Manual Integration 2012 [Compatibility Mode]

8/11/2019 Manual Integration 2012 [Compatibility Mode]

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MANUAL INTEGRATION OF CHROMATOGRAPHIC PEAKS

Understanding The Requirements For Performing Manual Integration


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Manual Integration and Technical Ethics

Improper Manual Peak Integration Can Be Interpreted As

Data Fraud. Laboratories Are Required To Provide and Document

Analysts Training on Manual Peak Integration. Accutests Ethical Conduct Agreement States:

I shall not intentionally modify data values unless themodification can be technically justified through ameasurable analytical process

Analysts must be trained to perform manual integrationusing a technically justifiable, measurable analyticalprocess


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Recognizing Unethical Manual Integration

Prohibited Cases

Prohibited for symmetrical peaks that exhibitacceptable baseline to baseline machine integration.

Prohibited for the sole purpose of adding area to meetQC acceptance criteria.

Prohibited for the sole purpose of subtracting area tomeet QC acceptance criteria.


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Understanding Chromatographic Peak Integration

Machine Integration Parameters

Peak Area Reject. Establishes the detection threshold in areaunits for detecting and integrating a chromatographic peak.

Peak Width. Establishes a minimum half height peak width

(in scans or minutes) for detecting a chromatographic peak.Establishing the width at too large a value filters out narrow peaks.

Initial Threshold. The slope value selected that indicates the beginning (start threshold) or end (stop threshold) of achromatographic peak. Selecting too low of a threshold valueresults in integration of background noise.


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Machine Integration Parameters (contd)

Shoulder Detection. In off mode, does not drop baselinetangents for unresolved chromatographic peaks. Manualintegration will always be required.

Data Point Sampling. The number of data points persampling used to perform any integration relateddetermination. Peaks from capillary columns usually employone data point per sampling.

Smoothing. The smoothing option prevents the splitting of bumpy chromatographic peaks and eliminates the integrationof noisy baselines. Smoothing is always turned on.

Understanding Chromatographic Peak Integration


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The Machine Integrator Sometimes Does Not Integrate

Appropriately in Situations Where Peak Integrity Is Marginal .

Missed Peak: Software does not integrate a peak, or integratesthe wrong peak. May be due to low concentration or peaks

with similar retention times

Overlapping Chromatographic Peaks. The retention time oftwo compounds are close to each other causing an overlap ofthe two. Both peaks may be integrated as one.

Acceptable Cases for Manual Integration


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Split Peak: Inflection point causes software to split the peak

resulting in incomplete integration

Acceptable Cases for Manual Integration

Poorly Defined Baselines: Matrix effects cause an erratic baseline that results in too little or too much peak area being

selected. Commonly occurring in PAH analysis.

Baseline Ripple: A sudden spike or dip in a normal baseline thatcauses integration difficulties, usually in ECD or HPLC analysis.

Poor Instrument Integration: Improper integration that cannot be defined by any of the above reasons


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A Word of Caution* Excessive amounts of Manual integrations, evenif they are done properly and for the right reasonsmay be a cause for suspicion. Manual integrationsshould be kept to a minimum whenever possible.

Manual integrations should not be used as asubstitute for proper instrument maintenance,updating retention times or proper configuration ofmachine integration parameters.

Manual integrations in your calibration standard

may be an indication of instrument problems.


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Use The Same Techniques For Manual Integration Thatare Automatically Used For Machine Integration .

Baseline to Baseline Integration . The peak integration baselineis a continuation of the existing baseline. Begin integrationwhen the signal first begins to rise above the baseline and endwhen the signal returns to the original baseline.

Baseline to Valley Integration . (Baseline Projection) Thisintegration is used for two overlapping chromatographic peaks.

Draw a vertical line from the valley of the two peaks to theoriginal baseline. Integrate the first peak from when the baselinestarts to rise, and end at the vertical line. Integrate the second

peak from the vertical line to when the signal returns to theoriginal baseline.

Drawing A Manual Baseline


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Baseline to Valley Integration . (Tangent Skim) Thisintegration is used for two overlapping chromatographic peaks.Draw a diagonal line from the valley of the two peaks to the

baseline. Integrate the first peak from when the baseline starts torise, and end at the valley. Integrate the second peak from thevalley to when the signal returns to the original baseline.When deciding whether to use baseline projection or tangentskim, operator judgment and experience are critical. Check tosee how the calibration standards were integrated and use thesame method. If unsure always ask your supervisor.

Drawing A Manual Baseline


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Drawing a Manual Baseline

Valley to Valley Integration . This integration is usedfor multiple overlapping chromatographic peaks. Atangent is dropped from the valley of each intersecting

peak to the original baseline. The first and last peaks areintegrated baseline to valley as before. All other peaksare integrated Valley to Valley.

Consistency of Execution . Manual integration can besubjective. It is imperative that manual integration is performed using a consistent approach. Standards,Samples and QC must be integrated the same way.


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Common Integration Situations: Easy Hard

Simple Integration

Examples of Unacceptable Integration

Overlapping Peaks

Multiple Overlapping Peaks


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Single Peak


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Baseline to Baseline Integration


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Incorrect Integrations


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Overlapping Peaks


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Incorrect Integration


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Baseline to Valley Integration/ Tangent Skim


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Baseline to Valley Integration/Baseline Projection


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Split Peak


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Correct Integration


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Baseline Ripple


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Correct Integration


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Overlapping Peak/ Shoulder


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Correct Integration


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Multiple Overlapping Peaks


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Valley to Valley Integration


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Both an initial and a final Extracted Ion Current Profile(EICP) must be generated for any peak that is manuallyintegrated.

The Analyst must acknowledge the Manual Integration inone of two ways:If a hard copy is printed then the analyst must initial, dateand list the justification for the manual integration or If electronic chromatograms are used the analyst must list the

peak number and justification in LIMS.

Review and Documentation


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The following list of codes may be used for justification:#1 Poorly Defined Baseline#2 Split Peaks

#3 Overlapping Peaks#4 Missed Peak #5 Baseline Ripple#6 Poor Instrument Integration#7 Add on Compound Quantitation



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Manually integrated peaks must be reviewed andacknowledged by the supervisor or team leader to ensurethey are correct and justified.

The chromatograms are stamped indicating the reviewersidentity and date and time of approval.


Documents

Manual Integration 2012 [Compatibility Mode]