Liquid Chromatography Mass Spectrometry Platforms

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Liquid Chromatography –Mass Spectrometry PlatformsNew Developments in LC/MSDr Shaun Bilsborough

Agilent’s 35 Years in Mass SpectrometryNearly 40,000 systems – Applied to Chromatographic Analysis5930A19715992A19765970B MSD19865971 - 59751988 - present6100 Single Quad4500, 7500 Series ICP/MS2006, 20071994-PresentLC/MSD Series1997-20056410 , 6430, 6460 Triple QuadESI-TOF Series2003-Present2006, 2007, 20086510, 6520, 6530, 6540 QTOF2006, 2007, 2008, 2009

Core LC/MS Platforms and TechnologiesMaximum Performance for our ApplicationsSelect Mass Spec Technologies for both Quantitative andQualitative applications.– Quadrupole technology is the ideal combination ofsensitivity, dynamic range, reliability and cost for targetcompound analysis– Time-of-Flight technology is the best choice for high massaccuracy, resolving power, sensitivity, dynamic range andspeed and vacuum requirements as applied to qualitativemass analysis– Ion Source development has long been realized as the mostcritical contributor to sensitivity, both in ion generation andsampling. JetStream ESI, APCI, Multimode, APPI,Nanospray CHIP, AP-MALDI.

Relentless Innovation6460 LCMS QQQ System

Cutting Edge 6460 QQQ PerformanceWith Agilent Jet Stream TechnologyUnmatched sensitivityWorkflow improvementsFaster Method developmentThe industry’s mostsensitive QQQ 1 pg on-column 1,000:1 S:N sensitivity and less than 10% RSD !!! Fast Pos/Neg switching (30 msec) More MRMs per time segment and faster MRMs (1 msec dwell) New Optimizer software enables faster Method development

NEW: Agilent Jet Stream Ion GenerationGas Dynamics ViewEnhanced efficiency nebulizerNebulizing gasSuper-heated sheath gasNozzle voltageHeated drying gasThe super-heated sheath gascollimates the nebulizer spray andcreates a dramatically “brighter source”Patent PendingResistive samplingcapillary

Agilent 6460 QQQ PerformanceShattering the Femtogram Barrier – 500 Attograms1.E 07R2 0.999Peak Area, counts1.E 06Verapamil1.E 05femtogrambarrier1.E 041.E 031.E 021.E 01500attogram1.8 mm C18, 2.1 x 50mm0.4 mL/min1.E 000110100100010000100000Amount, fgFive Decades of Linearity6460 triple quad with Agilent Jet Stream technology exhibits outstanding performance with 5 decades oflinearity from sub-femtogram to 100 picograms of verapamil injected on-column.

MassHunter Software‘Batch ata Glance’Curve-Fit Assistant

Parameter-Less Integrator & Peak ValidationTraditional derivative-basedMissed PeaksIntegratorParameter-LessMSMS IntegratorAddition.PeaksBad Baseline Traditional derivative-based integrators need tedious adjustment of integration parameters perform poorly near the Lower Limit of Quantitation need to apply smoothing to work

Parameter-Less Integrator & Peak Validation New parameter-less MSMS Integratorvalidates Peak Quality based onMergePeakAcceptedMergeInspectValleyPeak Peak height Peak areaHeightHeight Peak width15 %39 % Peak symmetry Merged peaks to the right or left Level of spikes on a peak Classifies peak quality as Accepted Inspect Rejected Let’s you focus on peaks in questionValleyMergePeakValleyRejectedHeight54%

MassHunter OptimizerAutomated MRM Method DevelopmentSoftware for 6400 series Triple Quad LC/MSCompound-specific optimization for MRM experiments Selection and optimization of precursor ion and fragment ions Support of optimization with multiple methods (e.g. pos/neg) Creation of a compound database with optimized resultsWITHOUT Optimizer: Manual optimization for multiple compounds WEEKS of tedious interactive work!WITH Optimizer: Optimization can be fully automated One or two days unattended work!

