Coupling three-phase solvent system high-speed countercurrent chromatography to ESI-HRMS/MS and NMR for metabolite profiling of Syzygium malaccense

Syzygium malaccense (L.) Merr. & Perry (Myrtaceae), usually called as “Malay apple”, is native of Malaysia; cultivated in other parts of tropical Asia [1]. In Brazil, S. malaccense, known as red jambo, is a tree used for ornamental purposes or for fruit production, it is commonly found in the northern, north-eastern and south-east regions of Brazil [2]. In this work, metabolite profiling of S. malaccense was performed by countercurrent chromatography (CCC) with fractions analyses by off-line ESI-HRMS/MS and NMR devices. CCC is a liquid-liquid partition technique in which the stationary phase is retained in the apparatus using centrifugal force only [3]. The fundamental principle of separation involves the partition of a solute between two immiscible liquid phases. Due to the wide polarity range of sample compounds, it was possible to use a three-phase solvent system (TPSS) mimicking a gradient elution mode. Few TPSSs are described as a result of the limited combinations of solvents that form three stable phases in a convenient volume percentage [4, 5]. The TPSS n-hexane – ethyl acetate – acetonitrile – H2O (2:1:1:1, v/v) was used in a normal phase gradient elution mode to fractionate about 0.8 g of S. malaccense EtOH leaves extract using a preparative 95 mL column HSCCC system (Quattro). The obtained fractions were analyzed in flow injection mode in the sequence of CCC recovery by a high-resolution tandem mass spectrometer (Thermo QExactive Plus) with an electrospray ion source operating in both positive and negative modes in the scan-range of m/z 100–2000 and also by a NMR equipment (Bruker). Mass spectrometry dereplication studies were performed comparing MS and MS/MS data to those available in public (MoNA) and commercial (NIST MSMS) databases as well as previously isolated compounds from Syzygium genus. This methodology enabled the rapid fractionation and detection of at least 100 compounds. NMR data provided confirmation of structures and substitution position, in some cases. It was possible to classify S. malaccense existing substances in primary and secondary metabolites with flavonoids, organic acids, terpenoids and glycerides as major compound classes (in this order). The combination of preparative purification by gradient CCC and analytical ESI-HRMS/MS and NMR fractions analyses facilitates and innovates the phytochemical study by the simultaneously fractionation (along with occasionally isolation) of different polarity compounds and identification of substances in a complex mixture.

References

[1] P. Sujanapal and C. Kunhikannan. “The Genus Syzygium in Western Ghats,” in The Genus Syzygium: Syzygium cumini and Other Underutilized Species, Boca Raton, CRC Press, 2017, pp. 15-56.
[2] G. F. Silveira, et al. Forest Pathology, 12408, 48, 2018.
[3] Y. Ito. Journal of Biochemical and Biophysical Methods, 5, 105-129, 1981.
[4] Y. Shibusawa, et al. Journal of Chromatography A, 1133, 119-125, 2006.
[5] A. Yanagida, et al. Journal of Chromatography A, 1151, 74-81, 2007.
[6] B. Arumugam, et al. LWT - Food Science and Technology, 59, 707-712, 2014.