Towards eco-friendly secondary plant metabolite quantitation: Ultra high performance supercritical fluid chromatography applied to common vervain (Verbena officinalis L.).

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.

Development and validation of a rapid ultra-high performance liquid chromatography diode array detector method for Verbena officinalis L.

This study presents a fast and validated ultra-high performance liquid chromatography diode array detector (UHPLC-DAD) method for the simultaneous determination of the major compounds in Verbena officinalis L. (Verbenaceae), a medicinal plant listed in the European, British, and, Chinese Pharmacopoeias. In order to get reference substances, nine compounds, belonging to iridoids, flavonoids, and phenylpropanoid glycosides, were isolated from the herb extract. Two of them, namely cistanoside D and leucosceptoside A, were found in this plant species for the first time. Chromatographic separation was achieved in less than 7 min on a Kinetex 1.7 u XB-C18 (50 × 2.10 mm) column by using a solvent gradient of water-acetonitrile modified with 0.1% formic acid. Method validation confirmed the assays sensitivity, linearity (R2 ≥ 0.997), precision (intraday precision ≤ 1.71%; interday precision ≤ 1.46%) and accuracy (recovery rates between 93.9% and 108.8%) for the quantitative analysis of the eight selected marker compounds. Identity and peak purity of the analytes was confirmed by coupling the UHPLC instrument to a quadrupole time-of-flight mass spectrometer via an electrospray ionization interface (ESI-QTOF-MS) operating in the negative ionization mode. Finally, the applicability of the developed UHPLC-DAD method was successfully proven for the sensitive quantitation of the major compounds in Verbena herb extracts, thereby providing a reliable tool for its rapid quality control.