Liquid chromatography-electrospray ionization mass spectrometry analysis of limonoids and flavanois in seeds of grapefruits, other citrus species, and dietary supplements

Authors: AVULA, BHARATHI - University Of Mississippi; SAGI, SATYANARAYANARA - University Of Mississippi; WANG, YAN-HONG - University Of Mississippi; WANG, MEI - University Of Mississippi; GARNER, STEFAN - Botanical Society Of America; Manthey, John; KHAN, IKHLAS - University Of Mississippi

Submitted to: Planta Medica
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/15/2016
Publication Date: 5/25/2016
Citation: Avula, B., Sagi, S.J., Wang, Y., Wang, M., Garner, S., Manthey, J.A., Khan, I.A. 2016. Liquid chromatography-electrospray ionization mass spectrometry analysis of limonoids and flavanois in seeds of grapefruits, other citrus species, and dietary supplements. Planta Medica. 82:1058-1069. https://doi.org/10.1055/s-0042-107598.
DOI: https://doi.org/10.1055/s-0042-107598

Interpretive Summary: Grapefruit seed extract (GFSE) is obtained from the seeds of grapefruit (Citrus paradisi Macf., Rutaceae). GFSE products are widely used as preservatives in the food and cosmetic industries. However, some commercially available products are not entirely natural. An analytical method was developed to screen grapefruit seeds, and other citrus seed samples for limonoids and flavonoids found in GFSE. The method was successfully applied to the analysis of various commercial extracts and dietary supplements claiming to contain GFSE. Limonoids were found to be the predominant component in all tested samples containing “inner seed only”. Limonoids and flavonoids were detected in one inner seed with outer seed coat and two outer seed coat samples. Many commercial products contained large numbers of flavonoids, indicating the use of peel, pulp or seed coat. Out of the 17 commercial products analyzed, two contained the synthetic antimicrobial agent benzethonium chloride as main constituent. The results showed that this current method can be used to identify various classes of compounds from commercial GFSE.

Technical Abstract: A selective ultra-high performance liquid chromatography-didode array detector-quadrapole time of flight-mass spectrometry (UHPLC-DAD-QToF-MS) method has been developed to screen grapefruit seeds, and other citrus seed samples for limonoid aglycones, limonoid acids, limonoid glucosides and flavonoids. The separation by UHPLC was achieved using C8 column and a gradient of water/acetonitrile each containing formic acid as the mobile phase. Accurate mass ToF spectrometry was used to distinguish the various compounds. The structural characteristics of compounds in methanolic extracts of grapefruit seed and dietary supplements have been identified in both negative and positive ion modes over a mass-to-charge (m/z) range from 100–1500. The fragmentation patterns of reference standards were studied and the presence of compounds in the extracts and dietary supplements were identified or tentatively characterized with reference to retention times and MS data. The method was successfully applied to the analysis of various citrus seed samples, commercial extracts, and dietary supplements claiming to contain grapefruit seed extract (GFSE), or extracts made from the seed and other fruit parts such as the peel or the pulp. Limonoids, oils and lipids are important constituents of citrus seeds, and limonin was found to be the major limonoid in most of the citrus seeds. Many commercial products contained large numbers of flavonoids, indicating the use of peel, pulp or seed coat. The method also permitted for detection of synthetic preservatives such as benzethonium chloride, methylparaben and triclosan in commercial GFSE products. Out of the 17 commercial products analyzed, two contained the synthetic antimicrobial agent benzethonium chloride as main constituent. No other synthetic antimicrobial compounds were detected.