Table 2.
Summary of the proposed metrics for system suitability test, with proposed acceptance criteria and possible troubleshooting targets. The thresholds of 15% for CV and +/− 20% for relative quantitation are adapted from current FDA recommendations34
| Metric | Purpose | Definition | Recommended Threshold | Possible Cause for Failure |
|---|---|---|---|---|
| CV of Peak Area | Check signal stability | %CV of peak area of EIC at 100% | <15% | Instable signal, likely an issue at the electrospray |
| Protein Sequence Coverage | Check method settings for identification capability | Determined from search algorithm based on the confidence level chosen | Near complete is ideal | Acquisition setting, intrinsic sensitivity, LC separation |
| Average Mass Error | Report overall mass accuracy | Average of absolute ppm error of all identified peptides with high confidence (or known peptides) | From system specification <5 ppm or lower (High resolution) | Temperature change, invalid calibration, incorrect method settings (e.g., lock mass, AGC) |
| Mass Resolution | Check instrument resolution | Full width at half maximum mass peak resolution at 100% concentration | Within instrument specification (instrument and operation mode dependent) | Invalid instrument tuning/qualification, instrument malfunctioning |
| Ion Source Settings | Minimize non-native species generated in source | Monitor the relative abundance of a fragile peptide in the standard | Maintain sensitivity while minimize excess activation of fragile species | Source tuning, source acceleration voltage too high |
| S/N of EIC | Defining LOD | S/N of target peptide EIC | > 3 | Bad instrument tuning, bad EIC peak shape, bad spraying condition, intrinsic sensitivity limit of system |
| MS2 Identification Score | Check MS2 quality at different concentrations | Target peptide score/cutoff threshold for intra and inter-scan | ≥1 | Acquisition setting, intrinsic sensitivity, LC separation |
| Mass Accuracy/Resolution Change | Evaluate confidence of mass measurement at low concentrations | Compare mass error/resolution at low and 100% concentration | Ideally minimal change at low concentrations | Instrument specific and can be normal to see mass error/resolution change at different signal intensities. Need to adjust data analysis procedure accordingly. |
| Accuracy of Relative Quantitation | Check accuracy and dynamic range of quantitation | Percent accuracy of the experimental result relative to the expected result | 80% –120% | Bad EIC extraction method, co-eluting species, instrument intrinsic limit |
| CV of Relative Quantitation Result | Check precision of quantitation | %CV of peak area ratios of a target peptide pair | <15% unless only intended for semi-quantitation | Low sensitivity/bad peak shape at the concentration, incorrect data processing, interfering co-eluting species |
| Differential Peak Sampling | Check potential error in quantitation due to stochastic sampling across EIC peak | Ratio of relative quantitation results between the proposed method and the MS1 only method | 0.8∼1.2 Use the MS1 only method for quantitation if exceeds the range | Improper MS2 settings such as the selection threshold, MS1 and MS2 scan rate, etc. |