Seeing the Invisible; Ion Suppression Resulting from Discrete Pharmaceuticals and Metabolites Anna Miller (Presenter) MedTox, Labcorp

Authors: Anna M. Miller, Gregory C. Janis
MedTox, LabCorp

Introduction:

Ion suppression in LC-MS/MS is often viewed as a phenomenon resulting from uncontrolled matrix components found within a sample. However, biologically occurring matrix components are not the only source of ion suppression. Most any substance co-eluting from the analytical column can suppress analyte ionization depending on the concentration and ionization profile of the co-eluting substance. Due to their infrequent and seemingly variable nature, ion suppression resulting from other drugs or metabolites that co-elute with a targeted analyte can be difficult to identify and challenging to eliminate. This poster details an example of the major and minor metabolites of a common pharmaceutical that both dramatically suppresses the signal of a targeted analyte and present conventional interference peaks. Additionally, steps which were taken to identify and eliminate the culprits are presented.

Problem:

Within multiple reverse phase methodologies on two distinct mass spectrometry platforms, dramatic signal suppression was seen when measuring norfentanyl in urine samples. The loss of signal for all norfentanyl and d5-norfentanyl transitions observed impacted approximately 6% of a small sample set. The infrequent but near complete level of suppression hinted that the culprit was not simply matrix components.

Troubleshooting techniques:

Using an untargeted analysis of impacted samples, the presence of venlafaxine was identified as a common component of samples displaying the characteristic suppression of norfentanyl. The presence of desmethylvenlafaxine was also common across impacted samples. Desmethylvenlafaxine was determined to be the metabolite present in impacted samples causing the signal suppression of norfentanyl. In addition, a minor metabolite, didesmethylvenlafaxine was identified as the component responsible for the peak interfering with norfentanyl. Further testing of the suspect samples showed desmethylvenlafaxine at concentrations approximately 10,000 times higher than the target concentration of norfentanyl. Lower concentrations of drug or metabolite may not cause the same ion suppression issues.

Multiple potential solutions were evaluated to reduce and resolve the ion suppression. Modifications to mass spectrometer source parameters were evaluated as a potential solution. Unfortunately, under such an overwhelming concentration of desmethylvenlafaxine the suppression could not be reduced by mass spectrometry techniques. Furthermore, modifications to the sample extraction procedure could not selectively separate the compounds with their similar structures and functional moieties. Likewise, simple modifications of the chromatographic parameters could not completely resolve the analyte from the suppressing metabolite.

A more dramatic shift in chromatography was necessary. Changing the separation mechanism from a conventional C18 reverse phase mechanism to the more complex mechanisms of a diphenyl column was determined to be the best solution. The separation change enabled the exclusion of both the suppression caused by desmethylvenlafaxine and the false peak from didesmethylvenlafaxine.

Outcome:

Armed with knowledge of the source of the suppression, samples containing high levels of venlafaxine were selected to challenge the revised assay and acceptable results were generated.

Problem

Method Information

Troubleshooting Steps

Outcome