Study
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.