Multidimensional preparative liquid chromatography to isolate flavonoids from bergamot juice and evaluation of their anti-inflammatory potential

Abstract

Important objectives of a high-performance liquid chromatography preparative process are: purity of products isolated, yield, and throughput. The multidimensional preparative liquid chromatography method used in this work was developed mainly to increase the throughput; moreover purity and yield are increased thanks to the automated collection of the molecules based on the intensity of a signal generated from the mass spectrometer detector, in this way only a specific product can be targeted. This preparative system allowed, in few analyses both in the first and second dimensions, the isolation of eight pure compounds present at very different concentration in the original sample with high purity (>95%) and yield, which showed how the system is efficient and versatile. Pure molecules were used to validate the analytical method and to test the anti-inflammatory and antiproliferative potential of flavonoids. The contemporary presence, in bergamot juice, of all the flavonoids together increases the anti-inflammatory effect with respect to the single compound alone.

Advances in extraction and purification of citrus flavonoids

“…Russo et al. (2015) isolated flavonoids in bergamot juice by PREP‐HPLC, and attempted to completely purify all flavonoids contents in bergamot juice by collecting the fractions of the first dimension, reinjecting them into the second dimension and conducting automatic collection. Since the automated collection of flavonoids in this method is based on the signal intensity generated by the mass spectrometer detector and only specific products can be detected by localization, the purity and yield were improved.…”

Recent trends in multidimensional countercurrent chromatography

“…MDCCC involves integrating CCC with other separation techniques, like high-performance liquid chromatography (HPLC), mass spectrometry (MS), and gas chromatography (GC). The CCC method is a useful preparative chromatographic instrument that complements HPLC due to its high selectivity and good retention of the stationary phase, even though HPLC's efficiency (regarding theoretical plate number) outclasses it [5,6].…”

Methodology for dyes purification by preparative bidimensional offline reversed‐phase high‐performance liquid chromatography separation based on mobile phase pH change: Case study of Justicia spicigera

“…RP-HPLC is frequently chosen for that purpose [9] and for complex samples, multidimensional separation is often required to obtain a higher purity of the collected products. The most popular bidimensional preparative approach is to use two different stationary phases to change the selectivity between both dimensions: Hydrophilic interaction liquid chromatography-RPLC [10][11][12][13][14] or RPLC-RPLC with two stationary phases [15] set-ups are reported. Another option is to work with the same stationary phase in both dimensions and provide orthogonality using a distinct mobile phase.…”