd 21Department of Chemistry, University of Warwick, Coventry, CV4 7AL, UK2School of Physical Sciences, University of Kent, Canterbury, Kent CT2 7NH, UKPhase-space methods have long been successfully used to investigate the theoretical performance ofquadrupole mass fi lters, and related devices. Here we present further investigations into the effect thatnon-linear resonances can have on modelled ion trajectories, visualised in phase-space and show that socalledphase-space ellipses should more accurately be parametrically described as Lissajous curves of twoFourier series. These effects become more pronounced for quadrupole designs which deviate from idealfields; this is a key feature of the majority of real quadrupoles. When such a non-ideal quadrupole is operatedaway from resonant conditions, only the fundamental term of the Fourier series are signifi cant and the iontrajectories will appear elliptical in phase space. However, when the quadrupole is operated at, or near,relevant non-linear resonances then the higher order terms in the Fourier series cease to be insignifi cantand can have a pronounced effect on the resultant phase-space “ellipses”.Keywords: quadrupole, resonance, phase space, modellingPOSTER 60METABOLOMIC LIPID PROFILING BY LC-MS; IDENTIFYING CHANGES IN ETHANOL METABOLISMAND ENDOGENOUS METABOLITES FOLLOWING THE ADMINISTRATION OF ALCOHOL AS ALIQUID DIET TO MICEAlan Barnes 1 , Simon Ashton 1 , Neil J Loftus 1 , Ian D Wilson 2 , Filippos Michopoulos 2 and Ji Cheng 31Shimadzu MS/BU, Manchester, UK;2Astra Zeneca, Alderley Park, Cheshire, UK;3University of Southern California, Los Angeles, CAThe harmful effects of alcohol in humans has been well documented, however, through using differentanimal models a greater understanding of metabolic stress responses to alcohol can be monitored.Alcohol can be delivered via intragastric cannulas or through liquid diets. In the intragastric feedingmodel, animals can be given alcohol for several months and high blood alcohol levels can be maintained.However, in this study a four week ethanol enriched liquid diet model was evaluated as an alternativevehicle. To assess the impact of ethanol exposure, a systems based approach was taken measuringurine, plasma and liver tissue to characterise both ethanol metabolites and endogenous lipid changes.Samples were analysed by Nexera UHPLC coupled to LCMS-IT-TOF (Shimadzu Corporation, Japan) toacquire high mass accuracy MSn data.In this study ethylated lipid marker compounds were detected, consistent to work published from intragastricfeeding models. Interestingly the profound lipid differences observed from the intragastric feeding modelwere markedly reduced in the current liquid diet study with the emergence of ethylated lipid markers at week4 but not at week 2. These included ethyl arachidonate, ethyl linolate and ethyl DHA. Retinol palmitate andretinol were also signifi cantly reduced in ethanol treated animals.Keywords: LC-MSn, metabolomics, profi ling, ethanol, metabonomics68 BMSS © 2012

PostersPOSTER 61SINGLE PASS PRINCIPAL COMPONENT ANALYSIS ALGORITHM FOR USE ON LARGE MASSSPECTROMETRY IMAGING DATA SETSAlan Race 1,2 , Andrew D Palmer 1 , Rory T Steven 1 , Joscelyn Sarsby 1 , Rian L Griffi ths 1 , Iain Styles 2 , JosephineBunch 1 .1School of Chemistry, University of Birmingham, Birmingham B15 2TT, UK2School of Computer Science, University of Birmingham, Birmingham B15 2TT, UKMass spectrometry imaging produces data sets which are typically too large to load into RAM. The mostcommon approach to reducing the data size is to bin the data, however this causes a loss of information.Principal component analysis (PCA) is performed to reduce the dimensionality of mass spectrometry datawhile maintaining the variance as a pre-cursor to clustering algorithms. PCA is performed on mean centreddata, requiring at least two passes over the data, which can be a considerable time cost if the data is notstored in memory. We present a single pass PCA algorithm applied to mass spectrometry imaging datawithout requiring binning.Data from MALDI (matrix-assisted laser desorption/ionization) imaging and LESA (liquid extraction surfaceanalysis) experiments were converted to an