The inherent sensitivity and specificity of LC/MS/MS has made it the technique of choice for bioanalysis. However, as compounds become more potent and are dosed at lower levels, the resultant circulatory levels also fall. This requires accurate and precise methods capable of analyzing analyte concentrations in the pg/mL range in plasma.
The development of a bioanalytical method at these low levels can be a time-consuming process, requiring the careful balance of mass spectrometry, chromatographic separation, and sample preparation. In early discovery, the development of such a method may not present too much of an issue due to the higher dosing levels and reduced assay-validation needs. As compounds progress into development, however, the need for a more sensitive, robust assay increases and method development becomes critical.
The development of a bioanalytical method is normally an iterative process. It is often necessary to adjust one part of the method to take account or address a finding from other stages. For example, if analyte response in the mass spectrometer is significantly greater at one pH or in one organic solvent than another, then it is appropriate to perform the chromatography using these conditions.
Additionally, if there is an interference that cannot be removed by solid-phase extraction (SPE) or liquid/liquid extraction, then it may be necessary to modify the chromatography conditions or employ a different MS multiple reaction monitoring (MRM) transition.
All of these individual components in a bioanalytical assay have to be evaluated and often require both MRM and full-scan MS data to identify and resolve issues. With a conventional tandem quadrupole mass spectrometer, this often requires two or more separate analytical runs as the collision cell must be emptied for full-scan MS data acquisition and be filled with collision gas for MRM analysis.
Waters’ Xevo™ TQ MS is a tandem quadrupole mass spectrometer equipped with a collision cell that is continuously filled with collision gas, allowing for the simultaneous collection of MRM and full-scan MS data. This capability can be used to simplify the bioanalysis method-development process, allowing both the analyte’s response to changes in methodology to be monitored, and also any background interferences to be detected and identified. The rapid data-acquisition rates of the Xevo TQ MS allow full-scan MS data to be acquired while still collecting a sufficient number of points across the analyte peak, in MRM mode, for accurate quantification.