Quantitation of endogenous GnRH by validated nano-HPLC-HRMS method: a pilot study on ewe plasma

Abstract

Gonadotropin-releasing hormone isoform I (GnRH), a neuro-deca-peptide, plays a fundamental role in development and maintenance of the reproductive system in vertebrates. The anomalous release of GnRH is observed in reproductive disorder such as hypogonadotropic hypogonadism, polycystic ovary syndrome (PCOS), or following prenatal exposure to elevated androgen levels. Quantitation of GnRH plasma levels could help to diagnose and better understand these pathologies. Here, a validated nano-high-performance liquid chromatography–high-resolution mass spectrometry (HPLC-HRMS) method to quantify GnRH in ewe plasma samples is presented. Protein precipitation and solid-phase extraction (SPE) pre-treatment steps were required to purify and enrich GnRH and internal standard (lamprey-luteinizing hormone-releasing hormone-III, l-LHRH-III). For the validation process, a surrogate matrix approach was chosen following the International Council for Harmonisation (ICH) and FDA guidelines. Before the validation study, the validation model using the surrogate matrix was compared with those using a real matrix such as human plasma. All the tested parameters were analogous confirming the use of the surrogate matrix as a standard calibration medium. From the validation study, limit of detection (LOD) and limit of quantitation (LOQ) values of 0.008 and 0.024 ng/mL were obtained, respectively. Selectivity, accuracy, precision, recovery, and matrix effect were assessed with quality control samples in human plasma and all values were acceptable. Sixteen samples belonging to healthy and prenatal androgen (PNA) exposed ewes were collected and analyzed, and the GnRH levels ranged between 0.05 and 3.26 ng/mL. The nano-HPLC-HRMS developed here was successful in measuring GnRH, representing therefore a suitable technique to quantify GnRH in ewe plasma and to detect it in other matrices and species. Graphical abstract

Rosuvastatin Potentiates Gonadotropin-Lowering Effects of Metformin in Postmenopausal Women: A Pilot Study

“…The main sources of plasma AMPK are peripheral organs and tissues (the liver, muscles, and adipose tissue), and measurements of AMPK in peripheral blood are markedly influenced by cellular energy status [ 42 ]. In turn, owing to short half-life, pulsatile secretion, and releasing to the hypophyseal portal system, peripheral levels of gonadotropin-releasing hormone are undetectable in most subjects, while in the remaining ones they do not reflect its hypothalamic secretion [ 43 ]. Thus, random measurements of circulating AMPK and gonadotropin-releasing hormone levels are poor indicators of AMPK activity and gonadotropin-releasing hormone transmission in the pituitary and hypothalamus.…”