Study of Isobaric Interference in Quantification of Citrulline by Parallel Fragmentation Monitoring

Parallel Fragmentation Monitoring (PFM), which is an analogue of selected reaction monitoring (SRM), is a recently developed method for quantification of small molecules by MALDI-TOF/TOF mass spectrometry (MS). It is well known that isobaric interference substances can be occasionally present in complex biological samples, and affect the accuracy of measurement by SRM. Unfortunately, by design it is not possible to assess whether isobaric interference happens in a SRM analysis. In contrast, the unique design of PFM should allow quick inspection for isobaric interference and subsequent correction. In this study, using arginine as an example, interference effect of isobaric structural analogs on the quantification of citrulline by PFM was evaluated. Our results showed that the presence of arginine affected the measured concentrations of citrulline standard solutions in a concentration dependent manner. Such interference could be observed readily in the MS/MS spectra, and contributed by [arginine+H-NH3](+) fragment ion. Because of having highly similar mass, (13)C-isotope of [arginine+H-NH3](+) fragment ion overlapped with monoisotope of [citrulline+H-NH3](+) fragment ion, which was used as the report ion for quantification. However, such interference could be mathematically eliminated or minimized through estimation of the signal intensity of the (13)C-isotopic peak of [arginine+H-NH3](+) from the intensity of the corresponding monoisotopic peak according to isotope distribution of elements. Furthermore, the presence of interfering fragment ions could be avoided by optimizing MALDI ionization condition. In conclusion, isobaric interference can happen in PFM, but can be easily identified in the mass spectra and eliminated (minimized) with simple methods.