High-Sensitivity Micro LC-MS/MS Assay for Serum Estradiol without Derivatization

Abstract

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.

Systemic estradiol levels with low-dose vaginal estrogens

“…More specific assays (ie, LC or GC with MS) have less cross-reactivity with other steroidal-like molecules and typically yield lower estradiol values than less specific assays with inherently greater cross-reactivities (ie, RIA or ELISA). 9,54 Our current review is consistent with previous findings demonstrating that many of the most recently developed highly specific assays can detect or quantify systemic estradiol as low as 0.1 to 2.7 pg/mL using LC/MS/MS or LC/MS, [37][38][39][40][41]43,44,[55][56][57][58][59][60][61] and 1 to 5 pg/mL using GC/MS/MS or GC/MS. 26,28,29,42 Indeed, the goal of sensitivity has been SYSTEMIC ESTRADIOL WITH VAGINAL ESTROGENS to develop assays detecting <5 pg/mL of estradiol for measuring estradiol in serum of postmenopausal women.…”

The Golden Age Revisited via Estradiol and LC-MS/MS

“…The study by Yi et al (1) affirms my confidence in stating that the second golden age of clinical chemistry is upon us once again. I am excited and grateful because, 30 years after starting my career in clinical chemistry, I am back in the real chemistry game, and patients will benefit from these efforts.…”

The Effect of the Testis on the Ovary: Structure-Function Relationships in a Neonate with a Unilateral Ovotestis (Ovotesticular Disorder of Sex Development)

“…Serum LH and FSH were measured by immunofluorometric assays (Delphia®, Wallach, Finland). Serum testosterone was measured by direct radioimmunoassay (Diagnostic Products Corporation, DPC, Coat-A-Count®, Los Angeles, CA, USA), estradiol by a post-extraction radioimmunoassay (Pantex®, Santa Monica, CA, USA), and 17-hydroxyprogesterone (17OHP) by post-chromatographic radioimmunoassay in the University of Chicago Hospital Laboratories (UCHL) [11]; these steroid radio-immunoassays have been validated against liquid chromatography-mass spectrometry methods [12, 13]. Other hormone levels were measured by commercially available assays at UCHL, Mayo Medical Laboratories (Rochester, MN, USA), or Esoterix (Calabasas Hills, CA, USA).…”