Endocrine Abstracts

Liquid Chromatography tandem mass spectrometry (LC–MS/MS) is gold-standard for androgen analysis in biological fluids, superseding immunoassays in specificity, particularly at low concentrations. While LC–MS/MS is well established for analysis of testosterone (T) and androstenedione (A4), 5α-dihydrotestosterone (DHT) presents greater analytical challenges. DHT circulates at low nanomolar concentrations in men and lower in women, ionising inefficiently. Thus, even using current LC–MS/MS technology, relatively large plasma volumes (>0.5 ml) are required for detection, undesirable clinically and unsuitable for animals. This study investigated stable derivatisation approaches using hydrazine-based reagents to enhance ionisation efficiency and sensitivity of analysis of DHT by LC–MS/MS. Derivatisation of DHT using 2-hydrazino-1-methylpyridine (HMP) and 2-hydrazino-4-(trifluoromethyl)-pyrimidine (HTP) were compared. A LC–MS/MS method was validated a solid-phase extraction (SPE) LC–MS/MS method using an Acquity-QTrap5500, analysing extracts of human plasma (male, pre and post-menopausal women), following SPE using Oasis^® HLB (1 cc/10 mg). HMP derivatives were selected for validation being more sensitive than those formed with HTP. HMP derivatives were detected by selected reaction monitoring (DHT-HMP m/z 396 → 108; T-HMP m/z 394 → 108; A4-HMP m/z 392 → 108). Chromatographic separation of androgen derivatives was optimised, carefully separating isobaric interferents. Limits of detection on column were 0.2, 0.4 and 0.2 pg and quantitation were 0.4, 0.8 and 0.5 pg for DHT-HMP, T-HMP and A4-HMP respectively. HMP derivatives of all androgen could be detected in small plasma volumes: male (100 µL) and female (200 µL), and derivatives were stable over 30 days. In conclusion, HMP derivatisation, in conjunction with LC–MS/MS, is suitable for quantitative analysis of DHT and T in small plasma volumes, offering clear advantages in sensitivity over current methodologies. Concomitant analysis of A4-HMP offers similar sensitivity to the underivatized steroid.