LC-MS method development is often a crucial and challenging process. If the LC-MS method requirements are more complex , the process may turn out to be time-consuming. Ideally, adequate selectivity and sensitivity are the two most necessary aspects of LC-MS/MS method development. The selectivity and sensitivity of an lc ms ms analysis may be affected at each phase of the assay development process. Therefore, a step-wise approach to LC-MS method development can ease the pressure and help develop a robust LC-MS/MS analysis.
Steps for developing a reliable LC-MS/MS analysis
Research and planning is the primary step in LC-MS method development. It won’t be surprising if scientists and technicians find any vital information during planning that saves tons of time in the laboratory. This information usually includes type and sample conditions, structural and chemical characteristics , data about related assay methods, and regulatory and industrial guidelines. Although generally, these guidelines are the same for all compounds, finer details in LC-MS method development can ensure unique requirements are fulfilled. Hence, robust research and assay planning set the tone for the later stages of the method development process.
The next step in the process is to determine the ionization potential of the analyte of interest. Usually, researchers test a stream of analytes and identify crucial parameters such as ionization voltage and gas flow that can guide them to tune the analyte for optimum assay conditions.
Developing the chromatography method is arguably the most decisive factor in LC-MS method development. If a new method is developed, ideally, researchers and scientists begin with the logD protocol. With LC-MS/MS analysis, it is beneficial to monitor 2-3 multiple reaction monitoring transitions for each analyte. It gives the space to identify the correct analyte, and the different multiple reaction monitoring transitions can later be used in sample analysis. After developing the chromatography conditions, researchers ideally retune the mass spectrometer to re-ensure assay sensitivity and reproducibility.
Method sensitivity is hugely based on the chromatography conditions, mass spectrometer response, and extraction efficiency. Ideally, sensitivity is determined through the signal-to-noise ratio, and therefore it is crucial to identify the analyte concentration that gives the required signal-to-noise ratio.
The next step in method development is the sample preparation and extraction step. It is one of the most challenging steps in LC-MS method development. The ultimate goal is to reduce interference and maximize analyte recovery. Solid-phase extraction, solvent extraction, and protein precipitation are some standard techniques for sample extraction. Moreover, identifying the matrix effect is crucial during the sample preparation and extraction stage. Two primarily employed methods include the qualitative post-column infusion method and post-extraction spike method.
Conclusion
When to end an LC-MS method development can be tricky. There will always be a room where some factors can be fine-tuned or modified for a more robust LC-MS method development. Hence, it becomes crucial to understand the point when extra efforts in method development turn into a waste of time. Once researchers adequately develop an LC-MS method, they should move on to the next step of method validation.
