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Agilent MassHunterQuantitative DataAnalysisPresenters: Howard SanfordStephen HarnosMassHunter Quantitation:Batch Table, CompoundInformation Setup,Calibration Curve andGlobals SettingsMassHunter Webinar Series9/13/20171

MassHunter Quantitative SoftwareReview and Quant Method OptimizationTopics Brief Review Batch Table Navigation Compound Information- Data review and manual integration Calibration Curve Working in Compounds At a Glance Method Editor Globals SettingMassHunter Webinar Series9/13/20172

ReviewThree major views in MassHunter Quantitative Analysis. Batch At a Glance Method Editor Compounds At a GlanceHandling MS/MS data QQQ and QTOF.Handling accurate mass data.MassHunter Webinar Series9/13/20173

MassHunter Quantitative SoftwareBatch-at-a-Glance ViewBatch TableCompound InformationCalibration CurveMassHunter Webinar Series9/13/20174

MassHunter Quantitative SoftwareMethod Editor ViewMethod Edit menu or F10 keyMethod TableSampleInformationMethodTasksCompound InformationMassHunter Webinar Series9/13/20175

MassHunter Quantitative SoftwareCompounds at a Glance ViewView Compounds-at-a-GlanceMassHunter Webinar Series9/13/20176

Editing a Quantitation Method for TOFMass Extraction SetupMethod Edit Advanced Tasks Mass Extraction SetupAllows for a mass range for the extraction of the accurate mass (MZ).Available MZ Extraction Window UnitsMassHunter Webinar Series9/13/20177

Editing a Quantitation Method for QTOFCompound Setup & Mass Extraction SetupQTOF is a combination of MS/MS and accurate mass dataName – Compound nameTS – time segments may be multipleTransition – Precursor ion Product ion scanScan – Product IonType – Target, ISTD, Surrogate or Matrix SpikePrecursor Ion – mass of the ionProduct Ion – mass of the ion for the target ion to monitorRT – retention time of compound of interestIon Polarity – usually positiveCriteria – Close RT, Close RT with Qualifiers, Greatest Response orGreatest Q-ValueMassHunter Webinar Series9/13/20178

Batch TableNavigationClick in a sample row to display data on that sampleClick on Next and Previous icons to move through a batch or useHotkeys:Next Sample Alt DownNext Compound Alt RightPrevious Sample Alt UpPrevious Compound Alt LeftCompound list are frequently long, try the Compound drop down list to godirectly to the desired compound.MassHunter Webinar Series9/13/20179

Batch TableAlphabetizing the Compound ListFrom within the Batch Tableright click, select ArrangeCompounds By NameMassHunter Webinar Series9/13/201710

Batch TableMessages and OutliersSelectOutliersfor DisplayRed Outlier – High(above upper limit)Blue Outlier – Low(below lower limit)MessagesOutliersHover cursor over the outlier or message to display detailsMassHunter Webinar Series9/13/201711

Batch Table Layout ModificationAdd/Remove/Move ColumnsUse the Add/Remove Columns function tocustomize the Batch table.Note that the columns of data in the BatchTable are organized into sections.Columns can be moved only within a section.Select correct table.MassHunter Webinar Series9/13/201712

Batch Table Layout ModificationSingle and Multiple Compound ModesSingle Compound ModeFlat Table compoundsacross the top.Multiple Compound ModeMassHunter Webinar Series9/13/201713

Batch Table Layout ModificationCompound Table ModesCompound Table lists by compound rather than sample.Select Flat Table to see list by sample.MassHunter Webinar Series9/13/201714

Batch TableCompounds GroupsIt may be useful to group compounds to organize Batch Table.Look at parent compound and metabolites.Group hydrocarbons to Group 1, aromatics to Group 2 and so forth.Compound groups are generated in the Method Editor.MassHunter Webinar Series9/13/201715

Batch TableCompounds GroupsActivated with a right clickto the right of the ‘flags’ or View Toolbars Filtering.Yields an Additional Toolbar.MassHunter Webinar Series9/13/201716

