Presentation for the integration of chromatographic peaks with the basics , regulatory approach with warning letters, 483s and references.

1. Deepak Mishra
9/16/2015 1

2.  Peak Integration & Processing
 Integration Process
 Types of Peak Integration
 Controlling the Integration Process
 Challenges of Integration in Chromatography
 Auto Integration v/s Manual Integration
 Regulatory Perspective
 Peak Integration & Data Integrity
 Peak Integration inWarning Letters
 References….
9/16/2015 2

3.  Integration - Process of calculating an area
that is bounded in part or in whole by a
curved line.
 The goal of chromatographic peak
integration is to obtain retention times,
heights and areas of these peaks.
9/16/2015 3

4. 9/16/2015 4

5.  Processing – Process that measures data to
determine the identities and/or amounts of
separated components.
 Processing methods define how software
detects, integrates, calibrates and
quantitates unprocessed, raw data from a 2D
channel.
9/16/2015 5

6. 9/16/2015 6
 The Integration Process consists of the following:
 1) Defines the initial baseline.
 2) Continuously tracks and updates the baseline.
 3) Identifies the start time for a peak and marks this
point with a vertical tick mark.
 4) Finds the apex of each peak, creates a parabolic fit
for the peak top, and stores the retention time.
 5) Identifies the end time for the peak, and marks this
point with a vertical tick mark.
 6) Constructs a baseline.
 7) Calculates the area, height, and peak width for each
peak

7. 9/16/2015 7

8.  Common Integration Baseline Options :
1. Drop Perpendicular
2. Valley toValley
3. Tangential Skim
4. Exponential Skim
5. Gaussian Skim
9/16/2015 8

9.  Figure 1. Baseline Profiles: 1. Drop Perpendicular, 2.Valley to Valley, 3.Tangential Skim, 4.
Exponential skim, 5. Gaussian Skim
9/16/2015 9

10.  Drop Perpendicular :
The addition of a vertical
line from the valley
between the peaks to the
horizontal baseline.
The vertical line is drawn
between the start and
stop points of the peak
group.
9/16/2015 10

11.  Valley toValley
sets start and
stop points at
the valley
between the
peaks, thus
integrating
each peak
separately.
9/16/2015 11

12.  Tangential Skim
 Separate the small peak
from the Parent peak
with separate baseline.
 Parent peak is integrated
from its starting point to
the apparent end of the
peak group.
9/16/2015 12

13.  Small peak baseline starts at the valley
between the peaks & ends where the signal
nears the baseline.
 Area under the skimmed peak is added to the
parent peak not to the skimmed peak.
 Small peak labeled as skim, Shoulder or Rider
peak.
9/16/2015 13

14.  Exponential Skim :
Used to create curvature
in the skim line to
approximate the
underlying baseline of
the parent peak.
9/16/2015 14

15.  Gaussian Skim:
Also referred as new
exponential skim method,
intent to reproduce the
Gaussian shape of the
parent peak.
9/16/2015 15

16.  Following parameters used by processing software :
 PeakThreshold(Detection Threshold or Slope
Sensitivity) - to determine if peak is detected or
not.
 Decreasing slope sensitivity will result in detecting
smaller and broader peaks.
 Height Reject - to set noise rejection.
 All peaks whose heights are below this value will not
be reported.
9/16/2015 16

17.  Peak width (Sampling rate) – sampling rate
(number of data points per second) that the
detector signal is sampled & to set an initial
sampling interval for the integrator to distinguish
peaks from baseline noise.
 Controls the ability of the integrator to distinguish
peaks from baseline noise. In general, increasing the
peak width will result in broader peaks.
 Faster chromatography needs higher sampling rate.
9/16/2015 17

18.  Area Reject- to filter small peaks.
 All peaks whose areas are below this value will not
be reported.
 Shoulder - to specify the algorithm for shoulder
detection.
 shoulders detected using the second derivative of
Peak.
 Shoulders occur when two peaks are so close
together that no valley exists between them.
9/16/2015 18

19.  Bunching (Smoothing)
 Adding several consecutive data points to obtain
an average time slice value equivalent to slower
sampling rate.
 Can also be used to reduce noise in
chromatogram.
9/16/2015 19

20.  Selection of Integration
baseline options.
 Setting initial
integration
events.
9/16/2015 20

21.  Clear separation of peaks – a fundamental
requirement of most accurate integration of
chromatographic peaks.
 If clear separation not achieved, an intelligent
approach of selecting the baseline options thus
maintaining the accuracy of Quantitation to the
most.
 Integration errors calculated using reference
calibration injections.
9/16/2015 21

22. Integration
 Most important step in data analysis part of
Chromatography yet no clear Guidelines available.
 Various errors can occur during integration which
include, but are not limited to, peak splitting,
adding area due to a coeluting interferant, failure to
detect a peak, excessive peak tailing due to failure
of the instrument response to return to baseline or a
rise in the baseline, and failure to separate peaks
9/16/2015 22

23.  The drop and Gaussian skim methods produce
the least error in all situations.
 The valley method consistently produces
negative errors for both peaks.
 The skim method generates a significant
negative error for the shoulder peak.
 Peak height also shown to be more accurate
than peak area
9/16/2015 23

24.  However, the results may vary depending
upon
The resolution between peaks
 Area and height of the peaks
 Position of the peaks relative to principle
peak or with respect to each other.
Peak size
Complex Baseline
9/16/2015 24

