Agilent Technologies, Inc. · NYSE:A
Agilent Technologies — Technical Presentation (July 2023)
70 slides from Agilent Technologies's technical presentation, July 2023
Slide 1: High Throughput Quantitative Analysis
High Throughput Quantitative Analysis MassHunter Quantitative Analysis Webinar Series Howard Sanford Stephen Harnos With Tom Barrett & Matt Leyden & Kevin Costalunga 1 High Throughput Quantitative Analysis
Slide 2: What is quantitative analysis?
MassHunter Quantitative Analysis Software Review and Quant Method Optimization What is quantitative analysis? In analytical chemistry, quantitative analysis is the determination of the absolute or relative abundance (often expressed as a concentration) of one, several or all particular substance(s)
Slide 3: Target Compound Analysis
Target Compound Analysis • A given compound will come out at a specified time under Retention Time a given set of chromatographic conditions. • Dual column analysis with columns of different polarities. • Must be unique to the compound of interest in the time Target Ion range of interest. • Must be
Slide 4: Analysts are buried in data!!!
Analysts are buried in data!!! Analysts are being overwhelmed with data … hundreds of compounds… multiple signals per compound…10’s or even hundreds of samples in a batch. Add an initial calibration, a continuing calibration or a QC and other data review functions and the analyst is overwhelmed with
Slide 5: Quant Method Check List
Quant Method Check List • Extract the compounds signals in the correct RT Window. • Utilize the correct integrator and settings for each compound. • Reduce unwanted peaks with • Reference and Non Reference Windows • Integration parameters – Peak Filters – Peak Filter Area Threshold – Zero Peaks Belo
Slide 6: Retention Time Setup
Retention Time Setup RT Delta Left and Right RT Delta determines the time range over which the specified signal is extracted. RT Delta Units • Percent – Uses a percentage of RT, good for long runs where retention time shifts can be larger late in the run. • Minutes - Absolute minutes. Default is 1 m
Slide 7: Retention Time Setup
Retention Time Setup Criteria When multiple peaks are found in RT window, Criteria decides which peak to use. • Close RT • Close RT with Qualifiers • Greatest Response • Greatest Q-Value High Throughput Quantitative Analysis 7
Slide 8: Reference & Non Reference Window
Reference & Non Reference Window Definitions Defined in Globals Setup in the Method Editor Reference Window • Applies only to compounds labeled as Time Reference. • Only ISTDs can be labeled as Time Reference. • Algorithm looks for ISTDs first, then target compounds related to that ISTD. Non Referen
Slide 9: Reference & Non Reference Window
Reference & Non Reference Window 1 Restricts peak selection to a smaller RT window. 2 3 9 High Throughput Quantitative Analysis
Slide 10: Non Reference Window
Non Reference Window Initial Adjusted Final Values Values Values Reduce the Non Reference Window from the default 200% to 10%. This helps eliminate false positives and reduced data review and “zero peak” work. Or, if you prefer, switch to absolute minutes. Though keep in mind this setting is for all
Slide 11: Properties Window
Properties Window Right click Properties > Fill Colors. 11 High Throughput Quantitative Analysis
Slide 12: Integrators
Integrators Choose the Right Integrator • Method >Edit >Advanced Tasks > Integration Parameters Setup • Each compound can have its own integrator. • Choose the one best suited for the compound’s chromatography. • Start with Agile2 (parameter-less) and move to other integrators if they work better. •
Slide 13: Integrators
Integrators Agile2 • 3rd generation parameter-less integrator • Default Integrator • Better baselines, higher sensitivity to smaller peaks Agile • 2nd generation parameter-less integrator Universal • 1st generation ChemStation integrator • Familiar to GC LC ChemStation users General (RTE) • Familiar
Slide 14: Integrators
Integrators General, Universal, Spectrum Summation Adjust parameters as needed for each compound to get optimal integrations. Timed Events 14 High Throughput Quantitative Analysis
Slide 15: Integrators
Integrators Qualifier Integration Control Qualifiers use the same integrator as quantifier. If needed each qualifier may have separate parameters to optimize integration but the default is to use quantifier parameters. 15 High Throughput Quantitative Analysis
Slide 16: Spectrum Summation Integrator
Spectrum Summation Integrator • Integrator designed for situations where compounds are poorly separated or peak shape is highly irregular such as …. – PCB mixtures – Fraction cut in hydrocarbons – Flow injection analysis (FIA) • Sums signal over a time range. • Exclude signal below threshold. • Alwa
