Key points
- Carryover in a blank injected after the highest standard: at most 20% of the LLOQ analyte response and 5% of the internal standard response.
- If carryover cannot be avoided, the run design must prevent it from affecting study samples.
- IS responses are monitored in every run against criteria pre-defined in an SOP.
Carryover limits
Carryover is analyte (or internal standard) from a previous injection that appears in the next one. ICH M10 limits it by injecting a blank after the highest-concentration calibration standard (ULOQ): the response in that blank should not exceed 20% of the analyte response at the LLOQ and 5% of the internal standard response.
Testing in every run
- Place at least one blank directly after the ULOQ standard in each run, and assess it against the limits above.
- If carryover is known to occur, place blanks after high-concentration study samples and define in the SOP how samples injected after a high sample are assessed or re-analysed.
- Avoid randomising study samples in a way that places low concentrations directly after very high ones without a blank.
Causes and investigation
| Where | What to check |
|---|---|
| Autosampler | Needle wash solvent strength and volume, wash sequence, needle seat, injection valve rotor seal |
| Column | Strongly retained analyte; add a high-organic wash at the end of the gradient or a longer re-equilibration |
| Tubing and fittings | Dead volumes and adsorptive surfaces; sticky (basic or lipophilic) analytes |
| Ion source | Contamination of the cone or capillary; clean and re-check |
| Method | Upper range too high for the system; consider a lower ULOQ with dilution of high samples |
Internal standard response
The internal standard corrects for variability in extraction, injection and ionisation - but only if it behaves consistently. ICH M10 expects the IS response of study samples to be monitored in each run to detect systematic variability. The guideline does not set a numerical limit: the laboratory defines its criteria in an SOP before analysis. A common approach, consistent with the FDA's 2019 question-and-answer document on internal standard responses, is to compare the IS response of each study sample with the range seen in the calibration standards and QCs of the same run, and to define when a sample is re-analysed.
Investigating IS variability
- Single samples low or missing: pipetting error, IS not added, injection problem - typically re-analysis per SOP.
- Drift across the run: source contamination, temperature, autosampler stability - check the QCs bracketing the drift.
- Study samples differ from standards and QCs: matrix effects (ion suppression), co-eluting metabolites or concomitant medication - assess whether the analyte-to-IS ratio is still reliable.
- Document the assessment: a well-tracked IS investigation is one of the clearest signs of data integrity in an audit.
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.