Background: Specific measurement of low serum/plasma concentrations of 17β-oestradiol (E2) is important in pre-pubertal and pubertal children in routine paediatric endocrinology. The role of oestrone (E1) and oestriol (E3) is not as comprehensively well understood in different pediatric endocrine disease states. We describe a method for high sensitivity analysis of estradiol (E2), oestrone (E1) and oestriol (E3) using LC-MS/MS.
Objective and hypotheses: Determination of E2, E1 and E3 in serum/plasma derived from prepubertal and pubertal children. Implementation of a high sensitive LCMSMS method in daily routine and establish reference ranges of E2, E1 and E3 for children.
Method: 0.1 ml serum and plasma samples respectively, were combined with stable isotope-labeled internal standard and subsequently, estrogens were extracted by SPE (solid phase extraction). The solvent was evaporated, estrogens were derivatized to form dansyl derivatives, the samples were analyzed using UPLC-MS/MS in positive MRM mode.
Results: The method was linear from 0.01 nmol/l (3 pg/ml) up to 50 nmol/l (13 600 pg/ml) for E1, E2 and E3. The lowest limit of quantification was 0.018 nmol/l (4.8 pg/ml). Preliminary method-specific reference ranges for children, age, sex and pubertal stage were established. Also preliminarily but expectedly, LC-MS/MS-determined E2 in girls with untreated precocious puberty was significantly higher compared with age-matched controls in contrast to E1 and E3. Obese girls and obese boys (BMI>97th centile respectively, did not show significantly higher E1, E2, and E3 levels than controls.
Conclusion: We developed a robust, fast and reliable method for analyzing the major estrogens in the daily pediatric endocrine routine. Our LC-MS/MS-triple-assay will help to get more insights into the differential roles of these three oestrogens in paediatric endocrine development and disease.
01 Oct 2015 - 03 Oct 2015