By Harika Vemula
Analytical assay development is an important part of drug discovery for finding new drug entities and supporting research and development from preclinical to clinical studies. As a Ph.D. student, it is important to understand and design new LC-MS assays for research. During this process, the most common questions for a new user are: Should I use high pressure liquid chromatography (HPLC) or liquid chromatography coupled mass spectrometry (LC-MS) for designing an assay ? Are chromatographic conditions more important vs. mass spectrometry parameters for sample analysis? These are interesting questions, and I would like to share my perspective as a Ph.D. student with a focus in bioanalytical method development.
The simple answer to the first question is: It depends on the objective of the experiment. Each method (HPLC and LC-MS) has its own pros and cons to analyze the samples. HPLC method development and validation includes selection of parameters specific for analyte resolution and retention on HPLC column, such as column type, dimensions and bonded phase of the column, mobile phase solvents, sample preparation, wavelength used (UV-Vis/ fluorescence/radioactivity). However, LC-MS includes a typical LC separation and optimization of MS or MS/MS parameters (declustering potential, collision energy, entrance potential, ionization type, etc.) selective to the analytes of interest.
For non-targeted global analyses in human serum, we have used LC-MS over HPLC analysis. This is because MS can effectively identify multiple analytes in a single run quantitatively, whereas it needs an extra layer of LC optimization to identify diverse analytes through HPLC only. Further, sample complexity determines the type of assay to be used. For example, for analysis of human blood, plasma, or bacteria extracts a good LC-MS method will help analyze even low concentrations of analyte, given the high sensitivity and specificity of mass spectrometry instruments. Additionally, we also modified the LC conditions to avoid matrix effects and analyte resolution.
Another important biological application of LC-MS is the ability to determine the active state/folded state of a protein. Unlike conventional techniques like ELISA or western blot, which utilize quantitative/qualitative analysis, we designed a LC-MS assay to determine the percentage of active protein for Type-II diabetes by bottoms-up approach. This is a high-level technique that needs isotope labeled proteins to quantitate the active protein from biological matrices.
Sometimes, HPLC can be the only stand-alone technique for sample analysis. Selecting the right column, mobile phase solvents, and chromatographic conditions (gradient, temperature, etc.) is extremely useful, especially for isomeric separations. In chiral or geometric isomers where m/z are same, MS alone cannot help differentiate the isomers. For these analyses, we derivatized the samples with a chiral reagent and designed specific HPLC methods that can chromatographically resolve a mixture of isomers.
To conclude, with most recent advances in technology, LC-MS analysis can provide double confirmation of the analyte by HPLC detection and MS analysis; whereas, HPLC conditions are more important in cases like chiral separation and isomeric separations.