Endocrine Abstracts

Physiological circulating concentrations of estrogens are very low in men and postmenopausal women (<50 pg/ml), which presenting analytical challenges. Immunoassays can detect as low as 30 pg/ml, but cross-reactivity is a concern. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) offers greater specificity than immunoassays, but ionisation of estrogens is inefficient. Derivatisation, which introduces charged moieties, may enhance ionisation. Dansylated derivatives of estrogens have been reported previously but these generate non-specific product ions originating from the “reagent” group in MS/MS. Therefore, a derivative with product ions specific to individual estrogens was sought.

Estrogens were extracted from human plasma using solid phase extraction and derivatised using 2-fluoro-1-methylpyridinium-p-toluenesulfonate (FMP). Estrogen FMP derivatives were quantified using triple quadrupole MS in positive electrospray ionisation mode, following LC separation. Reaction conditions were optimised and the assay validated for linearity, precision, accuracy, and stability. Limits of detection and quantitation (LOD and LOQ) were evaluated.

Transitions for the FMP derivatives of estrone (E₁) and estradiol (E₂) were compound specific (m/z 362→238 and m/z 364→110 respectively). The LOD was 2 pg/ml and the method was linear across the range 5–200 pg/ml. The values of intra- and inter-assay variables were acceptable at LOQ (5 pg/ml), precision (intra): 12%, 11% (inter): 15%, 13%; accuracy (intra): ±18%, ±17%, (inter): ±19%, ±19% for E₁, E₂ respectively. The derivatives demonstrated suitable stability over 24 h, (7–9% degradation). Using this approach, E₁ and E₂ were detected in human plasma (0.5 ml female, 1 ml male) at levels (7–9), (55–59) pg/ml in males and (78–84), (204–216) pg/ml in females for E₁ and E₂ respectively with signal to noise of 5–10.

Here, we have demonstrated that FMP derivatisation in conjunction with LC–MS/MS is suitable for quantitative analysis of low abundance estrogens in biological fluids. The assay offers clear advantages in specificity and sensitivity over immunoassay and existing MS techniques for estrogens analysis.