Abstract
This report presents the first ultra high performance supercritical fluid chromatography diode array detector based assay for simultaneous determination of iridoid glucosides, flavonoid glucuronides, and phenylpropanoid glycosides in Verbena officinalis (Verbenaceae) extracts. Separation of the key metabolites was achieved in less than 7 min on an Acquity UPC2 Torus Diol column using a mobile phase gradient comprising subcritical carbon dioxide and methanol with 0.15% phosphoric acid. Method validation for seven selected marker compounds (hastatoside, verbenalin, apigenin‐7‐O‐glucuronide, luteolin‐7‐O‐glucuronide, apigenin‐7‐O‐diglucuronide, verbascoside, and luteolin‐7‐O‐diglucuronide) confirmed the assay to be sensitive, linear, precise, and accurate. Head‐to‐head comparison to an ultra high performance liquid chromatography comparator assay did prove the high orthogonality of the methods. Quantitative result equivalence was evaluated by Passing‐Bablok‐correlation and Bland‐Altman‐plot analysis. This cross‐validation revealed, that one of the investigated marker compound peaks was contaminated in the ultra high performance liquid chromatography assay by a structurally related congener. Taken together, it was proven that the ultra high performance supercritical fluid chromatography instrument setup with its orthogonal selectivity is a true alternative to conventional reversed phase liquid chromatography in quantitative secondary metabolite analysis. For regulatory purposes, assay cross‐validation with highly orthogonal methods seems a viable approach to avoid analyte overestimation due to coeluting, analytically indistinguishable contaminants.
Highlights
Modern phytoanalysis is an ever challenging application field for latest developments in analytical instrumentation technology
Towards eco-friendly secondary plant metabolite quantitation: Ultra high performance supercritical fluid chromatography applied to common vervain (Verbena officinalis L.)
As the therapeutic effects of Verbenae herba are attributed to one, but three different classes of secondary metabolites, their simultaneous analysis is indispensable for adequate quality assessment
Summary
Modern phytoanalysis is an ever challenging application field for latest developments in analytical instrumentation technology. Ultra high performance liquid chromatography (UHPLC) paired with sub-2 μm packing columns became swiftly a key separation technology in modern phytoanalysis [3]. A different approach towards ecofriendly secondary plant metabolite quantitation is ultra high performance supercritical fluid chromatography (UHPSFC). In contrast to early SFC approaches, where pure carbon dioxide (CO2) in supercritical condition was used as mobile phase, modern UHPSFC applies mobile phases consisting of CO2 and organic solvents (mostly an alcohol). This results in mixtures that are not necessarily supercritical but rather subcritical fluids [4,5,6]. The implementation of cross-validations to compare the performance of different assays and to prove thereby the validity of obtained quantitative results is still hardly performed in phytoanalysis
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