Design, Implementation and Multisite Evaluation of a System Suitability Protocol for the Quantitative Assessment of Instrument Performance in Liquid Chromatography-Multiple Reaction Monitoring-MS (LC-MRM-MS)

Multiple reaction monitoring (MRM) mass spectrometry coupled with stable isotope dilution (SID) and liquid chromatography (LC) is increasingly used in biological and clinical studies for precise and reproducible quantification of peptides and proteins in complex sample matrices. Robust LC-SID-MRM-MS-based assays that can be replicated across laboratories and ultimately in clinical laboratory settings require standardized protocols to demonstrate that the analysis platforms are performing adequately. We developed a system suitability protocol (SSP), which employs a predigested mixture of six proteins, to facilitate performance evaluation of LC-SID-MRM-MS instrument platforms, configured with nanoflow-LC systems interfaced to triple quadrupole mass spectrometers. The SSP was designed for use with low multiplex analyses as well as high multiplex approaches when software-driven scheduling of data acquisition is required. Performance was assessed by monitoring of a range of chromatographic and mass spectrometric metrics including peak width, chromatographic resolution, peak capacity, and the variability in peak area and analyte retention time (RT) stability. The SSP, which was evaluated in 11 laboratories on a total of 15 different instruments, enabled early diagnoses of LC and MS anomalies that indicated suboptimal LC-MRM-MS performance. The observed range in variation of each of the metrics scrutinized serves to define the criteria for optimized LC-SID-MRM-MS platforms for routine use, with pass/fail criteria for system suitability performance measures defined as peak area coefficient of variation <0.15, peak width coefficient of variation <0.15, standard deviation of RT <0.15 min (9 s), and the RT drift <0.5min (30 s). The deleterious effect of a marginally performing LC-SID-MRM-MS system on the limit of quantification (LOQ) in targeted quantitative assays illustrates the use and need for a SSP to establish robust and reliable system performance. Use of a SSP helps to ensure that analyte quantification measurements can be replicated with good precision within and across multiple laboratories and should facilitate more widespread use of MRM-MS technology by the basic biomedical and clinical laboratory research communities. Multiple reaction monitoring (MRM) mass spectrometry coupled with stable isotope dilution (SID) and liquid chromatography (LC) is increasingly used in biological and clinical studies for precise and reproducible quantification of peptides and proteins in complex sample matrices. Robust LC-SID-MRM-MS-based assays that can be replicated across laboratories and ultimately in clinical laboratory settings require standardized protocols to demonstrate that the analysis platforms are performing adequately. We developed a system suitability protocol (SSP), which employs a predigested mixture of six proteins, to facilitate performance evaluation of LC-SID-MRM-MS instrument platforms, configured with nanoflow-LC systems interfaced to triple quadrupole mass spectrometers. The SSP was designed for use with low multiplex analyses as well as high multiplex approaches when software-driven scheduling of data acquisition is required. Performance was assessed by monitoring of a range of chromatographic and mass spectrometric metrics including peak width, chromatographic resolution, peak capacity, and the variability in peak area and analyte retention time (RT) stability. The SSP, which was evaluated in 11 laboratories on a total of 15 different instruments, enabled early diagnoses of LC and MS anomalies that indicated suboptimal LC-MRM-MS performance. The observed range in variation of each of the metrics scrutinized serves to define the criteria for optimized LC-SID-MRM-MS platforms for routine use, with pass/fail criteria for system suitability performance measures defined as peak area coefficient of variation <0.15, peak width coefficient of variation <0.15, standard deviation of RT <0.15 min (9 s), and the RT drift <0.5min (30 s). The deleterious effect of a marginally performing LC-SID-MRM-MS system on the limit of quantification (LOQ) in targeted quantitative assays illustrates the use and need for a SSP to establish robust and reliable system performance. Use of a SSP helps to ensure that analyte quantification measurements can be replicated with good precision within and across multiple laboratories and should facilitate more widespread use of MRM-MS technology by the basic biomedical and clinical laboratory research communities. Targeted analysis by liquid chromatography-stable isotope dilution-multiple reaction monitoring-MS (LC-SID-MRM-MS) 1The abbreviations used are:LC-SID-MRM-MSLiquid chromatography-stable isotope dilution-multiple reaction monitoring-MSCVcoefficient of variationLOQlimit of quantificationSSPsystem suitability protocolFWHMfull width half maximalRTretention 1The abbreviations used are:LC-SID-MRM-MSLiquid chromatography-stable isotope dilution-multiple reaction monitoring-MSCVcoefficient of variationLOQlimit of quantificationSSPsystem suitability protocolFWHMfull width half maximalRTretention to as the for precise quantification of peptides in the of basic biological studies quantification of proteins and by quantification of the by peptides and mass spectrometric quantification of with Multiple reaction of proteins in and for of in clinical assays for low proteins in by targeted mass spectrometry and stable isotope spectrometric quantification of in of a by and mass of in by targeted mass spectrometry and stable isotope of in with by in liquid mass quantification of proteins in targeted a quantitative LC-SID-MRM-MS the precision of analyte sample data is in the range of the limit of quantification (LOQ) and the when stable isotope are The precision is to LC-SID-MRM-MS and data are assays in which more a are the the to be quantification of proteins and by quantification of the by peptides and mass spectrometric quantification of with in and quantification of in is on multiple the standard and analyte the and peptides that are in mass the and the the in the analyte and standard analyte on the of assays are when sample stable isotope with by are the assays for low proteins in by targeted mass spectrometry and stable isotope assays for and in by and targeted mass spectrometric of peptides and proteins and by and for high and multiple reaction monitoring mass quantification of can be a LC-SID-MRM-MS scheduling that of data of the analyte peptides different time in LC-MRM-MS analysis quantification of proteins in targeted of proteins by multiple reaction monitoring of a retention time for more targeted of of and high quantitative quantitative LC-SID-MRM-MS for a clinical The of mass spectrometry in the clinical and of mass spectrometry to the clinical clinical a robust be in to ensure that performance of LC-SID-MRM-MS instrument is the of a for of chromatography-stable isotope dilution-multiple reaction monitoring-MS coefficient of variation limit of quantification system suitability protocol width half retention chromatography-stable isotope dilution-multiple reaction monitoring-MS coefficient of variation limit of quantification system suitability protocol width half retention on the of standardized protocols that the for for of the of of and of high performance liquid chromatography systems interfaced to triple quadrupole mass the of LC-SID-MRM-MS LC-SID-MRM-MS studies for of in by targeted mass spectrometry and stable isotope of a by and mass 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systems in sample in mass of systems stable in is a need for standardized to demonstrate that LC-SID-MRM-MS analysis platforms are performing the approaches developed for evaluation are to LC-SID-MRM-MS which for system performance the and evaluation of to system suitability protocol to performance metrics of triple instrument 11 laboratories 15 platforms different including different of mass in and evaluation of SSP, including and LC and MRM-MS chromatographic and MS metrics including peak width, chromatographic resolution, peak and the variability in peak area and retention time to of of a mixture of that the SSP and of system that a of performance in of precision and of and of the and use of data analysis including for and targeted for of and data in across multiple for of variability different The SSP was to be of in of variation across multiple laboratories performing protocol as well as for instrument of The effect of a marginally performing LC-SID-MRM-MS system on by for is and 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for of instrument performance. 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