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

Ann Clin Biochem 2008;45:210-212
© 2008 Association for Clinical Biochemistry

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Short Report

K Duxbury 1 ,
L Owen 1,
S Gillingwater 2 and
B Keevil 1

1 University Hospital of South Manchester NHS Foundation Trust, Southmoor Road, Manchester M23 9LT, UK;
2 Clinical Applications Group, Waters Ltd, Manchester, UK

Corresponding author: Miss K Duxbury. Email: k.j.duxbury.00{at}

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. 2H (D, deuterium) are often used as internalstandards.

Methods: Possible interference by naturally occurring isotopes of ananalyte in the internal standard channel in a liquid chromatographytandem MS assay was assessed using cortisol and its deuteratedinternal standard, D2-cortisol, as an example. Mass spectrawere analysed and standard curves were prepared with varyingconcentrations of internal standard to determine the extentof any interference.

Results: The mass spectra showed that a naturally occurring isotope ofcortisol at m/z 365 acts in the same way as D2-cortisol andfragments to give a daughter ion of the same m/z. Cortisol-365can therefore falsely, but significantly, increase the amountof internal standard detected, and this will concomitantly decreasethe relative response for cortisol. The standard curves withvarying concentrations of internal standard showed that thisphenomenon can affect the linearity of an assay.

Conclusions: Our results show that care is needed in assay development whendoubly deuterated internal standards are used. Interferenceby naturally occurring isotopes of the analyte of interest inthe internal standard transition is possible and it is importantto ensure that an appropriate internal standard concentrationis chosen that permits linearity of the assay over the requiredrange.

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