aspects of hplc analysis (system suitability) · • •detector noise - the root mean square (rms)...

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


System Suitability

• Uses standard laboratory practices and calculations– United States Pharmacopoeia XXVIII, 2005– European Pharmacopoeia– Japanese Pharmacopoeia


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


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


Sample Sets

System Suitability Samples

Assay Samples


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.


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


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.


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)


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)


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)


Processing Method – Noise and Drift


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.


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.


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)


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


Standard Accuracy

Avg Area Std1Avg Area Std2

Ratio must meet certain

criteria


Suitability Acceptance Fields

% Agree of SS check

and 1st Std

Overall%RSD of

Stds

% RSD of Sys Suit Checks


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


Click on Results Window icon to display results Information

Click on Peaks Tab to view Peak information, note the values outside limits


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


Reviewing Results

Peaks Tab allows you to easily see your peak results without opening Review


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


Individual System Suitability Report

Peaks table showing system suitability values and flagging any outside Limits


Repeatability of Injection Report

Peaks table and Bar Graph showing Retention times of all Standard A injections


Repeatability of Injection Report

Peaks table and Line/Scatter Graph showing Area of all Standard A injections - note the Outside Limit for %RSD


Flagging Values Outside Limits

With a Table

With a Graph


Processing Method Report

System suitability Limits are shown in a table


Self Checking Suitability Reporting


Self Checking Suitability Reporting

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aspects of hplc analysis (system suitability) · • •detector noise - the root mean square (rms)...

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