Potential problems with using deuterated internal standards for liquid chromatography-tandem mass spectrometry

We read with interest the recent article by Owen et al. ¹ on the importance of selecting the most suitable internal standard when validating a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. We are in agreement that any internal standard used should be thoroughly investigated during method development. Our recent experience in measuring 5-hydroxyindole acetic acid (5-HIAA) by LC-MS/MS using a d5 deuterated internal standard has highlighted a further issue not reported by Owen et al. ¹

It is considered best practice to use a deuterated analogue of the analyte being measured as they are proposed to have similar extraction recovery, ionization response in electrospray ionization mass spectrometry and the same chromatographic retention time. Moreover, these analogues should co-elute with the analyte to be quantified and contain enough mass increase to show a signal outside the natural mass distribution of the analyte.

An important consideration with lyophilized internal standards is the solvent used for reconstitution and storage. Recently in our laboratory we changed from using a d2 (5-hydroxyindole-acetic-2,2-d₂ acid) to a d5-labelled internal standard (5-hydroxyindole-4,6,7-d₃-3-acetic-2,2-d₂ acid) for the measurement of 5-HIAA. This was done to increase the signal outside the natural mass distribution of the analyte (i.e. the carbon 13 isotope of 5-HIAA). The d5-labelled internal standard (QMX Laboratories Ltd, Essex, UK) supplied contained no reconstitution or storage condition instructions. It was reconstituted and stored as a 10 g/L stock standard in 0.1 mol/L hydrochloric acid, as previously described by Perry et al. ² All stocks and dilutions were stored at −20℃. Serial dilution with 50:50 methanol:water allowed the d5 internal standard to be tuned (197.0>149.9) at a concentration of approximately 200 nmol/L and it was successfully introduced into our laboratory protocol. Within three months of introduction internal standard areas had dropped to 10% of the original peak area and external quality assessment data showed that our laboratory method had a positive bias. Evaluation of a parent ion scan revealed that the predominant m/z (mass to charge ratio) was 195.0 suggesting loss of two deuterium atoms (ion transition 195.0>147.0). Furthermore, a parent ion scan four weeks later revealed equal peak areas at both 194.0 (d2) and 195.0 (d3) parent masses. Personal communication with CDN isotopes suggested that the deuteriums at positions 4 and 6 were exchangeable. The loss of a third deuterium may be presumed to be at position 7. The total loss of three deuteriums resulted in the same parent mass as the previously used d2 5-HIAA.

The mechanism for the loss of deuterium labels appears to be back-exchange of the label for hydrogen ions, present in the 0.1 mol/L hydrochloric acid used to reconstitute the original d5 5-HIAA internal standard.

This phenomenon has not previously been reported in the literature for d5 5-HIAA internal standard. In our experience the d2 5-HIAA internal standard was more robust and was shown to be stable in 0.1 mol/L hydrochloric acid over an 18 month period. It is our recommendation that the solvent used to reconstitute deuterated internal standards be thoroughly investigated to avoid unnecessary loss in sensitivity. In this instance we recommend that d5 5-HIAA is reconstituted and stored in acetonitrile. Discussion with the supplier revealed the stability and storage data provided is only valid for the lyophilized product.