LC-MS/MS detection of increased androstenedione levels in patients receiving danazol therapy.

Androstenedione is measured as part of a steroid hormone panel. Samples were prepared as per previously published method ( 6 , 7 ). An Agilent 6460 triple quadropole mass spectrometer (Agilent, USA) equipped with an atmospheric pressure photoionization was used, employing isotope dilution with deuterium labeled internal standard for each analyte. Quantitation by multiple reaction monitoring (MRM) was performed in the positive ion mode. The quantifier MRM transition for androstenedione used was 287.2> 97.1 and the qualifier MRM of 287.2 >109 was used to confirm. The ratio of response of the two MRMs used, ranged from 56-58%. Retention times (RT) for AND were 6.540 -6.556 minutes for patient samples and 6.529- 6.554 minutes for the accompanying internal standard (IS). The ratio of androstenedione RT to internal standard RT for each specimen run was 1.00, thus confirming the identity of the peak. The common metabolites of danazol present in the serum namely, ethisterone and 2-hydroxymethyl- ethisterone (2-HME) have been reported to affect immunoassay measurements of androgens due to cross reactivity with assays or by affecting hormone binding protein concentrations ( 8 ). The molecular weight of each metabolite is 312.45 and 342.47 respectively and thus unlikely to interfere with LC –MS/MS measurement of androstenedione which has different MRM's. One of the important advantages of mass spectrometry analysis is the greater specificity of mass spectrometry measurement over immunoassay based testing. In the above described cases. Use of two MRM transitions (quantifier and qualifier) enabled the laboratory to confirm the presence of elevated androstenedione and exclude the likelihood of an interference.

Danazol increased the concentrations of androstenedione in all 8 patients with all being elevated to levels >97.5 th percentile. Similar increases were not observed for the other androgens measured. Two of the 3 males had significant estradiol levels after treatment while all the females studied had normal estradiol concentrations during therapy. This finding raises interesting questions as to the effect of danazol on estrogen concentrations.