Download Developing a Guided Procedure for Troubleshooting HPLC and

Developing a Guided Procedure for
Troubleshooting HPLC and UHPLC Systems
Susanne Fabel,1 Rainer Bauder, 2 and Frank Steiner1
1
Thermo Fisher Scientific, Germering, Germany, 2Thermo Fisher Scientific, Chelmsford, MA, U.S.A.
Overview
TABLE 1. Investig
Purpose: Develop a robust, user-friendly, and fast generic
approach for troubleshooting pharmaceutical HPLC and
UHPLC assays.
Retention time window
Isocratic test (Fig. 1)
Retention time RSD
Methods: Isocratic and gradient test mixtures are
separated on a reference column for assessing
performance of instrument and separation column.
Peak asymmetry
Results: The chromatographic performance is evaluated
by an application independent separation.
Chromatographic performance indicators are compared
with reference values and troubleshooting hints and
recommendations are given automatically by using the
capabilities of the Thermo Scientific™ Dionex™
Chromeleon™ Chromatography Data System (CDS)
software.
Capacity factor
Introduction
During sample analysis and interpretation things can go
wrong. Problems can occur strarting with sampling and
sample preparation. Also during the actual analysis and
reporting things can go wrong and might require corrective
action. For methodical troubleshooting it is important to
follow a systematic approach for finding the origin of the
observed deviation from the expected performance. The
presented work focuses on a troubleshooting solution
which can easily differentiate between instrument or
chemistry (i.e. column or mobile phase) issues. The
concept of our new methodical troubleshooting solution is
to identify key parameters of chromatographic
performance using standardized test mixes run with
standardized reference methods and a selected reference
column. The test methods are uploaded as electronic
workflows providing a completely setup system within a
few mouse clicks. A fully automated and preprogrammed
custom report offers immediate interpretation of the
investigated troubleshooting parameters. Electronic
workflows and reports are part of the software tools
provided with the described troubleshooting solution.
Peak area RSD
Theoretical plates
Generated back press
Number of peaks
All chromatograms
calculated results a
which are embedde
operation of all inst
appropriate fluidic c
report is generated
Results
Example: Out-of
Nevirapine is a non
with activity agains
(HIV-1). The impuri
active pharmaceuti
(3), B(1) and C(4).
Nevirapine formula
the area of one imp
area of the 25% for
compared to the in
FIGURE 3. Out-of
1.6
AU
0
0.9 AU
Method
Liquid Chromtaography System
Thermo Scientific™ Dionex™ UltiMate™ 3000 RS system
with: degasser SRD-3400, binary pump HPG-3400RS,
sampler WPS-3000TRS, column thermostat TCC-3000RS
and UV- detector VWD-3400RS.
2
Thermo Scientific XPert Troubleshooting Solution (PN
3200.0001) consisting of Reference Column (Thermo
Scientific™ Accucore™ XL C18, 3 x 100 mm, 4 m , PN
Developing a Guided Procedure for Troubleshooting
HPLC and UHPLC
Systems
74104-103030),
XPert
isocratic test mix , XPert gradient
test mix, and eWorkflows™.
0
0
The interpretation o
methodical troubles
FIGURE 4. Troub
(U)HPLC results
questionable
workflows and reports are part of the software tools
provided with the described troubleshooting solution.
1
0
0.9 AU
2
Method
Liquid Chromtaography System
Thermo Scientific™ Dionex™ UltiMate™ 3000 RS system
with: degasser SRD-3400, binary pump HPG-3400RS,
sampler WPS-3000TRS, column thermostat TCC-3000RS
and UV- detector VWD-3400RS.
1
0
0
The interpretation of the r
methodical troubleshootin
Thermo Scientific XPert Troubleshooting Solution (PN
3200.0001) consisting of Reference Column (Thermo
Scientific™ Accucore™ XL C18, 3 x 100 mm, 4 m , PN
74104-103030), XPert isocratic test mix , XPert gradient
test mix, and eWorkflows™.
FIGURE 4. Troubleshoo
Data Analysis
Chromeleon CDS 7.2 with service release package SR1.
