LC-MS method development
Mass spectrometry is a highly selective and sensitive technique. Today, several bioanalytical applications require isolating target analytes from complex biological samples. Generally, mass spectrometers can only differentiate between compounds based on their mass-to-charge ratio. However, this feature is insufficient for most practical biomedical applications. For example, more than 1000 compounds may have a molecular mass of around 250 Da. Therefore, scientists require an additional separation tool before passing the analytes into the mass spectrometer. Here, liquid chromatography comes into play.
Liquid chromatography-mass spectrometry (LC-MS) combines two selective techniques for isolating and measuring target analytes in complex biological samples. Liquid chromatography differentiates compounds based on their physical properties and interaction with the stationary phase, while mass spectrometry differentiates compounds by mass-to-charge ratio. This dual selectivity enhances the performance of LC-MS systems. Mass spectrometers are detector units. However, they can also provide data about individual species that correspond to chromatography peaks. The current article dives into LC-MS method development and is a brief guide for mass spectrometry service companies and mass spectrometry testing services.
LC-MS method development for mass spec services
Firstly, mass spec services should understand that developing an LC-MS method is an iterative process that can often be time-consuming and challenging. Achieving adequate selectivity and sensitivity are the two primary requirements for LC-MS method development. These factors are influenced at every stage of the assay development.
The first step of LC-MS method development is research and planning. The primary information includes structure and chemical data on analytes, information about the target, sample data regarding previously developed assays, and regulatory or industry guidelines.
Identifying whether the target analytes are ionizable is the primary requirement for mass spectrometers. Researchers introduce a stream of analytes using an infusion pump into the ion source to determine the ionization potential of the target analytes. This step allows fine-tuning of instrumental parameters such as ionization voltage and gas flow.
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Assay sensitivity and selectivity often depend on a potent chromatography method. Hence, a robust chromatography method is critical for accurate and reliable research. Generally, if researchers do not have any previous LC-MS method for comparison, one of the most sensible methods is to use the logD of the target analyte as a starting point.
After determining the LC parameters, researchers should return to the MS unit as the ion source elements are interconnected with the flow rate and composition of the mobile phase. Optimizing MS units ensures the reproducibility and sensitivity of the developed assay.
Extracting the sample is one of the most technically demanding steps in LC-MS method development. The goal is to remove interference in the matrix while maximizing the amount of analytes in the sample. For low-concentration analytes, one may introduce a concentration step. Some extraction techniques are solvent extraction, protein precipitation, and solid-phase extraction. Besides, including the post-extraction spike method and qualitative post-column infusion method help assess and reduce matrix effects.
However, before employing the LC-MS method to measure study samples, it is paramount to evaluate the overall method performance against multiple critical parameters.
