title: Development and validation of a rapid ultra-high performance liquid chromatography diode array detector method for Vitex agnus-castus.
abstract: A rapid ultra-high performance liquid chromatography diode array detector (UHPLC-DAD) method was developed and validated for the simultaneous determination of all classes of non-volatile phytochemicals (iridoids, flavonoids and diterpenes) in Vitex agnus-castus (Lamiaceae) fruits, a traditional medicinal plant used against premenstrual symptoms (PMS) and other disorders. Seven marker compounds, 3,4-dihydroxybenzoic acid, p-hydroxybenzoic acid, agnuside, 5-hydroxykaempferol-3,6,7,4'-tetramethylether, 1,2-dibenzoic acid glucose, methoxy-vitexilactone, and vitetrifolin D were isolated from the methanol extract of V. agnus-castus to be used as reference substances. Chromatographic separation was performed on a Zorbax Eclipse XDB-C18 (50mm×2.1mm) UHPLC column with 1.8μm particle size, within 20min. A solvent gradient from 0.5% acetic acid to acetonitrile at a flow rate of 0.6mL/min was used as mobile phase. Analyte detection and quantification was realized at 210nm and 260nm. The UHPLC-DAD assay was validated for the quantitative analysis of agnuside, isovitexin, casticin, 5-hydroxykaempferol-3,6,7,4'-tetramethylether and vitetrifolin D. It was found to be specific, accurate, precise, and reproducible for the quantification of these compound within a concentration range of 0.7-500.0μg/mL for casticin and 5-hydroxykaempferol-3,6,7,4'-tetramethylether, 1.4-1000.0μg/mL for isovitexin and agnuside, and 12.4-1000.0μg/mL for vitetrifolin D. Intra- and inter-day variations showed relative standard deviations (RSD) of less than 3.9% and 6.4%, respectively. Tentatively assignment of 62 chromatographic features found in the UHPLC-DAD assay was carried out by coupling the UHPLC instrument to a quadrupole time-of-flight mass spectrometer via an electrospray ionization interface (ESI-QTOF-MS) operated in positive and negative ion mode. By using the established quantitative UHPLC-DAD assay to asses agnuside, isovitexin, casticin, 5-hydroxykaempferol-3,6,7,4'-tetramethylether and vitetrifolin D in V. agnus-castus derived preparations as extracts, tinctures and tablets, the applicability of the developed assay to phytopharmaceuticals was successfully proven.
authors.affiliation: Institute of Pharmacy/Pharmacognosy, CCB - Centrum of Chemistry and Biomedicine, University of Innsbruck, Innrain 80-82, A-6020 Innsbruck, Austria.
authors.forename: C
authors.initials: C
authors.lastname: Högner
authors.forename: S
authors.initials: S
authors.lastname: Sturm
authors.forename: C
authors.initials: C
authors.lastname: Seger
authors.forename: H
authors.initials: H
authors.lastname: Stuppner
mesh_terms: D001565:Benzoates / Q000032:analysis / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004224:Diterpenes / Q000032:analysis / Q000737:chemistry; D005419:Flavonoids / Q000032:analysis / Q000737:chemistry; D016014:Linear Models; D010936:Plant Extracts / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D020950:Vitex / Q000737:chemistry*
chemical_list: D001565:Benzoates; D004224:Diterpenes; D005419:Flavonoids; D010936:Plant Extracts