Details

Autor(en) / Beteiligte

• Giraldo, Jeniffer Calderón

Titel

Analysis of Estrogen Profiles Including Methoxyestrogen Glucuronides: Method Validation and Applicability to Human Plasma and Breast Tissue

Ort / Verlag

ProQuest Dissertations & Theses

Erscheinungsjahr

2020

Quelle

ProQuest Dissertations & Theses A&I

Beschreibungen/Notizen

• Estrogens play a key role in breast cancer development. However, not all estrogen metabolites have the same effect. Therefore, the knowledge on the profile of circulating estrogens in healthy women is of importance. However, most methods used for the analysis of estrogens do either not target conjugated estrogens or detect only the sum of the respective free and conjugated form. Accordingly, this work aimed to evaluate the possibility to include methoxyestrogen glucuronides into an existing method for the analysis of estrogens, and therewith, complement the analysis of estrogen profiles in human plasma and breast tissue from women without breast cancer.First, microsomal protein fractions from rat liver were used to biosynthesize six different methoxyestrogen glucuronides and their corresponding deuterium analogues to serve as reference compounds. Deuterated analogues were synthesized in vitro by methylation and glucuronidation of deuterated hydroxyestrogens. Identities of the products were verified by UHPLC-tandem mass spectrometry in enhanced product ion scan mode. Quantification using the peak area ratio of estrone and 17β-estradiol to their corresponding glucuronides and calibration curves of their methoxylated derivates confirmed amounts of reference compound synthesized in the microgram range which were sufficient for further analysis. For these references, the most intense precursor and fragment ions were determined and their respective peak intensities and chromatographic separation were optimized.For the validation of the method, the mixture of deuterated analogues (internal standards) was separated exhibiting a good chromatographic resolution of the peaks as well as no interference with possible isotopes. The specificity of the analytical method was ensured by the specific substance- and source-dependent parameters and the selectivity by quantifier/qualifier ratios. The recoveries were between 51% (4- methoxyestrone-3-glucuronide) and 62% (2-methoxyestradiol-3-glucuronide). All conjugates exhibit a proportional analytical response except for 2-methoxyestradiol17-glucuronide. The limits of detection and quantification in plasma were between 66 fmol/mL (4-methoxyestrone-3-glucuronide) and 1022 fmol/mL (2-methoxyestradiol-17- glucuronide) and between 155 fmol/mL (4-methoxyestrone-3-glucuronide) and 3636 fmol/mL (2- and 4- methoxyestradiol-17-glucuronide), respectively. At the suggested limits of quantification, intra- and inter-day accuracies of 93% (2-methoxyestradiol-3- glucuronide) to 110% (4-methoxyestrone-3-glucuronide) and intra- and inter-day precisions of 2% (2-methoxyestradiol-3-glucuronide) to 15% (4-methoxyestrone-3- glucuronide) were achieved. For 2- and 4-methoxyestradiol-17-glucuronide, accuracies and precisions up to 188% and 33%, respectively, were out of specification. Biosynthesized references were stable at the storage conditions for at least five months, as well as in spiked plasma samples stored in the autosampler for one day, and after freeze/thaw cycles during four days. Since accuracies and precisions are in line with the FDA (2018) recommendations, and because of the recoveries above 50%, the limits of detection in the range of tens to hundred fmol/mL, and the proportional analytical response of the references, the extended method could be applied for the quantitative analysis of 2- and 4-methoxyestrone-3-glucuronide and 2- and 4- methoxyestradiol-3-glucuronide in human plasma. Because of accuracies and precisions out of specification, poor analytical response and higher limits of detection, the extended method was not optimum for the quantitative analysis in plasma of 2- and 4-methoxyestradiol-17-glucuronide, but the qualitative analysis could be performed.