Chromatography troubleshooting is a key part of a chromatographer’s daily work. Here, we present articles and protocols to help a chromatorgher achieve this efficiently.
A stable isotope labeled internal standard (SIL-IS) corrects LC-MS/MS results only for disturbances it shares with the analyte. Extraction losses that happen after it is added, injection variability and ion suppression in a shared retention window cancel in the analyte/IS ratio. Losses before it is added, retention differences, cross-signal contribution, IS-channel interference and detector saturation …
A post-extraction spike experiment measures how much co-eluting matrix changes LC-MS/MS response, separately from how much analyte the sample preparation loses. Three matched preparations – a neat reference, extracted blank matrix spiked after extraction and matrix spiked before extraction – give the matrix factor, extraction recovery and process efficiency. It is the quantitative half of …
Post-Extraction Spike in LC-MS/MS: Matrix Factor, Recovery and Process Efficiency Read More »
Post-column infusion is a qualitative LC-MS experiment that shows where in a chromatographic run co-eluting matrix components suppress or enhance ionization. A constant stream of analyte is teed into the column effluent while an extracted blank matrix is injected, and dips or rises in the otherwise steady signal mark the affected retention times. It is …
Post-Column Infusion for LC-MS Matrix-Effect Diagnosis: Setup, Interpretation and Limits Read More »
Phospholipid interference in LC-MS/MS happens when phospholipids carried through sample preparation co-elute with an analyte and change its ionization. In a quantitative assay this can shift analyte and internal-standard responses, widen lot-to-lot variability or bias results. A phospholipid marker at the analyte retention time is a clue; it does not establish the cause. Locate the …
Phospholipid Interference in LC-MS/MS: How to Detect, Remove and Control It Read More »
A failed qualifier/quantifier ion ratio means the relative responses of two monitored transitions differ from an appropriate reference. The deviation can reveal a false identification or a biased quantifier, but low qualifier signal and data-processing choices can also make a real analyte fail. The acceptance rule depends on the method and its regulatory scope; there …
Why Does My LC-MS/MS Qualifier/Quantifier Ion Ratio Fail? Read More »
If the concentration you calculate for the original sample changes with the dilution factor, the result is a pattern, not yet a cause. Before altering the method, verify the dilution arithmetic and confirm that every injected aliquot lies inside the calibrated range. Then compare matrix-matched dilution QCs, neat controls, analyte and internal-standard (IS) responses, and …
Why Does My LC-MS/MS Result Change After Dilution? Read More »
An LC-MS/MS peak area that climbs across an injection sequence is a pattern, not a diagnosis. The rise can come from the analyte, sample preparation, the matrix, the flow path, the ion source, detector response, or the relationship between one injection and the next. The fast route to the cause is to characterize the trend, …
