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Mass spectrometric determination of gonadotrophin‐releasing hormone (GnRH) in human urine for doping control purposes by means of LC–ESI‐MS/MS
Author(s) -
Thomas Andreas,
Geyer Hans,
Kamber Matthias,
Schänzer Wilhelm,
Thevis Mario
Publication year - 2008
Publication title -
journal of mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1076-5174
DOI - 10.1002/jms.1453
Subject(s) - chemistry , chromatography , mass spectrometry , orbitrap , luteinizing hormone , urine , high performance liquid chromatography , detection limit , solid phase extraction , extraction (chemistry) , ion suppression in liquid chromatography–mass spectrometry , hypothalamus , hormone , liquid chromatography–mass spectrometry , endocrinology , biochemistry , medicine
Abstract The decapeptide gonadotrophin‐releasing hormone (GnRH) is endogenously produced in the hypothalamus and secreted into the microcirculation between hypothalamus and pituitary gland. Here, the bioactive hormone is responsible for the release of luteinizing hormone (LH) and follicle‐stimulating hormone (FSH) into the systemic circulation. Because an intermittent application of exogenous GnRH in young males increases the testosterone plasma level by stimulation of the Leydig cells, the potential misuse of the administered substance offers a reasonable relevancy for doping controls and is prohibited in accordance to the list of banned substances of the World Anti‐Doping Agency (WADA). The presented method provides a mass spectrometric approach to determine the nondegraded hormone in regular doping control samples by utilizing a sample preparation procedure with solid phase extraction, immunoaffinity purification and a subsequent separation by liquid chromatography with ESI‐MS/MS detection. For liquid chromatography/mass spectrometry two alternative instrumental equipments were tested: the first consisted of an Agilent 1100 liquid chromatograph coupled to an Applied Biosystem Q Trap 4000 mass spectrometer, the second equipment was assembled by a Waters Aquity nano‐UPLC coupled to a Thermo LTQ Orbitrap high resolution/high accuracy mass spectrometer. In urine specimens provided from healthy volunteers GnRH was not detected in accordance to the recent literature, but in postadministration samples urinary concentrations between 20 to 100 pg/ml of the intact peptide were determined. The method offered good validation results considering the parameter specificity, linearity (5–300 pg/ml), limit of detection (LOD, approx. 5 pg/ml), precision (inter/intraday, < 20%) and accuracy (105%) using Des‐pGlu 1 ‐GnRH as internal standard to control each sample preparation step. Copyright © 2008 John Wiley & Sons, Ltd.