Matrix effects are regarded as the Achilles’ heel of clinical liquid chromatography tandem mass spectrometry (LC-MS/MS). Our laboratory has been developing clinical LC-MS/MS assays since 2006. To aid with assay validation, a protocol was established that included a serious investigation of matrix effects. To date, fourteen assay panels have successfully progressed through the validation protocol with most issues arising during the analytical measurement range or precision experiments. However, there are a few cases where the assay fails the matrix effect experiment. Failures at this stage are usually due to differences in ionization between the analyte of interest and the analyte’s internal standard.  Currently, this issue is thought to be resolved with the use of an isotope-replaced internal standard (IRIS). From our experience here, we note that this is not always true.

A particularly difficult case of matrix effects was identified when we were developing an LC-MS/MS assay for plasma metanephrines which use an IRIS. When performing mixing studies, we could not find a suitable dilution matrix where the analyte and internal standard recovery matched that of plasma for our assay. We investigated the potential cause(s) of the matrix effects upon the plasma metanephrines assay. The literature revealed a possible cause: differential matrix effects occurring due to slight retention time differences between the analyte and the deuterated internal standards in areas of changing ion suppression. The proposed mechanism for retention time differences was a consequence of the deuterium substitution of hydrogen leading to an acidity change of the compound. Was our IRIS deuterated? Yes, we were using a deuterated internal standard. Did we see ion suppression in the elution region for the metanephrines? Yes, we did. Were the retention times different? No, they were not different when dwell time and scanning rate were taken into account. If it is due to acidity changes, then a carbon-13 labeled internal standard should fix the problem. However, we have yet to locate a non-deuterated carbon-13 labeled metanephrine standard so we have yet to test this hypothesis. Since we were unable to establish a root cause, we continued our search until a suitable dilution matrix was identified based on our mixing studies.

So, what have we learned? 1) We are able to identify differential matrix effects during validation and find suitable dilution matrices using the mixing study. 2) There may be a second mechanism not reliant on retention time, which explains differential matrix effects. 3) (Most importantly) Deuterium replaced internal standards do not always correct for ion suppression.


Bunch DR, M. El-Khoury J, Gabler J, Wang S. Do deuterium labeled internal standards correct for matrix effects in LC-MS/MS assays? A case study using plasma free metanephrine and normetanephrine. Clinica Chimica Acta 2014;429(0):4-5 doi:[published Online First: Epub Date]