Developing a Guided Procedure for Troubleshooting HPLC and
Contents
1. 40 C Injection Volume 1 uL Detection 254 nm 25 Hz data collectiion rate FIGURE 1 Isocratic reference chromatogram 140 mAU 4 5 0 I min 206 2 0 40 45 FIGURE 2 Gradient reference chromatogram 300 mAU WLU 0 min 50 0 2 0 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 Xylene peak 5 Figure 1 is investigated For 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 TABLE 1 Investigated performance indicators 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 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 fin2. 6 000 8 000 Passed 7 298 Number of executed test cases 9 Total Result Passed Step Il 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 lsocratic test with application column Failing column LAA 0 1 2 3 4 Replacement column 5 I min 1 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 Xpert Performance Test Result a z ie Name Ret Value 1 Ret Value 2 Test Result Eval Resun 1 Pump Pressure Stabilty 594 000 E Passed 542 467 2 Pump Pressure Lmt 70 000 600 000 Passed 540 492 3 Peak Number Total 5 000 ina Failed 3 000 4 Retention Time 2 000 2 000 Passed 2 325 5 RSD of Peak Retention Tif 0 500 ina Passed 0 460 6 Peak Asymmetry 0 950 1200 NA gt Failed na 7 RSD of Peak 2 000 na Passed 1 984 8 Theoretical
3. Plates USP _ 100000 000 na NA gt Failed na je Capacity Factor 2 800 5 900 Failed 13 377 Number of executed test cases 9 Total Result Possible Cause Solution 1 Pumo Pressure Sti 2 Pumo Pressure Peak Number Total2 Split or double peaks 2 Split or double peaks Contamination on column or guard Flush column with strong mobile phase inlet back flush to waste replace column 3 Sample solvent too strong prepare sample in mobile phase Worn out rotor seal replace rotor seal with extreme pH Packing integrity loss UHPLC applications check compatibility of the seal applications polymer A Retention Time 5 RSD of Peak Retention Time g Peak Asvmmetrv RSD of Peak Areas_1 7 Theoretical Plates 8 USP _1 9 Capacity Factor Column degradation Replace column I Reviewer s signature Date Operator s signature Date Step Ill 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 Troubleshoot
4. Developing a Guided Procedure for Troubleshooting HPLC and UHPLC Systems Susanne Fabel Rainer Bauder and Frank Steiner Thermo Fisher Scientific Germering Germany Thermo Fisher Scientific Chelmsford MA U S A Overview Purpose Develop a robust user friendly and fast generic approach for troubleshooting pharmaceutical HPLC and UHPLC assays Methods Isocratic and gradient test mixtures are separated on a reference column for assessing performance of instrument and separation column 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 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 met
5. Replace column Instrument rerun XPert to Service benchmark or standard application amp preparation system Thermo Scientific Poster Note PN71313 ISC EN 0914S 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 LL ction Details Injection Name XPert RP gradient test mix hi Run Time min 6 50 a Vial Number RA3 Injection Volume 1 00 a Injection Type Check Standard Channel uv vVIs_1 Injection Counter 161 kl Wavelength 245 0 3 Instrument Method XPert_RP_RS_HPG_VWD k Bandwidth n a 3 Processing Method Accucore_XL_C18_100x3_gradient_HPG_RS hi Dilution Factor 1 0000 a Injection Date Time 07 Okt 13 14 17 b Sample Weight 1 0000 Performance Test Result Name Ref Value 1 Ref_ Value 2 Test Result Eval Result 1 Pump Pressure Stability 127 600 na Passed 117 030 2 Peak Number Total 10 000 n a Passed 10 000 3 Retention Time 3 000 4 000 Passed 3 543 4 RSD of Peak Retention 7 0 100 na Passed 0 022 5 Resolution USP 3 000 n a Passed 4 016 6 Peak Asymmetry 10 950 1 200 Passed 1 019 7 RSD of Peak Areas_1 0 500 na Passed 0 340 8 Peak Width 50 0 500 n a Passed 0 022 fe Capacity Factor
6. al 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 FIGURE 3 Out of spec result in impurity analysis 1 65 AU In spec result 07 min 0 9 AU 2 Out of spec result 1 0 min 0 0 25 0 5 The interpretation of the result is performed according to the methodical troubleshooting workflow of Fig 4 FIGURE 4 Troubleshooting workflow Install XPert U HPLC results column load questionable standards and eWorkflow mobile phase Review all details of XPert report applying system and system setup troubleshooting rerun XPert if recommended perform maintenance or repair Perform validation or SST according Investigate Initiate to your SOPs application and verify sample Run isocratic or gradient method gt immediately review tips in report Run XPert standards with original column modify method accordingly method transfer tools
7. hodical 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 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 Thermo Scientific XPert Troubleshooting Solution PN 3200 0001 consisting of Reference Column Thermo Scientific Accucore XL C18 3 x 100 mm 4 um PN 74104 103030 XPert isocratic test mix XPert gradient test mix and eWorkflows Data Analysis Chromeleon CDS 7 2 with service release package SR1 Isocratic Test Conditions Mobile Phase Water Acetonitrile 50 50 v v Flow rate 640 uL min Temperature 30 C Injection Volume 1 uL Detection 254 nm 25 Hz data collection rate 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 uL min Temperature
8. ing Solution FIGURE 8 Using Chromeleon CDS 7 2 trendplot functionality for performance monitoring 245 Ret Time 0 Xylene FE Pe a amp amp 8 8 8 amp R A amp 8 E ES pe ul a ae ea ae 5 RY Ry A RR ee A TA me oes amp So ES Se Se a le S ae ae Injections Pressure Average Retention Time Plates Asymmetry AN B K www thermoscientific com 2014 Thermo Fisher Scientific Inc All rights reserved ISO is a trademark of the International Standards Organization All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries This information is presented as an example of the capabilities of Thermo Fisher Scientific products It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others Specifications terms and pricing are subject to change Conclusion and Outlook e 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 e 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
9. while making the user aware of key performance indicators and introducing performance monitoring for an early intervention if things go wrong e 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 Thermo Fisher Scientific Sunnyvale CA USA is ISO 9001 2008 Certified Not all products are available in all countries Please consult your local sales representative for details 30 9001 Africa 43 1 333 50 34 0 Denmark 45 70 23 62 60 Japan 81 6 6885 1213 Russia CIS 43 1 333 50 34 0 Australia 61 3 9757 4300 Europe Other 43 1 33350340 Korea 82 2 3420 8600 Singapore 65 6289 1190 Austria 43 810 282 206 Finland 358 9 3291 0200 Latin America 1 561 688 8700 Sweden 46 8 556 468 00 Belgium 32 53 73 42 41 France 33 1 60 92 48 00 Middle East 43 1 333 50 340 Switzerland 41 61 716 77 00 Brazil 55 11 3731 5140 Germany 49 6103 408 1014 Netherlands 31 76 579 55 55 Taiwan 886 2 8751 6655 Canada 1 800 530 8447 India 91 22 6742 9494 New Zealand 64 9 980 6700 UK Ireland 44 1442 233555 China 800 810 5118 free call domestic Italy 39 02 950 591 Norway 46 8 556 468 00 USA 1 800 532 4752 400 650 5118 PN71313 EN 0914S Thermo SCIENTIFIC A Thermo Fisher Scientific Brand
Download Pdf Manuals
Related Search