Resolving Drug Metabolites
Monitoring matrix components is but one factor that needs to be considered when developing an LC/MS/MS method for bioanalytical assays; it is also critical that the assay is capable of resolving the drug metabolites from the analyte requiring quantification. The co-elution of drug metabolites with the analyte could also result in matrix effects and reduced assay performance.
Metabolites such as glucuronides and N-oxides metabolites can undergo thermal degradation in the source of the mass spectrometer and revert to the parent analyte. If the metabolite is not chromatographically resolved from the parent, this conversion will result in an overestimation of the concentration of the parent compound.
To ensure the assay specificity, it is necessary to monitor drug metabolites and ensure that they are resolved from the parent drug. Metabolite detection can be achieved by the use of full-scan MS or common fragment ion detection. The collision cell design of the Xevo TQ MS allows for the simultaneous collection of MRM, full-scan MS, and common fragment ion data. The data displayed in Figure 3 shows the simultaneous acquisition of MRM, full-scan MS, and common fragment ion data for the analysis of alprazolam in plasma.
Using this approach, the α-hydroxy metabolite of alprazolam was clearly detected by the MRM with a retention time of 1.55 minutes. This metabolite was also detected using the common fragment m/z 281. The full-scan MS data was used to confirm the identity of the metabolite. The data in Figure 3 confirms that the metabolite is well-resolved from the parent drug using a generic UPLC gradient, thus eliminating the possibility of an overestimation of drug compound.
In summary, utilizing the Xevo TQ MS’s dual scan MRM mode allowed for the detection of coeluting analytes from the plasma matrix in a single injection, reducing time and the need for additional analytical runs during the method-development process. Also employing Oasis MCX effectively isolated alprazolam from interfering components in the plasma matrix.
Endogenous metabolite information was obtained in a single injection with dual scan MRM mode, and the major metabolite of alprazolam was well resolved from the parent drug utilizing a generic UPLC® gradient. Dual scan MRM mode allowed full-scan data to be acquired simultaneously with MRM, which enabled matrix monitoring for method development as well as discovery of nontargeted compounds.