Test fails
Isocratic Test Conditions
Mobile Phase: Water/Acetonitrile (50/50 v/v)
Flow rate: 640 L/min
Temperature: 30°C
Injection Volume: 1 L
Detection: 254 nm, 25 Hz data collection rate
Review all details of XPert re
applying system and system
setup troubleshooting,
rerun XPert, if recommended
perform maintenance or repa
Gradient Test Conditions
Mobile Phase: A-Water, B-Acetonitrile
Gradient: 0–3.8 min 40–95% B, 3.8–4.5 min 95% B, 4.5–
6.5 min 40 %B
Flow rate: 800 L/min
Temperature: 40°C
Injection Volume: 1 L
Detection: 254 nm, 25 Hz data collectiion rate
Test fails
Initiate
Instrument
Service
FIGURE 1. Isocratic reference chromatogram.
140 mAU
4
1
3
2
5
0
-20 0
2.0
FIGURE 2. Gradient reference chromatogram.
300 mAU
2
1
0
-50
0
4.0
min
4.5
5
6
2.0
7
8
9
Test pas
Perform valid
or SST acco
to your SOPs
rerun XPert t
benchmark
application &
system
Step I – System Check:
separated on the referenc
installation using the eWo
assed by an report, show
status( Fig. 5)
FIGURE 5. Automaticall
operable system.
3
4
I
c
s
e
p
(U)HPLC results
questionable
10
min
4.0
Key Performance Indicators
Chromatographic parameters of the reference separation
in isocratic and gradient mode are processed
automatically by the CDS. The investigated key indicators
for judging the chromatographic performance are listed in
table 1. For the isocratic test mode the o-XyleneThermo
(peakScientific
5, Poster Note
Figure 1) is investigated. For the gradient test mode the
•
PN71313-ISC-EN 0914S 3
a Guided Procedure for Troubleshooting
Key Performance Indicators
HPLC Systems
Chromatographic parameters of the reference separation
0
-50
0
min
4.0
2.0
in isocratic and gradient mode are processed
automatically by the CDS. The investigated key indicators
for judging the chromatographic performance
are listed in
1
table 1. For the isocratic test mode the o-Xylene (peak 5,
Figure 1) is investigated. For 2
the gradient test mode the
indicators of octanophenone (peak 10 , Figure 2) and
butyrophenone and benzophenone as critical peak pair
(peak 5/6) are evaluated
Rainer Bauder2, and Frank Steiner
cientific, Germering, Germany, Thermo Fisher Scientific, Che
TABLE 1. Investigated performance indicators.
riendly, and fast generic
maceutical HPLC and
est mixtures are
or assessing
paration column.
rformance is evaluated
aration.
icators are compared
hooting hints and
matically by using the
c™ Dionex™
ata System (CDS)
retation things can go
ng with sampling and
e actual analysis and
might require corrective
oting it is important to
nding the origin of the
ted performance. The
bleshooting solution
een instrument or
hase) issues. The
ubleshooting solution is
matographic
est mixes run with
nd a selected reference
oaded as electronic
setup system within a
d and preprogrammed
erpretation of the
meters. Electronic
he software tools
eshooting solution.
Isocratic test (Fig. 1)
Gradient test (Fig. 2)
Retention time window
Retention time window
Retention time RSD
Retention time RSD
Peak asymmetry
Peak asymmetry
Peak area RSD
Peak area RSD
Theoretical plates
Resolution of critical peak pair
Capacity factor
Capacity factor
Generated back pressure
Pressure maximum
Number of peaks
Number of peaks
Step II – Colum
operability the a
by running the tr
transferred from
application colum
tool which is par
The resulting ch
separation is sh
FIGURE 6. Isoc
All chromatograms are integrated automatically. The
calculated results are checked against specifications values
which are embedded in the sequence table. The correct
operation of all instrument parts and the characterization of
appropriate fluidic connections is evaluated. Finally a final
report is generated.
