Use of a structural analogue versus a stable isotope labeled internal standard for the quantification of angiotensin IV in rat brain dialysates using nano‐liquid chromatography/tandem mass spectrometry

Abstract

Quantifying low concentrations of neuropeptides in microdialysates requires a selective and sensitive analysis technique, such as nano-liquid chromatography/electrospray ionization tandem mass spectrometry (nanoLC/ESI-MS/MS). However, we observed reduced accuracy of the method due to matrix effects. Indeed, ESI-MS detection is known to be sensitive to matrix effects. Moreover, dialysates are complex mixtures of small molecules, peptides and other matrix compounds that can influence the ionization efficiency of the neuropeptide of interest and the stability of the peptide in the samples. In the study reported in this paper, we investigated whether the use of an internal standard (IS) can correct for these matrix effects. As a model compound for neuropeptides we used angiotensin IV (Ang IV). We compared the use of a structural analogue (norleucine1-Ang IV) with a stable isotope labeled (SIL) analogue. Linearity of the method was improved when either of the proposed ISs were applied. Only when using the SIL-IS could the repeatability of injection and the method's precision and accuracy be improved. Finally, the IS was able to correct for degradation of Ang IV in dialysates, prolonging the possible storage period of the samples. We conclude that the structural analogue is not suited as an IS and that the application of a SIL analogue is indispensable when quantifying Ang IV in dialysates using nanoLC/ESI-MS/MS detection.