Slide 17: Integrators
Integrators Peak Filters Default Setting for both Target and Qualifiers is Peak Area >= 5 % of largest peak. • Available on all integrators including parameter-less ones. • Separate Peak Filters for quantifier and qualifiers. • Peak Filter is run after integrators create a peak list and then removes
Slide 18: Peak Filter Area
Peak Filter Area Typical noise or matrix peaks may be picked up by the integrator. They are far too small relative to the response of the lowest level Calibrator to be reported. Normally we would need to “zero peak” each one. Instead we can use peak filters in the method to remove the unwanted peaks
Slide 19: Adjusting Peak Filter Area Thresholds
Adjusting Peak Filter Area Thresholds Manual Setting the peak area threshold for each compound would be a slow and tedious process… 19 High Throughput Quantitative Analysis
Slide 20: Adjusting Peak Filter Area Thresholds
Adjusting Peak Filter Area Thresholds Automated To automate the process, while in the Method Editor view, use the “Set Peak Filter Area Threshold” script to set each compound to a percent of its lowest Calibrator in the batch. High Throughput Quantitative Analysis 20
Slide 21: Adjusting Peak Filter Area Thresholds
Adjusting Peak Filter Area Thresholds Example Before After Typical noise or matrix peaks are removed and require no data review or “zero peak” work. High Throughput Quantitative Analysis 21
Slide 22: Zero Peak Below LOD
Zero Peak Below LOD Method Editor > Outlier Setup Tasks > Limit of Detection. • Eliminate peaks based on concentration rather than area. • Must define the Limit of Detection outlier value for each compound. High Throughput Quantitative Analysis 22
Slide 23: Zero Peak Below LOD
Zero Peak Below LOD Tools > Actions > Zero Peak Below LOD • Accessed from Batch Table View or Method Editor View. • Zeros compounds if the Calculated Concentration is less than the LOD. 23 High Throughput Quantitative Analysis
Slide 24: Correlation Window
Correlation Window The retention time difference limit of target ions to one or more qualifiers. • Defines the maximum allowable variation of multiple extracted ion peak retention times before they are considered a single peak. • Default time of 2.00 min is rather wide. Typically 0.01 to 0.05 min (0
Slide 25: Correlation Window
Correlation Window Default value = 2.0 minutes. Note that the retention times for target and qualifiers are different! High Throughput Quantitative Analysis 25
Slide 26: Retention Time Drift
Retention Time Drift Retention times will Drift • GC ‒ Can use RTL locking to mitigate drift. ‒ Use time reference compound in the middle of chromatographic run. ‒ Make a single compound standard in the mid range of concentration. ‒ Lock the method. ‒ Relock the method as necessary. • LC ‒ Utilize a
Slide 27: Retention Time Drift: Update Retention Times
Retention Time Drift Update Retention Times Update Retention Times is available in Batch Table View and Method Editor View. Caveat - the compound must be found to update it. High Throughput Quantitative Analysis 27
Slide 28: Update Retention Times from ISTD
Retention Time Drift Update Retention Times from ISTD • Update Retention Times from ISTD is a Method Editor feature. • Particularly useful with isotopically labeled ISTD. 28 High Throughput Quantitative Analysis
Slide 29: Average Retention Times
Retention Time Drift Average Retention Times • Average Retention Times is a Method Editor feature. • Allows choice of Cals or QCs or both. • Includes a weighted average. High Throughput Quantitative Analysis 29
Slide 30: Shift Retention Time
Retention Time Drift Shift Retention Time • Shift Retention Time is a Method Editor feature. • Allows an Absolute Shift in minutes or a Relative Shift in percentage. • Would be most applicable when changing columns. High Throughput Quantitative Analysis 30
Slide 31: Qualifier Ion Ratios
Qualifier Ion Ratios Based on the sample currently loaded in Method Editor. Update Qualifier Ratios Average Qualifier Ratios Or Can use Cals and/or QCs. 31 High Throughput Quantitative Analysis
Slide 32: Update Mass Assignments
Update Mass Assignments • Update Mass Assignments is a Method Editor feature. • It is based on currently selected sample in Batch Table. • Useful with high resolution data (TOF and QTOF). 32 High Throughput Quantitative Analysis
Slide 33: Reference Library Globals Settings Option
Reference Library Globals Settings Option • Activated from Method > Edit > Library > Setup Reference Library • Reference Library may be obtained from a sample, preferably a calibrator, or from a small user generated library or a small subset library. • Reference Library name is auto populated in Glo
