TIC TAC TIC TAC T ET ETIC, TAC, TIC, TAC, ToEToE

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

g g

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

p

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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Reporting/Project Viewingp g/ j g

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A Tour of the Console

Signal filtration utility

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A Tour of the Console

Component Table

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A Tour of the Console

ExperimentsExperiments

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A Tour of the Console

Composite

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Chromatogram

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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Functional Links

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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

p

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.

Extracted Component Spectrap p

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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

Acknowledgementsg

Patrik Petersson, AstraZenecaPatrik Petersson, AstraZenecaGang Xue, PfizerGang Xue, Pfizerg ,g ,Rudy Sneyers and Willy Janssens, Johnson Rudy Sneyers and Willy Janssens, Johnson and Johnsonand Johnson

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