Since 1980, the applications of LC-MS/MS analysis have grown exponentially in biomedical and pharmaceutical domains. The increased applications of this method are due to its enhanced selectivity, sensitivity, and rapid analysis. LC-MS assays have been employed widely by mass spectrometry services. Tandem mass spectrometry was thus an evident extension of LC-MS assays.
From rapid data generation in preclinical studies to screening early drug candidates, LC-MS analysis has several applications in drug discovery and development. Successful implementation of LC-MS analysis requires an in-depth understanding of analyte extraction and the underlying mechanism of mass spectrometry and chromatography. Similar to LC-MS method development and validation, LC-MS analysis requires adequate method development, validation, and troubleshooting initiatives. The current article discusses troubleshooting issues with LC-MS/MS analysis.
Troubleshooting LC-MS/MS analysis procedure
Sample preparation approaches are crucial in LC-MS analysis. The primary step is removing unwanted compounds without losing the target analytes from study samples. The second step includes separating the target analytes from the unwanted compounds. However, extracting unwanted compounds poses a significant challenge for researchers and LC-MS practitioners. In the LC-MS mass spectrometry interface, unwanted interferences may compete with the target analytes for ionization. This may result in inconsistent matrix effects and affect quantitative LC-MS analysis. Hence, researchers must take care while analyzing LC-MS data and identify unseen MS peaks that may correlate to contaminants in the sample.
Drug product analytes may be subjected to degradation. Endogenous enzymes present in biological matrices can speed up the process of analytic degradation. Hence, adequate data on degraded analytes during method development is vital. Approaches to stabilize the analytes present in the sample may include choosing an adequate enzyme inhibitor, pH, or anticoagulant. If researchers cannot stabilize the analytes, then they can employ in-situ derivatization of analytes in biological matrices.
Plasma anticoagulants can significantly stabilize the target analytes. For example, analytes containing ester are unstable in plasma with sodium heparin. They can be relatively stable with sodium chloride anticoagulants in plasma samples. Besides, researchers should establish the stability of the target analyte in the study sample matrix as early as possible during method development.
Today, rapid LC-MS systems have resulted in the application of smaller dimension columns. Compared to conventional columns, smaller dimension columns offer benefits, including better concentration, sensitivity, faster analysis time, and lower solvent consumption. They can be easily disturbed. Misalignment between the mobile phase and injection solution is one of the common problems with compounds having early elution peaks.
Researchers can increase sensitivity by selecting a reconstitution solvent compatible with the mobile phase. One may enrich the influence of injection solution on chromatography efficiency and peak shape by injecting a solution with the sample eluting from the mobile phase. Researchers can achieve maximum chromatography efficiency by employing injection solutions with the weakest elution strength. However, considering practicality, one may obtain better chromatography efficiency by increasing the injection volume and diluting the sample with a weaker mobile phase component.
Conclusion
LC-MS/MS analysis is a robust technique for separating and detecting analytes of interest in complex biological matrices. However, an in-depth understanding of separation science and spectrometry detection is crucial for troubleshooting common issues that emerge during the LC-MS/MS analysis procedure.
