Heart-Cutting Multidimensional Liquid Chromatography For The Determination Of Chiral Coumarins And Furocoumarins In Citrus Essential Oils

Coumarins and psoralens (furocoumarins) play an important role for the characterization of Citrus essential oils, in fact have received considerable attention for their chemical, biological and pharmacological properties. Some of the coumarins and furocoumarins present in Citrus essential oils have, in their structure, chiral centers, hence a separation of enantiomers could be an important tool to improve knowledge on these oils. However, up to now the enantiomeric distribution of coumarins and psoralens in citrus oils have never been reported. The recent introduction of new chiral stationary phases for liquid chromatography contributed to the development of efficient methods for the separation of enantiomers. In this work, the coupling of two columns: the first one a micro-silica (250 x 1.0 mm, 5μm) column, and the second one a column packed with cellulosic-based chiral stationary phase (250 x 4.6mm, 5μm), has provided an excellent baseline separation of the enantiomers in Citrus essential oils. The two columns were connected by using a manually controlled two-position 10-port valve, placed between the two LC systems. Only five of the ten ports present in the valve were used, the other five ports were plugged. The NP-LC-LC instrumentation was equipped with a photodiode array detector. Chiral coumarins and furocoumarins, used to optimize the NP-HPLC method, were obtained by synthetic process and natural compound were isolated from lemon (oxypeucedanin, byakangelicol) and grapefruit (epoxybergamottin, meranzin and epoxyaurapten) essential oil residues by preparative RP-HPLC. In the heart-cutting multidimensional system developed in this research, peaks of our interest were transferred from the first to the second dimension by means of the manual controlled 2-position ten ports switching valve. Chiral coumarins or furocoumarins eluted in D1 are cut at maximum absorption time and transferred in D2 for the enantiomeric separation. The NP-LC-LC-/PDA method provided a good baseline separation of chiral coumarins (meranzin and epoxyaurapten) and furocoumarins (epoxybergamottin, byakangelicol, oxypeucedanin) present in cold-pressed Citrus essential oils without any sample pretreatment. Essential oils obtained during a whole production season using different extraction technologies were analysed to detect possible modification of the enantiomeric ratios. In addition oils from different geographical origin were also studied.