25.  Integration options are likely to generate
significantly different analytical results.
 Analysts must decide which approach provides
better accuracy. However the approach must be
documented in respective SOP with a sound
scientific judgment.
 Proper judgment is expected in selecting the
methodology for peak integration.
9/16/2015 25

26.  Select smallest peak for Integration.
 Set Minimum Area, Minimum height, peak slice,
Tailing/fronting sensitivity factor, valley to valley,
peak to valley etc.
 Set minimum area to about 90% of the area of
smallest peak.
9/16/2015 26

27.  Set Sensitivity value to about 33% of the peak
height of smallest peak that needs to be
integrated.
 Set peak slice to about 20% of the width
(baseline width) of the smallest peak.
 Peak slice parameter determine the width
from which several successive data points are
interpreted as peak or as noise.
9/16/2015 27

28.  Set inhibit integration “ON” from start to the
end of the void volume & “OFF” after the void
volume.
 Inhibit Integration detection parameter
serves to fade out certain chromatograms
area when set peak detection is disabled
9/16/2015 28

29.  Other Integration parameters are :
 Negative Peaks
 Front rider to main peak
 Lock Baseline
 RiderThreshold
 Rider skimming
 VoidVolume treatment
 Sensitivity
9/16/2015 29

30.  For peaks with excessive tailing or broader peaks,
base to valley and valley to base to be used as
integration parameter.
 Same integration parameter to be used through out
the sequence having same concentration of sample
and reference. However may vary from sample to
sample depending upon different peaks observed.
9/16/2015 30

31.  Use Auto Integration as far as possible since manual
integration Not accepted by regulatory bodies
unless necessary.
 Under no circumstances should manual
integration(i.e. peak shaving or peak
enhancement) be performed solely for the
purpose of meeting quality control criteria.
9/16/2015 31

32.  The automatic integration may fail mainly for small
peaks close to the LLOQ.
 Integrate manually as in line with your SOP.
 Proper training required to analyst to comply with
the expectations of manual integration
9/16/2015 32

33.  Acceptable cases for Manual Integration
 Peak Missed
 Poorly defined baseline
 Peak splitting
 Complicated chromatography due to sample
matrix interferences
 Poor instrument integration
9/16/2015 33

34.  Acceptable approach to manual integration :
 Should document both the original and
manually integrated chromatograms
 Analyst’s signature with date clearly specifying
the reason of manual integration.
 Reviewer’s Signature with date
 Review of hard raw data against the electronic
raw data.
9/16/2015 34

35.  Expectations of Regulatory bodies :
 Controlling of chromatographic peaks by
appropriate policies and standard operating
procedures with sound scientific approach.
 Changing of Integration parameters to comply
with Quality control requirements is
unacceptable.
 Use of same suitable Integration parameters for
a validated Analytical method as far as possible.
9/16/2015 35

36. 9/16/2015 36
manipulation in
integration either
intentionally or
malpractice
Data
falsification
Data
integrity

37. 9/16/2015 37
Avoiding Data Integrity in your laboratory…??
 Have a defined procedure in your laboratory
containing methods and procedures with the
recommended HPLC integration parameters.
Any manual integration should be approved
by the laboratory management.

38. HPLC Processing methods (including integration
parameters) and re-integration are executed
without a pre-defined, scientifically valid
procedure.Your analytical methods are not
locked to ensure that same integration
parameters are used on each analysis. A QC
operator interviewed during the inspection
stated that the integration are performed and
re-performed until the chromatographic peaks
are “good” but was unable to provide an
explanation for the manner in which integration
is performed.
9/16/2015 38

39. • The raw data retained does not include the
run sequence or the processing method used
to perform the peak integration.Your QC
personnel performed peak integration based
on analyst’s experience rather then by an
approved procedure.
9/16/2015 39

40. • Chromatography raw data does not include
the processing method used to produce the
final analytical result ; therefore it would not
be possible to detect if any modification to
the processing method is done.
9/16/2015 40

41.  In addition, our investigators documented many
instances wit extensive manipulation of data with
no explanation regarding why the manipulation was
conducted.This manipulation would include
changing integration parameter or relabeling peaks
such that previously resolved peaks would not be
integrated and included in the calculation for
impurities.
9/16/2015 41

42. • HPLCs did not have the audit trails enabled;
some audit trails missing when peaks were
manually integrated, no SOP to describe
when manual integration is acceptable.
• And the series continues……………………
9/16/2015 42

43.  USEPA Region 9 SOP #835,Chromatographic Integration Procedure,@
Revision 0, July 1, 1998.
 Questions of Quality :Where Can I draw the line? By R.D. McDowall,
LCGC Europe,Volume 28, Issue 6.
 Warning letters by FDA.
 Taking the Pain Out of Chromatographic Peak Integration By Shaun
Quinn,1 Peter Sauter,1 Andreas Brunner, shawnAnderson,2 Fraser
McLeod1, 1Dionex Corporation,Germering, Germany; 2Dionex
Corporation, Sunnyvale, CA, USA.
 Integration Errors in ChromatographicAnalysis, Part I: Peaks of
Approximately Equal Size, LCGC.
 Integration Errors in ChromatographicAnalysis, Part II: Large Peak Size
Ratios, LCGC.
9/16/2015 43