Quantitative Analysis of Prenylated Constituents in Commercial Hops Samples Using Ultrahigh-Performance Supercritical Fluid Chromatography

Abstract

The importance of hops (the flowers of Humulus lupulus) as food and an herbal remedy is reflected by a large number of analytical methods published. However, supercritical fluid chromatography, a highly efficient, rapid, and “green” separation technique, has not been considered for hops samples so far. This prompted us to establish the first supercritical fluid chromatography-based protocol for the separation, identification, and quantitation of five prenylated constituents of hops. Hulupinic acid (1), a prominent oxidation product of hop acids, three flavanones, i.e., 8-prenylnaringenin (2), 6-prenylnaringenin (3), and isoxanthohumol (4), as well as the chalcone xanthohumol (5) could be baseline separated in less than 5 minutes using a Viridis BEH 2-EP column (3.0 × 100 mm; 1.7 µm particle size) and a mobile phase consisting of CO2 and isopropanol. Good results regarding selectivity, accuracy (recovery rates: 85.0 – 113.1%), precision (intra-day ≤ 2.1%, inter-day ≤ 3.5%), and linearity (R2 ≥ 0.99) were obtained for both photodiode array and mass detection. The lowest detection limit at 220 nm was at 0.1 µg/mL (1, 3, and 4), with mass detection even at 0.001 µg/mL (4). As an application example of the validated method, the five hops constituents were quantified in three dietary supplements, one herbal medicinal product, and two batches of hop flowers (Lupuli flos). In most samples analyzed, the major component was 5 (0.01 – 1.02%), whereas the major component in Lupuli flos samples was compound 1 (0.12 – 0.21%). This protocol offers a fast and environmentally friendly alternative to liquid chromatography for the quality control of hops.

The Potent Phytoestrogen 8-Prenylnaringenin: A Friend or a Foe?

“…Apart from MS, HPLC methods with ultraviolet (UV) or UV/diode array detector (DAD) detection have been developed [ 18 , 25 , 31 , 32 ]. Some modifications of these techniques include ultra-high-pressure LC–tandem MS (UHPLC–MS/MS), ultra-high-performance supercritical fluid chromatography (UHPSFC; with either UV or MS detection), and the application of secondary standards in HPLC–UV/DAD [ 23 , 25 ]. Antibody-based methods have also found their way to 8-PN quantification.…”

“…None of the samples contained measurable levels of 8-PN, 6-PN, or 6,8-diprenylnaringenin. In a more recent study, Schretter et al (2020) [ 23 ] were unable to detect 8-PN in two out of the three hop dietary supplements analyzed. In contrast, the third product would provide 144 µg 8-PN and 1740 µg IX at the recommended daily dosage, indicating huge differences among these products.…”

“…In the study by Schretter et al (2009) [ 23 ], a herbal medicinal product and two batches of the herbal drug “Lupuli flowers” (in pharmacopeia quality) proved to encompass similar quantities of IX and 8-PN, and the recommended daily dose for the medicinal product would lead to the intake of ~45 µg 8-PN and ~255 µg IX. Dhooghe et al (2010) studied hop-derived capsules, tablets, and drops on the market in Belgium for their 8-PN content [ 25 ].…”

Ultra High-Performance Supercritical Fluid Chromatography for the Quantitation of Diterpene Resin Acids in Norway Spruce Samples

“…A reduced environmental impact due to shortened run times and the omission of organic solvents as mobile phase, is an additional benefit. Today, SFC and the advanced ultra-high performance SFC (UHPSFC) constitute a versatile technology providing high separation efficiency, access to a broad range of compound classes, with various stationary phase chemistries available and high compatibility with numerous detectors ( Schretter et al, 2020 ; Ganzera and Zwerger, 2021 ; Langeder and Grienke, 2021 ). For these reasons, this first UHPSFC method for the quantitative analysis of DRAs is preferable to the previously suggested techniques.…”