open mass spectrometry (mzML) using msconvert (ProteoWizard).The mzML data were then converted to the open mass spectrometry imaging format (imzML) developedby the COMPUTIS project using custom in-house software. MATLAB was used for implementation of thesingle pass algorithm for use on the imzML data.Keywords: MSI PCA single pass largePOSTER 62MALDI MS IMAGING OF ON-TISSUE DIGESTED PROTEINS AIDED BY HIGH EFFICIENCY IONMOBILITY SEPARATION.Ian Edwards, Emmanuelle Claude and James Langridge.Waters Corporation, Manchester, M22 5PP, UKMALDI imaging mass spectrometry (MSI) (Caprioli et al, 1997) enables label free multiplexed molecularanalysis (i.e. lipids, sugars & proteins) in one MS experiment. Protein analysis is challenging, often reportedwithout annotation. Direct on-tissue enzymatic digestion is one strategy for protein identification; as the lowerm/z of peptides makes them more manageable for MS/MS.Post ionization separation by high efficiency ion mobility separation (IMS) coupled to time-offlightmass spectrometry is a means of selectively decongesting peptide complexity by mobility,without sacrifi cing analysis of compounds such as lipids, or isobaric species with differing mobility.Tryptic digestion and CHCA matrix application was performed on rat brain sections by spray depositionusing the SunCollect (SunChrom, Germany). Data was acquired on a MALDI SYNAPT G2 HDMS system(Waters Corporation, Manchester, UK) operated in HDMS mode (m/z 700 - 3000) with a laser repetition rateof 1000 Hz. Data was processed and visualised using High Defi nition Imaging (HDI) software (Waters) withfull ion mobility data integration. Identifi cation of peptides by MS/MS was performed directly on adjacenttissue sections.IMS is shown to provide a dimension of post ionisation separation advantageous to MALDI imagingexperiments. Ion images from peptides and lipids separated by ion mobility will be presented.Keywords: MALDI imaging mass spectrometryBMSS © 2012 69

PostersPOSTER 63PROXIMITY EFFECTS IN THE FRAGMENTATION OF IONISEDDIHALOGENOSTYRYLBENZOXAZOLESStephen Ayrton 1 , Jetakerina Panova 1 , Richard D Bowen 1 , Adam R Michalik 1 and Richard T Gallagher 21Chemical and Forensic Sciences, School of Life Sciences, University of Bradford BD7 1DP, England;2AstraZeneca CVGI DMPK, Alderley Park, Macclesfi eld, Cheshire SK10 4TG, EnglandBenzoxazoles are an important class of biologically active heterocycles. Elimination of a hydrogen orhalogen atom from the ortho position of ionised halogenosubstituted 2-styrylbenzoxaoles [XC6H3(NOC)CH=CHC6H4Y; X, Y = H, F, Cl or Br] may occur by a proximity effect, in which ring closure by attachment ofthe nitrogen atom to the pendant [C6H4Y] ring is followed by elimination of Y. to form an exceptionally stabletetracyclic aromatic cation. The competition between direct elimination of a halogen atom, X., by simplecleavage of the C-Halogen bond in the benxoxazole ring and loss of a halogen atom, Y., from the pendantring via the proximity effect has been studied by varying the position and nature of X and Y. Complementaryinformation has been acquired by considering the competition between loss of a different halogen atomfrom various positions in the pendant ring of dihalogenostyrylbenzozazoles [C6H4(NOC)CH=CHC6H3XY] inwhich both halogen atoms are in the pendant ring. These studies refi ne the analytical value of the proximityeffect in determining the position of the halogen atom in the pendant ring.Keywords: Benzoxazoles, Proximity Effect, Rearrangement, EliminationPOSTER 64USING ELECTRON INDUCED DISSOCIATION TO CHARACTERISE POLYKETIDES ANDPOLYKETIDE SYNTHASE INTERMEDIATESRebecca H Wills, Manuela Tosin and Peter B O’ConnorDepartment of Chemistry, University of Warwick, Coventry, CV4 7AL, UKPolyketides are an important class of natural products, particularly in the drug discovery process, as theyare one of the main sources of medicinal compounds. The identifi cation of their biosynthetic pathwaysand the characterisation of their structures is of interest to chemists and biochemists who wish to preparethem on a large scale and investigate their bioactivity. 