Results
Example: Out-of-Spec Result in Quality Control (QC)
Nevirapine is a non-nucleoside reverse transcriptase-inhibitor
with activity against human immunodeficiency virus type 1
(HIV-1). The impurity analysis (see Figure 3) separates the
active pharmaceutical ingredient (API, 2) from its impurities A
(3), B(1) and C(4). The trendplot of an impurity control of a
Nevirapine formulation shows out-of spec results related to
the area of one impurity. The outlier result shows an relative
area of the 25% for impurity B (Figure 3, Peak 1, bottom)
compared to the in spec result of 0.03%.
1
2
1
2
0
The specificatio
immediately a fa
column failure a
replace the colu
FIGURE 7. Faile
mismatch.
FIGURE 3. Out-of-spec result in impurity analysis.
1.6
AU
2
2
1
0
0.9 AU
1
Systems
4 Developing a Guided Procedure for Troubleshooting HPLC and UHPLC 2
3
4
In spec result
3
4
min
Out-of spec result
Step III – Repla
ooting solution is
phic
es run with
lected reference
s electronic
ystem within a
preprogrammed
tion of the
Electronic
ware tools
ng solution.
area of the 25% for impurity B (Figure 3, Peak 1, bottom)
compared to the in spec result of 0.03%.
FIGURE 3. Out-of-spec result in impurity analysis.
1.6
AU
2
1
1
0
0.9 AU
3
In spec result
2
3
4
4
min
Out-of spec result
2
1
00 RS system
PG-3400RS,
tat TCC-3000RS
Solution (PN
mn (Thermo
m, 4 m , PN
XPert gradient
0
min
0.5
0.25
0
The interpretation of the result is performed according to the
methodical troubleshooting workflow of Fig. 4.
FIGURE 4. Troubleshooting workflow.
Install XPert
column, load
standards and
eWorkflow, mobile
phase
(U)HPLC results
questionable
e package SR1.
Run isocratic or
gradient method =>
immediately review
tips in report
Test fails
Test passes
Review all details of XPert report
applying system and system
setup troubleshooting,
rerun XPert, if recommended:
perform maintenance or repair
Run XPert standards with
original column, modify
method accordingly (method
transfer tools)
/v)
rate
min 95% B, 4.5–
Test fails
Initiate
Instrument
Service
rate
ogram.
5
ogram.
8
9
4.0
min
4.5
Test passes
Perform validation
or SST according
to your SOPs,
rerun XPert to
benchmark
application &
system
Test fails
Replace
column
min
4.0
After replacing the colu
the operability of the co
The interpretation of the
impurity can be assess
Step IV – Performance
Long term monitoring o
indicators is implement
periodically running the
column allowing an ear
done without exporting
settings of the XPert Tr
FIGURE 8. Using Chro
functionality for perfo
Test passes
Investigate
application and
verify sample
or standard
preparation
Step I – System Check: First the isocratic test mix is
separated on the reference column by easy method
installation using the eWorkflow. The results are immediately
assed by an report, showing that the system is in an operable
status( Fig. 5)
FIGURE 5. Automatically evaluated parameters show
operable system.
10
Step III – Replacing th
Conclusion an
• A standardized check
reference test mixture
chromatographic para
troubleshooting indepe
• Automatically evaluat
provide helpful indica
suggested actions to
simplified for LC begi
knowledge and practi
while making the use
and introducing perfo
intervention if things g
Thermo Scientific Poster Note • PN71313-ISC-EN 0914S 5
• The XPert troublesho
hromatogram.
application &
system
5
hromatogram.
4.0
min
4.5
Conclusio
preparation
Step I – System Check: First the isocratic test mix is
separated on the reference column by easy method
installation using the eWorkflow. The results are immediately
assed by an report, showing that the system is in an operable
status( Fig. 5)
FIGURE 5. Automatically evaluated parameters show
operable system.
ubleshooting
7
8
9
10
min
4.0
© 2014 Thermo Fisher Scientific
its subsidiaries. This information i
intellectual property rights of othe
rmo Fisher Scientific, Chelmsford, MA, U.S.A.
nt test (Fig. 2)
n time window
n time RSD
ymmetry
ea RSD
on of critical peak pair
y factor
Step II – Column Check: After checking the system
operability the application column performance is evaluated
by running the troubleshooting methods. The methods are
transferred from the reference column dimension to the
application column dimension by using the method transfer
tool which is part of the Chromeleon CDS.
