Key points
- Accuracy within ±15% (±20% at the LLOQ); precision ≤ 15% (≤ 20% at the LLOQ).
- Accuracy and precision: LLOQ, low, medium and high QCs, at least 5 replicates per run, in at least 3 runs on at least 2 days.
- Interference in blanks: ≤ 20% of the LLOQ analyte response and ≤ 5% of the IS response.
Full, partial and cross validation
- Full validation for a new method or a new analyte.
- Partial validation for changes to a validated method - for example a new laboratory, a change of anticoagulant, a change in sample processing or a new species. The extent depends on the change.
- Cross validation when data from different methods or laboratories are combined or compared within a study or across studies.
Selectivity and specificity
Analyse blank matrix from at least six individual sources (non-haemolysed, non-lipaemic). The response of interfering components at the analyte retention time should be ≤ 20% of the LLOQ response, and at the IS retention time ≤ 5% of the IS response. Include lipaemic and haemolysed matrix (at least one source each) and, where relevant, matrix from special populations. Specificity covers interference from metabolites, concomitant medications and degradation products.
Matrix effect
Low and high QCs, at least three replicates each, in matrix from at least six sources: accuracy within ±15% and precision ≤ 15% in each source. See matrix effect in LC-MS/MS for how to measure and reduce it.
Calibration curve and range
- A blank, a zero sample and at least six non-zero concentration levels including the LLOQ and ULOQ.
- The simplest adequate regression model and weighting, justified during validation.
- Back-calculated concentrations within ±15% (±20% at the LLOQ); at least 75% of the standards, with at least six levels, must meet this.
Accuracy and precision
| Design | Acceptance |
|---|---|
| QCs at LLOQ, low (≤ 3× LLOQ), medium (30-50% of range) and high (≥ 75% of ULOQ) | - |
| Within-run: at least 5 replicates per level in one run | Mean accuracy ±15% (LLOQ ±20%); CV ≤ 15% (LLOQ ≤ 20%) |
| Between-run: at least 3 runs on at least 2 days | Same limits for the overall mean and CV |
Carryover
A blank after the ULOQ: ≤ 20% of the LLOQ response and ≤ 5% of the IS response. See carryover and IS response for causes and investigation.
Dilution integrity
Spike above the ULOQ, dilute with blank matrix by the factors that will be used (at least five replicates per dilution factor). Mean accuracy within ±15% and precision ≤ 15%.
Stability
Low and high QCs (at least three replicates) under conditions that cover the study: stock and working solutions, whole blood where relevant, bench-top, freeze-thaw, long-term and processed-sample stability. Mean accuracy within ±15% of nominal. See stabilising unstable analytes.
Documentation
A validation plan before the work starts, raw data and audit trails for every run (including rejected runs, with reasons), and a validation report that summarises each parameter against its acceptance criterion - the first document a sponsor or inspector will read.
Educational summary for chromatographic (LC-MS/MS) bioanalysis. Always apply the current ICH M10 text, the expectations of the regulator concerned and your laboratory's SOPs. Ligand-binding assays have wider limits in ICH M10.
ARISENTO Insights is published by Autofai Technologies Private Limited, the makers of ARISENTO bioanalytical software.