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Sample preparation and liquid chromatography are sometimes ignored during method development and validation given the highly selective Multiple Reaction Monitoring (MRM) in LC – MS analysis. For a rugged method, however, we must pay attention to all sample cleanup and chromatography steps. Additionally, this approach safeguards reliability and minimizes tweaking while transitioning from study-to-study all the way up to clinical development.

Mostly, only limited information on drug metabolism is available at the early stages of drug development. If highly metabolized, it’s possible for the drug and its metabolite to have similar transition in the Mass Spectrometer. This introduces complexity 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 metabolite 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. We have hands-on, in-depth experience in the aforementioned from hundreds of GLP and non-GLP bioanalytical studies. Moreover, our audit history with regulatory authorities and client inspectors is virtually spotless. We have successfully completed and responded to FDA audit observations as early as 2016. Ultimately, we appreciate good science and high quality achieved through carefully coupling selective and sensitive chromatography with mass spectrometry (LC–MS/MS).


In this case, one of our clients sought help with method development, 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 scientists confirmed 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, we successfully validated and applied the method 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 we should not compromise on sample cleanup and Chromatographic separation. We should allow reasonable retention time to separate early eluting endogenous compounds and metabolites 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 team follows Standard Operating Procedures (SOPs) to develop, optimize, and validate bioanalytical methods. We review and update these SOPs regularly to meet regulatory and client expectations and provide reliable, high-quality data.

We are committed to provide our sponsors with high-quality bioanalytical services and support. Please contact us for any
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