The resulting chromatogram of the isocratic test mix
separation is shown in Fig. 6 (blue).
FIGURE 6. Isocratic test with application column.
e maximum
of peaks
matically. The
specifications values
able. The correct
e characterization of
ated. Finally a final
uality Control (QC)
• Automatically
provide helpfu
suggested act
simplified for L
knowledge an
while making t
and introducin
intervention if
• The XPert trou
automated tro
and builds on
Chromeleon 7
e reference separation
processed
estigated key indicators
erformance are listed in
the o-Xylene (peak 5,
radient test mode the
10 , Figure 2) and
e as critical peak pair
ndicators.
• A standardized
reference test m
chromatograph
troubleshooting
1
2
3
4
1
2
3
4
5
Failing column
Replacement column
5
min
0
1
2
3
The specification of “Peak number total” is not fulfilled and
immediately a failed test report is given announcing the
column failure as a cause and giving the recommendation to
replace the column (Fig.7).
FIGURE 7. Failed test result due to specification
transcriptase-inhibitor
mismatch.
ciency virus type 1
ure 3) separates the
2) from its impurities A
mpurity control of a
ec results related to
a Guided Procedure for Troubleshooting HPLC and UHPLC Systems
6 Developing
ult shows
an relative
3, Peak 1, bottom)
haracterization of
d. Finally a final
ity Control (QC)
nscriptase-inhibitor
ncy virus type 1
3) separates the
om its impurities A
urity control of a
results related to
shows an relative
eak 1, bottom)
min
0
1
2
3
The specification of “Peak number total” is not fulfilled and
immediately a failed test report is given announcing the
column failure as a cause and giving the recommendation to
replace the column (Fig.7).
FIGURE 7. Failed test result due to specification
mismatch.
y analysis.
In spec result
3
4
min
Out-of spec result
min
0.5
d according to the
. 4.
Run isocratic or
gradient method =>
immediately review
tips in report
Test passes
Step III – Replacing the Column
After replacing the column rerunning the isocratic test mix,
the operability of the column is given again (Fig. 6, black).
The interpretation of the out-of spec result of the Nevirapine
impurity can be assessed as column failure.
Step IV – Performance Monitoring
Long term monitoring of chromatographic performance
indicators is implemented in the routine workflow by
periodically running the test methods on the separation
column allowing an early intervention. The visualization is
done without exporting by just using the predefined data view
settings of the XPert Troubleshooting Solution.
FIGURE 8. Using Chromeleon CDS 7.2 trendplot
functionality for performance monitoring.
ert standards with
column, modify
accordingly (method
tools)
fails
ce
n
Test passes
Investigate
application and
verify sample
or standard
preparation
Conclusion and Outlook
Thermo Scientific Poster Note • PN71313-ISC-EN 0914S 7
Investigate
application and
verify sample
or standard
preparation
Conclusion and Outlook
• A standardized check run consisting of reference column and
reference test mixture generates all fundamental
chromatographic parameters required for system and column
troubleshooting independent from the originally used method.
est mix is
method
are immediately
is in an operable
• Automatically evaluated data with instant customized report
provide helpful indication on the source of problems and
suggested actions to remedy them. Troubleshooting is
simplified for LC beginners and allows building own
knowledge and practical troubleshooting competence,
while making the user aware of key performance indicators
and introducing performance monitoring for an early
intervention if things go wrong.
meters show
• The XPert troubleshooting solution is the next generation of
automated troubleshooting and performance monitoring
and builds on the high degree of functionality of the
Chromeleon 7.2 CDS software.
© 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and
its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe the
intellectual property rights of others.
PO71313-EN 0814S
www.thermoscientific.com
©2014 Thermo Fisher Scientific Inc. All rights reserved. ISO is a trademark of the International Standards Organization.
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