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hwww.acdlabs.comwww.acdlabs.com
Composite Chromatograms as a Summary of the Contents of a Sample or Set of
Samplesp
Mike McBrien, Graham McGibbon, and Mike McBrien, Graham McGibbon, and Andrey VazhentsevAndrey VazhentsevAndrey VazhentsevAndrey Vazhentsev
PittconPittconMarch 9, 2009March 9, 2009
Advanced Chemistry Development, Inc. (ACD/Labs)
Outline
Scope of applicationScope of applicationScope of applicationScope of applicationSummarizing elution:Summarizing elution:
the downside of overlaysthe downside of overlaysthe downside of overlays…the downside of overlays…An extension of overlays An extension of overlays –– the composite the composite chromatogramchromatogramchromatogramchromatogramThe power of linked chromatogramsThe power of linked chromatogramsIllustrative examplesIllustrative examplesIllustrative examplesIllustrative examplesA project viewer for chromatographic A project viewer for chromatographic systemssystems
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systemssystems
Why do we inject?y j
Routine quantitationRoutine quantitationf lf l
outside scopeoutside scope
Comparison of samplesComparison of samplesDetection of unknowns/Detection of unknowns/knownsknownsMethod developmentMethod development
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How do we (typically) display chromatographic information?chromatographic information?
Peak tablesPeak tablesPeak tablesPeak tablesTotal Ion Chromatogram Total Ion Chromatogram –– TICTICT t l Ab b Ch t T t l Ab b Ch t TACTACTotal Absorbance Chromatogram Total Absorbance Chromatogram -- TACTACOverlays of chromatogramsOverlays of chromatogramsIt can be difficult to see the links between It can be difficult to see the links between peak tables and overlaid chromatograms peak tables and overlaid chromatograms –– 3 3 detectors and 7 related samples = 21 detectors and 7 related samples = 21 detectors and 7 related samples = 21 detectors and 7 related samples = 21 traces!traces!
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Linking Chromatograms
Typical Chromatography Data Systems link Typical Chromatography Data Systems link
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Typical Chromatography Data Systems link Typical Chromatography Data Systems link chromatograms for a given injection.chromatograms for a given injection.Multiple injections may have equivalent Multiple injections may have equivalent Multiple injections may have equivalent Multiple injections may have equivalent value.value.Visualization of the links can be very Visualization of the links can be very yyeffective.effective.Functional application of the links can Functional application of the links can facilitate decisionfacilitate decision--making.making.
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Again - Why Do We Inject?g y j
C ll t d t t t t i f tiC ll t d t t t t i f tiCollect data to extract information:Collect data to extract information:Peak Peak ttRRss, widths, areas, asymmetries., widths, areas, asymmetries.Extracted spectra for components.Extracted spectra for components.
Processing converts data to info:Processing converts data to info:PeakPeak--pickingpickingCalibrationCalibrationLabelingLabelingExtraction of spectra for peaks.Extraction of spectra for peaks.
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a o o sp a o p a sa o o sp a o p a s
Chromatographic MethDev
Some LC/MS/UV projects can be 2 gigabytes Some LC/MS/UV projects can be 2 gigabytes
g p
Some LC/MS/UV projects can be 2 gigabytes Some LC/MS/UV projects can be 2 gigabytes or more.or more.Most of this data is highMost of this data is high--frequency and lowfrequency and low--Most of this data is highMost of this data is high frequency and lowfrequency and lowfrequency noisefrequency noiseKey information may be very limited Key information may be very limited –– ttRRss, , y y yy y y RR ,,widths, asymmetries, spectrawidths, asymmetries, spectraWe need a fast way to get access to all of We need a fast way to get access to all of the extracted info without browsing through the extracted info without browsing through gigabytes of material.gigabytes of material.
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The Composite ChromatogramChromatogram
“Total of Experiments” for “Total of Experiments” for “Total of Experiments” for “Total of Experiments” for a given set of conditionsa given set of conditionsConcise report on the Concise report on the KnowledgeConcise report on the Concise report on the pertinent information pertinent information regarding a sample or a regarding a sample or a
g
regarding a sample or a regarding a sample or a set of related samplesset of related samplesAtAt--aa--glance view of glance view of
Information
ggsituationsituation--specific infospecific infoCustomizable to the Customizable to the
Data
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question at hand.question at hand.
