Regulatory Aspects of HPLC analysis (System Suitability)
Regulatory Aspects of HPLC analysis (System Suitability)
©2005 Waters Corporation
Introduction
What is System Suitability?
• A way of checking that an entire chromatography system is working within acceptable limits– For a single day, or tracking and trending over time
• Set of Samples to test the system at the point of use– Part of the Assay Sample Set– A separate Sample Set
• To determine parameters such as– Injector performance– Column Performance– Preparation of mobile phase– Preparation of standards
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System Suitability
• Uses standard laboratory practices and calculations– United States Pharmacopoeia XXVIII, 2005– European Pharmacopoeia– Japanese Pharmacopoeia
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System Suitability
• Many calculations automatically performed– Tailing Factor– Resolution– Plate Count– Signal to Noise and EP Signal to Noise
• Custom Calculations can be created– Injection Ratio– 2 % Area difference comparison– % RSD (plus stop on fault)– Detection Limit (report either Below Detection limit or Amount)
• Viewed in Review• Reported in Preview
– Trend Plots (Bar, Line, Scatter)– % RSD Plots– Overall % RSD Plots– Peak Plots
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Sample Sets
Sample sets typically contain:
• Blank, Standard A, Standard B, Standard Check
• Can be run as part of the routine sample set or as separate from routine sample set
• If run separate, the sample sets can be queued to run sequentially with no analyst intervention
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Sample Sets
System Suitability Samples
Assay Samples
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Processing Methods
• Enabling System Suitability
• If you want system suitability to be calculated you will need to tell the processing Method to Calculate system suitability
• Open the processing Method and select the Suitability Tab
• Check the “Calculate Suitability Results” box.
• Complete the other sections as required.
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Processing Methods
Select Suitability Tab
Check Box Calculate Suitability Box
If you want to calculate baseline noise, type the time you want to record noise
Select which Pharmacopoeia you wish to use
Select to calculate S/N or EP S/N
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Noise and Drift
• Baseline Noise – obtained by averaging the maximum change in signal over two sections of baseline (specified by the user in the Processing Method) and is expressed in millivolts (mV).
• Baseline Drift – the signal value at the “Baseline Start Time” subtracted from the signal value at the “Baseline End Time” – also expressed in mV.
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Automating System Suitability
• Limits
• User-defined acceptance limits can be set on any results
• Results outside of the range will be displayed as ‘faulted’– Fault field set– Fields are red (customizable) in the Project Window and Review– Fields are bolded on reports (tabular and graphical data)
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Setting Limits
• Open the processing Method and select the Limits Tab
• Check the “Flag Values outside Limits” box. This will highlight the Faulted values in the review tables
• Select the component you wish to apply a Limit(s) to.
• Select the parameter(s) you wish to monitor and assign a value(s) to that parameter(s)
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Processing Method - Limits
Select Limits Tab
Check “Flag Values outside Limits” Box
Select which Component you wish to apply Limit(s) to
Select the parameter(s) you wish to monitor and assign a value(s) to that parameter(s)
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Processing Method – Noise and Drift
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Noise and Drift Calculations
• These calculations attempt to mimic the ASTM method for Detector Noise and Drift measurements which is typically performed by hand.
• A least-squares (“best fit”) line is fitted to the whole time interval (specified in the processing method) of the data and is used to perform all calculations.
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Noise and Drift Calculations
• •Detector Noise - The root mean square (RMS) noise of the data expressed in detector units.
• Peak-to-Peak Noise - the algebraic difference of the maximum and minimum residuals between each data point and the least-square line expressed in detector units.
• Detector Drift - the slope of the least-squares line expressed in detector units per hour.
• Average Detector Noise - Average Detector Noise is calculated bydividing the data into segments (specified in the processing method) and averaging the values for each segment.
• Average Peak-to-Peak Noise - Average Peak to Peak Noise is calculated by dividing the data into segments (specified in the processing method) and averaging the values for each segments.
• Average Drift - Drift is expressed in detector units per hour. Average Drift is calculated by dividing the data into segments (specified in the processing method) and averaging the values for each segments.
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Calculations
• Empower calculates over 50 system suitability results– Resolution– USP Tailing– Symmetry Factor– EP Plate Count– Width determinations
• These are documented in Help and the System Suitability Quick Reference Guide (available on our website)
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System Suitability Custom Fields
• Custom Fields allow you to calculate many values specific to the way you work
• Examples– Sample Injection Repeatability
%RSD of AreasStop on Fault, Reinject on Fault, etc.
– EP Signal to Noise using a blank injectionHeight/Baseline Noise of blank injection
– % Area of Active after rejection of peaks < 0.05% of total area– Standard Accuracy
Average peak area of Std 1 (for multiple injections) / Average peak area of Std 2 (for multiple injections) * 100
– Suitability Acceptance Fields
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Standard Accuracy
Avg Area Std1Avg Area Std2
Ratio must meet certain
criteria
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Suitability Acceptance Fields
% Agree of SS check
and 1st Std
Overall%RSD of
Stds
% RSD of Sys Suit Checks
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Review
• Reviewing System Suitability values
• Take the processed results in to review
• Click on the Peaks Tab– View the Peaks information
ie Tailing factor, Resolution etc
• Click on the Results Window– View the Results information
Basline Noise
• Click on the Processing Method– View the Prcessing Method and modify if required
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Click on Results Window icon to display results Information
Click on Peaks Tab to view Peak information, note the values outside limits
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Click on Processing Method Window icon to display Processing Method Information
Results information such as Baseline noise
Peak and calibration Information is also displayed in this window
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Reviewing Results
Peaks Tab allows you to easily see your peak results without opening Review
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Preview - Reporting
• Reporting• System Suitability reports
– Tailing Factor – Repeatability of injection– etc
• Trend Plots– Tailing Factors, Resolution over the life of the Column– Assay Values
Showing Limits, % RSDs, Overall % RSDsBar Plots, Line Plots, Scatter Plots
• Methods
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Individual System Suitability Report
Peaks table showing system suitability values and flagging any outside Limits
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Repeatability of Injection Report
Peaks table and Bar Graph showing Retention times of all Standard A injections
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Repeatability of Injection Report
Peaks table and Line/Scatter Graph showing Area of all Standard A injections - note the Outside Limit for %RSD
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Flagging Values Outside Limits
With a Table
With a Graph
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Processing Method Report
System suitability Limits are shown in a table
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Self Checking Suitability Reporting
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Self Checking Suitability Reporting
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