title: Unexpected retention behavior of baicalin: Hydrophilic interaction like properties of a reversed-phase column.
abstract: The original aim of this study was to develop a method for the determination of baicalin from membrane vesicles. The unconventional chromatographic separation ("inverse gradient elution" on a reversed phase column) was due to a lucky chance, which is detailed and discussed in this study. The validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is proved to be sensitive, rapid and selective. Chromatographic separation was performed on a Zorbax SB-C8 column (250 mm × 4.6 mm, i.d.; 5 μm) with 0.1% formic acid in water and methanol by linear gradient elution. Quantification of baicalin was determined by multiple reaction monitoring (MRM) mode using electrospray ionization (ESI). The calibration curve was linear (r = 0.9987) over the concentration range from 1 to 1000 nM. The coefficient of variation and relative error of baicalin for intra- and inter-assay at three quality control (QC) levels were 2.0-10.2% and -6.1 to 6.7%, respectively. The lower limit of quantification (LLOQ) for baicalin was 1 nM (0.446 ng/ml), without preconcentration of the sample. This method was subsequently applied to vesicular transport assays of baicalin in membrane vesicles successfully. The developed method can open up new area of research in the chromatographic separation of flavonoids and their glucuronides.
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: Zoltán
authors.initials: Z
authors.lastname: Márta
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: Department of Pharmaceutics, Semmelweis University, Hőgyes St 7, H-1092 Budapest, Hungary.
authors.forename: Bernadett
authors.initials: B
authors.lastname: Kalapos-Kovács
authors.affiliation: Department of Pharmaceutics, Semmelweis University, Hőgyes St 7, H-1092 Budapest, Hungary.
authors.forename: Imre
authors.initials: I
authors.lastname: Klebovich
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. Electronic address: szabo.pal@ttk.mta.hu.
authors.forename: Pál T
authors.initials: PT
authors.lastname: Szabó
mesh_terms: D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods; D004365:Drugs, Chinese Herbal / Q000737:chemistry*; D005419:Flavonoids / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D004365:Drugs, Chinese Herbal; D005419:Flavonoids; C038044:baicalin
keywords: Baicalin; Hydrophilic interaction chromatography; LC–MS/MS; Reversed-phase chromatography; Vesicular transport assay