title: Charged derivatization and on-line solid phase extraction to measure extremely low cortisol and cortisone levels in human saliva with liquid chromatography-tandem mass spectrometry.
abstract: The aim of this study was to develop a sensitive, reliable and high-throughput liquid chromatography - electrospray ionization - mass spectrometric (LC-ESI-MS/MS) method for the simultaneous quantitation of cortisol and cortisone in human saliva. Derivatization with 2-hydrazino-1-methylpyridine (HMP) was one of the most challenging aspects of the method development. The reagent was reacting with cortisol and cortisone at 60°C within 1h, giving mono- and bis-hydrazone derivatives. Investigation of derivatization reaction and sample preparation was detailed and discussed. Improvement of method sensitivity was achieved with charged derivatization and use of on-line solid phase extraction (on-line SPE). The lower limit of quantitation (LLOQ) was 5 and 10pg/ml for cortisol and cortisone, respectively. The developed method was subsequently applied to clinical laboratory measurement of cortisol and cortisone in human saliva.
authors.affiliation: MS Metabolomics Laboratory, Core Facility, Research Centre for Natural Sciences, Hungarian Academy of Sciences (IOC RCNS HAS), H-1117 Budapest, Magyar Tudósok Blvd 2, Hungary.
authors.forename: Balázs
authors.initials: B
authors.lastname: Magda
authors.affiliation: Institute of Organic Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences (IOC RCNS HAS), H-1117 Budapest, Magyar Tudósok Blvd 2, Hungary.
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Dobi
authors.affiliation: Department of Laboratory Medicine, Semmelweis University, Budapest, Hungary; "Lendület" Hereditary Endocrine Tumours Research Group, HAS-SE, Budapest, Hungary.
authors.forename: Katalin
authors.initials: K
authors.lastname: Mészáros
authors.affiliation: Egis Pharmaceuticals PLC, H-1106 Budapest, Keresztúri Street 30-38, Hungary.
authors.forename: Éva
authors.initials: É
authors.lastname: Szabó
authors.affiliation: MS Metabolomics Laboratory, Core Facility, Research Centre for Natural Sciences, Hungarian Academy of Sciences (IOC RCNS HAS), H-1117 Budapest, Magyar Tudósok Blvd 2, Hungary.
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Márta
authors.affiliation: MS Metabolomics Laboratory, Core Facility, Research Centre for Natural Sciences, Hungarian Academy of Sciences (IOC RCNS HAS), H-1117 Budapest, Magyar Tudósok Blvd 2, Hungary.
authors.forename: Tímea
authors.initials: T
authors.lastname: Imre
authors.affiliation: MS Metabolomics Laboratory, Core Facility, Research Centre for Natural Sciences, Hungarian Academy of Sciences (IOC RCNS HAS), H-1117 Budapest, Magyar Tudósok Blvd 2, Hungary. Electronic address: szabo.pal@ttk.mta.hu.
authors.forename: Pál T
authors.initials: PT
authors.lastname: Szabó
mesh_terms: D002853:Chromatography, Liquid; D003348:Cortisone; D006801:Humans; D006854:Hydrocortisone; D012463:Saliva; D052616:Solid Phase Extraction; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D003348:Cortisone; D006854:Hydrocortisone
keywords: 2-Hydrazino-1-methylpyridine; Column-switching; Cortisol; Cortisone; LC–MS/MS