Key points
- Matrix effect is a change in ionisation caused by co-eluting components of the sample - usually suppression in ESI.
- ICH M10: low and high QCs, at least 3 replicates in matrix from at least 6 individual sources; accuracy within ±15% and precision ≤ 15% in each source.
- The strongest fixes are cleaner extraction, moving the analyte away from the suppression zone and a stable-isotope-labelled internal standard.
What matrix effect is
In electrospray ionisation the analyte competes with everything else that reaches the droplet surface at the same moment. When co-eluting matrix components take the charge or change droplet evaporation, fewer analyte ions reach the detector - ion suppression. Less often the response increases - ion enhancement. Neither shows up in a clean standard solution, which is why a method can look perfect in solvent and still fail in plasma.
The danger is not the effect itself but its variability: if one subject's plasma suppresses the signal by 40% and another's by 10%, an internal standard that does not experience the same change will give wrong concentrations.
Where it comes from
- Phospholipids (glycerophosphocholines, lysophosphatidylcholines) - the most common cause in plasma after protein precipitation. They often elute late and can wrap around into the next injection.
- Salts and polar endogenous compounds eluting near the void volume.
- Anticoagulants and additives (for example lithium heparin, EDTA salts), dosing vehicles such as PEG 400 or polysorbate in early-phase studies.
- Concomitant medication and metabolites that co-elute with the analyte.
- Haemolysed and lipaemic samples - different matrix, different effect.
How to measure it
Post-column infusion shows where suppression happens. Infuse the analyte continuously after the column while injecting an extracted blank: dips in the baseline mark the retention times to avoid. It is a development tool, quick and visual.
Post-extraction spiking shows how much. Compare the response of analyte spiked into extracted blank matrix with the same amount in neat solution. The ratio is the matrix factor (MF); dividing the analyte MF by the IS MF gives the IS-normalised matrix factor. Values near 1 with low variability across lots mean the IS compensates well.
| Approach | Answers | When |
|---|---|---|
| Post-column infusion | Where in the chromatogram is the suppression? | Method development |
| Post-extraction spike (matrix factor) | How large is the effect, and does the IS compensate? | Development, troubleshooting |
| QCs in individual matrix lots (ICH M10) | Is the method accurate and precise in different sources? | Validation |
What ICH M10 expects
ICH M10 evaluates matrix effect through accuracy and precision in individual matrix sources: analyse at least three replicates of low and high QCs, each prepared in matrix from at least six different sources or lots. In each individual source, the accuracy should be within ±15% of nominal and the precision (CV) not greater than 15%.
The guideline also expects matrix effect to be evaluated in relevant special matrices - for example haemolysed and lipaemic plasma, and matrix from special populations such as renally or hepatically impaired patients when they are part of the study.
How to reduce it
- Cleaner extraction. Protein precipitation leaves most phospholipids in the extract. Liquid-liquid extraction, solid-phase extraction or phospholipid-removal plates usually reduce matrix effect substantially.
- Move the peak. Change the gradient, column chemistry or mobile-phase pH so the analyte elutes away from the suppression zone you mapped by post-column infusion. Add a strong organic wash so late phospholipids do not appear in the next injection.
- Use a stable-isotope-labelled IS (deuterated or 13C/15N). It co-elutes and experiences the same suppression. Watch for deuterium isotope effects that shift retention slightly in sharp gradients.
- Dilute. If sensitivity allows, diluting the extract reduces matrix load.
- Change the source. APCI is generally less prone to suppression than ESI for compounds that ionise well in it; lower flow rates and source settings also help.
- Monitor phospholipids during development (for example the m/z 184 product ion) to see where they elute.
Watching for it in study runs
Validation proves the method in six lots; a study may contain hundreds of subjects. Internal standard response is the everyday indicator: a group of study samples with clearly lower IS response than the standards and QCs of the same run often points to a matrix effect - for example a concomitant medication or a dosing vehicle. Investigate before reporting, and keep the assessment with the run.
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.