Slide 34: Reference Library Globals Settings Option
Reference Library Globals Settings Option Appears in Compound Information window. Customizable in right click > Properties > Compound Information (2) tab. High Throughput Quantitative Analysis 34
Slide 35: Reference Pattern Library High Resolution Data
Reference Pattern Library High Resolution Data • Activated from Method > Edit > Library > Setup Reference Pattern Library. • Can be obtained from a calibrator or from another library. • Method must contain molecular formula. • Reference Pattern Library name is populated in Globals. Select the adduct
Slide 36: Reference Pattern Library High Resolution Data
Reference Pattern Library High Resolution Data • Right click Properties > Compound Information (2) > Reference pattern spectrum • Isotopic abundance and pattern appears in Spectrum window. 36 High Throughput Quantitative Analysis
Slide 37: Demo time
Demo time 37 High Throughput Quantitative Analysis
Slide 38: Data Review Check List
Data Review Check List Filter on Filter on Filter on Sample Sample Compound Auto Review. Type. Group. Group. 38 High Throughput Quantitative Analysis
Slide 39: Data Review: Filter on Sample Type
Data Review Filter on Sample Type Review Data in stages (Calibrators, then QC, then Samples). Focus on only the calibrators or samples or blanks, etc. 39 High Throughput Quantitative Analysis
Slide 40: Data Review: Sample Type
Data Review Sample Type • The Type or Sample Type is a parameter available in the sequence or worklist. • Should be specified when data is acquired, but if necessary it can be entered in the Batch Table. • In Sequence Table in GCMS software. • In Worklist in LC MassHunter software. 40 High Throughpu
Slide 41: Data Review: Filter on Sample Group
Data Review Filter on Sample Group Sample Group is activated by right clicking on the toolbar. • This is helpful with large batches that contain several sample types. • Sample Group is a column that can be added to the Worklist or Sequence Table . 41 High Throughput Quantitative Analysis
Slide 42: Data Review: Filter on Compound Group
Data Review Filter on Compound Group Compound groups are assigned in the Method Editor. • Compound groups are useful for parent compound and metabolites. • Reviewing Aroclor congeners by group (PCB). 42 High Throughput Quantitative Analysis
Slide 43: Data Review: Filter on Compound Group
Data Review Filter on Compound Group Compounds may be assigned to more than one group by separating the group names using commas. Review Compounds by Group (using Compound Table View). 43 High Throughput Quantitative Analysis
Slide 44: Data Review: Compound Information
Data Review Compound Information Compound groups are shown in the Compound information window. BHC Isomers (3) Manual Integration turned on. 44 High Throughput Quantitative Analysis
Slide 45: Data Review: Chromatogram Information
Data Review Chromatogram Information • Accessed from View > Chromatogram Information. • Useful to compare multiple chromatograms. • Useful to compare patterns. • Create Compounds GC Data only Method Editor. • Available for GC and MS Quantitative Analysis only. 45 High Throughput Quantitative Analy
Slide 46: Data Review: Chromatogram Information
Data Review Chromatogram Information Head to head view. Can anchor a chromatogram. 46 High Throughput Quantitative Analysis
Slide 47: Data Review: Chromatogram Information
Data Review Chromatogram Information Head to tail view. Colors are changeable. 47 High Throughput Quantitative Analysis
Slide 48: Data Review: Chromatogram Information
Data Review Chromatogram Information Overlay view. Many other options are available. 48 High Throughput Quantitative Analysis
Slide 49: Auto Review
Auto Review Auto Review Samples • Displays sample by sample. Auto Review Compounds • Displays compound by compound. • Available in Flat Table or Compound Table. • Stop, pause, continue and variable intervals. 49 High Throughput Quantitative Analysis
Slide 50: Demo time
Demo time 50 High Throughput Quantitative Analysis
Slide 51: Outlier Check List
Outlier Check List An outlier is a result that is outside the range of acceptable values for a given parameter. What outliers are important in the workflow? • Retention Time… • Limit of Detection, Quantitation, Method Detection Limit. • Qualifier Ratio. • ISTD Response or ISTD Response Percent Devia
Slide 52: Outliers Setup Tasks
Outliers Setup Tasks Method > Edit >Outlier Setup Tasks Outliers are setup in the Method Editor and are part of quantitation method. 52 High Throughput Quantitative Analysis
