If you're observing area variation in linearity solutions while the standard area remains consistent, this points to some potential root causes specific to the linearity sample preparation or handling. Here's a breakdown of what could be causing this issue and how to troubleshoot it: Possible Reasons & Solutions for Area Variation in Linearity Solutions1. Pipetting/Volumetric ErrorIssue: Inaccurate pipetting during dilution steps leads to inconsistent concentration.Solution: Use calibrated pipettes and class A volumetric flasks. Repeat dilution steps with special attention to technique.2. Improper Mixing or Incomplete DissolutionIssue: Linear samples may not be fully dissolved or homogenized, leading to inconsistent injection results.Solution: Ensure thorough mixing at each dilution level; use sonication or vortexing as required.3. Weighing ErrorsIssue: If linearity solutions are prepared by weight, small errors in weighing the API or excipients can cause variation.Solution: Verify the balance calibration. Use higher sample weights to reduce relative error.4. Solution StabilityIssue: If linearity solutions are not freshly prepared, degradation or evaporation might occur.Solution: Prepare fresh solutions and inject immediately. Validate the short-term stability of solutions.5. Glassware Cross-contaminationIssue: Residue in glassware between dilutions can introduce variation.Solution: Rinse thoroughly and ensure glassware is clean before use.6. Injection Volume InconsistencyIssue: Autosampler may not be consistent for all injections.Solution: Check injection reproducibility by multiple injections of the same sample. Recalibrate the injector if needed.7. Matrix or Mobile Phase MismatchIssue: If linearity solutions are not matrix-matched with the standard, it may impact area.Solution: Ensure the same diluent and mobile phase are used for both standard and sample prep.

Just to complete and additional to Mr Pawar comment, if you are using a Waters LC make sure that the rinse solution has the propper organic:aquous ratio. This is more likely to be a sample preparation issue (specifically extracción/dissolution conditions/diluent election), or a filtering issue (i.e. non filtering the standard solutions vs filtering the linearity solutions)