Agilent MassHunter Quantitative Data Analysis

1 Agilent MassHunter Quantitative Data AnalysisPresenters: Howard SanfordStephen HarnosMassHunter quantitation :Batch and Method SetupOutliers, Data Review, Reporting1/10/2018 MassHunter Webinar Series1 MassHunter Quantitative Analysis SoftwareReview and Quant Method OptimizationTopics Batch Definitions Three main views in MassHunter Quantitative Analysis Developing a quantitation methodMethod Editor tasks Analyze the batch Outlier setup Advanced Tasks Integration parameters Data review Generating a report, modifying graphic output1/10/2018MH QQQ Webinar Series2 DefinitionsBatch-A set of data files that may include calibrators, samples, blanks, and QC samples.

2 The data in the batch will be operated on by one method. The method and calculated results are contained in the batch. Quant Method A list of target compounds with quantifier and qualifier ions specified for each compound, retention times, ISTD info, calibrator concentrations, outliers, integration parameters, and much more. Results After the batch has been analyzed, results are generated and contained within the batch (xml file). These results are displayed in a tabular format in the Batch Table and graphically in either the Batch View or the Compounds at a Glance View. Batch ContentsAcquired Data Quant MethodResults1/10/2018 MassHunter Webinar Series3 DefinitionsSQ Single quadrupole instrument low or unit mass resolution data.

3 Can be scan data or Triple quadrupole or tandem quadrupole. Composed of a Q1, collision cell (Q2) and Q3. Usually acquired in multiple reaction monitor (MRM). MS/MS Time of flight High resolution mode. Gives exact mass to 4 decimal places. Data acquired in scan TOF Quadrupole combined with high resolution TOF. MS/MS QQQ Webinar Series4 MassHunter Quantitative SoftwareThere are 15 possible Quantitative Analysis icons 1/10/2018 MassHunter Webinar Series5 Use the icon applicable to the instrument and dataset for the UV signals (UV only)can be processed with the GC icon or as part of an MS core software different Data Folder StructureFour Basic Components to the Batch Directory1/10/2018 MassHunter Webinar Series6 Use the icon applicable to the instrument and dataset for the , samples, blanks.

4 QC and CC samples comprising the fileMassHunter Quantitative SoftwareBatch-at-a-Glance View1/10/2018 MassHunter Webinar Series7 Batch TableCalibration CurveCompound InformationMethod TasksMassHunter Quantitative SoftwareMethod Editor ViewMethod > Edit menu orF10 key1/10/2018 MassHunter Webinar Series8 Method TableSample InformationCompound InformationMassHunter Quantitative SoftwareCompounds at a GlanceView > Compounds-at-a-Glance1/10/2018 MassHunter Webinar Series9 Important MassHunter Quant Concepts and RulesBatch SetupA Batch is a file which contains all the Quant results from a set of data files and the Quant method used. All the data files in a Batch must reside in a single directory, so put them all together before creating a New the Batch name within the batch the single batch concept allows for easy archiving and retrieving of information about the batch is stored in the QuantResults\ file1/10/2018 MassHunter Webinar Series10 Important MassHunter Quant Concepts and Rules:Quant MethodsThe most common method problem: Calibration/QC level names in Batch and Quant method do not match, : levels are 1,2,3,4,5-Batch: levels are L1,L2,L3,L4,L5 Second most common problem.

5 A hidden column in the Method with a key parameter, Ion Polarity if method created Quant parameters can be applied to multiple compounds with the Fill Down feature or Apply to All Webinar Series11 Creating a Batch for ProcessingBatch-at-a-Glance1) File > New ) Create the batch in the directory in which the data files reside3) Add Samples4) 5) Method > Open Method from Existing Method > Open Method from Existing Webinar Series12 Let s take a moment for questions on batch definitions and setting up a batch. Up Next:Setting up a quantitation 10, 201813 Short DemoCreating a quantitation Method for SQMethod > NewNew Method from Acquired SIM available from MS Quantitative Analysis in QQQ but not as Method from Acquired Scan (with Library Search)Use Library Method to identify and create up unwanted ions choices (Selected ions may not be the best ions).

