Leveraging Ion Ratio for Analyte Confirmation in Mass Spectrometry

Analysis of chemical compounds by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in complex samples presents unique challenges that should be considered, from ion suppression due to matrix effects, to lack of available stable isotopically labeled standards for accurate quantification. Understanding the caveats of LC-MS/MS is important for assessing samples with varying matrix complexity.

Triple quadrupole MS achieves high selectivity and sensitivity through selected reaction monitoring (SRM). In SRM, a precursor ion is isolated in the first quadrupole and fragmented in the collision cell, followed by isolating selected product ions in the third quadrupole. Despite this excellent selectivity, there is still a possibility of a compound other than the target analyte producing a signal with a similar m/z value to the qualifying or quantifying ion at the same retention time. This can result in a significant deviation in the expected ion ratio for the monitored fragment ions during the two SRM transitions.

To confirm the presence of a compound, the chromatographic peak must exhibit both the quantitative and qualitative ion transitions, aligning with the retention times of the standard analyte (Figure 1).

Figure 1

Furthermore, the ion ratio of the two SRM transitions serves as an additional confirmation criterion, as outlined in legal documents from esteemed organizations such as the European Union (EU) and the US Food and Drug Administration (US FDA). These guidelines provide essential insights for analyzing official samples. By monitoring the ion ratios, we can enhance confidence in reporting analyte concentrations, minimizing the occurrence of false positives and false negatives, which have been documented in the literature. However, doing an additional calculation can be time consuming, especially when it involves multiple compounds and samples. To aid in this additional work, we created a template that automatically calculates the ion ratios and flags samples that fail.

Ion Ratio Calculator Preview

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