Background: There are considerable demands to accurately measure estradiol (E 2 ) at low concentrations (<20 pg/mL) in postmenopausal women, men, pediatric patients, and patients receiving breast cancer treatment. Most current high-sensitivity LC-MS/MS E 2 methods require large sample volumes and involve complex sample preparations with dansyl chloride derivatization. Our study aims to develop a high-sensitivity, underivatized method using micro LC-MS/MS to reliably measure E 2 concentrations below 5 pg/mL by the use of low sample volume. Methods: A total of 290 μL of sample was mixed with internal standard (IS), E 2 -d4, and extracted with a mixture of hexane/ethyl acetate (90/10) (v/v). After extraction, sample was separated by Eksigent Ekspert™ micro LC 200 system with a flow rate of 35 μL/min in a total run time of 3.5 min and detected by SCIEX QTRAP 6500 mass spectrometer in a negative mode using transitions: 271/145 (quantifier) and 271/143 (qualifier). In this method, it was crucial to use HPLC columns with stability at a pH >10. Results:The validation study demonstrated broad linear ranges (3.0 -820.0 pg/mL) with r 2 > 0.999. Total precision was below 15% at all QC levels, and limit of quantification (LOQ) was 3.0 pg/mL. Our method showed good correlation with E 2 RIA (r 2 = 0.96, bias = −1.0 pg/mL) and modest correlation with E 2 Roche Cobas automated immunoassay (r 2 = 0.86, bias = 6.0 pg/mL). Conclusions:In conclusion, we developed and validated a routinely applicable micro LC-MS/MS method without derivatization for E 2 in blood samples with an LOQ of 3.0 pg/mL. IMPACT STATEMENTPatients under assessment of puberty delays, pubertal growth, gynecomastia, and efficiency of aromatase inhibitor (AI) treatment will benefit from the information presented here. Evidence presented on high-sensitivity LC-MS/MS assay for E 2 will allow better characterization of E 2 concentration in blood in the low measurement range. Knowledge in the field of LC-MS/MS method development for E 2 will be advanced by the information presented.

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Objectives Analysis of platelet functional responses to stimuli is presently quite limited with respect to measurement of dense granule secretion. We sought to develop a nonradioactive assay of stimulated serotonin release using liquid chromatography tandem mass spectrometry (LC-MS/MS). Methods Citrated whole blood (200 μL) was incubated with deuterated serotonin (d45-HT). Following uptake by platelets, blood was diluted 10-fold and aliquots were incubated with platelet stimuli. Following stimulation, blood was further diluted, centrifuged, and supernatant was assayed for released d45-HT by micro-LC–MS/MS. Results This study demonstrated a broad linear range of 50 to 2,000 pg/mL d45-HT, with a total precision of less than 15.0% coefficient of variation at all quality control levels and a limit of quantitation of 50 pg/mL. Conclusions Quantification of d45-HT by micro-LC–MS/MS assay offers a highly sensitive, nonradioactive methodology for quantitating platelet serotonin uptake and dense granule secretion, requiring only small volumes of patient blood.