A-172 Development of high Sensitivity Quantitative Measurement of Estrogen in Human serum by Liquid Chromatography Tandem Mass Spectrometry

Abstract

Measurement of serum estrogen provides invaluable information in the assessment of reproductive function in females and in the assessment of infertility, oligo-amenorrhea, and menopausal status. High-sensitivity measurement of serum estrogens is particularly important in adult and pediatric endocrinology and oncology. We developed and validated high Sensitivity LC-MS/MS based assay for the simultaneous measurement of Estrone (E1), Estradiol (E2) and Estriol (E3) in human serum. Serum sample is mixed with 13C3 labelled E1 and E2 in Acetonitrile to precipitate protein followed by solid liquid extraction in organic solvent using ISOLUTE SLE+ Solid Liquid Extraction (Biotage). The extract was dried under N2 stream and derivatized with densyl chloride into corresponding densyl complex and analyzed using AB Sciex 6500+ mass spectrometer coupled with Shimadzu LC. Calibrators prepared ultra-low hormone & steroid, lipid free serum (Mass Spect Gold serum, GoldenWest). The assay is validated by evaluating linearity and AMR, LOD and LOQ, accuracy and precision, interference and ion suppression, specificity, carryover and correlation. The assay is linear from 4 to 800 pg/mL with LOD of 1.3 pg/mL. Inter and Intra assay imprecision is within 6.5% with mean recovery within 95 - 105%. The correlation co-efficient is 0.9988 in 22 serum sample compared to Siemens Atellica immunoassay. The correlation co-efficient is 0.9959 in 80 samples analyzed with CDC reference method obtained through CDC steroid hormone standardization program with bias <20% in over of 97.5% samples. %bias was within 15% in all 12 CAP proficiency testing samples. A simple high sensitivity LC-MS/MS based assay suitable for clinical use was developed for measurement of estrogen in the human serum