Data Hierarchyy
ProjectProjectM h d M h d
jj
WavesWavesWaves
Method Method DevelopmentDevelopment
SpecificSpecificWavesWavesWaves pp
ExperimentsExperimentsExperiments
SubsamplesSubsamplesSubsamples
‹#›DetectorsDetectors
Data Hierarchyy
ProjectProject
ExperimentsExperimentsExperiments
Collapse into Collapse into
SubsamplesSubsamplesSubsamples
Collapse into single trace Composite
Collapse into single trace Composite
DetectorsDetectors
Composite ChromatogramComposite Chromatogram
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The “SpecMan Console”p
Ch t / t l ViCh t / t l ViChromatogram/spectral ViewerChromatogram/spectral ViewerViews only the relevant information Views only the relevant information –– the the
it h tit h tcomposite chromatogramcomposite chromatogram
ExperimentsExperimentsAll sets of chromatographic conditions All sets of chromatographic conditions investigatedinvestigated
Component TableComponent TableAll the components across the projectAll the components across the project
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The Component Tablep
S f ll t i th S f ll t i th Summary of all components in the Summary of all components in the project.project.Reject/relevant/irrelevant.Reject/relevant/irrelevant.For a given detector trace, the For a given detector trace, the g ,g ,component may/may not be detected.component may/may not be detected.
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Subsamples
It is commonplace to inject multiple vials It is commonplace to inject multiple vials
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It is commonplace to inject multiple vials It is commonplace to inject multiple vials under a set of conditions:under a set of conditions:
Reaction mixtures/starting materialsReaction mixtures/starting materialsReaction mixtures/starting materialsReaction mixtures/starting materialsForced degradationForced degradationMetabolism Metabolism Standards/BlanksStandards/Blanks
It’s implied that components will be shared It’s implied that components will be shared p pp por unique.or unique.Chromatographers are typically interested in Chromatographers are typically interested in
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a summary of all of the contents of the vials.a summary of all of the contents of the vials.
Hyperlinks to Raw Data
Data processing inherently removes some Data processing inherently removes some
yp
Data processing inherently removes some Data processing inherently removes some data.data.Information can be lost in any filtration Information can be lost in any filtration Information can be lost in any filtration Information can be lost in any filtration process.process.The user must retain access to the data until The user must retain access to the data until The user must retain access to the data until The user must retain access to the data until the project is complete.the project is complete.The drillThe drill--down approach can also offer full down approach can also offer full The drillThe drill down approach can also offer full down approach can also offer full access to the hyphenated data.access to the hyphenated data.
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Interactivity of the Composite Chromatogram
Th it h t i Th it h t i
Chromatogram
The composite chromatogram is a The composite chromatogram is a dynamic view:dynamic view:
Reject componentsReject componentsSelect/unselect detectorsSelect/unselect detectorsSelect/unselect subsamplesSelect/unselect subsamples
View extracted spectraView extracted spectraTrigger new processes and view resultTrigger new processes and view result
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ExamplesBl k
Probably the most common subsampleProbably the most common subsample
Blanks
Probably the most common subsample.Probably the most common subsample.Components attributed to the dissolving Components attributed to the dissolving medium or artifacts attributed to the medium or artifacts attributed to the medium, or artifacts attributed to the medium, or artifacts attributed to the method can be identifiedmethod can be identifiedIn method development these components In method development these components In method development, these components In method development, these components can be designated “irrelevant” and modeling can be designated “irrelevant” and modeling can take this into account.can take this into account.In some workflows, formulation components In some workflows, formulation components will be included with the blank.will be included with the blank.
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ExamplesP St d d
In reaction monitoring and many MethDev In reaction monitoring and many MethDev
Pure Standards
In reaction monitoring and many MethDev In reaction monitoring and many MethDev projects, pure standards will be availableprojects, pure standards will be availableConfirmation of identity is helpful in peak Confirmation of identity is helpful in peak Confirmation of identity is helpful in peak Confirmation of identity is helpful in peak tracking, etc.tracking, etc.MethDev (and visualization) can be done MethDev (and visualization) can be done MethDev (and visualization) can be done MethDev (and visualization) can be done without all anticipated compounds present in without all anticipated compounds present in the sample at hand.the sample at hand.pp
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ExamplesF d D d tiForced Degradation
Typically, 3Typically, 3--7 subsamples.7 subsamples.Blank, several sets of degradation conditions.Blank, several sets of degradation conditions.(Typically) elaborate method development (Typically) elaborate method development projects.projects.UV, MS peak tracking.UV, MS peak tracking.UV area is used to filter MS components.UV area is used to filter MS components.Shared components are critical.Shared components are critical.
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ExamplesM t b li
Typically an array of metabolism conditions Typically an array of metabolism conditions
Metabolism
Typically an array of metabolism conditions Typically an array of metabolism conditions will be examined under a set of conditions:will be examined under a set of conditions:
Feces blood urineFeces blood urineFeces, blood, urine…Feces, blood, urine…Shared components across subsamples?Shared components across subsamples?M ltiple detecto sM ltiple detecto sMultiple detectorsMultiple detectors
Selected Ion Monitoring tracesSelected Ion Monitoring tracesMS/MS d MS/MS d di tdi tMS/MS and MS/MS and radiotracesradiotraces
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Hyphenated Data Managementyp g
Links to full datafiles are retainedretained
Peak table information is extracted directly
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extracted directly…
Hyphenated Data Managementyp g
…and summarized in an overall peak table.
