The continuing adoption of LC-MS/MS technologies in clinical diagnostics requires improvements in throughput, scope and simplification. This presentation will highlight efforts within our laboratory to address these challenges. Key technologies employed in delivering ultra high-throughput quantitative LC-MS/MS (>2000 samples/system/day) will be described in detail. Brief details of automated sample preparation technologies will be shown. The primary focus of the presentation will center on maximizing MS detection rate and automated data review/release technologies applied in a high volume clinical diagnostics laboratory. Data generation using multiplexed 4-channel (1-6 assays/MS system) and 8-channel (8-16 assays/MS) with daily ultilized capacities >2500 samples/24 hours will be described. Automated data review technologies involving multi-factorial event flagging for streamlined data review will also be described.
Automated sample preparation was performed using Tecan EVO/Beckman FX 8-tip/96-tip liquid handlers for plate formatting/processing, import lists and operational scheduling. Staggered-parallel multiplexed sample introduction via LC, 2D-LC and TFC-LC was realized in standard 4-channel and in-house developed open-access 8-channel modes using Aria Transcend TX4 systems with active wash autosamplers. Analyte detection in SRM mode was performed using Sciex API5000 mass spectrometers with either ESI or APCI interfaces. Raw data files were exported to Rubicon processing software to generate final quantified results for upload and reporting. Multiple outlier events such as transition ratio variance/calculated concentration were generated in multi-factorial mode to enable streamlined technical review.
Previously employed ARIA TX4-API4000 LC-MS/MS systems enabled 800-1000 sample/day capacity in our laboratories, however, the increased volume of testing required a complete remodelling of processes and throughput. Sample tube to plate formatting and plate processing provided the required throughput to prepare >10000 samples per day for direct-injection, protein precipitation, SPE or partial automation of LLE based assays. To achieve >2000 samples/MS system/day capacity, the LC method for each assay in 4-channel mode must be <3 minutes with inject to inject cycles of <60 seconds for each of the 2-injector arms and acquisition cycle times of 40 seconds. Use of low dead-volume agilent 1200SL binary pumps and minimized dead-volume enabled LC cycle times of 2-3 minutes in high flow gradient modes (LC), method folding in 2D-LC/TFC-LC modes and the required reproducibility to reduce acquisition windows to <30 seconds. Active wash systems reduced carry-over and enabled 50 second inject to inject cycles for all assays. The turbo V source of the API5000 system enabled eluent flow rates >2ml/min to be introduced. Data review of multiple outlier events enabled technical review to be reduced by >50%. Small highly-polar analytes such as metanephrine, epinephrine and homovanillic acid are assayed using high flow HILIC gradient separations in ESI mode. Neutral analytes (25-hydroxyvitamin D, testosterone) are assayed via highly selective RPLC separations in either 1D or TFC-LC mode using miniaturized extraction columns to further reduce dead-volume. Drug assays such as amitryptiline, chlordiazepoxide and warfarin are analyzed using TFC-LC strategies involving generic column chemistries with class specific gradient programming. Examples of analytical cassetting, polarity and functionality based assay standardization, generic direct-injection column formatting and 8-channel STAT testing applications will be shown as examples of how ultra high-throughput LC-MS/MS can be realized.
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