LC-MS combines two robust techniques for isolating and measuring analytes in complex biological matrices. These two techniques include liquid chromatography and mass spectrometry. The LC component differentiates compounds based on their physicochemical properties, while the MS unit differentiates by mass-to-charge ratio. This duality makes LC-MS assay a powerful analytical tool in drug discovery and development. Besides, the MS unit not only acts as a detector system but also identifies chromatographic peaks and corresponds them to unique species in the compound.
Today, tandem MS/MS systems provide enhanced sensitivity and selectivity. However, irrespective of the system used, LCMS testing and LC-MS/MS method development and validation will remain crucial for generating reliable and accurate results.
Factors necessary for successful LC-MS testing in drug discovery and development
Different manufacturers provide LC and MS systems with particular features and operating conditions. However, they all follow the same assay principle. Hence, below are some crucial considerations for successful LC-MS testing and analysis during drug discovery and development.
Matric effects
Matric effects are due to disturbances to the ionization process because of components in biological samples. Usually, these components tend to co-elute with the analyte of interest, affecting assay precision. However, there are approaches to reduce the matrix effect. They are:
- Researchers can physically separate the interferent and analytes on the chromatography column.
- Researchers can selectively remove the interference. Generally, during sample preparation.
- Or use a stable isotopically labeled standard.
Sample collection
A homogenous sample that is adequately represented must be used for analysis. This criterion ensures a reliable quantitative analysis. Besides, researchers must control the conditions between sampling and analysis to ensure the integrity of study samples.
Calibration and quality control samples
Researchers prepare samples with known concentrations to quantify the unknown concentrations of target analytes. This process typically involves adding a concentration range to produce calibration solutions for generating a calibration line or plot. The blank samples must closely relate to the matrix of the study samples. Generally, QC samples are prepared in bulk and are tested at regular intervals. Moreover, today researchers commonly retest a small portion of study samples to demonstrate assay precision.
Sample preparation and extraction
Although direct LC-MS analysis is possible, researchers have to manually remove the interference or concentrate the sample in case of low analyte concentration. Scientists generally add an internal standard to monitor assay variation. However, the extraction endpoint must be compatible with the liquid chromatography method.
Analysis
Researchers inject calibration standards, samples, and QCs into the LC-MS system during LC-MS analysis. The LC-MS assay must be sensitive enough to detect samples with low concentration and selective enough to avoid interferences. Hence, a system suitability test is usually performed before analyzing study samples. This test involves running a known solution and comparing the data with previous results. Besides, selectivity can be demonstrated by analyzing study samples and blank samples containing internal standards. Also, ensure that LC-MS systems are linked to modern quantification software capable of allowing direct export of study data into spreadsheets or word processing software.
