Planta Med 2020; 86(15): 1140-1147
DOI: 10.1055/a-1130-0590
Natural Product Chemistry and Analytical Studies
Original Papers

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

Carmen Schretter
Department of Pharmacognosy, University of Vienna, Vienna, Austria
Julia Langeder
Department of Pharmacognosy, University of Vienna, Vienna, Austria
Victoria Freisinger
Department of Pharmacognosy, University of Vienna, Vienna, Austria
Judith M. Rollinger
Department of Pharmacognosy, University of Vienna, Vienna, Austria
Ulrike Grienke
Department of Pharmacognosy, University of Vienna, Vienna, Austria
› Author Affiliations


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.

# Dedicated to Professor Dr. Wolfgang Kubelka on the occasion of his 85th birthday.

Supporting Information

Publication History

Received: 15 January 2020

Accepted: 27 February 2020

Article published online:
17 March 2020

© 2020. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

  • References

  • 1 Lin M, Xiang D, Chen X, Huo H. Role of characteristic components of Humulus lupulus in promoting human health. J Agric Food Chem 2019; 67: 8291-8302
  • 2 European Medicines Agency. Herbal Medicine: Summary for the Public – hop Strobile. London: European Medicines Agency; 2016: 304459
  • 3 Li J, Li N, Li X, Chen G, Wang C, Lin B, Hou Y. Characteristic alpha-acid derivatives from Humulus lupulus with antineuroinflammatory activities. J Nat Prod 2017; 80: 3081-3092
  • 4 Bolton JL, Dunlap TL, Hajirahimkhan A, Mbachu O, Chen SN, Chadwick L, Nikolic D, van Breemen RB, Pauli GF, Dietz BM. The multiple biological targets of hops and bioactive compounds. Chem Res Toxicol 2019; 32: 222-233
  • 5 Chen SF, Chen PY, Hsu HJ, Wu MJ, Yen JH. Xanthohumol suppresses Mylip/Idol gene expression and modulates LDLR abundance and activity in HepG2 cells. J Agric Food Chem 2017; 65: 7908-7918
  • 6 Aghamiri V, Mirghafourvand M, Mohammad-Alizadeh-Charandabi S, Nazemiyeh H. The effect of hop (Humulus lupulus L.) on early menopausal symptoms and hot flashes: A randomized placebo-controlled trial. Complement Ther Clin Pract 2016; 23: 130-135
  • 7 Ferk F, Misik M, Nersesyan A, Pichler C, Jäger W, Szekeres T, Marculescu R, Poulsen HE, Henriksen T, Bono R, Romanazzi V, Al-Serori H, Biendl M, Wagner KH, Kundi M, Knasmüller S. Impact of xanthohumol (a prenylated flavonoid from hops) on DNA stability and other health-related biochemical parameters: Results of human intervention trials. Mol Nutr Food Res 2016; 60: 773-786
  • 8 Pichler C, Ferk F, Al-Serori H, Huber W, Jäger W, Waldherr M, Misik M, Kundi M, Nersesyan A, Herbacek I, Knasmüller S. Xanthohumol prevents DNA damage by dietary carcinogens: results of a human intervention trial. Cancer Prev Res 2017; 10: 153-160
  • 9 Teuscher E, Melzig MF, Lindequist U. Biogene Arzneimittel: ein Lehrbuch der Pharmazeutischen Biologie, 7th edition. Stuttgart: Wissenschaftliche Verlagsgesellschaft mbH; 2012: 321
  • 10 van Wyk BE, Wink C, Wink M. Handbuch der Arzneipflanzen. 3rd edition. Stuttgart: Wissenschaftliche Verlagsgesellschaft mbH; 2015: 262
  • 11 Nikolic D, van Breemen RB. Analytical methods for quantitation of prenylated flavonoids from hops. Curr Anal Chem 2013; 9: 71-85
  • 12 Prencipe FP, Brighenti V, Rodolfi M, Mongelli A, dallʼAsta C, Ganino T, Bruni R, Pellati F. Development of a new high-performance liquid chromatography method with diode array and electrospray ionization-mass spectrometry detection for the metabolite fingerprinting of bioactive compounds in Humulus lupulus L. J Chromatogr A 2014; 1349: 50-59
  • 13 Vazquez Loureiro P, Hernandez Jimenez I, Sendon R, Rodriguez-Bernaldo de Quiros A, Barbosa-Pereira L. Determination of xanthohumol in hops, food supplements and beers by HPLC. Foods 2019; 8: 435
  • 14 Hartmann A, Ganzera M. Supercritical fluid chromatography – theoretical background and applications on natural products. Planta Med 2015; 81: 1570-1581
  • 15 Saito M. History of supercritical fluid chromatography: instrumental development. J Biosci Bioeng 2013; 115: 590-599
  • 16 Klesper E, Corwin AH, Turner DA. High pressure gas chromatography above critical temperatures. J Org Chem 1962; 27: 700-701
