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

Area of Science:

• Phytochemistry
• Analytical Chemistry
• Pharmacognosy

Background:

• Vitex agnus-castus (Lamiaceae) is a traditional medicinal plant used for premenstrual symptoms (PMS).
• Accurate quantification of its non-volatile phytochemicals (iridoids, flavonoids, diterpenes) is essential for quality control and therapeutic understanding.
• Existing analytical methods may lack the speed and comprehensive profiling required for complex plant matrices.

Purpose of the Study:

• To develop and validate a rapid ultra-high performance liquid chromatography-diode array detector (UHPLC-DAD) method for simultaneous determination of phytochemical classes in Vitex agnus-castus fruits.
• To establish a reliable analytical tool for quantifying key marker compounds in phytopharmaceutical preparations.

Main Methods:

• Development and validation of a UHPLC-DAD method using a C18 column and a gradient elution with acetic acid and acetonitrile.
• Isolation of seven marker compounds for use as reference standards.
• Quantification of agnuside, isovitexin, casticin, 5-hydroxykaempferol-3,6,7,4'-tetramethylether, and vitetrifolin D within specified concentration ranges.
• Method validation for specificity, accuracy, precision, and reproducibility.
• Tentative identification of 62 chromatographic features using UHPLC coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS).

Main Results:

• The UHPLC-DAD method demonstrated specificity, accuracy, precision, and reproducibility for quantifying five key marker compounds.
• Validated concentration ranges were established for each quantified compound, with relative standard deviations (RSD) for intra- and inter-day variations below 3.9% and 6.4%, respectively.
• The method was successfully applied to analyze Vitex agnus-castus derived preparations (extracts, tinctures, tablets), proving its applicability in phytopharmaceuticals.

Conclusions:

• A rapid and validated UHPLC-DAD method enables simultaneous determination of major phytochemical classes in Vitex agnus-castus.
• The developed assay provides a reliable tool for quality control and standardization of Vitex agnus-castus based phytopharmaceuticals.
• This method supports further research into the therapeutic efficacy of Vitex agnus-castus for conditions like PMS.