Batch TableSamples GroupsSample Groups are specified in the Batch Table through Add/RemoveColumns.Each Sample isassigned to agroup, then onlysamples specificto a group aredisplayed andevaluated.Samples groups are distinctively different from compound groups.MassHunter Webinar Series9/13/201717

Batch Table Layout ModificationFormat ColumnsHow to change thenumber of decimalplaces.Alter numberformats1) Exponential2) Fixed point3) GeneralAlter Date formatsMassHunter Webinar Series9/13/201718

Batch Table Layout ModificationPropertiesChange the Font sizeChange the Color schemeMassHunter Webinar Series9/13/201719

Export Batch TableEasy to layout columns and format data, then export into Excel.MassHunter Webinar Series9/13/201720

Let’s take a momentfor questions on BatchTable navigation.Next up:Compound InformationMassHunter Webinar Series9/13/201721

Compound InformationDisplay and access one compound in one sample at a sISTD QualifiersTargetSpectrumISTDSpectrumMassHunter Webinar Series9/13/201722

Compound Information - ToolbarUn-zooms,Peak scaling,Auto Scales xand y axesSampleandCompoundNavigationNext DManual IntegrationFunctionsMassHunter Webinar Series9/13/201723

Manual Integrations and MI ToolbarClick on the Manual Integration icon to place Compound Information inManual Integration mode.Each signal is placed in its own window (no overlay of qualifiers).MassHunter Webinar Series9/13/201724

Manual Integration ToolbarEnable Manual Integration – thistool activates the manual integrationtoolbar.Split Peak and Pick Right/Left –this tool splits the peak and thenselects the right or left peak.Clear Manual Integration – this toolbecomes active once a manualintegration is present.Snap Baseline – this tool placesstart/stop integration points on thebaseline.Zero Peak – this tool sets start andstop to the same value.Drop Baseline – this tool finds thelowest end of the peak, then dropsthe baseline on the other side of thepeak from it, thereby creating a flatbaseline that avoids negative area.Merge Right/Left Peak – this toolmerges the peak to the right/left ofthe selected peak.MassHunter Webinar Series9/13/201725

Compound InformationPeak AnnotationActivated from Tools Add-Ins NI – The peak was not integrated at all by thecomputer software.LT – The peak in question was inappropriatelyintegrated to an area less than what it should be(e.g., Peak area was cut).GT - The peak in question was inappropriatelyintegrated to an area greater than what it should be(e.g., Peak Tailing).BA – The baseline had to be adjusted correctly by the analyst.CO –The analyst had to split to co-eluting peaks apart that were not (or couldnot be) separated by the computer system.RT – The retention time for the peak in question has shifted from the expectedretention time.INT – There was electronic interference (e.g., Noise).MassHunter Webinar Series9/13/201726

Compound InformationPeak AnnotationThe editable file is located in nt\bin\AddIns\PeakAnnotations.xml.MassHunter Webinar Series9/13/201727

Compound InformationPeak AnnotationThe Peak Annotation can also be displayed in the Batch Table.When the batch is saved, the values are retained.The MI flag is checked in the CompoundResults.MassHunter Webinar Series9/13/201728

Compound InformationContext Menu Integration ParametersRight click to expose thecontext menu.Most of the features are toolbaricons.Integration Parameters can beused to change the integrationparameters.Applies ONLY to this compoundin this sample.Restore Integration Parametersreverts to the methodintegration values.MassHunter Webinar Series9/13/201729

Compound InformationContext Menu PropertiesProperties allows forcustomization of the display.Can change Fill colors.Can change Peak labels.Can change Titles.MassHunter Webinar Series9/13/201730

Compound InformationContext Menu PropertiesQualifier Properties can bechanged.Spectrum Properties can bealtered.Manual integrationparameters can be varied.MassHunter Webinar Series9/13/201731

