GUIDANCE FOR SPECTRAL ANALYSIS USING Waters PDA WITH  Empower

Written by Dr. Shulamit Levin

Waters Israel.

Definitions:

                The spectral contrast angle  measures the shape difference between two spectra. It is the basis for all spectral  comparisons in the software:




                The Purity angle is a measure of the spectral heterogeneity of a peak based on the comparison of spectra over all the peak, using the spectral contrast angle

                The non-ideal effects are quantified and provided as a value of the Threshold Angle.

                When the peak is pure, the Purity Angle < Threshold Angle and Match Angle < Match Threshold

 

1)    SPECTRAL ANALYSIS Non Regulated

(Not for validation of stability indicating chromatographic method!).

1-1 Instrumental Considerations:

                   Choose the spectral resolution preferred for the particular compounds.  Preferably use 1.2 nm resolution to distinguish between fine features of the UV-VIS spectrum.  PDA Peak Purity measurements are not recommended for poor UV spectral shaped molecules.  .

                   Choose the spectral range for the acquisition (nm).  When Peak Purity is measured for Assay methods, make sure that it includes at least 10 x wavelength resolution values below and above the assay's wavelength.

                   Select the appropriate acquisition rate (Number of spectra per minute) to make sure that there will be at least 15 points across the peaks.  For HPLC, generally 1-5 spectra/sec is sufficient, however, when fast chromatography is used 5-20 spectra/sec might be preferable (note the double storage memory needed).

 

1-2 Experimental Considerations

                   If available, make sure to have a standard injected at concentrations that will give 0.5-1 AU in the MaxPlot chromatogram, to assure linear relation between concentration and Absorbance.  Make sure that the sample gives peaks at similar responses if available. 

                   The standards and the unknowns should be injected in the same session under the same conditions exactly, to be able to evaluate the values of  Threshold angle and Purity Angle.

                   Make sure to include a blank run, to be able to subtract the 3D data in case of problematic results.

1-3 Software Considerations

                    Create a method set that uses Maxplot derived channel and apply it for all the channels under study.

                    Check carefully the entire chromatogram for peak-less time interval in the chromatogram, minimum of 30 points or seconds long, to assign as the Noise Interval.

                    Create an appropriate PDA Processing Method that enables Peak Purity, set the appropriate wavelength range (cut the noisy low UV range or too quiet high UV range).   Set the Noise Interval and the criteria for Threshold Determination.  The Auto-threshold will be applicable to most cases.

1-4 Results can be observed in the following places:

Peaks tab at the bottom table in the main window of the Review.

Results window, the top table, use the Peak Purity tab.  Also, select the Purity Plot at the bottom, to observe the results graphically.

Using the Result view use the Preview tool, select an existing PDA report or create one with the report wizard, in which you can select the PDA Default Report.

 


 

2)    SPECTRAL ANALYSIS- Regulated

(For validation of stability indicating chromatographic method!).

2-1 Instrumental Considerations:

                   If available, make sure to have a standard injected at concentrations that will give 0.5-1 AU in the MaxPlot chromatogram, to assure linear relation between concentration and Absorbance.  Make sure that the sample gives peaks at similar responses if available. 

                   The standards and the unknowns should be injected in the same session under the same conditions exactly, to be able to evaluate the values of  Threshold angle and Purity Angle.

                   Make sure to include a blank run, to be able to subtract the 3D data in case of problematic results.

2-2 Experimental Considerations: Sensitive Threshold Determination

                     Make sure to have 5-6 replicates of the standard for the Peak Purity analysis and 4 duplicates of the standard at 50%, 75% , 100% and 120% of the assay's concentration for the spectral matching.

                     The unknowns are stressed samples of the main component's API as well as the formulation (if applies).  It is recommended to inject duplicates.  The samples stressed by heat, light, acid, base and oxidizing agents according to current regulations.  Make sure to include their blank medium.

                     The standards and the unknowns should be injected in the same session, under the same conditions exactly, to be able to evaluate the values of  Threshold angle from the Purity Angles of the standards. 

                     Make sure to include a blank run (diluent, mobile phase and medium of the stress) as well as the placebo, to be able to subtract the 3D data in case of problematic results.

2-3 Software Considerations

                   Create a method set that uses Maxplot derived channel and apply it for all the channels under study. 

                   Apply the method set on all the runs, superimpose the chromatograms in the Results view window, and check carefully the entire chromatogram for Peak-less baseline interval in all the chromatograms, minimum of 30 points or seconds long, to assign as the Noise Interval for all the runs. 


 

2-4 Procedure: Peak Purity

Create an appropriate PDA Processing Method that enables Peak Purity, set the appropriate wavelength range (cut off the noisy low UV range or straight line at the high end of the UV-VIS range).  

Set the Noise Interval and the criteria for Threshold Determination in the Purity tab.  The Auto-threshold might not be applicable to all cases.  For method validation in a regulated environment it is recommended to use the sensitive Threshold angle determination:

1.    Select the 6 standard replicates and process them, using the method set. Check the results for Purity Angle of all 6 runs.  Select the maximum value and set it in the processing method under Solvent and Noise criteria for Threshold determination.

2.    Process the 6 replicates again and verify that all standards came out pure (Purity Angle < Purity Threshold Angle).

3.    Process the unknowns and use an appropriate PDA report to report their results.  You can use the Purity Plots in the report.  If the Purity angle is smaller than the Threshold angle, the peak is pure.

2-5 Procedure: Spectral Matching

       1.           A UV-VIS library MUST be created for EACH experiment.  Select one of each duplicate of standard concentrations (50%, 75%, 100% and 120%), and create a new spectral library.  Make sure to indicate the standard source and level or concentration in its name for information. 

       2.           Mark the library name in the Processing Method at the PDA Spectral match tab.  Select in this window the depth of search 3 and the degree of match (in deg).  Indicate if you are interested in RT presearch.

       3.           Select the second channel in the duplicates from each standard concentration and process it using the method set with the updated processing method.  Check Match 1 angle of all 4 channels and select the maximum value from all match angles (3 results each channel), and use it in the Processing Method as the Threshold angle for the Spectral Matching.  Sometimes it is better to use also Match 2 or Match 3 angles.

       4.           Process the 4 Channels again, using the method set with the updated Processing Method.  Verify that the results indicate that they all are identified appropriately (Match angle<Threshold angle).

       5.           Process all the unknowns using the same method set with the Processing Method with the sensitive Thresholds in both Purity and Spectral Match tabs.  When Purity Angle is lower than Purity Threshold Angle, as well as Match angle is smaller than the Match Threshold angle the Peak is considered as Pure and appropriate for stability studies.

 

 

2-6 Results can be observed in the following places:

Peaks tab at the bottom table in the Main window of the Review.

Results window, the top table, use the Library match tab.  Also, select the Match Plot at the bottom, to observe the results graphically.

Using the Result view use the Preview tool, select an existing PDA report or create one with the report wizard, in which you can select the PDA Default Report.

Points to Note:

        The Noise interval must be free of any peaks in all the chromatograms.

        The noise interval in gradients must be near the studied peak.

        Non linearity of Beer's law will result in wrong results and unusual behavior.

        Integration events can affect the results; make sure to use them appropriately.

        Compounds without UV-VIS featured spectrum might provide false results of peak purity.

 

References:

Detecting Coeluting Impurities by Spectral Comparison, Gorenstein, Marc V. et al, LC/GC, 1994   Volume:   October