title: A liquid chromatography-electrospray ionization-mass spectrometry method for quantification of cyclotides in plants avoiding sorption during sample preparation.
abstract: Cyclotides are plant-produced, bioactive, cyclic mini-proteins with interesting pharmaceutical and agricultural applications. A reverse phase liquid chromatography electrospray ionization mass spectrometry (RP-LC-ESI-MS) method for analysis of cyclotides in plant materials with a minimum of sample pre-treatment is presented. Three exemplary cyclotides (kalata B1, kalata B2 and cycloviolacin O2) were used as reference substances for the method development. Linearity (r(2)>0.99) was achieved in the concentration range 0.05-10 mg/L and the limit of detection was 1.7-4.0 μg/L. The present study is the first to demonstrate that cyclotides dissolved in water sorb to glass vials, but the addition of 15% of acetonitrile or 40 mg/L of bovine serum albumin is sufficient to keep the cyclotides in solution. Cyclotides were extracted from candied violets, violet tea, and the plants Oldenlandia affinis and Viola odorata using 70% methanol containing 0.1% formic acid (v/v). The plant content was determined to be 23.5-14,200 μg/g (dry weight). The highest content of cyclotide was found in wild Danish V. odorata, and it is the highest content of cyclotide in a plant reported hitherto. Candied violets contained 0.00-8.66 μg/g (dry weight), while no cyclotides were detected in commercial violet tea.
authors.affiliation: Department of Basic Sciences and Environment, Faculty of Life Sciences (LIFE), University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark. rgo@dsr.life.ku.dk
authors.forename: Rikke G
authors.initials: RG
authors.lastname: Ovesen
authors.forename: Ulf
authors.initials: U
authors.lastname: Göransson
authors.forename: Steen H
authors.initials: SH
authors.lastname: Hansen
authors.forename: John
authors.initials: J
authors.lastname: Nielsen
authors.forename: Hans Christian B
authors.initials: HC
authors.lastname: Hansen
mesh_terms: D000327:Adsorption; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D047169:Cyclotides / Q000032:analysis* / Q000302:isolation & purification; D059625:Liquid-Liquid Extraction; D008958:Models, Molecular; D008969:Molecular Sequence Data; D032067:Oldenlandia / Q000737:chemistry; D010936:Plant Extracts / Q000737:chemistry*; D010940:Plant Proteins / Q000032:analysis* / Q000302:isolation & purification; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D012710:Serum Albumin, Bovine / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D032413:Viola / Q000737:chemistry
chemical_list: D047169:Cyclotides; D010936:Plant Extracts; D010940:Plant Proteins; D012710:Serum Albumin, Bovine