Peak table information is extracted directly
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extracted directly…
Composite Chromatogram Viewp g
S b l 1S b l 1Subsample 2Subsample 2Subsample 3Subsample 3Composite SampleComposite SampleSubsample 1Subsample 1Subsample 2Subsample 2Subsample 3Subsample 3Composite SampleComposite Sample
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The Power of Linked ChromatogramsChromatograms
Decisions are rarely made based on a single Decisions are rarely made based on a single ec s o s a e a e y ade based o a s g eec s o s a e a e y ade based o a s g epiece of information; e.g.:piece of information; e.g.:
Does this MS peak have a significant UV Does this MS peak have a significant UV ??area?area?
Is there a peak on the Is there a peak on the radiotraceradiotraceassociated with this TIC peak?associated with this TIC peak?associated with this TIC peak?associated with this TIC peak?Is this component also present in my Is this component also present in my blank?blank?blank?blank?Are these isotope patterns coeluted in Are these isotope patterns coeluted in other runs?other runs?
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Reconcile Detectors
Components may have signal in multiple Components may have signal in multiple Components may have signal in multiple Components may have signal in multiple detectors. detectors. How do we link the peaks between How do we link the peaks between How do we link the peaks between How do we link the peaks between detectors?detectors?Manually: Copy/paste label in each traceManually: Copy/paste label in each traceManually: Copy/paste label in each traceManually: Copy/paste label in each traceAutomatically: reconcile based on retention Automatically: reconcile based on retention index/detector lagindex/detector lagindex/detector lag.index/detector lag.If sufficient data is contained in the project, If sufficient data is contained in the project, detector lag can be automatically obtained.detector lag can be automatically obtained.
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detector lag can be automatically obtained.detector lag can be automatically obtained.
Detector Lag
Even with parallel detectors there is some Even with parallel detectors there is some
g
Even with parallel detectors, there is some Even with parallel detectors, there is some offsetoffsetAccurate reconciliation necessitates Accurate reconciliation necessitates Accurate reconciliation necessitates Accurate reconciliation necessitates determination of this volumedetermination of this volumeWith a sufficient number of peaks every With a sufficient number of peaks every With a sufficient number of peaks, every With a sufficient number of peaks, every injection of every subsample can create a injection of every subsample can create a powerful capacity to determine detector lagpowerful capacity to determine detector lagp p y gp p y g
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Reconcile Subsamples
Which components are shared between the Which components are shared between the
p
Which components are shared between the Which components are shared between the vials?vials?Question applies to:Question applies to:Question applies to:Question applies to:
Method developmentMethod developmentMetabolismMetabolismMetabolismMetabolismForced degradationForced degradation
Console looks for coConsole looks for co--elution across all elution across all Console looks for coConsole looks for co elution across all elution across all experiments, like detector reconciliation…experiments, like detector reconciliation…
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Extracted Spectra
Hyphenated detection offers spectral Hyphenated detection offers spectral
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Hyphenated detection offers spectral Hyphenated detection offers spectral information for every peak.information for every peak.The value/accuracy of the spectra can be The value/accuracy of the spectra can be The value/accuracy of the spectra can be The value/accuracy of the spectra can be impaired:impaired:
CoCo--elutionelutionCoCo elutionelutionLowLow--frequency noise due to gradientfrequency noise due to gradient
Typical approach is careful userTypical approach is careful user--directed directed Typical approach is careful userTypical approach is careful user directed directed “combine and subtract” “combine and subtract” –– laborlabor--intensiveintensiveChemometrics can extract pure spectra and Chemometrics can extract pure spectra and
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p pp pproject management can store.project management can store.
Filtration
Whi h k h d b t Whi h k h d b t Which peaks are shared between my Which peaks are shared between my detectors? Between the vials?detectors? Between the vials?
Linking peak tables can be extremely Linking peak tables can be extremely powerful.powerful.MS t b filt d b UV MS t b filt d b UV MS components can be filtered by UV MS components can be filtered by UV areas. areas. Blank peaks can be designated i ele antBlank peaks can be designated i ele antBlank peaks can be designated irrelevant.Blank peaks can be designated irrelevant.Peaks that correspond with standards can Peaks that correspond with standards can be instantly labeledbe instantly labeled
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be instantly labeled.be instantly labeled.
Conclusions
Composite chromatograms can provide a Composite chromatograms can provide a Composite chromatograms can provide a Composite chromatograms can provide a fast, clear communication of the postfast, clear communication of the post--processing results of a set of experiments.processing results of a set of experiments.p g pp g pUnder a given set of chromatographic Under a given set of chromatographic conditions, they can relate detectors and conditions, they can relate detectors and samples in a e po e f l mannesamples in a e po e f l mannesamples in a very powerful manner.samples in a very powerful manner.Views can be quickly customized to provide Views can be quickly customized to provide the answer to a question at handthe answer to a question at handthe answer to a question at hand.the answer to a question at hand.Hyperlinks can provide ready access to raw Hyperlinks can provide ready access to raw data for detailed investigation.data for detailed investigation.
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gg