Signal to NoiseFive algorithms are availableConsult online help for moreinformation on the algorithms.Noise regions can beautomatically determined orindividually specified.The Noise regions can bedisplayed in the CompoundInformation window underProperties CompoundInformation (1) BaselineCalculation Points.In this example, 2 noise regionswere determined—one beforethe peak and the other after.MassHunter Webinar Series9/13/201732

Let’s take a momentfor questions onCompound InformationNext up:Calibration Curve andCurve Fit AssistantMassHunter Webinar Series9/13/201733

Calibration CurveView ISTD ResponsesFit toLevels(Scaling)Change Curve Fit.R2CC LevelsQC SamplesConcentration can be set as relative (to ISTD) or actual.MassHunter Webinar Series9/13/201734

Calibration CurveDisable calibration pointsClick on calibration point once to disable and remove from curve.MassHunter Webinar Series9/13/201735

Curve Fit AssistantMassHunter Webinar Series9/13/201736

Curve Fit AssistantSorting ColumnsBest curve fit may be ranked using byR2, Standard Error, or Max % Residual.Curve Fit Assistant calculates the mathematical curves.MassHunter Webinar Series9/13/201737

Curve Fit AssistantDisabled Points# of Disabled Points defaults to 3 but column can be filtered.MassHunter Webinar Series9/13/201738

Curve Fit AssistantConfidence BandOriginal Fit(black)Best Fit(blue)ConfidenceBand(blue)MassHunter Webinar Series9/13/201739

Calibration CurveLog Log PlotLogarithmicScaleMassHunter Webinar Series9/13/201740

Curve Fit AssistantAccept Assistant CurveMassHunter Webinar Series9/13/201741

Calibration CurvePropertiesMassHunter Webinar Series9/13/201742

Let’s take a momentfor questions onCalibration Curve andCurve Fit AssistantNext up:Compounds at a GlanceMassHunter Webinar Series9/13/201743

Compounds-at-a-GlanceHigh throughput data review environment.View compounds across multiple samples.View all compounds within a sample.MassHunter Webinar Series9/13/201744

Compounds-at-a-GlanceTo start, select View Compounds-at-a-Glance Choose Layout Predefined LayoutMassHunter Webinar Series9/13/201745

Compounds-at-a-GlanceSetup Graphics WizardTo customize select Layout Setup Specify Samples to view inCompounds-at-a-Glance thenCompounds. By default, all samplesand all compounds are selected.Specific samples and order can bemodified.MassHunter Webinar Series9/13/201746

Compounds-at-a-GlanceSetup Graphics WizardDefine how to Organize the selectedcompounds and samples.Define the Overlay mode.Review ModeSample by SampleCompound by CompoundCompound Group by CompoundGroupDisplay OptionsMassHunter Webinar Series9/13/201747

Compounds-at-a-GlanceSetup Graphics WizardFinally, define which Outliersshould be highlighted Outliers can also be filtered byPanes without outliersPanes with outliersMassHunter Webinar Series9/13/201748

Compounds-at-a-GlanceOutliersOutliers arehighlighted in red.MassHunter Webinar Series9/13/201749

Compounds-at-a-GlanceManual Integration Pop UpDouble click forsingle paneaccess.Can be accessedwithout manualintegration beingactivated.MassHunter Webinar Series9/13/201750

Compounds-at-a-GlancePrint PreviewSelect File Print Preview to create a chromatogram report.Export Graphicsgive a graphicimage in variousformats.MassHunter Webinar Series9/13/201751

Compounds-at-a-GlancePropertiesFrom thecontext menuselectPropertiesMassHunter Webinar Series9/13/201752

Compounds-at-a-GlanceSave LayoutSome settings in theLayout screens are notsaved such ascompounds, samples, etc.MassHunter Webinar Series9/13/201753

Let’s take a momentfor questions onCompounds at aGlanceNext up:Global SettingsMassHunter Webinar Series9/13/201754

Globals SettingsComponent of Method Editor.Global parameters are critical to proper quantitation.Globals are method parameters.Globals apply to the whole batch – all samples and all compounds.MassHunter Webinar Series9/13/201755