Re: Characterization of Bioactive Constituents in Hops Cones and Pellets

Hops (Humulus lupulus, Cannabaceae) Mass Spectrometry Direct Infusion Two-dimensional Liquid Chromatography Date: 10-15-2018 HC# 031871-602 Sommella E, Pagano F, Salviati E, et al. Chemical profiling of bioactive constituents in hop cones and pellets extracts by online comprehensive two-dimensional liquid chromatography with tandem mass spectrometry and direct infusion Fourier transform ion cyclotron resonance mass spectrometry. J Sep Sci. April 2018;41(7):1548-1557. doi: 10.1002/jssc.201701242. Hops (Humulus lupulus, Cannabaceae) cones not only give beer flavor and bitterness, they also have sedative, antibacterial, and phytoestrogenic properties. They may also prevent cell proliferation, which could have applications in cancer treatment. However, the many bioactive compounds of hops are difficult to characterize. The purpose of this study was to maximize characterization by using two complementary techniques. The authors used "online comprehensive two-dimensional liquid chromatography [(2D LC)] with tandem mass spectrometry and direct infusion Fourier transform ion cyclotron mass spectrometry [(FT-ICR-MS)]," as well as "a reversed phase x reversed phase approach with a shifted gradient in the second dimension." Together, these methods could be used to discover and identify hops compounds, distinguish cultivars, and monitor storage and processing. The different constituents of hops exhibit a diverse array of properties depending on the variety, harvest time, and storage of the herb. Previous methods have been unable to identify all compounds, particularly minor or novel ones. The authors sought to overcome this limitation by combining the "increased peak capacity and selectivity" of 2D LC with the "high mass accuracy and resolution of direct infusion FT-ICR-MS." The AEFFE craft brewery company in Castel San Giorgio, Salerno, Italy donated the hops cones and pellets, variety Hallertauer Magnum T90, used in the chemical profiling. Using 2D LC the authors identified 83 compounds, which is reportedly more than previously identified using MS or combined techniques. In addition, no particular extraction process or fractionation was required, and "multiple compound classes were simultaneously identified and separated" including 10 hydroxycinnamic acids, 18 flavonols, 24 procyanidins, 22 bitter acids, and nine prenylflavonoids. The following compounds were detected: hydroxycinnamic acids including chlorogenic acid isomers, cis- and trans-3'-coumaroylquinic acid isomers, cis- and trans-4'-coumaroylquinic acid, cis- and trans-3'-feruloylquinic acid, and flavonol derivatives including quercetin and kaempferol glycosides. One of those, tentatively identified as quercetin 3-O-(malonyl-hexoside)-O-rhamnoside, was identified for the first time in hops using LC-MS methods. Also identified were procyanidin isomers, (epi) catechin dimers, trimers, tetramers, and pentamers, alpha and beta bitter acids (five beta bitter acids and one alpha bitter acid were proposed as previously unknown acids), prenylated compounds, xanthohumol, isoxanthohumol, oxidized derivatives of xanthohumol, and 8- and 6-prenylnaringenin. The authors point out that "resveratrol and other stilbenes were not detected," which they think may not have been due to the absence of the compounds but rather due to either low concentrations of the compounds or instrumentation used to quantify the substances. Using the 2D LC method, the authors were able to identify multiple compounds at a time and find novel compounds that could remain hidden using one dimensional liquid chromatography separations. The authors report that FT-ICR MS is useful for metabolomics and profiling studies because of its "high mass accuracy, resolution, and wide dynamic range." The high mass accuracy enables molecular formula confirmation which can help identify unknown compounds. Using FT-ICR MS, the authors were able to tentatively identify the unknown bitter acids derivatives. [Note: Other complementary techniques such as NMR spectroscopy are required for definitive confirmation.] It also works rapidly with an analysis time under one minute, uses much less solvent than 2D LC, and is very sensitive, allowing for small sample amounts. However, "the isomers that are resolved by chromatography could not be differentiated," and some compounds were not detected. In conclusion, combining LC x LC-MS/MS with FT-ICR-MS improved the identification of compounds in hops. Together, 101 compounds were identified including previously unknown bitter acids and a novel quercetin derivative. The liquid chromatography mass spectrometry method has "separation capability," and distinguishes isomeric compounds and compound classes. It also enabled the rapid identification of 83 compounds in 70 minutes including six previously unknown bitter acids and a novel quercetin derivative. FT-ICR MS offers high mass accuracy and resolution and provides critical information to elucidate molecular formulas. The authors do not explicitly address conflict of interest. They report that "there are no funding sources for this paper." —Heather Anderson, MD