  • 17 West C. Current trends in supercritical fluid chromatography. Anal Bioanal Chem 2018; 410: 6441-6457
  • 18 Sanchez-Camargo AD, Parada-Alfonso F, Ibanez E, Cifuentes A. On-line coupling of supercritical fluid extraction and chromatographic techniques. J Sep Sci 2016; 40: 213-227
  • 19 Scheuba J, Wronski VK, Rollinger JM, Grienke U. Fast and green – CO2 based extraction, isolation, and quantification of phenolic styrax constituents. Planta Med 2017; 83: 1068-1075
  • 20 Murauer A, Ganzera M. Quantitative determination of major alkaloids in Cinchona bark by supercritical fluid chromatography. J Chromatogr A 2018; 1554: 117-122
  • 21 Qi NL, Gong X, Feng CP, Wang XX, Xu YW, Lin LJ. Simultaneous analysis of eight vitamin E isomers in Moringa oleifera Lam. leaves by ultra performance convergence chromatography. Food Chem 2016; 207: 157-161
  • 22 Grienke U, Zwirchmayr J, Peintner U, Urban E, Zehl M, Schmidtke M, Rollinger JM. Lanostane triterpenes from Gloeophyllum odoratum and their anti-influenza effects. Planta Med 2019; 85: 195-202
  • 23 Song W, Qiao X, Liang WF, Ji S, Yang L, Wang Y, Xu YW, Yang Y, Guo DA, Ye M. Efficient separation of curcumin, demethoxycurcumin, and bisdemethoxycurcumin from turmeric using supercritical fluid chromatography: From analytical to preparative scale. J Sep Sci 2015; 38: 3450-3453
  • 24 Zhao Y, McCauley J, Pang X, Kang LP, Yu HS, Zhang J, Xiong CQ, Chen R, Ma BP. Analytical and semipreparative separation of 25 (R/S)-spirostanolsaponin diastereomers using supercritical fluid chromatography. J Sep Sci 2013; 36: 3270-3276
  • 25 Yang W, Zhang Y, Pan H, Yao C, Hou J, Yao S, Cai L, Feng R, Wu W, Guo D. Supercritical fluid chromatography for separation and preparation of tautomeric 7-epimeric spiro oxindole alkaloids from Uncaria macrophylla . J Pharm Biomed Anal 2017; 134: 352-360
  • 26 Lemasson E, Bertin S, West C. Use and practice of achiral and chiral supercritical fluid chromatography in pharmaceutical analysis and purification. J Sep Sci 2016; 39: 212-233
  • 27 Murauer A, Ganzera M. Quantitative determination of lactones in Piper methysticum (Kava-Kava) by supercritical fluid chromatography. Planta Med 2017; 83: 1053-1057
  • 28 Pfeifer I, Murauer A, Ganzera M. Determination of coumarins in the roots of Angelica dahurica by supercritical fluid chromatography. J Pharm Biomed Anal 2016; 129: 246-251
  • 29 Winderl B, Schwaiger S, Ganzera M. Fast and improved separation of major coumarins in Ammi visnaga (L.) Lam. by supercritical fluid chromatography. J Sep Sci 2016; 39: 4042-4048
  • 30 Xu R, Chen X, Wang X, Yu L, Zhao W, Ba Y, Wu X. Development and validation of an ultra-high performance supercritical fluid chromatography-photodiode array detection-mass spectrometry method for the simultaneous determination of 12 compounds in Piper longum L. Food Chem 2019; 298: 125067
  • 31 Wu W, Zhang Y, Zhang F, Liu J, Ren Z, Xu Y, Liu T, Zhou W, Li H, Zhang C. An analytical strategy for accurate, rapid and sensitive quantitative analysis of isoflavones in traditional Chinese medicines using ultra-high performance supercritical fluid chromatography: Take Radix Puerariae as an example. J Chromatogr A 2019; 1606: 460385
  • 32 Gagnon D, Wendakoon C, Smith R, Leker J. Stability of active constituents of hops (Humulus lupulus) strobiles and their ethanolic extracts during storage. Eur J Med Plants 2014; 4: 1302-1312
  • 33 ICH (The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use). Guideline: Validation of analytical Procedures: Text and Methodology, Q2(R1). Geneva: ICH; 2005: 1-13
  • 34 Dhooghe L, Naessens T, Heyerick A, De Keukeleire D, Vlietinck AJ, Pieters L, Apers S. Quantification of xanthohumol, isoxanthohumol, 8-prenylnaringenin, and 6-prenylnaringenin in hop extracts and derived capsules using secondary standards. Talanta 2010; 83: 448-456
  • 35 Stevens JF, Page JE. Xanthohumol and related prenylflavonoids from hops and beer: To your good health!. Phytochemistry 2004; 65: 1317-1330
  • 36 Sticher O, Heilmann J, Zündorf I. Pharmakognosie Phytopharmazie, 10th edition. Stuttgart: Wissenschaftliche Verlagsgesellschaft mbH; 2015: 974-975
  • 37 Biendl M. A milestone in Xanthohumol research: effects confirmed in first studies on humans. Hopfen-Rundschau Internat Ed 2017; 1: 72-76