Isotopically labelled testosterone derivatives as internal standards in liquid chromatography-tandem mass spectrometry

Bui et al. ¹ raise some interesting considerations in response to our recent manuscript. ² To clarify, the same D2 testosterone was used as described in the original method paper. ³ Although we agree that isotopically labelled compounds can elute away from their non-labelled counterparts, particularly when using ultrapressure liquid chromatography (UPLC), this was not the case in our investigation. We used high-performance liquid chromatography rather than UPLC conditions for our assay and the D2, D5 and C13 testosterone all co-eluted with endogenous testosterone.

As detailed in the discussion part of our original paper, we accept that different results to ours may be achieved using alternative extraction procedures, columns and chromatography conditions. However the aim of our publication was to stress that internal standards may not be interchangeable within one assay system and that particular attention should be paid to the combination of chromatography conditions, sample preparation and ion suppression during the method development phases to prevent this phenomenon. Particular care should be taken if liquid chromatography-tandem mass spectrometry (LC-MS/MS) users adopt the sample preparation and chromatography of an existing assay such as our own but opt to change the internal standard. Careful selection of sample preparation and chromatography conditions with respect to different internal standards will result in an assay in which this phenomenon does not occur such as in the method of Bui et al. ⁴ This is a problem that has developed recently due to the wide variety of internal standards now available, whereas D2 testosterone was the only one available during our method development phase.

Both ourselves and Bui et al. ¹ have had the opportunity to validate our assays against a reference method. This is not always possible and in these instances laboratories should try to run Certified Reference Materials when available or compare their method with well validated LC-MS/MS procedures using clinical samples.