Endocrine Abstracts

Introduction: Testosterone measurement by liquid chromatography tandem mass spectrometry (LC-MS/MS) is well accepted as the preferred technique for the analysis of serum testosterone in both males and particularly females. However variation is seen between LC-MS/MS assays and is most likely to be due to method differences between laboratories. One area of inconsistency amongst routine LC-MS/MS assays is the choice of internal standard. We investigated the effects of three internal standards on the results obtained.

Methods: Stable isotopes of testosterone were prepared in methanol. Testosterone with two deuterium (D2), five deuterium (D5) and three carbon thirteen enrichment (C13) were separately assessed. Sera, calibrators and quality controls (100 µL) were extracted using 0.5 mL of ether following the addition of 10 µL of internal standard. All aliquots were prepared in triplicate, one for each type of internal standard. After mixing the ether was transferred to the wells of a 96-deep well block then evaporated to dryness. Extracts were reconstituted with 100 µL of 50% mobile phases and analysed using a Waters Acquity LC and Quattro Premier tandem mass spectrometer. This method had previously shown to have excellent agreement with a reference method using the D2 internal standard.

Results: Lower results were obtained when using D5 testosterone when compared to D2 testosterone. The Passing-Bablock regression equation was; testosterone (D5) nmol/L = 0.86 × testosterone (D2) + 0.04. The C13 internal standard also gave lower results but was closer than the D2 target than the D5 internal standard. The Passing-Bablock regression analysis was; testosterone (C13) nmol/L = 0.90 × testosterone (D2) + 0.02.

Discussion: The choice of internal standard alone can have a significant affect on the results obtained by LC-MS/MS assays for testosterone using this chromatography. The effects of the combination of chromatography and internal standard choice should be investigated during method development.

Declaration of interest: There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding: No specific grant from any funding agency in the public, commercial or not-for-profit sector.