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

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 syndrom...

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Autores principales: Mecarelli, Enrica, Aigotti, Riccardo, Asteggiano, Alberto, Giacobini, Paolo, Chasles, Manon, Tillet, Yves, Dal Bello, Federica, Medana, Claudio
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer Berlin Heidelberg 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9587114/
https://www.ncbi.nlm.nih.gov/pubmed/36063171
http://dx.doi.org/10.1007/s00216-022-04293-z
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author Mecarelli, Enrica
Aigotti, Riccardo
Asteggiano, Alberto
Giacobini, Paolo
Chasles, Manon
Tillet, Yves
Dal Bello, Federica
Medana, Claudio
author_facet Mecarelli, Enrica
Aigotti, Riccardo
Asteggiano, Alberto
Giacobini, Paolo
Chasles, Manon
Tillet, Yves
Dal Bello, Federica
Medana, Claudio
author_sort Mecarelli, Enrica
collection PubMed
description 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: [Image: see text]
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spelling pubmed-95871142022-10-23 Quantitation of endogenous GnRH by validated nano-HPLC-HRMS method: a pilot study on ewe plasma Mecarelli, Enrica Aigotti, Riccardo Asteggiano, Alberto Giacobini, Paolo Chasles, Manon Tillet, Yves Dal Bello, Federica Medana, Claudio Anal Bioanal Chem Research Paper 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: [Image: see text] Springer Berlin Heidelberg 2022-09-05 2022 /pmc/articles/PMC9587114/ /pubmed/36063171 http://dx.doi.org/10.1007/s00216-022-04293-z Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Research Paper
Mecarelli, Enrica
Aigotti, Riccardo
Asteggiano, Alberto
Giacobini, Paolo
Chasles, Manon
Tillet, Yves
Dal Bello, Federica
Medana, Claudio
Quantitation of endogenous GnRH by validated nano-HPLC-HRMS method: a pilot study on ewe plasma
title Quantitation of endogenous GnRH by validated nano-HPLC-HRMS method: a pilot study on ewe plasma
title_full Quantitation of endogenous GnRH by validated nano-HPLC-HRMS method: a pilot study on ewe plasma
title_fullStr Quantitation of endogenous GnRH by validated nano-HPLC-HRMS method: a pilot study on ewe plasma
title_full_unstemmed Quantitation of endogenous GnRH by validated nano-HPLC-HRMS method: a pilot study on ewe plasma
title_short Quantitation of endogenous GnRH by validated nano-HPLC-HRMS method: a pilot study on ewe plasma
title_sort quantitation of endogenous gnrh by validated nano-hplc-hrms method: a pilot study on ewe plasma
topic Research Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9587114/
https://www.ncbi.nlm.nih.gov/pubmed/36063171
http://dx.doi.org/10.1007/s00216-022-04293-z
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