Naturally occurring isotopes of an analyte can interfere with doubly deuterated internal standard measurement

Background: Internal standards are essential in quantitative mass spectrometry (MS) assays to correct for variability in sample extraction and ionization at the source. In liquid chromatography MS assays, analogues of the analyte with several atoms replaced by their stable isotopes, e.g. H-2 (D, deuterium) are often used as internal standards. Methods: Possible interference by naturally occurring isotopes of an analyte in the internal standard channel in a liquid chromatography tandem MS assay was assessed using cortisol and its deuterated internal standard, D2-cortisol, as an example. Mass spectra were analysed and standard curves were prepared with varying concentrations of internal standard to determine the extent of any interference. Results: The mass spectra showed that a naturally occurring isotope of cortisol at m/z 365 acts in the same way as D2-cortisol and fragments to give a daughter ion of the same m/z. Cortisol-365 can therefore falsely, but significantly, increase the amount of internal standard detected, and this will concomitantly decrease the relative response for cortisol. The standard curves with varying concentrations of internal standard showed that this phenomenon can affect the linearity of an assay. Conclusions: Our results show that care is needed in assay development when doubly deuterated internal standards are used. Interference by naturally occurring isotopes of the analyte of interest in the internal standard transition is possible and it is important to ensure that an appropriate internal standard concentration is chosen that permits linearity of the assay over the required range.