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Endocrine Abstracts (2018) 59 P065 | DOI: 10.1530/endoabs.59.P065

SFEBES2018 Poster Presentations Clinical biochemistry (10 abstracts)

An oestrogen profiling mass spectrometry method using N-Methyl Pyridine-3-sulfonyl chloride derivatisation

Jamine Johal , George Jolly , Lorna C Gilligan & Angela E Taylor


Insitute of Metabolism and Sytems Resaerch, Birmingham, UK.


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.

Volume 59

Society for Endocrinology BES 2018

Glasgow, UK
19 Nov 2018 - 21 Nov 2018

Society for Endocrinology 

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