6 New Method from Acquired Chromatographic for 2D Data (UV, FID, ECD, etc.).Gives only Compound_1, etc. based on Method using Manual SetupManually enter compound data (RT, concentration, masses, etc.).Can be tedious for large number of Method from CEF file generated in Qualitative a small user generated Webinar Series14 Editing a quantitation Method Step through Method Setup TasksApplies to SQ, TOF, QQQ MRM SetupAllows setup of transitions with precursorand product Webinar Series15 Method Editor-Method Tasks Method Tasks organizes the method actions and parameters into related when you cannot add compounds or qualifiers method editing content.

7 Save and applyQuant Webinar Series16 Editing a quantitation Method for SQCompound SetupName compound nameTS time segment usually 1 for scan datamay be many time segments for SIM and MRM dataType Target, ISTD, Surrogate or Matrix SpikeMZ mass of target or quantifier ionRT retention time of compound of interestIon Polarity may be Positive, Negative, Unassigned or Both-in most applications, it will be positive Criteria Close RT, Close RT with Qualifiers, Greatest Response or Greatest Q-value1/10/2018 MassHunter Webinar Series17 Editing a quantitation Method for All InstrumentsRetention Time SetupRT retention time of compound of interestLeft RT Delta represents the left side of the EIC window around the expected retention RT Delta-represents the right side of the EIC window around the expected retention time RT Delta Units typically in Webinar Series18 Editing a quantitation Method for All InstrumentsISTD SetupDefines the ISTD and associates each ISTD with one or more target ISTDs can be assigned in a Toggle this flag on for all ISTDsType-Compounds assigned as an ISTD

8 Become available ISTD-Compound Name Establishes concentration levelsISTD Conc. a concentration must assigned for each ISTDTime Reference Flag only ISTD can be assigned as time reference compoundsA Time Reference ISTD adjusts the RT of all target compounds related to a given ISTD due to slight RT shifting due to extraneous factors such as matrix effects. This is done a on a per sample basis. 1/10/2018 MassHunter Webinar Series19 Editing a quantitation Method for All Instruments Concentration SetupDefines the concentration of each compound in the in Method must match Level in Batch table!If Unchecked a level can be disabled and not appear in the calibration Webinar Series20 Editing a quantitation Method for All InstrumentsConcentration SetupCalibration levels can be created manually or in levels and associated concentrations.

9 Manual SetupSetup Levels from Calibrators, QCs & CCs in BatchLevel IDs are filled in from the Calibrators, QCs & CCs in the must be updated! Click to add calibration Webinar Series21 Creating a quantitation Method for All InstrumentsConcentration Setup Create Levels from Calibration Samples1/10/2018 MassHunter Webinar Series22 Then Method > Editand then Method > Create Levels from Calibration a quantitation Method for All Instruments Concentration SetupIf concentrations are the same, they can be copied to the other Webinar Series23 Editing a quantitation Method for All InstrumentsQualifier Setup Right click Qualifier may be added interactively1/10/2018 MassHunter Webinar Series24 Editing a quantitation Method for All InstrumentsQualifier Setup If checked.

10 Qualifier area is added to Qualifier Ion response as Percentage of Quant Ion, and Percentage relative Response with 20% UncertaintyAbsolute = 80 +/-20 = range of 60-100 relative = 80 +/-(80*20/100) = range of 74 -961/10/2018 MassHunter Webinar Series25 Editing a quantitation Method for All InstrumentsCalibration Curve Curve Fit Linear. Quadratic. Power. 1storder ln. 2ndorder ln. Average response Fit Origin Ignore Include. Force. Blank Fit Weight None (equal wt.) 1/x, 1/x2 1/y,1/y2 Log 1 : Calibration Curve not available for viewing until batch is Webinar Series26 Editing a quantitation Method for All InstrumentsGlobals SettingsApply Multiplier s To avoid flagging target compounds that absentNone or Overlapped or Sample GroupPeak identification within the extraction windowAssociates target and qualifiers in minSemi quant relative to ISTDQ uantitate with Standard AdditionPeak identification within the extraction window(ISTD with Time Reference Flag checked)Spectral Reference Library (.)