1 Electron Induced Dissociation (EID) is an MS/MStechnique used on Fourier Transform Ion Cyclotron Resonance (FTICR) mass spectrometers and involvesthe fragmentation of species using electrons with typical energies of between 10 and 20 eV. Its potentialfor unveiling structural information has previously been demonstrated on different classes of biomoleculesincluding peptides 2 and fatty acids. 3 Herein, EID has been used in tandem with Collision Induced Dissociation(CID) in order to characterise the structures of known polyketides, namely erythromycin A, lasalocid A andiso-lasalocid A, as well as of putative trapped intermediates in their biosynthesis. 4,5 In a comparison withCID, EID caused fragmentation through multiple pathways, providing complementary structural informationpreviously inaccessible through the use of CID alone. The use of EID in tandem with CID has thereforeproved to be a valuable tool for determining detailed structural information on polyketides, and can assist inthe characterisation of intermediate structures, helping to identify their biosynthetic pathways.1. Weissman, K. J., Introduction to polyketide biosynthesis. In Complex enzymes in microbial naturalproduct biosynthesis, part b: Polyketides, aminocoumarins and carbohydrates, Hopwood, D. A., Ed. 2009;Vol. 459, pp 3-16. 2 . Lioe, H.; O’Hair, R. A. J., Anal. Bioanal. Chem. 2007, 389 (5), 1429-1437. 3 . Yoo, H. J.;Hakansson, K., Anal. Chem. 2010, 82 (16), 6940-6946. 4 . Tosin, M.; Demydchuk, Y.; Parascandolo, J. S.;Per, C. B.; Leeper, F. J.; Leadlay, P. F., Chem. Commun. 2011, 47 (12), 3460-3462. 5 . Tosin, M.; Smith, L.;Leadlay, P. F., Angew Chem Int Edit 2011, 50 (50), 11930-11933.Keywords: Polyketides, EID70 BMSS © 2012

PostersPOSTER 65TOWARDS TOP-DOWN IDENTIFICATION, DETERMINATION OF SEQUENCE TERMINI OF INTACTPROTEINS IN MIXTURES OF MODERATE COMPLEXITYLaura Main 1 , Anja Resemann 2 , Eckhardt Belau 2 and Detlev Suckau 21Bruker UK Ltd, Banner Lane, Coventry, United Kingdom2Bruker Daltonik GmbH, Fahrenheitstraße 4, 28359 Bremen, GermanyTop-Down mass spectrometric sequence analysis of undigested proteins by MALDI in-source decay (ISD)can provide for identifi cation and for terminal analysis in one step. Information about the PTM status and ofisoforms/truncation variants can be obtained from Top-Down data. Traditionally, MALDI-ISD was applied toside product characterization of recombinant proteins. We applied Protein-LC-MALDI-Top-Down Sequencing(LC-MALDI-TDS) to the characterization of moderately complex protein mixtures of different origin.We used proteins such as composed mixtures of several model proteins or commercial proteins such ascarbonic anhydrase containing several contaminating proteins, to identify many of them and to assign thestatus of their protein termini including terminal modifi cations. The protein mixtures were well separatedin a single Top-Down LC separation on a monolithic column (Dionex Pepswift) and TDS spectra acquired.In mixtures of 10 proteins, 9 of them were identifi ed and their terminal modifi cation status was confi rmed,e.g. N-terminal acetylation or pyroglutamylation. Typically, sequence information was obtained in the rangeof 10-70 N- as well as C-terminal residues and identifi cation was achieved with a dedicated Mascot search.For unusual PTMs, the Mascot strategy was complemented by de novo sequencing and MS-BLASThomology searching. All standard search methods were also available to the Top-Down protein identification.The new approach overcomes the limitation of MALDI-ISD that prevents precursor ion selection and providesprotein separation chromatographically and represents the most likely candidate for a truly Top-Downproteomics scenario.Keywords: MALDI, LC-MALDI-TDS, Top-down, protein identifi cation,POSTER 66MASS SPECTRA OF TRICYCLIC INDOLES, KETONINDOLES AND INDOLENINESAmie Saidykhan 1 , William Martin 1 , Richard D Bowen 1 , Richard T Gallagher 21Chemical and Forensic Sciences, School of Applied Sciences, University of Bradford,Bradford BD7 1DP, England;2AstraZeneca CVGI DMPK, Alderley Park, Macclesfi eld, Cheshire SK10 4TG, UKThe value of mass spectrometry as a means of detecting and identifying indoles, indolenines and ketoindolesthat could serve as biomarkers of extinct or extant life in extreme environments has been probed byinvestigating selected deuterium labelled and methyl substituted analogues of tricyclic heterocycles, includingexamples with 6,5,5-, 6,5,6-, 6,5,7- and 6,5,8- fused-ring systems. Electron impact and electrospray ionisationmethods are shown to provide complementary information on the fragmentation of molecular ions [M+.]and protonated molecules [MH+], respectively, thus enhancing the analytical value of mass spectrometryin this context.Keywords: Indoles, Indolenines, Ketoindoles, Deuterium Labelling, Fragmentation PatternsBMSS © 2012 71

