Identifying And Resolving Metabolite Interference


In industry practice, sample preparation and chromatography are sometimes ignored during the highly selective Multiple Reaction Monitoring (MRM) in LC/MS/MS analysis. To develop a rugged method, however, critical attention must be paid to all sample cleanup and chromatography steps. One distinct advantage of carefully establishing such methods is higher reliability and minimal tweaking while transitioning from study-to-study up to clinical development ultimately.

Mostly, only limited information on drug metabolism is available at the early stages of drug development. If the parent compound is highly metabolized, there is a possibility that one of the metabolites has similar transition as the drug in the Mass Spectrometer. This introduces complexity in the analysis as the Mass Spectrometer can’t distinguish between two entities with same mass and daughter ion. Therefore, the separation of these analytes by chromatography becomes essential to avoiding interference.

Quality and Compliance

Per FDA guidelines, method development and validation for a bioanalytical assay should include demonstrations of i) selectivity, ii) accuracy, precision, and recovery, iii) the calibration curve, iv) sensitivity, v) reproducibility, and iv) stability of analyte in spiked samples. Our scientists and technical staff have in-depth, hands-on experience in this domain from hundreds of GLP and even more non-GLP bioanalytical studies. Our audit history with regulatory authorities and client inspectors is spotless, with the most recent FDA audit completed successfully as early as 2016. One key way by which we emphasize “good science” and high quality in our laboratory is through carefully coupling selective and sensitive liquid chromatography with tandem mass spectrometry (LC–MS/MS) methods.


In this case, one of our clients sought help with method reproduction, optimization, and validation. During sample analysis, we noticed a small peak appearing as a shoulder to the analyte peak. The analyte at hand had a retention time of 1.5 minutes and our experimental approach was to increase that and separate analyte peak from endogenous material eluting earlier as sample preparation was based on protein precipitation. We were aiming to achieve this by changing chromatographic conditions which included change of mobile phase and columns. Our analysts confirmed upon further analysis that this was not a split peak due to improper chromatography or interference in control plasma rather due to another compound present in plasma with same MS transition. Thus, we hypothesized and started investigating whether this issue was due to a metabolite with similar transition.

In our process of separating the two peaks through chromatography, we found that the intensity of the interfering peak was changing from sample to sample. This clearly established that the noise was emanating from a metabolite. We further estimated this metabolite to be a glucuronide conjugate, given the peak under consideration was eluting earlier than the analyte’s. We, however, had no available reference standard for glucuronide at that time. At last, we were able to confirm that this peak was a glucuronide conjugate upon examining the plasma samples using enzymatic hydrolysis. Consequentially, the method was validated and was applied successfully thereon to analyze plasma samples.


Glucuronide Conjugate metabolite of analyte was present in clinical samples and chromatographic separation was essential to avoid this interference.

Mass spectrometry is a great tool for selectivity, but sample cleanup and Chromatographic separation should not be compromised. Furthermore, we strongly recommend allowing reasonable retention time for the analyte on the column such that early eluting endogenous compounds, including any metabolites are separated from the analyte.

NorthEast Biolab offers a comprehensive solution suite of bioanalytical services, including the following –

  • Clinical LC / MS / MS method development, validation, and analysis
  • Nonclinical LC/MS/MS bioanalytical support including Dose range finding (DRF) studies
  • GLP toxicology method development, validation, and analysis

Our procedures for developing, reproducing, and validating bioanalytical methods are documented in Standard Operating Procedures (SOPs). These SOPs are reviewed regularly, and as appropriate, updated to ensure that we exceed regulatory and client expectations for reliable high-quality data.