Separation of cucurbitane triterpenoids from bitter melon drinks and determination of partition coefficients using vortex-assisted dispersive liquid-phase microextraction followed by UHPLC analysis

Authors: HU, SHUANG - University Of Mississippi; WANG, YAN-HONG - University Of Mississippi; AVULA, BHARATHI - University Of Mississippi; WANG, MEI - University Of Mississippi; KHAN, IKHLAS - University Of Mississippi

Submitted to: Journal of Separation Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/10/2017
Publication Date: 5/11/2017
Citation: Hu, S., Wang, Y., Avula, B., Wang, M., Khan, I.A. 2017. Separation of cucurbitane triterpenoids from bitter melon drinks and determination of partition coefficients using vortex-assisted dispersive liquid-phase microextraction followed by UHPLC analysis. Journal of Separation Science. 40(10):2238-2245.

Interpretive Summary: Momordica charantia L. has been used as a traditional herbal medicine in Asian and African, and well known for anti-diabetic purpose. Cucurbitane-type triterpenoids and related glycosides in M. charantia L. are the major contributors to the anti-diabetic effects. Consuming M. charantia L. in raw or juice form can be more efficacious than as tablets or capsules in lowering blood-glucose levels. However, the amount of triterpenoid glycosides in bitter melon drinks is usually low that the quantitation of the target compounds is hard to be accomplished. The aim of the present work is to develop a vortex-assisted liquid–liquid microextraction (VALLME) method for simultaneous separation and enrichment of trace amounts of cucurbitane-type triterpenes and triterpene glycosides from bitter melon juices, followed by the UHPLC-ELSD/MS analysis. The developed method is simple, low consumption of organic solvents and operation time, achieving good reproducibility, recoveries, high enrichment factor and sensitivity (low LODs) for the tested cucurbitane triterpenoids. The microextraction method was applied to determine solvent-water partition coefficient values for the target compounds. The results show that the VALLME is an effective approach to determine values with short equilibration time. The presented method illustrated that it may be potentially used for the extraction and analysis of cucurbitane triterpenoids in other aqueous matrices without dilution or further pre-treatment procedure.

Technical Abstract: A rapid, effective technique applying vortex-assisted liquid–liquid microextraction (VALLME) prior to ultra high performance liquid chromatography-evaporating light scattering detectection/ mass spectroscopy (UHPLC-ELSD/MS) determination was developed for the analysis of four cucurbitane triterpenoids (momordicoside L, momordicoside K, momordicoside F2, and 3ß,7ß,25-trihydroxy cucurbita-5, 23(E)-dien-19-al) in bitter melon juices. Variables affecting the extraction efficiency including different extraction solvents, volume of extraction solvent, salt amount, acid condition (pHs), and vortexing speed and time were investigated and optimized thoroughly. Under the optimum conditions, recoveries are between 95.7% and 106.1% with relative standard deviations (RSDs) less than 2.1% (n=3). Precision was determined by the intra- and inter-day tests in a range of 1.0%-5.4% (RSD) and 2.9%-3.8% (RSD), respectively. Enrichment factors of four target compounds were 27 – 63 times. Therefore, the limits of detection (LODs) and limits of quantitation (LOQs) were 0.8-1.9 ng mL-1 and 4.0-9.3 ng mL-1, respectively, for MS detection. While using ELSD, LODs and LOQs were 0.3-0.4 µg mL-1 and 0.5-0.7 µg mL-1 for different compounds. The calibration curves showed good linearity with square correlation coefficient of 0.9936-0.9991 (ELSD) and 0.9858-0.9989. The proposed method was used to determine the apparent solvent/water partition coefficients (K org/aq) of analytes, and the values calculated within the range of 53-120. The developed method can effectively enrich and quantitate cucurbitane triterpenoids from bitter melon drinks.