Slide 53: Outliers Setup Tasks
Outliers Setup Tasks ~ 48 Outliers are available. Outliers are not calculated unless values have been set up. Outliers are used to perform automated quality checks. Aids in data review by highlighting problem areas. Increases confidence in data integrity by utilizing outliers. Which outliers are imp
Slide 54: Outliers
Outliers • Most Outliers have both a low and high limit. • Both limits need to be set for the outlier to be calculated. • Some outliers are one dimensional. • A few only have a single limit. 54 High Throughput Quantitative Analysis
Slide 55: Outliers: Batch Table
Outliers Batch Table Icons on the toolbar. Select Outliers Turn off outlier filter Display rows that have High/Low outliers Display rows that have High outliers Display rows that have Low outliers Display rows that have no outliers 55 High Throughput Quantitative Analysis
Slide 56: Outliers: Messages and Outliers
Outliers Messages and Outliers Select Outliers for Display Red Outlier – High Blue Outlier – Low (above upper limit) (below lower limit) Messages Outliers Hover cursor over the outlier or message to display details 56 High Throughput Quantitative Analysis
Slide 57: Outliers: Filter on Outliers in Batch Table
Outliers Filter on Outliers in Batch Table By default all outliers are displayed but it is possible to limit this to a subset or single outlier for a more targeted data review pass. Remember, an outlier is not active unless a limit has been specified for it in the Method Editor . 57 High Throughput
Slide 58: Outliers: Default outliers
Outliers Default outliers By default these outliers are enabled. If an outlier is not enabled, it is not calculated. Accuracy = Calculated/Expected x 100 58 High Throughput Quantitative Analysis
Slide 59: Outlier Help
Outlier Help Many Outliers also have Quant videos. 59 High Throughput Quantitative Analysis
Slide 60: Metric Plot
Metric Plot Right click (on header) > Plot this Column. Right click (in the plot window) > Show Average/Std Dev lines. 60 High Throughput Quantitative Analysis
Slide 61: Compounds at a Glance
Compounds at a Glance View > Compounds-at-a-Glance Custom layouts can be loaded and saved. 61 High Throughput Quantitative Analysis
Slide 62: Compounds at a Glance
Compounds at a Glance Layout > Setup Layout Select various Qualifiers Show panes with or without outliers High Throughput Quantitative Analysis 62
Slide 63: Compounds at a Glance
Compounds at a Glance Data Review Sample Approved box for QA. Display outliers by category. 63 High Throughput Quantitative Analysis
Slide 64: Compounds at a Glance
Compounds at a Glance Review Sample by Sample • Can be reviewed Sample by Sample. • Optional Pane Dimensions • Can scroll through the samples. • Once the layout is saved it can be loaded time after time. • Numerous Predefined Layouts 64 High Throughput Quantitative Analysis
Slide 65: Compounds at a Glance
Compounds at a Glance Review Compound by Compound • Layout > Setup Layout or from • Layouts can be customized. • Can be reviewed Compound by Compound. • Various Overlay modes. • Various Display Options. • Can synchronize Compounds at a Glance with Quantitative Analysis. • Right click Properties > Sy
Slide 66: Demo time
Demo time 66 High Throughput Quantitative Analysis
Slide 67: Summary
Summary • Target Compound Analysis • Quant Method Checklist • RT setup and RT Criteria • Reference and Non-Reference windows • Integrators • Peak Filters • Adjusting Peak Filter Area Thresholds Zero Peak Below LOD • Correlation window • Data Review • Filtering on Sample Type, Sample Group and Auto R
Slide 68: Training Resources
Training Resources Available Training Resources Convenient Training In our classrooms, at your site or online. From a team of industry experts that deliver a high quality learning experience. Classroom Training Introductory level to in-depth, hands-on for laboratory instrumentation and software. Cus
Slide 69: Agilent University
Agilent University Access From Home Page Upgraded customer experience Search and find courses that meet your interests and needs in the format they require. Introduce new eLearning capabilities Recorded and video-based learning Virtual online classes Expanded portfolio Foundational subjects Intermed
Slide 70: Agilent Community
Agilent Community Agilent Community community.agilent.com Get answers. Share insights. Build connections. Collaborate - Ask and answer questions. Connect - Interact with other Agilent users. Discover - Find relevant discussions, documents, and videos. Share - Contribute your insights. 70 High Throug
Agilent Technologies — Technical Presentation (July 2023) on 6ix