Development and validation of LC-ESI-MS/MS methods for quantification of 27 free and conjugated estrogen-related metabolites

• Published in: Analytical biochemistry 2020-02, Vol.590, p.113531-113531, Article 113531
• Main Authors: van der Berg, Carien, Venter, Gerda, van der Westhuizen, Francois H., Erasmus, Elardus
• Format: Article
• Language: English
• Subjects: Adolescent; Adult; Biotransformation; Breast Neoplasms - metabolism; Chromatography, High Pressure Liquid - methods; Dansylation; Estrogen metabolism; Estrogens - urine; Estrogens, Conjugated (USP) - urine; Female; Healthy Volunteers; Humans; LC-MS/MS; Method development; Tandem Mass Spectrometry - methods; Young Adult
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2019.113531

An imbalance in the estrogen metabolism has been associated with an increased risk of breast cancer development. Evaluation of the estrogen biotransformation capacity requires monitoring of various estrogen metabolites. Up to now, only some estrogen metabolites could be measured in urine. However, in order to offer tailor made nutritional support or therapies, a complete estrogen metabolite profile is required in order to identify specific deficiencies in this pathway for each patient individually. Here, we focused on this need to quantify as many as possible of the estrogen-related metabolites excreted in urine. The method was developed to quantify 27 estrogen-related metabolites in small urine quantities. This entailed sample clean-up with a multi-step solid phase extraction procedure, derivatisation of the metabolites in the less water-soluble fraction through dansylation, and analyses using liquid chromatography–tandem mass spectrometry (LC-MS/MS). The metabolites accurately quantified by the method devised included parent estrogens, hydroxylated and methylated forms, metabolites of the 16α-hydroxyestrogen pathway, sulphate and glucuronide conjugated forms, precursors and a related steroid hormone. This method was validated and enabled quantification in the high picograms and low nanograms per millilitre range. Finally, analyses of urine samples confirmed detection and quantification of each of the metabolites. [Display omitted] •Imbalance in estrogen biotransformation is associated with breast cancer risk.•Devised method enables analysis of free and conjugated estrogen-related metabolites.•Requires sample fractionation during SPE and derivatisation of only one fraction.•Detection and quantification of 27 metabolites confirmed in urine samples.