Optimization of LC‐ESI‐MS Quantification of Ventromedial Hypothalamic Nucleus Estradiol Content by Central Composite Design

Abstract

Brain structures exhibit characteristic patterns of estrogen receptor expression and regulatory functions and local capacity to generate neuroestradiol, necessitating quantification of estradiol content of individual loci. Estradiol acts on ventromedial hypothalamic nucleus (VMN) substrates to control behavior and glucostasis. Current research utilized Analytical Quality of Design to optimize uHPLC‐electrospray ionization‐mass spectrometry (LC‐ESI‐MS) sensitivity for analysis of estradiol in VMN samples obtained by high‐resolution microdissection techniques. A Design of Experiments matrix was developed to assess effects of critical independent variables on estradiol chromatogram area. A second‐order polynomial equation was generated by statistical response surface methodology using central composite design to identify synergistic versus antagonistic factors and to predict singular/combinatory effects on response area. Estradiol, estrone, and estriol were used as optimizing compounds; ethynyl estradiol served as internal standard. Lysates of VMN tissue harvested by micropunch dissection were transferred to methanol‐water conditioned solid phase extraction syringe cartridges and treated with 2% formic acid. Estradiol content was extracted with methanol, then vacuum concentrated. Samples were treated with 2‐fluoro‐1‐methylpyridinium‐p‐toluenesulfonate (FMP‐TS) prior to application to a C18 column [0.2ml/min mobile phase (25:75, v/v) flow rate; 0.1% formic acid, acetonitrile], followed by spectrometric analysis of estradiol and internal standard responses at m/z 364.2 and 388.2, respectively. Optimized mass spectrometric vaporizer (320°C) and ion transfer tube (266 °C) temperatures and sheath (22 psig), auxiliary (5 psig), and sweep (0.7 psig) gas pressures resulted in maximum tissue estradiol response. Outcomes validate a novel combinatory methodology for estradiol quantification in precisely‐defined brain components.Support or Funding InformationNational Institutes of Health DK‐109382