Table 2 Quality assurance of LC–MS/MS assays [17, 26, 33, 35, 36]

System suitability assessment (SSA): System suitability material (e.g. mixture of internal standards and/or target analytes in pure solvent) is analyzed following system equilibration and priming to ensure acceptable LC–MS system performance prior to commencement of an analytical run. SSA acceptance criteria established during method validation should be met for pre-defined chromatography metrics such as peaks eluting at expected retention times, adequate analyte and background signal, etc

Calibrator accuracy and calibration curve metrics: Each point in the calibration curve should meet pre-specified allowable bias metrics (e.g. ± 15% of target value). Additional calibration curve metrics include the slope, and r² supporting the linear response between analyte response (peak area) to that of SIS (y-axis) and known analyte concentrations (x-axis). The metrics should be defined based on variation observed during method validation and informed by clinical guidelines, and regulatory requirements

Quality control monitoring: Quality control samples containing known analyte concentrations should be included throughout the batch and yield concentrations within an established range (e.g. mean ± 2 standard deviations). The ranges are established for each lot and level of quality control through repeat analysis within and between multiple runs. Runs are rejected in case of unacceptable quality control. Quality control samples should span clinically relevant concentrations

Internal standard monitoring: Ensures internal standard peak area for each sample is within acceptable recovery limits established during method development. The SIS peak area should be within acceptable limits for calibrators, quality control and patient samples. If outside limits, specimens are to undergo repeat analysis and be rejected from being reported to the patient chart if this does not resolve the issue

Ion ratio monitoring: Ion ratios, the ratios of the peak area of the qualifying ion transitions to the peak area of the quantifying ion transitions, should be reviewed for each patient sample and IS. Patient samples with ion ratios falling outside of of predefined limits should undergo troubleshooting as they may have interferences preventing reliable quantification. The criteria for the predefined limits are determined during method development, prior to method validation

Retention time monitoring: Analyte and SIS peaks are checked for chromatographic retention times during and between analytical runs to ensure they are constant and within acceptable tolerance limits. Retention times between analyte and IS in patient samples and QC should be similar to the standards

Lot change evaluation: In order to ensure consistency of patient results and avoid introduction of significant analytical biases, prior to implementation of new lots, patient samples (n ≥ 5) should be analyzed on new and current lots of critical reagents and consumables and values should compare within a pre-specified allowable bias limit. Critical reagents include new lots of calibrators, internal standards, calibrator matrix, and any other reagents that may impact analytical performance. Consumables include LC columns