Endocrine Abstracts

Objectives: Oestrogen analysis using liquid chromatography mass spectrometry is problematic, as oestrogens do not readily ionise. This coupled with low concentrations in men, pre-pubertal and post-menopausal women provides an analytical challenge. We investigated N-Methyl Pyridine-3-sulfonyl chloride (NMPS) derivatisation, as described by Wang et al. (Steroids 2015 Apr;96:140-152) to improve sensitivity of 11 oestrogens; oestrone (E1), oestradiol (E2), 2-hydroxy-oestrone, 4-hydroxy-oestrone, 16-hydroxy-oestrone (oestriol-E3), 2-methoxy-oestradiol, 2-hydroxy-oestradiol, 4-hydroxy-oestradiol, 2-methoxy-oestrone, 11β-hydroxy-oestradiol.

Methods: NMPS derivatisation is a two-step process. A pyridine sulfonyl group is first added to hydroxyl groups on the aromatic ring, then treatment with iodomethane adds the N-methyl group, the new molecule termed an oestrogen-NMPS. We used a Waters Xevo-XS with Acquity UPLC, a HSS T3, 1.8 μm, 1.2×50mm column with water and methanol (both with 0.1% formic acid) as elution solvents over five minutes.

Results: Six of the non-derivatised oestrogens were chromatographically separated. Both the 2- and 4-hydroxy metabolites of E1 and E2 co-eluted. LOQ ranged from 0.05 to 0.2ng/mL. Following NMPS derivatisation sensitivity for most analytes at least doubled with LOQ ranging from 0.025 to 0.2 ng/mL. Again, six of the 11 oestrogens chromatographically separated. However, both single and double derivatised products of the 2 and 4-hydroxy oestrogens were observed, adding to the complexity of the method. Excluding these analytes the method was reproducible with repeatability measured as relative standard deviation of less than 10%. Matrix effects were less than ±20%, process efficiency and absolute recovery were between 30 and 50%. Further optimisation of the derivatisation procedure is required to improve recovery and to produce only the double derivatives.

Conclusions: NMPS derivatisation improves oestrogen sensitivity in mass spectrometry, and could provide the sensitivity required for low concentration oestrogen analysis in numerous conditions.