title: Pushing quantitation limits in micro UHPLC-MS/MS analysis of steroid hormones by sample dilution using high volume injection.
abstract: Ultratrace analysis of sample components requires excellent analytical performance in terms of limits of quantitation (LoQ). Micro UHPLC coupling with sensitive tandem mass spectrometry provides state of the art solutions for such analytical problems. Decreased column volume in micro LC limits the injectable sample volume. However, if analyte concentration is extremely low, it might be necessary to inject high sample volumes. This is particularly critical for strong sample solvents and weakly retained analytes, which are often the case when preparing biological samples (protein precipitation, sample extraction, etc.). In that case, high injection volumes may cause band broadening, peak distortion or even elution in dead volume. In this study, we evaluated possibilities of high volume injection onto microbore RP-LC columns, when sample solvent is diluted. The presented micro RP-LC-MS/MS method was optimized for the analysis of steroid hormones from human plasma after protein precipitation with organic solvents. A proper sample dilution procedure helps to increase the injection volume without compromising peak shapes. Finally, due to increased injection volume, the limit of quantitation can be decreased by a factor of 2-5, depending on the analytes and the experimental conditions.
authors.affiliation: Institute of Organic Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences (IOC RCNS HAS), Magyar Tudósok Blvd 2, H-1117 Budapest, Hungary; Department of Inorganic and Analytical Chemistry, Faculty of Chemical Technology and Biotechnology, Budapest University of Technology and Economics, Szent Gellért Sq 4, H-1111 Budapest, Hungary.
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Márta
authors.affiliation: Department of Inorganic and Analytical Chemistry, Faculty of Chemical Technology and Biotechnology, Budapest University of Technology and Economics, Szent Gellért Sq 4, H-1111 Budapest, Hungary.
authors.forename: Balázs
authors.initials: B
authors.lastname: Bobály
authors.affiliation: Department of Inorganic and Analytical Chemistry, Faculty of Chemical Technology and Biotechnology, Budapest University of Technology and Economics, Szent Gellért Sq 4, H-1111 Budapest, Hungary.
authors.forename: Jenő
authors.initials: J
authors.lastname: Fekete
authors.affiliation: Institute of Organic Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences (IOC RCNS HAS), Magyar Tudósok Blvd 2, H-1117 Budapest, 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), Magyar Tudósok Blvd 2, H-1117 Budapest, Hungary.
authors.forename: Tímea
authors.initials: T
authors.lastname: Imre
authors.affiliation: "Momentum" Hereditary Endocrine Tumours Research Group, Hungarian Academy of Sciences - Semmelweis University, Szentkirályi St 46, H-1088, Budapest, Hungary.
authors.forename: Katalin Viola
authors.initials: KV
authors.lastname: Mészáros
authors.affiliation: Institute of Organic Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences (IOC RCNS HAS), Magyar Tudósok Blvd 2, H-1117 Budapest, Hungary. Electronic address: szabo.pal@ttk.mta.hu.
authors.forename: Pál Tamás
authors.initials: PT
authors.lastname: Szabó
mesh_terms: D000735:Androstenedione / Q000032:analysis* / Q000097:blood; D002851:Chromatography, High Pressure Liquid / Q000379:methods / Q000592:standards; D012739:Gonadal Steroid Hormones / Q000032:analysis* / Q000097:blood; D006801:Humans; D006854:Hydrocortisone / Q000032:analysis* / Q000097:blood; D053719:Tandem Mass Spectrometry / Q000379:methods* / Q000592:standards
chemical_list: D012739:Gonadal Steroid Hormones; D000735:Androstenedione; D006854:Hydrocortisone
keywords: High volume injection; LC–MS/MS; Microflow liquid chromatography; Steroid hormone