PostersPOSTER 67IDENTIFICATION OF CRIP1 AS A NOVEL PROTEIN BIOMARKER FOR HER2-POSITIVE BREASTCANCERSandra Rauser 1 , Claudio Marquardt 1 , Benjamin Balluff 1, 2 , Detlev Suckau 3 , Katja Specht 4 , Matthias P. Ebert 2 ,Manfred Schmitt 5 , Michaela Aubele 1 , Heinz Höfl er 1,4 , Axel Walch 1 and Julia Smith 61Institute of Pathology, Helmholtz Zentrum München - German Research Center forEnvironmental Health, Neuherberg, Germany2Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, Munich, Germany3Bruker Daltonik GmbH, Bremen, Germany4Institute of Pathology, Technische Universität München, Munich, Germany5Department of Obstetrics and Gynecology, Klinikum rechts der Isar, Technische UniversitätMünchen, Munich, Germany, 6 Bruker UK Ltd, Banner Lane, Coventry, United KingdomClinical laboratory testing for HER2 status in newly diagnosed, primary breast cancer tissues is criticallyimportant for therapeutic decision making. Matrix-assisted laser desorption/ionization (MALDI) imaging massspectrometry (IMS) is a powerful tool for investigating proteins through the direct and morphology-drivenanalysis of tissue sections. Unlike immunohistochemistry (IHC), MALDI-IMS enables the acquisition ofcomplex protein expression profi les without any labeling. We hypothesized that MALDI-IMS may determineHER2 status directly from breast cancer tissues.We found that specific protein/peptide expression changes strongly correlated with the HER2 over expression(m/z 4740, 8404, 8419, 8455, 8570, 8607, 8626). Among these, we identifi ed m/z 8404 as Cysteine-richintestinal protein 1 (CRIP1). Of particular note, the proteomic signature was able to accurately defi ne HER2-positive from HER2-negative tissues achieving high values for sensitivity of 83%, for specifi city of 92% andan overall accuracy of 89% (95% CI: 65% to 99%).Our results underscore the potential of MALDI-IMS proteomic algorithms for morphology-driven tissuediagnostics such as HER2 testing and show that MALDI-IMS can reveal biologically signifi cant moleculardetails from tissues which are not limited to traditional high-abundance proteins. CRIP1 is a cytosolic proteinthat is potentially useful for serum based diagnostics of HER2 if tissue leakage can be demonstrated.Keyword: Biomarker, MALDI,Ion trap, Top-down, Clinical.POSTER 68POST-TRANSLATIONAL MODIFICATIONS TO TRANSLATION INITIATION FACTORS AS ARESPONSE TO STRESSJemma Keenan, Paul F. G. Sims, Mark P. AsheThe Faculty of Life Sciences, The University of Manchester.Fusel alcohols signal nitrogen scarcity to elicit a range of responses in the yeast Saccharomyces cerevisiae.These alcohols activate pseudohyphal growth and cause rapid inhibition of translation initiation. The precisemechanism through which this inhibition occurs is unknown, however translation initiation factors eIF2B andeIF5A have been implicated. A number of post-translational modifi cation sites within eIF2B and eIF5A havebeen previously identifi ed. This study aims to characterise the effects of fusel alcohols on these factors andidentify modifi cations, which may be responsible for protein synthesis inhibition.eIF2B and eIF5A have been purifi ed and successfully identifi ed using mass spectrometry. Biochemicaltechniques have been used to investigate these factors and mass spectrometric analysis has been conductedto identify modifi ed peptide residues and characterise the nature of any post-translational modifi cations.Data obtained indicates that these factors are extensively modifi ed and the goal now is to establish whetherfusel alcohol treatment impacts upon this.Keywords: Yeast, initiation factors, modifi ed peptides72 BMSS © 2012