MassHunter MS/MS Method OptimizerBasic StepsRun 1: The precursor adducts (H , Na , etc) andcorresponding Fragmentor value are optimized. Basedon this, the optimum precursor mass and Fragmentorvalue are set.Run 2: Coarse product ion scan finds the largest 4 productions with corresponding Collision Energy. As a default0, 10, 20, 30, 40 volt CE are surveyed.Run 3: Fine adjustment of each product ion to establishoptimal Collision Energy.

MassHunter OptimizerStep 1 – Setup Compounds Setup compounds manually or via import from XLS Provide either formula or molecular weight Optionally assign compounds to groups

MassHunter OptimizerStep 2 – Setup Precursor Ion Selection Define up to 4 adducts and/or charge states for each polarity Define exclusion of ions based on m/z or intensity

MassHunter OptimizerStep 3 – Setup Product Ion Selection Optimizer will find the 4 most abundant product ions Define minimum product ion mass Define exclusion of ions based on m/z, intensity or neutral losses

MassHunter OptimizerStep 4 – Setup Optimization Parameters Setup Sample introduction mode– Manual or automatic infusion, Injection with or without column Select data file directory Select range and step size for Fragmentor and CE coarse and fine

MassHunter OptimizerStep 5 –View Results for Project Results for the project can be immediately viewed after optimization Results get also automatically written into the compound database

Agilent 1290 Infinity LC- Infinite possibilities 1290 Infinity Binary Pumpwith integrated degasser 1290 Infinity Autosampler 1290 Infinity FlexCube(extend the autosampler possibilites) 1290 Infinity TCC 1290 Infinity Diode-Array Detector Chemstation Software Control Third party SW control High Pressure RRHD columns Diagnostics capabilities

1290 Infinity – Compatible with any HPLC and UHPLCA new power range providing maximum performance,flexibility, compatibility and investment protectionbar12001290 Infinity1000Vendor A800Vendor D600Agilent RRLCVendor B400Vendor C200Standard LC0012345ml/min

Agilent 1290 Infinity LC- Separation Power per time*Pc based on 2nd peak W(1/2). Tranquilizer Sample0.095sec PW0.140secPWColumn: Superficially porous50 x 2.1mm, 2.7umFlow 3.5ml/min, 700 barGradient 2-92% ACN in 0.4minT 80 C

MassHunter “Dynamic MRM”Allows 10,000 MRMs per analysis!A simple comparison of MRM and Scheduled MRMTime (min)Time Segment 11 2 3 4 56Time Segment 2Time Segment 3Time Segment 47 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25MRMCompounds (10/block)508010070Cycle Time (sec)0.50.810.7Max Coincident20404030Cycle Time (sec)0.20.40.40.3Scheduled MRM 2 x shorter cycle times supports narrow chromatographicpeaks, more analytes or longer dwell per analyte.

Analysis of 300 Compounds in 15 min.Agilent 1290 Infinity LC 6460A Triple Quad with Agilent Jet StreamTechnology 600 Dynamic MRM Transitions9 replicates analyzed at 200 pg levelMean area %RSD 3.2, Mean height %RSD 3.6

Dynamic MRM Targets the Analyte when it ElutesEIC’s of 40 Compounds Eluting in a 1 min. RT Window

LCMS Analysis: What is NanosprayType of ElectrosprayFlow RateSheath GasConventional50 – 1000 uL/minYesMicrospray1 – 20 uL/minUsuallyNanospray0.01 to 0.5 uL/minNot usually

Nanospray: The concentration advantage!ColumnIDFlow RateRelative [C]Standard4.6 mm1 mL/min1Microbore1 mm50 uL/min21Capillary0.3 mm5 uL/min206Nanobore0.075 mm250 nL/min3750(75 um) Nanospray must be used for proteomics BUT difficult and unreliableFrom Tomer & Mosely, Mass Spec. Rev., 1994, 13:431

HPLC-Chip Platform for Nanospray LC/MSHPLCChip/MSinterface1200 NanoLC System6000 Series Mass Spectrometer(SQ, QQQ, TOF, Q-TOF)

Application: Protein Biomarker ProfilingHPLC-Chip designµ-Fluidic 6 Port ValveSeparation Column75x50 µm id, 150 mm lengthµ-filterLaser Ablated Nanospray Tip160 nL Enrichment ColumnTip Channel10x14 µm

