Fast chromatographic separation for the quantitation of the main flavone dyes in Reseda luteola (weld)

In the past decades, there has been a renewed interest in the use of natural dye plants for textile dyeing, e.g. Reseda luteola (weld). Its main yellow dye constituents are the flavones luteolin-7,3′-O-diglucoside, luteolin-7-O-glucoside and luteolin. The aim of this work was to develop a simple validated industrially usable quantitative method to assess the flavone content of R. luteola samples. The flavones were overnight extracted from the dried and ground aerial parts of the plant at room temperature via maceration with methanol–water 8:2. Afterwards, they were quantified through internal standardisation against chrysin by RP-HPLC–UV at 345 nm. The efficiency of the one-step extraction was 95%. The limits of detection (LOD) and quantitation (LOQ) were ≤1 ng and ≤3 ng, respectively, providing ample sensitivity for the purpose. The precision expressed as relative standard deviation of the entire method was <6.5% for the combined content of luteolin-7,3′-O-diglucoside, luteolin-7-O-glucoside and luteolin. The average absolute recovery (accuracy) at three spiking levels was 102% (range: 98–107%) and the relative recovery ranged from 99 to 102%. The separation was initially carried out on a traditional 250 mm × 4.6 mm 5 μm HPLC column (80 min run time, 35.9 mL MeOH). It was then speeded up by the use of a 50 mm × 3.0 mm 1.8 μm UHPLC column (5 min run time, 1.4 mL MeCN), while still using a conventional HPLC system. Whereas, the retention times on the UHPLC column were relatively less reproducible, cross-validation showed that the quantitation of luteolin-7,3′-O-diglucoside, luteolin-7-O-glucoside and luteolin was not statistically significantly different, with comparable precision. The method using the UHPLC column is more sensitive. The analytical method described meets the demand for a very small manpower input per sample and uses standard laboratory equipment. Usage of short UHPLC columns opens up interesting possibilities for modernising HPLC-based phytochemical analyses.

► Quantitative method for the three main flavones in Reseda luteola (weld).
► Thorough validation: extraction, recovery, peak purity, accuracy and precision.
► Method runs on both HPLC and UHPLC columns, cross-validation between them.
► Fast UHPLC separation (5 min runs) on HPLC equipment.
► Advantages are small manpower input per sample and low solvent consumption.