Rapid Nanoflow LC/MS – 3 Minute Analysis21 MRM Transitions From 7 Peptides

Reproducibility of MRM response of HSA Peptide from10 amol to 200 fmol: (n 6)

SM-Chip/QQQ in DMPKApplicationdeveloped with

Chip/QQQxxxxPK-profilexIdea Achieve substantialsavings in the DMPK Lab Mice cheaper to keep and breed Mice require less active pharma ingredient than dogs or rats.Problem Mice cannot donate as much blood Serial bleeding, which increases the result-quality small sample-VChip solution Sensivity, allows to handle smallest sample quantitiesxtime

Ultra High Capacity (UHC)-SM-Chip Main Objective Enrichment of small molecules comprising a wide range of polarities500nL enrichment columnSeparation Column 150mm x 75µm ID456312

ESI versus SM-Chip104102ESIChipAmount inj. 5pgAmount inj. 1pgArea: 932Area: 85403 400 times higher!C18 150mm x 4.6mm; 5µmC18 43mm x 75µm; 5µm (40nl)1ml/min400nl/minMeOH/water 0.1% ammoniaMeOH/water 0.1% ammoniaInj-V 5µlInj-V 1µl

10fg on Column Level in Plasma 1µl injection following protein precipitation LOD not reached8x10 2290.1 - 124.1 , 290.1 - 93.0x10 2Counts3.4 MRM (290.1 - 124.1) 10fg 300nl 2.d281.2 - 58.0 , 290.1 - 86.0x10 2Countsx10 2CountsCounts MRM (281.2 - 58.0) 10fg 300nl 0.410.40.40.20.50.20.200010.4 10.6 10.81111.2 11.4 11.6Acquisition Time (min)10.410.610.81111.2 11.4 11.6Acquisition Time (min)088.28.48.68.899.2Acquisition Time (min)88.28.48.68.899.2Acquisition Time (min)

Atenolol4logP 0 21 Atenolol2 Caffein3 Quinine4 Atropine5 Metoprolol6 Propranolol7 Imipramine3ResponsesIntensityQuantitative enrichment on the UHC-SM-Chip5x1062.82.6Atropine - 6 Levels, 6 Levels Used, 12 Points, 12 Points Used, 0 QCsR 2 0.9905Inj.-V: 6µl2.462.22ImipraminelogP 41.8(A)1.61.41.210.80.6Inj.-V: 1µl0.40.6 0.8Time11.2 1.4 1.61.822.2 2.4 2.62.833.23.4 3.6 3.844.2 4.44.6 4.855.2 5.4 5.6 5.8 66.2 6.4Concentration (ng/ml)

Precision, reproducibility and life-timex10 498.5AtropinePrecision%RSD RT0.08%8%RSD 00fg 0.21%0.10%4.10%5.00%77.588.599.5Counts vs. Acquisition Time (min)1010.51111.512Test for:No of ChipsResultChip-to-chip reproducibility110.7 % RT-RSDSpray tip lifetime2 2 weeks continuous sprayLifetime with 4-compound mix4 500 injectionsLifetime with 4-compound mix in plasma 2after protein precipitation200-500 injections12.5

Agilent 6540 Ultra High Definition QTOF

Ultra High DefinitionOptimizing all Analytical Dimensions Sensitivity Dynamic RangeSignal Response Linearity Separation Speed Peak CapacityMass Spectrum Mass Accuracy Resolving Power Acquisition Rate

Agilent’s New 6540 Ultra High DefinitionAccurate Mass Q-TOFExceptional accurate mass, sensitivity,dynamic range and resolution . perfectmatch for 1290 Infinity UHPLC 500 ppb mass accuracy Femtogram sensitivity 5 decades dynamic range 40,000 resolving power 20 Spectra/sec Excellent Linearity and IsotopicFidelity Supports Agilent Jet Stream andHPLC-Chip

6540 Ultra High Definition QTOFMass Accuracy – 10 Repetitive Injections ESI EIC(609.28066)Scan Frag 240.0V Reserpine .751Counts vs. Acquisition Time 040.300.25x10 51.2609.2808110.8IsotopeObs %Calc %Obs m/zCalc 609610611612613.29210613614Counts vs. Mass-to-Charge (m/z)250 ppb mass accuracy calibration and very accurate isotopic ratiosDiff (ppm)

Why Accurate Mass?C12 H14 N9 ClC13 H23 N3 P S ClMSUnit accuracy instrumente.g. ion trap, QQQ, QC14 H24 N2 S2 ClC14 H22 N O3 S ClC14 H16 N6 O ClC15 H25 O P S ClC16 H19 N3 P ClLow Mass Accuracym/zC17 H20 N2 S ClC18 H21 O P ClC13 H23 N3 P S ClMSHigh mass accuracyinstrumente.g. QTOF, TOFC14 H24 N2 S2 ClC14 H22 N O3 S ClC14 H16 N6 O ClC15 H25 O P S ClC16 H19 N3 P Clm/zHigh Mass AccuracyC17 H20 N2 S ClC18 H21 O P Cl

Identify Compounds Not in DatabaseMolecular Formula GenerationScoring algorithm considersaccurate mass and abundancesof all isotopesx10 2 Scan (0.859-0.957 min, 7 scans) sulfas 10ng 12.2312.4Counts (%) vs. Mass-to-Charge 4314.6314.8

Data Mining - Molecular Feature ExtractionRaw dataBackground noiseremoved Map signalsIndividual m/z peaksgrouped into isotopeclustersIsotope clustersgrouped intomolecularfeatures Remove noise Identify all mass signals with a common RT Combine mass signals with common RT and chemical relation (isotope, adduct,dimer, different charge state) Create Extracted Compound Chromatograms (ECC) and Compound Mass Spectrafrom ions associated to a molecular feature (compound)

Easily ID 400 Compounds in Complex Sample in 5 min. !List of compoundswith calculatedMolecular FormulasOverlaid compoundchromatograms showea individualcompound foundDetailedFormulaGenerationResultsDetailed MolecularFormula CalculationResultsCompound massspectrum withtheoretical isotopepattern overlaid

6540 Ultra High Definition QTOFMaintaining Resolving Power – Across the Mass Range622.00822m/z 622 and isotopes9225622x10 5.5R 42424623.01059R 397025624.0121912214.5625.01471R 43674Counts vs. Mass-to-Charge (m/z)415213.5R 42750321212.518212R 41825R 404052421R 200240026002800Counts vs. Mass-to-Charge (m/z)Scan Rate Independent

Novel Ion Beam Compression (IBC)* TechnologyDrives higher resolution without requirement for long flight tubeCompressed and cooled ion beam ensures the best sensitivityperformance in high resolution modeExit from collision cellInto pulser regionNarrowed beam enables mass resolutionof 40KUp to 10-fold ion beam compression* Patent pending

New High Speed 32 Gbit/sec Acquisition SystemProvides up to 5 Decades of In-Spectrum Dynamic Range(no beam attenuation required, unlike TDC-based TOF)Max Abd3x1079x106 cts250 ctsNiacinamide, 10 ng/µL[M H] 123.055289 m/zError 0.8 ppmIn-Scan Dynamic RangeThis Example9x106 / 250 3.6104Maximum3x107 / 250 1.2x 105Erythromycin, 500 fg/µL[M H] 734.468518 m/zError 0.6 ppm

Accuracy, Resolution & Speed Simultaneously10 spectra/second1 spectrum/second-0.60 ppm-0.37 ppmR 40,600R 41,400ONH3COOCH3OONNazoxystrobin

Agilent’s Forensic Personal Compound DatabaseDatabase containing 8000 Forensic AnalytesControlled substances, poisons, explosives, accelerants

Agilent’s Pesticide Personal Compound Database 1,500 Pesticides

Agilent’s METLIN Personal Database 23,000 metabolites

Agilent 1290 Infinity LC - Infinite possibilities 1290 Infinity Binary Pump with integrated degasser 1290 Infinity Autosampler 1290 Infinity FlexCube (extend the autosampler possibilites) 1290 Infinity TCC 1290 Infinity Diode-Array Detector Chemstation Software

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